Session 467 Autoimmunity

ARVO 2016 Annual Meeting Abstracts
467 Autoimmunity / AMD
Wednesday, May 04, 2016 3:45 PM–5:30 PM
Skagit 4/5, TCC Paper Session
Program #/Board # Range: 5235–5241
Organizing Section: Immunology/Microbiology
Program Number: 5235
Presentation Time: 3:45 PM–4:00 PM
Mincle activation and Syk/Card9 signaling axis are central to
development of autoimmune disease of the eye
Holly L. Rosenzweig1, 3, Brieanna Brown1, Paige Snow1,
Emily Vance1, 3, Phyllis Silver2, Rachel R. Caspi2, Ellen J. Lee1, 3.
1
Portland VA Medical Center, Portland, OR; 2Laboratory of
Immunology, NEI, Bethesda, MD; 3Molecular Microbiology &
Immunology, Oregon Health & Science University, Portland, OR.
Purpose: The innate immune receptors responsible for induction of
eye-specific autoreactive T cells remain poorly defined. We recently
identified Mincle, a C-type lectin receptor (CLR) vital to host defense
that signals through Syk and Card9 to mediate innate immune cell
activation, as essential in induction of ocular autoimmunity, using
experimental autoimmune uveitis (EAU). Here, we further delineate
molecular and cellular factors involved in orchestrating this Mincledependent mechanism.
Methods: EAU was induced in C57BL/J mice deficient in
Card9 or Mincle (encoded by Clec4e) by immunization with
interphotoreceptor retinoid-binding protein (IRBP). Uveitis was
evaluated by fundus imaging and histology. The synthetic Mincle
agonist TDB (trehalose-6-6’-dibehenate) was used in lieu of CFA.
Syk was pharmacologically inhibited by piceatannol (given i.p., q4d,
d0-16). To define the cellular source of Card9 and Mincle-activated
responses, studies with bone marrow (BM) chimeras were performed.
In addition, Cre-Lox recombination was used to generate conditional
KO mice with CD11c+ dendritic cells (DCs) lacking Syk.
Results: Mice immunized with Mincle agonist TDB developed
uveitis (p<0.05; n=8 mice/group). The effect of TDB required
Mincle and its downstream CLR signaling adaptor, Card9, as
EAU was significantly reduced in the absence of either molecule
(p<0.05, n=10-15 mice/group). Likewise, inhibition of Syk, an
upstream kinase of Card9, resulted in abrogation of TDB-induced
EAU (p<0.05 vs. vehicle control; n=15 mice/group). These
results demonstrate that Mincle signaling is both necessary and
sufficient to support EAU. BM chimera experiments showed that
Card9 expression was required in hematopoietic, rather than nonhematopoietic cells (p<0.05; n=10 mice/group). Since Card9 is
primarily expressed by myeloid cells, contribution of DC-specific
Syk was investigated. Syk-deficiency in DCs alone resulted in
significantly reduced disease in early induction of EAU (p<0.05 vs
controls; n=6-10 mice/group).
Conclusions: These studies uncover an essential role for Mincle and
the Syk/Card9-coupled signaling axis in autoimmune uveitis. This
could provide novel targets for treatment of ocular inflammation.
Commercial Relationships: Holly L. Rosenzweig, None;
Brieanna Brown; Paige Snow, None; Emily Vance, None;
Phyllis Silver, None; Rachel R. Caspi, None; Ellen J. Lee, None
Support: NIH/NEI (grant EY025250), the Department of Veterans
Affairs Biomedical Laboratory (I01 BX002180), and NEI intramural
support (Project # EY00184).
Program Number: 5236
Presentation Time: 4:00 PM–4:15 PM
IL-17A is dispensable for spontaneous autoimmune uveitis and
negatively regulates the Th17 cytokine program
Wai Po Chong, Reiko Horai, Phyllis Silver, Yingyos Jittayasothorn,
Chi-Chao Chan, Rachel R. Caspi. National Eye Institutes, Bethesda,
MD.
Purpose: The Th17 response has been associated with host defense
as well as with autoimmune diseases in patients and in experimental
animal models. Th17 cells cause tissue damage by producing
a stereotypic profile of pro-inflammatory cytokines, including
Interleukin (IL)-17A, IL-17F, Granulocyte macrophage colonystimulating factor (GM-CSF) and IL-22. IL-17A is recognized as the
Th17 signature cytokine and IL-17-producing T cells are pathogenic
effectors in models of autoimmunity, including experimental
autoimmune uveitis induced by immunization with the retinal
protein IRBP in complete Freund’s adjuvant. Paradoxically, however,
injection of exogenous IL-17 was shown to ameliorate the disease
(Ke et al., JI 2009).
Methods: Using a recently developed model of spontaneous
uveitis in R161H mice, which express a transgenic T cell receptor
specific for IRBP, we investigated the susceptibility to disease on an
IL-17A deficient background. Additionally, IRBP-specific T cells
from IL17A sufficient and deficient R161H mice were polarized
under Th17 conditions and were adoptively transferred to WT
recipient to investigate their cytokine profile and their ability to
induce uveitis.
Results: We found that IL-17A deficiency did not reduce the severity
of uveitis in R161H mice. Furthermore, IL-17A deficient R161H
T cells polarized under Th17 conditions and adoptively transferred
into wild type recipients induced similar disease to IL-17A sufficient
R161H T cells. Interestingly, IL-17A deficient R161H T cells
polarized under Th17 conditions produced elevated amounts of other
Th17-related cytokines, i.e. IL-17F, GM-CSF and IL-22. Conversely,
supplementing Th17 polarization cultures of IL-17A deficient T cells
with recombinant IL-17A, reduced the elevated production of those
cytokines.
Conclusions: These data suggest that: (a) IL-17A itself is not
necessary for the pathogenicity of uveitogenic Th17 cells in this
model, and (b) that IL-17A exerts feedback inhibition on Th17 cells
to control their expression of other Th17-related cytokines.
Commercial Relationships: Wai Po Chong, None; Reiko Horai;
Phyllis Silver, None; Yingyos Jittayasothorn, None; ChiChao Chan, None; Rachel R. Caspi, None
Program Number: 5237
Presentation Time: 4:15 PM–4:30 PM
The lectin but not classical pathway of activation is important
for complement to regulate the development of experimental
autoimmune uveitis
Lingjun Zhang1, Brent A. Bell2, Yan Li1, Xiaomin Zhang3, John Fung4,
Rachel R. Caspi5, Feng Lin1. 1Immunology, Cleveland Clinic,
Cleveland, OH; 2Cole Eye Institute, Cleveland Clinic, Cleveland,
OH; 3Eye Institute, Tianjin Medical University Eye Hospital, Tianjin,
China; 4Digestive Diseases Institute, Cleveland Clinic, Cleveland,
OH; 5National Eye Institute, National Institutes of Health, Bethesda,
MD.
Purpose: Complement needs to be activated to function. Although
the alternative pathway has been found important in regulating T cell
responses, the potential roles of the other two complement activation
pathways, the classical and the lectin pathways in this process remain
unclear. To address this issue, we studied mice deficient of C4 (both
classical and lectin pathways deficient) or C1q (classical pathway
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ARVO 2016 Annual Meeting Abstracts
deficient) in the development of experimental autoimmune uveitis
(EAU).
Methods: WT, C4 and C1q knockout (KO) mice (all C57BL/6J
background) were immunized with interphotoreceptor retinoid
binding protein (IRBP) peptide to induce EAU following an
established protocol. After immunization, the development of EAU
was monitored by indirect ophthalmoscopy, and clinical scores were
assigned according to previously published criteria. Two weeks
after immunization, mice were also examined by scanning laser
ophthalmoscopy (SLO) and spectral-domain optical coherence
tomography (OCT). Three weeks after immunization, mice were
euthanized for retinal histopathological analysis, for numbers of
antigen-specific CD4+ T cells comparison by tetramer staining, and
for antigen-specific Th1/Th17 responses assessment by recall assays.
Results: After immunization, C4 KO mice developed significantly
milder EAU than WT controls while C1q KO mice developed
comparable EAU to WT mice as judged by clinical scores, retinal
histopathological scores, SLO and OCT analyses. Consistent with the
imaging results, C4 KO mice in EAU also showed reduced numbers
of IRBP-specific CD4+ T cells and had decreased IRBP-specific
Th1 and Th17 responses compared with WT mice while C1q mice in
EAU did not show any significant difference from WT mice in these
immunological assays.
Conclusions: Complement classical pathway of activation is not
important for the development of EAU, while the lectin pathway of
complement activation is required for the pathological T cell response
development in EAU. These data suggest that the lectin pathway
of complement activation should be a novel target to suppress
pathogenic T cell responses for treating EAU, and potentially, for
treating autoimmune uveitis.
Commercial Relationships: Lingjun Zhang; Brent A. Bell,
None; Yan Li, None; Xiaomin Zhang, None; John Fung, None;
Rachel R. Caspi, None; Feng Lin, None
Program Number: 5238
Presentation Time: 4:30 PM–4:45 PM
Microglia and monocyte-derived macrophage responses to light
damage differ across mouse strains
Emily Okoren, Rose Mathew, Daniel R. Saban. ophthalmology, Duke
University, Durham, NC.
Purpose: Non-redundant functions between resident microglia
and recruited monocyte-derived macrophages (mo-MFs) in
neuroinflammation are ill-defined, even though it is now firmly
established that these are distinct myeloid populations. We have
recently reported that the microglia phenotype of CD45lo CD11clo
I-A/Elo F4/80lo is conserved in retinal inflammation, whereas acutely
recruited mo-MFs are CD45hi CD11chi I-A/Ehi F4/80hi, which we
validated using in vivo fate-mapping. Here, we took advantage of
this approach to determine their respective responses in light-induced
retinal degeneration. We used mouse strains, C57Bl/6J (pigmented),
BALB/c (albino), and their F1 progeny, CB6F1J (pigmented), as the
latter two harbor increased light susceptibility due to RPE65 genetic
variation.
Methods: Dark-adapted mice were exposed to white-light LEDs
for 4 hr at 40k lux or 8hr at 60k lux on day 0. Mice were then
returned to normal housing with normal 12 hr light cycles until
euthanasia on day 5. Neuroretinas were harvested for 10-color flow
cytometry, which included: viability, CD45 (APC), CD45 (APC-cy7),
CD11b, CD11c, I-A/E, Ly6G, Ly6C, F4/80, CD64. Contralateral
eye were cryosectioned to assess ONL thinning (DAPI) and
immunofluorescence (CD11b, Iba-1).
Results: LED-induced light damage in B6J mice required a 60k lux
/8 hr exposure, whereas only 40k lux/4 hr was required in BALB/c
and CB6F1J mice. CB6F1J appeared to have greater ONL thinning
relative to their BALB/c counterparts. Transmigration of Iba-1+
cells to the photoreceptor layers was readily detectable in damaged
CB6F1J mice, whereas transmigration was unremarkable in BALB/c
counterparts. Likewise in CB6F1J mice, presence of mo-MF recruits
was robust, whereas only microglia were detectable in BALB/c
counterparts.
Conclusions: Our findings may suggest that augmented
photoreceptor degeneration in CB6F1J could be associated with
mo-MF recruitment. These data may therefore implicate mo-MFs in
secondary damage that results in greater photoreceptor loss.
Commercial Relationships: Emily Okoren, None; Rose Mathew,
None; Daniel R. Saban, None
Support: NEI-R01EY021798 and Research to Prevent Blindness
[RPB] Career Development Award and P30EY005722 (Duke
Ophthalmology Department; Durham, NC, USA).
Program Number: 5239
Presentation Time: 4:45 PM–5:00 PM
Permeability of Bruch’s membrane to complement proteins:
Implications for translational medicine in AMD
Simon J. Clark1, 2, Selina McHarg1, Nicole Brace1, Paul N. Bishop1, 2.
1
Faculty of Medicine and Human Sciences, University of Manchester,
Manchester, United Kingdom; 2Central Manchester University
Hospitals NHS Foundation Trust, Centre for Advanced Discovery
and Experimental Therapeutics, Manchester, United Kingdom.
Purpose: The dysregulation of the complement system is implicated
in the pathogenesis of age-related macular degeneration (AMD),
where variants in genes encoding complement proteins are known
to greatly modify risk of disease. Complement proteins are mainly
synthesized in the liver, but can also be made locally by retinal
pigment epithelial (RPE) cells. The relative contribution made
between systemic and locally produced complement on immune
homeostasis at the RPE cell/Bruch’s membrane interface, and the
underlying choriocapillaris, remains unknown. Here, we investigate
the ability of complement proteins to diffuse across Bruch’s
membrane and reach their respective site of function within the eye.
Methods: Using modified Ussing chambers and Bruch’s membrane
enriched from human donor eyes, we assess the passive diffusion
of complement across Bruch’s membrane. Western blots identify
the presence of complement components in the diffusate chamber.
Functional tests (i.e. C3b breakdown assays) are used to assess if
protein below our level of detection is still capable of diffusing across
Bruch’s membrane and mediating C3b breakdown. Also, the local
expression of complement proteins by RPE cells is investigated using
primary adult RPE cells harvested from postmortem eyes.
Results: We demonstrate for the first time that specific complement
proteins cannot diffuse through Bruch’s membrane in either
direction. In some cases this may be due to the glycosylation state
of the complement protein. Factor I is not able to diffuse across
Bruch’s membrane and mediate C3b breakdown. Furthermore, we
demonstrate local expression of complement proteins necessary for
maintaining immune homeostasis on Bruch’s membrane by primary
adult RPE cells.
Conclusions: While certain systemic complement proteins will
contribute to immune homeostasis of the choriocapillaris and
surrounding stroma, they cannot help protect from the natural
tick-over of the alternative pathway of complement on the Bruch’s
membrane interface with the RPE cell monolayer, and vice versa.
This means that targeting complement turnover at this site (i.e. the
site of drusen formation) must be directed via RPE cell synthesis
directly. Similarly, readdressing complement turnover in the
choriocapillaris must be mediated via the choroid. These findings
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
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ARVO 2016 Annual Meeting Abstracts
must be taken into account when developing any complement
mediated therapeutic strategies for treating AMD.
Commercial Relationships: Simon J. Clark, None;
Selina McHarg, None; Nicole Brace, None; Paul N. Bishop, None
Support: MRC Career Development Fellowship MR/K024418/1
Program Number: 5240
Presentation Time: 5:00 PM–5:15 PM
Combination of Factor H Mutation and Properdin Deficiency
Causes Subretinal and Sub-RPE Deposition
Delu Song1, Imran Mohammed2, Takshi Miwa2, Allison Wlliams2,
Damodar Gullipali2, Sayaka Sato2, Wenchao Song2,
Joshua L. Dunaief1. 1Scheie Eye Institute, University of Penn,
Philadelphia, PA; 2Pharmacology, University of Pennsylvania,
Philadelphia, PA.
Purpose: Both factor H (fH) and properdin (fP) regulate
complement. fH is a complement inhibitor, while fP activates the
alternative pathway (AP). Mutations in fH are associated with several
human kidney diseases and macular degeneration. Surprisingly,
knocking out fP converted the mild C3 glomerulonephritis of a
fH-mutant mouse to a lethal C3 glomerulonephritis with features
of human dense deposit disease (DDD)1. Here we characterized its
retinal phenotype.
Methods: Mice with a fH C-terminal mutation leading to SCR 1920 truncation (fHm/m) were crossed with fP knockout mice (fP-/-) to
generate fHm/m/fP−/− mice. Electroretinography (ERG) and fundus
imaging were performed to assess retinal function and appearance
in fHm/m/fP−/−, fHm/m, fP−/− mice and wild-type (WT) mice.
After euthanasia, at age 2-3 months, eyes were used for electron
microscopy (EM), histology, immunolabeling with C3 antibody and
rhodamine-conjugated peanut agglutinin lectin (PNA).
Results: ERG showed decreased rod a-waves in fHm/m/fP−/− mice,
but not in fHm/m, fP−/− mice and WT mice. Similarly, fundus
images showed more hypopigmented lesions in fHm/m/fP−/− mice
than other genotypes. Thinning of the ONL was observed in fHm/m/
fP−/− mice. EM showed subretinal and sub-RPE deposits in fHm/m/
fP−/− mice, combined with thickening of Bruch’s membrane and RPE
hypertrophy. Increased C3 staining in Bruch’s membrane and fewer
PNA staining cones were found in fHm/m/fP−/− mice compared with
other genotypes.
Conclusions: Our results indicate that the combination of a fH
mutation in the C terminus of the mouse fH gene with properdin
knockout leads to subretinal and sub-RPE complement-containing
deposits, as well as photoreceptor and RPE degeneration. The cooccurrence of C3 glomerulopathy and AMD-like retinal degeneration
in this mouse model recapitulates the clinical observations of
retinopathy in human C3 glomerulopathy patients. The fHm/m/fP−/−
mouse represents a new mouse model of complement-mediated rapid
onset dense deposit disease with retinal manifestations related to
AMD, and could be useful in exploring the pathogenic mechanism
and therapy of AMD and AMD-like retinopathies.
Electron microscopy shows lipid droplets in RPE cell, sub-RPE
depostion and thickening of Bruch’s membrane in fHm/mfP-/-
Immunostaining of C3 and RPE65 in RPE cell and Bruch’s
membrane
Commercial Relationships: Delu Song, None; Imran Mohammed,
None; Takshi Miwa, None; Allison Wlliams, None;
Damodar Gullipali, None; Sayaka Sato; Wenchao Song, None;
Joshua L. Dunaief, None
Support: NIH EY015240, NIH EY023709, Research to Prevent
Blindness
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 5241
Presentation Time: 5:15 PM–5:30 PM
The aged Cxcr5 knockout mice develop the features of agerelated macular degeneration
Hu Huang1, WEN LI1, 2, Gerard A. Lutty1. 1Ophthalmology, Johns
Hopkins Wilmer Eye Inst, Baltimore, MD; 2Ophthalmology, 3.
Aier School of Ophthalmology, Central South University, ChangSha,
China.
Purpose: The purpose of this study is to investigate the role of the
chemokine receptor Cxcr5 in the pathogenesis of age-related macular
degeneration (AMD).
Methods: The 9~15-month-old Cxcr5 knockout (-/-) mice were
examined for Crb1 (or rd8) gene genotype and fundus appearance.
The cryopreserved and ultrathin eye sections were made for
histopathological assessments using H&E staining, light and
transmission electron microscopy (TEM). Retinal pigmented
epithelium (RPE) flatmounts were prepared for the staining of tight
junction zonula occludens (ZO)-1 and macrophage/microglia marker
F4/80. Laser-induced choroidal neovascularization (CNV) areas and
retinal layers were isolated by laser capture micro-dissection (LCM)
for gene expression analysis.
Results: The aged Cxcr5-/- mice developed several features of agerelated macular degeneration (AMD): drusen-like spots, lipofuscin and
lipid deposits, RPE death, phagocytosis defect, and inflammatory cell
accumulation. These AMD-like features were not caused by retinal
degeneration because Cxcr5-/- mice did not have rd8 mutation in Crb1
gene. The fundus of aged Cxcr5-/- mouse had numerous white spots,
which appeared to be drusen. These drusen-like spots became bigger and
merged with age. Hyperpigmention, hypopigmention, and geographic
atrophy were detected at the aged Cxcr5-/- mice. The protein level of
tight junction ZO-1 was reduced in the RPE of aged Cxcr5-/- mice.
TEM revealed the undigested photoreceptor outer segments and many
vacuoles in the RPE. Additionally, F4/80 (+) microglia or macrophages
accumulated in the sub-retinal space. The Cxcr5 (+) inflammatory cells
migrated to the photoreceptors in the laser-induced CNV. Cxcl13, the
ligand for Cxcr5, was expressed by photoreceptors and the cells of inner
nuclear layer in the proximity to CNV.
Conclusions: The results suggest that Cxcl13-Cxcr5 axis plays a
critical role in the pathogenesis of AMD, perhaps by controlling
the migration of inflammatory cells to and/or from the RPE and
photoreceptors.
Commercial Relationships: Hu Huang; WEN LI, None;
Gerard A. Lutty, None
Support: Brightfocus foundation
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.