UNIVERSIDADE ESTADUAL DE FEIRA DE SANTANA PROGRAMA

Transcription

UNIVERSIDADE ESTADUAL DE FEIRA DE SANTANA
PROGRAMA DE PÓS-GRADUAÇÃO EM BIOTECNOLOGIA
JULIANA FRAGA VASCONCELOS
AVALIAÇÃO DOS EFEITOS DO TRATAMENTO COM O FATOR ESTIMULADOR
DE COLÔNIAS DE GRANULÓCITOS (G-CSF) NA CARDIOPATIA CHAGÁSICA
CRÔNICA EXPERIMENTAL
Salvador, BA
2012
JULIANA FRAGA VASCONCELOS
AVALIAÇÃO DOS EFEITOS DO TRATAMENTO COM O FATOR ESTIMULADOR
DE COLÔNIAS DE GRANULÓCITOS (G-CSF) NA CARDIOPATIA CHAGÁSICA
CRÔNICA EXPERIMENTAL
Tese apresentada ao Programa de Pós-graduação em Biotecnologia, da Universidade
Estadual de Feira de Santana como requisito parcial para obtenção do título de Doutor
em Biotecnologia.
Orientadora: Profa. Dra. Milena Botelho Pereira Soares
Salvador, BA
2012
AGRADECIMENTOS
À Dra Milena Botelho Pereira Soares, chefe do Laboratório de Engenharia Tecidual e
Imunofarmacologia, por todo apoio e compreensão, pelo convívio e pela confiança em mim
depositada. Agradeço também pelo tempo investido, as horas de discussão e pela orientação
concedida.
À Dra Simone Garcia Macambira, pela amizade, pelas discussões científicas, pelo carinho e
pelo grande incentivo durante toda a minha formação.
Ao Dr Ricardo Ribeiro-dos-Santos, chefe do Centro de Biotecnologia e Terapia Celular, pelo
incentivo durante cada etapa do trabalho.
Agradeço ao apoio dos companheiros do LETI e do CBTC, amigos de tantas agruras, por toda
amizade, e pela colaboração durante cada etapa do trabalho. Em especial a Alice Kiperstock,
Daniele Brustolim, Fabrício Souza, Flávia Maciel, Fernando Costa, Matheus Sá, Ricardo
Lima, Sheilla Andrade e Siane Campos.
Ao amigo Bruno Solano pela indescritível ajuda na finalização dos últimos experimentos.
À Adriano Alcântara, Geraldo Pedral, Carine Machado, Carla Kaneto e Cássio Meira pela
ajuda técnica e científica durante o desenvolvimento do trabalho.
Agradeço em particular as alunas de iniciação científica que me acompanharam ao longo dos
experimentos e me deram um grande apoio, Fabiana Mascarenhas, Letícia Garcia e Thaise
Lins.
Agradeço também às amigas que, mesmo indiretamente, participaram desse trabalho, Sheila
Resende e Camila Vasconcelos, pois me suportaram nos piores momentos.
À coordenação da pós-graduação em Biotecnologia pelo apoio e pela preocupação na
formação de recursos humanos.
Ao secretário da PPgBiotec, Helton Ricardo, pela atenção, disponibilidade, dedicação,
organização e pela enorme paciência e gentileza para com os alunos.
À Lucyvera Imbroinise em especial, pelo trabalho de secretaria do LETI e pelo ombro amigo
sempre encontrado por mim.
Aos colegas da Pós- graduação em Biotecnologia.
Agradeço a Universidade Estadual de Feira de Santana pela excelência de seus professores.
Ao Centro de Pesquisa Gonçalo Moniz da Fundação Oswaldo Cruz e ao Centro de
Biotecnologia e Terapia Celular do Hospital São Rafael, pela disponibilidade de excelente
estrutura física e material.
À FAPESB e CAPES pela concessão das bolsas, imprescindíveis para realização deste
trabalho.
AGRADECIMENTOS ESPECIAIS
À Deus, que me presenteia a cada dia com força, paciência e perseverança.
Aos meus pais por proporcionarem minha formação intelectual e moral.
À minha avó Maria Janete Fortunato Fraga, um exemplo de vida.
Às minhas filhas, Giulia e Rafaela, pela compreensão e amor incondicional, pelo apoio e pela
eterna cobrança de quando essa tese ia acabar...
À Claudio Roberto Costa por sempre estar ao meu lado, nos momentos alegres ou tristes, e
por me acalmar nos momentos de surtos apocalípticos.
Nesse momento me alegro em dizer que esta conquista é nossa!
"Não existe um caminho para a felicidade. A felicidade é o caminho."
Mahatma Gandhi
RESUMO
A cardiopatia chagásica crônica (CChC) acomete cerca de 30% dos indivíduos
infectados pelo Trypanosoma cruzi, e resulta da destruição progressiva do miocárdio, cujos
pacientes evoluem para insuficiência cardíaca e óbito. A escassez de alternativas terapêuticas
e a importância socioeconômica da CChC ressaltam a necessidade da busca de novos
tratamentos para esta doença. O Fator Estimulador de Colônias de Granulócitos (G-CSF) é
uma citocina já utilizada na clínica que tem sido estudada quanto ao seu potencial terapêutico
em modelos experimentais de outras doenças cardíacas. Neste trabalho avaliamos os efeitos
do tratamento com G-CSF em um modelo experimental de CChC. Camundongos C57BL/6
chagásicos crônicos, seis meses pós- infecção, foram tratados com G-CSF por 3 ciclos (200
µg/kg/dia por 5 dias), com intervalos de uma semana entre cada ciclo, após aprovação pelo
comitê de Ética local. Os animais infectados apresentaram alterações cardíacas graves, que
foram parcialmente revertidas pelo uso do G-CSF, além de melhora da função
cardiorrespiratória avaliada por ergoespirometria. Observamos a redução do infiltrado
inflamatório com redução da produção de CXCL12, ICAM-1 e syndecam-4 e do aumento da
apoptose de leucócitos no coração dos animais tratados com G-CSF. Demonstramos que tanto
a presença de macrófagos quanto a produção de galectina-3 foi reduzida no grupo G-CSF, o
que esteve associado com a diminuição da fibrose. O tratamento com G-CSF modulou a
produção de IFN-γ e TNFα, com redução da expressão gênica de Tbet, além de proporcionar
pequena redução de IL-17, sem modificar significativamente a produção de IL-4 ou GATA-3.
O tratamento com G-CSF induziu o aumento da migração de células Treg da medula óssea
para a periferia, fato demonstrado pelo aumento dessa população celular no baço e no coração
dos animais tratados. A presença de células Foxp3+ produtoras de IL-10, assim como a
produção elevada de IL-10 no coração e no baço dos animais tratados, parece contribuir para
a modulação da resposta inflamatória. Apesar da redução da resposta inflamatória, também
observamos a redução da carga parasitária no coração dos animais tratados com G-CSF. A
atividade do G-CSF sobre o parasito foi confirmada em ensaios in vitro, onde foi encontrada a
redução da viabilidade de tripomastigotas e a redução do número de macrófagos infectados e
de amastigotas/macrófago.
Em conclusão, o G-CSF exerce efeitos múltiplos em
camundongos infectados por T. cruzi, atuando como agente modulador da resposta imune e
promovendo a redução da carga parasitária, possibilitando a melhora da função cardíaca no
modelo de cardiopatia chagásica crônica, o que encoraja o estudo da avaliação do seu
potencial terapêutico em pacientes chagásicos.
Palavras-Chave: Cardiopatia chagásica crônica. G-CSF. Imunomodulação. Células T
regulatórias. Trypanosoma cruzi.
ABSTRACT
Chronic Chagasic cardiomyopathy (CChC) affects approximately 30% of individuals
infected with Trypanosoma cruzi, and results from a progressive destruction of the
myocardium, causing the development of heart failure and death of the patients. The lack of
therapeutic alternatives and the socio-economic impact of the CChC emphasize the
importance of developing new treatments for this disease. The G-CSF, a cytokine already
used in clinical practice, has been studied for its therapeutic potential in other experimental
models of cardiac diseases. In this study we evaluated the therapeutic effects of G-CSF in a
model CChC. After approval by the local ethics committee, C57BL/6 mice infected with T.
cruzi for six months were treated with G-CSF in 3 courses (200 µg / kg / day for 5 days) with
an interval between the cycles of one week. Infected animals had severe conduction
disturbances, partially reversed by the use of G-CSF. In addition, an improvement of the
cardiorespiratory function was observed, as assessed by spirometry. We observed a reduction
of the inflammatory infiltrate associated with reduced production of SDF-1, ICAM-1 and
syndecam-4 and increasing apoptosis of leukocytes in the hearts of the animals treated with
G-CSF. We also found that both macrophages, as well as the production of galectin-3, were
reduced in the group G-CSF, which is associated with the decrease of fibrosis in the group.
Treatment with G-CSF modulates the production of IFN-γ and TNFα, reduced the gene
expression of Tbet, while providing a slight reduction in IL-17 without any significant
changes in IL-4 or GATA-3. Treatment with G-CSF induced the migration of Treg cells from
the bone marrow to the periphery, as demonstrated by increasing this population in spleen and
heart of the treated animals. The presence of IL-10-producing Foxp3+ cells, as well as an
increased production of IL-10 on heart and spleen, suggests that this cytokine contributes to
the modulation of the inflammatory response. Despite the reduction of the inflammatory
response, we also observed a reduction in parasitic load in the hearts of the animals treated
with G-CSF. The activity of G-CSF on the parasite was confirmed in experiments in vitro, in
which we found a reduction in the viability of trypomastigotes and in the number of infected
macrophages and amastigotes/macrophage. In conclusion, G-CSF exerts multiple effects in
mice infected with T. cruzi, acting as a modulatory agent in the immune response and
promoting the reduction of parasite load, allowing the improvement of cardiac function in
chronic Chagas disease model, results which encourage the study of its therapeutic potential
in patients with Chagas disease.
Keywords: Chronic Chagasic Cardiomiopathy. G-CSF. Immunomodulation. Treg cells.
Trypanosoma cruzi.
LISTA DE FIGURAS
Figura 1
Figura 2
Figura 3
Figura 4
Figura 5
Figura 6
Figura 7
Representação esquemática das formas evolutivas do T. cruzi no
hospedeiro invertebrado e vertebrado.
Imagens representativas de alterações patológicas encontradas em
pacientes chagásicos crônicos.
A resposta imune nas formas cardíaca e indeterminada da doença de
Chagas
Modelo computacional proposto para a estrutura tridimensional da
molécula do G-CSF humano.
Influência das citocinas durante o desenvolvimento das células
sanguíneas na medula óssea.
Efeitos do G-CSF que sustentam sua ação na tolerância central e
periférica.
Representação esquemática da ação direta e indireta do G-CSF sobre o
tecido cardíaco promovendo modulação da resposta inflamatória local
em modelo de CChC experimental.
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15
17
25
26
28
83
LISTA DE ABREVIAÇÕES
AMPc
ANOVA
APC
A-V
BMSC
BSA
CChC
CD
cDNA
CSF
CT
Cx43
CXCR
DAPI
ECA
ECG
EDTA
ELISA
EPO
FDA
FITC
G-CSF
G-CSFR
GM-CSF
HE
HLA
IC
ICAM-1
ICC
IFN-
Ig
IL
JAK
Kda
LPS
MCP
MHC
MIP
NK
OMS
PAF
PBS
PMSF
ROS
RT-PCR
SDF-1
Monofosfato de adenosina cíclico
Análise de variância
Célula apresentadora de antígeno
Átrio-ventricular
Células-tronco da medula óssea
Albumina de soro bovino
Cardiopatia Chagásica Crônica
Células dendríticas
DNA complementar
Fator estimulador de crescimento de colônias
Células-tronco
Conexina 43
Receptor de quimiocina CXC
4´,6-diamidino-2-feninlidole
Enzima conversora da angiotensina
Eletrocardiografia
Ácido etilenodiamino tetra-acético
Ensaio imunoenzimático
Eritropoietina
Órgão regulador da administração de alimentos e medicamentos norteamericano
Isotiocianato de fluoresceína
Fator estimulador de colônias de granulócitos
Receptor do G-CSF
Fator estimulador de colônias de granulócitos e macrófagos
Hematoxilina e Eosina
Antígeno leucocitário humano
Insuficiência cardíaca
Molécula de adesão intercelular 1
Insuficiência cardíaca congestiva
Interferon gama
Imunoglobulina
Interleucina
Proteína janus quinase
QuiloDalton
Lipopolisacarídeo
Proteína quimiotática de monócitos
Complexo de histocompatibilidade principal
Proteína inflamatória de macrófagos
Célula matadora natural
Organização Mundial de Saúde
Fator ativador de plaquetas
Solução tampão fosfato
Fenilmetilsulfonilfluorido
Espécies reativas de oxigênio
Reação em cadeia da polimerase via transcriptase reversa
Fator derivado do estroma 1
SMA
STAT
TGF-β
Th
TMB
TNF
Treg
TUNEL
VCO2
VO2
Actina de músculo liso
Fator de transdução e ativação da transcrição
Fator transformador do crescimento β
Linfócito T auxiliar
Tetrametilbenzodine
Fator de necrose tumoral
Linfócito T regulatório
Ensaio de marcação de desoxinucleotidiltransferase terminal dUTP
Produção de dióxido de carbono
Consumo de oxigênio
SUMÁRIO
1 INTRODUÇÃO GERAL .............................................................................
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OBJETIVOS .................................................................................................
32
2 CAPÍTULO I ................................................................................................
33
2.1
INTRODUÇÃO ................................................................................................
34
2.2
MATERIAIS E MÉTODOS ...............................................................................
34
2.3
RESULTADOS ................................................................................................
36
2.4
DISCUSSÃO ....................................................................................................
37
REFERÊNCIAS ................................................................................................
40
3 CAPÍTULO II ................................................................................................. 42
3.1
INTRODUÇÃO ................................................................................................... 45
3.2
MATERIAIS E MÉTODOS ...............................................................................
46
3.3
RESULTADOS ...................................................................................................
53
3.4
DISCUSSÃO .....................................................................................................
66
REFERÊNCIAS ................................................................................................
71
4 DISCUSSÃO GERAL ....................................................................................
77
5 CONCLUSÕES .............................................................................................
85
6 REFERÊNCIAS .............................................................................................
86
ANEXOS .......................................................................................................
101
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1 INTRODUÇÃO GERAL
1.1 A DOENÇA DE CHAGAS
A doença de Chagas, uma antropozoonose antes restrita às Américas, tem impacto
sanitário e socioeconômico significativo no continente americano. Dados da Organização
Mundial de Saúde (OMS) em 2002 indicam a presença de 17 a 18 milhões de indivíduos
infectados no mundo, porém, números recentes da própria OMS (2010) estimam a redução
nesse número para aproximadamente 10 milhões de pessoas infectadas. De acordo com
Siqueira-Batista e colaboradores (2011), as ações de controle da doença de Chagas
desenvolvidas ao longo do século XX, especialmente a redução da transmissão vetorial em
diversos países latino-americanos, têm permitido a transição epidemiológica ao menos em
determinadas regiões do continente, sendo observada tendência à queda do número de casos
em alguns países, tais como Argentina, Brasil, Chile, Uruguai e Venezuela, justificando a
redução do número de infectados. A implantação de programas de controle da transmissão
vetorial e em bancos de sangue foi responsável pela redução da morbidade e mortalidade
decorrente da forma crônica da doença. Entretanto, a OMS alerta que ainda existem cerca de
25 milhões de pessoas expostas ao risco de infecção na América Latina e, mesmo que a
transmissão fosse interrompida, restariam milhões de pacientes com doença de Chagas sob o
risco de morte. Desses pacientes, dois milhões se encontram na fase crônica sintomática da
doença de Chagas e correm risco de irem a óbito pela falta de acesso a ferramentas adequadas
para o diagnóstico e o tratamento desta doença (MONCAYO, 2003; WHO, 2010).
No ano de 2009 foi comemorado o centenário da descoberta da tripanosomíase
americana por Carlos Chagas, que descreveu o agente etiológico, o vetor, os sinais clínicos
em humanos e, ainda, espécies animais que funcionam como reservatórios naturais da doença.
A importância médica dos triatomíneos como vetor da doença de Chagas é reconhecida,
possuindo a família Reduviidae, subfamília Triatominae, mais de cem espécies das quais
várias são vetores ou vetores potenciais da doença, sendo as espécies de maior importância na
transmissão vetorial o Triatoma infestans, T. dimidiata, T. sórdida, Rhodnius prolixus e
Panstrongylus megistus (GARCIA, GONZALEZ &AZAMBUJA, 2000). Embora a doença de
Chagas tenha sido descrita pela primeira vez em 1909, um estudo recente realizado por
paleoparasitologistas revelou a presença de DNA de T. cruzi em múmias colombianas de
cerca de 9000 anos de idade, mostrando que essa é uma doença que aflige a população
humana há milhares de anos (ARAÚJO et al., 2009). Em decorrência de migrações
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populacionais, a doença de Chagas, classicamente considerada como uma enfermidade rural
passou a atingir centros urbanos (COSTA & LORENZO, 2009).
1.1.1 O Trypanosoma cruzi
O Trypanosoma cruzi é um hemoflagelado pertencente à família Trypanosomatidae cuja
principal característica é a presença de flagelo e de uma mitocôndria modificada denominada
cinetoplasto (SOUZA, 2000). A observação, por microscopia óptica, do parasito permite a
identificação de três formas evolutivas bem definidas, de acordo com a posição flagelar:
tripomastigota e amastigota durante seu ciclo de vida nos hospedeiros mamífero e
invertebrado, e a forma epimastigota no tubo digestivo do vetor. A figura 1 ilustra o ciclo do
T. cruzi tanto no hospedeiro invertebrado quanto no vertebrado, representando suas formas
evolutivas. A forma epimastigota se multiplica por divisão binária no trato gastrointestinal do
triatomíneo e se diferencia na forma tripomastigota metacíclica, que é a forma infectante do
hospedeiro vertebrado. Esta é eliminada junto com as fezes e urina do vetor triatomíneo, sobre
a pele do hospedeiro vertebrado durante o repasto sanguíneo, podendo penetrar através do
local da picada ou mucosas e invadir células nucleadas. No interior das células do hospedeiro
vertebrado, os tripomastigotas metacíclicos se diferenciam na forma amastigota, que se
replica por fissão binária. Após alguns ciclos de multiplicação, os amastigotas se diferenciam
em tripomastigotas sanguíneos, ocorrendo então o rompimento das células e liberação dos
parasitos para o meio extracelular ou na corrente sanguínea, podendo assim migrar e invadir
novas células do hospedeiro ou serem sugados pelo inseto vetor, reiniciando o ciclo do
parasito (DIAS & COURA, 1997; BRENER, ANDRADE & BARRAL-NETO, 2000).
Figura 1. Representação esquemática das formas evolutivas do Trypanosoma cruzi no hospedeiro
invertebrado e vertebrado.
Fonte: Adaptada de Buscaglia et al.,2006.
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1.1.2 Formas clínicas
A interação parasito-hospedeiro é bastante dinâmica na doença de Chagas, sendo o
resultado da associação de fatores múltiplos relacionados ao T. cruzi, tais como a cepa, a
virulência e o tamanho do inóculo, assim como a características do hospedeiro (seres
humanos), como idade, sexo e raça, além de fatores ligados ao ambiente, como condição
física da habitação e o grau de preservação do ambiente natural. De uma maneira geral, após a
infecção pelo T. cruzi em humanos, há o desenvolvimento da fase aguda da doença, muitas
vezes subclínica, que é transitória e caracterizada por parasitemia patente, onde são
encontradas formas tripomastigotas no sangue periférico e formas amastigotas se
multiplicando intracelularmente (BRENER, ANDRADE & BARRAL-NETO, 2000). No
local da picada pode-se desenvolver uma lesão volumosa, o chagoma, local eritematoso e
edematoso, se a picada for perto do olho é frequente a conjuntivite com edema da pálpebra,
também conhecido por sinal de Romaña (PUNUKOLLU et al., 2007; DUTRA et al., 2009). A
duração da fase aguda é, em média, de 2 a 4 meses, e pode ocorrer o aparecimento de outros
sintomas, tais como febre, linfadenopatia, anorexia, hepatoesplenomegalia, miocardite
brandas e, mais raramente, meningoencefalite (PUNUKOLLU et al., 2007; MAYA et al.,
2010; PARKER & SETH, 2011).
Dos indivíduos infectados cronicamente pelo T. cruzi, cerca de 70% são assintomáticos
e não apresentam alteração cardíaca ou digestiva. Estes pacientes apresentam a forma
indeterminada da doença de Chagas, caracterizada pela ausência de manifestações clínicas
relevantes. Como esses pacientes não exibem alterações eletrocardiográficas significativas ou
dilatação do coração, esôfago ou cólon, observado no exame de raios-X, em geral são
diagnosticados apenas por triagem em banco de sangue por apresentar testes sorológicos
positivos para o T. cruzi. Os indivíduos infectados evoluem, em cerca de 30% dos casos, para
uma forma sintomática, que apresenta sintomas cardíacos e/ou digestivos. Desses pacientes,
cerca de 8-10% desenvolvem a forma digestiva da doença de Chagas, com dilatação do
esôfago e cólon e é, supostamente, o resultado da destruição neuronal do trato gastrointestinal
(DUTRA, ROCHA & TEIXEIRA, 2005). Os 20-25% restantes evoluem para um
acometimento cardíaco de maior ou menor gravidade, desenvolvendo, em graus clínicos
variados, a cardiomiopatia chagásica crônica (CChC) que é a principal causa de morte em
indivíduos chagásicos (DIAS & COURA, 1997).
Na figura 2 são ilustradas algumas imagens de alterações patológicas encontradas em
pacientes com CChC com forma cardíaca ou com mega-síndromes. A CChC pode ocorrer em
15
períodos que variam de 5 a 30 anos após a infecção primária e é caracterizada por uma
resposta inflamatória intensa e destruição progressiva do tecido cardíaco, causando
anormalidades da condução cardíaca e arritmias (DIAS & DIAS, 1989). O aneurisma apical é
característico da cardiopatia chagásica, secundário ao comprometimento do sistema de
condução, pela presença de áreas inativadas ou áreas aonde o estímulo elétrico chegaria com
atraso, as quais ficariam mais susceptíveis aos efeitos da pressão intraventricular durante a
sístole (ANDRADE, 1983). Paralelamente, ocorre o afinamento progressivo do músculo
cardíaco, com dilatação das cavidades do coração, tendo como consequência a insuficiência
cardíaca (IC) – incapacidade de bombear adequadamente o sangue para o organismo que,
frequentemente, tem um curso fatal (PRATA, 2001).
Figura 2.Imagens representativas de alterações patológicas encontradas em pacientes chagásicos
crônicos. A) Cardiomegalia moderada; B) Cardiomegalia grave com congestão pulmonar; C)
Aneurisma apical; D) Afinamento da parede do ventrículo esquerdo; E) Megaesôfago e
megaestômago; F) Megaesôfago e megaduodeno; G e H) Megacólon.
Fontes: Higuchi et al., 2003; Tarleton et al., 2007; Coura & Borges-Pereira, 2010;
http://www.isradiology.org ; http://www.fiocruz.br/chagas.
1.1.3 Imunopatogênese
Todos os organismos vivos possuem mecanismos adaptativos para responder a
estímulos agressivos no sentido de manter o equilíbrio homeostático. Esta resposta complexa
inclui uma série de alterações bioquímicas, fisiológicas e imunológicas, coletivamente
denominada inflamação, sendo sua principal função o encaminhamento de leucócitos à lesão e
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ativação para que desempenhem suas funções normais de defesa do hospedeiro (KUMAR,
ABBAS & FAUSTO, 2005). Na doença de Chagas não é diferente, e durante a fase aguda da
doença há forte ativação da resposta imune inata como sendo a primeira linha de defesa contra
a invasão do microorganismo, com participação de macrófagos, células natural killer (NK),
eosinófilos e mastócitos (BRENER & GAZZINELLI, 1997; JANEWAY et al., 2001). As
células ativadas do sistema fagocítico mononuclear iniciam a cascata de eventos de fase
aguda, secretando, citocinas da família da interleucina (IL) -1 e do fator de necrose tumoral
(TNF). Estas moléculas têm ação pleiotrópica, tanto a nível local como sistêmico,
estimulando a produção de IL-6 e IL-8 e das proteínas inflamatória de macrófago 1 (MIP1/CCL3) e quimiotática (MCP/CCL2) de monócitos, além de aumentar também a própria
síntese de IL-1 e TNF (ABBAS, LICHTMAN & PILLAI, 2007; BAUMANN & GAULDIE,
1994). O TNF e a IL-1, sinergisticamente ativam a ciclooxigenase 2 com geração de
prostaglandina E2, capaz de causar vasodilatação e aumento da percepção da dor, além de
aumentarem a expressão do fator tecidual e fator ativador de plaquetas (PAF), aumentando o
risco de distúrbios de coagulação.
Na infecção pelo Trypanosoma cruzi, as respostas imune inata e adaptativa
coordenadas, efetuadas por macrófagos, linfócitos B e linfócitos T tendem a controlar a
parasitemia na fase aguda da doença. As células do sistema fagocítico mononuclear, os
monócitos e macrófagos, são fundamentais no controle do parasito no sangue e nos tecidos.
Entretanto, apesar de servirem como células efetoras do sistema imune, realizando a
fagocitose, servem também como células hospedeiras responsáveis pela disseminação do
parasito. Após a ativação linfocitária, os macrófagos se tornam elementos fundamentais no
controle do parasito, através da produção de citocinas pró-inflamatórias e substâncias
microbicidas. As células NK possuem papel fundamental no controle da infecção por ser
fonte primária inicial de interferon- (IFN-) na fase inicial da infecção, aumentando a síntese
de IL-12 por macrófagos. A IL-12 estimula a produção de IFN- e induz a diferenciação de
linfócitos T helper (Th) 0 em Th1, promovendo maior resistência a infecção aguda (REED et
al., 1994). No homem, bem como em modelos experimentais, a infecção pelo T. cruzi
mobiliza diferentes compartimentos do sistema imune, induzindo respostas humorais e
celulares específicas contra o parasito (BRENER & GAZZINELLI, 1997). A estimulação da
resposta imune é crucial na redução da carga parasitária, mas, por outro lado, pode contribuir
para o agravamento dos sintomas clínicos.
A fase crônica sintomática do processo inflamatório se caracteriza pela presença de
infiltrado celular mononuclear, incluindo macrófagos, linfócitos e plasmócitos, levando a
17
destruição tecidual (Figura 3) pela persistência do agente nocivo, das células inflamatórias ou
ambos. Já na forma indeterminada há a formação de um ambiente regulador que proporciona
o equilíbrio dinâmico entre a resposta inflamatória protetora e o parasito de maneira a
restringir a lesão tecidual. A compreensão dos mecanismos relacionados ao estabelecimento
de diferentes formas clínicas da doença de Chagas é um ponto crucial para nortear futuras
intervenções
profiláticas
ou
terapêuticas.
Apesar
dos
processos
que
levam
ao
desenvolvimento da forma crônica da doença não estarem completamente esclarecidos, é
indiscutível o papel da resposta inflamatória na CChC (ANDRADE, 1983; MORRIS et al.,
1990). Histologicamente, a CChC é caracterizada pela presença de infiltrados de células
mononucleares compostos por macrófagos, células B, e células T, frequentemente aderidas a
miócitos e que induzem a miocitólise (DIAS & COURA, 1997). Vários estudos indicam a
participação de linfócitos Th1 e produção aumentada de IFN-, semelhante a uma reação de
hipersensibilidade tardia (SOARES et al., 2001; DUTRA, ROCHA & TEIXEIRA, 2005). De
fato, a associação entre progressão para formas crônicas cardíacas e a produção aumentada de
IFN- foi observada em pacientes chagásicos, bem como uma diminuição na produção de IL10 (CORREA-OLIVEIRA et al., 1999; GOMES et al., 2003).
Figura 3. A resposta imune nas formas cardíaca e indeterminada da doença de Chagas
Fonte: http://www.fiocruz.br/chagas/cgi/cgilua.exe/sys/start.htm?sid=170
Durante algum tempo, acreditava-se na ausência de parasitos teciduais durante a fase
crônica da doença (HIGUCHI, 1999), porém os avanços de técnicas de biologia molecular
permitiram a detecção da presença do DNA do T. cruzi em biópsia de corações humanos
(AÑEZ et al., 1999; BENVENUTI et al., 2008), indicando que a persistência do parasito pode
18
ser um fator importante para o desenvolvimento da CChC. A presença do parasito ou de seus
antígenos indefinidamente nos tecidos do hospedeiro estimula constantemente a resposta
imune específica, desencadeando o processo inflamatório exacerbado com conseqüentes
efeitos deletérios ao hospedeiro. Tanto a resposta imune ao T. cruzi quanto a auto-reatividade
a componentes cardíacos já foram apontadas como possíveis mecanismos desencadeadores do
processo patológico observado na CChC (SOARES, PONTES-DE-CARVALHO &
RIBEIRO-DOS-SANTOS,
2001;
KIERSZENBAUM,
2003;
DUTRA,
ROCHA
&
TEIXEIRA, 2005), mas nenhuma se mostrou suficiente para explicar cada caso. Tem sido
dada atenção especial à hipótese do mimetismo molecular, onde a presença de auto-anticorpos
que reagem contra antígenos cardíacos, como os receptores β1 adrenérgico e M2 muscarínico
por homologia a antígenos do T. cruzi, promovem anormalidades na condução elétrica no
coração(BORDA et al., 1984; STERIN-BORDA et al., 1991; CUNHA-NETO, 1995;
MASUDA et al., 1998; HERNANDEZ et al., 2003; MACIEL et al., 2012). Rocha e
colaboradores (2006) sugerem que a infecção crônica pelo T. cruzi promove alterações na
densidade desses receptores cardíacos, que em conjunto com a fibrose observada, promovem
distúrbios de condução cardíaca.
A análise da expressão diferencial de extensa quantidade de genes por microarranjo de
DNA em corações de animais chagásicos crônicos demonstrou que a infecção crônica pelo T.
cruzi induz produção aumentada de diversos genes associados ao processo inflamatório e a
fibrose em si, quando comparados aos animais normais, evidenciando a importância tanto da
inflamação quanto da fibrose na patogênese da CChC (SOARES et al., 2010, 2011). Além do
próprio dano causado pela infecção do T. cruzi em células cardíacas e a persistência da
estimulação antigênica que mantém a resposta imune local, a hipótese neurogênica também é
proposta como agravante dos distúrbios de condução apresentados por pacientes com CChC.
Segundo essa hipótese, a desnervação autonômica devida à destruição de células ganglionares
parasimpáticas no coração causaria os distúrbios cardíacos apresentados, porém esses dados
são controversos, já que não há correlação entre a lesão neuronal cardíaca e as manifestações
clínicas da doença (MARIN-NETO et al., 1999).
A existência de epítopos antigênicos compartilhados entre o T. cruzi e células
mamíferas é bem descrita na literatura, sugerindo a quebra de tolerância por reatividade
cruzada por componentes do hospedeiro, como miosina cardíaca, receptor adrenérgico β1 e
muscarínico M2 (RIZZO, CUNHA-NETO & TEIXEIRA, 1989; CUNHA-NETO et al.,1995;
CUNHA-NETO et al., 1996; ROCHA et al., 2006). Para comprovar a reatividade cruzada
entre antígenos do parasito e antígenos cardíacos, Girones & Fresno (2003) purificaram
19
células T de animais infectados com T. cruzi e as injetaram em camundongos singenéicos
não-infectados, observando o desenvolvimento de uma cardite semelhante à encontrada nos
animais chagásicos, relacionada com a interação dessas células T ativadas e antígenos
cardíacos. Desta forma, o T. cruzi deve funcionar como um gatilho, sendo capaz de estimular
a resposta imunológica através da presença de parasitos ou de seus peptídeos imunogênicos
que iniciariam o processo inflamatório.
1.1.4 Tratamento da doença de Chagas
1.1.4.1 Quimioterápicos
A doença de Chagas é considerada uma doença tropical negligenciada, assim como a
malária, a dengue e a leishmaniose, apresentando-se como um grave problema de saúde
pública. Embora exista financiamento para pesquisas relacionadas a essas doenças,
infelizmente o conhecimento produzido não se reverte em avanços terapêuticos, como, por
exemplo, novos fármacos, métodos diagnósticos e vacinas (FRANCO-PAREDES,
BOTTAZZI & HOTEZ, 2009; SIQUEIRA-BATISTA et al., 2011). Segundo o Ministério da
Saúde (2010), uma das razões para esse quadro é o baixo interesse da indústria farmacêutica
nesse tema, justificado pelo potencial reduzido de retorno lucrativo para a indústria, uma vez
que a população atingida é de renda baixa e presente, em sua maioria, nos países em
desenvolvimento. As doenças negligenciadas não só prevalecem em condições de pobreza,
mas também contribuem para a manutenção do quadro de desigualdade, já que representam
forte entrave ao desenvolvimento (de MEIS et al., 2009). Os custos associados à doença de
Chagas são variáveis nos diferentes países devido às diferenças nos custos de atendimento
médico. Como ela é uma doença debilitante e incapacitante e que em geral os pacientes
chagásicos são de nível socioeconômico menos privilegiado, os custos de atendimento
acabam recaindo sobre o estado (SCHMUÑIS, 2000).
O fato da doença de Chagas ser uma doença negligenciada é ilustrado pela falta de
novas drogas, já que por mais de três décadas, somente dois medicamentos, nifurtimox e
benzonidazol, foram disponibilizados para seu tratamento (MARIN-NETO et al., 2009).
Segundo Costa e colaboradores (2011), nenhum destes compostos é ideal por que: (i) não são
ativos durante a fase crônica da doença e apresentam sérios efeitos colaterais, (ii) requerem
administração por longos períodos de tempo sob supervisão médica, (iii) há grande variação
na susceptibilidade de isolados do parasito a ação destas drogas, (iv) populações de parasitos
resistentes a ambos compostos têm sido relatadas, (v) apresentam alto custo, e (vi) não há
20
formulações pediátricas. Esses compostos apresentam mecanismos de ação citotóxicos, que
inicialmente envolvem uma ou mais reações de redução do grupo nitro, seguida por inibições
de várias enzimas, que são necessárias às exigências energéticas do parasito e produção de
radicais livres (TROSSINI, 2004). No entanto entre os dois quimioterápicos, o nifurtimox
(Lampit, Bayer 2503) por produzir metabólitos de oxigênio reduzidos altamente tóxicos, não
é mais utilizado em diversos países, incluindo o Brasil. O benzonidazol se torna, portanto, o
medicamento de escolha utilizado para o tratamento da Doença de Chagas, cuja ação
tripanocida é capaz de eliminar o parasito quando administrado durante a fase aguda da
doença, curando cerca de 75% dos pacientes tratados (SOSA-ESTANI & SEGURA, 1999;
RASSI et al., 2007; MARIN-NETO et al., 2009).
Apesar da doença de Chagas ser de notificação compulsória (Consenso Brasileiro em
Doença de Chagas, 2005), muitas vezes os sintomas de fase aguda são confundidos com um
simples resfriado, não sendo o paciente diagnosticado nessa fase, o que proporciona o
desenvolvimento da fase crônica, dificultando o seu tratamento (BILATE & CUNHA-NETO,
2008). Como o benzonidazol tem ação tripanocida, e na fase crônica da doença não há muitos
parasitos circulantes, sua utilização após a fase aguda da doença foi questionada por muito
tempo (BRITO et al., 2001; CANÇADO et al., 2002; CALDAS et al, 2008). Uma das
primeiras publicações sugerindo a importância dessa droga na melhora da CChC foi descrita
por Garcia e colaboradores (2005), que demonstraram uma relação direta entre a diminuição
do parasitismo tecidual e a diminuição do infiltrado inflamatório e do percentual de área
ocupada por tecido fibrótico, bem como uma melhora funcional, avaliado por
eletrocardiografia e ergoespirometria, em animais tratados com benzonidazol em fase crônica.
Recentemente foi iniciado um estudo internacional, multicêntrico e duplo-cego, visando
avaliar a eficácia da utilização do benzonidazol na fase crônica da doença de Chagas
(MARIN-NETO et al., 2009).
1.1.4.2 Tratamento da insuficiência cardíaca
Uma vez que a grande maioria dos pacientes não é tratada durante a fase aguda da
doença de Chagas, os pacientes que evoluem para a fase crônica sintomática precisam de
tratamento que, apesar de não ser capaz de reverter as causas das alterações cardíacas, é
importante para o retardo da progressão da CChC. O tratamento da insuficiência cardíaca
crônica é voltado para o alívio dos sintomas e melhora da qualidade de vida do paciente,
envolvendo medidas não-farmacológicas, farmacológicas e cirúrgicas, dependendo do estágio
da síndrome. A identificação da etiologia e a remoção da causa subjacente são o tratamento
21
mais desejável, como a correção cirúrgica de malformações congênitas ou o tratamento da
hipertensão arterial, o que não é possível com a IC de etiologia chagásica. O tratamento
medicamentoso tem apresentado evidências consistentes de redução da morbimortalidade
desses pacientes. Aconselhamento, instruções de saúde e programas de avaliação constante
ajudam os pacientes a se tratar e a lidar com seu regime de tratamento (PORTH, 2004).
Entre as medicações usadas no tratamento da IC, estão os diuréticos, digoxina,
inibidores da enzima conversora de angiotensina (ECA) e β-bloqueadores. De acordo com as
Diretrizes da Sociedade Brasileira de Cardiologia para o diagnóstico e tratamento da
Insuficiência Cardíaca, revisada em 2002, os inibidores ECA são o grupo de fármacos de
maior importância, sendo extensa a lista de inibidores da ECA disponíveis para uso clínico,
incluindo captopril, enalapril e quinapril, dentre outros. A IC acarreta a diminuição do fluxo
sanguíneo renal, causando aumento da produção de renina pelos rins, juntamente com o
aumento da concentração de angiotensina II, contribuindo para vasoconstricção excessiva e
consequente aumento da pressão arterial. A angiotensina II também proporciona a regulação
da pressão arterial à longo prazo, pois estimula a produção de aldosterona pela supra-renal,
com aumento da retenção de sal e água pelos rins (GUYTON, 1988). Ambos os mecanismos
tornam maior a carga de trabalho do coração, justificando a terapia com inibidores da ECA
para aliviar os sintomas e aumentar a sobrevida do paciente.
Os digitálicos permanecem como o agente mais comumente prescrito para o tratamento
da ICC e modulam a ativação neuro-hormonal, reduzem a atividade simpática e estimulam a
ação vagal, diminuindo a frequência cardíaca. Os diuréticos são outra classe de medicamento
usada para redução de volume intravascular, do volume ventricular, da pré-carga e
conseqüentes sintomas da IC, sempre em associação com um inibidor da ECA. Outra ação
terapêutica é a utilização de bloqueadores β-adrenérgicos, a fim de inibir a ativação simpática
e os níveis plasmáticos elevados de noradrenalina que desempenham papel primário na
progressão da IC. Os agentes vasodilatadores tendem a melhorar o desempenho cardíaco,
sendo também usado na IC. De qualquer forma, todos esses tratamentos são paliativos na IC
de etiologia chagásica já que nos pacientes que evoluem para a forma crônica da doença, há
persistência da resposta inflamatória e progressão da destruição do músculo cardíaco
(BARRETO et al., 2002).
O transplante cardíaco constitui, na atualidade, a modalidade de tratamento estabelecida
para os pacientes com IC grave e refratária ao tratamento clínico otimizado. Em 1968, Zerbini
& Decourt realizaram o primeiro transplante cardíaco da América Latina e o décimo sétimo
no mundo, em um paciente com miocardiopatia dilatada. O desconhecimento técnico sobre a
22
rejeição e a ausência de medicações eficazes para seu controle, associada a complicações
infecciosas levaram ao descrédito crescente do procedimento em virtude de resultados
desfavoráveis (FIORELLI et al., 2008). Ao longo do tempo os resultados dos transplantes
cardíacos foram gradativamente melhorando, com o refinamento das técnicas e das drogas
utilizadas para imunossupressão do paciente, com diminuição do risco de rejeição. Entretanto,
nos pacientes chagásicos, a utilização de imunossupressores implica no agravante de uma
possível reagudização da doença, pela redução da resposta imune e aumento consequente da
parasitemia (BOCCHI et al., 1996; PARKER & SETH, 2011). Apesar do grande êxito
alcançado durante a realização dos transplantes, o grande limitante – no Brasil e no mundo – é
a pouca disponibilidade de doadores de órgãos. O Brasil dispõe do maior programa público de
transplantes do mundo, com taxa de 5,4 doadores por milhão de habitantes/ano e aumento
expressivo do número de transplantes, entretanto, existem mais de 70 mil brasileiros ainda na
fila de espera de doação de órgãos (MARINHO, 2004; MATTIA et al., 2010). Por essa razão,
há um grande número de pesquisas relacionadas ao tratamento da IC que não envolvem o
transplante cardíaco (HELITO et al., 2009).
1.2 A TERAPIA CELULAR
As células-tronco (CT) são células indiferenciadas com capacidade de auto-renovação
por toda a vida e de diferenciação em um ou mais tipos celulares especializados. De acordo
com a sua capacidade de se diferenciar em todos, muitos ou alguns tipos celulares diferentes,
quando estimuladas, as CT podem ser nomeadas como totipotentes, pluripotentes, ou
multipotentes, respectivamente (KRAMPERA et al., 2007). Quanto mais indiferenciada a
célula for, maior será sua plasticidade e consequente potencial de diferenciação (ORLIC,
2004). Elas podem ser classificadas em três grandes categorias de acordo com seu estágio de
desenvolvimento, como embrionárias, fetais ou adultas (THOMSON et al., 1998). De maneira
distinta das CT embrionárias, que são definidas pela sua origem, as CT adultas, embora
mantenham a capacidade de diferenciação em tipos celulares diversos e de auto-renovação,
carecem de melhor caracterização.
A manutenção do comportamento das CT depende de reguladores autônomos próprios,
intrínsecos das células, que são modulados por sinais externos ou do microambiente. O
controle intrínseco inclui proteínas responsáveis pela regulação de fatores nucleares
envolvidos na modulação da expressão gênica da célula (BONNET, 2002). Desta forma, as
células progenitoras estão sob forte influência de citocinas, cada uma apresentando ações
múltiplas mediadas por receptores que sinalizam desde a sobrevivência, diferenciação,
23
maturação até sua ativação (ABBAS, LICHTMAN & PILAI, 2007; HELD & GUNDERTREMY, 2009). Entre as CT adultas, encontramos as hematopoiéticas e as mesenquimais. As
CT hematopoiéticas apresentam um potencial proliferativo elevado, o que possibilita a sua
diferenciação em células progenitoras de todas as linhagens sangüíneas e a reconstituição da
população hematopoiética a partir de uma única célula.
Com seu potencial elevado de diferenciação, as CT possibilitam a produção de células
especializadas que não são rejeitadas nos transplantes autólogos, por serem oriundas do
próprio receptor. Essas células são usadas na chamada medicina regenerativa, permitindo ao
próprio organismo reparar tecidos e órgãos lesados (DOMEN, WAGERS & WEISSMAN,
2006). Podemos constatar a ideia da pluripotência das CT de medula óssea através do trabalho
publicado por Deb e colaboradores (2003), os autores observaram a presença de
cardiomiócitos diferenciados a partir de células transplantadas da medula óssea, evidenciados
pela presença do cromossomo Y nestas células, em biópsias de coração de pacientes do sexo
feminino que receberam células de medula óssea de doadores do sexo masculino.
Alguns dos alvos terapêuticos foram órgãos considerados por muito tempo como
incapazes de desenvolver quaisquer processos de regeneração, como o cérebro e o coração.
Dessa forma, foram abertas perspectivas inovadoras para o tratamento de doenças crônicodegenerativas, como a doença de Chagas. As CT migram por quimiotaxia em direção ao
estímulo lesivo, onde fatores, tais como citocinas, fatores de crescimento e de proliferação
celular, são secretados pelas células. O contato célula-célula, ou interação da célula com a
matriz extracelular mediada por moléculas de adesão e quimiocinas também são fatores
importantes que irão determinar não só a migração dessas células como também a
permanência delas nos seus microambientes particulares (WATT et al, 2000; KIROUAC et al,
2009). Em modelos experimentais de cardiopatia tem sido demonstrado o papel promissor das
células de medula óssea na promoção de neovascularização, formação de cardiomiócitos e
melhora funcional do coração em animais tratados (ORLIC et al, 2001; KOCHER et al, 2001;
TOMA et al, 2002; ASSMUS et al, 2002; ROTA et al., 2007).
Nos últimos anos as CT mesenquimais têm merecido destaque, pois podem ser isoladas
de praticamente todos os tecidos adultos e preservam as suas características após um cultivo
padrão, além de apresentarem propriedades imunomodulatórias como as CT hematopoiéticas
(GUIMARÃES, 2010). Hoje existem evidências que as CT mesenquimais estão presentes em
diversos tecidos e órgãos do indivíduo adulto, tais como a pele, o fígado, a polpa de dente de
leite, o sangue do cordão umbilical, o tecido adiposo, o sangue menstrual, entre outros
(POULSOM et al., 2002; BONNET, 2002; CASE et al., 2008; WILSON, BUTLER &
24
SEIFALIAN, 2010; GUIMARÃES et al., 2010; MARUYAMA et al., 2010). Diversos autores
demonstraram que células já diferenciadas podem proliferar em resposta a agressões
teciduais, indicando que o processo de regeneração ocorre também a partir do próprio tecido,
dependendo do estímulo recebido (PITTENGER et al., 1999; KRAUSE, 2002; QUAINI et al.,
2002; LERI et al., 2002; LONGO et al., 2010). Steele e colaboradores (2005), por exemplo,
demonstraram a capacidade, do que ele chamou de células semelhantes a progenitoras
cardíacas, têm de serem mobilizadas e migrarem para a área da lesão induzida.
O primeiro trabalho demonstrando a possibilidade de utilização das CT hematopoiéticas
na regeneração do miocárdio em modelo murino de lesão isquêmica foi apresentado por Orlic
e colaboradores (2001). A partir de então, diversos estudos comprovaram a capacidade dessa
células em reparar o tecido cardíaco (KOCHER et al., 2001, WANG et al., 2001, TOMA et
al., 2002). Os resultados de estudo realizado em modelo animal de cardiopatia chagásica
crônica demonstraram que o transplante com células mononucleares da medula óssea
proporcionou a melhora da função cardíaca, com redução de inflamação e fibrose, sugerindo a
possibilidade de utilização da terapia celular na doença de Chagas (SOARES et al., 2004). A
partir desses e de outros dados experimentais, foram realizados estudos clínicos de fase I e II,
para investigar os efeitos do transplante autólogo de células da medula óssea em pacientes
com insuficiência cardíaca com etiologia chagásica.
Os resultados apresentados
demonstraram tanto a ausência de efeitos adversos quando a melhora funcional desses
pacientes até sessenta dias pós-transplante, com aumento da distância percorrida em 6
minutos, aumento da fração de ejeção ventricular esquerda, mostrando que além da segurança
do procedimento, existiam potenciais benefícios associados à terapia (VILAS-BOAS et al.,
2004; VILAS-BOAS et al., 2006; VILAS-BOAS et al., 2011).
No entanto, os resultados do primeiro estudo clínico randomizado em pacientes
chagásicos crônicos que realizaram transplante autólogo com células mononucleares da
medula óssea foram discordantes dos resultados relatados anteriormente por Vilas-Boas e
colaboradores. Nesse estudo foram analisados 234 pacientes entre julho de 2005 e outubro de
2009, que receberam o transplante ou que participaram como grupo controle, sem a injeção de
células nas coronárias, não havendo diferença entre os grupos tanto na função cardíaca quanto
na avaliação de questionários de qualidade de vida (RIBEIRO-DOS-SANTOS et al., 2012).
Dessa maneira, apesar de promissora, a terapia celular ainda carece de aprimoramento, como
por exemplo, a padronização do número de células, a via de administração e investigação dos
seus mecanismos de ação.
25
1.3 O FATOR ESTIMULADOR DE COLÔNIA DE GRANULÓCITOS (G-CSF)
1.3.1 O G-CSF na resposta imune inata
O G-CSF é uma glicoproteína com peso molecular de 24-25 kDa que consiste de quatro
α-hélices antiparalelas (Figura 4), contendo uma molécula de ácido neuramínico e, no
mínimo, uma ponte dissulfeto interna, necessária para sua atividade biológica(DEMETRI &
GRIFFIN, 1991). O G-CSF recombinante humano (Filgrastim) é uma glicoproteína produzida
através da tecnologia do DNA recombinante, descrita há mais de vinte anos, que tem seu uso
aprovado pelo órgão regulador da administração de alimentos e medicamentos norteamericano (FDA) desde o ano de 1991(SOLAROGLU et al., 2006; MASET, DUARTE &
GRECO, 2007). A proteína recombinante pode ser expressa na forma glicosilada (peguilada)
em células da linhagem derivada de ovário de Hamster chinês (CHO) e na forma nãoglicosilada, com uma metionina extra na posição N-terminal em Escherichia coli. A forma
nativa do G-CSF produzida naturalmente é glicosilada, o que confere resistência à degradação
por proteases e pode influenciar toda a estrutura da proteína. A glicosilação também parece
prevenir a agregação da proteína pelo aumento da solubilidade e estabilidade desta molécula
que é altamente hidrofóbica em pH neutro (OH-EDA et al., 1990). Apesar do aumento da
atividade no G-CSF peguilado, tanto a forma nativa quanto a não glicosilada apresentam
atividade biológica.
Figura 4. Estrutura do G-CSF recombinante humano determinada por cristalografia.
Fonte: http://maptest.rutgers.edu/drupal/?q=node/113
Durante algum tempo acreditava-se que citocinas específicas atuavam induzindo a
diferenciação das CT hematopoiéticas ao longo de determinada via. Atualmente parece que
cada célula está intrinsecamente predisposta a uma linhagem ou outra, cumprindo as
citocinas, o papel de promover a sua sobrevida (PARSLOW et al., 2004). Na figura 5 são
ilustradas algumas citocinas que possuem essa função, dentre elas estão os fatores
26
estimuladores de colônias (CSFs), tais como o fator estimulador de colônias de granulócitos
(G-CSF), o fator estimulador de colônias de granulócitos e macrófagos (GM-CSF), a
interleucina 3 (IL-3) e a eritropoietina (EPO) (DEMETRI & GRIFFIN, 1991). Os vários CSFs
não exibem nenhuma relação estrutural entre si e ambos se ligam a receptores distintos, no
entanto apresentam ações que se sobrepõem como é o caso do G-CSF e do GM-CSF, sendo o
significado biológico dessa redundância ainda não estabelecido (ABBAS, LICHTMAN &
PILLAI, 2007).
Figura 5. Influência das citocinas durante o desenvolvimento das células sanguíneas na medula óssea.
Fonte: Dale, 2008.
O G-CSF é um polipeptídico inicialmente identificado como fator de estimulação para
neutrófilos, capaz de induzir sua proliferação e maturação (DEMETRI & GRIFFIN, 1991).
Sua utilização na clínica é bem conhecida para prevenção da granulocitopenia induzida por
tratamento, a principal causa de morte entre pacientes com câncer submetidos à quimioterapia
ou radioterapia, e a sua administração causa raros efeitos colaterais, tais como náusea, vômito,
dor no estômago e sintomas semelhantes aos de um resfriado (DOMEN, WAGERS
&WEISSMAN, 2006; HUTTMAN et al, 2006). A terapia com G-CSF, a fim de mobilizar CT
da medula óssea, é uma estratégia utilizada na clínica para aumentar a proliferação e a
migração dessas células para a circulação com o objetivo de coleta e uso dessas células no
transplante de medula óssea autóloga (ANDERLINI et al., 1999; CASHEN et al., 2004;
ANDERLINI & CHAMPLIN, 2008).
27
A habilidade da célula em responder as citocinas depende da presença de receptores
para esses fatores na sua superfície. Dessa forma, os efeitos biológicos do G-CSF são
mediados via ativação do seu receptor (G-CSFR), um membro da superfamília de receptores
de citocinas tipo l. O receptor de G-CSF humano é conhecido por CD114, sendo expresso
primeiramente em progenitores neutrofílicos e granulócitos maduros, transmitindo
principalmente sinais de proliferação, diferenciação e sobrevivência destas células
(BONEBERG et al., 2000). Também pode ser encontrado em monócitos, com a função de
atenuar a liberação de citocinas pró-inflamatórias quando estas células se encontram ativadas
e de potencializar a fagocitose, além de ter sido identificado em plaquetas maduras e várias
outras células não hematopoiéticas, incluindo células endoteliais e placenta (DEMETRI &
GRIFFIN, 1991; BOBER et al., 1995). A expressão do G-CSFR também foi descrita em
linfócitos T e B em humanos, após a exposição com o G-CSF, sugerindo a ação dessa citocina
na modulação da resposta imune adaptativa (FRANZKE et al., 2003; FRANZKE, 2006).
1.3.2 Regulação da resposta imune adaptativa pelo G-CSF
Muitas rotas de sinalização intracelular se iniciam a partir da interação entre moléculas
e seus ligantes, tais como as citocinas e seus receptores de superfície. Essa ligação
desencadeia uma cascata de sinais intracelulares direcionadas ao núcleo, onde modulam a
transcrição gênica. O receptor de G-CSF está associado à tirosina-quinases citoplasmáticas,
chamadas Janus quinases (Jaks) que se autofosforilam e ativam um grupo de proteínas
reguladoras gênicas denominadas STATs (fator de transdução e ativação da transcrição) que,
nessa condição, migram para o núcleo e estimulam a transcrição de genes-alvo específicos
(JANEWAY et al., 2001; ALBERTS et al., 2002; FRANZKE, 2006). Dependendo do
conjunto de proteínas fosforiladas, a ligação das STATs nas sequências de DNA alvo
proporcionam efeitos diversos, como a ativação de GATA3 e a modulação de linfócitos T por
ativação da apoptose via Fas/FasL em linfócitos, já em neurônios e miócitos o G-CSF
apresenta efeito antiapoptótico, através do aumento de Bcl2 (REYES et al., 1999;
SOLAROGLU et al., 2006; TOH et al., 2009; MILBERG et al., 2011).
Na revisão feita por Franzke (2006) são apontados alguns resultados obtidos em estudos
in vitro, em modelos experimentais e dados clínicos, que evidenciam o papel do G-CSF na
regulação da resposta imune adaptativa, conforme sumarizado na figura 6. Foi observada a
redução da proliferação de linfócitos T em sangue periférico de pacientes tratados com GCSF, após a estimulação por mitógenos in vitro. Alguns autores sugerem que esta redução é
decorrente da regulação do ciclo celular do próprio linfócito T, enquanto que outros autores
28
sugerem que o G-CSF modula a função de monócitos, regulando os linfócitos T por ação
indireta, por diminuição de moléculas co-estimulatórias, aumento de IL-10 e diminuição na
produção de IL-12 e TNF (MIELCAREK et al., 1998; RUTELLA et al., 1998; REYES et al.,
1999; RUTELLA et al., 2000; NAWA et al., 2000).
Figura 6. Efeitos do G-CSF que sustentam sua ação na tolerância central e periférica.
Fonte: modificada de Franzke, 2006.
Além de alterar a função de linfócitos T, o G-CSF também exerce efeito modulatório
sobre as células dendríticas. As células dendríticas (CD) são células apresentadoras de
antígenos profissionais (APC) que têm a capacidade de migrar para diferentes
compartimentos corporais através do sistema linfático, e que estão envolvidas com a resposta
inicial dos linfócitos T culminando em sua ativação (JANEWAY et al., 2001). De acordo com
sua habilidade de induzir a diferenciação de linfócitos T em células efetoras Th1 ou Th2, duas
linhagens distintas de células dendríticas têm sido descrita em humanos: a linhagem mielóide
(CD1) e a linhagem linfóide (CD2). A CD1, originada de precursores mielóides e que
precisam de GM-CSF para sua sobrevivência, produz uma maior quantidade de IL-12 quando
estimulada por TNF-α ou CD40L e direciona a diferenciação para Th1, já as células
dendríticas linfóides (CD2) são também chamadas de células plasmocitóides e são capazes de
induzir a diferenciação de células Th2 após sua ativação (ROBINSON et al., 1999).
Arpinati e colaboradores (2000) demonstraram que o tratamento com G-CSF aumenta
seletivamente o número de células CD2 no sangue periférico de pacientes, induzindo uma
29
maior diferenciação de células Th2, com produção elevada de IL-4 e IL-10. Acredita-se que o
estado de ativação/maturação das CDs pode ser um ponto de controle para a indução de
tolerância periférica através da modificação do estado de ativação dos linfócitos T. Quanto
imaturas, as CDs, não só podem induzir a diferenciação de células Treg, como também podem
apresentar antígenos num contexto de expressão de CTLA-4, promovendo um estado
tolerogênico (KLANGSINSIRIKUL & RUSSEL, 2002; RUTELLA et al., 2002; AGNELLO
et al., 2004; RUTELLA & LEMONI, 2004; VLAD, CORTESINI & SUCIU-FOCA, 2005).
Estudos in vitro também demonstraram que o G-CSF tem a capacidade de aumentar a geração
de células T regulatórias (Treg) produtoras de IL-10 e TGF-β (RUTELLA et al., 2004).
Sakaguchi e colaboradores (1995) foram os pioneiros a descrever uma população de
células caracterizadas pela presença de marcadores de superfície CD4 e CD25, com
capacidade regulatória, conhecidas atualmente como Treg naturais. Uma vez que animais
atímicos recebiam transplante de uma população de células depletadas dessa subpopulação, os
mesmos desenvolviam resposta autoimune grave em diversos órgãos, ao passo que, quando
essas células eram transplantadas, suprimiam a doença. As células Treg naturais também
expressam o fator de transcrição Foxp3, sendo hoje conhecida a sua capacidade de controle da
intensidade da resposta inflamatória, seja contra auto-antígenos, seja contra antígenos
estranhos, de modo a evitar lesão tecidual (HSIEH, LEE & LIO, 2012). O envolvimento de
tais células tem sido implicado na homeostasia de linfócitos T promovido pelo tratamento
com G-CSF, através do controle da resposta imune autoreativa (SAKAGUCHI, 2000).
Diversos estudos sugerem mecanismos de ação múltiplos das células Treg que as
possibilitam agir como moduladores da resposta imunológica, sendo organizados em três
modelos básicos:
- 1. Dependente de contato célula-célula: supressão da célula-alvo com liberação de
fatores de supressão incluindo o monofosfato de adenosina cíclica (AMPc), citocinas
supressivas como o TGF–β, citólise direta ou sinalização negativa através da molécula
CTLA-4;
- 2. Dependente de fatores de supressão solúveis como citocinas IL-10, TGF-β e IL-35
ou secreção de fatores supressivos pelas APC como adenosina;
- 3. Por competição: competição por citocinas que sinalizam através de receptores da
cadeia γ comum (IL-2, IL-4 e IL-7) (READ, MALMSTROM & POWRIE, 2000; SOJKA,
HUANG & FOWELL, 2008; de ARAÚJO et al., 2011).
Outra subpopulação de Treg é formada na periferia e é conhecida como Treg induzida,
pois seu fenótipo, apesar de não ser o de Treg naturais pela não expressão de CD25 e de
30
foxp3, é funcionalmente o de células capazes de assumir um perfil tolerogênico induzido por
TGF- β, IL-2 e ácido retinóico (CHEN et al., 2003; BENSON et al., 2007). Contrastando com
as Treg naturais, originadas do timo, as células Tr1 possuem atividade imunoreguladora e são
induzidas por estimulação antigênica na periferia de maneira dependente de IL-10. Roncarolo
e colaboradores (2006) propõem a utilização da nomenclatura de células Tr1 para todas as
células T produtoras de IL-10 que são induzidas pela própria IL-10 a apresentarem atividade
regulatória. Linfócitos T CD4+ mobilizados com o uso de G-CSF de doadores saudáveis
proporcionam in vitro a supressão de resposta proliferativa contra aloantígenos de maneira
dependente de contato, produzindo citocinas em um perfil semelhante a Tr1. No entanto, a
exposição direta ao G-CSF in vitro não foi capaz de induzir esse mesmo fenótipo Tr1
(NAWA et al., 2000; RUTELLA et al., 2002).
Morris e colaboradores (2004) demonstraram que, apesar dos dados controversos dos
experimentos in vitro, a administração de G-CSF em transplante alogênico murino reduziu a
reação de rejeição do enxerto x hospedeiro através da produção de IL-10 por LT,
provavelmente com o fenótipo Tr1. Independente de qual população de célula
imunoreguladora esteja atuando, o tratamento com G-CSF também causou o aumento do
número de células T reguladoras, produtoras de TGF-β em modelo animal de diabetes
(KARED et al., 2005).Na medula óssea, a expressão do fator derivado do estroma 1 (SDF1/CXCL12), que é o ligante do receptor CXCR4, possibilita a permanência dessas células
nesse compartimento. A terapia com G-CSF proporcionou a redução da expressão de
CXCL12, o que está associado com a migração de células Treg da medula para a periferia, a
fim de cumprir seu papel na tolerância periférica (ZOU et al., 2004).
1.3.3 O G-CSF e o coração
O uso de fatores de crescimento para mobilização de CT abriu perspectivas novas para o
tratamento de doenças crônico-degenerativas com um custo e risco menor haja vista ser esse
um tratamento menos invasivo. Dessa forma, as pesquisas envolvendo a terapia com o G-CSF
vêm crescendo a fim de avaliar sua participação na modulação da resposta inflamatória e seu
envolvimento no reparo tecidual. Dentre os modelos de doenças cardiovasculares que usam o
G-CSF para a terapia celular, podemos citar o trabalho de Orlic e colaboradores (2001) que
demonstraram a melhora da função ventricular pós-infarto do miocárdio, após o tratamento,
resultando na regeneração do músculo cardíaco. Esse estudo foi uma das primeiras
publicações demonstrando a eficácia do uso do G-CSF na mobilização de CT de medula óssea
em modelo experimental de lesão isquêmica no miocárdio, causando a redução da
31
mortalidade dos animais e levando ao remodelamento cardíaco. Esses resultados foram
confirmados por Minatoguchi e colaboradores (2004) em um modelo de infarto em coelhos,
no qual verificaram o aumento da fração de ejeção ventricular e diminuição do
remodelamento após o tratamento com G-CSF. Em modelo de cardiopatia diabética em ratos
com diabetes tipo 2, foi demonstrado que o tratamento com G-CSF causa uma melhora da
disfunção diastólica nos animais, além de promoção da diminuição da fibrose perivascular e
intersticial (LIM et al., 2011), o que corrobora o trabalho de Sugano e colaboradores (2005),
que descrevem o G-CSF como uma citocina reguladora da síntese reparadora de colágeno em
corações infartados.
O G-CSF foi inicialmente utilizado experimentalmente em modelos de doenças
cardiovasculares por sua capacidade de recrutamento de CT, no entanto estudos mais recentes
apontam a ação do G-CSF diretamente sobre cardiomiócitos promovendo o aumento da sua
sobrevida (MINATOGUCHI et al., 2004). Atualmente sabemos que tanto o G-CSF quanto o
seu receptor são expressos em cardiomiócitos durante todo o período embrionário, sugerindo
sua participação no desenvolvimento desse órgão. A adição de G-CSF a cultura de CT
embrionárias ou a CT induzidas derivadas de cardiomiócitos não só aumentou a proliferação
de cardiomiócitos como também aumentou a expressão do marcador cardíaco Nkx2.5,
confirmando o papel do G-CSF na cardiogênese (SHIMOJI et al., 2010). Um estudo recente
demonstrou, por exemplo, que o G-CSF é capaz de ativar diversas vias de sinalização
intracelular, como a ativação de Akt e da via Jak2-STAT3, em cardiomiócitos, prevenindo
dessa forma o remodelamento cardíaco associado com infarto do miocárdio (TAKANO et al.,
2007).
Apesar do conhecimento da ação imunomoduladora do G-CSF, os mecanismos
envolvidos na melhora da função cardíaca após a administração de G-CSF ainda não estão
totalmente esclarecidos. Levando–se em consideração que a doença de Chagas é uma das
principais causas de falência cardíaca na América Latina, e que existem poucos recursos
terapêuticos para a forma crônica sintomática da doença, o uso terapêutico do G-CSF na
cardiopatia chagásica se torna atraente não só por ser um tratamento não invasivo quando
comparado ao transplante celular ou cardíaco, mas também por ser uma terapia inovadora em
modelo de CChC.
32
OBJETIVOS
OBJETIVO GERAL:
Investigar a eficácia do tratamento com G-CSF recombinante, por três ciclos, na
melhora da insuficiência cardíaca de etiologia chagásica e seus possíveis mecanismos de ação,
em modelo experimental de camundongos cronicamente infectados por Trypanosoma cruzi da
cepa Colombiana.
OBJETIVOS ESPECÍFICOS

Analisar a função cardiorespiratória dos animais chagásicos crônicos tratados ou não
com G-CSF;

Avaliar os efeitos do G-CSF na modulação da resposta inflamatória e da fibrose no
coração de camundongos chagásicos crônicos;

Avaliar os efeitos do tratamento com G-CSF no parasitismo na fase crônica da infecção
por T. cruzi;

Avaliar os efeitos do tratamento com G-CSF na resposta imune através da comparação
da produção de citocinas e anticorpos entre os camundongos na fase crônica da
infecção;

Investigar a produção de moléculas de adesão e de moléculas pró-inflamatórias em
corações dos camundongos infectados e tratados ou não com G-CSF;

Investigar a ação do G-CSF sobre o recrutamento de células T regulatórias nos animais
chagásicos crônicos;

Avaliar a ação direta do G-CSF sobre o T. cruzi in vitro.
33
2. CAPÍTULO I
“Granulocyte Colony-Stimulating Factor treatment in Chronic Chagas Disease:
Preservation and Improvement of cardiac structure and function”, publicado no periódico The
FASEB Journal, no ano de 2009.
The FASEB Journal article fj.09-137869. Published online July 16, 2009.
The FASEB Journal • Research Communication
Granulocyte colony-stimulating factor treatment in
chronic Chagas disease: preservation and
improvement of cardiac structure and function
Simone G. Macambira,*,† Juliana F. Vasconcelos,* Claudio R. S. Costa,*
Wilfried Klein,‡ Ricardo S. Lima,* Patrícia Guimarães,* Daniel T. A. Vidal,*
Lucas C. Mendez,* Ricardo Ribeiro-dos-Santos,*,§ and Milena B. P. Soares*,§,1
*Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil; †Instituto de
Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil;
‡
Instituto de Biologia, Universidade Federal da Bahia, Salvador, Bahia, Brazil; and §Hospital São
Rafael, Salvador, Bahia, Brazil
ABSTRACT
This study investigates the effects of
granulocyte colony-stimulating factor (G-CSF) therapy in experimental chronic chagasic cardiomyopathy. Chagas disease is one of the leading causes of
heart failure in Latin America and remains without an
effective treatment other than cardiac transplantation.
C57BL/6 mice were infected with 103 trypomastigotes
of Trypanosoma cruzi, and chronic chagasic mice were
treated with G-CSF or saline (control). Evaluations
following treatment were functional, immunological,
and histopathological. Comparing hearts of G-CSFtreated mice showed reduced inflammation and fibrosis
compared to saline-treated chagasic mice. G-CSF treatment did not alter the parasite load but caused an
increase in the number of apoptotic inflammatory cells
in the heart. Cardiac conductance disturbances in all
infected animals improved or remained stable due to
the G-CSF treatment, whereas all of the saline-treated
mice deteriorated. The distance run on a treadmill and
the exercise time were significantly greater in G-CSFtreated mice when compared to chagasic controls, as
well as oxygen consumption (V̇O2), carbon dioxide
production (V̇CO2), and respiratory exchange ration
(RER) during exercise. Administration of G-CSF in
experimental cardiac ischemia had beneficial effects on
cardiac structure, which were well correlated with improvements in cardiac function and whole animal performance.—Macambira, S. G., Vasconcelos, J. F.,
Costa, C. R. S., Klein, W., Lima, R. S., Guimarães, P.,
Vidal, D. T. A., Mendez, L. C., Ribeiro-dos-Santos, R.,
Soares, M. B. P. Granulocyte colony-stimulating factor
treatment in chronic Chagas disease: preservation and
improvement of cardiac structure and function. FASEB
J. 23, 000 – 000 (2009). www.fasebj.org
Key Words: chagasic cardiomyopathy 䡠 inflammation 䡠 arrhythmias 䡠 treadmill performance
Chagas disease, caused by Trypanosoma cruzi infection, is
one of the main causes of death due to heart failure in
0892-6638/09/0023-0001 © FASEB
Latin American countries. About 25% of chagasic individuals develop a chronic chagasic cardiomyopathy (CChC),
the most severe form of disease. The chemotherapy used
in chagasic patients is highly toxic and has limited efficacy,
especially in the chronic disease. The unique definitive
treatment for CChC aggravated by severe heart failure is
heart transplantation.
Studies establishing therapies to restore the cardiac
function using stem cells or the administration of
growth factors have been developed. Bone marrow
stem cells (BMSCs) differentiate into cardiomyocytes
and endothelial cells and may participate in the regeneration of cardiac lesions (1– 4). Granulocyte colonystimulating factor (G-CSF) increased the number of
peripheral granulocytes (5) and induced the mobilization of BMSCs to the periphery (6). This property and
regenerative capacity of BMSCs justify the efforts to
prove the efficacy of this therapy.
The beneficial effects of G-CSF in the treatment of
cardiac ischemia lesions have been shown (7–9). The
therapeutic use of G-CSF is attractive because it is
already employed in clinical practice, has mild side
effects, and is a less invasive treatment than bone
marrow aspiration and cell transplantation. We have
previously shown that therapy with BMSCs decreases
heart inflammation and fibrosis in experimental CChC
(10). Here, we investigated the effects of G-CSF on
cardiac alterations in a model of CChC.
MATERIALS AND METHODS
Animals
Two-month-old male C57BL/6 mice, raised and maintained in the animal facilities at the Gonçalo Moniz
1
Correspondence: Milena Botelho Pereira Soares, Centro
de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz. 121,
Rua Waldemar Falcão, Candeal, Salvador, Bahia, Brazil,
40.296-710. E-mail: [email protected]
doi: 10.1096/fj.09-137869
1
Research Center (Fiocruz, Rio de Janeiro, Brazil) were
used in the experiments, and were provided with rodent
diet and water ad libitum. All animals were sacrificed under
anesthesia by intraperitoneal injection of xylazine at 10
mg/kg body wt and ketamine at 100 mg/kg body wt, and
handled according the National Institutes of Health guidelines for ethical use of laboratory animals.
T. cruzi infection and treatment with G-CSF
Mice were infected by intraperitoneal injection of 1000
trypomastigote forms of Colombian strain T. cruzi (11) obtained by in vitro infection of LCC-MK2 cell line. Parasitemia
was evaluated at different time points after infection by
counting the number of trypomastigotes in peripheral blood
aliquots (12). Groups of chronic chagasic mice (6 mo after
infection) were treated with human recombinant G-CSF
(Granulokine 30; Hoffman la Roche, Switzerland) with 200
␮g/kg/d during 5 consecutive days with 3 cycles of administration or with 5% glucose saline solution in the same
regimen.
Histopathological analysis
Hearts from G-CSF-treated mice and untreated controls were
removed and fixed in buffered 10% formalin. Sections of
paraffin-embedded tissue were stained by standard hematoxylin-and-eosin (H&E) and Sirius red staining for evaluation of
inflammation and fibrosis, respectively, by optical microscopy. Images were digitized using a color digital video camera
(CoolSnap, Montreal, QC, Canada) adapted to a BX41 microscope (Olympus, Tokyo, Japan). The images were analyzed using Image Pro 5.0 (Media Cybernetics, San Diego, CA,
USA), to integrate the number of inflammatory cells counted
by area. Ten fields per heart were counted from every mouse
of each group.
Parasite quantification by immunofluorescence analysis
Frozen heart sections (5 ␮m thick) were prepared in a
cryostat in poly-l-lysine-coated slides and fixed with cold
acetone. Sections were incubated with PBS 5% BSA for 30
min, followed by overnight incubation with rat serum anti-T.
cruzi (1:400). After washing with PBS, sections were incubated
for 1 h with FITC-conjugated rabbit anti-rat IgG 1:100 (Sigma,
St. Louis, MO, USA). Sections were washed 3 times, counterstained with Evans blue, and mounted with Vectashield
(Vector, Burlingame, CA, USA). Images were digitized using
a color digital video camera (DP-70; Olympus) adapted to an
AX-70 microscope (Olympus). The images were analyzed
using Image Pro, and the numbers of parasite foci were
counted (10 fields/heart, 5 mice/group) and integrated by
area.
Stroma-derived factor-1 (SDF-1) assessment in total-protein
heart extracts
Heart proteins were extracted at 100 mg of tissue/ml of PBS
to which 0.4 M NaCl, 0.05% Tween 20 and protease inhibitors
(0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM
EDTA, and 20 KI aprotinin A/100 ml) were added. The
samples were centrifuged for 10 min at 3000 g, and the
supernatant was frozen at ⫺70°C for later quantification.
SDF-1 levels were estimated using a commercially available
Immunoassay ELISA kit (R&D Systems, Minneapolis, MN,
USA), according to the manufacturer’s guidelines.
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Vol. 23
November 2009
Apoptosis assay
Apoptosis in heart sections fixed using 4% formaldehyde was
evaluated by terminal uridine deoxynucleotidyl transferase
dUTP nick end labeling (TUNEL) assay, performed using
in situ DeadEnd Colorimetric TUNEL System kit (Promega,
Madison, WI, USA), according to the manufacturer’s instructions. Images were digitized, and quantitative analysis was
performed using Image Pro. Results were expressed as the
number of positive cells per square millimeter of 10 sections
from 4 animals/group. The positive controls were nucleasetreated slides.
ECG analysis
Electrocardiography was performed using the Bio Amp
PowerLab System (PowerLab 2/20; ADInstruments, Castle
Hill, NSW, Australia), recording the bipolar lead I. All
animals were anesthetized by intraperitoneal injection of
xylazine at 10 mg/kg body weight and ketamine at 100 mg/kg
body weight to obtain the records. All data were acquired for
computer analysis using Chart 5 for Windows (PowerLab).
Records were bandpass filtered (1 to 100 Hz) to minimize
environmental signal disturbances. The sampling rate was 1
kHz. The ECG analysis included heart rate, PR interval, P
wave duration, QT interval, QTc, and arrhythmias. Wave
durations (ms) and heart rate were calculated automatically
by the software after the cursors were placed. The QTc was
calculated as the ratio of QT interval by square roots of RR
interval.
Treadmill
A motor-driven treadmill chamber for one animal (LE 8700;
Panlab, Barcelona, Spain) was used to exercise the animals.
The speed of the treadmill and the intensity of the shock
(mA) were controlled by a potentiometer (LE 8700 treadmill
control; Panlab). Room air was pumped into the chamber at
a controlled flow rate (700 ml/min) by a chamber air
supplier (Oxylet LE 400; Panlab). Outflow was directed to an
oxygen and carbon dioxide analyzer (Oxylet 00; Panlab) to
measure consumption of oxygen (V̇O2), production of carbon dioxide (V̇CO2), and the respiratory exchange ratio
(RER). The mean room temperature was maintained at 21 ⫾
1°C. After an adaptation period of 40 min in the treadmill
chamber, the mice exercised at 5 different velocities (7.2,
14.4, 21.6, 28.8 and 36.0 m/min), with increasing velocity
after 10 min of exercise at a given speed. Velocity was
increased until the animal could no longer sustain a given
speed and remained ⬎10 s on an electrified stainless-steel
grid, which provided an electrical stimulus to keep the mice
running. After reaching exhaustion, animals were left undisturbed in the treadmill chamber for 30 min. V̇O2 and V̇CO2
were determined using the program Metabolism for 2 channel PowerLab (ADInstruments), analyzing the last 5 min
recorded at rest and at each speed. During recovery, V̇O2 and
V̇CO2 were determined during a 3-min period following
immediately after cessation of exercise (recovery 1; exhaustion) and at the end of the 30-min recovery period (recovery
2). Subsequently, RER was calculated. Total running distance
and running time were recorded. To determine peak oxygen
consumption, we measured the greatest value in oxygen
consumption shown by each mouse during exercise.
Statistical analyses
All continuous variables are presented as means ⫾ se. Morphometric and cytokine levels were analyzed using 1-way
The FASEB Journal 䡠 www.fasebj.org
MACAMBIRA ET AL.
ANOVA, followed by Newman-Keuls multiple-comparison
test. Cardiopulmonary parameters were analyzed using
Student’s t-test and Mann Whitney U test with Prism 3.0
(GraphPad Software, San Diego, CA, USA). Degree of
severity was analyzed using a 2-way ANOVA, followed by a
Bonferroni post-test. Treadmill data were analyzed applying a repeated-measures 1-way ANOVA, followed by an
all-pairwise multiple comparison procedure (Student-Newman-Keuls method) using Prism 3.0. All differences were
considered significant at values of P ⱕ 0.05.
RESULTS
Decreased myocarditis and fibrosis after G-CSF
treatment
A marked decrease in the number of inflammatory cells
was observed 2 mo after G-CSF treatment, compared
with saline-treated chagasic mice (Fig. 1A, C). Morphometric analysis showed a significant reduction in the
number of inflammatory cells after G-CSF treatment
(Fig. 2A). In addition, hearts of G-CSF-treated mice had a
reduced area of fibrosis (Fig. 1B, D), statistically different from saline-treated mice (Fig. 2B). The number of
inflammatory cells undergoing apoptosis was ⬃7-fold
greater in hearts of G-CSF-treated mice compared to
those of saline-treated mice (Fig. 2C; P⬍0.0001). The
levels of the chemokine SDF-1 (CXCL2) in hearts of
saline-treated, but not of G-CSF-treated mice, were
significantly higher than those of normal mice (Fig.
2D). Hearts from both saline- and G-CSF-treated mice
had similar numbers of parasite foci 2 mo after therapy
(2.4⫾0.4 and 2.0⫾0.6 parasite foci/mm2, respectively;
P⬍0.05).
G-CSF treatment ameliorates cardiac electrogenesis in
chronic chagasic mice
All infected mice showed severe cardiac conduction
disturbances in ECG records, such as AV blockage,
intraventricular conduction disturbances, and abnormal cardiac rhythm 6 mo after infection, compared to
normal mice (Fig. 3). In saline-treated mice (n⫽7),
none improved cardiac function, and 5 became worse
after therapy. In contrast, in the G-CSF-treated group
(n⫽6), 2 animals improved cardiac conduction (Fig. 4),
and 4 had no alterations (Fig. 3).
G-CSF treatment improves exercise capacity
Among the saline-treated infected mice, only 2 of 6
were able to keep up with a belt speed of up to 14.4
m/min. The other 4 untreated infected mice were
not able to run on the treadmill. All animals in the
G-CSF-treated group (n⫽7) and in the normal control group (n⫽10) were able to exercise on the
treadmill. All of the G-CSF-treated infected mice
sustained locomotion at a speed of 14.4 m/min, but
3 of 7 animals were able to perform at 21.6 m/min.
These differences resulted in significantly greater
running times and distances covered by G-CSFtreated mice when compared to saline-treated mice,
although G-CSF-treated mice still performed at lower
capacity than normal mice (Fig. 5).
Figure 1. Histopathological analysis in heart
sections of T. cruzi-infected mice. Heart sections
of saline-treated (A, C) or G-CSF-treated (B, D)
T. cruzi-infected mice were analyzed 2 mo after
therapy. Staining: H&E (A, B; ⫻400); Sirius red
(C, D; ⫻200).
G-CSF EFFECT IN EXPERIMENTAL CHAGAS DISEASE
3
Figure 2. Alterations in inflammation, fibrosis, and SDF-1 production in
hearts of G-CSF-treated mice. A, B) Quantification of inflammatory cells (A)
and fibrosis area (B) in heart sections of normal and chagasic mice treated
with saline or G-CSF; 6 –10 mice/group. C) Numbers of apoptotic inflammatory cells in heart sections were quantified by TUNEL assay; 4 mice/group.
D) Levels of SDF-1 in heart homogenates were determined by ELISA; 7–10
mice/group. Values are means ⫾ se.
Under resting conditions, the respiratory exchange
ratio was significantly different between each group,
whereas V̇O2 and V̇CO2 showed no differences (Fig. 6).
During exercise stages 1 and 2, V̇CO2 was significantly
greater in the normal mice compared with G-CSFtreated mice, and respiratory exchange ratio was significantly greater in normal mice at exercise stage 2 when
compared with the G-CSF group. Shortly after exercise,
V̇O2 and V̇CO2 were significantly greater in normal
mice when compared with G-CSF-treated mice, and
V̇CO2 remained significantly greater during recovery
when compared with both other groups. Within the
normal mice, V̇O2, V̇CO2, and RER rose significantly
above resting values during exercise, reaching greatest
V̇O2 shortly after cessation of exercise, with decreasing
respiratory variables during the recovery phase. The
G-CSF-treated mice showed no significant elevation of
V̇O2 and V̇CO2 during exercise when compared with
resting values, but V̇O2 and V̇CO2 shortly after exercise
were significantly greater than the values measured
during the other stages. Peak oxygen consumption of
normal mice (8068 ml O2/min/kg) was significantly
greater than that of the G-CSF-treated group (6561 ml
O2/min/kg).
DISCUSSION
Figure 3. Graph representing cardiac conduction disturbances in arbitrary units in C57BL/6 mice infected with T.
cruzi before and 2 mo after administration of saline or G-CSF.
Degree of severity: 0, no cardiac conduction disturbances; 1,
first-degree atrium-ventricular block; 2, intraventricular conduction disturbance; 3, atrium-ventricular dissociation; 4,
death. Dashed line indicates median of G-CSF treated mice;
solid line indicates median in saline-treated mice. Before
treatment, both medians are identical. ***P ⬍ 0.001 between
groups after treatment with G-CSF.
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Vol. 23
November 2009
G-CSF is a cytokine known to improve cardiac function
and recovery in models of ischemic disease (7, 13, 14).
In this study, we demonstrated that repeated administration of G-CSF induces beneficial effects on cardiac
structure, such as reduction of inflammation and fibrosis, that were well correlated with improvements in
cardiac function in an experimental model of CChC
that closely resembles the human disease (15, 16). One
may question the low number of infected animals used
in this study, and consequently the significance of our
results. However, the fact that all of the G-CSF-treated
animals improved their performance on the treadmill,
when compared with saline-treated mice, shows that
G-CSF has a profound effect on chronic chagasic mice.
Regarding the cardiac conduction disturbances studied
here, 6 of 6 infected animals showed improvements or
remained stable due to the G-CSF treatment, whereas
all of the saline-treated mice deteriorated; even mice
with only a slight first-degree AV blockage evolved
MACAMBIRA ET AL.
Figure 4. Reversion of cardiac disturbance after
G-CSF treatment. ECG of a mouse before infection (A); 6 mo after infection, before G-CSF
treatment (B); and 2 mo after G-CSF treatment
(C). , P wave; ‚, QRS complex; ⴱ, A-V dissociation.
toward an AV dissociation. G-CSF-treated mice, on the
other hand, improved cardiac conductance and never
surpassed a first-degree AV blockage. These facts, taken
together, indicate that G-CSF has a significant therapeutic effect on mice with chronic Chagas disease.
One of the main features of CChC is the presence of
prominent inflammation with participation of an autoimmune component (17–19) that causes destruction of
myofibers and fibrosis deposition. We have recently
shown that autologous BMC transplant modulates the
myocarditis in experimental CChC, an effect associated
with apoptosis of inflammatory cells (10). In the current study, we also found that the decreased inflammation after G-CSF therapy correlated with an increase in
apoptosis of inflammatory cells. Thus, the benefits of
G-CSF therapy may result, in part, from regulation of
pathological immune responses. In fact, recent reports
have demonstrated that G-CSF inhibits T cells by stimulating apoptosis (20, 21).
Heart SDF-1 levels were increased in CChC. The
modulation of heart inflammation by G-CSF therapy
also correlated with a reduction in SDF-1 in chagasic
hearts. This chemokine promotes the recruitment of
inflammatory cells, including T cells, and of stem cells,
which express its receptor CXCR4 (22), and may play a
role in other inflammatory processes in the heart (23,
G-CSF EFFECT IN EXPERIMENTAL CHAGAS DISEASE
24). The fact that G-CSF mobilizes stem/precursor cells
to the periphery suggests that these cells migrate to the
inflamed myocardium and contribute to tissue regeneration, since it has been shown before that mobilized
BMSCs repair the damaged myocardium (25). In fact,
we have previously found that transplanted BMSCs
migrate to and differentiate into cardiomyocytes (10).
Sugano et al. (13) described an accelerated healing
process due to increased reparative collagen synthesis
in affected areas after G-CSF administration, in an
experimental myocardial infarction. A reduction in
fibrosis was observed after a long-term treatment using
low doses of G-CSF after myocardial infarct (26). Here,
we also found a significant reduction in heart fibrosis
after G-CSF treatment.
Another feature of chronic Chagas disease is the
scarce parasitism found in this phase of infection.
Several studies have demonstrated that T. cruzi parasites
or antigens can be found, although rarely, in individuals with chronic infection (27, 28). Although the inflammatory response was modulated after G-CSF therapy, the residual parasite load in our model was not
affected by this treatment.
It has been proposed that the main effect of G-CSF
after severe cardiac injury is to induce the proliferation
of cardiac stem cells rather than BMSC migration and
5
is necessary to attach many proteins to the cell membrane, including proteins related to gap junction formation. Conexin 43 is responsible for cell-cell communication, and thereby allows the appropriate conduction of
cardiac impulses through the whole heart, avoiding arrhythmias. The expression of this protein is reduced by
T. cruzi infection (35) and may contribute to the
arrhythmias. Thus, the beneficial effects of G-CSF treatment on cardiac electrogenesis may be related to an
increase of conexin 43 expression. Kuhlmann et al. (33)
proposed that the enhanced expression of G-CSF receptor in cardiomyocytes and other cell types of the
Figure 5. G-CSF treatment improves the capacity of infected
mice to perform exercise. Time (A) and distance run (B) on
a motorized treadmill by normal (noninfected) mice (n⫽10)
and chronic chagasic mice treated with saline (n⫽2) or with
G-CSF (n⫽7). Data are means ⫾ se. Note that running time
and distance of saline-treated mice are highly influenced by 2
of the 6 animals tested that were able to exercise on the
treadmill, whereas the other 4 saline-treated animals were not
able to run on the treadmill.
proliferation. Kanellakis et al. (29) demonstrated, in a
model of acute myocardial infarction, that G-CSF/SCF
therapy improved cardiac function, increasing the
number of blood vessels and cells of the cardiomyogenic lineage. However, differently from previous studies (30, 31), they demonstrated that these cells were of
myocardial rather than of bone marrow origin. They
also provided evidence that the effects were due to
G-CSF alone, since the addition of SCF to G-CSF
provided little additional benefit at the functional level.
Brunner et al. (32) demonstrated that treatment with
G-CSF after myocardial infarct reduces the migratory
capacity of bone marrow cells into ischemic tissue, but
increases the number of resident cardiac cells.
In our study, we showed that G-GSF administration
can avoid the aggravation of cardiac disturbances associated with chronic Chagas disease and, more important, seems to reverse some of the severe pathologies
associated with CChC. On the other hand, the untreated group aggravated the cardiac abnormalities.
These results were in agreement with other preclinical
studies that investigated the antiarrhythmic effects of
G-CSF and showed an increase in protein expression
levels of ␤-catenin and connexin 43 (33, 34). ␤-Catenin
6
Vol. 23
November 2009
Figure 6. Cardiopulmonary function amelioration during
physical effort after G-CSF administration. Oxygen consumption (A), carbon dioxide release (B), and respiratory exchange ratio (C) in normal mice (solid columns), salinetreated chagasic mice (dashed columns), and G-CSF-treated
chagasic mice (open columns) during resting conditions,
exercising at 4 different velocities (7.2, 14.4, 21.6, and 28.8
m/min) on a motorized treadmill, immediately (recovery 1)
and 30 min after exercise (recovery 2). Data are means ⫾ sd;
6 –10 mice/group. Note that only 2 of 6 saline-treated infected animals were able to run at a velocity of 14.4 m/min;
therefore, no data are given for saline-treated infected mice
during exercise.
MACAMBIRA ET AL.
infarcted myocardium indicates a sensitization of the
heart to direct influences of this cytokine.
Appropriate cardiac contractility is necessary to allow
adjustments of cardiac output during physical exertion,
thereby guaranteeing an adequate oxygen supply during any kind of physical effort. In our study, we observed an increase in V̇O2 in G-CSF-treated chagasic
mice during exercise, showing values of V̇O2 similar to
the levels reached by the uninfected control group,
which indicates adequate adjustments of cardiac output
during light exercise regarding oxygen transport. Values of peak V̇O2 during exercise, however, where
significantly lower in G-CSF mice compared with normal mice, indicating that recovery caused by G-CSF is
not complete. Carbon dioxide release, on the other
hand, showed a significantly different pattern. Whereas
V̇CO2 increased in normal mice at the lower velocities,
reaching a plateau in the subsequent exercise velocities
and the first recovery period, CO2 release in G-CSFtreated chagasic mice seemed to be significantly impaired. The reasons for such a discrepancy, animals
matching their V̇O2 demands but not being able to
appropriately release CO2 during light exercise remain
unknown. RER ratio was significantly greater in untreated chagasic mice compared with both other
groups. This could be due to a reduced capacity of
chagasic mice to carry oxygen to the tissues, resulting in
elevated anaerobic metabolism and increased release of
CO2. The performance improvement of G-CSF-treated
mice can be attributed solely to the beneficial effect of
G-CSF on cardiac structure, improving cardiac efficiency.
Li et al. (36) showed that administration of G-CSF in
experimental chronic heart failure improved the myocardium contractility by avoiding the systolic and diastolic dysfunction through the changes in the geometry
of the infarcted heart to short and thick, induced
hypertrophy among surviving cardiomyocytes, and reduced myocardial fibrosis. These effects could be explained by a direct effect of G-CSF on cardiomyocytes
that could lead to the activation of an intracellular
signal, since the expression of G-CSF receptor was
confirmed in failing hearts and was up-regulated by
G-CSF treatment (8, 13). Our results are in agreement
with Li et al. (36), as G-CSF administration caused a
reduction in functional impairment and partial recovery of heart structure.
Chronic heart failure remains a leading cause of
mortality due to the absence of an efficient therapy that
avoids structural and electrical cardiac remodeling.
The only option for these patients remains heart transplantation. Besides limitations of available donated
organs, in the specific case of Chagas disease, the use of
immunosupressive drugs following heart transplantation can affect the latent parasitism. On the basis of the
beneficial effects of G-CSF shown in the present study,
we conclude that this may be a promising therapy for
the treatment of patients with heart failure due to
Chagas disease. An ongoing phase I/II clinical trial of
G-CSF therapy with multiple administrations in chaG-CSF EFFECT IN EXPERIMENTAL CHAGAS DISEASE
gasic patients (37) may indicate the benefits of this
therapy in humans.
This work was supported by grants from the Brazilian
Ministry of Science and Technology [Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq)], Financiadora de Estudos e Projetos (FINEP), Fiocruz, and Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB).
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(2006) Granulocyte colony-stimulating factor and stem cell
factor improve endogenous repair after myocardial infarction.
Cardiovasc. Res. 70, 117–125
Laflamme, M. A., Myerson, D., Saffitz, J. E., and Murry, C. E.
(2002) Evidence for cardiomyocyte repopulation by extracardiac progenitors in transplanted human hearts. Circ. Res. 90,
634 – 640
Müller, P., Pfeiffer, P., Koglin, J., Schäfers, H. J., Seeland, U.,
Janzen, I., Urbschat, S., and Böhm. M. (2002) Cardiomyocytes
of noncardiac origin in myocardial biopsies of human transplanted hearts. Circulation 106, 31–35
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M., David, R., Zaruba, M. M., Vallaster, M., Rischpler, C., Wilke,
A., Gerbitz, A., and Franz, W. M. (2008) G-CSF treatment after
myocardial infarction: Impact on bone marrow derived vs
cardiac progenitor cells. Exp. Hematol. 36, 695–702
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J., Fabritz, L., Stelljes, M., Tian, W., Zwiener, M., Mueller, M.,
Kienast, J., Breithardt, G., and Nikol, S. (2006) G-CSF/SCF
reduces inducible arrhythmias in the infarcted heart potentially
via increased connexin 43 expression and arteriogenesis. J. Exp.
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through recruitment of ␤-catenin and cadherin. FEBS Lett. 581,
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Nazareth Meirelles, M., and Spray, D. C. (2008) Trypanosoma
cruzi induces changes in cardiac connexin43 expression. Microbes Infect. 10, 21–28
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Received for publication June 1, 2009.
Accepted for publication June 25, 2009.
MACAMBIRA ET AL.
42
3 CAPÍTULO II
“Modulation of myocarditis after therapy with G-CSF in a mouse model of chronic Chagas
disease is associated with an increase in regulatory T cells”, em fase de submissão.
43
Modulation of myocarditis after therapy with G-CSF in a mouse model of
chronic Chagas disease is associated with an increase in regulatory T cells
Juliana Fraga Vasconcelos,1,2 Bruno Solano de Freitas Souza,1,2 Thayse Fernanda da Silva
Lins,1 Letícia Maria da Silva Garcia,2 Carla Martins Kaneto,2 Geraldo Pedral Sampaio,2
Adriano Alcântara,2 Cássio Santana Meira,1 Simone Garcia Macambira,1,2 Ricardo Ribeirodos-Santos,2 and Milena Botelho Pereira Soares1,2
1
Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, BA, Brazil; 2Centro
de Biotecnologia e Terapia Celular, Hospital São Rafael, Salvador, BA, Brazil.
Running title: Immunomodulatory effects of G-CSF in Chagas disease
Corresponding author: Milena B. P. Soares, Centro de Pesquisas Gonçalo Moniz, Fundação
Oswaldo Cruz, Rua Waldemar Falcão, 121, Candeal, Salvador, BA, Brazil. CEP: 40296-710.
Phone: (55) (71) 3176-2260; Fax: (55) (71) 3176-2272
E-mail address: [email protected]
44
Abstract
Chagas disease, caused by Trypanosoma cruzi infection, is a leading cause of heart failure in
Latin American countries. In a previous study we showed beneficial effects of granulocytecolony stimulating factor (G-CSF) administration in the heart function of mice with chronic T.
cruzi infection, we investigated here the mechanisms by which this cytokine exerts its
beneficial effects. Mice chronically infected with T. cruzi were treated with human
recombinant G-CSF (3 courses of 200 g/kg/day for 5 days). A reduction of inflammation
and fibrosis was observed in the hearts of G-CSF-treated mice, compared to vehicle-treated
mice, which correlated with decreased syndecan 4, ICAM 1 and galectin 3 expressions. A
marked reduction of IFNγ and TNFα, as well as an increase of IL-10 and TGFβ, was found
after G-CSF administration. Since the therapy did not induce a Th1- to Th2-immune response
deviation, we investigated the role of regulatory T cells. A significant increase in
CD4+FoxP3+ cells was found in hearts of G-CSF-treated mice. In addition, a reduction of
parasitism was observed after G-CSF treatment. Our results indicate a role of Treg in the
immunosuppression induced by G-CSF treatment and reinforces its potential use as treatment
for Chagas disease patients.
45
Introduction
Chagas disease, caused by infection with Trypanosoma cruzi, is considered a
neglected tropical disease endemic of Latin American countries, where it mainly affects the
poorest populations.1,2 Although recent data indicate a reduction in the number of infected
people as a result of vector transmission control, it is estimated the presence of 10 million
infected people and 25 million people at risk of contracting the disease.3,4 About 70% of
infected individuals remain asymptomatic, whereas 30% will develop the chronic
symptomatic form of Chagas disease. Chronic chagasic cardiomyopathy (CChC), the most
common symptomatic form of the disease, is one of the leading causes of heart failure. The
only available treatment is heart transplantation, a high cost procedure, limited by organ
donation, and with severe complication in Chagas disease patients due to reactivation of
infection following immunosuppressant administration. 5
Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine which
stimulates the production of neutrophils and the release of bone marrow stem cells into the
peripheral circulation. It has been in clinical use for nearly two decades, mainly as an
adjunctive medication to chemotherapy or to mobilize stem cells for bone marrow
transplantation.6 In addition to neutrophils and their precursors, monocytes are direct target
cells of G-CSF action.7,8 The administration of G-CSF in models of cardiac ischemic diseases
has also shown the potential use of this cytokine in regenerative medicine. 9-11
Evidence is now accumulating that G-CSF has also immunomodulatory effects on
adaptive immune responses by several mechanisms, including activation of T regulatory
(Treg) cells.12 Treg cells express the regulatory lineage factor Foxp3, comprise 5 to 10% of
peripheral CD4+ T cells and are known as natural regulatory T cells. 13 CD4+ T cells from GCSF-mobilized stem cell donors are able to suppress allo-proliferative responses of
autologous T cells in a cell contact-independent manner, by acquiring a Treg like cytokine
46
profile.14 G-CSF drives the in vitro differentiation of human dendritic cells that express
tolerogenic markers involved in Treg cell induction. 15
We have previously demonstrated that administration of G-CSF in mice with chronic
heart lesions caused by T. cruzi infection improved the heart structure and function. 16 Here we
investigated the immunomodulatory effects of G-CSF in a mouse model of chronic Chagas
disease, by investigating the modulation of key inflammatory mediators and the participation
of T regulatory cells.
Materials and methods
Animals
Four week-old male C57BL/6 mice were used for T. cruzi infection and as normal
controls. All animals were raised and maintained at the Gonçalo Moniz Research
Center/FIOCRUZ in rooms with controlled temperature (22 ± 2ºC) and humidity (55 ± 10%)
and continuous air flow. Animals were housed in a 12 h light/12 h dark cycle (6 am - 6 pm)
and provided with rodent diet and water ad libitum. Animals were handled according to the
NIH guidelines for animal experimentation. All procedures described had prior approval from
the local animal ethics committee under number L-002/11 (Fiocruz, Bahia).
Trypanosoma cruzi infection and G-CSF administration
Trypomastigotes of the myotropic Colombian T. cruzi strain
17
were obtained from
culture supernatants of infected LLC-MK2 cells. Infection of C57BL/6 mice was performed
by intraperitoneal injection of 100 T. cruzi trypomastigotes in saline. Parasitemia of infected
mice was evaluated at various times after infection by counting the number of trypomastigotes
in peripheral blood aliquots. Groups of chronic chagasic mice (6 months after infection) were
treated i.p. with 3 courses of administration with human recombinant G-CSF (Filgastrim; Bio
47
Sidus S.A., Argentina) consisting of 200 µg/kg/day during 5 consecutive days with an interval
of 9 days. Control chagasic mice received saline solution in the same regimen.
Morphometric analysis
Groups of mice were euthanized two months after the therapy under anesthesia, 5%
ketamine (Vetanarcol®; Konig, Avellaneda, Argentina) and 2% xylazine (Sedomin®; Konig)
and hearts were removed and fixed in 10% buffered formalin. Heart sections were analyzed
by light microscopy after paraffin embedding, followed by standard hematoxylin/eosin
staining. Inflammatory cells infiltrating heart tissue were counted using a digital
morphometric evaluation system. Images were digitized using a color digital video camera
(CoolSnap, Montreal, Canada) adapted to a BX41 microscope (Olympus, Tokyo, Japan).
Morphometric analyses were performed using the software Image Pro Plus v.7.0 (Media
Cybernetics¸ San Diego, CA). The inflammatory cells were counted in ten fields (400x
magnification) per heart. The percentage of fibrosis was determined using Sirius red-stained
heart sections and the Image Pro Plus v.7.0 Software to integrate the areas, 10 fields per
animal were captured using a 200x magnification. All the analyses were done double-blinded.
Confocal immunofluorescence analyses
Frozen or formalin-fixed paraffin embedded hearts were sectioned and 4 µm-thick sections
were used for detection of syndecan-4, ICAM-1, galectin 3, CD3, Foxp3 and IL-10 expression
by immunofluorescence. First, paraffin embedded sections were deparaffinizated and a heatinduced antigen retrieval step by incubation in citrate buffer (pH=6.0) was performed. Then,
sections were incubated overnight with the following primary antibodies: anti-syndecan-4,
diluted 1:50 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-ICAM-1, 1:50 (BD
Biosciences), anti-CD3, diluted 1:400 (BD Biosciences), anti-Foxp3, 1:400 (Dako, Glostrup,
Denmark) or anti-IL-10, diluted 1:100 (BD Biosciences). On the following day, sections were
48
incubated, for 1 h, with Alexa fluor 633 or 488 conjugated Phalloidin, diluted 1:200, mixed
with one of the secondary antibodies: Alexa fluor 594-conjugated anti-goat IgG, 1:200 or
Alexa fluor 488-conjugated anti-rabbit IgG, 1:200 (Molecular Probes, Carlsbad, CA, USA).
Nuclei were stained with 4,6-diamidino-2-phenylindole (VectaShield Hard Set mounting
medium with DAPI H-1500; Vector Laboratories, Burlingame, CA). The presence of
fluorescent cells was determined by observation on a FluoView 1000 confocal microscope
(Olympus). Quantifications of galectin-3+ cells, syndecan-4+ blood vessels and ICAM-1+
percentual area were performed in ten random fields captured under 400x maginification,
using the Image Pro Plus v.7.0 software.
Cytokine assessment
Cytokines concentrations were measured in total spleen or heart protein extracts.
Tissue proteins were extracted at 50 mg of tissue/500 ml of PBS to which 0.4 M NaCl, 0.05%
Tween 20 and protease inhibitors (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM
EDTA and 20 KIU aprotinin A/100 ml) were added. The samples were centrifuged for 10 min
at 3000 g and the supernatants were immediately used for ELISA assays or frozen at -70o C
for later quantification. IFN-γ, TNF-α, TGF-β, IL-4, IL-10 and IL-17 were quantified in tissue
extracts from individual mice with an enzyme immunoassay determined by ELISA using
specific antibody kits (R&D System, Minnesota, MN), according to manufacturer's
instructions. Briefly, 96-well plates were blocked and incubated at room temperature for 1 h.
Samples were added in duplicates and incubated overnight at 4º C. Biotinylated antibodies
were added and plates were incubated for 2 h at room temperature. A half-hour incubation
with streptavidin–horseradish peroxidase conjugate was followed by detection using 3,3’,5,5’tetramethylbenzidine peroxidase substrate and read at 450 nm.
Real time reverse transcription polymerase chain reaction (RT-qPCR)
49
Total RNA was isolated from heart samples with TRIzol reagent (Invitrogen,
Molecular Probes, Oregon, USA) and concentration was determined by photometric
measurement. High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster
City, CA, USA) was used to synthesize cDNA of 1µg RNA following manufacturer’s
recommendations. RTq-PCR assays were performed to detect the expression levels of Tbet
(Mm_00450960_m1), GATA3 (Mm_00484683_m1) and G-CSF (Mm 00438334_m1). The
qRT-PCR amplification mixtures contained 20 ηg template cDNA, Taqman Master Mix (10
µL) (Applied Biosystems) and probes in a final volume of 20 µL. All reactions were run in
duplicate on an ABI7500 Sequence Detection System (Applied Biosystems) under standard
thermal cycling conditions. The mean Ct (Cycle threshold) values from duplicate
measurements were used to calculate expression of the target gene, with normalization to an
internal control (GAPDH) using the 2–DCt formula. Experiments with coefficients of
variation greater than 5% were excluded. A non-template control (NTC) and non-reverse
transcription controls (No-RT) were also included.
Flow cytometry analysis
Quantitative analysis of Treg cells was performed in the bone marrow and spleen of
G-CSF- or saline-treated chronic chagasic mice, by flow cytometry. Briefly, mice were
treated with 1 course of G-CSF or saline and were euthanized under anesthesia the day after
the last dose. Bone marrow cells were obtained from femurs of mice. The bone marrow from
each bone was collected by flushing the bone with DMEM medium and after that the cells
were purified by centrifugation in Ficoll (Histopaque 1119 and 1077, 1:1; Sigma, St. Louis,
MO) gradient at 1,000 g for 15 minutes. The spleens were collected, washed in DMEM and
homogenized by pressing through a 40 mm cell strainer. Bone marrow and spleen cells were
counted and ressuspended in FACS buffer (1% fetal bovine serum in PBS). For flow
cytometry, cells were stained with APC labeled anti CD4 and CD25 antibodies (BD
50
Biosciences) for 20 min, at room temperature. Cells were washed and analyzed using a cell
analyzer (LSRFortessa, BD Biosciences) with FACSDiva software.
Quantification of parasite load
T. cruzi DNA was quantified in heart samples by qPCR analysis. For DNA extraction, heart
fragments were submitted to DNA extraction using the NucleoSpin Tissue kit (MachenereyNagel, Düren, Germany), as recommended by the manufacturer. Briefly, 10 mg of each heart
sample were submitted to DNA extraction and the DNA amount and purity (260/280 nm)
were analyzed by Nanodrop 2000 spectrophotometry (ThermoFisher Scientific, Waltham,
MA). Kapa Probe Fast Universal 2X qPCR master mix was used to perform the qPCR, in 20
µL reactions, including rox low as the passive reference, as recommended by the
manufacturer (Kapa Biosystems Inc., Woburn, MA). Primers were designed based on the
report by Schijman et al
(primer
1
18
and the amounts used per reaction were 0.4 µM of both primers
5’-GTTCACACACTGGACACCAA-3’
and
primer
2
5’-
TCGAAAACGATCAGCCGAST-3’) and 0.2 µM of the probe (SatDNA specific probe 5’/56-FAM/AATTCCTCC/ZEN/AAGCAGCGGATA/3IABkFQ/-3’), all included in a mini
PrimeTime qPCR Assay (IDTDNA, Coralville, USA). One microliter of each point of the
standard curve, samples and controls were applied to different wells of a PCR microplate
(Axygen, Union City, USA), film sealed and submitted to amplification. Cycles were
performed in an ABI 7500 (Applied Biosystems, Carlsbad, USA) as follows: firstly, 3
minutes at 95ºC for Taq activation; and secondly, 45 cycles at 95ºC for 10 seconds followed
by 55ºC for 30 seconds. To calculate the number of parasites per mg of tissue, each plate
contained an 8-log standard curve of DNA extracted from trypomastigotes of Colombian T.
cruzi strain (ranging from 4.7x10-1 to 4.7x106) in duplicate. Data were analyzed using the
7500 software 2.0.1 (Applied Biosystems, Carlsbad, USA).
Assessment of trypanocidal activity
51
T. cruzi epimastigotes (Colombian strain) were maintained at 26 °C in LIT medium
supplemented with 10% fetal bovine serum (FBS; Cultilab, Campinas, Brazil), 1% hemin
(Sigma), 1% R9 medium (Sigma), and 50 µg/mL of gentamycin (Novafarma, Anápolis,
Brazil). Parasites were counted in a hemocytometer and then dispensed into 96-well plates at
a cell density of 5 x 106 cells/mL in the absence or presence of the human recombinant G-CSF
at 3, 10 or 30 µg/mL, in triplicate. The plate was incubated for 5 days at 26 oC and aliquots of
each well were collected and the number of viable parasites was counted in a Neubauer
chamber. Trypomastigote forms of T. cruzi (Colombian strain) were obtained from
supernatants of LLC-MK2 cells previously infected and cultured in 96-well plates at a cell
density of 2 x 106 cells/mL in RPMI-1640 medium (Sigma) supplemented with 10% FBS and
50 µg/mL of gentamycin in the absence or presence of the human recombinant G-CSF. After
24 h of incubation, the number of viable parasites, based on parasite motility, was assessed in
a Neubauer chamber and compared to untreated parasite culture to calculate the percentage of
inhibition. Benznidazole (30 µg/mL) was used as positive control. For in vitro infection,
peritoneal macrophages obtained from C57BL/6 mice were seeded at a cell density of 2 x 105
cells/mL in a 24 well-plate with rounded coverslips on the bottom in RPMI supplemented
with 10% FBS and 50 µg/mL of gentamycin and incubated for 24 h. Cells were then infected
with trypomastigotes (1:10) for 2 h. Free trypomastigotes were removed by successive washes
using saline solution. Cultures were incubated in complete medium alone or with G-CSF (3 or
10 µg/mL) or benznidazole (10 µg/mL) for 6 h. The medium was then replaced by fresh
medium and the plate was incubated for 3 days at 37oC. Cells were fixed in absolute alcohol
and the percentage of infected macrophages and the mean number of amastigotes/100 infected
macrophages was determined by manual counting after hematoxylin and eosin staining using
an optical microscope (Olympus). The percentage of infected macrophages and the number of
amastigotes were determined by counting 100 cells per slide.
52
Evaluation of anti-T. cruzi antibodies
T. cruzi-specific, total IgG, IgG1 and IgG2 antibodies were detected in the sera of naive, GCSF- or saline-treated chronic chagasic mice by ELISA. Microtiter plates were coated
overnight at 4°C with T. cruzi trypomastigote antigen (3 µg/ml) in 50 µl of carbonatebicarbonate buffer (pH 9.6). The plates were washed three times with PBS containing 0.05%
Tween 20 and then blocked by incubation at room temperature for 1 h with PBS–5% nonfat
milk. After washing, the plates were incubated with 50 μl of a 1:200 (IgG) or 1:100 (IgG1 or
IgG2a) dilution of each serum sample at 37°C for 2 h. The plates were washed, and a 1:1000
dilution of goat anti-mouse IgG (Sigma), rat anti-mouse IgG1 or IgG2a (BD Biosciences)
were incubated for 1 h at room temperature. After washing, peroxidase conjugated anti-mouse
polyvalent immunoglobulins (Sigma) diluted 1:1000 was dispensed into each well and the
plate was incubated for 30 min at room temperature followed by detection using 3,3’,5,5’tetramethylbenzidine peroxidase substrate and read at 450 nm.
All continuous variables are presented as means ± SE. Morphometric and cytokine
levels were analyzed using 1-way ANOVA, followed by Newman-Keuls multiple-comparison
test with Prism 3.0 (GraphPad Software, San Diego, CA, USA). All differences were
considered significant at values of p < 0.05.
Results
Administration of G-CSF reduces inflammation and fibrosis in hearts of chronic
chagasic mice
53
Multifocal inflammation, mainly composed by mononuclear cells, and fibrosis were
found in the hearts of T. cruzi-infected mice during the chronic phase of the disease (Figures
1A-B). Administration of G-CSF reduced the number of inflammatory cells and the area of
fibrosis in chronic chagasic hearts (Figures 1C-D). Morphometric analysis showed a
statistically significant reduction of inflammation and fibrosis area after G-CSF treatment,
when compared to saline-treated controls (Figures 1E-F).
54
Figure 1: Reduction of inflammation and fibrosis in the hearts of chagasic mice after GCSF administration. Groups of C57BL/6 mice in the chronic phase of infection (6 months)
were treated with saline (A and B) or G-CSF (C and D). A and C, heart sections stained with
H&E; B and D, heart sections stained with Sirius red. E, Quantification of inflammatory cells
was performed in heart sections of naive mice or saline-treated or G-CSF-treated chagasic
mice and integrated by area. F, Fibrotic area was represented by percentage of collagen
deposition in heart sections. Bars represent the mean±SEM of 9-10 mice/group. *** p<0.001.
55
Reduction of syndecan-4, ICAM-1 and galectin 3 expression in the hearts of G-CSFtreated mice
We have previously shown the overexpression of syndecan-4, ICAM-1 and galectin 3
in the hearts of chronic chagasic mice. 19 To evaluate the effects of G-CSF on the expression
of these inflammation markers, we performed analysis by confocal microscopy in heart
sections of mice of the three different groups. A marked decrease in syndecan-4 production,
highly expressed in blood vessels of chagasic hearts, was seen after G-CSF treatment (Figures
2A-B). Morphological analyses showed a difference of statistical significance (Figure 2C).
Similarly, the expression of ICAM 1, mainly expressed in inflammatory cells and
cardiomyocytes in hearts of chronic chagasic mice, was significantly decreased after G-CSF
treatment (Figures 2D-F). Moreover, the high expression of galectin 3 on inflammatory cells
was down-modulated in hearts of mice treated with G-CSF, correlating with the decrease of
inflammation (Figures 2G-I).
56
Figure 2: Reduction of Syndecan-4, ICAM-1 and galectin 3 in the hearts of chronic
chagasic mice after G-CSF administration. Heart sections of saline-treated (A, D and G) or
G-CSF-treated (B, E and H) mice were stained with anti-syndecan-4 (green; A and B), antiICAM-1 (green; D and E) or anti-galectin 3 (red; G and H) antibodies. F-actin stained with
phalloidin 633 (red, A, B, D and E) or 488 (green, G and H). All sections were stained with
DAPI for nuclear visualization (blue). Scale bars represent 50 µm. Morphometric analyses (C,
F and I) in heart sections of naive mice or chagasic mice treated with saline or G-CSF. Bars
represent the mean±SEM of 3 animals/group. *** p < 0.001.
57
Modulation of cytokine production after G-CSF administration
Chronic chagasic cardiomyopathy has been associated with increased production of
IFN-γ and TNFα both in mice, as well as in humans.19,20 The concentrations of these two proinflammatory cytokines were increased in heart extracts of saline-treated chagasic mice,
compared to normal mice. The administration of G-CSF promoted a statistically significant
reduction in the concentrations of both cytokines (Figures 3A-B). In contrast, a significant
increase in TGFβ and IL-10 concentrations was found in the hearts of G-CSF-treated chagasic
mice, compared to saline-treated controls (Figures 3C-D). No significant differences were
found in IL-17 and IL-4 concentrations (Figures 3E-F). Moreover, the analysis by RT-qPCR
showed a significant decrease in Tbet gene expression after G-CSF administration (Figure
3G), but no significant alterations in GATA3 gene expression were observed in the hearts of
chronic chagasic mice (Figure 3H).
The systemic effects of G-CSF on cytokine production were also investigated. G-CSF
reverted the overexpression of TNFα and IL-17 in the spleens of chagasic mice, when
compared to uninfected and saline-treated mice (Figures 4A-B). This effect was also
correlated with an increase in IL-10 production promoted by G-CSF (Figure 4C).
Additionally, G-CSF administration also induced the increase in G-CSF gene expression in
the spleens of chagasic mice (Figure 4D).
58
59
Figure 3: Modulation of cytokine production after G-CSF treatment. The concentrations
of IFN- (A), TNF-α (B), TGF-β (C), IL-10 (D), IL-17 (E) and IL-4 (F) were determined in
heart homogenates from naive (n=3) or chagasic mice treated with saline (n=7) or G-CSF
(n=10), by ELISA. The analysis of Tbet (G), Gata-3 (H) was performed by quantitative real
time RT-PCR using cDNA samples prepared from mRNA extracted from hearts of naive and
chronic chagasic mice treated with saline or G-CSF (n=9-10 mice/group). Values represent
the mean±SEM. * p<0.05; ** p<0.01; *** p<0.001.
60
Figure 4: Modulation of cytokine production in the spleens of chronic chagasic mice
treated or not with G-CSF. The concentrations of TNFα (A), IL-17 (B) and IL-10 (C) were
determined in spleen homogenates from naive (n=3) and chagasic mice treated with saline
(n=7) or G-CSF (n=10), by ELISA. (D), G-CSF mRNA expression was determined by
quantitative real time RT-PCR using cDNA samples prepared from mRNA extracted from
spleens of naive and chronic chagasic mice treated with saline or G-CSF. Values represent the
mean±SEM. * p<0.05.
61
G-CSF therapy increases the percentage of Treg cells in the hearts of chagasic mice
Next, we examined whether G-CSF administration altered the number of Treg cells in
chagasic mice. G-CSF caused a decrease on the percentage of CD4+CD25+ cells in the bone
marrow of chagasic mice (Figures 5A-C). The expression of Foxp3 was analyzed in CD3+
cells in the hearts by immunofluorescence. A higher percentage of CD3+ Foxp3+ T cells
expressing was found in the hearts of G-CSF-treated mice, compared to saline-treated mice
(Figures 5D-F). A partial recovery of CD4+CD25+ cells in the spleens of chagasic mice was
found after G-CSF administration (Figure 5G), and splenic Foxp3+ cells co-expressed IL-10
(Figure 5H).
62
Figure 5: Mobilization of Treg cells after G-CSF administration. Flow cytometry analysis
of bone marrow cells obtained from chagasic mice treated with saline (A) or one course of GCSF (B). C, Quantification of CD4+ CD25+ cells was evaluated in the bone marrow of naive
and chagasic mice treated with saline or G-CSF. Foxp3 expression (red) in CD3+ cells (green)
was evaluated in heart sections of mice treated with saline (D) or G-CSF (E) by
immunofluorescence (n=4/group). Nuclei (blue) were stained with DAPI. (F) Quantification
of CD3+FoxP3+ cells in hearts of naive and chagasic mice treated with saline or G-CSF. (G),
Quantification of CD4+ CD25+ cells was evaluated in the spleens of naive and chagasic mice
treated with saline or G-CSF. Bars represent the mean±SEM. (H), Spleen section of a G-CSFtreated mouse, stained with antiFox3 (green) and anti-IL-10 (red) antibodies. Nuclei (blue)
were stained with DAPI. ** p<0.01; *** p<0.001.
63
Effects of G-CSF administration on the infection by T. cruzi
In order to investigate whether the supressive response induced by G-CSF treatment
affected the immune response against the parasite, we first analyzed the levels of parasitespecific antibodies in the sera of mice from the different experimental groups. The levels of
total IgG anti-T. cruzi antibodies were similar between G-CSF and saline-treated chagasic
mice (Figure 6A). A significant increase in IgG1, but not in IgG2 anti-T. cruzi antibodies, was
found in the group treated with G-CSF compared to saline-treated controls (Figures 6B-C).
To evaluate whether G-CSF administration in chronic chagasic mice affected the
residual T. cruzi infection, we performed a qRT-PCR analysis to quantify the parasite load in
the hearts of mice. As shown in figure 6D, a significant reduction in the parasite load was
observed in the hearts of G-CSF-treated mice when compared to saline-treated controls. To
determine whether G-CSF has a direct action on the parasite, we analyzed the effects of GCSF on T. cruzi cultures. Addition of different concentrations of G-CSF in axenic cultures of
T. cruzi epimastigotes had little effect on its viability at 30 µg/mL (Table 1). In contrast, a
concentration-dependent trypanocidal effect was seen in cultures of isolated trypomastigotes
(Table 1). In addition, when G-CSF was added to cultures of macrophages infected with T.
cruzi, a significant decrease in the percentage of infected cells, as well as in the number of
intracellular amastigotes, was observed (Figures 6E-F).
64
Figure 6: Effects of G-CSF in the production of anti-T. cruzi antibodies and in the
parasite. A-C, Serum from normal and T. cruzi-infected mice treated with saline or G-CSF (3
courses) were obtained two months after treatment. The levels of anti-T. cruzi total IgG (A),
IgG1 (B) and IgG2 (C) antibodies were determined by ELISA. Bars represent the mean±SEM
of 5-10 mice/group. (D), Heart fragments obtained from normal and T. cruzi-infected mice
treated with saline or G-CSF (3 courses) were used for DNA extraction and RT-qPCR
analysis for quantification of the parasite load. E and F, Mouse peritoneal macrophages were
infected with T. cruzi and treated with G-CSF (3 and 10 µg/mL) or benznidazole (10 µg/mL),
a standard drug. The number of infected cells (E) and amastigotes (F) were determined by
counting hematoxylin and eosin-stained cultures. Values represent the mean±SEM of
triplicate obtained in one of two experiments performed. * p<0.05; ** p<0.01; *** p<0.001
compared to the other groups.
65
Table 1. Effects of G-CSF in axenic cultures of T. cruzi (Colombian strain).
Drug
G-CSF
Benznidazole
Concentration
% Inhibition
Epimastigotes
Trypomastigotes
3 µg/mL
0.52 (±0.52)
1.90 (± 0.90)
10 µg/mL
2.58 (± 0.66)
12.85 (± 1.43)*
30 µg/mL
9.60 (± 1.34)**
24.28 (± 2.51)***
30 µg/mL
96.78 (± 0.06)***
100***
Values are mean ± SEM of three independent experiments. * p<0.05; ** p<0.01; ***
p<0.001.
66
Discussion
The hallmark of CCHC is the presence of a multifocal inflammatory response mainly
composed by lymphocytes and macrophages, which promote myocytolysis, fibrosis
deposition and myocardial remodeling.21 This is a progressively debilitating disease which
occurs in a phase of the disease when parasitism is very scarce. Although the mechanisms of
pathogenesis are still a matter of debate, 22 the correlation of severity of the disease and IFNγ
production has been well demonstrated.23,24 The fact that the parasite persists in T. cruziinfected individuals renders any immunosuppressive condition of risk for reactivation of
parasitemia.25,26
In the present study we have demonstrated that systemic administration of G-CSF, a
cytokine widely used in the clinical setting, modulates the inflammatory response, decreasing
the production of key inflammatory mediators such as IFNγ and TNFα, in the main target
organ, the heart. Different cytokine profiles are involved in the control of both the immune
response and pathology during T. cruzi infection. The control of parasitism during the acute
phase of Chagas disease is critically dependent on effective macrophage activation by
cytokines such as IFN-γ and TNFα, crucial for limiting parasite replication.20,27 On the other
hand, an intense Th1 response will enhance heart inflammation. 23 An exacerbated production
of IFN-γ against T. cruzi antigens favors the development of a strong Th1 response in
symptomatic cardiac patients, which leads to progression of heart disease. 20,28 Despite the
suppression of IFNγ and TNFα seen after G-CSF administration in chagasic mice, this therapy
did not increase the parasite load,17 suggesting a partial or selective downregulation of
immune responses in chronic chagasic mice. Moreover, G-CSF did not interfere with
macrophage and lymphocyte activation in vitro, suggesting that this cytokine does not
modulate directly the production of pro-inflammatory mediators (data not shown).
67
Previous reports have shown immune deviation induced after treatment with G-CSF.29
In our model of Chagas disease, we did not observe an increase in IL-4, a marker of Th2-type
responses. In addition, we found a significant decrease on the mRNA for Tbet, a
transcriptional factor essential for Th1-polarized immune responses, but we did not find a
significant difference on the gene expression levels for GATA3, essential for Th2 responses,
in the hearts of G-CSF-treated mice. Therefore, our results do not indicate a shift towards a
Th2 profile after G-CSF administration in chagasic mice, but rather a suppression of the Th1
type immune response found in chronic chagasic mice.
In addition to cytokines, adhesion molecules important to cell migration, such as
syndecan 4 and ICAM 1, already shown to be elevated in the hearts of chronic chagasic
mice,19,29 were downmodulated after G-CSF therapy. TNFα induces ICAM-1 expression thus
increasing the adhesiveness of endothelium for leukocytes. 30 Syndecan 4 is a transmembrane
heparan sulfate proteoglycan that acts cooperatively with integrins in generating signals
necessary for the assembly of actin stress fibers and focal adhesions.31-33 Since TNFα
upregulates syndecan 4 expression,34 the decrease on its expression in endothelial cells in the
hearts of chagasic mice may be a consequence of the TNFα downregulation observed after GCSF administration. Altogether, the decrease of syndecan 4 and ICAM1 may contribute to a
reduction of cell migration into the myocardium and, consequently, of inflammation.
Another important action of G-CSF in the model of Chagas disease, as well as in other
models of ischemic heart disease, is the induction of fibrosis reduction.17,35 This may be due
to the modulation of fibrogenic mediators in the heart, such as galectin-3, which has been
shown to play important roles in fibrosis deposition, as well as in heart failure.36 Galectin-3
expression was found in activated myocardial macrophages and is increased by IFNγ.37,38 In
addition, the administration of recombinant galectin-3 in rats induced cardiac fibroblast
proliferation, collagen production and left ventricular. 37 Moreover, galectin-3 is known to
68
play important roles in the regulation of inflammatory responses, including suppression of T
cell apoptosis .38,39 In fact, in our previous study we observed an increase of apoptosis in the
hearts of chagasic mice treated with G-CSF,17 correlating with the decrease of galectin 3
found herein.
The suppression of inflammatory mediators observed herein after G-CSF
administration was accompanied by an increase of IL-10 production in the hearts and spleens
of chagasic mice. This is an important regulatory cytokine already shown to be associated
with a better evolution of chronic chagasic patients. 20 The co-expression of IL-10 in Treg
cells, which are increased in percentage after G-CSF treatment in the present study, suggests
these cells as one source of IL-10. In fact, the production of IL-10 has been described as one
of the mechanisms by which Treg cells exert their suppressive activity.40
Regulatory T cells (Treg) constitute an antiinflammatory T cell population, associated
with immune regulation, which may prevent tissue damage caused by parasite-triggered
immune responses.41 Treg cells expressing CD4+CD25+ cells have been recently associated
with the expression of the regulatory lineage factor Foxp3 and are responsible for maintaining
self-tolerance.42,43 The increased percentage of Treg cells in the spleens and lymph nodes of
G-CSF-treated mice by mobilization of bone marrow resident Treg cells has been previously
described.44 This mobilization of Treg cells after G-CSF administration seems to be due to a
reduced expression of SDF1, the CXCR4 ligand, in the bone marrow. Rutella and coauthors.45
have reported that G-CSF induces an increase of Treg cells in the peripheral blood of normal
human recipients. Recent investigations evaluating the frequency of Treg cells during early
and late indeterminate Chagas disease have shown the correlation between the severity of the
chronic chagasic cardiomyopathy with a lower frequency or suppressive activity of
CD4+CD25+ cells.46-48 Thus, our data corroborate with these findings, since an inverse
69
correlation between the percentage of Tregs and inflammation cells and cytokines in the heart
of chagasic mice was found when G-CSF and saline-treated mice were compared.
One important issue raised was whether the suppressive effects of G-CSF could
interfere with the control of T. cruzi infection. The levels of anti-T. cruzi IgG antibodies in the
sera of chagasic mice were not reduced by treatment with G-CSF. More importantly, by using
a very sensitive quantification method,18 we found a decrease in the parasite load in the hearts
of chronic chagasic mice treated with G-CSF when compared to saline-treated mice,
suggesting that this cytokine could have a direct effect on parasite elimination. To address this
question, we evaluated the effects of G-CSF in vitro, in the three forms of the parasite.
Although G-CSF had little effect on the viability of epimastigote form, it did affect
significantly the forms found in the mammalian hosts (trypomastigotes and amastigotes) in a
concentration-dependent manner. In fact, previous reports have shown that other cytokines,
such as GM-CSF and TNFα, can have direct effects on the parasite and interfere with T. cruzi
infection.49 Altogether, our data indicate that reduction of parasitism may be a positive effect
of G-CSF therapy.
In conclusion, the present study reinforces our previous work, in which we
demonstrated improvement of cardiopulmonary function after G-CSF administration, by
revealing its potent antiinflammatory properties in a model of parasite driven heart disease.
More importantly, we evidenced the modulation of pathogenic immune responses without
affecting the control of infection, a feature highly desired in the clinical setting. Altogether,
our results reinforce the possibility of clinical applications of G-CSF, a well-tolerated drug
with low side effects, in the treatment of Chagas cardiomyopathy patients.
70
Acknowledgments
J.F.V. was recipient of a CAPES doctoral scholarship (UEFS/PPgBIOTEC). B.S.F.S. is
holding a CNPq doctoral scholarship and C.S.M. is holding a FAPESB master scholarship.
M.B.P.S. and R.R.S. are recipients of a CNPq senior fellowship.
Funding
This work was supported by grants from the Brazilian Ministry of Sciences and Technology
(MCT), Conselho Nacional de Pesquisas (CNPq), Financiadora de Estudos e Projetos
(FINEP), Fundação Oswaldo Cruz (FIOCRUZ), and Fundação de Amparo as Pesquisas da
Bahia (FAPESB).
Authorship
Contribution: M.B.P.S., R.R.S., B.S.F.S., and J.F.V. designed the study. J.F.V., B.S.F.S,
T.F.S.L., L.S.G., C.M.K., G.P.S., A.A., C.S.M., and S.G.M. performed experiments, supplied
reagents, tools, laboratory analyses, and analyzed the data. J.F.V. and M.B.P.S. wrote the
paper. All authors revised the paper.
Conflict of interests
The authors declare no competing financial interests.
71
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77
4 DISCUSSÃO GERAL
A doença de Chagas é uma doença debilitante e incapacitante, com alternativas
terapêuticas limitadas na atualidade, o que torna premente o desenvolvimento de novas
terapias para essa doença que acomete mais de dez milhões de pessoas no mundo. A única
terapia para os pacientes chagásicos crônicos com insuficiência cardíaca avançada é o
transplante cardíaco. Neste estudo descrevemos, pela primeira vez na literatura, a utilização
de um hormônio celular, o G-CSF, no tratamento da CChC experimental, bem como a
investigação de seus mecanismos de ação na doença.
Para o tratamento de doenças crônicas em que a cura ainda não é possível de ser
alcançada, a indústria farmacêutica tem apostado no uso de citocinas, tais como os IFNs e a
eritropoietina (EPO), como agentes modificadores da resposta biológica. O desafio para a
utilização clínica dessas moléculas se deve, em parte, às suas características, em especial ao
pleiotropismo e a redundância, que resultam em uma complexa rede de cascata de citocinas e
circuitos
reguladores,
com
efeitos
de
retroalimentação
(THOMAS
&
BECK-
WESTERMEYER, 2007; FELDMANN et al., 2008). Dentre as citocinas usadas
terapeuticamente, o G-CSF recombinante humano tem uma utilização menos restrita por
apresentar menos efeitos colaterais e por ser mais bem tolerado pelos pacientes. O G-CSF
recombinante humano é empregado em larga escala na prática clínica há mais de duas
décadas, apresentando atividade benéfica em várias situações clínicas, tais como neutropenia
idiopática, mielossupressão induzida por quimioterapia, recuperação da aplasia póstransplante de medula óssea e mobilização de células progenitoras hematopoiéticas para a
circulação periférica (ANDERLINI & CHAPLIN, 2008). Dada à larga experiência préexistente da sua utilização clínica e segurança da sua utilização, pode-se propor a transposição
dos resultados obtidos em nosso estudo para a realização de estudos clínicos em pacientes
chagásicos crônicos.
O modelo de CChC é bem descrito na literatura, onde camundongos da linhagem
C57Bl/6 infectados com T. cruzi da cepa Colombiana desenvolvem, após seis meses de
infecção, um quadro característico de CChC com inflamação e fibrose no coração, semelhante
ao observado em humanos (SOARES et al., 2004; ROCHA et al., 2006). Nesse modelo,
observamos que o tratamento por três ciclos de G-CSF proporcionou diversos efeitos
benéficos, dentre eles a melhora da função cardiorespiratória dos animais. O consumo
máximo de oxigênio, o maior volume de oxigênio por unidade de tempo que um indivíduo
consegue captar durante o exercício, é alcançado quando se atingem níveis máximos de débito
78
cardíaco e é considerado um índice muito empregado para classificar a capacidade funcional
cardiorrespiratória do indivíduo. Os animais tratados com G-CSF apresentaram melhor
desempenho em esteira ergométrica já que foram capazes de se exercitar por mais tempo, o
que possibilitou que percorressem uma distância maior do que os animais tratados apenas com
salina. Demonstramos aqui que o tratamento com G-CSF causou uma melhora funcional,
também avaliada pelo aumento do consumo de VO2 e CO2 nos animais tratados com G-CSF.
Quanto mais grave for a insuficiência cardíaca nos pacientes com CChC pior será sua
capacidade de exercício, acarretando ao longo do tempo a perda da capacidade laboral,
apresentando o G-CSF um efeito protetor.
A CChC é caracterizada por uma variedade de anormalidades estruturais que geram
desvio de eixo e condução lenta, além do aparecimento de arritmias ventriculares que podem
levar à morte súbita (RASSI et al., 2001). Os camundongos que receberam G-CSF
apresentaram uma diminuição significativa no percentual de fibrose no coração, associada à
indução de efeitos benéficos funcionais no coração, conforme observado através de avaliação
por eletrocardiografia (MACAMBIRA et al., 2009). Todos os animais chagásicos crônicos
apresentaram alterações elétricas que variaram de bloqueio átrio-ventricular de primeiro grau
a dissociação átrio-ventricular. Comparativamente, enquanto os animais tratados com salina
permaneceram com os distúrbios de condução ou agravaram seu quadro, os animais tratados
com G-CSF tiveram a gravidade de seus distúrbios diminuída e alguns reverteram
completamente o distúrbio de condução antes apresentado.
A origem das arritmias parece ser dependente de alteração nas conexinas, proteínas
presentes nas junções comunicantes, e uma vez que haja menor acoplamento elétrico entre os
cardiomiócitos, há falta de coordenação entre as células cardíacas. A infecção pelo T. cruzi
está associada a uma diminuição da expressão de Cx43 em cardiomiócitos em modelo animal
e em humanos (KUHLMANN et al., 2006; WAGHABI et al., 2009). A utilização de G-CSF
em modelo de infarto do miocárdio também proporcionou a reversão de arritmias, fato este
que foi associado o aumento da expressão de Cx43 como uma das possíveis causas da
reversão dos distúrbios elétricos encontrados (KUHLMANN et al., 2006; KUWABARA et
al., 2007). De maneira discordante, não encontramos diferença na expressão do mRNA da
Cx43 nos corações dos grupos experimentais e nem tampouco na presença da Cx43 no tecido,
não nos permitindo associar a melhora dos distúrbios de condução nos animais tratados com
G-CSF com alterações na expressão gênica ou produção de Cx43 (dados não mostrados).
Dados de cultura de cardiomiócitos de rato infectados com T. cruzi sugerem que nem a
transcrição nem a tradução da conexina 43 (Cx43) são alteradas, porém foi observada uma
79
alteração na localização subcelular dessa proteína, com conseqüente desorganização da junção
comunicante e perda do acoplamento elétrico entre tais células (CAMPOS-DE-CARVALHO
et al., 1994).
De maneira indireta, nossos dados corroboram a hipótese da importância do
mimetismo molecular para o desencadeamento das arritmias cardíacas em modelos de CChC,
já que demonstramos que a diminuição da presença de anticorpos anti-β1 e anti-M2, assim
com a redução de anti-P2β nos animais tratados com G-CSF estão associados à melhora da
função cardíaca desses animais. Apesar de seus mecanismos de ação não estarem
completamente esclarecidos, estudos indicam que esses anticorpos podem ativar vias
intracelulares por ativação de proteína G e consequente alteração na eletrogênese cardíaca
(MACIEL et al., 2012) e, portanto, a redução da presença de tais anticorpos nos animais
tratados com G-CSF pode estar associada à melhora dos distúrbios arritmogênicos.
A resposta inflamatória é um marco central nas lesões cardíacas durante a CChC,
sendo a dinâmica desse processo capaz de induzir ao remodelamento do miocárdio, com
especial participação de linfócitos T e macrófagos como agentes indutores de miocardite,
hipertrofia dos cardiomiócitos, culminando no processo de fibrose (ANDRADE, 1983;
HIGUCHI et al., 2003). A perda de cardiomiócitos durante a infecção pelo T. cruzi também
contribui para o agravamento das lesões no miocárdio e consequente disfunção cardíaca.
Harada e colaboradores (2005) não observaram a proliferação de cardiomiócitos após o
tratamento com G-CSF em camundongos infartados, induzindo os autores a atribuir a melhora
observada na função cardíaca à ação direta do G-CSF como agente anti-apoptótico, tanto em
cardiomiócitos quanto em células endoteliais.
A ligação do G-CSF com seu receptor em cardiomiócitos ativa a via JAK2/ STAT1 e
3, induzindo a produção de Bcl2, proteína anti-apoptótica, protegendo dessa maneira as
células cardíacas. Esse mesmo efeito anti-apoptótico do G-CSF também foi descrito em
neurônios e neutrófilos por diversos mecanismos tais como a produção de survivina, da
proteína anti-apoptótica Mcl-1, bloqueio da translocação de Bax e inibição da ativação da
caspase 9/3 (MAIANSKI et al., 2002; MAIANSKI et al., 2004; XIAO et al., 2007; JUN et al
2011). Ao contrário da doença isquêmica, na CChC observa-se uma baixa taxa de
cardiomiócitos apoptóticos (da SILVA & GORDON, 1999), entretanto esse possível efeito
benéfico do G-CSF na diminuição da apoptose de cardiomiócitos não foi analisado em nosso
modelo. Novos estudos deverão ser realizados com o objetivo de entender se o tratamento
com G-CSF no modelo de CChC promove a proliferação ou a geração de cardiomiócitos.
80
A comparação entre a expressão gênica em corações de animais normais e chagásicos
possibilitou a observação de diferença significativa na expressão de genes associados tanto
com a resposta imune-inflamatória quanto com a fibrose, com o aumento de expressão de
moléculas de adesão e de fatores quimiotáticos em corações chagásicos crônicos (SOARES et
al., 2010). Nesse trabalho demonstramos que o tratamento com G-CSF reduziu a produção de
ICAM-1 e syndecan-4 no coração, contribuindo para um menor aporte de células
inflamatórias no coração. A produção de CXCL12, uma quimiocina com ação recrutadora de
células-tronco, foi diminuída no coração dos animais após o tratamento com G-CSF, o que,
segundo Mieno e colaboradores (2006), também está relacionado a um menor recrutamento
de células inflamatórias para o coração. De maneira semelhante, no modelo de CChC, o
aumento de CXCL12, do ICAM-1 e de syndecan-4 nos animais chagásicos crônicos tratados
com salina proporcionam um maior recrutamento de células inflamatórias, contribuindo para
a manutenção do infiltrado inflamatório observado nesse modelo, sendo o G-CSF capaz de
inibir esse processo e, consequentemente, de controlar a lesão tecidual.
A possibilidade de reversão dos danos causados pela resposta inflamatória no coração
tem sido objeto de intensa investigação nas últimas décadas. Além do menor aporte e
permanência de leucócitos no coração, demonstramos também uma maior taxa apoptótica de
leucócitos no coração dos animais tratados. Esses resultados corroboram os dados de Rutella e
colaboradores (2001), nos quais observou-se que a indução da apoptose de linfócitos T pelo
G-CSF por consequência do aumento da expressão da bax, desregulação do potencial das
membranas mitocondriais e clivagem da caspase-3 com conseqüente fragmentação do DNA.
Durante o processo de apoptose, as células mortas são eliminadas por fagocitose sem
provocar uma resposta inflamatória nociva, eliminando desta forma, linfócitos indesejáveis a
fim de evitar respostas patológicas. Por ser capaz de induzir a apoptose das células
inflamatórias no miocárdio chagásico, que é composto principalmente por células
mononucleares, o G-CSF tem desta forma, uma ação inibitória da progressão da lesão.
Diversos grupos demonstraram que o G-CSF induz uma mudança de resposta imune
para o perfil Th2 da resposta imune com aumento da produção de IL-4 e IL-10, acompanhado
por um decréscimo na produção de IFN-γ in vitro (PAN et al., 1995; SLOAND et al., 2000).
No modelo de CChC, ao invés de uma forte resposta Th2, descrevemos aqui a inibição do
perfil Th1 pelo G-CSF associada ao controle da resposta inflamatória. Não foi observado o
aumento de IL-4 nos animais tratados, sendo a produção de IFN-γ, que é a principal citocina
Th1, reduzida após o tratamento com G-CSF, assim como a expressão de Tbet, um fator
transcricional essencial para a diferenciação de células Th1. Além disso, observamos uma
81
redução de TNF-α, que é uma citocina com múltiplos efeitos no coração após interação com
seu receptor tipo 1 (TNFR1), como indução do aumento da expressão endotelial das
quimiocinas CCL3 e CCL2, como também a expressão de moléculas de adesão em
cardiomiócitos resultando em aumento do infiltrado leucocítico no coração (HIGUCHI et al.,
2004; BILATE et al., 2007).
Na doença de Chagas a persistência do infiltrado macrofágico no coração, está
associado a indução de cronicidade, sendo evidenciado nesse trabalho, o aumento do número
de macrófagos nos animais tratados apenas com salina, associado a uma maior expressão de
galectina-3. A galectina-3 é uma molécula pleiotrópica que age como um potente mitógeno
para fibroblastos in vitro e tem sido envolvida com uma variedade de processos biológicos
incluindo a proliferação, adesão e sobrevivência celular, bem como a desregulação da
produção de colágeno (KUWABARA & LIU, 1996; AKAHANI et al., 1997; HSU et al.,
1999; HENDERSON & SETHI, 2009; WANG et al., 2000; OLIVEIRA et al., 2011). A
galectina 3 está especificamente aumentada em camundongos com falência cardíaca
descompensada, fato este que tem sido associado à ativação de fibroblastos e macrófagos,
células características do remodelamento cardíaco (de BOER et al., 2011). Segundo Liu e
colaboradores (1995), quanto mais diferenciado e ativado for o macrófago, tanto maior será a
produção de galectina-3, o que sugere que o G-CSF pode estar diminuindo a ativação de
macrófagos, já que esse grupo produziu menos galectina-3, o que justificaria a redução da
área ocupada por tecido fibrótico.
Recentemente, o paradigma Th1/Th2 tem sido expandido pela descoberta de outros
subgrupos como as células Th17 e as células Treg, que exibem funções distintas das de
células Th1 e Th2. Embora as células Th17 possam estar associadas à eliminação de
patógenos que não foram eliminados adequadamente por células Th1 ou Th2, a ativação das
células Th17 células parece contribuir significativamente para a destruição tecidual (KORN et
al., 2009). A participação dessas células na imunopatologia da doença de Chagas ainda não é
muito clara. Observamos que animais chagásicos apresentaram um aumento da produção de
IL-17 quando comparados a camundongos normais, e o grupo tratado com G-CSF teve uma
redução significativa da produção desta citocina no baço, mas não no coração. As células
Th17 dependem da produção de TGF-β e IL-6 para ativação do fator de transcrição RORγT e
produção de IL-17, a falta de TGF-β impede a diferenciação Th17 (LI et al., 2007). Apesar da
produção diminuída de IL-17 no coração e baço de animais tratados, não observamos
diferença na expressão gênica de RORγT entre os grupos (dados não apresentado), sugerindo
82
que o tratamento com G-CSF não altera de forma significativa a sub-população de células
Th17.
As células Treg constituem uma linhagem anti-inflamatória de linfócitos T associada à
regulação do sistema imune, que atua regulando as respostas inflamatórias para evitar dano
causado por intensa ativação celular (BELKAID, BLANK & SUFFIA, 2006). A frequência de
células Treg CD4+ CD25+no sangue periférico de pacientes chagásicos crônicos que
apresentam a forma indeterminada da doença é maior do que naqueles pacientes que
desenvolvem a forma sintomática, com manifestações clínicas da doença (VITELLIAVELAR et al., 2005, de ARAÚJO et al., 2011). Também já é conhecida a ação do G-CSF na
promoção do aumento do número de células CD4+ CD25+ Foxp3+ produtores de IL-10 e
TGF-β (RUTELLA et al., 2005; TOH et al., 2009), dessa forma decidimos investigar a
participação de células Treg na regulação da resposta inflamatória nos animais tratados com
G-CSF.
Observamos uma menor frequência de células CD4+CD25+ na medula óssea de
animais tratados com G-CSF em relação aos chagásicos injetados com salina, coerente com a
ação descrita do G-CSF no recrutamento de tais células para a circulação periférica (KARED
et al., 2005; RUTELLA et al., 2005). Por ser o baço um órgão de passagem importante para as
células presentes na circulação, investigamos a presença de células Treg nesse tecido. Dessa
maneira, observamos a presença elevada de células Treg no baço dos animais tratados com GCSF, bem como o aumento da produção in situ de IL-10. Embora outros tipos celulares
produzam IL-10, é possível que as Treg estejam contribuindo de maneira mais evidente para
este aumento uma vez que observamos a presença de células Foxp3 +IL-10+ nesse órgão. O
aumento de células Treg na circulação propicia sua maior chegada ao coração dos animais
chagásicos crônicos tratados com G-CSF, conforme demonstramos através da quantificação
de células CD3+ Foxp3+. Portanto, é possível que o aumento no percentual de células Treg no
coração contribua com a redução da resposta inflamatória.
Os resultados descritos nesse estudo estão sumarizados na figura 7, onde apresentamos
a mobilização de células Treg da medula óssea pela administração do G-CSF in vivo
proporcionando um efeito modulador da resposta imune a nível sistêmico e local, em uma
doença caracterizada por intensa agressão tecidual pela resposta inflamatória.
83
Figura 7. Representação esquemática da ação direta e indireta do G-CSF sobre o tecido cardíaco
promovendo modulação da resposta inflamatória local em modelo de CChC experimental.
Uma preocupação inerente à imunomodulação proporcionada pelo tratamento com GCSF é a possibilidade de reativação dos parasitos restantes, refletido em um aumento da carga
parasitária. Para excluir tal possibilidade analisamos a presença de antígenos e DNA de T.
cruzi no coração dos animais dos diferentes grupos experimentais. Utilizando uma técnica
mais sensível (qPCR), demonstramos uma redução significativa na carga parasitária após o
tratamento por 3 ciclos de G-CSF sugerindo a possibilidade de atividade tripanocida dessa
citocina. Em cultura axênica de epimastigotas não observamos efeito do G-CSF em ensaio
cinético (dados não apresentados), no entanto observamos diminuição da viabilidade de
tripomastigotas e uma redução significativa no número de macrófagos infectados, assim como
na quantidade de formas amastigotas presentes por macrófago infectado in vitro. Esses dados
indicam que esta citocina possa estar contribuindo para a eliminação das formas intracelulares
84
do T. cruzi, o que de toda forma favorece a redução do estímulo inflamatório. A manutenção
da produção de anticorpos também contribui para a não reagudização da doença. Outras
citocinas, tais como GM-CSF e TNF, são capazes de agir diretamente sobre as formas
tripomastigotas, promovendo sua lise ou reduzindo sua capacidade infectiva (OLIVARESFONTT et al., 1998). Novos estudos estão sendo desenvolvidos visando esclarecer os
mecanismos envolvidos na redução do parasitismo tecidual em nosso modelo.
Dessa forma, apresentamos mecanismos múltiplos de ação do G-CSF que propicia a
melhora do quadro de cardiopatia chagásica crônica em camundongos infectados pela cepa
Colombiana de T. cruzi. Os resultados obtidos com o uso do G-CSF em modelo animal de
CChC encorajam a sua avaliação terapêutica na doença de Chagas. Por fim, outros estudos
devem ser realizados para que haja uma melhor compreensão dos efeitos desta citocina, tanto
no parasito quanto no sistema imune do hospedeiro, visando um melhor uso do G-CSF na
clínica para o tratamento de doenças crônicas.
85
5 CONCLUSÕES

O tratamento por três ciclos de G-CSF em modelo experimental de CChC
proporcionou modulação da resposta imune sistêmica com participação das células T
regulatórias;

A modulação da resposta imune-inflamatória dos animais nesse modelo proporcionou
a melhora funcional dos animais tratados com G-CSF por três ciclos, sem
proporcionar a reagudização da doença de Chagas, o que encoraja a sua utilização
terapêutica nessa doença.
86
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ANEXOS
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4. Milena BP Soares, Ricardo S Lima, Bruno SF Souza, Juliana F Vasconcelos, Leonardo L
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5. Milena Botelho Pereira Soares, Ricardo Santana de Lima, Leonardo Lima Rocha, Juliana
Fraga Vasconcelos, Silvia Regina Rogatto, Ricardo Ribeiro dos Santos, Sanda Iacobas,
Regina Coeli Goldenberg, Dumitru Andrei Iacobas, Herbert Bernard Tanowitz, Antonio
Carlos Campos de Carvalho, David Conover Spray. Gene expression changes associated with
myocarditis and fibrosis in hearts of mice with chronic chagasic cardiomyopathy. J Infect
Dis. Author manuscript; available in PMC 2011 August 15. Published in final edited form as:
J Infect Dis. 2010 August 15; 202(3): 416–426. doi: 10.1086/653481.
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6. Longo B, Romariz S, Blanco MM, Vasconcelos JF, Bahia L, Soares MB, Mello LE,
Ribeiro-dos-Santos R. Distribution and proliferation of bone marrow cells in the brain after
pilocarpine-induced status epilepticus in mice. Epilepsia. 2010 Aug;51(8):1628-32.
7. Brustolim D, Vasconcelos JF, Freitas LA, Teixeira MM, Farias MT, Ribeiro YM,
Tomassini TC, Oliveira GG, Pontes-de-Carvalho LC, Ribeiro-dos-Santos R, Soares MB.
Activity of physalin F in a collagen-induced arthritis model. J Nat Prod. 2010 Aug
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8. Vasconcelos JF, Teixeira MM, Barbosa-Filho JM, Agra MF, Nunes XP, Giulietti AM,
Ribeiro-Dos-Santos R, Soares MB. Effects of umbelliferone in a murine model of allergic
airway inflammation. Eur J Pharmacol. 2009 May 1;609(1-3):126-31. Epub 2009 Mar 14.
Cell Transplantation
Transplantation of Stem Cells Obtained from Murine Dental
Pulp Improves Pancreatic Damage, Renal Function and
Painful Diabetic Neuropathy in Diabetic Type 1 Mouse Model
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Journal:
Manuscript ID:
Manuscript Type:
Complete List of Authors:
CT-0385.R2
Original Article
n/a
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Date Submitted by the Author:
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Guimarães, Elisalva; Fundação Oswaldo Cruz, Laboratório de Engenharia
Tecidual e Imunofarmacologia
Cruz, Gabriela; Fundação Oswaldo Cruz, Laboratório de Engenharia
Almeida, Tiago; Fundação Oswaldo Cruz, Laboratório de Engenharia
Souza, Bruno; Hospital São Rafael, Centro de Biotecnologia e Terapia
Celular
Kaneto, Carla; Hospital São Rafael, Centro de Biotecnologia e Terapia
Celular
Vasconcelos, Juliana; Fundação Oswaldo Cruz, Laboratório de Engenharia
dos Santos, Washington; Fundação Oswaldo Cruz, Laboratório de
Engenharia Tecidual e Imunofarmacologia
dos Santos, Ricardo; Hospital São Rafael, Centro de Biotecnologia e
Terapia Celular; Fundação Oswaldo Cruz, Laboratório de Engenharia
Villarreal, Cristiane; Universidade Federal da Bahia, Faculdade de Farmácia
Soares, Milena; Fundação Oswaldo Cruz, Laboratório de Engenharia
Tecidual e Imunofarmacologia; Hospital São Rafael, Centro de
Biotecnologia e Terapia Celular
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Keywords:
Abstract:
stem cells, Dental pulp, Streptozotocin, Neuropathic pain, Hyperglycemia
Abstract.doc
http://mc.manuscriptcentral.com/cogcom-ct
Page 1 of 30
Transplantation of Stem Cells Obtained from Murine Dental Pulp Improves Pancreatic
Damage, Renal Function and Painful Diabetic Neuropathy in Diabetic Type 1 Mouse
Model
Elisalva Teixeira Guimarãesa,b, Gabriela da Silva Cruza,c, Tiago Farias de Almeidaa,
Bruno Solano de Freitas Souzac, Carla Martins Kanetoc, Juliana Fraga Vasconcelosa,
Washington Luis Conrado dos Santosa, Ricardo Ribeiro-dos-Santosa,c, Cristiane Flora
Villarreala,d and *Milena Botelho Pereira Soaresa,c.
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a
Centro de Pesquisas Gonçalo Moniz, FIOCRUZ, Bahia, Brazil.
b
Universidade do Estado da Bahia, Salvador, Bahia, Brazil
c
Centro de Biotecnologia e Terapia Celular, Hospital São Rafael, Salvador, Bahia,
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Brazil.
d
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Universidade Federal da Bahia, Salvador, Bahia, Brazil
*Corresponding author. Centro de Pesquisas Gonçalo Moniz. Rua Waldemar Falcão,
121 - Candeal - Salvador, BA, Brazil. 40296-710.
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Tel: 55-71-3176-2292, ext. 260/272; FAX: 55-71-3176-2272
e-mail: [email protected]
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1
Abstract
Diabetes Mellitus (DM) is one of the most common and serious chronic diseases in the
world. Here we investigated the effects of mouse dental pulp stem cells (mDPSC)
transplantation in a streptozotocin (STZ)-induced diabetes type 1 model. C57BL/6 mice
were treated intraperitoneally with 80 mg/kg of STZ and transplanted with 1 x 106
mDPSC or injected with saline, by endovenous route, after diabetes onset. Blood and
urine glucose levels were reduced in hyperglycemic mice treated with mDPSC when
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compared to saline-treated controls. This correlated with an increase in pancreatic islets
and insulin production 30 days after mDPSC therapy. Moreover, urea and proteinuria
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levels normalized after mDPSC transplantation in diabetic mice, indicating an
improvement of renal function. This was confirmed by histopathological analysis of
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kidney sections. We observed the loss of the epithelial brush border and proximal tubule
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dilatation only in saline-treated diabetic mice, which is indicative of acute renal lesion.
STZ-induced thermal hyperalgesia was also reduced after cell therapy. Three days after
transplantation, mDPSC-treated diabetic mice exhibited nociceptive thresholds similar
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to that of non-diabetic mice, an effect maintained throughout the 90-day evaluation
period. Immunofluorescence analyses of the pancreas revealed the presence of GFP+
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Page 2 of 30
cells in or surrounding pancreatic islets. Our results demonstrate that mDPSC may
contribute to pancreatic β-cell renewal, prevents renal damage in diabetic animals, and
produces a powerful and long-lasting antinociceptive effect on behavioral neuropathic
pain. Our results suggest stem cell therapy as an option for the control of diabetes
complications such as intractable diabetic neuropathic pain.
Key words: Stem cells; Dental pulp; Streptozotocin; Neuropathic pain; Hyperglycemia.
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INTRODUCTION
Diabetes Mellitus (DM) is one of the most common and serious chronic diseases in
the world. While type 1 diabetes (DM1) is characterized by the destruction of the
insulin–producing β cells in the pancreas mediated by autoreactive T cells, type 2
diabetes (DM2) is associated with peripheral resistance to insulin and impaired insulin
secretion (9,27). The excess of glucose is responsible for most of the complications of
DM1, which include blindness, kidney failure, amputations and heart disease.
Neuropathy is a frequent microvascular complication of diabetes, with patients often
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suffering from severe pain that can be acute of onset or chronic with symptoms lasting
for many years. Patients with diabetic neuropathy can exhibit a variety of aberrant
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sensations, including spontaneous pain, allodynia, hyperalgesia, and loss of stimulusevoked sensation (29).
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All devastating complications of DM1 can be prevented by the normalization of
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blood glucose levels. Conventional treatment includes the dependence on daily
injections of insulin and routine monitoring of blood glucose levels. Other therapies
such as transplantation of pancreatic islets, can be applied to treat this disease, but its
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widespread use is hampered by a shortage of donor organs or a recurrent attack by
underlying autoimmunity against islets (4,23). Moreover, there are no treatments that
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restore nerve function and the usual therapeutic strategies for neuropathic pain aim to
reduce the pain. Unfortunately, most of the available analgesic drugs appear to be
relatively ineffective in controlling diabetic neuropathic pain (15,26). Thus, there is a
clear need for new disease-modifying therapeutic approaches. In this context, cell-based
therapies represent a promising alternative. Several reports have suggested that
embryonic or adult bone marrow stem cells contribute to β-cell renewal and possess
great potential for damaged nervous system repair (11,14,21,24,26). In fact,
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transplantation of bone marrow mononuclear cells significantly reduced the pain
behavior following peripheral nerve injury and is a renewable alternative source of
insulin-producing cells in human or mouse models (10,12,16,19,30).
We have recently described the isolation, characterization, and differentiation of
stem cells obtained from mouse dental pulp (mDPSC) (10). We observed that mDPSC
is a population of stem cells with mesenchymal and embryonic characteristics, since
they express mesenchymal cell surface molecules such as CD90, CD73, Ly6a/Sca-1,
and the embryonic markers SSEA-1, Pou5f1/Oct-4 and alkaline phosphatase (10). This
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mesenchymal/embryonic mixed profile of stem cells obtained from human dental pulp
has been previously described by other authors (13,16). Moreover, mDPSC also has
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differentiation potential in osteocytes, adipocytes and chondrocytes (10). In the current
study, we evaluated the therapeutic potential of mDPSC in important complications of
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diabetes, namely pancreatic damage, renal function alterations, and diabetic peripheral
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neuropathy. For this, we studied the effects of mDPSC a model of pancreatic damage
induced by streptozotocin (STZ), an antibiotic isolated from Streptomyces
achromogenes widely used to create rodent models of type 1 diabetes (2).
Mice
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MATERIALS AND METHODS
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Specific-pathogen-free, 8-week-old female C57BL/6 and male C57BL/6 enhanced GFP
mice were maintained at the animal facilities at the Gonçalo Moniz Research CenterFIOCRUZ (Salvador, Bahia, Brazil) and provided with rodent diet and water ad libitum.
Animals were used only once in a given experiment. All experiments were carried out
in accordance with the recommendations of the International Association for the Study
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of Pain (IASP) Committee for Research and Ethical Issues Guidelines, and were
approved by the local Animal Ethics Committee.
Isolation and culture of mDPSC
The incisors teeth were dissected carefully from the mandibles of male EGFP transgenic
C57BL/6 mice. Dental pulp tissue was gently collected and explants were cultured into
24-well plates (Nunc A/S, Roskilde, Denmark) in DMEM medium supplemented with
10% FBS (Cultilab, Campinas, SP, Brazil), 23.8 mM sodium bicarbonate (Sigma, St.
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Louis, Mo., USA), 10 mM Hepes (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1
mM sodium pyruvate (Sigma), 2 mM L-glutamine (Sigma), 0.05 mM 2-ME, 50 µg/ml
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gentamycin (Sigma), and incubated at 37oC/5% CO2. When confluence was achieved
usually after 15-20 days, the adherent cells were released with 0.25% trypsin solution
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(Invitrogen/Molecular Probes, Oregon, USA) and used to treat diabetic mice, as
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described below. Cells until the fifth passage were used for transplant.
Streptozocin injection and mDPSC transplantation model
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β-cell injury was induced with streptozotocin (STZ) injection. Female C57BL/6 mice
were injected intraperitoneally (i.p.) with 80 mg/kg STZ (Sigma) diluted in a citrate
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buffer (pH = 4.5). Negative control group received vehicle only. Mice were considered
diabetic if glycemia were above 250 mg/dl. About 70% of the animals developed
hyperglycemia. After ten days, hyperglycemic mice (n = 6/group) were transplanted by
orbital plexus injection with 1 x 106 cells/mouse in a final volume of 200 µl. Control
group received saline only (n = 6/group). Blood glucose levels were measured weekly
with the glucometer system Accu-Chek (Roche Diagnostic, Mannheim, Germany).
Animals were housed individually in metabolic cages for 24 h. Urine was collected and
5
levels of proteinuria, glycosuria, urea, and creatinine were measured by standard
biochemical kits (Biosystems, Barcelona, Spain). The experimental design is shown in
figure 1.
Histopathologic evaluation
Kidneys and pancreas were removed after 30 days of experiment and immediately
placed in Bouin solution and formol, respectively. After 24 h of fixation, tissues were
washed, embedded in paraffin, and sections 3 and 5 mm-thick were stained with
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conventional hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS) stainings. The
number of islets was determined by manual counting of whole pancreas sections stained
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with H&E digitalized with ScanScope, a digital slide scanner (Aperio, Vista, CA). A
mean area of 102.5 mm2 ± 21.8 SD of pancreatic tissue was analyzed per mouse.
Assessment of nociception: tail flick test
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The tail flick test of D’amour and Smith (6) modified for mice was used. The mice were
habituated in a Plexiglas cylindrical mouse restrainer 10 min daily for one week before
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starting the experiments. To measure the latency of the tail flick response, mice were
gently placed in the restrainer and the tails (2 cm from the tip) were immersed in a water
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bath maintained at 54.0 ± 0.2°C. The monitored response was a curling of the tail. A
cutoff latency of 20 s was used to minimize the probability of skin damage. Baseline
withdrawal latencies were obtained from mice before diabetes induction.
Immunofluorescence analysis
Pancreas were removed from mice euthanized 30 days after the first STZ
administration, fixed with 4% paraformaldehide and embedded in paraffin. Pancreatic
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sections (4 µm-thick) were obtained, deparaffinizated and submitted to a heat-induced
antigen retrieval step in citrate buffer (pH = 6.0). Sections were then incubated
overnight with primary antibodies guinea pig anti-insulin (Invitrogen) diluted 1:50 and
chicken anti-GFP (Aves Biotech) diluted 1:100. The next day, the sections were
incubated with the secondary antibodies for 1 hour at room temperature. The following
secondary antibodies were used: goat anti-guinea pig IgG Alexa Fluor conjugated with
Alexa Fluor 568 and anti-chicken IgG conjugated with Alexa Fluor 488 (Molecular
Probes, Carlsbad, CA), both diluted 1:200. Nuclei were stained with 4,6-dismidino-2-
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phenylindole and coverslips were mounted on glass slides (VectaShield Hard Set
mounting medium with DAPI H1500, Vector Laboratories, Burlingame, CA). Images
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were analyzed using a confocal microscope Fluoview 1000 (Olympus).
For quantification of insulin production, images of pancreas sections were digitally
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captured in 200x magnification using a digital color camera adapted to an AX70
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microscope with an epifluorescence system plus grid to enhance the fluorescence
resolution (Optigrid, structured light imaging system; Thales Optem Inc., Olympus,
Center Valley, PA, USA). Ten randomic fields were captured per pancreas and analyzed
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using Image Pro Plus v. 7.0 (Media Cybernetics), which automates the analysis by
segmentation, converting all immunolabeled elements that fall within a threshold range
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into red pixels and the rest of the image into yellow pixels. The same threshold range
that defined positivity was used in all images analyzed. The software then calculates the
percentage of red and yellow, allowing for the comparison of the pixel values between
the groups, defined as the percentage of stained area.
Real time polymerase chain reaction (qPCR)
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Total RNA was isolated from kidney samples with TRIzol reagent (Invitrogen) and
concentration was determined by photometric measurement. High Capacity cDNA
Reverse Transcription Kit (Applied Biosystems) was used to synthesize cDNA of 1 µg
RNA following manufacturer’s recommendations. RT-PCR assays were performed to
detect the expression levels of eGFP. qRT-PCR amplification mixtures contained 20 ηg
template cDNA, Taqman Master Mix (10 µL) (Applied Biosystems) and probes for
eGFP (assay by design) in a final volume of 20 µL. All reactions were run in duplicate
on an ABI7500 Sequence Detection System (Applied Biosystems, Foster City, CA,
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USA) under standard thermal cycling conditions. The mean Ct (cycle threshold) values
from duplicate measurements were used to calculate expression of the target gene, with
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normalization to an internal control (GAPDH) using the 2–DCt formula. Experiments
with coefficients of variation greater than 5% were excluded. A no-template control
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(NTC) and no-reverse transcription controls (No-RT) were also included.
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Statistical analysis
Comparisons across three groups were made using one-way ANOVA with Tukey’s post
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hoc test or repeated measures two-way ANOVA with Bonferroni’s post hoc test, when
appropriate. All data were analyzed using the Prism 5.01 computer software (GraphPad,
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Page 8 of 30
San Diego, USA). Statistical differences were considered to be significant at p < 0.05.
RESULTS
Establishment of diabetes model
STZ was used to produce hyperglycemia in C57Bl/6 mice. Several STZ
concentrations (40, 80 and 120 mg/kg) were used in the standardization of this
experimental model (data not shown), but only the concentration of 80 mg/kg
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administrated in the three consecutive days were able of to induce hyperglycemia in the
animals. Ten days after the first STZ dose, mice presented about 2.6 times more blood
glucose when compared with non-treated mice (Fig. 2A). A morphologic assessment of
hematoxylin and eosin stained pancreas sections showed insulitis (Fig. 2C), and
reduction of insulin levels as observed by immunofluorescence (Fig. 2E). Moreover,
STZ-treated mice presented deposition of glycogen in the tubules (Armanni-Ebstein
change), a renal lesion characteristically associated with diabetes (Fig. 2G).
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Glucose levels and renal function
Mice treated with STZ became apathic and presented polyuria when compared
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to mice that received citrate buffer only. After transplantation, the body weight variation
in diabetic mice that received mDPSC was similar to that observed in non diabetic mice
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during all of the experimental period. In contrast, diabetic mice treated with saline
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exhibited a significant body weight loss (Fig. 3). Moreover, 30 days after
transplantation, blood glucose levels were reduced in hyperglycemic mice transplanted
with mDPSC when compared to those of saline-treated mice (Fig. 4).
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Correlating with the improvement of glucose levels, diabetic mice transplanted
with mDPSC presented a higher number of pancreatic islets when compared with
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diabetic mice non-transplanted (Fig.5A). Immunostaining for insulin confirmed the
increase in insulin-producting area in the pancreas of mDPSC-transplanted mice (Fig.
5B).
To evaluate the renal function, the urine levels of glucose, protein, urea, and
creatinine were measured. Diabetic mice treated with mDPSC showed significantly
reduced glucose and protein levels (Fig. 6A and B) and higher urea (Fig. 6C) when
compared to saline-treated diabetic mice. Creatinine levels were also higher in diabetic
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mice treated with mDPSC compared with saline-treated diabetic mice, although the
differences were not statistically significant (p = 0.0582) (Fig. 6D). Moreover, diabetic
mice did not develop morphologic alterations in glomerulli or vascular areas, probably
due to the short time of disease (30 days). However, we observed the loss of the
epithelial brush border and proximal tubule dilatation in saline-treated diabetic mice,
which is indicative of acute renal lesion. In contrast, these alterations were not observed
in mice transplanted with mDPSC, which had renal parenchyma similar to those of nondiabetic mice. Armanni-Ebstein change, a deposition of glycogen in the tubules, was
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more severe in saline-treated than in mDPSC-treated diabetic mice (Table 1). No GFP+
cells were found in the kidneys of mDPSC-transplanted mice, both by
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immunofluorescence as well as by PCR analyses (data not shown).
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Effect of mDPSC transplantation in the STZ-induced painful neuropathy
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Sensory testing results from tail flick assay are shown in figure 7. Baseline
response latencies before treatment were similar for all groups. Four days after STZ or
saline treatment, the response latencies were significantly lower (p < 0.001) for the
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diabetic groups relative to the saline-treated group, indicating the development of a
marked thermal hyperalgesia. Three days after mDPSC transplantation, diabetic mice
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treated with mDPSC exhibited nociceptive thresholds similar to the non-diabetic mice.
This effect was maintained throughout the testing period. We observed hyperalgesia (p
< 0.001) in diabetic mice from one week following the induction of diabetes that
progressed to hypoalgesia (p < 0.05). However, neither hyperalgesia nor hypoalgesia
were observed in diabetic mice after the mDPSC transplantation.
Pancreatic engraftment of mDPSC
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To examine whether mDPSC migrate to and engraft in the damaged pancreas
after STZ treatment, pancreas sections were immunostained with anti-insulin antibody
and analyzed by confocal microscopy. Insulin-production cells in the pancreatic islets
were observed, some of them co-expressing GFP (Fig. 8). Some GFP+, insulin negative
cells were found surrounding the islets (Fig. 8D).
DISCUSSION
In the present study, using an experimental model of Diabetes Mellitus, we
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demonstrated that the administration of mDPSC improves pancreatic damage, renal
function, and diabetic neuropathic pain.
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We used a model of Diabetes Mellitus type 1 induced by STZ in mice to assess
whether mDPSC can home and repair injured tissue in vivo because insulitis,
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hyperglycemia, and neuropathy are well documented in this model, mimicking the
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human diabetes (8,28). In our study, thermal hyperalgesia, severe hyperglycemia, and
renal dysfunction were observed ten days after the first STZ administration in C57BL/6
mice, and no spontaneous recovery was observed. Previous studies have shown
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reduction, but not complete reversion, of hyperglycemia after transplantation with a
single dose of bone marrow-derived stem or endothelial cells in diabetic mice (8,11).
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Similarly, in the present study we observed that a single mDPSC administration into
diabetic mice resulted in reduction of glucose levels. However, hyperglycemia increased
gradually with time, even in mDPSC-treated mice, suggesting the need of additional
doses to sustain the effects. Thus, the number of cells and doses to achieve a better
therapeutic effect is parameter to be evaluated.
Moreover, we observed GFP and insulin co-expression in the pancreatic islets.
Cells only expressing GFP were found less frequently in a peri-islet distribution. These
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data correlate with reports showing that stem cells obtained from other sources, such as
bone marrow, can engraft and transdifferentiate into a pancreatic endocrine β cell
phenotype in vivo (7) or surround pancreatic islets and ducts and produce factors that
stimulate resident cells of the pancreas in recipient mice in vivo (11). In contrast,
Lechner et al. (18) did not find a significant engraftment of bone marrow-derived βcells in the pancreatic islet after partial pancreatectomy or STZ administration.
Differences in the model and mouse strain used, STZ dosage, route of administration,
the number and source of transplanted cells may therefore influence the fate of the cells.
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We also observed improvement of renal function in transplanted mice with
mDPSC by evaluation of the biochemical parameters. Ezquer et al. (8) suggested that
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therapeutic effects of mesenchymal stem cells in the kidney include the modulation of
the
inflammatory
process
and
neovascularization
after
kidney
damage.
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Microalbuminuria reversion and the absence of renal histopathologic alterations were
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observed in diabetic mice transplanted with mesenchymal stem cells obtained from
bone marrow (8). In addition, Sca-1+ cells isolated from adult mouse bone marrow are
mobilized into the circulation by transient renal ischemia, specifically home to injured
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regions and differentiate into renal tubular epithelial cells (13). In our model, GFP+
mDPSC were not observed in the kidney of transplanted diabetic mice by
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immunofluorescence analyses 48 hours and 30 days after transplantation (data not
shown). Therefore, we suggest that improvements in kidney function are a consequence
of lower glucose levels promoted by mDPSC transplantation.
It has been suggested that diabetic neuropathy is causally related to the reduction
of blood flow and conduction velocity in the nerve, impaired angiogenesis, and
deficient growth factors (3,17, 20). A recent study demonstrated that the transplantation
of bone marrow-derived endothelial progenitor cells in STZ-induced diabetic mice
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improves the nerve conduction velocity, blood flow, and capillary density in the sciatic
nerve (12). In addition, the authors observed increased angiogenic and neurotrophic
factors in sciatic nerves of transplanted mice. In the present study, we demonstrated, for
the first time, that transplantation of dental pulp stem cells produces a powerful and
long-lasting antinociceptive effect in mice with diabetic neuropathic pain. Interestingly,
the observed antinociception was not only rapid but also irreversible, an effect that is
hardly reached by analgesics clinically used. The mechanism underlying the
effectiveness of mDPSC transplantation for diabetic neuropathic pain is unknown;
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however, it may result from secretion of antiinflammatory, angiogenic, and
neurotrophic factors by transplanted cells.
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In conclusion, our study supports the concept that mDPSC could reverse
frequent complications of diabetes, such as pancreatic β-cell destruction, renal damage,
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and neuropathic pain, and suggest that dental pulp is an innovative source of insulin-
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producing cells and could represent an option for the control of intractable diabetic
neuropathic pain.
ACKNOWLEDGEMENTS
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This work was supported by CNPq, FAPESB, FINEP, and FIOCRUZ.
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Table 1. Morphologic alterations in the kidney after diabetes induction and mDPSC
transplantation.
Experimental
group
Glomerular
and vascular
changes
Deposition of
glycogen in
the tubules
Loss of brush
border
Tubular
dilatation
Non-diabetic
mice
-
-
-
-
Diabetic nontransplanted
mice
-
+++/ +++
++/+++
++/+++
Diabetic mice
transplanted
with mDPSC
-
+/+++
-
-
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(-), absent; (+), present in less than 25% of the analyzed fields; (++), present in 50%;
(+++) present in 100%.
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FIGURE LEGENDS
Figure 1. Experimental design. Diabetes was induced in mice by three daily
administrations of streptozotocin in identical concentrations. Control mice received
three daily injections of citrate buffer. On day 10 following the STZ administrations,
mice received injection of 106 mDPSC or saline by endovenous route (through the
orbital plexus).
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Figure 2. Establishment of diabetes model induced by streptozotocin administration.
(A) Increased blood glucose levels correlated with the presence of (C) insulitis in
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pancreas sections stained with H&E and (E) with the decreased of insulin-expressing
cells (red) analyzed by immunofluorescence in STZ-treated mice, in contrast to
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untreated C57Bl/6 mice (B and D). Histological examination of kidney sections by light
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microscopy. (F) Normal kidney. (G) Glycogen deposition was observed in diabetic mice
(arrows).
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Figure 3. Body weight variation in STZ-diabetic mice after mDPSC transplantation.
Body weight was evaluated before (A) and one week after (B) streptozotocin
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administration. Groups of mice treated with streptozotocin and treated with mDPSC
(STZ+ mDPSC+) or saline (STZ+ mDPSC-) and non-diabetic mice (STZ-) are
represented in the figure. The body weight is shown only for 20 (C), 30 (D), 60 (E), and
80 (F) days after mDPSC transplantation. Data are expressed as means±SEM (n=6). * P
< 0.05 compared to the control group (STZ-); # P < 0.05 compared with remaining
groups; tested by two-way ANOVA with Bonferroni’s post hoc.
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Figure 4. Transplantation of mDPSC reduces blood glucose levels in STZ-diabetic
mice. Comparison of blood glucose levels in STZ-treated mice injected with saline or
transplanted with 106 cells (n = 6/group). Data are expressed as mean±SEM. * P <
0.001 and # P < 0.05 compared to the STZ+ mDPSC+ vs. STZ+ mDPSC- group, using
two-way ANOVA with Bonferroni’s post hoc.
Figure 5. Quantification of pancreatic islets and insulin production after mDPSC
transplantation. The number of islets was determined in H&E stained pancreatic
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sections of STZ-treated mice transplanted or not with mDPSC (A). Quantification of
insulin-producing area by immunofluorescence analysis (B).*P < 0.05, Mann Whitney’s
test.
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Figure 6. Improvement of the renal function in transplanted mice with mDPSC. Thirty
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days after the mDPSC transplant, urine was collected and the levels of glucose (A)
protein (B), urea (C), and creatinine (D) were measured. Data are expressed as
mean±SEM (n=6). * P < 0.05 compared with the control group (STZ-); # P < 0.05
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compared with remaining groups; tested by one-way ANOVA with Tukey’s post hoc.
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Figure 7. Prolonged antinociceptive effect of a single transplantation of mDPSC in
STZ-diabetic mice. Baseline response latencies (B) were performed before the
treatments for all groups. The tail flick test was carried out daily during all of the
experimental period, but the figure shows only data of selected days. Data are expressed
as mean±SEM (n=6). * P < 0.01 compared with the control group (STZ-); # P < 0.01
compared to the remaining groups; tested by two-way ANOVA with Bonferroni’s post
hoc.
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Figure 8. Transplanted mDPSC transdifferentiate into insulin-producing cells and
surround STZ-damaged pancreas. Pancreas sections of mDPSC-transplanted mice were
analyzed 30 days after the first STZ administration and subjected to staining with antiinsulin antibody (red; A). GFP+ cells were detected (green; B). Nuclei were stained with
DAPI (blue; C). Merged images are shown in D.
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Cytotherapy, 2012; Early Online: 1–11
Transplantation of bone marrow cells decreases tumor necrosis
factor-α production and blood–brain barrier permeability and
improves survival in a mouse model of acetaminophen-induced
acute liver disease
Cytotherapy Downloaded from informahealthcare.com by 189.48.72.147 on 07/23/12
For personal use only.
BRUNO SOLANO DE FREITAS SOUZA1,2, RAMON CAMPOS NASCIMENTO3,4,
SHEILLA ANDRADE DE OLIVEIRA1,5, JULIANA FRAGA VASCONCELOS1,2,
CARLA MARTINS KANETO2, LIAN FELIPE PAIVA PONTES DE CARVALHO2,
RICARDO RIBEIRO-DOS-SANTOS1,2, MILENA BOTELHO PEREIRA SOARES1,2 &
LUIZ ANTONIO RODRIGUES DE FREITAS3,4
1Laboratório
de Engenharia Tecidual e Imunofarmacologia, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo
Cruz, Salvador, BA, Brazil, 2Centro de Biotecnologia e Terapia Celular, Hospital São Rafael, Salvador, BA, Brazil,
3Laboratório de Patologia e Biointervenção, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador,
BA, Brazil, 4Faculdade de Medicina, Universidade Federal da Bahia, Salvador, BA, Brazil, and 5Laboratório de
Imunopatologia e Biologia Molecular, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, PE, Brazil
Abstract
Background aims. Acute liver failure (ALF), although rare, remains a rapidly progressive and frequently fatal condition.
Acetaminophen (APAP) poisoning induces a massive hepatic necrosis and often leads to death as a result of cerebral edema.
Cell-based therapies are currently being investigated for liver injuries. We evaluated the therapeutic potential of transplantation of bone marrow mononuclear cells (BMC) in a mouse model of acute liver injury. Methods. ALF was induced in
C57Bl/6 mice submitted to an alcoholic diet followed by fasting and injection of APAP. Mice were transplanted with 107
BMC obtained from enhanced green fluorescent protein (GFP) transgenic mice. Results. BMC transplantation caused a
significant reduction in APAP-induced mortality. However, no significant differences in serum aminotransferase concentrations, extension of liver necrosis, number of inflammatory cells and levels of cytokines in the liver were found when BMCand saline-injected groups were compared. Moreover, recruitment of transplanted cells to the liver was very low and no
donor-derived hepatocytes were observed. Mice submitted to BMC therapy had some protection against disruption of the
blood–brain barrier, despite their hyperammonemia, and serum metalloproteinase (MMP)-9 activity similar to the salineinjected group. Tumor necrosis factor (TNF)-α concentrations were decreased in the serum of BMC-treated mice. This
reduction was associated with an early increase in interleukin (IL)-10 mRNA expression in the spleen and bone marrow
after BMC treatment. Conclusions. BMC transplantation protects mice submitted to high doses of APAP and is a potential
candidate for ALF treatment, probably via an immunomodulatory effect on TNF-α production.
Key Words: acetaminophen, acute liver failure, bone marrow mononuclear cells, brain edema, cell therapy, tumor necrosis factor
Introduction
Acute liver failure (ALF) is caused by liver cell
dysfunction, leading to coagulopathy, hepatic
encephalopathy and death in previously healthy
patients. The main cause of this illness is poisoning
with acetaminophen (N-acetyl-paraaminophen;
APAP) through unintentional overdoses or suicide
attempts (1). This drug induces severe centrolobular hepatocellular necrosis and increases serum
transaminase levels, in a dose-dependent manner. The extent of APAP-induced hepatic lesions
is increased by fasting and alcohol consumption,
both in animals and humans (2,3).
APAP is metabolized by cytochrome P450 to form
N-acetyl-p-benzoquinone imine (NAPQI). This reactive metabolite depletes glutathione and covalently
binds to cysteine groups on proteins, leading to inhibition of mitochondrial respiration, mitochondrial
permeability transition, hepatic necrosis and inflammatory response (4,5). The consequent massive
death of hepatocytes is followed by disturbances in
the hepatic cycle of urea and an increase in serum
Correspondence: Luiz A. R. de Freitas, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Rua Waldemar Falcão, 121, Candeal, Salvador, BA,
CEP 40296–710, Brazil. E-mail: [email protected].
(Received 15 February 2012; accepted 1 April 2012)
ISSN 1465-3249 print/ISSN 1477-2566 online © 2012 Informa Healthcare
DOI: 10.3109/14653249.2012.684445
2
B. S. de Freitas Souza et al.
ammonia levels. In the brain, the ammonia excess
is incorporated as glutamate by astrocytes to form
glutamine, an organic osmolyte, which leads to oxidative/nitrosative stress, blood–brain barrier (BBB)
disruption and increased cerebral blood flow (6,7).
All of these features induce encephalopathy and
brain edema, the main cause of death in humans
after an APAP overdose. Furthermore, the systemic
production of the pro-inflammatory cytokine tumor
necrosis factor (TNF)-α is increased in patients with
acute liver failure, and a growing body of evidence
indicates this cytokine to be a central mediator promoting the development of encephalopathy in this
condition (8).
The definitive treatment for APAP-induced
acute liver failure is liver transplantation, limited
by the shortage of donors and surgical risks (9).
Research for therapeutic approaches has focused on
the prevention of liver damage with anti-oxidants,
control of brain edema and hepatic support with
bio-artificial livers or hepatocyte transplantation
(10). Cell-based therapies have also been investigated as treatments for liver lesions. Transplantation of bone marrow mononuclear cells (BMC) has
been shown to ameliorate dysfunction in different
organs, such as the heart, brain and liver. BMC are
easy to isolate and comprise different cell populations that can produce anti-inflammatory molecules
related with hepatic protection in acute liver failure
(11,12). In this study we evaluated the therapeutic potential of BMC transplantation in a model
of APAP-induced hepatotoxicity potentiated by
alcohol consumption.
Methods
Animals and acute liver failure induction
Animals were handled according to the Fundação
Oswaldo Cruz (FIOCRUZ) guidelines for animal
experimentation and the experimental protocols
were approved by local animal ethics committees.
Four to 6-week-old male C57Bl/6 mice weighing
approximately 20 g were raised and maintained
at the Gonçalo Moniz Research Center/Oswaldo
Cruz Foundation (Salvador, Brazil) in rooms with
controlled temperature (22 2°C) and humidity
(55 10%) and continuous air renovation. Animals
were maintained with 10% alcohol solution in water
for 3 weeks. Before APAP administration, they were
fasted for approximately 12 h with free access to
pure water. Fresh suspensions of 16 mL/kg APAP
(All Chemistry, São Paulo, Brazil) were prepared in
warm saline (40°C) and given at 300 mg/kg intraperitoneally (corresponding to a lethal dose of 70%).
Control mice received the same volume of saline
solution.
Transplantation of BMC
Bone marrow cells were obtained from femurs and
tibiae of 4–6-week-old enhanced green fluorescent
protein (EGFP)-transgenic C57Bl/6 mice.The mononuclear cell fraction was purified by centrifugation in
a Histopaque gradient at 1000 g for 25 min at 25°C
(Histopaque 1119 and 1077, 1:1; Sigma-Aldrich, St
Louis, MO, USA). The mononuclear cell fraction
was collected and washed three times in Dulbecco’s
modified Eagle medium (Sigma-Aldrich). Cell suspensions were filtered over nylon wool and diluted
in saline (5 107 cells/mL), and their viability was
evaluated by trypan blue exclusion (Sigma-Aldrich).
The cells were injected into the peripheral circulation 3 h after APAP injection (1 107 cells/mouse).
In addition, BMC samples were analyzed by flow
cytometry in a FACScalibur flow cytometer using
conjugated antibodies (Becton Dickinson, San Diego,
CA, USA), showing that 96.5 1.3% expressed
green fluorescent protein (GFP) and 96.3 3.1%
expressed CD45. Hematopoietic progenitor markers Sca-1, CD34 and CD117 were expressed by
0.11 0.03%, 0.20 0.05% and 0.17 0.04% of
the cells, respectively.
Tissue preparation and evaluation of liver injury
Mice from BMC- and saline-treated groups were
anesthetized with ketamine (115 mg/kg) and xylazine
(10–15 mg/kg) at different time-points after APAP
injection. The animals were immediately perfused
transcardially with 50 mL cold 0.9% saline, followed by 100 mL cold 4% paraformaldehyde (Merk,
Darmstadt, Germany) in phosphate-buffered saline
(PBS), pH 7.2. Livers were excised and left lobes
were fixed in 10% formalin and paraffin-embedded.
Four micrometer-thick paraffin-embedded sections
were stained with hematoxylin-eosin (H&E). Analyses were performed on whole liver sections after slide
scanning using an Aperio ScanScope system (Aperio Technologies, Vista, CA, USA). The images were
analyzed using the Image Pro program (version 7.0;
Media Cybernetics, San Diego, CA, USA). Hepatic
injury was determined as a percentage of necrotic
tissue, and a mean area of 24 676 428 μm2/mouse
was analyzed.
Quantification of EGFP cells in the liver by
immunofluorescence
After transcardiac perfusion with paraformaldehyde,
non-left hepatic lobes were additionally fixed in 4%
paraformaldehyde at 4°C for 24 h. These samples
were then incubated overnight in 30% sucrose solution in PBS at 4%, embedded in medium for congeal
tissue and frozen at –70°C. Four-micrometer thick
Therapy with bone marrow cells in acute liver failure
sections were stained using a rabbit anti-human
albumin (DAKO, Glostrup, Denmark) followed by
anti-rabbit IgG conjugated with Alexa Fluor 568
(Invitrogen, Carlsbad, CA, USA), and mounted with
VectaShield Hard Set medium with 4¢,6-Diamidino2-Phenylindole (DAPI) for nuclear staining (Vector,
Burlingame, CA, USA). GFP cells were quantified
in five randomly selected areas per animal at a magnification of 200 .
Biochemical analyses
Blood was carefully collected in lithium heparinized
tubes at different time-points immediately before
killing, to avoid hemolysis. Plasma was obtained
by centrifugation of blood for 10 min at 9000 g
and stored at 4°C. Ammonia, Aspartate transaminase (AST) and ALT were evaluated within 2 h of
plasma isolation using commercial kits (Vitros, New
Jersey, USA, and Roche/Hitachi, Tokyo, Japan).
Quantification of inflammation
Quantification of infiltration of inflammatory cells
was performed in 5-mm thick liver sections frozen in Optimal Cutting Temperature Compound
(OCT). The analysis was performed in sections
obtained from the left hepatic lobe. For microscopic
analysis, an Olympus FluoView 1000 confocal laser
scanning microscope (Olympus, Tokyo, Japan) was
used for image acquisition. Sections were immunostained with primary antibody against mouse CD45,
diluted 1:100 (Caltag, Buckingham, UK) overnight
at 4°C. The next day sections were washed in PBS
and incubated for 1 h at room temperature with the
secondary antibody against rat IgG conjugated with
Alexa Fluor 568. A total of five images per mouse
was obtained in random fields. A magnification of
200 was used in order to cover an area of 1 mm2.
Numbers of CD45 cells were counted manually using Image-Pro Plus v.7.0 software (Media
Cybernetics). In order to avoid overestimation
because of counting partial cells that appeared
within the section, only cells with intact morphology
and a visible nucleus were counted.
3
10 min at 3000 g and the supernatant was frozen at
–70°C for later quantification. Cytokine levels were
estimated using commercially available immunoassay enzyme-linked immunosorbent assay (ELISA)
kits for mouse TNF-α, IL-6 and IL-10 (BD, Franklin Lakes, NJ, USA), according to the manufacturer’s instructions. Briefly, 96-well plates were blocked
and incubated at room temperature for 1 h. Samples
were added in duplicates and incubated overnight at
4°C. Biotinylated antibodies were added and plates
were incubated for 2 h at room temperature. A
half-hour incubation with streptavidin–horseradish
peroxidase conjugate at a dilution of 1:200 was followed by detection using 3,3,5,5- tetramethylbenzidine (TMB) peroxidase substrate and reading at
450 nm.
Real-time polymerase chain reaction data
RNA was harvested and isolated with TRIzol reagent
(Invitrogen) and the concentration was determined
by photometric measurement. The RNA quality
was analyzed in a 1.2% agarose gel. A high-capacity
cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA) was used to synthesize
cDNA of 2 μg RNA following the manufacturer’s
recommendations.
Real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays were performed to detect the expression levels of TNF-α
(Mm 00443258_m1), IL-10 (Mm 00439616_
m1), endothelin-1 (EDN-1; Mm 00438656_m1),
adrenomedullin (ADM) (Mm00437438_g1) and
GFP (assay by design) using hydrolysis probes
from Applied Biosystems. Quantitative (q)RT-PCR
amplification mixtures were made with Universal
master mix (Applied Biosystems) and a 7500 realtime PCR system (Applied Biosystems). The cycling
Analysis of cytokine production
TNF-α, interleukin (IL)-6 and IL-10 levels were
measured in total liver extracts and serum. Liver
proteins were extracted from 100 mg tissue/mL
PBS to which 0.4 M NaCl, 0.05% Tween-20 and
protease inhibitors (0.1 mM phenylmethanesulfonyl
fluoride; (PMSF), 0.1 mM benzethonium chloride,
10 mM Ethylenediamine tetraacetic acid (EDTA) and
20 Kallikrein Inhibitor Unit (KIU) aprotinin A/100
mL) were added. The samples were centrifuged for
Figure 1. Effects of BMC therapy in APAP-induced lethality. Mice
received saline or BMC 3 h after APAP injection. Survival was
followed for 5 days. Results shown are from one experiment of
four performed, each one with 15 mice/group. ∗P 0.05.
4
Figure 2. Liver necrosis after APAP injection and BMC transplantation. (A, B) Liver section of a mouse killed after 3 weeks of an alcoholic diet
without APAP treatment, showing an absence of significant alteration of hepatic parenchyma. (C, D) Extensive centrolobular necrosis 24 h after
APAP injection in the liver of saline-injected mice. (E) Quantification of the percentage of necrotic area in the liver parenchyma of saline-treated
or BMC-treated mice. Results shown are from one of three experiments performed, each one with 3–5 mice/group/time-point.
conditions comprised 10 min polymerase activation at 95°C and 40 cycles at 95°C for 15 s and
60°C for 60 s. Normalization was made with the
target internal control glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) using the cycle threshold
method, and analysis of variance followed by the
Tukey test.
in medium for congeal tissue (Tissue-Tek) and frozen at –70°C. Five-micrometer thick sections were
prepared, fixed in paraformaldehyde 4%, and nuclei
stained with DAPI. EB cells were quantified in
five randomly selected areas per animal at a magnification of 20 .
Metalloproteinase zymogram
Evaluation of BBB integrity
Evans blue (EB) has been used widely to assess brain
edema (13). Mice were injected intravenously with 4
mL/kg 2% (w/v) EB solution. Thirty minutes later,
the animals were killed with a ketamine overdose and
brains were dissected away from the brainstem, cerebellum and meninges. Brain samples were embedded
Serum samples diluted 1:150 in non-reducing sample buffer (Tris–HCl buffer 500 mM/L, 10% glycerol,
4% Sodium Dodecyl Sulfate (SDS) w/v, 0.04% bromophenol blue w/v, pH 6.8) were loaded onto 7.5%
SDS polyacrylamide gels containing type A gelatin
from porcine skin 0.2% w/v (Sigma, St. Louis, MO,
USA) and electrophoresis was performed at 100 V for
5
Parametric data were analyzed with t-test or ANOVA.
Survival curves were evaluated by log-rank (Mantel–
Cox) test, using Prism Software (version 5.0; GraphPad Software, San Diego, CA, USA). A P-value less
than 0.05 was considered statistically significant.
Results
Transplantation of BMC reduces APAP-induced
mortality without affecting hepatic necrosis
Figure 3. Measurement of serum levels of transaminases. (A) AST
and (B) ALT serum levels of BMC- and saline-treated mice were
determined at different time-points after APAP injection. Data
obtained from one of two experiments performed represent the
means SEM of 3–5 mice/group/time-point. ∗P 0.05.
3 h under cooling conditions (FB300 eletrophoresis
power supply; Fisher Scientific, Waltham, MA, USA).
Gels were then washed twice in 2.5% Triton X-100
solution for 30 min at room temperature with gentle
agitation to remove SDS, and incubated at 37°C for
24 h in substrate buffer (Tris–HCl buffer 10 mM/L,
1.25% Triton X-100, CaCl2 5 mM/L, ZnCl2 1 μM/L,
pH 7.5). Thereafter, gels were stained with a 0.5%
w/v Coomassie blue R250 (Sigma-Aldrich) solution
in 50% methanol, 10% acetic acid for 18 h and then
unstained with the same solution without dye. Gelatinase activity was detected as unstained bands in a
blue background, representing areas of proteolysis of
the gelatin. After lamination and image acquisition,
densitometric semi-quantitative analysis was performed using Scion image software (Image Program,
National Institutes of Health, Scion Corporation,
Frederick, MD, USA).
Results were expressed as mean SEM. Statistical
comparisons were performed using Mann–Whitney
or Kruskal–Wallis tests for non-parametric data.
To evaluate the effect of BMC therapy in APAP-induced mortality, animals were injected with saline or
BMC suspensions by an intravenous route 3 h after
APAP injection. Five days later, the survival rate of
the BMC-treated group was 60%, significantly higher
than the 20% observed in the saline-injected group
(Figure 1). On the other hand, when the same dose
of BMC was given 9 h after APAP injection, this protective effect was not observed (data not shown).
Alcoholic diet alone did not result in any
morphologic alterations visible by optic microscopy
(Figure 2A, B). However, APAP administration after
an alcoholic diet resulted in extensive necrosis in the
centrolobular liver zone (Figure 2C, D). We did not
observe any differences in necrosis extension (Figure
2E) and serum transaminase levels (Figure 3A, B)
between BMC- and saline-treated groups at different
time-points after APAP injection. To correct a possible
selection bias in necrosis analysis as a result of mortality, an experiment was performed in which mice
were organized in pairs containing one BMC- and one
saline-treated mouse. Mortality was evaluated every
3 h after APAP injection. Livers from paired animals
were immediately harvested after the death of any animal from each pair, and even then we did not observe
differences in hepatic damage between BMC-treated
and saline-injected mice (data not shown).
Transplanted BMC migrate to the liver but
do not differentiate into hepatocytes or reduce
local inflammation
Liver sections from BMC-treated mice were processed
for analysis by immunofluorescence microscopy in
order to track the presence of GFP cells. Despite
the inflammatory cell infiltrate in centrolobular areas
observed in H&E sections, there was little recruitment
of transplanted cells to hepatic parenchyma. The maximum engraftment occurred 24 h after APAP injection,
when about 13 cells/mm2 were found in liver sections
(Figure 4B). Moreover, GFP cells did not express
albumin and did not show hepatocyte-like polygonal
morphology at the time-points evaluated (Figure 4A).
GFP cell were also found in the spleens of BMCtransplanted mice (Figure 4C). The analysis of GFP
mRNA expression in the bone marrow, spleens and
livers of BMC-transplanted mice demonstrated that
6
Figure 4. Migration of BMC to different organs with low recruitment to the injured liver. (A) Immunofluorescence of liver section showing
a centrolobular zone from a BMC-treated mouse 5 days after APAP injection. Presence of GFP cells (green) in sections stained with
DAPI (blue) for nuclei visualization and with anti-albumin antibody (red). GFP cells (green) on liver parenchyma did not express albumin
and did not assume hepatocyte-like polygonal morphology. The section was counterstained with DAPI for nuclei visualization (blue). (B)
Quantification of GFP cells in livers of BMC-treated mice at different time-points after APAP injection. Data represent the means SEM
of 3–5 mice/group/time-point. (C) The presence of GFP cells (green) in the spleen section of a BMC-transplanted mouse. The section
was counterstained with DAPI for nuclei visualization (blue). Bar 50 μm. (D) GFP gene expression in bone marrow, spleen and liver of
mice 6 h after APAP injection, showing increased recruitment of transplanted BMC to the bone marrow, followed by spleen and liver.
Data represent mean SEM of seven mice/group. Scale bars represent 50 μm. ∗P 0.05; ∗∗P 0.001; ∗∗∗P 0.0001.
most of the cells migrated to the bone marrow and the
spleen, and very few cells were retained in the liver 6
h after APAP injection (Figure 4D).
To evaluate the number of inflammatory cells,
liver sections were submitted to immunofluorescence analysis using an antibody against the panleukocyte marker CD45. Mice injected with APAP
had CD45 cell infiltrates of mild intensity after 6 h
(Figure 5A), which increased after 14 h (Figure 5B).
The percentage of CD45 cells did not differ significantly between BMC-treated and saline-injected
groups (Figure 5C).
BMC transplantation protects the BBB integrity
We next evaluated leakage in the BBB by EB injection
18 h after APAP injection. The number of EB-stained
neurons was significantly attenuated in brains of
mice from the BMC-treated group in comparison
with saline-injected mice (Figure 6A–C). However,
we did not observe migration of GFP cells to the
brains of BMC-transplanted mice by fluorescence
microscopy.
Because hyperammonemia is implicated in the
pathogenesis of brain edema in acute liver damage (7),
we determined the serum ammonia levels after APAP
injection in cell- and saline-treated mice. Ammonia
levels were similar in both groups (BMC, 680 30.4
μg/dL; saline, 730 57.7 μg/dL; P 0.4859). These
data indicated that the reduction in the brain vessel
permeability associated with BMC transplantation
was probably because of an ammonia-independent
mechanism.
Another factor that may contribute to disruption
of the BBB is the increased activity of metalloproteinases (MMP (14–16). We evaluated the activity
of MMP-9 in the sera of mice submitted to APAPinduced injury. No significant differences in the activity of MMP-9 were found when BMC-treated and
saline-injected mice were compared (Figure 6D). We
7
Figure 5. BMC transplantation does not affect the number of inflammatory cells in the liver. (A) Immunofluorescence showing mild
infiltration of CD45 cells (red) into the injured liver 6 h after APAP injection. (B) Infiltration of CD45 cells into the liver increases 14
h after APAP injection. (C) Quantification of CD45 cells in the liver, showing similar numbers of infiltrating cells in both BMC- and
saline-treated groups. Scale bars represent 50 μm. The data represent means SEM of seven mice/group.
then hypothesized that BMC could act by modulating
the expression of adrenomedullin and endothelin-1,
two peptides known for their role in the regulation of BBB permeability (17). Although the gene
expression of adrenomedullin and endothelin-1 was
found to be elevated in the brains of mice 14 h after
APAP injection, no statistically significant difference
was found between BMC- and saline-treated mice
(Figure 6E, F).
BMC-transplanted and saline-injected mice were
not significantly different (Figure 7B). However,
when the expression of IL-10 mRNA was evaluated,
a marked increase was observed in the spleen and
bone marrow of BMC-transplanted mice 6 h after
APAP injection (Figure 8A–D). Fourteen hours after
APAP injection, gene expression of TNF-α was suppressed in the spleen, while IL-10 was up-regulated
(Figure 8E, F).
Modulation of APAP-induced cytokine production
after BMC transplantation
Discussion
Because increased production of TNF-α is strongly
associated with hepatic encephalopathy in acute
liver failure (8), we measured the concentrations
of TNF-α in the liver and serum of mice submitted to APAP-induced injury, as well as in normal
controls. Saline-injected mice had increased TNF-α
concentrations in the liver (Figure 7A) and serum
(Figure 7C) when compared with normal controls.
When BMC-treated mice were evaluated 14 h after
APAP injection, we observed a significant reduction
of TNF-α in the serum, but not in the liver, compared with saline-injected mice (Figure 7A, C).
To investigate the mechanisms by which BMC
modulate TNF-α production, we measured the
concentrations of the anti-inflammatory cytokine
IL-10. IL-10 concentrations in the livers of
We have demonstrated a protective effect of BMC
transplantation in mice with ALF induced by APAP.
This was evidenced by a reduction in mortality, modulation of the production of inflammatory mediators
and decreased damage to the BBB. This was only
achieved when the cells were transplanted early (up
to 6 h after APAP challenge), indicating that early
events modulated by the cell therapy may be determinants of disease outcome.
The protective effect of BMC transplantation
observed in our study did not correlate with an
improvement in liver necrosis or function (ALT,
AST and ammonia). However, in a rat model of
ALF, improvement of liver function and decreased
area of necrosis were observed after transplantation
of BMC (18). This difference may be because of
a slower evolution of the disease in the rat model
8
Figure 6. Evaluation of brain edema 18 h after APAP injection. Mice were injected with EB and killed 30 min later. Brains were processed
as described in the Methods. (A, B) Fluorescence microscopy of brain sections obtained from saline- and BMC-treated mice, respectively.
BMC induced low impregnation of cells with EB (red). Nuclei were stained with DAPI (blue). (C) EB staining cells were quantified in
brain sections, and were reduced in BMC-treated mice. Data represent the means SEM of 3–5 mice/group. ∗P 0.05. (D) Zymogram
for detection of serum MMP-9 activity 14 h after APAP injection, showing similar lysis area between BMC- and saline-treated groups.
Data represent one of two experiments, presented as the means SEM of 12–14 mice/group. (E, F) qPCR analysis of gene expression of
endothelin-1 (E) and adrenomedullin (F) in the brains of mice 14 h after APAP injection. Data represent the means SEM of 5–7 mice/
group. ∗∗∗P 0.0001.
compared with the mouse model used in our study;
most of the mice had died 24 h after APAP injection,
whereas in the rat model mortality started after the
first 24 h. Thus it is possible that, in our study, the
fast evolution of the disease renders it difficult for
the transplanted cells to promote any improvement
of liver regeneration.
A few transplanted cells migrated to the liver,
and no contribution of GFP cells to hepatocyte
formation was observed during all the time-points
analyzed. Thus it is likely that the transplanted
BMC act via a paracrine effect, causing the modulation of cytokine production in this model. In
fact, a decrease in serum TNF-α concentrations
and an increase in IL-10 mRNA expression in
the bone marrow and spleen were observed after
BMC transplantation in our model of ALF. The
modulation of cytokine production and inflammatory response by BMC transplantation has been
observed in other disease models, such as chronic
chagasic cardiomyopathy (19), epilepsy (20) and
chronic liver diseases (21).
Figure 7. Assessment of cytokines in the liver and serum 14 h after
APAP injection. Concentrations of TNF-α (A, C) and IL-10 (B)
were determined in liver extracts (A, B) and serum (C) in salineand BMC-treated mice 14 h after APAP injection. Data represent
the means SEM of 5–7 mice/group. ∗∗∗P 0.0001.
The increase in TNF-α during the course of
ALF seems to be a crucial event determining the
disease outcome (22,23). TNF-α is a pleiotropic
cytokine the effects of which may contribute in
different ways to disease progression, including
apoptosis induction, endothelial cell and platelet activation, increased vascular permeability
and induction of soluble mediators (24). TNF-α
elevation in ALF is indicated as one of the main
causes of hepatic encephalopathy, and the severity
9
of encephalopathy correlates with TNF-α serum
concentration in patients with ALF (25). Elevated
serum levels of TNF-α has been shown previously to correlate with increased permeability of
the BBB in mice with APAP-induced ALF (26).
Furthermore, Bemeur et al. (27), using an experimental model of ALF induced by azoxymethane
in mice, have shown successfully that TNFR1
knockout mice are protected against the onset of
coma and brain edema. Thus it is possible that the
modulation of TNF-α production by transplanted
BMC causes a decreased leakage in the BBB, consequently decreasing the development of hepatic
encephalopathy and death rate of mice after APAP
challenge.
TNF-α production was modulated in the
serum but not in the liver of mice submitted to
BMC transplantation after APAP challenge. This
suggested a systemic immunomodulatory effect of
the transplanted cells, and in fact an increase in
IL-10 mRNA expression was observed both in the
spleen as well as the bone marrow of BMC-treated
mice 3 h after cell transplantation. The spleen and
bone marrow are sites in which transplanted cells
are preferentially retained (28), and this was confirmed in our study by qRT-PCR analysis. The
production of IL-10, a potent anti-inflammatory
cytokine with well-known suppressive activities
over TNF-α production, has been described previously to occur in BMC cultures in vitro and
in vivo in a model of myocardial infarct (29). Thus
it is possible that both cell populations, transplanted cells as well as resident cells in the spleen
and bone marrow, contribute to the increase in
IL-10 mRNA expression.
In summary, we observed that peripheral transplantation of BMC reduced mortality in an APAPinduced model of acute liver failure potentialized
by ethanol without improving liver damage. Transplanted cells did not differentiate into hepatocytes
and did not up-regulate the liver function, as
showed by serum ammonia dosage. Nonetheless,
mice that received BMC transplantation showed
less BBB injury. Studies are needed to evaluate the
therapeutically active subpopulations in the BMC
fraction and to describe the protective mechanisms
involved.
Acknowledgments
The authors would like to acknowledge Dr Washington Luis Conrado dos Santos for his comments and
discussions; Adriano Alcântara, Joselli Santos Silva,
Elton Sá Barreto and Carine Machado Azevedo
for technical assistance; CNPq, FINEP, MCT and
FAPESB for financial support; LACEN, Salvador,
10
Figure 8. Measurement of cytokine gene expression in the bone marrow and spleen. (A, B) Analysis of IL-10 (A) and TNF-α (B) mRNA
expression in the bone marrow 6 h after APAP injection. (C–F) Analysis of IL-10 (C, E) and TNF-α (D, F) mRNA expression in the
spleen 6 (C, D) and 14 (D, F) h after APAP injection. Data represent the means SEM of 6–7 mice/group. ∗P 0.05; ∗∗P 0.001.
BA, for transaminase evaluation; and the Laboratory of Clinical Analysis of the Hospital São Rafael,
Salvador, BA, for assessment of serum ammonia.
Conflict of interest statement: The authors claim
they have no conflict of interest in this study.
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Document downloaded from http://www.elsevier.es, day 17/07/2012. This copy is for personal use. Any transmission of this document by any media or format is strictly prohibited.
Rev Port Pneumol. 2012;18(3):128---136
www.revportpneumol.org
ORIGINAL ARTICLE
Alterations in pulmonary structure by elastase administration
in a model of emphysema in mice is associated with functional
disturbances
D. Vidal a,b , G. Fortunato a,c , W. Klein d,e,f , L. Cortizo a , J. Vasconcelos a,b ,
R. Ribeiro-dos-Santos a,b , M. Soares a,b , S. Macambira a,b,g,∗
a
Laboratório de Engenharia tecidual e Imunofarmacologia, Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz,
Salvador, BA, Brazil
b
Centro de Biotecnologia e Terapia Celular, Hospital São Rafael, Salvador, BA, Brazil
c
Programa de Pós-Graduação em Biotecnologia, Universidade Estadual de Feira de Santana, Feira de Santana, BA, Brazil
d
Faculdade de Biologia, Instituto de Biologia, Universidade Federal da Bahia, Salvador, BA, Brazil
e
Instituto de Biociências, Instituto Nacional de Ciência e Tecnologia em Fisiologia Comparada, UNESP, Rio Claro, SP, Brazil
f
Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, SP, Brazil
g
Laboratório de Eletrofisiologia Cardíaca ‘‘Prof Antônio Paes de Carvalho’’, Instituto de Biofísica Carlos Chagas Filho,
Universidade Federal do Rio de Janeiro, RJ, Brazil
Received 6 July 2011; accepted 23 December 2011
Available online 17 March 2012
KEYWORDS
Pulmonary
emphysema;
Ergometric test;
Porcine elastase;
C57Bl/6
∗
Abstract Several experimental studies of pulmonary emphysema using animal models have
been described in the literature. However, only a few of these studies have focused on the
assessment of ergometric function as a non-invasive technique to validate the methodology
used for induction of experimental emphysema. Additionally, functional assessments of emphysema are rarely correlated with morphological pulmonary abnormalities caused by induced
emphysema. The present study aimed to evaluate the effects of elastase administered by
tracheal puncture on pulmonary parenchyma and their corresponding functional impairment.
This was evaluated by measuring exercise capacity in C57Bl/6 mice in order to establish a
reproducible and safe methodology of inducing experimental emphysema. Thirty six mice underwent ergometric tests before and 28 days after elastase administration. Pancreatic porcine
elastase solution was administered by tracheal puncture, which resulted in a significantly
decreased exercise capacity, shown by a shorter distance run (−30.5%) and a lower mean
velocity (−15%), as well as in failure to increase the elimination of carbon dioxide. The mean linear intercept increased significantly by 50% in tracheal elastase administration. In conclusion,
application of elastase by tracheal function in C57Bl/6 induces emphysema, as validated by
Corresponding author.
E-mail address: simone@bahia.fiocruz.br (S. Macambira).
0873-2159/$ – see front matter © 2011 Sociedade Portuguesa de Pneumologia. Published by Elsevier España, S.L. All rights reserved.
doi:10.1016/j.rppneu.2011.12.007
129
morphometric analyses, and resulted in a significantly lower exercise capacity, while resulting
in a low mortality rate.
© 2011 Sociedade Portuguesa de Pneumologia. Published by Elsevier España, S.L. All rights
reserved.
PALAVRAS-CHAVE
Enfisema pulmonar;
ensaio ergométrico;
Elastase suína;
C57Bl/6
Alterações na estrutura pulmonar devido à administração de elastase num modelo
de enfisema em ratos encontram-se associadas a distúrbios funcionais
Resumo Vários estudos experimentais de enfisema pulmonar em modelos animais têm sido
descritos na literatura científica. No entanto, apenas alguns destes estudos têm sido concentrados na avaliação da função ergométrica como técnica não-invasiva para validar a metodologia
utilizada para a indução do enfisema experimental. Além disso, as avaliações funcionais de
enfisema raramente se encontram correlacionadas com anomalias morfológicas pulmonares
causadas por enfisema induzido. O presente estudo teve como objetivo avaliar os efeitos da
elastase administrada por punção traqueal no parênquima pulmonar e a sua disfunção funcional correspondente. Esta foi avaliada através da medição da capacidade de exercício em
ratos C57Bl/6, de forma a estabelecer uma metodologia reprodutível e segura de induzir o
enfisema experimental. Trinta e seis ratos foram submetidos a testes ergométricos antes e
28 dias após a administração de elastase. A solução de elastase pancreática suína foi administrada por punção traqueal, o que resultou numa diminuição significativamente da capacidade
de exercício, demonstrada pela diminuição da distância percorrida (menos de 30,5%) e por
uma velocidade média inferior (menos de 15%), assim como pela incapacidade de aumentar a
eliminação de dióxido de carbono. A intersecção linear média aumentou significativamente em
50% na administração traqueal da elastase. Em conclusão, a aplicação de elastase por punção
traqueal em ratos C57Bl/6 induz enfisema, conforme foi validado por análises morfométricas, e
resultou numa capacidade de exercício significativamente mais baixa, embora se tenha obtido
uma baixa taxa de mortalidade.
© 2011 Sociedade Portuguesa de Pneumologia. Publicado por Elsevier España, S.L. Todos os
direitos reservados.
Introduction
Emphysema is a chronic obstructive pulmonary disease
(COPD), limiting air flow during expiration, and is associated with an abnormal inflammatory response of the lungs to
noxious particles or gases (mainly cigarette smoke). This disease is characterized by a permanent abnormal dilatation of
alveolar spaces. The destruction of pulmonary parenchyma
impairs alveolar gas exchange, compromising the physical
capacity of a patient since it is associated with airflow limitations that are not fully reversible.
No therapy capable of reversing emphysematous tissue
lesions is available to date and, in severe chronic stages
the only treatment that remains is lung transplantation,
representing a procedure with high levels of morbidity and
mortality. Emphysema, together with other types of COPD,
are responsible for more than 2.5 million deaths every year,
representing the fifth leading cause of mortality in the
world.
The severity of its pathology together with the lack of
any effective treatment transforms emphysema into a great
medical challenge. There is a need for studies aiming to
understand the pathogenic cellular mechanisms causing tissue destruction in order to elucidate the disease and to
open new therapeutic avenues. Another aggravator is the
fact that a considerable variation exists in the course of the
disease among different smokers. Only about 15% of smokers
develop COPD.1,2
Animal models represent a fundamental instrument to
correlate pre-clinical research with clinical studies. The
experimental model allows the detailed investigation of different factors influencing emphysema. These factors include
inflammatory cell recruitment, genetic background, abnormal matrix repair, lung cell apoptosis, and misbalance
between apoptosis and replenishment of structural cells in
the lung. Additional factors are research of potential therapeutic agents and strategies, such as administration of stem
cells or different growth factors.3
Until now there are many experimental models of COPD,
each of them with advantages and disadvantages. In reality, a number of approaches have been tried because until
now none of them constitutes a model that exactly reproduces all phases of development and clinical features of
emphysema or any other abnormalities that make up this
clinical entity, the COPD. Other factor that affects the reproducibility of data is the differences in anatomical structure
with respect to development, maturation, structural organization of respiratory branches, and constituent cells and
vascularization.4 On the other side, some common characteristics can be observed between animal model and human
disease. For example, rodents develop the phenomenon of
metaplasia induced by reaction to injury,5 which is a frequently observed response to an insult in different organs
in humans, such as in respiratory tract, urinary bladder, and
esophagus.
The first animal model of emphysema was developed more than 40 years ago6 and it was evoked in
rats by intratracheal instillation of the plant proteinase
papain. The experimental models of emphysema are continuously improving and became a research tool for
130
several groups of scientists. These include enzyme airway administration model,7,8 tobacco smoking model,9,10
apoptosis induced emphysema by intratracheal instillation
of activated caspase-3,11 AIAT deficient animals,12 natural
mutants,13 knockout mice,14 and transgenic mice.15 Despite
the model chosen all of them present restrictions.16---22
Taking these informations into account, in the present
study we chose to reproduce human pulmonary emphysema
in mouse model using intratracheal instillation of porcine
pancreatic elastase because it is used for more than three
decades and has been well characterized.29 Until now it is
considered to be the most consistent and impressive model
of airspace enlargement.30 This is due to the exposure of
elastin fibers to porcine pancreatic elastase leading to acute
lung inflammation and pulmonary parenchyma destruction.
This model is very appropriate to investigate the relation
between structure and function abnormalities characteristics of emphysema progression, as well as elucidated factors
associated to these disturbances and collagen remodeling
failure related to this pathology.31 This model was used
to investigate and validate a new methodology to evaluate the dynamic lung mechanical function in experimental
model of emphysema.32 The pathogenesis for emphysema
includes many events such as inflammation, elastase, matrix
metalloprotease imbalance, apoptosis, and oxidative stress,
which are reproduced by elastase administration in animal
models.33 We chose to develop the model in female C57Bl/6
mice based on clinical evidences that point out women are
more susceptible to emphysema development than men34
and on pre-clinical assays that demonstrated the higher susceptibility of female A/J mice to emphysema.35 The use of
an appropriate model of emphysema is essential to investigate relevant questions involved in the pathophysiology of
the disease and to test the therapeutic potential of new
drugs. Another essential point is the use of methods to
evaluate the physiological parameters of respiratory in the
emphysema model.
Evaluation of pulmonary function and measurement of
physiological parameters are crucial to studies that investigate new therapeutic strategies, pathogenesis of disease
and toxicology. There are invasive and noninvasive pulmonary function tests available which are sensitive in
detecting abnormalities in mechanical respiratory. The
invasive methodology evolution allowed the evaluation
of relevant parameters transpulmonary pressure, expiratory and inspiratory flows, such as pulmonary resistance
and compliance. Although these procedures give precise
data, they involve invasive surgical procedures23 (orotracheal intubated animal) and anesthesia that impose
non-physiological conditions to experiment that may generate significant artifacts, such as decreased respiratory
frequency,24 besides the risk of death of animals due to
the procedure which implies the use of a larger number of animals per experimental group. The non-invasive
methodology started in the end of 70s25 and there are basically two types of plethysmograph: double-chamber and
single-chamber whole-body instrument. This noninvasive
serial measurements reduced the number of animal per
study, but long-term examination is not allowed due to the
stress imposed by of the neck collar restrained. Noninvasive
techniques to evaluate respiratory parameters in conscious
mice are easy, convenient, repetitive and reproducible,
D. Vidal et al.
appropriate for quick and repeatable screening of respiratory function in a large number of conscious animals,26
however are not so accurate as the invasive method, and
some important data about pulmonary function may be misleading.
This kind of evaluation is essential to reveal information about pulmonary function but does not provide data
on the degree of impairment of physical capacity of the animal. Lüthje et al.27 demonstrated exercise intolerance by
treadmill as a consequence of pulmonary emphysema and
its systemic consequence in elastase-induced-emphysema
model in transgenic mice.
Following this rational, we sought to evaluate a different
procedure to establish experimental pulmonary emphysema
in C57Bl/6 using elastase via tracheal puncture. To prove
the efficacy of emphysema induction, we investigated the
degree of impairment of physical capacity by the evaluation
of respiratory function during exercise testing on treadmill,
acquiring data about distance run, exercise time, oxygen
consumption, and carbon dioxide production. To evaluate
the structure alterations we measured LM, an objective
parameter that represents the average distance between
alveolar walls. The increase of Lm with age is relatively
small and it is independent of body size. This measurement
is easy to make and is independent of the observer. This
measurement is considered to be suitable for detailed studies that aim to correlate structure and function disturbances
in lungs.28
By the investigation of functional disturbances and
structural alterations associated with elastase-induced
emphysema in mice, we aimed to establish an experimental model with high reproducibility and low mortality. We
analyzed safety, feasibility and reproducibility of our model
applying intratracheally an intermediary dose of elastase to
produce the enlargement of alveolar space and its consequences on respiratory function.
Materials and methods
Animals
Two-month old female C57Bl/6 mice (n = 36), weighing
around 20 g, raised and maintained at the animal facilities at the Gonçalo Moniz Research Center, FIOCRUZ, were
used in the experiments, and were provided with rodent
diet and water ad libitum. All animals were euthanized in a
CO2 chamber, and handled according the NIH guidelines for
ethical use of laboratory animals.
Experimental groups
The animals were randomly divided into three groups: (A)
control: non-manipulated animals (n = 12); (B) intratracheal
elastase: 100 ␮l (n = 12); and (C) intratracheal PBS 100 ␮l
(n = 12).
Emphysema induction
For elastase instillation, mice were anaesthetized with
ketamine (Vetbrands) and atropine (Allergan), intubated
orotracheally, and ventilated using a ventilator/respirator
(Mini-Vent Small Animal Ventilators, Kent Scientific). Experimental animals received 2 U/100 g body weight porcine
pancreatic elastase (Sigma, Aldrich, Taufkirchen, Germany)
dissolved in 100 ␮l phosphate-buffered saline (PBS)solution
or 100 ␮l PBS alone. Control animals were not manipulated.
The respective solution was administered intratracheally
via tubing followed by 200 ␮l of air for an even distribution of the liquid throughout both lungs. The animals were
submitted to anterior cervical incision in order to visualize
the trachea and make the tracheal puncture to administer
one of the solutions. Afterwards, mice were sutured, kept
on a warm plate (30 ◦ C) until restoration of spontaneous
breathing and then were extubated.
Treadmill
A motor-driven treadmill chamber for one animal (LE 8700,
Panlab, Barcelona, Spain) was used to exercise the animals.
The speed of the treadmill and the intensity in milliamps of
the electric shock applied to an stainless steel grid at the
rear end of the treadmill were controlled by a potentiometer
(LE 8700 treadmill control, Panlab). Room air was pumped
into the chamber at a controlled flow rate (700 ml/min)
by a chamber air supplier (OXYLET LE 400, Panlab). Outflow was directed to an oxygen and carbon dioxide analyzer
(Oxylet 00; Panlab) to measure consumption of oxygen and
production of carbon dioxide. The mean room temperature
was maintained at 21 ± 1 ◦ C. After an adaptation period of
40 min in the treadmill chamber the mice were exercised
at different velocities, starting at 7.2 m/min and increasing
the velocity 7.2 m/min every 10 min. The inclination of the
treadmill was maintained at an uphill angle of 10◦ . Velocities were increased until the animal could no longer sustain
a given speed and remained for more than ten seconds on
the electrified grid, which provided an electrical stimulus
(1 milliamp) to keep the mice running. Total running distance and running time were recorded. Treadmill tests were
carried out on all mice before the induction of emphysema
and 28 days after experimental procedures.
131
Table 1 Percentage of mortality related to different
methodology to induce experimental pulmonary emphysema. (A) Control: non-manipulated animals; (B) intratracheal 100 ␮l of elastase administration; (C) intratracheal
100 ␮l of saline administration.
Group
Number of mice
Mortality
%
A
B
C
12
12
12
0
2
2
0
16.7
16.7
Total
36
4
11.1
lung sections of 5 ␮m and the number of intercepts crossing
the lines counted. The Lm was calculated from the length
of the lines multiplied by the number of the lines divided by
the sum of all counted intercepts.
Statistical analysis
Numeric values were expressed as mean ± SD unless stated
otherwise. Treadmill performance was analysed using a
paired t-test to compare groups before and after treatment
and morphometric data were compared applying a One-way
ANOVA followed by a Tukey test to identify groups that are
significantly different. A value of P < 0.05 was considered
statistically significant.
Results
Safety of the procedure --- mortality
Four out of 36 mice (11.1%) died during the experimental
procedure. The groups manipulated by intratracheal function showed a similar mortality (two death per group),
independent of the kind of solution administrated (Table 1).
Functional analysis --- ergometry
Histopathological analysis
Twenty-eight days after application of elastase or PBS,
the mice were euthanized using CO2 and the lungs were
exposed. The trachea was canullated with gelco number
18 and the lungs were perfused with buffered 4% formalin
applying a constant transpulmonary pressure of 20 cm H2 O
for 2 h. After this procedure the trachea was sutured in order
to hold the intrapulmonary pressure at 20 cm H2 O and the
entire cardiopulmonary tissue block was removed and fixed
in formalin (4%).
Morphologic examinations were performed following
Thurlbeck28 method modified. Briefly, the mean linear
intercept (Lm) of each lung was determined using light
microscopy on 20 randomly selected fields, originating from
randomly selected tissue samples covering the entire lung
and containing apical as well as basal areas of the organ.
Five pictures were taken of each lung. The Lm as indicator of air space size was calculated from counting lines of
defined length that were randomly placed on each of the 20
Maximum velocity and running distance reached by each
group before (pre) and after (post) administration of PBS
or elastase are summarized in Table 2.
Before the manipulation, all mice were capable of
exercising on the treadmill. After manipulation, intratracheal elastase administration (group B) showed a significant
decrease in the maximum exercise velocity being able to sustain treadmill velocities up to 15.1 m/min (Table 2). These
differences resulted in a significant reduction in distance
covered by tracheal elastase-treated mice when compared
to tracheal saline-treated mice, reducing running distance
by 30.5% in the former group (Table 2).
Concerning oxygen consumption and carbon dioxide production, all mice treated with elastase increased oxygen
consumption after induction when compared at the same
speed before and after administration of elastase (Fig. 1A).
In relation to the production of carbon dioxide, no significant difference was observed at the same speed before and
after administration of elastase (Fig. 1B).
132
D. Vidal et al.
Table 2 Mice performance during exercise on a motorized treadmill evaluated by maximum velocity reached and maximum
distance covered before (pre) and after treatment (post) and the respective percentage change (%). Groups as in Table 1.
Group
Velocity (m/min)
A
B
C
*
Distance (m)
Pre
Post
Pre
Post
19.8 ± 3.0
19.2 ± 2.7
18.0 ± 3.0
19.2 ± 3.2
16.2 ± 3.1*
18.0 ± 3.6
900 ± 124
810 ± 161
710 ± 157
810 ± 173
570 ± 45*
720 ± 201
Indicates a value significantly lower after treatment (P < 0.05).
Morphometric analysis --- mean linear interseptum
measurement
*
*
120
90
µm
The administration of 100 ␮l of elastase by the tracheal route significantly increased Lm (99.6 ± 2.9 ␮m) when
compared to intratracheal saline-treated (33.3 ± 0.6 ␮m)
and non-manipulated mice (25.6 ± 0.1 ␮m). A slight, but
*
150
60
A
10000
30
VO2(mL/Kg-1/h-1)
8000
*
**
6000
***
*
0
A
4000
2000
g
t in
es
R
8000
VCO2(mL/Kg-1/h-1)
Pre-induction
B
6000
C
Figure 2 Comparison of the mean linear intercept length in
three groups of C57Bl/6 mice treated in (A) non-manipulated
animals (n = 12), (B) animals receiving tracheal elastase (n = 12),
or (C) receiving tracheal saline (n = 12). Data are presented
as means ± SD. * Indicates a significant difference (P < 0.05)
between goups.
7.
2
m
/m
in
14
.4
m
/m
in
21
.6
m
/m
in
28
.8
m
/m
in
36
m
/m
Ex
in
ha
us
tio
n
R
ec
ov
er
y
0
B
Pos-induction
significant, increase in Lm was found comparing traqueal
application of saline with non-manipulated animals (Fig. 2).
Discussion and conclusion
4000
2000
R
es
tin
g
7.
2
m
/m
in
14
.4
m
/m
in
21
.6
m
/m
in
28
.8
m
/m
in
36
m
/m
Ex
in
ha
us
tio
n
R
ec
ov
er
y
0
Pre-induction
Pos-induction
Figure 1 Comparison of respiratory function during physical
effort in mice before and after the administration of elastase. Oxygen consumption (A) and carbon dioxide release (B),
during resting conditions, exercising at 5 different velocities
(7.2, 14.4, 21.6, 28.8 and 36 m/min) on a motorized treadmill,
immediately (Exhaustion) and 25 min after exercise (recovery).
Data are means ± SD; 10 mice/group. Note that mice elastasetreated were unable to run at a velocity of 36 m/min; therefore,
no data are given. *p < 0.05; **p < 0.01; ***p < 0.001.
In this work we induced emphysema by intratracheal instillation of porcine pancreatic elastase, which resulted in a
mean linear intercept of 99.6 ␮m. Ishizawa et al.29 applied
the same concentration of elastase by intranasal instillation
to induce the experimental emphysema in mice and, after
three weeks, they observed pulmonary abnormalities and a
mean linear intercept of 80 ␮m. The same group repeated
the same procedure in order to test the effects of Hepatocyte Growth Factor in injured lungs by elastase, but the
mean linear intercept was slightly smaller: 65 ␮m.30 Another
study,31 using both C57BL/6 mice and SP-C/TNF-a transgenic
mice, using similar dosage of elastase found a mean linear
intercept ranging between 60 and 85 ␮m. Comparing these
morphometric data suggests that intratracheal administration of elastase seems more efficient to induce emphysema,
coupled with a relatively low mortality. The experimental
procedures used in our study to induce emphysema can be
considered tolerable since the mortality index in all experimental groups remained low (Table 1). Ishizawa et al.29,30
did not mention the mortality rate observed in their studies.
Recently,27 emphysematous disturbances were induced
in a transgenic strain of mice (NMRI), which possesses a
higher endogenous anti-protease activity. Two experimental
groups were used to administer porcine pancreatic elastase
by intratracheal instillation: 5 U/100 g body weight, single administration and 3.3 U/100 g body weight, repetitive
administration. In both treatments the absolute mortality of
animals was much higher than that in our work (six and five
mice, respectively). However, the authors do not mention
the total number of animals used per group, but it seems
clear that the higher concentrations of elastase used in their
study resulted in a greater mortality rate among experimental animals when compared to the present study.27 These
results indicate that the experimental protocol used in our
study is more applicable to induce emphysema in mice.
Comparing the morphological parameters of our study
with the ones measured by Lüthje et al.,27 we observed that
the Lm was greater in the latter study (260.7 ␮m) when compared to our data (99.6 ␮m). The greater destruction of lung
parenchyma induced in the mice by Lüthje et al.27 can be
attributed to the greater concentration of elastase used, as
well as to the mouse strain used, which is more susceptible
to the development of pulmonary structure abnormalities.
The physical activity is significantly impaired in many
patients with COPD, significantly altering their quality of
life. The pathogenesis of physical incapacity is complex
and involves loss of respiratory muscle strength, changes in
gas exchange, and abnormalities in lung mechanics, which
reflects in exercise intolerance that is a direct consequence
of impairment in respiratory mechanics. However, the measurement of parameters related to respiratory mechanics
involved invasive or noninvasive methodology, which it is
not totally secure, requiring a large number of animals to be
used or a loss of accuracy of measurements of respiratory
parameters, since it does not reflect the physiological reality of an undisturbed animal. The invasive measurements
require that animals are anesthetized and ventilated,23 orotracheally or endotracheally intubated, tracheotomized32
or under spontaneous breathing. The advantages of these
methods include no stress for the animal and the evaluation of gold standard parameters, such as resistance and
compliance. However, the anesthesia could alter some physiological parameters and depending on methodology the
measurement cannot be repeated.33 Hantos et al.34 tried
to establish a link between the mechanical properties of
the respiratory system and absolute lung volumes in
elastase-induced emphysema in order to investigate how
these physiological findings are related to the pathophysiological changes in lung volumes and the associated
alterations in respiratory resistance and elastance in
patients. Thus, the tissue damage did not correlate with
airway dysfunction in this mouse model of emphysema.34
On the other side, noninvasive techniques are easy to
perform, allow spontaneously breathing, and can be done in
large numbers of conscious animal during the same period.
As previously mentioned, there are two types of plethysmograph: double-chamber and single-chamber whole-body
instruments. In the first one the animal is restrained at
the neck. The animals are placed in the body plethysmograph, while the head protruded through a neck collar
into a ventilated head exposure chamber.35,36 This apparatus which is attached to an amplifier is connected to a
133
pneumotachograph and a differential pressure transducer,
which allows airflow measurement, tidal volume, minute
volume and respiratory rate.37 These noninvasive serial measurements reduced the number of animal per study, but
long-term examination is not allowed due to the stress
imposed by the neck collar restrained. There is also a unrestrained double-chamber instrument where the animal’s
head and body are separated in a chamber by a rubber
collar around the neck, which creates two environments:
nasal chamber and thoracic chamber. In single-chamber
whole-body plethysmograph, no restraint is required and the
animals have free access to water and food. These recordings may be done all day long but physiological measurement
is indirect since it is done based on pressure changes in
single-chamber whole-body plethysmograph.38 The results
are reproducible despite of the chamber plethysmograph
employed. The single-chamber is appropriate to measure
airway resistance reactivity to drug administration, but not
to evaluate tidal volume. On other side, the double-chamber
is to provide accurate results about tidal volume and respiratory rate.37 When this technique is performed in a double
chamber with restrained animal records of inspiratory and
expiratory flows, tidal volume, minute volume and respiratory rate can be done directly, but it is not possible to
measure transpulmonary pressure or airway resistance. The
noninvasive measurements with unrestrained animal in a
single chamber allow physiological measurement based on
pressure alterations inside the chamber, thus the inspiratory and expiratory flows are evaluated indirectly, and the
transpulmonary pressure cannot be analyzed. The limitation
is the shortage of accuracy on the evaluation of physiological
parameters, such as respiratory rate and tidal volume.39
In addition to the reasons described above, that justify
the use of treadmill exercise to assess the functional limitations of the respiratory system induced by emphysema,29---31
there is the fact that acute physical exercise is known to
markedly increase the blood flow and oxygen uptake in
the lung. The severity of pulmonary parenchyma lesions on
exercise performance can be easily evaluated by ergometric tests, and in experimental models of emphysema this
fact may be evaluated by testing exercise capacity in treadmill, as performed by Lüthje et al.,27 which demonstrated a
reduction in relative run distance in emphysematous mice.
Here, not only the distance run and exercise time were
measured but the consumption of oxygen and carbon dioxide were evaluated. The results obtained demonstrated
that emphysema induction committed the exercise capacity probably due to an impairment in gas exchange (Fig. 1
and Table 2).
In our work, with a single intratracheal administration of
elastase and a small degree of inclination in the treadmill,
we observed a reduction in running distance of 30.5% when
compared to pre-treatment exercise capacity of emphysematous mice. The greatest decrease in maximum velocity
was also seen in the group treated with elastase by intratracheal route (Table 2). A reduction in maximum velocity
during treadmill exercise was also observed in a rat model of
emphysema after intratracheal administration of elastase.34
The treadmill performance of mice in the study by Lüthje
and co-workers revealed that there was no difference in
running distance between control and single intratracheal
administration of elastase groups, whereas the reduction in
134
running distance in the group with repetitive intratracheal
administration of elastase was 29.7%. Therefore, although
the pulmonary tissue destruction was more severe in the
study by Lüthje and co-workers, as shown by liner intercept
length, probably due to strain susceptibility and the number of elastase administration, our experimental procedure
resulted in the same level of serious functional impairment, as revealed by the exercise performance. Different
from the previous works, we analysed the dynamics of gas
exchange during exercise. The augment in oxygen consumption (Fig. 1A) could reflect the ability of the heart to increase
cardiac output in order to attend the metabolic demands
of animal during physical effort, trying to hold the blood
oxygen partial pressure in the face of blood carbon dioxide
partial pressure altered. The maintenance of carbon dioxide production (Fig. 1B) could mean the impairment of gas
exchange. These respiratory dysfunction is reflected in a
lower exercise capacity and could be consequence of pulmonary parenchyma lesion.
Up to date, all of the studies inducing emphysema in animal models are insufficiently capable of reproducing the
slow and progressive inflammatory process that leads to
chronic pulmonary injury in humans. However, they helped
to elucidate the pathogenic mechanisms and the development of new clinical therapeutic protocols. Different
animals such as hamsters, rats, and mice were used to
establish models of emphysema, which are based on genetic
manipulations (in mice), exposure to cigarette smoke, or
administration of proteinases into the airway. Knockout or
transgenic mice models for emphysema revealed different
genes that could be involved in the pathogenesis of emphysema, such as platelet derived growth factor A,40 fibroblast
growth factor receptors 3 and 4,41 metalloproteinase1 transgenic mice,15 platelet derived growth factor B
transgenic mice,42 macrophage elastase,43 and neutrophil
elastase,44 the last ones given resistance to smoke-induced
emphysema. However, it is noteworthy that these genetargeted mice may have alterations in lung structure, such as
alveolar enlargement, possibly associated to alterations in
lung development instead of emphysema development due
to destruction in pulmonary parenchyma, since these genes
are expressed throughout the tissue development, growth,
and maturation.45 The smoke-induced animal models of
COPD include nose-only exposure or whole body exposure.
This model may be developed in different animal species,
such as rat,46 guinea-pig,47 or mouse.48 The guinea-pig
reproduces the most appropriate model of emphysema, but
there are many problems related to the cost, shortage of
tools to investigate molecular mechanisms, and reduced
number of reagent specific to this animal species.4,49 Rats
are very resistant and if the time and volume of smoke
exposure are extrapolated these animals developed in specific abnormalities.50 With regards to mice, in addition
to the advantages described above, functional parameters such as compliance, can be accurately measured.4
However, the smoke-induced-emphysema model is not so
reproductible,51,52 the time for developing the disease is
long (about six months), produce only a mild disease and
after the cessation of smoke the lesions do not appear to
progress.4 Currently, mice are considered the gold standard to reproduce and study many human diseases, since
the mouse genome shows a great similarity to the human
D. Vidal et al.
genome, having about 300 genes uniquely present in one
species or the other.53 Furthermore, more than 10,000
genetic markers have been mapped in the mouse, a large
number of inbred strains and transgenic mice are available,
and data on the animals’ anatomy, biology, immunology, and
physiology are available.
The model tested in our study provides some advantages,
including the use of a lower elastase concentration when
compared to other studies. Nonetheless, it induced severe
structural abnormalities that may be responsible for functional disturbances characterizing pulmonary emphysema.
Furthermore, the emphysema could be induced with a very
low mortality rate and a high degree of reproducibility,
which could be functional analyzed by non-invasive technique that reflects the physical impairment associated to
structural abnormalities characteristic of pulmonary emphysema.
Conflicts of interest
The authors have no conflicts of interest to declare.
Acknowledgements
This work was supported by grants from the Brazilian Ministry of Science and Technology, CNPq, FINEP, FIOCRUZ, and
FAPESB.
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report
Report
Cell Cycle 10:9, 1-8; May 1, 2011; © 2011 Landes Bioscience
This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
Reversion of gene expression alterations in hearts
of mice with chronic chagasic cardiomyopathy
after transplantation of bone marrow cells
Milena B.P. Soares,1,2,* Ricardo S. Lima,3 Bruno S.F. Souza,1,2 Juliana F. Vasconcelos,1,2 Leonardo L. Rocha,1
Ricardo Ribeiro dos Santos,1,2 Sanda Iacobas,4 Regina C. Goldenberg,5 Michael P. Lisanti,9 Dumitru A. Iacobas,4
Herbert B. Tanowitz,6,7,* David C. Spray4,6 and Antonio C. Campos de Carvalho4,5,8
1
Centro de Pesquisas Gonçalo Moniz; Fundação Oswaldo Cruz; 2Hospital São Rafael; Salvador, Brazil; 3Departamento de Medicina; Universidade Federal do Vale do São
Francisco; Petrolina, Pe Brazil; 4Dominick P. Purpura Department of Neuroscience; Albert Einstein College of Medicine; Bronx, NY USA; 5Instituto de Biofísica Carlos Chagas
Filho; Universidade Federal do Rio de Janeiro; 8Instituto Nacional de Cardiologia; Rio de Janeiro, Brazil; 6Department of Medicine; 7Department of Pathology; Albert Einstein
College of Medicine; Bronx, NY USA; 9Department of Stem Cell Biology and Regenerative Medicne; Kimmel Cancer Center; Thomas Jefferson University; Philadelphia, PA USA
Key words: Chagas disease, Trypanosoma cruzi, cardiomyopathy, cell transplantation, microarray, inflammation, fibrosis
Abbreviations: BMC, bone marrow cell; G-CSF, granulocyte-colony stimulator factor; GO, gene ontology;
vWF, von willebrand factor
Chronic chagasic cardiomyopathy is a leading cause of heart failure in Latin American countries, being associated with
intense inflammatory response and fibrosis. We have previously shown that bone marrow mononuclear cell (BMC)
transplantation improves inflammation, fibrosis and ventricular diameter in hearts of mice with chronic Chagas disease.
Here we investigated the transcriptomic recovery induced by BMC therapy by comparing the heart transcriptomes of
chagasic mice treated with BMC or saline with control animals. Out of the 9,390 unique genes quantified in all samples,
1,702 had their expression altered in chronic chagasic hearts compared to those of normal mice. Major categories of
significantly upregulated genes were related to inflammation, fibrosis and immune responses, while genes involved
in mitochondrion function were downregulated. When BMC-treated chagasic hearts were compared to infected mice,
96% of the alterations detected in infected hearts were restored to normal levels, although an additional 109 genes
were altered by treatment. Transcriptomic recovery, a new measure that considers both restorative and side effects of
treatment, was remarkably high (84%). Immunofluorescence and morphometric analyses confirmed the effects of BMC
therapy in the pattern of inflammatory-immune response and expression of adhesion molecules. In conclusion, by using
large-scale gene profiling for unbiased assessment of therapeutic efficacy we demonstrate immunomodulatory effects
of BMC therapy in chronic chagasic cardiomyopathy and identify potentially relevant factors involved in the pathogenesis
of the disease that may provide new therapeutic targets.
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Introduction
The discovery of stem cells with potential therapeutic properties to
promote tissue regeneration recovery has opened new avenues for
the treatment of degenerative and traumatic disorders, including
heart failure. Patients with Chagas disease, one of the main causes
of heart failure in Latin American countries, could possibly benefit from a stem cell-based therapy aiming to ameliorate the heart
function deteriorated by the chronic infection with Trypanosoma
cruzi. Currently, heart transplantation is the only efficient therapy
for chagasic patients with heart failure, a procedure limited by the
scarcity of donated organs and complications when performed in T.
cruzi-infected patients due to the risk of a resurgence of parasitemia
and tissue parasitism upon administration of immunosuppressive
drugs. Therefore, instead of replacing the hearts of chagasic heart
failure individuals, the perspective of decreasing the damage of the
heart and restoring pre-infection status with cell-based therapies
presents an attractive option that should be investigated.1
Chronic chagasic cardiomyopathy is a progressive disease characterized by focal or disseminated inflammation causing myocytolysis, necrosis and progressive deposition of collagen.2 Transplantation
of bone marrow cells (BMC) was reported to reduce inflammation
and fibrosis in the heart. The decrease in inflammatory cells correlated with an increase in apoptotic cells in the inflammatory infiltrate.3 A regression of right ventricular dilation, typically observed
in a specific chagasic mouse model, was observed in mice treated
with BMC, as detected by magnetic resonance image analysis.4 In
addition, administration of granulocyte-colony stimulating factor
(G-CSF), a cytokine capable of mobilizing BMCs to the peripheral blood, to chagasic mice caused a decrease in inflammation
*Correspondence to: Milena B.P. Soares and Herbert B. Tanowitz; Email: [email protected] and [email protected]
Submitted: 03/12/11; Accepted: 03/15/11
DOI:
www.landesbioscience.com
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Figure 1. Transplantation of BMC decreases inflammation and fibrosis in hearts of C57Bl/6 mice chronically infected with Colombian strain T. cruzi.
Mice were infected with 1,000 trypomastigote forms of Colombian strain T. cruzi. Inflammation (A) and fibrosis (B) were quantified in heart sections of
normal mice, mice 8 months after infection injected with saline (Saline) or with bone marrow cells (BMC), stained with H&E and Sirius red. Bars represent the means ± SEM of 5–8 animals/group at six months after infection. *p < 0.05; ***p < 0.001. Heart sections of chagasic mice injected with saline
(C) or with BMC (D), stained with H&E (original magnification x40).
and fibrosis and improvement of cardiopulmonary function.5
Altogether, these results suggest a paracrine effect of BMC therapy,
modulating the immune response that causes tissue aggression and
fibrosis deposition in the heart.1
The identification of the mechanisms by which BMCs modulate the immune-inflammatory response and promote improvement of cardiac damage in infected mice may contribute to the
development of more efficient therapies for chagasic patients. We
have previously shown that a number of genes related to inflammation and fibrosis are altered in the hearts of chagasic mice
compared to normal controls.6 In this study we have used cDNA
microarray analysis as an unbiased, high throughput approach
to evaluate the efficacy of transplantation of BMC to restore the
normal transcriptome in the myocardium of mice chronically
infected with T. cruzi. Our results show a marked transcriptomic
recovery caused by the BMC therapy in the hearts of mice with
chronic chagasic cardiomyopathy and suggest that such an analytical approach may be useful to evaluate efficacy of other treatments for various pathological conditions.
Results
Injection of BMCs in mice with chronic chagasic cardiomyopathy reduces inflammation and fibrosis. Mice infected with
2
the Colombian strain of T. cruzi develop a progressive myocarditis accompanied by fibrosis during the chronic phase of infection. Treatment with BMCs caused a decrease in the number of
inflammatory cells (Fig. 1A) and a reduction in fibrosis (Fig. 1B)
compared to saline-treated controls. An intense multifocal
inflammatory response composed mainly by mononuclear cells,
associated with myocytolysis, was observed in sections of salinetreated chagasic mice (Fig. 1C). Smaller and less frequent foci of
inflammatory cells were found in heart sections of chagasic mice
treated with BMCs (Fig. 1D).
Gene regulation by BMC transplantation. We compared transcriptomic changes in the BMC-treated and untreated infected
mouse hearts with respect to the uninfected hearts, defining as
significantly regulated those genes whose mean expression level
changed by more than 1.5-fold and that were different at the 0.05
level of significance when the variation among the four biological
replicas was considered. Microarray results have been deposited
in www.ncbi.nlm.nih.gov/gds as GSE24088. Two months after
transplantation of BMC (at eight months after infection), 9,390
genes were quantifiable on all twelve arrays (from uninfected
controls, infected and saline-treated and BMC treated infected
hearts); of these, 480 (5.12%) were downregulated and 1,222
(13.01%) were upregulated in the infected saline-treated hearts.
GenMapp analysis was used to determine whether regulated
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genes encoding proteins performing specific functions were differentially affected. Of the upregulated genes, most prominent
Gene Ontology (GO) terms that corresponded to these pathways were immune system process/immune response, chemokine
receptor binding and chemokine activity, including inflammatory response and chemotaxis genes Ccl12, Ccl6, Ccl7, Ccl8
(50-fold upregulated), Ccl9 (40-fold upregulated) and Cxcl1.
Downregulated genes in the infected hearts were most prominently associated with mitochondria (cofactor binding, TCA
cycle, glycoloysis, oxidative phsophorylation, coenzyme biosynthesis, oxydoreductase activity, electron transport, NADH,
RNA biosynthesis), although other affected pathways included
axon guidance receptor (Ephb3, Q8c419), negative regulation
of BMP signaling (Htra1, Twsg1, Flt1), transmembrane receptor
tyrosine kinase activity (Erbb3, Ephb3, Q8c419), cell cycle arrest
(Ak1, Cdkn1b, Cdkn1c, Sesn1) and multidrug transport (Slc47a1).
In the infected hearts of mice treated with BMC, we found
that expression of 103 genes (1.21% of the quantified genes) were
upregulated compared to controls and 77 (0.91%) were downregulated. Most prominent categories of upregulated genes were
those associated with angiogenesis and blood vessel development
(Casp8, Col18a1, Dicer1, Myh9, Pdgfa, Tnfrsf12a, Cxcl12, Fgf8,
Serpinf1, Stab1, Btg1, Notch1) and endothelial cell development
(Vezf1), thiamin transport and folate carrier activity (Slc19a2)
and vitamin biosynthesis (Itgb1bp3), cyclin binding (Cdkn1a)
and purine catabolism (Ampd2). Downregulated gene categories
included nuclear transport (Rangrf, Akt1), cofactor metabolic
process (Akt1, Ndufs1, Ldhd, Acss1, Idh3a, Sdha, Sucla2, Suclg2,
Coq5, Coq6, Coq9) and isomerase activity (flbp10, hsd367, Fkpb10,
Hsd3b7, Tpi1), calcium ion binding (Eef2k, Fkbp10, Frem2) and
cell cycle arrest (Ak1, Cdkn1b). The remarkable restoration of the
control gene expression pattern by BMC therapy of infected mice
is summarized in Figure 2 and is quantified by the TRE score,
calculated as follows:
present in focal adhesions, was found highly expressed in endothelial cells in chagasic hearts compared to normal mice (Fig.
4B and D). Heart sections of BMC-treated mice showed a significant reduction in the intensity of syndecan-4 staining and in
the number of syndecan-4 + blood vessels (Figs. 3B, C and 4F).
In contrast, the total number of blood vessels was similar in the
three groups, as indicated by staining with antibodies against von
Willebrand Factor (Fig. 3D).
Discussion
Stem cell transplantation has become an attractive therapeutic
possibility for patients with cardiac diseases, including chronic
chagasic cardiomyopathy. The demonstration that BMC transplantation has beneficial effects in hearts of mice with chronic
chagasic cardiomyopathy has raised several questions and the
determination of the molecular and cellular mechanisms by
which these cells exert their action may contribute to the development of new therapeutic strategies for chronic chagasic cardiomyopathy based on cell transplantation and/or cell factors.
Here we performed a microarray analysis in an attempt to understand some of the mechanisms involved in the activity of BMC
in the mouse model of chronic chagasic cardiomyopathy. Gene
expression analysis was carried out comparing hearts of mice
with chronic chagasic cardiomyopathy treated or not with BMC
and non-infected controls. We have found that most of the genes
altered by infection are modulated by BMC therapy, indicating a
dramatic effect of these cells in the mechanisms of pathogenesis
of the disease. The use of large-scale gene profiling for unbiased
assessment of therapeutic efficacy is thus a major contribution of
the present work to cell based therapies.
Previously we have shown that a significant number of
genes with expression altered in the heart by infection with the
Colombian T. cruzi strain are related to inflammation and fibrosis.6 As expected, since BMC therapy causes a significant decrease
in inflammation and fibrosis, we found that most of these upregulated genes in chronic chagasic cardiomyopathy have their
expression in the heart decreased after cell therapy.
The decrease in inflammation observed two months after BMC
therapy (at a total of eight months post-infection) was previously
associated with an increased number of apoptotic cells compared
to untreated chagasic controls.3 In contrast to the high number of
inflammatory cells undergoing apoptosis during the indeterminate stage,7 a reduced number (about 0.5%) of cells in the inflammatory infiltrate were found undergoing apoptosis in hearts of
individuals with chronic chagasic cardiomyopathy.8 We found
several genes related to apoptosis altered by T. cruzi infection and
modulated by BMC therapy. Analysis such as the one used in this
study will detect global alterations in gene expression, representing all cell types present in the heart, including cardiomyocytes
and inflammatory cells. Thus, additional studies are needed to
clarify which mechanisms are involved in the prevention of apoptosis of inflammatory cells in chronic chagasic cardiomyopathy, as
well as those promoting apoptosis after BMC therapy.
The inflammatory infiltrate present in hearts of mice with
chronic chagasic cardiomyopathy is mainly composed by
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where initials above the numbers indicate whether the genes
were down (D), up (U) or not (X) regulated in the infected and
saline-treated hearts (first letter) or BMC-treated hearts (second
letter) with respect to control hearts. Note that only 70 genes
(of 1,702) retained their up (31) or down (39) regulation in the
chagasic heart following BMC therapy and only a single gene was
switched between up and downregulated (UD).
Alterations in the expression pattern of galectin-3 and syndecan-4 in hearts of mice with chronic chagasic cardiomyopathy after BMC transplantation. The expression of two proteins
encoded by genes upregulated by T. cruzi infection and modulated
by BMC therapy was investigated by confocal analysis. Galectin-3
was found highly expressed in macrophages of the inflammatory
infiltrate in chagasic hearts, when compared to non-infected controls (Figs. 3A, 4A and C). Upon BMC treatment, the number
of galectin-3 positive cells was significantly reduced in heart sections of chagasic mice (Figs. 3A and 4E). Syndecan-4, a heparansulfate proteoglycan that regulates cell-matrix interactions and is
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Figure 2. Significantly regulated genes in BMC-untreated (injected with saline) (A) and BMC-treated (B) infected hearts with respect to controls. A substantial reduction in the number of regulated genes was observed in treated hearts. Significantly regulated immune response genes in untreated
(vertical axis) and treated (horizontal axis) (C). Symbols of certain regulated genes were written close to their representative points. Note that, while
the upregulation of ifitm1 and psca was reduced by treatment, the regulation of all other immune response gene was fully recovered. However,
lfi2712a and lfi2712b, whose expression was not significantly altered by the infection, were found as downregulated in treated hearts.
mononuclear cells found adhered to myofibers, many of them in
process of myocytolysis.2 Macrophages are one of the main populations found in the inflammatory site, and are highly activated
by IFNγ and TNFα, two cytokines produced in the hearts of
chagasic mice.6 Here we found that the expression of galectin-3
(also known as Mac-2), a member of a large family of lectins that
are highly conserved throughout animal evolution, upregulated
in chagasic mice, is modulated by BMC therapy. By immunofluorescence analysis, we showed that this molecule is expressed
mainly in macrophages within the inflammatory infiltrate in the
hearts of chagasic mice. A previous study has demonstrated, in
4
a model of hypertrophied heart in rats, that galectin-3 was the
most overexpressed gene in failing versus functionally compensated hearts.9 Galectin-3 colocalized with activated myocardial
macrophages and treatment of rats with recombinant galectin-3
induced cardiac fibroblast proliferation, collagen production,
cyclin D1 expression and left ventricular dysfunction.9 In addition, galectin-3 is known to play important roles in inflammatory responses, including suppression of T-cell apoptosis and its
expression is induced by IFNγ in macrophages found in inflammatory infiltrates in the heart.10,11 Altogether, these data suggest
an important role of galectin-3 in the pathogenesis of chronic
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Figure 3. Quantification of galectin-3, syndecan-4 and vWF. Expression of galectin-3 (A), syndecan-4 (B and C) and vWF (D) were quantified by immunohistochemistry and morphometric analysis in heart sections of normal or eight month chagasic mice that had been injected at six months after
infection with saline (Saline) or with bone marrow cells (BMC). Bars represent the means ± SEM of 3 animals/group. *p < 0.05; **p < 0.01; ***p < 0.001.
chagasic cardiomyopathy, and indicate this molecule as a target
for development of new treatments for chronic chagasic cardiomyopathy. In fact, galectin-3 production has recently been
pointed out as a novel marker of heart failure in patients.12
Another molecule with its gene expression modulated by BMC
therapy was syndecan-4, a heparin sulfate-carrying cell surface
protein expressed by a number of different cell types, including
endothelial cells, smooth muscle cells and cardiac myocytes that
participates in processes of cell signaling, adhesion and migration.13,14 Little is known about the regulation of syndecan-4,
but it has been reported to increase after various forms of tissue
injury including vascular wall injury,13 or myocardial infarction.14
Syndecan-4 expression has been shown to be increased with migration of blood-derived macrophages after myocardial infarction.14
Zhang et al. (1999) have shown that TNFα is the principal factor produced by the ischemic myocytes responsible for induction
of endothelial cell syndecan-4 expression.15 To our knowledge,
this is the first study investigating the expression of syndecan-4
in Chagas disease. The fact that TNFα levels are increased in
chronic chagasic hearts may explain the finding of enhanced
expression of syndecan-4 observed herein.6 Furthermore, the
modulation of the chronic inflammatory response in chagasic
hearts induced by BMC therapy may explain the reduction of
syndecan-4 expression found in BMC-treated animals.
A correlation between intensity of expression of syndecan-4 in
endothelial cells was found in close association with inflammatory
infiltrates, but this was not due to an increase in the number
of vWF+ blood vessels. The microarray analysis suggested an
increase in angiogenesis in hearts of chronic chagasic mice after
BMC therapy, but this was not confirmed by the immunohistochemistry analysis. This apparent disparity may be explained by
the fact that the genes upregulated after BMC therapy, participate in a number of other biological processes in addition to those
related to blood vessel formation, such as apoptosis.16-18
We previously commented on the prominent upregulation of
immune response genes in the chagasic heart and validated substantial numbers of these gene products using ELISA, real-time
PCR and confocal microscopy.6 The identification of mitochondria-associated genes as one of the pathways that are most downregulated in the infected heart is entirely consistent with reports
by Garg and others indicating that mitochondrial function is a
prominent target of infection in acute and chronic states.19-21
Gene expression profiling with microarrays has been widely
used to characterize tissue and cell responses to various stimuli,
to identify disease biomarkers and to reveal components and
interplay within and between gene regulatory networks. The
application of this unbiased high throughput method to compare
a pathological situation with changes occurring after a therapeutic regimen here revealed a striking degree of recovery of control
gene expression status in infected mice treated with BMC. As
we previously showed in a study examining structural and physiological parameters of hearts of chagasic mice, the BMC therapy
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Figure 4. Expression of galectin-3 and syndecan-4 in hearts of chronic chagasic mice treated with bone marrow cells. Heart sections of normal mice
(A and B), of mice injected with saline (C and D) or with BMC (E and F). Sections were stained (red) with anti-galectin-3 (A, C and E) or anti-syndecan-4
(B, D and F). Nuclei (blue) were stained with DAPI. Scale bars: 50 μm (original magnification x40).
6
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not only prevents further damage over time but also reverses the
damage that was present before the stem cells were injected.4
In conclusion, this unbiased global gene expression analysis
indicated a potent restorative effect of BMC in the hearts of mice
with chronic chagasic cardiomyopathy, with over 90% recovery
of normal expression level of genes altered by T. cruzi infection.
This impressive effect is most probably mediated by a paracrine
effect through the secretion of soluble mediators. From this
global expression profile identification of such factors is possible
and may lead to the association or the replacement of cell therapy
by these cellular hormones in order to achieve the desired repair
to the damaged chagasic heart.
Materials and Methods
Animals. Four week-old female and male C57Bl/6 mice were
used for T. cruzi infection. All animals, weighing 20–23 g, were
raised and maintained at the Gonçalo Moniz Research Center/
FIOCRUZ in rooms with controlled temperature (22 ± 2°C)
and humidity (55 ± 10%) and continuous air flow. Animals were
housed in a 12 h light/12 h dark cycle (6 am–6 pm) and provided
with rodent diet and water ad libitum. Animals were handled
according to the NIH guidelines for animal experimentation. All
procedures described had prior approval from the local IACUC
(Albert Einstein College of Medicine and Fiocruz, Bahia).
Infection with Trypanosoma cruzi. Trypomastigotes of the
myotropic Colombian T. cruzi strain were obtained from culture
supernatants of infected LCC-MK2 cells. Infection of C57Bl/6
mice was performed by intraperitoneal injection of 1,000 T. cruzi
trypomastigotes in saline. Parasitemia of infected mice was evaluated at various times after infection by counting the number of
trypomastigotes in peripheral blood aliquots.
Bone marrow cell (BMC) transplantation. BMCs obtained
from femurs and tibiae of C57Bl/6 mice were used in transplantation experiments.3,4 Briefly, BMCs were purified by centrifugation in Ficoll gradient at 1,000 g for 15 minutes (Histopaque
1119 and 1077, 1:1; Sigma, St. Louis, MO). After two washings
in RPMI medium (Sigma), the cells were resuspended in saline,
filtered over nylon wool and injected intravenously in chagasic
mice (3 x 106 cells/mouse) six months after infection. Nontransplanted control mice received intravenous injections of the
same volume (200 μl) of saline.
Morphometric analysis. Groups of mice were sacrificed at
two months after BMC or saline injection (at eight months after
infection) and hearts removed and fixed in 10% buffered formalin. Heart sections were analyzed by light microscopy after paraffin embedding, followed by standard hematoxylin/eosin staining.
Inflammatory cells infiltrating heart tissue were counted using a
digital morphometric evaluation system. Images were digitized
using a color digital video camera adapted to a microscope. The
images were analyzed using the Image Pro Program (Media
Cybernetics, San Diego, CA), such that the inflammatory cells
were counted and integrated with respect to area. Ten fields per
section were counted in 5–10 sections per heart. The percentage
of fibrosis was determined using Sirius red-stained heart sections
and the Image Pro Plus v.7.0 Software to integrate the areas.
Immunofluorescence. Frozen or formalin-fixed paraffin
embedded hearts were sectioned and 4 μm-thick sections were
used for detection of galectin-3 and syndecan-4 expression by
immunofluorescence. First, paraffin embedded sections were
deparaffinizated and a heat-induced antigen retrieval step in
citrate buffer (pH = 6.0) was performed. Then, sections were
incubated overnight with the following primary antibodies:
anti-galectin-3, 1:50 (Santa Cruz Biotechnology), anti-syndecan-4, 1:50 (Santa Cruz Biotechnology) or anti-vWF, 1:100
(Dako). On the following day, sections were incubated with
Alexa fluor 633 conjugated Phalloidin, 1:50, mixed with one
of the secondary antibodies Alexa fluor 488-conjugated antigoat IgG, 1:200 or Alexa fluor 488-conjugated anti-rabbit IgG,
1:200 (Molecular Probes) for 1 hour. Nuclei were stained with
4,6-diamidino-2-phenylindole (VectaShield Hard Set mounting medium with DAPI H-1500; Vector Laboratories). The
presence of fluorescent cells was determined by observation on a
FluoView 1000 confocal microscope (Olympus). Approximately
10 random fields per animal were captured using a 40x objective. Morphometric analyses were performed using Image Pro
Plus v.7.0 software.
DNA microarray and data analysis. We compared RNA
samples extracted from whole hearts of 4 control, 4 chagasic
and 4 BMC-treated chagasic mice by analyzing hybridization
to MO30k microarrays printed by Duke University (www.
ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL8938) spotted
with 70-mer oligonucleotides (mouse Operon version 3.0).
The hybridization protocol has already been described in reference 6, the slide type and the scanner settings were uniform
throughout the entire experiment to minimize the technical
noise. Briefly, 20 μg total RNA extracted in Trizol from each of
the twelve samples (individual hearts) was reverse transcribed
in the presence of fluorescent Alexa Fluor ® 555- and Alexa
Fluor ®647-aha-dUTPs (Invitrogen, Carlsbad, CA) to obtain
labeled cDNA. Red and green labeled samples of biological
replicas were then co-hybridized (“multiple yellow” strategy22 )
overnight at 50°C. After washing (0.1% SDS and 1% SSC)
to remove the non-hybridized cDNA, each array was scanned
at 630 V (635 nm) and 580 V (532 nm) with GenePix 4100B
scanner (Axon Instruments, Union City, CA) and images were
primarily analyzed with GenePixPro 6.0 (Molecular Devices,
Sunnyvale, CA). Microarray data were processed as described
previously in reference 6. A gene was considered as significantly
up or downregulated when comparing four hearts from one
condition to those from another if the absolute fold change was
>1.5x and the p-vlaue of the Student’s heteroscedastic t-test of
equality of the means of the distributions with a Bonferronitype adjustment for each redundancy group (set of spots probing the same gene) was <0.05. GenMapp23 and MappFinder
(www.genmapp.org) software and associated databases were
used to identify the most affected GO (Gene Ontology) categories. In order to determine whether genes differentially
expressed in untreated and treated infected hearts with respect
to uninfected hearts were disproportionately affected in specific pathways, we used GenMAPP and MappFinder software
(Gladstone Institute; San Francisco: www.genmapp.org) to
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provide the statistics of affected GO categories. GO sets with
fewer than 10 analyzed members were excluded from this
analysis.
The novel parameter transcriptomic recovery efficacy (TRE)
was computed as:
percentage of not regulated genes in infected hearts whose expression changed due to treatment alone (XD & XU).
Statistical analysis. Morphometric data were analyzed using
Student’s t test or ANOVA followed by Turkey. Differences were
considered significant when p < 0.05.
Acknowledgements
where: D, U, X indicate whether the gene was down, up or not
regulated in the saline-treated (first position) or BMC-treated
(second position) infected hearts. TRE is thus the percentage
of up and downregulated genes in infected hearts whose expression level was restored to normal (UX & DX) penalized by the
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Volume 10 Issue 9
MAJOR ARTICLE
Gene Expression Changes Associated
with Myocarditis and Fibrosis in Hearts
of Mice with Chronic Chagasic Cardiomyopathy
Milena Botelho Pereira Soares,1,2 Ricardo Santana de Lima,1 Leonardo Lima Rocha,1 Juliana Fraga Vasconcelos,1
Silvia Regina Rogatto,3,4 Ricardo Ribeiro dos Santos,1,2 Sanda Iacobas,7 Regina Coeli Goldenberg,5
Dumitru Andrei Iacobas,7 Herbert Bernard Tanowitz,8,9 Antonio Carlos Campos de Carvalho,5,6
and David Conover Spray7,8
1
Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, and 2Hospital São Rafael, Salvador, 3Departamento de Urologia, Faculdade
de Medicina, Universidade do Estado de São Paulo, Botucatu, 4Hospital do Câncer A. C. Camargo, São Paulo, and 5Instituto de Biofı́sica Carlos
Chagas Filho, Universidade Federal do Rio de Janeiro, and 6Instituto Nacional de Cardiologia, Rio de Janeiro, Brazil; 7D. P. Purpura Department
of Neuroscience, 8Department of Medicine, and 9Department of Pathology, Albert Einstein College of Medicine, Bronx, New York
Chronic chagasic cardiomyopathy is a leading cause of heart failure in Latin American countries. About 30%
of Trypanosoma cruzi–infected individuals develop this severe symptomatic form of the disease, characterized
by intense inflammatory response accompanied by fibrosis in the heart. We performed an extensive microarray
analysis of hearts from a mouse model of this disease and identified significant alterations in expression of
∼12% of the sampled genes. Extensive up-regulations were associated with immune-inflammatory responses
(chemokines, adhesion molecules, cathepsins, and major histocompatibility complex molecules) and fibrosis
(extracellular matrix components, lysyl oxidase, and tissue inhibitor of metalloproteinase 1). Our results
indicate potentially relevant factors involved in the pathogenesis of the disease that may provide new therapeutic
targets in chronic Chagas disease.
Chagas disease, caused by infection with the protozoan
Trypanosoma cruzi, is still a major health problem in
Latin America, where it affects 16–18 million people
[1]. The most common chronic form, chagasic cardiomyopathy (CCM), is a fatal disease for which there is
no effective treatment available other than heart transplantation. CCM is characterized by focal or dissemi-
Received 22 April 2009; accepted 25 September 2009; electronically published
21 June 2010.
Potential conflicts of interest: none reported.
Presented in part: Third International Symposium on Advanced Therapies and
Stem Cells, Curitiba, Brazil, 2 September 2008.
Financial support: National Institutes of Health (grants HL-73732, HD-32573,
AI-076248, and AI-052739), Coordenação de Aperfeiçoamento de Pessoal de Nı́vel
Superior, Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico,
Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro, Fundação de Amparo
à Pesquisa do Estado da Bahia, and Fogarty International Center (training grant
D43TW007129 to R.C.G. and L.L.R.).
Reprints or correspondence: Dr Milena Soares, Rua Waldemar Falcão 121,
Candeal, Salvador, BA 40296-710, Brazil ([email protected]).
The Journal of Infectious Diseases 2010; 202(3):416–426
2010 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2010/20203-0012$15.00
DOI: 10.1086/653481
416 • JID 2010:202 (1 August) • Soares et al
nated inflammation causing myocytolysis, necrosis, and
progressive fibrosis [2–4].
The pathological basis of CCM is multifactorial [5,
6]. It may in part result from inflammatory responses
to T. cruzi antigen, to cardiac autoantigens, or to both
types of antigens [7]. A prominent role of parasite antigens in this pathology has been supported by the demonstration that a decrease in parasite load caused a
reduction in myocarditis and cardiac disturbances in
mice chronically infected with T. cruzi [8].
The identification of factors involved in the establishment of chronic heart lesions is of great interest for
the development of new therapeutic strategies for patients with this fatal disease. In this study we performed
a DNA microarray analysis to identify alterations in
gene expression in the myocardium of mice chronically
infected with the Colombian strain of T. cruzi, compared with uninfected counterparts. Our results indicate a profound effect on expression of a number of
genes related to inflammation and fibrosis in the hearts
of mice with CCM.
METHODS
Trypomastigotes of Colombian T. cruzi strain [9] were obtained
from culture supernatants of infected LCC-MK2 cells. C57Bl/
6 male and female mice were infected by intraperitoneal injection of T. cruzi trypomastigotes. Parasitemia was evaluated
at various times after infection by counting the number of
trypomastigotes in peripheral blood aliquots. Animals were
raised and maintained at the Gonçalo Moniz Research Center/
Fundação Oswaldo Cruz (FIOCRUZ) and provided with rodent
diet and water ad libitum. Animals were handled according to
the National Institutes of Health guidelines for animal experimentation. All procedures described here had prior approval
from the local animal ethics committee.
Mice were killed after 8 months of infection, and their hearts
removed and fixed in 10% buffered formalin. Morphometric
analyses were performed in hematoxylin-eosin– or Sirius red–
stained heart sections captured using a digital camera adapted
to a BX41 microscope (Olympus). Images were analyzed using
Image-Pro Program software (version 5.0; Media Cybernetics).
Frozen heart sections were used for detection of CD4, CD8,
CD11b, intercellular adhesion molecule 1 (ICAM-1), and major
histocompatibility complex (MHC) class II expression by immunofluorescence, using specific antibodies (BD Biosciences)
followed by streptavidin (Alexa Fluor 568; Molecular Probes).
The myocardium was stained with phalloidin (Molecular
Probes) or an anti–cardiac myosin antibody (Sigma). Nuclei
were stained with 4,6-diamidino-2-phenylindole (DAPI) (Vectashield HardSet mounting medium with DAPI H-1500; Vector
Laboratories). Sections were analyzed using a BX61 microscope
equipped with epifluorescence and appropriate filters (Olympus) and a system to enhance the fluorescence resolution
(OptiGrid; Thales Optem).
Stromal cell–derived factor 1 (SDF-1), tumor necrosis factor
(TNF) a, and interferon (IFN) g concentrations were measured
in total heart extracts. Heart proteins were extracted from 100
mg tissue/mL phosphate-buffered saline, to which 0.4 mol/L
sodium chloride, 0.05% Tween 20, and protease inhibitors (0.1
mmol/L phenylmethylsulfonyl fluoride, 0.1 mmol/L benzethonium chloride, 10 mmol/L ethylenediaminetetraacetic acid, and
20-KI aprotinin A/100 mL) were added. The samples were centrifuged for 10 min at 3000 g, and the supernatant was kept
frozen at ⫺70C. Cytokine levels were estimated using commercially available enzyme-linked immunosorbent assay kits for
mouse SDF-1, TNF-a, and IFN-g (R&D Systems), according
to the manufacturer’s instructions. Reaction was revealed after
incubation with streptavidin–horseradish peroxidase conjugate,
followed by detection using 3,3,5,5-tetramethylbenzidine peroxidase substrate and reading at 450 nm.
Hearts of normal and T. cruzi–infected mice were extracted
and quickly frozen in liquid nitrogen for 5 min. The material
was ground, and RNA extraction was performed using RNeasy
Mini Kit (Qiagen), following the manufacturer’s instructions.
After addition of 1 U/mL DNase I (Invitrogen), the complementary DNA (cDNA) was obtained using SuperScript II Reverse Transcriptase (Invitrogen) in a final volume of 30 mL.
Reaction cycles were performed on an Eppendorf Mastercycler
gradient for 1 h (42C for 60 min; 70C for 15 min). Polymerase
chain reaction (PCR) amplification was performed in an ABI
Prism 7000 Sequence Detection System (Applied Biosystems).
Primers and TaqMan probe for Timp1, the glyceraldehyde 3phosphate dehydrogenase control reference gene, were designed
and synthesized according to Assay-by-Design (Applied Biosystems). Quantitative data were analyzed using Sequence Detection System software (version 1.0; Applied Biosystems).
PCRs were carried out in a total volume of 25 mL, according
to the manufacturer’s instructions. The standard curves of the
target and reference genes showed similar results for efficacy
(190%). The relative quantification was given by the ratio between the mean values of the target gene and the reference
gene (Gapdh) in each sample. The relative amount of PCR
product generated from each primer set was determined on the
basis of the cycle threshold (Ct) value. The relative quantification was calculated by the 2⫺DDCt method (Ct, fluorescence
threshold value; DCt, the Ct of the target gene minus the Ct
of the reference gene; DDCt, the infected sample DCt minus
the reference sample DCt).
Total RNA (20 mg) extracted from each of the 4 control and
4 infected hearts was reverse transcribed into cDNA incorporating fluorescent Alexa Fluor_647 or Alexa Fluor_555–ahadUTPs (Invitrogen), by means of the SuperScript Plus Direct
cDNA Labeling System (Invitrogen). Differently labeled biological replicas were cohybridized overnight at 50C with
MO30N mouse oligonucleotide arrays spotted with 32,620
70mer Operon oligonucleotides (Duke Microarray Facility; version 3.0.1) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi
?accpGPL8938) using the “multiple yellow” strategy described
elsewhere [10]. In this strategy, differently labeled biological
replicas are cohybridized with the array. Thus, we have hybridized 2 arrays with samples from 4 control hearts and 2
other arrays with samples from 4 infected hearts. After washing
(0.1% sodium dodecyl sulfate and 1% saline-sodium citrate)
to remove the nonhybridized cDNAs, each array was scanned
with an Axon 4000B dual-laser scanner (MDS Analytical Technologies) and images were primarily analyzed with GenePix
Pro software (version 6.0; Axon Instruments). Locally corrupted or saturated spots, as well as those for which the foreground median fluorescence did not exceed twice the median
local background fluorescence in 1 sample, were eliminated
from analysis in all samples.
Microarray data were processed as described in our other
studies [10–12]. In brief, we used a normalization algorithm
that alternates intrachip and interchip normalization of the net
Gene Alterations in Chagas Heart Disease • JID 2010:202 (1 August) • 417
fluorescence (ie, background-subtracted foreground) signals of
the validated spots until the residual error is !5% in subsequent
steps. Intrachip normalization balances the averages of net fluorescence values in the 2 channels within each pin domain (subset of spots printed by the same pin), corrects the intensitydependent bias (usually referred as Lowess normalization), and
forces the standard distribution (mean, 0; standard deviation,
1) of log2 ratios (scale normalization) for net fluorescent values
in the 2 channels for each array. Interchip normalization assigns
a ratio between the corrected net fluorescence of each valid
spot and the average net fluorescence of all valid spots in both
control (C1, C2, C3, and C4) and infected (I1, I2, I3, and I4)
samples. The spots probing the same gene were organized into
redundancy groups, and their background-subtracted fluorescence was replaced by a weighted average value. A gene was
considered significantly up- or down-regulated in the comparison between 4 infected and 4 control hearts if the absolute
fold change was 11.5 and the P value was !.05 (Student’s heteroscedastic t test of equality of the mean distributions, with
Bonferroni-type adjustment for redundancy groups). GenMAPP [13] and MAPPFinder software (http://www.genmapp
.org) and databases were used to identify the most affected gene
ontology categories.
Morphometric, quantitative reverse-transcription PCR, and
cytokine data were analyzed using Student’s t test. Differences
were considered significant at P ! .05.
RESULTS
CCM caused by chronic infection with Colombian strain T.
cruzi in C57Bl/6 mice. On infection with 1000 trypomastigote forms of Colombian strain T. cruzi, C57Bl/6 mice develop
blood parasitemia peaking at ∼35 days after infection (Figure
1A). The mortality rate reached 28.5% during the first 100 days
(Figure 1B) and ∼31.4% after 8 months of infection. Progressive
myocarditis accompanied by fibrosis occurs after the acute
phase of infection. At 8 months of infection, heart sections
from chagasic mice revealed a multifocal inflammatory response composed mainly of mononuclear cells (Figure 1C and
1E) and exhibited areas of fibrosis (Figure 1D and 1F).
Global gene expression analysis. Microarray data from this
experiment have been deposited in GenBank (https://www
.ncbi.nlm.nih.gov/geo/query/acc.cgi?accpGSE17363). When
control hearts from C57Bl/6 mice were compared with those
from age- and sex-matched mice chronically infected with the
Colombian strain of T. cruzi, genes differentially expressed were
detected. Spots corresponding to 14,356 unigenes satisfied the
criteria of adequate quantitation for all 8 RNA samples. Of
these, 1221 (8.5%) were significantly up-regulated in the chagasic hearts and 494 (3.4%) were significantly down-regulated
(150% difference; P ! .05). A list of all genes that were found
to be differentially expressed is presented in Table 1, and subsets
of the genes showing higher fold change in expression ratio are
considered below.
Pathways of proteins encoded by genes that were significantly
affected by parasitic infection were determined using GenMAPP software (http://www.genmapp.org), in which significance is assessed by whether regulated genes are disproportionately represented within a gene ontology term. Pathways
of genes significantly up-regulated in infected hearts (P ! .05)
are listed in Table 2 and prominently include immune response
and related terms (eg, inflammatory response, intracellular signaling cascade, and chemokine and cytokine receptor activity).
Results of the GenMAPP analysis of these altered genes are
shown in Figure 2A. In addition, up-regulated pathways include
phosphate transport, cell proliferation, and actin binding (eg,
Arp2/3 protein complex and actin filament organization, cytoskeleton, and membrane ruffling). These genes related to the
actin cytoskeleton are illustrated in Figure 2B. In addition to
these well-represented pathways, smaller pathways showed
prominent perturbation, including genes involved in cardiac
differentiation (Tgfb2 and Itgb1) and regulation of action potential (Gnaq, Hexa, Hab1, and Cd9).
Pathways containing an overrepresentation of down-regulated genes (Table 2) included mitochondrion, enzymatic activity of several types, and tyrosine kinase signaling. Genes
down-regulated in less extensive pathways included negative
regulation of notch plus bone morphometric protein signaling (Htra1 and Twsg1) and regulation of vascular endothelial
growth factor receptor signaling (Flt1).
Mice chronically infected with the Colombian strain of T.
cruzi have intense myocarditis (Figure 1E). The inflammatory
infiltrate is mainly composed by mononuclear cells, including
CD4+ and CD8+ T lymphocytes (Figure 3A and 3B) and macrophages (Figure 3C). The analysis of genes that were up-regulated ⭓5-fold in the arrays showed alterations in a number
of genes related to inflammation and immune responses. Genes
coding for the macrophage cell surface marker CD68 and the
lymphocyte antigens CD38 and CD52 had their expression increased in chronic chagasic hearts (Table 3), a finding compatible with the presence of these cells in the inflammatory
infiltrate.
Up-regulation of genes coding for chemoattractant factors
Ccl2, Ccl7, Ccl8, and Ccl12 was observed (Table 3). Immunocytochemistry confirmed that the levels of Ccl12 (SDF-1) in
hearts of chronically chagasic mice were increased in comparison with those of normal mice (Figure 4A). In addition, the
expression of phospholipase A2, group VII (platelet-activating
factor [PAF] acetylhydrolase), and complement factor B genes
were highly increased (47.6- and 42.5-fold, respectively) by
chronic infection (Table 3).
The expression of genes coding for adhesion molecules, such
as galectin-3, P-selectin ligand (CD162), integrin b3 (CD61),
Figure 1. Infection of C57Bl/6 mice with Colombian strain Trypanosoma cruzi. Mice (n p 35 ) were infected with 1000 Colombian strain trypomastigotes. A and B, Parasitemia (A) and mortality (B ) evaluated during the acute phase of infection. Data in panel A represent medians for individual
parasitemia. C and D, Inflammation (C ) and fibrosis (D ) evidenced in heart sections from mice 8 months after infection, stained with hematoxylineosin and Sirius red. E and F, Morphometric quantification of inflammatory cells (E ) and fibrosis area (F ) in heart sections from normal mice (n p
4) and chagasic mice (n p 9; 8 months after infection with T. cruzi ). Bars represent means standard errors of the mean. ***P ! .05.
and ICAM-1 (CD54), was increased in hearts of chagasic mice
(Table 3). Immunostaining revealed that ICAM-1 is virtually
absent in control hearts, but in hearts of infected mice it is
found mainly in inflammatory and endothelial cells (Figure 3D
and 3E). The expression of genes coding for several cathepsins,
proteases important in lysosomal degradation, was also upregulated (Table 3). Of special interest is cathepsin S, which
mediates degradation of the invariant Ii chain in antigen-presenting cells [13]. The expression of genes coding for MHC
class II molecules IEb and IAa were highly altered. MHC class
II molecules were observed to be highly expressed in cells of
the inflammatory infiltrate in infected hearts (Figure 3F and
3G). In addition, the expression of genes encoding 2 proteasome subunits was also up-regulated (Table 3).
Cytokine-associated genes were differentially expressed in
hearts of chagasic mice (Table 3). Of special interest is upregulation of genes associated with 2 cytokines related to the
severe form of chronic CCM [14, 15], IFN-g (Igtp, Ifi30, Ifi47,
Irf1, and Irf5) and TNF-a (Tnfaip2, Tnfrsf1b, and Litaf). Although regulation of genes encoding IFN-g and TNF-a could
not be analyzed in this microarray data set owing to technical
problems, the protein levels of both cytokines were increased
Table 1. Genes Found to Be Differentially
Regulated
This table is available in its entirety in the online
version of the Journal of Infectious Diseases
Table 2. Up-regulated and Down-regulated Gene Ontology (GO) Categories in Trypanosoma cruzi–Infected Hearts
GO name
Type
No.
measureda
Change, %
Z score
Ruffle
Immune response
Chemokine activity
Lysosome
Endoplasmic reticulum
Intracellular signaling cascade
Chemotaxis
Actin cytoskeleton organization and biogenesis
Carboxypeptidase activity
Hydrolase activity, acting on glycosyl bonds
Actin cytoskeleton
Cell cortex
Actin filament organization
Amino acid–polyamine transporter activity
Myelination
Negative regulation of cell proliferation
Positive regulation of cell proliferation
Negative regulation on cell differentiation
Cell proliferation
Heparin binding
Inflammatory response
Cell surface
Phosphate transport
Hematopoietin/interferon class (D200 domain)
cytokine receptor activity
Branching morphogenesis of a tube
Cell fate commitment
Regulation of cell proliferation
Small GTPase-mediated signal transduction
C
P
F
C
C
P
P
P
F
F
C
C
P
F
P
P
P
P
P
F
P
C
P
F
21
108
19
44
305
169
31
32
11
25
40
10
15
11
13
45
56
13
55
28
67
43
40
16
38.10
19.44
36.84
31.82
14.43
12.43
25.81
12.50
27.27
28.00
10.00
20.00
26.67
18.18
23.08
15.56
16.07
7.69
16.36
21.43
11.94
18.60
17.50
25.00
5.039
4.817
4.592
4.487
3.806
3.302
3.254
3.232
3.22
3.172
3.09
3.035
2.914
2.91
2.806
2.67
2.669
2.603
2.588
2.585
2.582
2.347
2.18
2.18
!.001
P
P
P
P
12
16
17
83
33.33
12.50
0.00
10.84
2.123
2.113
2.055
2.055
.048
.038
.045
.05
Mitochondrion
Cytoplasm
Catalytic activity
Oxidoreductase activity
NADH dehydrogenase activity
FAD binding
Pyridoxal phosphate binding
Peroxisome
Electron transport
Electron carrier activity
Carbohydrate metabolic process
Ligase activity
Transaminase activity
Transmembrane receptor protein tyrosine kinase signaling pathway
Fatty acid metabolic process
Nucleotide binding
Biosynthetic process
Cell cycle arrest
Metabolic process
Lipid metabolic process
Transferase activity
C
C
F
F
F
F
F
C
P
F
P
F
F
P
351
613
104
208
10
34
31
51
189
60
81
121
10
30
17.38
2.61
5.77
6.73
30.00
17.65
16.13
13.73
7.41
8.33
6.17
8.26
20.00
13.33
13.839
5.628
5.241
4.835
4.599
4.541
3.871
3.839
3.727
3.349
3.169
3.123
2.947
2.801
!.001
P
F
P
P
P
P
F
33
715
31
24
288
87
558
12.12
3.78
12.90
12.50
8.68
8.05
4.84
2.735
2.698
2.511
2.43
2.319
2.041
1.995
.013
.007
.009
.034
.025
.043
.044
GOID
Up-regulated
1726
6955
8009
5764
5783
7242
6935
30036
4180
16798
15629
5938
7015
5279
42552
8285
8284
45596
8283
8201
6954
9986
6817
4896
48754
45165
42127
7264
Down-regulated
5739
5737
3824
16491
3954
50660
30170
5777
6118
9055
5975
16874
8483
7169
6631
166
9058
7050
8152
6629
16740
Permuted P
!.001
!.001
!.001
!.001
!.001
.004
.002
.007
.004
.007
.01
.015
.016
.024
.015
.014
.014
.012
.019
.008
.026
.039
.048
!.001
!.001
!.001
.005
!.001
.002
.002
!.001
.003
.001
.005
.017
.012
NOTE. C, cellular location; F, molecular function; FAD, flavin adenine dinucleotide; GOID, GO identification no.; NADH, nicotinamide adenine
dinucleotide, reduced; P, biological process.
a
No. of genes analyzed in that GOID.
Figure 2. Genes found to be altered within the category of immune
response and related terms from the GenMAPP database (Gladstone
Institute, University of California, San Francisco).
in the hearts of chagasic mice compared with uninfected controls (Figure 4B and 4C). The expression of genes coding for
surface receptors, such as C3a receptor 1, Fc receptors for immunoglobulin E (high affinity) and G (low affinity), and Tolllike receptor 2, was also elevated in chagasic hearts (Table 3).
Fibrosis is characteristic of hearts in chronically chagasic mice
(Figure 1F), and there was marked up-regulation of genes related to synthesis of extracellular matrix components (Table 3).
In addition, the gene expression of lysyl oxidase, an enzyme
that promotes the cross-linking of collagen fibers, was increased
(Table 3). The tissue inhibitor of metalloproteinase 1 (TIMP1), an inhibitor of collagen degradation, was also up-regulated
in chronic chagasic hearts (Table 3). Quantitative real-time PCR
analysis confirmed a significant overexpression in Timp1 in
hearts of chronically chagasic mice compared with normal controls (Figure 4D).
Figure 3. Analysis of heart sections from Trypanosoma cruzi–infected mice. Hearts from uninfected controls (D and F ) and chronically chagasic
mice (A–C, E, and G ) were compared. A, Presence of CD4+ cells (green) in infected myocardium. B, Section stained with anti-CD8 antibody (green)
and phalloidin (green). C, Presence of CD11b+ cells (red ) in the inflammatory infiltrate and phalloidin staining (green) reveal proximity of macrophages
to cardiac myocytes. D and E, Control (D ) and infected (E ) sections stained with an anti–intercellular adhesion molecule 1 antibody (red ) and phalloidin
(green), revealing up-regulation of this protein in the chagasic heart. F and G, Control (F ) and infected (G ) sections stained with an anti–major
histocompatibility complex (MHC) II (Ia/Ie) antibody (red ), showing the presence of MHC-II–expressing cells in the inflammatory infiltrate of chagasic
hearts. All sections were stained with 4,6-diamidino-2-phenylindole for nuclear visualization (blue).
Table 3. Selected Up-regulated (15-Fold) Genes
Gene name
Symbol
Cytokine-related genes
Chemokine (C-C motif) ligand 2
Fold regulation
Ccl2
26.5
Ccl7/MCP3
Ccl8
Ccr5
Cxcl12/SDF1
16.2
50.6
12.1
5.0
IFN-g–induced GTPase
IFN-g–inducible protein 30
Igtp
Ifi30
12.4
11.9
IFN-g–inducible protein 47
IFN regulatory factor 1
Ifi47
Irf1
11.1
7.7
IFN regulatory factor 5
IL-10 receptor, a chain
Irf5
IL-10ra
11.1
7.9
IL-18 binding protein
IL-4 receptor, a chain precursor
LPS-induced TNF
TNF-a–induced protein 2
IL-18bp
IL-4Ra
Litaf
Tnfaip2
6.6
9.2
9.0
6.2
TNF receptor superfamily, member 1b
TNF-a–induced protein 8–like
Tnfrsf1b
Tnf p8l
9.4
8.9
Immune response–related genes
CD38 antigen
CD52 antigen
CD68 antigen
Cd38
Cd52/B7
Cd68
7.0
21.6
8.9
Complement component 4B
Complement factor B
C4b
Cfb
6.0
42.5
Fc receptor, IgE, high affinity I, gamma polypeptide
Fc receptor, IgG, low affinity III
Histocompatibility 2, class II antigen A, a
Histocompatibility 2, class II, locus Mb1
Fcer1g
Fcgr3
H2-Aa
H2-DMb1
17.6
9.1
38.5
12.7
Histocompatibility 2, Q region locus 7
Histocompatibility 2, T region locus 10
Semaphorin 4A
T cell–specific GTPase
H2-Q7
H2-T10
SemaA4
Tgtp
23.7
5.0
8.9
5.7
Tcirg1, TIRC7
9.4
Tlr2
5.4
Galectin-3
Integrin b3
Integrin b1–binding protein 3
Gal3
Itgb3
Itgbp3
36.9
6.0
36.2
Intercellular adhesion molecule
Enzymes
Icam-1/Mala2
6.7
Cathepsin C
Cathepsin H
Ctsc
Ctsh
12.5
8.1
Cathepsin S
Cathepsin Z
Ctss
Ctsz
47.5
8.3
Lysozyme 1
Lysoyme 2
Lyz1
Lys2
8.7
7.0
Lox
Pla2g7
5.3
47.6
Chemokine
Chemokine
Chemokine
Chemokine
(C-C motif) ligand 7
(C-C motif) ligand 8
(C-C motif) receptor 5
(C-X-C motif) ligand 12
T cell, immune regulator 1, ATPase, H+ transporting, lysosomal
V0 protein A3
Toll-like receptor 2
Cell adhesion
Lysyl oxidase
Phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma)
Proteasome (prosome, macropain) subunit, b type 10
Proteasome (prosome, macropain) subunit, b type 8 (large multifunctional peptidase 7)
Psmb10
Psmb8
Matrix metalloproteinase 14
Mmp14
6.56
8.1
10.8
Table 3.
(Continued.)
Gene name
Symbol
Fold regulation
a3 Type IX collagen
ECM protein 1
Col9a3
Ecm1
7.3
5.5
Microfibrillar-associated protein 5
Procollagen, type I, a2
TIMP-1
TGF-b induced
Mfap5
Col1a2
Timp1
Tgfbi
8.0
6.0
49.6
15.4
ECM-related genes
NOTE. ECM, extracellular matrix; IFN, interferon; Ig, immunoglobulin; IL, interleukin; LPS, lipopolysaccharide; TGF,
transforming growth factor; TIMP, tissue inhibitor of metalloproteinase; TNF, tumor necrosis factor.
DISCUSSION
The factors responsible for the establishment of the symptomatic form of chronic Chagas heart disease are still not fully
understood. However, it is likely that the damage sustained by
the myocardium is derived from parasite as well as host factors
[7]. Here we have identified, by microarray analysis, a number
of genes up-regulated in hearts of chronically chagasic mice
that probably play a role in modulating inflammation and fibrosis in this phase of infection and thus may represent targets
for therapeutic intervention in this disease.
A significant proportion of cells in the inflammatory infiltrate
found in hearts of chronically chagasic mice are macrophages,
as shown here by the expression of CD11b and up-regulation
of CD68 gene expression. These cells are found in close contact
with myofibers and may directly contribute to their damage
through the secretion of TNF-a. In addition, macrophages are
in close contact with T lymphocytes in the inflammatory foci
presenting antigens by MHC II molecules to CD4+ T lymphocytes, which secrete IFN-g, increasing the cytotoxic potential
of macrophages as well as of CD8+ T cells present in the inflammatory foci. Interestingly, we found expression of the Tolllike receptor 2 gene (Tlr2) up-regulated in hearts of chronically
chagasic mice. T. cruzi molecules, such as glycosylphosphatidylinositol anchors and glycoinositolphospholipids, activate
macrophages to produce interleukin 12, TNF-a, and nitric oxide [16]. Thus, the residual parasitism found in the chronic
infection probably contributes directly to the maintenance of
TNF-a levels and indirectly to the maintenance of IFN-g levels
(through interleukin 12 production) in the hearts of chronically
chagasic mice.
Although a growing body of evidence indicates that TNF-a
contributes to the pathogenesis of heart failure [17], other reports have suggested beneficial effects of this cytokine in the
heart [18]. Cardiac myocytes express both TNF-a receptors, type
1 (TNFR1) and type 2 (TNFR2) [19], which mediate its functions. TNFR1 seems to mediate the majority of the deleterious
effects of TNF-a, such as TNF-a–induced cell death [20]. In
contrast, activation of TNFR2 appears to exert protective effects
against cardiac myocyte damage and apoptosis [21–23]. The
strong up-regulation of the TNFR2 gene (Tnfrsf1b) in the hearts
of chronically chagasic mice indicates that this receptor may
contribute to the low number of apoptotic cardiac myocytes
found during this phase of infection [24].
PLA2G7, another molecule secreted by monocytes and macrophages and found to be up-regulated in our study, degrades
PAF, a lipid mediator that activates various cell types and promotes inflammation. Mice lacking PAF receptor (PAFR) have
increased inflammation and parasitism in their hearts during
acute infection [25]. This may be due to decreased parasite
uptake and macrophage activation in the absence of PAFR activation, because PAF has been shown to mediate nitric oxide
production and resistance to T. cruzi infection in mice [26]. In
our model of chronic chagasic myocarditis, the role of PAF
degradation by PLA2G7 is unknown, but the reduction in PAF
accumulation may be related to the progressive damage of the
myocardium, because PAF was shown to have a cardioprotective effect in isolated hearts [27].
A number of molecules involved in the recruitment of inflammatory cells to the heart of chagasic mice, including adhesion molecules and chemoattractant factors, were found to
be up-regulated in our study. ICAM-1 expression in heart and
endothelial cells was also increased in chagasic hearts, as described elsewhere [28, 29]. TNF-a increases the adhesiveness
of endothelium for leukocytes and induces ICAM-1 expression
[30]. Thus, the proinflammatory cytokines produced at the
inflamed heart may be promoting the maintenance of inflammation by increasing the expression of ICAM-1. In agreement
with the present study, overexpression of galectin-3 has been
reported in T. cruzi–infected mice [31]. Galectin-3 binds to
extracellular matrix components and was shown to participate
in the adhesion of the parasite to coronary artery smooth muscle cells [32].
In the present study we found that chemokine genes encoding for CCL2, CCL8, and CCL7 (monocyte chemoattractant protein [MCP] 1, 2, and 3, respectively) are up-regulated in hearts
of chronically chagasic mice. A number of studies have shown
Figure 4. Increased production of stromal cell–derived factor 1 (SDF-1), interferon (IFN) g, and tumor necrosis factor (TNF) a and transcript levels
of Timp1 in hearts of chronically chagasic mice. A–C, Levels of SDF-1 (A), IFN-g (B ), and TNF-a (C ) identified in heart homogenates of normal mice
(n p 4) and chagasic mice (n p 9; 8 months after infection with Trypanosoma cruzi ), by enzyme-linked immunosorbent assay. *P ! .05 and **P !
.01. D, Timp1 analyzed by quantitative real-time reverse-transcription polymerase chain reaction, using complementary DNA samples prepared from
messenger RNA extracted from the hearts of normal (n p 5 ) or chronically chagasic (n p 5 ) mice. Data represent means standard errors of the
mean for values obtained from individual mice.
that T. cruzi infection stimulates the production of chemokines
by macrophages as well as by cardiomyocytes [33–35]. These
chemokines are known to recruit monocytes and T lymphocytes.
Other studies have demonstrated an association between the expression of MCP-1 and MCP-2 in the heart and both myocarditis
and heart dysfunction [36, 37], suggesting a role of these cytokines in the maintenance of chagasic myocarditis.
CCR5 is a receptor for several chemokines of the CC family,
including CCL3, CCL4, and CCL5, known to be up-regulated
by infection with the Colombian strain of T. cruzi [38], and
also for CCL8. Hearts of CCR5-deficient mice infected with T.
cruzi have reduced migration of T cells. Because this receptor
is predominantly expressed on the surface of Th1 cells [39],
and a type 1 response with production of IFN-g is associated
with severity of CCM, CCR5 may play an important role in
the pathogenesis of chronic chagasic myocarditis, as described
in a model of autoimmune myocarditis [40]. In fact, treatment
of chagasic mice with a selective CCR1 and CCR5 antagonist
(Met-RANTES) decreased heart inflammation and fibrosis [41].
A positive correlation between severity of cardiomyopathy and
the presence of CCR5+ IFN-g+ T cells was found in patients
with chronic Chagas disease [14].
We found that CXCL12 (SDF-1) expression is increased in
hearts of chronically chagasic mice. SDF-1 is a potent chemoattractant factor for lymphocytes [42], and therefore its expression may be relevant for the maintenance of immune-mediated heart destruction during the chronic phase of infection.
Conversely, because this chemokine is also a stem cell recruitment factor, its increased expression may contribute to tissue
regeneration of the damaged myocardium, as reported elsewhere in a model of myocardial ischemia [43]. In addition,
MCP-3 (CCL7) was recently shown to be a mesenchymal stem
cell homing factor for the myocardium [44]. Thus, the increased expression of these chemokines indicates that migration
of stem cells can be promoted in chronic chagasic myocarditis
by the presence of stem cell chemoattractant factors such as
SDF-1 and MCP-3. In fact, we have shown that intravenously
injected bone marrow cells migrate to the hearts of chronically
chagasic mice [45].
The myocardial interstitial collagen matrix surrounds and supports cardiac myocytes and the coronary microcirculation, and
its integrity is critical for the proper function of the heart. Thus,
alterations in the collagen matrix will disrupt myocardial mechanical properties and ventricular function [46]. In Chagas dis-
ease, as a consequence of the sustained inflammatory process
found in the myocardium during the chronic phase of infection,
fibrosis is evident and contributes to cardiac remodeling. We also
observed alterations in genes related to extracellular matrix deposition, such as extracellular matrix components and Timp1.
Plasma concentrations of TIMP-1 are significantly elevated in
patients with terminal heart failure compared with healthy controls [47], suggesting that this metalloproteinase inhibitor may
also play a role in the evolution of heart failure in chronic Chagas
disease. In addition, lysyl oxidase was increased in chagasic hearts.
This enzyme promotes the cross-linking of collagen fibers, irreversibly altering the structure and function of the extracellular
matrix proteins, causing dysfunction of the cardiomyocytes and,
consequently, of the heart [46]; it therefore probably plays an
important role in the evolution of fibrosis in chronic chagasic
hearts.
To understand the delicate balance of multiple factors involved in the pathogenesis of Chagas disease is a complex task.
Microarray approaches have been used before in mouse models
of T. cruzi infection (C3H/HeN mice infected with T. cruzi
Sylvio X10/4 strain [47] and C57Bl/6.129sv mice infected with
T. cruzi Brazil strain [48, 49], as well as in hearts of chronically
chagasic patients [50]. Using the model of infection with Colombian T. cruzi strain, we have identified new potentially important genes that may serve as a basis for therapeutic interventions in chronic Chagas heart disease.
Acknowledgments
We thank Carine Machado and Fabiola Encinas Rosa for technical
assistance.
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Epilepsia, 51(8):1628–1632, 2010
doi: 10.1111/j.1528-1167.2010.02570.x
BRIEF COMMUNICATION
Distribution and proliferation of bone marrow cells in the
brain after pilocarpine-induced status epilepticus in mice
*y1Beatriz Longo, y1Simone Romariz, yMiriam Marcela Blanco, *Juliana Fraga Vasconcelos,
*Luciana Bahia, *zMilena Botelho Pereira Soares, yLuiz E. Mello, and
*zRicardo Ribeiro-dos-Santos
*Laboratório de Engenharia Tecidual e Imunofarmacologia, CPqGM/FIOCRUZ, Bahia, Brazil; yLaboratório de Neurofisiologia,
Departamento de Fisiologia, UNIFESP, São Paulo, Brazil; and zCentro de Biotecnologia e Terapia Celular,
Hospital São Rafael, Salvador, BA, Brazil
SUMMARY
The distribution of bone marrow cells in brain areas during the acute period after pilocarpine-induced status epilepticus (SE) was investigated here. To achieve this, we
generated chimeric mice by engrafting bone marrow cells
from enhanced green fluorescent protein (eGFP) transgenic mice. GFP+ bone marrow–derived cells were found
throughout the brain, predominantly in the hippocampus.
The occurrence of immunologic dysfunction after seizures has been widely demonstrated (Bernardino et al.,
2005; Fabene et al., 2008). Studies from human and experimental epilepsy indicate that after status epilepticus (SE) or
epileptic seizures inflammatory cells within the bone marrow cell population have been shown to infiltrate and proliferate in the brain parenchyma, which suggests alterations in
immunologic function. Bone marrow–derived cells in the
central nervous system (CNS) contribute mostly to the generation of microglia, and the number of microglia that originate from bone marrow increases dramatically after brain
damage at the site of injury (Priller et al., 2001; Simard &
Rivest, 2004).
Because bone marrow is an accessible source of progenitor cells, the use of these cells has been speculated to treat
neurologic diseases in a number of clinical trials. Therefore,
the use of bone marrow cell as a possible treatment for neurologic disorders should be carefully evaluated. It is critical
to examine first how endogenous bone marrow cells behave
and function after the SE. In this study we investigated the
Accepted February 19, 2010; Early View publication April 8, 2010.
Address correspondence to Ricardo Ribeiro dos Santos, Centro de Biotecnologia e Terapia Celular, Hospital S¼o Rafael, Av. S¼o Marcos, 2152,
S¼o Marcos, CEP: 41.253-190, Salvador- Bahia, Brazil. E-mail: ricardo.
[email protected]
1
Longo and Romariz contributed equally to this work.
Wiley Periodicals, Inc.
ª 2010 International League Against Epilepsy
As expected, these cells exhibited the characteristics of
microglia. The pattern of distribution, proliferation, and
differentiation of GFP+ cells changes as a function of intensity and time following SE. This pattern is also a consequence of the inflammatory response, which is followed
by the progressive neuronal damage that is characteristic
of the pilocarpine model.
KEY WORDS: Hippocampus, Chimeric mice, Microglia,
Status epilepticus, GFP.
migration, proliferation, and microglial nature of bone marrow–derived cells present in the brain at various time points
after pilocarpine-induced SE in chimeric mice that were
engrafted with bone marrow cells expressing green fluorescent protein (GFP).
Methods
We transplanted bone marrow from the eGFP transgenic
mice into lethally irradiated adult male C57Bl/6 wild-type
mice (n = 59). Bone marrow cells were obtained from adult
eGFP-donor mice by flushing the femurs and tibiae.
Approximately 3 · 107 cells were administered into each
irradiated animal. One month after transplantation, a group
of chimeras (n = 44) was injected with pilocarpine
(280 mg/kg) to induce SE. Behavioral data were collected
during the SE, and the seizures were classified according to
a modified version of the Racine scale (Shibley & Smith,
2002). The animals were deeply anesthetized and perfused
with 4% paraformaldehyde at the following time points
post-SE: 2 h (n = 6), 24 h (n = 6), 48 h (n = 6), 72 h
(n = 6), 96 h (n = 6), 7 days (n = 7), or 15 days (n = 7).
Before the perfusion, the animals from the 24 h, 7 day, and
15 day groups were administered 5-bromo-2¢-deoxyuridine-5¢-monophosphate (BrdU, 50 mg/kg) once a day
beginning at 30 min of SE onset and ending 2 h before the
perfusion. Control chimeric animals (n = 15) were injected
with saline and also with BrdU. The brains were removed
1628
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Status Epilepticus Induces Migration of Bone Marrow Cells into the Brain
and processed for immunofluorescence. Sections were incubated with anti-GFP AlexaFluor 488–conjugated (1:600),
anti-BrdU (1:500), anti-NeuN (1:200), anti-GFAP (1:200),
and anti-Iba1 (1:2,000) antibodies. The sections were then
conjugated to the fluorochromes and analyzed using fluorescence and confocal microscopes.
GFP+ cells were counted in 10 random, nonoverlapping
fields for all of the time points post-SE (2 h, 24 h, 48 h,
72 h, and 96 h, 7 days, and 15 days) and controls. The same
counting protocol was used to estimate the percentage of
BrdU+ cells and double-stained GFP+/BrdU+, /Iba1+,
/NeuN+ and /GFAP+ cells in the control, 24-h, 7-d, and
15-d, groups. Cells were counted in six selected brain areas:
neocortex, piriform cortex, hippocampus, thalamus, subventricular zone in the lateral ventricle wall (SVZ), and choroid plexus. Analysis of variance (ANOVA) followed by
Tukey-Kramer or Mann-Whitney post hoc tests were used
for statistical analysis.
Results
Although there was a low number of GFP+ cells in the
control chimeric animals, a significant increase of the GFP+
cells was observed in the brain after SE (p < 0.05). The
number of these cells gradually increased at different time
points after SE (2 h, 24 h, 48 h, 72 h, 96 h, 7 days, and
15 days) (p < 0.001). The GFP+ cells began increasing in
frequency by 48 h and continued increasing until 15 days
after SE, which was the last time point examined (supporting information). The hippocampus had the largest number
of GFP+ cells, followed by the choroid plexus and thalamus
(p < 0.001). The GFP+ cells were concentrated in the hilus,
CA1, and CA3. In addition, more GFP+ cell clusters were
observed in caudal levels of the ventral hippocampus than
in the dorsal hippocampus. The number of bone marrow–
derived GFP+ cells in the brain was also influenced by the
severity of SE based on a Racine modified scale. Fifty-four
percent (precisely 54.3%) of the total number of GFP+ cells
was found in animals with the highest scores (4 and 5) and
25.5% was present in animals with a score of 3. Scores 1
and 2 were excluded from this analysis.
By counting the number of GFP+/BrdU+ and GFP)/
BrdU+ (only BrdU) cells in the control and SE animals at
24 h, 7 days, and 15 days after SE, we evaluated bone
marrow cells and endogenous cell proliferation, respectively. High levels of GFP)/BrdU+ cells were present in the
A
B
C
D
E
F
G
H
I
Figure 1.
GFP+ and BrdU+ stained cells in the hippocampus (CA1) of chimeric animals. Images show GFP+ cells in green, BrdU+ cells in red, and
double-stained GFP+/BrdU+ cells as merge images (orange) at 24 h (A–C), 7 days (D–F), and 15 days (G–I) after status epilepticus
(SE). Squares in DAPI images of the hippocampus indicate the level of GFP+/BrdU+ cells in CA1. Dashed lines indicate the CA1 pyramidal cell layer. Magnification of 20· in A-I and 2· for DAPI.
Epilepsia ILAE
Epilepsia, 51(8):1628–1632, 2010
doi: 10.1111/j.1528-1167.2010.02570.x
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B. Longo et al.
A
B
C
D
E
F
G
H
I
Figure 2.
GFP+ (green) in Iba1+ (red) in cell population in the hippocampus (CA3) of chimeric animals at 24 h (A–C), 7 days (D–F), and 15 days
(G–I) after status epilepticus (SE) (in A: detail of a control animal with very few GFP+ cells). Double-stained cells (Iba1+/GFP+) are
indicated in merge images (arrows). Cells were counterstained with DAPI (blue) for nuclear staining. Magnification of 20·.
Epilepsia ILAE
hippocampus, SVZ, and choroid plexus of SE mice at
7 days, but they decreased at 15 days (p < 0.001). In the
hippocampus, no GFP+/BrdU+ cells were present in the control group, very few were present in the 24 h group, and the
number of cells increased approximately 10-fold by days 7
and 15 (Fig. 1). Despite this increase, the GFP+/BrdU+ cells
represented a small proportion (approximately one-third) of
the total number of GFP+ cells. By 24 h after SE, the GFP+/
BrdU+ cells represented 12.8% of the total. By days 7 and
15, the GFP+/BrdU+ cells represented 28.5% and 23.7% of
the total, respectively, which indicates a delay in proliferation after bone marrow GFP+ cell migration.
Qualitative immunofluorescence analyses to evaluate
whether the GFP+ cells expressed microglia (Iba1), astrocyte (GFAP), or neuronal (NeuN) markers indicated that
endogenous microglia expressing Iba1 were detected in the
controls and in the SE animals. In the controls, no co-localization of Iba1+ with GFP+ cells was observed. However,
subsequent to SE, the percentage of double-stained GFP+/
Iba1+ cells increased over time, and coincided with the temporal pattern of bone marrow–derived GFP+ cell migration
and proliferation. At later time points, the majority of the
Epilepsia, 51(8):1628–1632, 2010
doi: 10.1111/j.1528-1167.2010.02570.x
GFP+ cells observed in the epileptic groups expressed Iba1
(Fig. 2). The percentages of GFP+/Iba1+ within the Iba1+
population at 24 h, 7 days, and 15 days were 2.8%, 23.1%,
and 44.7%, respectively. The same distribution pattern of
microglia and co-localized GFP+ cells was observed in the
hippocampus (0.3%, 8.9%, and 24.5%, respectively). We
did not observe GFP+ cells developing astrocytic or neuronal phenotypes, which would have been indicated by double-staining for GFP+ and GFAP+ or NeuN+.
Correlation analyses were performed between the time
after SE and the presence of GFP+, BrdU+, and Iba1+ cells
in the hippocampus. A significant correlation was noted
between GFP+ cells and time after SE. The number of GFP+
cells as well as double-stained GFP+/BrdU+ and Iba+/GFP+
cells in the hippocampus increased as time after SE
increased. Significant correlations were also observed
between SE intensity and the number of GFP+ and GFP+/
BrdU+ cells. The more severe the SE, the greater the number
of GFP+ and GFP+/BrdU+ cells. A negative correlation was
found between the time after SE and Iba1+; as time passed,
the number of Iba1+ stained cells decreased (supporting
information).
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Status Epilepticus Induces Migration of Bone Marrow Cells into the Brain
Discussion
The gradual migration of bone marrow–derived cells in
the chimeric brain began immediately after SE induction
and continued for at least 15 days, whereas the proliferation
of these cells and their microglial phenotype began later
(7 days and 15 days). At all times after SE the majority of
GFP+ cells concentrated in the hippocampus. This preferential concentration of bone marrow–derived cells may result
from the type of seizure induced by the pilocarpine model,
which damages the hippocampus predominantly (Mello
et al., 1993).
Interestingly, the majority of bone marrow–derived cells
present in the brain exhibited the characteristics of microglia during the first and second weeks following SE,
although the increase did not occur in the first 24 h. Endogenous microglia were already numerous a few hours after SE
(2–24 h) but they were not derived from the transplanted
bone marrow (GFP)). The bone marrow–derived cells
expressing the microglial marker (GFP+/Iba1+) appeared
7 days and 15 days following SE. These findings suggest a
biphasic nature of microglia according to which microglia
already present at the inflammation site are recruited during
the first 24 h after SE, followed by the migration of bone
marrow–derived microglia to the damaged tissue during the
first and second weeks after SE. Recent studies have shown
that bone marrow–derived and CNS resident microglia
cells possess different properties and kinetics (Napoli &
Neumann, 2009). Moreover, articles reviewing microglial
function in the CNS have proposed that microglia serve a
dual role in neurogenesis and inflammation depending upon
the balance between secreted molecules with pro- and antiinflammatory action and on the physiologic situation
(Monje et al., 2003; Ekdahl et al., 2009). There is evidence
of both protective (Albensi, 2001) and toxic (Vezzani et al.,
2000; Borges et al., 2004) roles of the inflammatory
response elicited following seizures to control the patterns
of neuronal loss and replacement associated with seizures.
In summary, the temporal migration, distribution, and
proliferation patterns of bone marrow–derived cells in the
brain after SE were modified as part of the inflammatory
response that occurs following progressive brain injury
characteristic to this model. Whether these bone marrow–
derived cells have degenerative or regenerative effects on
seizure-induced damage is still unclear. Our study helped
clarify some important issues regarding bone marrow–
derived cells in seizure and may contribute to develop novel
and reliable cell-based therapies in the future.
Acknowledgments
We apologize to all whose works were not cited due to space limitations.
We are grateful to Jaqueline Noronha, Maria Fernanda Valente, Enas Ferrazolli, and Thomas Perlaky for the technical support; Yiwei Jia and Willy
Hausner for the help with confocal images; and Dr. Daniel Peterson for the
critical comments. This work was supported by FAPESB, FAPESP, CNPq,
and FIOCRUZ.
Disclosure
We confirm that we have read the Journal’s position on issues involved
in ethical publication and affirm that this report is consistent with those
guidelines. None of the authors has any conflict of interest to disclose.
References
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Bernardino L, Ferreira R, Cristovao AJ, Sales F, Malva JO. (2005) Inflammation and neurogenesis in temporal lobe epilepsy. Curr Drug Targets
CNS Neurol Disord 4:349–360.
Borges K, McDermott D, Dingledine R. (2004) Reciprocal changes of
CD44 and GAP-43 expression in the dentate gyrus inner molecular
layer after status epilepticus in mice. Exp Neurol 188:1–10.
Ekdahl CT, Kokaia Z, Lindvall O. (2009) Brain inflammation and adult
neurogenesis: the dual role of microglia. Neuroscience 158:1021–1029.
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Bach S, Angiari S, Benati D, Chakir A, Zanetti L, Schio F, Osculati A,
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Osculati F, Sbarbati A, Butcher EC, Constantin G. (2008) A role for leukocyte-endothelial adhesion mechanisms in epilepsy. Nat Med
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Shibley H, Smith B. (2002) Pilocarpine-induced status epilepticus results in
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on intracerebral injection and astrocytic overexpression in mice. Proc
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Supporting Information
Additional supporting information may be found in the
online version of this article:
Table S1. Listing of correlations.
Figure S1. Quantitative assessment of green fluorescent
protein–positive (GFP+) cells in the brain of chimeric mice.
The increasing number of GFP+ cells in status epilepticus
(SE) (as black square) at each time point along 15 days after
SE—2 h (n = 6), 24 h (n = 6), 48 h (n = 6), 72 h (n = 6),
96 h (n = 6), 7 days (n = 7), or 15 days (n = 7)—was compared to the control (n = 15), which is represented as an
opened square (*p < 0.001 vs. control group).
Epilepsia, 51(8):1628–1632, 2010
doi: 10.1111/j.1528-1167.2010.02570.x
1632
B. Longo et al.
Figure S2. Distribution of degenerating neurons after status epilepticus (SE). Fluoro-Jade B–positive neurons in the
neocortex (A, D), pyramidal layer of CA1 in the hippocampus (B, E), and thalamus (C, F) of respectively 8 h, 7 days,
and 15 days after SE (magnification of 20· in A, B, and C
and 40· in D, E, and F).
Figure S3. Expression of glial and neuronal markers
7 days after status epilepticus (SE). Images show green
fluorescent protein–positive (GFP+) cells (green) counterstained with 4¢,6-diamidino-2-phenylindole (DAPI) (blue)
for the nuclear staining (A, C, D, F, G, and I) in the neocortex. CD11b+ (B and C), GFAP+ (E and F), and NeuN+ (H
and I) cells were stained in red. In C, F, and I images were
Epilepsia, 51(8):1628–1632, 2010
doi: 10.1111/j.1528-1167.2010.02570.x
merged. Arrows indicated GFP+CD11b+ cells in C. Magnification of 20·.
Figure S4. Confocal image of BrdU (red) and green fluorescent protein (GFP) (green) merged cells in the hippocampal area 7 days after status epilepticus (SE). Note microglial
morphology of donor-derived GFP+/BrdU labeled cells.
(magnification of 60·, Olympus FV100).
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the
article.
J. Nat. Prod. XXXX, xxx, 000
A
Activity of Physalin F in a Collagen-Induced Arthritis Model
Daniele Brustolim,† Juliana F. Vasconcelos,† Luiz Antônio R. Freitas,† Mauro M. Teixeira,‡ Marcel T. Farias,†
Yvone M. Ribeiro,§ Therezinha C. B. Tomassini,§ Geraldo G. S. Oliveira,† Lain C. Pontes-de-Carvalho,†
Ricardo Ribeiro-dos-Santos,†,⊥ and Milena B. P. Soares*,†,⊥
Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, SalVador, BA, Brazil, UniVersidade Federal de Minas Gerais, Belo Horizonte,
MG, Brazil, FarManguinhos, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil, and Hospital São Rafael, SalVador, BA, Brazil
ReceiVed October 28, 2009
The effects of physalin F (1), a steroid derivative purified from Physalis angulata, were investigated in models of
collagen-induced arthritis in DBA/1 mice and allergic airway inflammation in BALB/c mice. Oral treatment with 1 or
dexamethasone caused a marked decrease in paw edema and joint inflammation when compared to vehicle-treated
arthritic mice. In contrast, treatment with 1 had no effect in mice with allergic airway inflammation caused by ovalbumin
immunization, whereas dexamethasone significantly reduced the number of inflammatory cells and eosinophils in the
broncoalveolar lavage fluid and in lung sections of challenged mice. To further demonstrate that 1 acts through a
mechanism different from that of glucocorticoids, a nuclear translocation assay was performed of the glucocorticoid
receptor (GR) using COS-7 cells transfected with a plasmid encoding for a yellow fluorescent protein (YFP)-GR fusion
protein. Untreated or treated cells with 1 had YFP staining mainly in the cytoplasm, whereas in dexamethasone-treated
cells the YFP staining was concentrated in the nuclei. It is concluded that the mechanism of the immunosuppressive
activity of physalin F is distinct from that of the glucocorticoids.
Immunosuppressive substances are used widely to inhibit
undesired immune responses in autoimmune and allergic diseases,
such as rheumatoid arthritis and asthma. Although several immunosuppressive drugs are currently available, their prolonged use is
usually accompanied by undesired effects. An extensively utilized
class of immunosuppressive agents is comprised by the glucocorticoids, which possess undesirable side effects, mainly associated
with chronic use, such atherosclerosis, cataracts, fatty liver, growth
retardation, osteoporosis, and skin atrophy. In addition, other side
effects are common in patients with underlying risk factors, such
as acne vulgaris, diabetes, hypertension, and peptic ulcers.1 Glucocorticoids act via glucocorticoid receptor (GR) activation, which
mediates transactivation or transrepression of several genes, which
explains their anti-inflammatory effect and, possibly, their adverse
effects.2 Thus, the development of new immunosuppressors with
different mechanisms of action and reduced toxic effects is of great
interest.
Plant secondary metabolites are important for flavoring of food,
for their resistance against pests, and as drugs, including substances
having immunosuppressive activity.3,4 Examples of natural immunosuppressive compounds are lupeol and umbelliferone, which
inhibit allergic airway inflammation in a mouse model.5,6 Ursolic
acid7 and alkaloids from Radix linderae,8 the dry roots of Lindera
aggregata (Sims) Kosterm., possess antiarthritic effects, as shown
in experimental models of arthritis.
Physalis angulata L. (Solanaceae) is an herb widely used in
popular medicine as an analgesic and antirheumatic and to treat
sore throats and abdominal pain. It is considered as an antidiuretic,
an anti-inflammatory for hepatitis and cervicitis, an antinociceptive,
and an antipyretic.9,10 Physalins are modified steroids isolated from
Physalis species with suppressive activities on macrophage and
lymphocyte cultures in vitro and inhibit the production of proinflammatory mediators such as TNF.11,12 These molecules also
prevent mortality induced by a lethal injection of lipopolysaccharide
* Corresponding author. Tel: +55 71 3176 2260. Fax: +55 71 3176
2272. E-mail: [email protected].
†
Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz,
Salvador.
‡
Universidade Federal de Minas Gerais, Belo Horizonte.
§
FarManguinhos, Fundação Oswaldo Cruz, Rio de Janeiro.
⊥
Hospital São Rafael, Salvador.
10.1021/np900691w
(LPS)11 and inhibit rejection of allogeneic transplants in mice.12
In addition, physalins have other potent anti-inflammatory activities,
decreasing injuries following intestinal ischemia and reperfusion
in mice.13 This activity of physalins in the intestinal ischemia and
reperfusion model, but not in in vitro assays, was blocked by the
GR antagonist RU486.11-13 In the present study, the effects of
physalin F (1) in a collagen-induced arthritis and allergic airway
inflammation model in mice were determined and compared to the
effects of dexamethasone, a gold standard glucocorticoid.
Results and Discussion
To test the effects of physalin F (1) in a model of collageninduced arthritis, mice were treated by the oral route after the onset
of paw edema. A significant decrease in paw edema was observed
in mice 20 days after being treated with 1 (Figure 1). Mice treated
with a standard drug, dexamethasone, exhibited a significant
reduction in paw edema compared to vehicle-treated mice starting
10 days after treatment (Figure 1). Twenty days after treatment,
the macroscopic evaluation of paws from vehicle-treated mice
showed swelling of the footpad and toes, whereas paws from 1and dexamethasone-treated mice appeared normal (Figure S1,
Supporting Information).
Histological evaluation performed 20 days after treatment showed
an inflammatory infiltrate of neutrophils into the articular space
and in the synovial tissue, with the formation of granulation tissue
and the presence of fibrin, as well as edema and congestion of
vessels, in paws of all vehicle-treated mice (Figure S1, Supporting
Information). In the group treated with 1, one mouse developed
 XXXX American Chemical Society and American Society of Pharmacognosy
B
Journal of Natural Products, XXXX, Vol. xxx, No. xx
Figure 1. Treatment with physalin F (1) in mice with collageninduced arthritis (CIA). Measurement of paw volume of DBA/1
mice with CIA. Mice were immunized with type II collagen and
daily treated by oral route with vehicle, 1, or dexamethasone for
20 days. The paw volumes were measured every five days using a
plethysmometer. Values are expressed as means ( SEM of 6 or 7
mice per group, in one of two experiments performed. (*p < 0.05;
**p < 0.01; ***p < 0.001 compared to the control group).
synovial cell hyperplasia and the presence of fibrotic tissue and
the deposition of fibrin in the interarticular space. The other mice
in the group treated with 1 and all the mice treated with
dexamethasone had paws with intact joint space, normal synovial
tissue, and preserved cartilage (Figure S1, Supporting Information).
Previous work by our group has shown that 1 inhibits macrophage activation and nitric oxide production and prevents death
induced by a lethal dose of LPS.10 A similar effect was found with
physalin B, which also inhibited the secretion of pro-inflammatory
cytokines, such as TNF-R, which is a pivotal cytokine in collageninduced and rheumatoid arthritis.10 In addition, physalin F suppressed the activation and proliferation of T lymphocytes.11 Thus,
the beneficial effects of 1 in the model of collagen-induced arthritis
may be explained by its action in both macrophages and T
lymphocytes, which play important roles in the pathogenesis of
arthritis.
The effects of treatment with 1 in lung inflammation were
evaluated by comparing bronchoalveolar lavage fluid cytology of
mice treated with 1 to those of mice treated with vehicle. The total
number of cells (Figure 2B), the ratio of eosinophils per 200 cells
(Figure 2 A), and the absolute number of eosinophils (not shown)
in the bronchoalveolar lavage fluid were not reduced after treatment
with 1 compared to vehicle-treated mice. A significant inhibition
was observed in mice treated with dexamethasone. To further
characterize the changes in the lung caused by antigen challenge
of immunized mice, lung sections were examined after hematoxylin
and eosin staining. Ovalbumin challenge caused an intense cell
infiltrate containing many lymphocytes, macrophages, and eosinophils. Mice treated with 1 exhibited a discrete reduction in lung
inflammation, whereas dexamethasone had very potent inhibitory
effects (Figure 2C). Lung sections from control mice revealed
discrete alcian blue staining in the respiratory epithelium compared
to the saline-treated group. Treatment with 1 at 60 mg/kg was not
capable of modulating mucus production, since it did not decrease
the number of alcian blue+ cells in the lung compared to salinetreated mice (Figure 2D). The injection of the mice with allergic
airway inflammation with 60 mg/kg of 1 by the intraperitoneal route
(which leads to an amount of circulating drug that is higher than
that obtained by oral administration) also did not lead to any
protective effect (unpublished data).
The production of Th2 cytokines was quantified in the bronchoalveolar lavage fluid of individual mice from each group. As
expected, the concentrations of IL-4 and IL-13 were increased in
ovalbumin-immunized mice, when compared to naive mice. Treatment with 1 did not cause a reduction in the concentrations of Th2associated cytokines in BAL fluid, which were similar to those of
Brustolim et al.
the saline-treated group. In contrast, dexamethasone treatment
caused a marked reduction in IL-4 and IL-13 production (Figure
S2, Supporting Information).
Physalin B, another steroid isolated from P. angulata with
immunosuppressive activities, also did not decrease inflammation
in this model (data not shown). The lack of anti-inflammatory
activity by the physalins was associated with a lack of inhibition
of Th2-associated cytokines crucial for the development of this
pathological response. The treatment protocol consisted of five daily
oral doses of 60 mg/kg of 1, with each dose preceding the allergenic
challenge, a fact that should favor the unveiling of any beneficial
effect. This daily dose was indeed 3-fold higher than the daily dose
that effectively reduced joint inflammation. It seems, therefore, that,
while 1 has a potential therapeutic effect on experimental arthritis,
it does not have a significant effect on experimental respiratory
allergy, even following a protocol that would be better than the
one usually used to treat asthmatic patients with oral corticosteroids
(in which the drugs are usually given after the onset of the asthmatic
crisis). It is possible, therefore, that 1 has a preferential effect on
both innate, macrophage-mediated immune responses and on Th1mediated immune responses, when contrasted with corticosteroids,
which would also readily inhibit Th2-mediated immune responses.
Previous studies by our group have shown that the in vitro inhibitory
activities of physalins B and F on macrophage and lymphocyte
activation are not blocked by the glucocorticoid receptor antagonist
RU486.11,12 In contrast, RU486 administration in mice treated with
the physalins blocked their protective effects in the development of
ischemia and reperfusion injury in an experimental model.13 These
apparently conflicting results motivated the present investigation on
whether 1 activated the glucocorticoid receptor (GR), and a nuclear
translocation assay of GR was performed. COS-7 cells transfected with
a plasmid coding for a YFP-GR fusion protein had YFP staining
mainly in the cytoplasm (Figure 3A). Transfected cells treated with
dexamethasone exhibited YFP staining in the nuclei, indicating GR
activation and translocation (Figure 3B). In contrast, treatment with 1
in a concentration chosen in accordance to the biological activity in
vitro did not induce translocation of YFP-GR from the cytoplasm to
the nucleus (Figure 3C).
The inhibitory activity of RU486 on the effects of physalins on
ischemia and reperfusion injury13 can be explained by a possible
dependency of the physalins on endogenous corticoids to produce
those particular effects, so that the blockage of the glucocorticoid
receptor by RU486 would render the animals more susceptible. The
lack of action of physalins by activation of the glucocorticoid
receptor was confirmed in the present work by showing that 1 did
not suppress inflammation in the allergic airway inflammation
model, in which the glucocorticoid receptor is known to act. In
addition, 1 failed to induce the nuclear translocation of the
glucocorticoid receptor in vitro, as described above, which is
required for its action as a transcription factor.
A recent report has shown that physalins B and F isolated from
Witheringia solanacea inhibit the activation of NF-κB, a key
transcription factor regulating immune-inflammatory responses.14
Vandenberghe et al. suggested that physalin B acts as an inhibitor
of the ubiquitin-proteasome pathway.15 These authors showed that
this inhibition may produce an accumulation of ubiquitinated
proteins and consequently inhibit NF-κB activation induced by
TNF.15 Thereby it is possible that the immunosuppressive effects
of physalins B and F are at least partly dependent on this mechanism
of action.
The fact that physalins B and F exert their inhibitory action by
a different mechanism from that of glucocorticoids may be of
relevance, since the long-term use of glucocorticoids in chronic
diseases such as reumathoid arthritis has several undesired side
effects that perhaps 1 may lack.1 The present results also reinforce
the importance of screening natural products with the particular
aim of developing new immunomodulatory agents.
Antiarthritis ActiVity of Physalin F
Journal of Natural Products, XXXX, Vol. xxx, No. xx C
Figure 2. Effects of physalin F (1) treatment in mice with allergic airway inflammation. Mice were sacrificed 24 h after the last challenge
with ovalbumin. The cellularity in BAL fluid and lung morphometric analysis from naive or ovalbumin-challenged mice treated with
saline, dexamethasone (Dexa), or 1 were evaluated. (A) Number of eosinophils in 200 cells. (B) Total cell counts. (C) Intensity of inflammation
on hematoxylin- and eosin-stained lung sections. (D) Analysis of mucus production on alcian blue-stained lung sections. Values are expressed
as means ( SEM of 6 or 7 mice per group, in one of two experiments performed (*p < 0.05; ***p < 0.001 compared to the control group).
Figure 3. Physalin F (1) does not activate the nuclear translocation
of the glucocorticoid receptor. Nuclear translocation assay of
glucocorticoid receptor was performed in COS-7 cells transfected
with a plasmid coding for an YFP-GRR fusion protein. Transfected
cell cultures untreated (A) or treated with dexamethasone (B) or 1
(C) were observed in a confocal microscope to analyze the
YFP-GRR staining (yellow). Nuclei were stained with DAPI
(blue).
Experimental Section
General Experimental Procedures. Male, 9- to 11-week-old DBA/1
mice were used for induction of collagen-induced arthritis. Male
BALB/c mice, 4- to 6-weeks old, were used in the allergic airway
inflammation study. All mice were raised and maintained at the animal
facilities at Gonçalo Moniz Research Center, FIOCRUZ (Salvador,
Brazil), in rooms with controlled temperature (22 ( 2 °C) and humidity
(55 ( 10%) and continuous air renovation. Animals were housed in a
12 h light/12 h dark cycle (6 A.M. to 6 P.M.) and provided with rodent
diet and water ad libitum. Animals were handled according to the NIH
guidelines for animal experimentation. All procedures described herein
had prior approval from the local animal ethics committee.
Extraction and Isolation. Physalin F (1) was isolated from the stems
of Physalis angulata L. collected in December 2006 in Belém do Pará,
Brazil. The plant was identified by Dr. Lucia Carvalho from the
Botanical Garden of Rio de Janeiro. A voucher specimen is held under
number RFA 23907/8 at the Federal University of Rio de Janeiro,
Brazil, as described previously.12 The procedure used for purification
used has been described.16,17 The purity of the compound was greater
than 97%. The sample of 1 was dissolved in dimethyl sulfoxide
(DMSO) and diluted in culture medium or saline for use in the assays
constituting the present study.
Induction of Collagen-Induced Arthritis and Treatment. Mice
were randomized into groups of six and immunized by intradermal
injection at the base of the tail with 100 µg/mouse of bovine type II
collagen (Chondrex, Redmond, WA), in complete Freund’s adjuvant
(CFA; Sigma, St Louis, MO). After an interval of 20 days, the animals
received another injection of 100 µg/mouse of collagen in CFA. The
mice were evaluated every five days by macroscopic observation and
measurement of the footpad volume using a plethysmometer (Ugo
Basile, Comerio, Italy). After the appearance of swelling in the joints,
approximately 10 days after the second injection of collagen, the animals
were treated daily by gavage with 20 mg/kg of 1, 2.5 mg/kg of
dexamethasone (Sigma), or vehicle (10% DMSO in saline), orally, for
20 days.
Sensitization and Challenge with Ovalbumin and Treatment.
Allergic airway inflammation was induced as described before.5 Groups
of seven mice received systemic immunization by subcutaneous
injection of 10 µg of chicken egg ovalbumin (Grade V, >98% pure;
Sigma, St Louis, MO) adsorbed on 50 µL of 2 mg/mL alum
(AlumImject; Pierce, Rockford, IL) followed by a booster injection at
day 14. A nasal challenge was performed starting at day 28, by
inhalational exposure to aerosolized ovalbumin for 15 min/day, on five
consecutive days. Exposures were carried out in an acrylic box. A
solution of 1% ovalbumin in saline was aerosolized by delivery of
compressed air to a sidestream jet nebulizer (Respiramax NS, São Paulo,
Brazil). Two hours before each aerosol delivery, mice were treated
orally with 1 (60 mg/kg), dexamethasone (30 mg/kg), or vehicle (10%
DMSO in saline).
Collection of Blood and Bronchoalveolar Lavage. Twenty-four
hours after the last inhalational exposure, mice were sacrificed by a
lethal dose of thiopental (Thiopentax; Cristália, São Paulo, Brazil).
Bronchoalveolar lavage (BAL) was performed twice by intratracheal
D
Journal of Natural Products, XXXX, Vol. xxx, No. xx
instillation of 1 mL of 0.15 M phosphate-buffered saline, pH 7.2 (PBS).
The first lavage fluid was centrifuged, and aliquots of the supernatant
were kept at -70 °C until use for cytokine measurements. The cell
pellets from the two lavages were pooled by resuspension in 1 mL of
PBS, and the number of total leukocytes in bronchoalveolar lavage
fluid was estimated in a Neubauer chamber. A differential count of
200 cells was made in a blind fashion and according to standard
morphologic criteria using panotic-stained cytospin preparations.
Histopathological and Morphometric Analyses. In the collageninduced arthritis model, the animals were sacrificed with a lethal
injection of barbituric anesthetic (Thiopentax; Cristália). The hind
footpads were removed, fixed in 10% formalin for 48 h, and
subsequently decalcified in 7% nitric acid for 3 days, in a mechanical
shaker. For the removal of excess acid, the legs were washed for 5
days with distilled water, and then sections including joints were
dehydrated in graded alcohol, clarified in xylene, and embedded in
paraffin. Sections of 4-6 µm thick were obtained and stained with
hematoxylin and eosin. For the allergic airway inflammation model,
the right lobe of the lungs from each animal was removed for
histological analysis. The lung was inflated via the tracheal cannula
with 4% buffered formalin, fixed in the same solution, and routinely
processed so as to be embedded in paraffin. Sections were stained with
hematoxylin and eosin for quantification of inflammatory cells by optical
microscopy. For each lung, 10 fields (400×) were analyzed per section,
and the data used to calculate the mean number of cells per mm2. Mucus
production was analyzed in alcian blue-stained sections. All images
were digitalized using a color digital video camera (CoolSnap cf)
adapted to a BX41 microscope (Olympus, Tokyo, Japan) calibrated
with a reference measurement slide and were analyzed using the Image
Pro image program (version 6.1; Media Cybernetics, San Diego, CA).
Measurement of Cytokines. Concentrations of interleukin (IL)-4
and IL-13 in bronchoalveolar lavage fluid were also determined by
ELISA using specific antibody kits (R&D Systems, Minnesota, MN),
according to the manufacturer’s instructions. Briefly, 96-well plates
were coated with the capture antibody overnight and treated by
incubation with block buffer at room temperature for 1 h. Samples
were added in duplicate and incubated overnight at 4 °C. Biotinylated
antibodies were added and plates were incubated for 2 h at room
temperature. A half-hour incubation with streptavidin-HRP was followed by detection using 3,5,3′,5′-tetramethylbenzidine (TMB) and
H2O2 as substrate and read under a 450 nm wavelenght light beam.
Cell Culture and Transfection. COS-7 cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum. One day before transfection, cells were transferred
to 8-well Labtek dishes (2 × 104 cells per well). Cells were transfected
using lipofectamin (Invitrogen, Carlsbad, CA) with the YFP-hGRR
expression vector (0.2 µg of plasmid DNA/well), kindly donated by
Dr.JohnA.Cidlowski.Aftera4hincubationwiththelipofectamin-plasmid
mixture in serum-free DMEM, cells were refed with supplemented
DMEM. One day after transfection, cells were treated with dexamethasone (1 µM), 1 (3.8 µM), or medium for 1 h. Cells were fixed in
cold acetone, and slides were prepared using Vectashield with 4,6diamidino-2-phenylindole (VectaShield Hard Set mounting medium
with DAPI H-1500; Vector Laboratories, Burlingame, CA). Cells were
observed by using an Olympus confocal laser scanning microscope (FV
1000 Olympus).
Statistical Analysis. Results were expressed as means ( SEM of 6
or 7 mice per group. Differences between groups in the collageninduced arthritis model were analyzed using multiple comparison test
(Tukey-Kramer) and GraphPad InStat software version 3.05. For the
Brustolim et al.
allergic airway inflammation model, statistical comparisons between
groups were performed by analysis of variance (ANOVA) followed
by a Newman-Keuls multiple comparison test, using the GraphPad
InStat program (Software Inc., San Diego, CA). Results were considered
to be statistically significant when p was e0.05.
Acknowledgment. The authors thank Dr. R. D. Couto for assistance
with statistical analysis and L. Ramires Santos for technical assistance.
This work was supported by FIOCRUZ/PDTIS, CNPq, Institutos do
Milênio, IMSEAR, MCT, FINEP, and RENORBIO.
Supporting Information Available: Figures S1 and S2. This
material is available free of charge via the Internet at http://pubs.acs.org.
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NP900691W
European Journal of Pharmacology 609 (2009) 126–131
Contents lists available at ScienceDirect
European Journal of Pharmacology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Effects of umbelliferone in a murine model of allergic airway inflammation
Juliana F. Vasconcelos a,e, Mauro M. Teixeira b, José M. Barbosa-Filho c, Maria F. Agra c, Xirley P. Nunes d,
Ana Maria Giulietti e, Ricardo Ribeiro-dos-Santos a,f, Milena B.P. Soares a,f,⁎
a
Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, 40296-750, Salvador, BA, Brazil
Instituto de Ciências Biomédicas, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte, MG, Brazil
Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, 58051-970, João Pessoa, PB, Brazil
d
Colegiado do Curso de Medicina, Universidade Federal do Vale do São Francisco, 56306-410, Petrolina, PE, Brazil
e
Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, 44031-640, Feira de Santana, BA, Brazil
f
Hospital São Rafael. Av. São Rafael, 2152. São Marcos 41253-190, Salvador, BA, Brazil
b
c
a r t i c l e
i n f o
Article history:
Received 7 December 2008
Received in revised form 19 February 2009
Accepted 3 March 2009
Available online 14 March 2009
Keywords:
Umbelliferone
Coumarin
Typha domingensis
Bronquic asthma
(Mouse)
a b s t r a c t
The therapeutic effects of umbelliferone (30, 60 and 90 mg/kg), a coumarin isolated from Typha domingensis
(Typhaceae) were investigated in a mouse model of bronchial asthma. BALB/c mice were immunized and
challenged by nasal administration of ovalbumin. Treatment with umbelliferone (60 and 90 mg/kg) caused a
marked reduction of cellularity and eosinophil numbers in bronchoalveolar lavage fluids from asthmatic mice. In
addition, a decrease in mucus production and lung inflammation were observed in mice treated with umbelliferone.
A reduction of IL-4, IL-5, and IL-13, but not of IFN-γ, was found in bronchoalveolar lavage fluids of mice treated with
umbelliferone, similar to that observed with dexamethasone. The levels of ovalbumin-specific IgE were not
significantly altered after treatment with umbelliferone. In conclusion, our results demonstrate that umbelliferone
attenuates the alteration characteristics of allergic airway inflammation. The investigation of the mechanisms of
action of this molecule may contribute for the development of new drugs for the treatment of asthma.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Coumarins constitute a very large class of compounds present in
several species belonging to different botanical families, which are
widespread in the world. They are secondary metabolites naturally
occurring in different parts of the plants, such as roots, flowers and fruits
(Leal et al., 2000; Ribeiro and Kaplan, 2002; Razavi et al., 2008). The search
for useful pharmaceutical has led to a resurgence of interest in coumarins
because these substances display potent and relevant pharmacological
activities that are structure-dependent, while at the same time appearing
to lack toxicity in mammalian systems (Hoult and Payá, 1996).
Among the biological effects of coumarins are anti-microbian (Thati
et al., 2007), anti-viral (Mazzei et al., 2008), anti-thrombotic, vasodilatory
(Casley-Smith et al., 1993), antitumoral, antineoplasic, antiinflammatory
(Hoult and Payá, 1996; Lino et al., 1997), and antimetastatic properties
(Jiménez-Orozco et al., 2001; Finn et al., 2004; Finn et al., 2005; ElinosBáez et al., 2005), and inhibition of acetylcholinesterase and angiotensin
converting enzyme (Barbosa-Filho et al., 2006a,b). In humans, coumarins
have a short half-life, and its major biotransformation product (75%) is
umbelliferone (Hoult and Payá, 1996; Egan et al., 1997).
Umbelliferone or 7-hydroxycoumarin is a widespread natural product
of the coumarin family (Fig. 1). It occurs in many familiar plants from the
⁎ Corresponding author. Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz,
Laboratório de Engenharia Tecidual e Imunofarmacologia. Rua Waldemar Falcão, 121,
Candeal. 40296-710, Salvador, Bahia, Brazil. Tel.: +55 71 3176 2260; fax: +55 71 31762272.
E-mail address: milena@bahia.fiocruz.br (M.B.P. Soares).
0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2009.03.027
Apiaceae (Umbelliferae) family such as carrot, coriander and garden
angelica, as well as plants from other families such as the mouse-ear
hawkweed. It is a yellowish-white crystalline solid which has a slight
solubility in hot water, but high solubility in ethanol (Dean, 1963).
Asthma is a chronic inflammatory disorder of the airways in which
many cells and elements play a role, associated with increased airway
hyperresponsiveness, leading to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing (Barnes, 2001). This chronic
inflammation is characterized by infiltration of eosinophils, neutrophils,
mast cells and T cells (Casale et al., 1987; Wenzel, 2003), and is promoted
by Th2 type immune responses (Barnes, 2001; El Biaze et al., 2003; Reed,
2006). In the current study we aimed to evaluate the effects of
umbelliferone, a coumarin purified from Typha domingensis (Typhaceae),
in a mouse model of asthma, as a potential anti-asthmatic agent.
2. Materials and methods
2.1. Plant material
The aerial parts of T. domingensis Pers. were collected for first
extracts in March, 2002 in Bravo, State of Bahia, in temporary shallow
lake in Dryland “Caatinga” Bioma and after in December, 2005 near
the city of Santa Rita, State of Paraíba, Brazil. The plant was identified
by Ana Maria Giulietti and Maria de Fátima Agra and the voucher
specimens (Giulietti 2035 et al. and Agra 5520 et Góis — JPB) were
deposited in the Herbaria HUEFS and JPB.
J.F. Vasconcelos et al. / European Journal of Pharmacology 609 (2009) 126–131
127
2.4. Sensitization and challenge with ovalbumin and treatment
Fig. 1. Chemical structure of umbelliferone.
2.2. Extraction and isolation of umbelliferone
The aerial parts (2000 g) of T. domingensis were extracted with 95%
ethanol at room temperature. The extract (150 g) was evaporated under
vacuum to yield a brown residue which was suspended in methanol:
H2O (3:7 v/v) and fractionated with chloroform and ethyl acetate. The
chloroform phase (25 g) was subjected to column chromatography over
silica gel, eluted with hexane, chloroform and methanol mixtures in an
increasing order of polarity, yielding three coumarins and one cinnamic
acid derivative. The ethyl acetate phase (25 g) was subjected to
chromatography column over Sephadex LH-20 using methanol yielding
two flavonoids. The isolated compounds were identified as coumarin
(0.0044%), scopoletin (0.0014%) (Brateoeff and Perez-Amador, 1994),
umbelliferone (0.007%) (Cussans and Huckerby, 1975), p-coumaric acid
(0.0016%), (Pouchert, 1992), quercetin (0.0028%) (Williams et al., 1971)
and isorhamnetin-3-O-glucoside (0.0039%) (Bilia et al., 1994) based on
1
H NMR, 13C NMR, COSY, HMQC and HMBC spectroscopic data and
comparison with those reported in the literature.
2.3. Animals
Male BALB/c mice, 4–6 weeks old, were used in the experiments.
All animals were raised and maintained at the animal facilities of the
Gonçalo Moniz Research Center, FIOCRUZ/BA, in rooms with controlled temperature (22 ± 2 °C) and humidity (55 ± 10%) and
continuous air renovation. Animals were housed in a 12 h light/12 h
dark cycle (6 am–6 pm) and provided with rodent diet and water ad
libitum. Animals were handled according to the NIH guidelines for
animal experimentation. All procedures described here had prior
approval from the local animal ethics committee.
Allergic airway inflammation was induced as described before
(Vasconcelos et al., 2008). Groups of seven mice received systemic
immunization by subcutaneous injection of 10 μg of chicken egg
ovalbumin (Grade V, N98% pure; Sigma, St Louis, MO) diluted in
2 mg/ml alum (AlumImject; Pierce, Rockford, IL) followed by a booster
injection at day 14. A nasal challenge was performed starting at day 28,
by inhalational exposure to aerosolised ovalbumin for 15 min/day, on
five consecutive days. Exposures were carried out in an acrylic box. A
solution of 1% ovalbumin in saline was aerosolised by delivery of
compressed air to a sidestream jet nebuliser (RespiraMax, NS, Brazil).
Two hours before each aerosol delivery, mice were treated orally with
umbelliferone (30, 60, and 90 mg/kg), dexamethasone (30 mg/kg) or
vehicle (10% DMSO in saline).
2.5. Collection of blood and bronchoalveolar lavage
Twenty four hours after the last inhalational exposure, mice were
anesthetized and bled via the brachial plexus for collection of blood
samples used to estimate the IgE production. Bronchoalveolar lavage
(BAL) was performed twice by intratracheal instillation of 1 ml of PBS.
The first lavage fluid was centrifuged, and aliquots of the supernatant
were kept at 70 °C until use for cytokine measurements. The second
lavage fluid was centrifuged and the two cell pellets were resuspended in a PBS final volume of 1 ml. The number of total leukocytes
in bronchoalveolar lavage fluid was estimated in a Neubauer chamber.
Differential counts were obtained using panotic-stained cytospin
preparations. A differential count of 200 cells was made in a blinded
fashion and according to standard morphologic criteria.
2.6. Histopathological and morphometric analysis
The right lobe of the lungs from each animal was removed for
histological analysis. The lung was inflated via the tracheal cannula
with 4% buffered formalin, fixed in the same solution, and embedded
in paraffin. Sections were stained with hematoxylin and eosin for
quantification of inflammatory cells by optical microscopy. For each
lung 10 fields (400×) were analyzed per section, and the data used to
calculate the mean number of cells per mm2. Mucus production was
Fig. 2. Total cell counts and leukocyte quantification in BAL samples obtained from mice submitted to different treatments. Mice were sacrificed 24 h after the last challenge with
ovalbumin. The cellularity in BAL fluid from control or ovalbumin-challenged mice treated with saline, dexamethasone (dexa) or umbelliferone (umb) was evaluated. A, Total cell
counts. B, Number of neutrophils in 200 cells. C, Number of eosinophils in 200 cells. D, Number of mononuclear cells in 200 cells. Values are expressed as means ± S.E.M. of 6–7 mice
per group, in one of two experiments performed. ⁎⁎⁎P b 0.001.
128
analyzed in alcian blue-stained sections. All images were digitalized
using a color digital video camera (CoolSnap cf) adapted to a BX41
microscope (Olympus, Tokyo, Japan) calibrated with a reference
measurement slide and were analyzed using Image Pro image
program (version 6.1; Media Cybernetics, San Diego, CA, USA).
(IFN)-γ in bronchoalveolar lavage fluid were also determined by ELISA
using specific antibody kits (R&D System, Minnesota, MN, USA),
according to manufacturer's instructions.
2.7. Ovalbumin-specific IgE antibody levels and cytokine production
Results were expressed as means ± S.E.M. of 7 mice per group.
Statistical comparisons between groups were performed by analysis of
variance (ANOVA) followed by Newman–Keuls multiple comparison
test, using GraphPad InStat program (Software Inc., San Diego, CA, USA).
Results were considered to be statistically significant when P b 0.05.
IgE antibody levels to ovalbumin in sera samples from individual
animals were quantified using an enzyme immunoassay modified from
Jungsuwadee et al. (2004). Briefly, microplate wells coated with 2 µg/ml
of anti-mouse IgE (Pharmingem, San Diego, CA) were incubated
overnight with dilutions of the test samples in PBS containing 5% nonfat milk. Detection of bound anti-ovalbumin IgE was performed by
sequential ovalbumin incubation followed by biotinylated anti-ovalbumin antibody (Fitzgerald, Concord, MA, USA). After incubation with
streptoavidin-peroxidase conjugate, the reaction was developed using
3,3′,5,5′-tetramethylbenzidine (TMB) peroxidase substrate and read at
450 nm. Concentrations of interleukin (IL)-4, IL-5, IL-13, and interferon
2.8. Statistical analysis
3. Results
3.1. Treatment with umbelliferone reduces lung inflammation
Mice sensitized and challenged with ovalbumin have increased the
number of inflammatory cells in bronchoalveolar lavage fluid, compared
to control mice — sensitized and challenged with saline (Fig. 2A). The
Fig. 3. Histopathology of lungs from control and asthmatic mice. Lung sections of control (A–C) and asthmatic mice treated with vehicle (D–F), dexamethasone (G–I), or
umbelliferone 90 mg/ml (J–L). Narrow arrows indicate areas of alcian blue+ cells; large arrows indicate inflammatory cells. A, D, G, and J: Alcian blue staining. B, C, E, F, H, I, K and L:
H&E staining.
129
Fig. 6. Ovalbumin-specific IgE antibodies in asthmatic mice. The levels of anti-OVA IgE
antibodies were determined in the sera of individual mice by ELISA. The data are
expressed as means ± S.E.M. of 7 mice per group, in one of two experiments performed.
⁎⁎P b 0.01 compared to saline-treated mice.
potent reduction in the number of eosinophils (Fig. 2C). The number of
mononuclear cells was significantly altered only by treatment with
umbeliferone at 60 mg/ml and by dexamethasone (Fig. 2D).
Fig. 4. Quantification of inflammation and mucus production in lungs of mice. A, Intensity
of inflammation in lungs of mice treated with saline, dexamethasone (dexa) or
umbelliferone 60 mg/ml (umb) compared to control animals. The number of
inflammatory cells was evaluated on H&E-stained sections. B, Analysis of mucus
production on alcian blue-stained lung sections. The area of alcian blue staining was
estimated by morphometric analysis. Data are expressed as means± S.E.M. of 7 mice per
group, in one of two experiments performed. ⁎⁎⁎P b 0.001 compared to saline-treated mice.
effects of umbelliferone treatment in lung inflammation were evaluated
comparing bronchoalveolar lavage fluid cytology of umbelliferonetreated mice to that of mice treated with vehicle. The number of total
cells and of neutrophils in the bronchoalveolar lavage fluid was
significantly reduced after umbelliferone treatment compared to vehicle-treated mice (Fig. 2A and B). A significant inhibition was observed in
mice treated with dexamethasone, a gold standard drug. Treatment with
umbelliferone at 60 and 90 mg/ml, but not at 30 mg/ml, decreased
significantly the number of eosinophils found in the bronchoalveolar
lavage fluid of challenged mice, although dexamethasone caused a more
3.2. Histological evaluation of lungs from umbelliferone-treated mice
To characterize further the changes in lung pathology caused by
antigen challenge of immunized mice, we examined lung sections stained
with H&E. Ovalbumin challenge caused an intense cell infiltrate containing
many lymphocytes, macrophages, and eosinophils (Fig. 3E and F). Mice
treated with umbelliferone at 60 and 90 mg/ml, but not at 30 mg/ml, had
reduced inflammation, particularly reduced eosinophil infiltration,
although some less dense inflammatory foci remained (Fig. 3K and L;
Fig. 4A). Dexamethasone treatment almost completely eliminated the
inflammatory infiltrate, in particular eosinophils (Fig. 3H and I; Fig. 4A).
Lung sections were stained with alcian blue to analyze mucus
overproduction. Lungs from control mice revealed rare alcian blue+
cells in the respiratory epithelium compared to saline-treated group,
which had high alcian blue+ staining (Fig. 3A and D; Fig. 4B). Similar
to dexamethasone, umbelliferone treatment at 60 mg/ml was
capable of modulating mucus production, decreasing the intensity
of alcian blue+ staining observed in the saline-treated group by 38%
Fig. 5. Cytokine production in umbelliferone-treated mice. Cytokine levels in individual mice from each experimental group were determined in BAL samples by ELISA. A, IL-4. B, IL-5.
C, IL-13. D, IFN-γ. Data are expressed as means ± S.E.M. of 6–7 mice per group. ⁎⁎P b B0.01; ⁎⁎⁎P b 0.001 compared to saline-treated mice.
130
and a more potent effect after umbelliferone treatment at 90 mg/ml
(Fig. 3G and J; Fig. 4B).
3.3. Treatment with umbelliferone modulated T-helper type 2 cytokine
response but not IgE production
The production of cytokines was studied in bronchoalveolar lavage of
individual mice from each group. As expected, the concentrations of IL-4,
IL-5, and IL-13 were increased in ovalbumin-immunized, compared to
control mice (Fig. 5A–C). Treatment with umbelliferone caused a
reduction in the levels of Th2-associated cytokines, compared to salinetreated group, an effect similar to that of dexamethasone. The levels of
Th1-associated cytokine IFN-γ were not increased by ovalbumin
challenge, but were reduced by dexamethasone. In contrast, umbelliferone did not alter the production of IFN-γ (Fig. 5D).
When levels of ovalbumin-specific IgE antibodies were analyzed,
ova-immunized mice treated with vehicle had high levels of serum
anti-ovalbumin IgE antibodies compared to control mice (Fig. 6). A
significant reduction in ovalbumin-specific IgE antibodies was
observed in mice treated with dexamethasone, but not with
umbelliferone, compared to saline-treated controls (Fig. 6).
4. Discussion
In the present study we demonstrated a potent anti-inflammatory
activity of umbelliferone in an allergic airway inflammation model
induced by ovalbumin administration in mice. This effect was evidenced
by a marked reduction of inflammation both in bronchoalveolar lavage
fluid and in the lung, especially in neutrophil and eosinophil numbers, in
mucus production and in the levels of Th2-associated cytokines in
bronchoalveolar lavage after umbelliferone treatment.
Umbelliferone is one of several coumarins with anti-inflammatory
activity already reported (Hoult and Payá 1996; Lino et al., 1997). In our
study it was extracted from T. domingensis (Typhaceae), a species widely
distributed in north-east Brazil, especially found in damp areas and in
shallow water, lakes and river margins, used for water and soil metal
decontamination (Taggart et al., 2005; França, 2006; Maine et al., 2007).
We have isolated three coumarins from T. dominguensis. The most
abundant was identified as coumarin or 1,2-benzopyrone with effects
already described in an experimental asthma model (Marinho et al.,
2004; Biavatti et al., 2004; Zhang et al., 2005; Dos Santos et al., 2006;
Werz, 2007). The other two were scopoletin, isolated in small amounts,
and umbelliferone, which has not been studied in animal models of
asthma before.
Umbelliferone is present in Typhae Pollen, a traditional Chinese herbal
medicine with demonstrated immunomodulatory activity (Park et al.,
2004; Qin & Sun, 2005). Thus, umbelliferone may be one of the substances
responsible for the anti-inflammatory effects of this extract. A recent
study of some plant species largely used in north-eastern Brazil for
respiratory tract diseases has shown they have in common coumarins as
one of their active principles (Leal et al., 2000; Agra et al., 2007).
Ramanitrahasimbola et al. (2005) showed that the bronchodilator effect
after treatment with Phymatodes scolopendria extract, a plant used to treat
respiratory disorders, is mainly caused by coumarins. Our results support
the protective role of coumarins in asthma.
Studies on the pharmacokinetics of coumarin in man have shown
there is extensive first-pass metabolism after oral administration
(Ritschel et al., 1979; Ritschel and Hoffmann, 1981; Ritschel, 1984).
This, and other pharmacokinetic data in man, has led to suggestions
that coumarin is a prodrug, most likely active as umbelliferone or,
possibly, even as the 7-hydroxyglucuronide form after metabolization
in the liver (Hoult and Payá, 1996). Baccard et al. (2000) also
suggested that umbelliferone was vasodilating activity. Our results
demonstrate that the effects of umbelliferone go beyond a bronchodilating effect, since it has also potent immunomodulatory effects,
downregulating cytokine and mucus production and inflammation.
The effects of umbelliferone in the reduction of histological
alterations and cellularity in the bronchoalveolar lavage fluid were
similar to those of dexamethasone, a synthetic glucocorticoid
commonly used as a gold standard anti-inflammatory drug. The side
effects after chronic use of corticoids, such as osteopenia, poor wound
healing, hyperglycemia, hypertension, and cataracts limit their
systemic administration (Barnes, 2001; Schimmer & Parker, 2001),
and therefore the search of substances with similar immunosuppressive activities, free or with lower toxicity, is of great interest (Marinho
et al., 2004; Bezerra-Santos et al., 2006; Costa et al., 2008a,b).
Although reports of adverse effects in humans resulting from
coumarin administration are rare (Casley-Smith et al., 1993; Lake,
1999), it is acutely toxic to laboratory animals in chronic oral gavage
administration at doses equal or higher of 200 mg/kg (Born et al.,
2003). There are marked differences in coumarin metabolism
between susceptible rodent and other species, including humans.
According to Lake (1999) the 7-hydroxylation pathway of coumarin
metabolism, which generates umbelliferone, is usually the major
pathway in humans but not in rats and mice, in which it represents
only a minor pathway of metabolism. In contrast, the major route of
coumarin metabolism in murines is by a 3,4-epoxidation pathway,
which results in the formation of toxic metabolites. In our study we
did not observe any signs of toxicity in umbelliferone-treated mice.
Carrageenan stimulates the release of several inflammatory mediators
such as histamine, serotonin, bradykinin and prostaglandins. Nonsteroidal anti-inflammatory drugs (NSAID) block the synthesis of
prostaglandins by inhibiting cyclooxygenase (COX) (Hoult et al., 1994).
Murakami et al. (2000) demonstrated umbelliferone does not suppress
O−
2 generation in vitro nor attenuates lypopolysaccharide-induced nitric
oxide synthase (NOS)/COX-2 expression and TNF-α release. Interesting,
umbelliferone inhibits prostaglandin biosynthesis, which involves fatty
acid hydroperoxy intermediates, indicating that this substance has a
mechanism of action similar to NSAID in a carrageenan-induced
inflammation model (Fylaktakidou et al., 2004). This is an unlikely
mechanism of action of umbelliferone in our system, NSAID are not shown
to inhibit effectively the migration of eosinophils and mucus production in
murine models of allergic inflammation.
There are certain pharmacologic compounds that inhibit eosinophil function, eosinophil infiltration, or both, which is desirable for the
treatment of patients with atopy/ allergic disorders (Barnes, 2001).
The inhibition of Type-II cytokine production by umbelliferone
suggests an inhibition of T cells and may be underlining the effects
of the drug on the system, as allergic airway inflammation is shown to
depend on the action of IL-4, IL-5 and IL-13 (Bodey et al., 1999; Boyton
and Altmann, 2004). It has also been shown that IL-5 enables terminal
differentiation and proliferation of eosinophil precursors, as well as
the eosinophil activation effect (Barnes, 2001). The reduction of
eosinophil infiltrates found in umbelliferone-treated asthmatic mice
may be due to IL-5 reduction, since it promotes the differentiation to
and activation of eosinophils. Further studies are clearly needed to
understand further the ability of umbelliferone to inhibit the synthesis
of these cytokines.
Ovalbumin-specific IgE production was not significantly altered after
treatment with umbelliferone, suggesting that this compound did not
affect the production of IgE by activated B cells, in contrast to
dexamethasone, which caused a decrease on the levels of IgE. This
effect of dexamethasone on IgE levels in the mouse model of allergic
airway inflammation has been demonstrated before (Zhao et al., 2007).
In allergic asthma, the inhibition of macrophages, T lymphocytes,
eosinophils, mast cells, and mucus secretion leads to an improvement of
respiratory flow. Altogether, our results show that umbelliferone
attenuates airway inflammation in a murine model of asthma. The
effects described herein, as well as those observed by other investigators, together with the broad spectrum of the biological effects of this
substance, strongly suggest the therapeutic potential of umbelliferone
for the treatment of asthma and other allergic diseases.
Acknowledgements
This work had the financial support of FIOCRUZ, CNPq, Institutos
do Milênio, IMSEAR, MCT, FINEP, RENORBIO, and CAPES. The authors
wish to thank Ricardo Santana de Lima and Daniele Brustolim for their
technical assistance.
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