docLactobacilli- and Staphylococcus aureus mediated
download 
Report Transcription
Lactobacilli- and Staphylococcus aureus mediated
Doctoral thesis from the Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Sweden Lactobacilli- and Staphylococcus aureus mediated modulation of immune responses in vitro Yeneneh Eshetu Haileselassie Stockholm University Stockholm 2016 About the cover: As the micro-environment in the gut influences the development and response of the immune cells, our surroundings (the macro-environment) have influence on our development and success. All previously published papers were reproduced with the permission from the publishers ©Yeneneh Haileselassie 2016 ISBN 978-91-7649-365-6 Printed in Sweden by Holmbergs, Malmö 2016 Department of Molecular Biosciences, The Wenner-Gren Institute POPULÄRVETENSKAPLIG SAMMANFATTNING Vår tarmflora påverkar tarmens funktioner och ”hjälper till” med olika viktiga metabola funktioner. Därtill kommunicerar tarmfloran med värden via olika utsöndrade ämnen och kan därmed nå organsystem utanför tarmen och påverka många olika funktioner i kroppen. De senaste årens forskning har visat att tarmfloran spelar en betydande roll för immunsystemets utveckling och funktion i allmänhet. En rubbad tarmflora, t.ex. genom behandling med bredspektrum antibiotika tidigt i livet, kan således få stora konsekvenser. En obalans i vår tarmflora är förknippad med många olika typer av ohälsa, inklusive allergi, metabola sjukdomar och cancer. Bakteriesläktet Lactobacillus (L.) innehåller flera stammar med probiotiska egenskaper. En tidig närvaro av laktobaciller i tarmen är exempelvis kopplat till en minskad allergiförekomst under barnaåren, medan förekomst av andra bakterier i tarmen tidigt i livet, t.ex. Staphylococcus (S.) aureus, har associerats både till en ökad och minskad allergirisk. Vår forskning har tidigare visat på tydliga samband mellan den mycket tidiga tarmflorans sammansättning, immunologisk profil och funktion under barnaåren och allergiutveckling. I min studie har jag undersökt hur utsöndrade ämnen från L. reuteri och S. aureus påverkar inflammatoriska och regulatoriska immunsvar in vitro. Jag har studerat effekterna av bakterieämnena på dendritiska celler, T celler och tarmepitelceller. Interaktionen mellan de dendritiska cellerna i tarmen, tarmepitelet och tarmfloran tros vara centrala för vilken typ av T cells svar som initieras vid efterföljande interaktioner mellan dendritiska celler och T celler. I Studie I kunde vi visa att S. aureus inducerar produktion av ett flertal inflammatoriskt aktiva cytokiner i immunceller (T celler), en produktion som dämpades av laktobaciller. Vi såg också att S. aureus också inducerade inflammatoriska svar hos tarmepitelceller, något som laktobacillerna inte kunde påverka, vilket tyder på att immunceller och epitelceller är olika reglerade. I Studie II undersökte vi hur mognadsprocessen hos dendritiska celler påverkades av närvaro av faktorer från L. reuteri och S. aureus, samt hur dessa olika typer av dendritiska celler sedan påverkade T celler att producera cytokiner. Här kunde vi se att laktobacillerna hade en tydlig effekt på de dendritiska cellernas fenotyp och funktion, men förhållandevis liten effekt på T cells svar, medan det omvända förhållandet gällde för S. aureus-exponerade dendritiska celler. i I Studie III, undersökte vi skillnaden mellan olika typer av dendritiska celler med avseende på gen- och proteinuttryck samt förmåga att inducera regulatoriska T celler. Här kunde vi se att dendritiska celler av den typ som anses finnas i tarmen, var mer ”tolerogena” och var bättre på att inducera regulatoriska T celler än vanliga dendritiska celler. Även här hade laktobacillerna reglerande effekter och förstärkte den ”tolerogena” fenotypen hos de dendritiska cellerna. Sammantaget har jag visat på betydande effekter av dessa två typer av bakterier på såväl epitel som immunceller, och framförallt dendritiska celler. Resultaten från mina studier är i linje med en immunreglerande roll hos laktobaciller. De effekter jag har observerat skulle kunna vara en bakomliggande orsak till varför förekomst av laktobaciller i tarmen tidigt i livet är associerat till en regulatorisk immunologisk profil och en minskad allergirisk hos barn. ii SUMMARY The human gut harbors a vast number of microbes. These microbes are not passive bystanders; rather they are actively participating in host metabolic activity, protecting the host from infection and maintaining the gut mucosal layer. Moreover, a vast number of clinical and experimental findings indicate the importance of microbes in modulating the immune system. We have previously shown that early colonization with lactobacilli and Staphylococcus (S.) aureus differentially associates with allergy development and/or immune profile at early ages. Here we focus on understanding how these microbes modulate the response of intestinal epithelial cells and immune cells in vitro. In paper I, we investigated the impact of UV-killed and/or cell free supernatant (CFS) of different Lactobacillus (L.) species and S. aureus strains on cytokine production from intestinal epithelial cells (IEC) and immune cells. Enterotoxinexpressing S. aureus 161:2-CFS triggered CXCL1/GROα and CXCL8/IL8 production by IEC. S. aureus-induced CXCL8/IL8 production was hampered by MyD88 gene silencing of IEC, indicating the importance of TLR signaling. Further, lactobacilli-CFS and S. aureus-CFS were able to induce the production of a number of cytokines by peripheral blood mononuclear cells (PBMC) from healthy donors, but only S. aureus triggered T-cell associated cytokines: IL2, IL17, IFNγ and TNFα; which were dampened by the co-treatment with S. aureus and any of the different Lactobacillus strains. Flow cytometry of the stimulated PBMC further verified IFN-γ and IL-17 production by T cells upon treatment with S. aureus-CFS, which also induced CTLA-4 expression and IL-10 production by Treg cells. In paper II, we investigated the influence of CFS of L. reuteri and S. aureus on the differentiation of monocyte to DC and subsequently how the generated DC influence T cell response. DC generated in the presence of L. reuteri exhibited an increase in expression of surface markers (HLA-DR, CD86, CD83, CCR7) and cytokine production (IL6, IL10 and IL23), but had a decreased phagocytic capacity compared with conventional Mo-DC, showing a more mature phenotype. However, upon LPS stimulation, DC generated in the presence of L. reuteri-CFS displayed a more regulatory phenotype, with a reduced cytokine response both at mRNA and protein levels. On the contrary, DC generated in the presence of S. aureus-CFS resembled the control Mo-DC both at mRNA and protein expression, but SA-DC was more efficient in inducing cytokine production in autologous T cells. In paper III, we studied the influence of L. reuteri-CFS on the retinoic acid (RA)-driven mucosal-like DCs’ phenotype and function to modulate T regulatory cells (Treg) in vitro. DC generated in the presence of RA showed a mucosal-like regulatory-DC phenotype with its CD103 expression, high IL10 production and decreased expression of genes associated with inflammation (NFκB1, RELB and TNF). Further, treatment with L. reuteri-CFS enhanced iii the regulatory phenotype of RA-DC by increasing the production of several chemokines, such as CXCL1, CXCL5, CCL3, CCL15 and CCL20, which are involved in gut homeostasis, while dampening the expression of most chemokine receptor genes. L. reuteri-CFS also increased CCR7 expression on RA-DC. RA-DC co-cultured with T cell increased IL10 and FOXP3 expression in Treg. However L. reuteri-CFS pre-conditioning of the RA-DC did not improve the Treg phenotype. In conclusion, bacteria-CFS can have an impact on the response of IEC, differentiation and function of DC and, subsequently the T cell response, when taken together in the context of gut; these can have an impact on the health and disease of the host. iv LIST OF PAPERS This thesis is based on the three original papers listed below, which will be referred to by their roman numerals in the text. I. Haileselassie Y, Johansson MA, Zimmer CL, Björkander S, Petursdottir DH, Dicksved J, Petersson M, Persson J-O, Fernandez C, Roos S, Holmlund U, Sverremark-Ekström E. Lactobacilli regulate Staphylococcus aureus 161:2-induced pro-inflammatory T-cell responses in vitro. Plos One 2013 8(10): e77893. II. Haileselassie Y, Navis M, Vu N, Qazi KR, Rethi B, Sverremark-Ekström E. The influence of Lactobacillus reuteri 17938 and Staphylococcus aureus on monocyte differentiation to DC in vitro. 2016; Submitted. III. Haileselassie Y, Navis M, Vu N, Qazi KR, Rethi B, Sverremark-Ekström E. Postbiotic modulation of retinoic acid imprinted mucosal-like dendritic cells by probiotic Lactobacillus reuteri 17938 in vitro. Front. Immunol. 2016 7(96);| doi: 10.3389/fimmu.2016.00096. v LIST OF PAPERS (not included in the thesis) The following original articles are relevant but not included in this thesis. IV. Holmlund U, Amodruz P, Johansson MA, Haileselassie Y, Ongoiba A, Kayentao K, Traorè B, Doumbo S, Scholin J, Doumbo O, Montgomery SM, Sverremark-Ekström E. Maternal country of origin, breast milk characteristics and potential influences on immunity in offspring Clinical & Experimental Immunology 2010; 162(3): 500-9 V. Johansson MA, Saghafian-Hedengren S, Haileselassie Y, Roos S, Troye-Blomberg M, Nilsson C, Sverremark-Ekström E. Early gut bacteria associate with IL-4, IL-10 and IFN-γ production at two years of age. PLoS ONE 2012; 7(11): e49315. VI. Khan Mirzaei M, Haileselassie Y, Navis M, Cooper C, Sverremark-Ekström E, Nilsson AS. Morphologically distinct Escherichia coli bacteriophages differ in their efficacy and ability to stimulate cytokine release in vitro. Front. Microb. 2016: Provisionally accepted) ( vi TABLE OF CONTENT POPULÄRVETENSKAPLIG SAMMANFATTNING............................................................. i SUMMARY ................................................................................................................................... iii LIST OF PAPERS ......................................................................................................................... v TABLE OF CONTENT .............................................................................................................. vii ABBREVIATIONS ...................................................................................................................... ix GENERAL INTRODUCTION .................................................................................................... 1 INTESTINAL EPITHELIUM ..................................................................................................... 2 GUT ASSOCIATED LYMPHOID TISSUES (GALT) ............................................................ 3 Peyer’s patches (PP) .................................................................................................................... 3 Cryptopatches (CP) and Isolated lymphoid follicles (ILF) ........................................................ 4 Intestinal Lamina propria (LP) .................................................................................................... 4 Mesenteric lymph node (MLN) .................................................................................................. 5 Innate immune system ................................................................................................................. 5 Intestinal dendritic cells (DC) ............................................................................................. 6 Intestinal macrophages (MФ) ............................................................................................. 7 Intestinal Monocytes ........................................................................................................... 8 Intestinal eosinophils ........................................................................................................... 9 Intestinal neutrophils ......................................................................................................... 10 Intestinal Mast cells........................................................................................................... 10 Adaptive immune system .......................................................................................................... 10 Conventional T cells in the intestine ................................................................................ 11 CD4+T cells in the intestine ......................................................................................... 11 CD8+ T cell in the intestine.......................................................................................... 13 Intestinal B cells ................................................................................................................ 13 Unconventional T cells and innate lymphoid cells in the intestine.......................................... 14 COLONIZATION OF THE GUT ............................................................................................. 15 Major factors that influence the microbiota composition ........................................................ 16 Methods for studying microbial composition in the gut .......................................................... 18 MICROBIOTA-HOST INTERACTION ................................................................................. 19 Microbiota-epithelial cell interaction ........................................................................................ 20 Epithelial cell-regulation of immune cell function ................................................................... 22 Microbiota-immune system interaction .................................................................................... 23 Effects of microbiota on mucosal immune cells .............................................................. 23 vii Effects of microbiota on systemic immune function ....................................................... 25 Lactobacilli and Staphylococcus aureus in the gut .................................................................. 25 The therapeutic use of lactobacilli as probiotics, prebiotics or postbiotics ............................. 26 PRESENT STUDY ...................................................................................................................... 28 General aim:............................................................................................................................... 28 Specific aims:............................................................................................................................. 28 Material and Methods ................................................................................................................ 29 Results and Discussion .............................................................................................................. 31 General conclusion .................................................................................................................... 40 FUTURE PERSPECTIVES ....................................................................................................... 41 ACKNOWLEDGEMENT .......................................................................................................... 43 REFERENCES ............................................................................................................................ 47 viii ABBREVIATIONS AMP APC APRIL BAFF BCR CBMC CFS CP DC FAE GF IBS IEL IFN Ig IL IDO IEC ILC ILF LI LP LPS LTi MФ MHC MAMP NEC NFκB NK cell NOD PBMC PGN PP PRR RA RORγt SI Tc cell Th cell TLR TNF Treg cell anti-microbial peptides antigen-presenting cell a proliferation-inducing ligand B-cell-activating factor B cell receptors cord blood mononuclear cells cell free supernatant crypt patches dendritic cells follicular associated epithelium germ free irritable bowel syndrome intraepithelial lymphocytes interferon Immunoglobulin interleukin indoleamine 2,3-dioxygenase intestinal epithelial cells innate lymphoid cells isolated lymphoid follicles large intestine lamina propria lipopolysaccharide lymphoid tissue inducer Macrophage major histocompatibility complex microbial-associated molecular pattern necrotizing enterocolitis nuclear factor-κB natural killer cell nucleotide oligomerization domain peripheral blood mononuclear cells peptidoglycan Peyer’s patches pattern recognition receptor Retinoic acid retinoic-acid-receptors γt small intestine T cytotoxic cell T helper toll-like receptor tumor necrosis factor T regulatory cell ix GENERAL INTRODUCTION The human gastro-intestinal tract is in direct contact with the external environment. It continuously encounters dietary products, environmental antigens, pathogens and commensal microbes. Given the huge antigenic load, a balance needs to be maintained between immunogenic and tolerogenic immune responses in the gut (1). The responsibility of conducting the appropriate response relies on the gut associated lymphoid tissues (GALT) (2). GALT is the largest immune organ, rich in both innate and adaptive immune cells. The innate cells consist of macrophages (MФ), mast cells, neutrophils, eosinophils and dendritic cells (DCs). They provide an immediate relatively non-specific response towards microbial products, by recognizing microbe associated molecular patterns (MAMP). Compared to innate immune cells, adaptive immune cells, which include B cells and T cells, are slower to act and recognize specific antigens, rather than common patterns. They respond by secreting antibodies, by cytolytic killing and/or by secreting cytokines that could facilitate in clearing out the pathogens (3). There is a strong intercommunication between the innate and adaptive immune cells. The innate immune cells trigger the adaptive immune response by processing and presenting antigens to adaptive immune cells, while cytokines and antibodies secreted by the adaptive immune cells regulate the phagocytic and killing activity of the innate immune cells. In addition to the hematopoietic cells, in the gut, there is a single layer of epithelium that lines between the luminal content and the immune cells. The epithelium not only serves as a physical barrier, but it also consists of epithelial cells and paneth cells which are involved in innate immune responses in the gut by secreting biological factors such as antimicrobial products and cytokines (4). As mentioned above, there is a load of luminal antigens in the gut that can interact with the immune cells. Most importantly, the commensal microbes play a crucial role in regulating the immune response of the host, interactions that could impact future health. As the focus of my PhD was to understand the interaction of these microbes and host immune responses, I will start by giving a background on the mucosal immune system in the gut, followed by a summary of recent findings on the microbial composition in the gut, its impact on host immune response and development and finally with a discussion about possible therapeutic benefits from these microbes. 1 INTESTINAL EPITHELIUM The intestinal epithelium is the largest surface area of the host, which is highly exposed to several types of foreign substances; including dietary products, environmental antigens, pathogens and commensal microbes (1). It is a single layer composed of polarized epithelial cells connected to each other by tight junction proteins, such as occludin, claudins, and zonula occludens, which regulate the paracellular transport of molecules (5). The intestinal epithelial cells (IEC) are heterogeneous. The stem cells of the epithelium, which are located at the bottom of the crypt, give rise to enterocytes, enteroendocrine-, goblet- and Paneth cells (4). The enterocytes are the most abundant in the epithelium and their main function is to serve as absorptive cells that control the transport of nutrients from the lumen into the body (1). In addition, enterocytes are equipped with poly-immunoglobulin (Ig) receptors that enable them to shuffle antibodies, such as IgA and IgM to the lumen. Paneth cells are present at the bottom of the crypt of the small intestine (SI), but are absent in the colon (6). They produce antimicrobial factors, such as defensins, lysozyme and phospholipases, which protect the host from invasive pathogens and uncontrolled expansion of the microbes in the gut (7). In the colon, antimicrobial peptides are secreted by enterocytes (4). Goblet cells are found throughout the epithelium, but more abundant in the colon (6). Their main function is to produce mucin, which is the building block of the mucous layer that serves as a barrier, but they also produce lysozyme and antimicrobial peptides (8). In addition, recent findings have indicated that goblet cells contribute in transporting antigens from the lumen to the underlying immune cells, such as dendritic cells (DC) (9) (10). Enteroendocrine cells are found throughout the epithelium, but less abundant in the colon. They play a major role in regulating the digestive function by secreting hormones. Enteroendocrine cells produce neurohumoral factors, such as substance P, which is responsible for inducing vomiting as a means of innate defense (11). The IEC express pattern recognition receptors (PRR) that enable them to sense conserved MAMP on gut microbes and respond by secreting cytokines and antimicrobial peptides in response to the interaction (7). IEC can express major histocompatibility complex (MHC) class I and II, and CD1d on their surface, which are important for antigen presentation (4) (12); however, they lack the expression of co-stimulatory molecules. The presence of MHC molecules on IEC help them to interact with intraepithelial lymphocytes (IEL), which are found in the epithelium (6). The IEL are found lying between epithelial cells (13). They are composed of heterogeneous effector T cells, which are mainly CD8+. IEL contribute in host defense and epithelial barriers’ maintenance. In addition, there are DC and MФ that resides between the epithelial cells, which 2 can directly sample luminal antigen without disrupting the epithelial barrier (14) (15). Moreover, the intestinal epithelium works closely with the underlying gut associated lymphoid tissues (GALT) to provide an efficient immune system (Figure 1). Figure1 Schematic representation of the intestinal immune system: A single layer of IEC separates luminal content from the underlying immune cells. There are goblet cells, enteroendocrine cells and paneth cells along the epithelium. The intestinal lumen contains nutrients, commensal bacteria and secretory IgA. Goblet cell-produced mucus layer covers the apical side of the epithelium. Beneath the IEC, effector immune cells are scattered sparsely throughout the lamina propria and epithelium. IEL and APC localize between the IEC. Specialized epithelium termed follicle-associated epithelium and M cells overlie the Peyer's patches. Reprinted with the permission from the Nature Reviews Immunology (200). GUT ASSOCIATED LYMPHOID TISSUES (GALT) The GALT is the primary immune organ in the mucosa of the gut as well as the largest immune organ. It is composed of both innate and adaptive immune cells, and encompasses 70% of the body's immunocytes (2), and can be divided into two parts consisting of inductive and effector sites. The inductive sites consist of: Peyer’s patches (PP), crypt patches (CP) and isolated lymphoid follicles (ILF). The effector sites include lymphocytes scattered throughout the epithelium, and the lamina propria (LP). Peyer’s patches (PP) PP are macroscopic lymphoid aggregates, which are distributed in the submucosa along the length of the SI (2). PP consist of B cell follicles and T cell areas, which are surrounded by a single layer of a particular epithelium, the follicle-associated epithelium (FAE) that separates 3 the GALT and the luminal microenvironment. The FAE clearly varies from the regular epithelium in the intestine. It has fewer and shorter microvilli and encompasses specialized M cells (for microfold) (16). These M cells are efficient in transporting luminal antigens towards the underlying immune cells, e.g. the DC, which in turn present the antigens to naïve lymphocytes in the PP. Once activated, the lymphocytes migrate to the draining mesenteric lymph nodes (MLN), an intersection between the peripheral and mucosal immune system, where they undergo further differentiation and maturation before they migrate into the systemic blood stream via the thoracic duct. Cryptopatches (CP) and Isolated lymphoid follicles (ILF) It was Kanamori and colleagues who first identified cryptopatches (CP) in the murine intestinal wall, as aggregates of innate lymphoid tissue cells surrounded by DC (17). In addition, CP harbors cells expressing stem cell growth factor receptor (C-kit) and they are negative for lineage markers. It was long believed that CP are absent in humans (18), but this idea was later challenged by Lügering and colleagues (19). CP has been considered to be essential for the generation of IEL in the epithelium, supported by the finding that tissue grafting of lin- C-kit+ CP cells into mice increased the number of IEL (20). However, the importance CP in the generation of IEL was later proved to be dispensable (18). The other inductive site in the GALT is the ILF. ILF are microscopic and found in the mucosal surface of both SI and large intestine (LI). Unlike CP and PP, ILF are devoid in germ free mice, which can indicate that ILF are microbiota-induced structures (21). ILF comprise B cell follicles, encompassed in an epithelium containing M cells (22). Intestinal Lamina propria (LP) The LP is the effector site of the intestinal immune system. The LP contains a loosely packed connective tissue that makes the scaffolding for the villus, which also contains blood supply, lymph drainage and nervous supply for the mucosa (6). In addition, the LP contains effector lymphocytes (B cells and T cells) and numerous innate immune cells (DC, MФ, eosinophils and mast cells). DCs and MФ in the LP can directly sample luminal antigen (23) (14). The overlying epithelium can also transfer luminal antigens to DC, which can then upregulate their chemokine receptor and migrate to the mesenteric lymph node (MLN) to present the antigen to naïve lymphocytes in the PP (9). 4 Mesenteric lymph node (MLN) MLN play a crucial role in the induction of mucosal immune responses. They are located within the layers of the mesentery of the intestine. MLN are connected to the PP and LP via afferent lymphatics (2). Lymphocytes from the circulation enter into the MLN via high endothelial venules (24). Like other lymph nodes, MLN has an inner medulla and an outer cortex that are enclosed in the fibrous capsule. The cortex contains B cell follicles and T cell area. The medulla is rich in MФ and plasma cells. Lymphocytes leave the MLN to the bloodstream via efferent lymphatic vessels in the medulla that are connected to the thoracic duct. Innate immune system The gastrointestinal tract has multiple layers of protection to prevent the pathogenic and commensal microbes from translocating into the body and elicit an excessive immune response. Together, the mucus layer and the single layer of the epithelial cells constitute a first physical barrier. However, since the epithelial layer of the intestine is the site of the body where the nutrients are taken up, it is semi-permeable, which make the mucosal site of the intestine at risk of invasion by microbes. Nonetheless, the mucosa of the intestine contains antimicrobial peptides that contribute to the innate immune response as a chemical barrier. If the microbe evades these protections, it will encounter the cellular component of the innate immune system, which includes monocytes, MФ, DC, mast cells, basophils, neutrophils, and eosinophils. Innate cells recognize patterns common to most microbes known as MAMP through their pattern recognition receptors (PRR) which are expressed in a secreted form, bound to the membrane of cells, or in intracellular compartments. The innate cells, depending on the signal, respond rapidly, either by killing using engulfing (phagocytosis) or secreting soluble factors (such as cytokines or antimicrobial factors). The major PRR are the TLR and the intracytoplasmic nucleotide-binding oligomerization domain (NOD) receptors. 13 murine and 10 human TLR have already been identified (3) (25). Each TLR shows specificity by recognizing distinct MAMP. For instance, TLR4 recognizes lipopolysaccharide (LPS), a molecule found in the outer membrane of Gram-negative bacteria, whereas TLR2 recognize peptidoglycan and lipoteichoic acid (LTA) from Gram-positive bacteria. However, a TLR can also form a hetrodimer with another TLR, which increases the PRR repertoire (26). Another group of 5 membrane-bound PRR are C-type lectin receptors (CLR). CLRs, such as DC-SIGN, are expressed by innate cells and interact with a wide array of microbes through the recognition of mannose and fucose carbohydrate (27). NOD receptors are intracellular receptors that recognize components of bacterial peptidogycan (28) (29). For instance, NOD2 recognize muramyl dipeptide, a peptidoglycan motif, which is common to both gram-positive and gramnegative bacteria. In the intestine, the PRR signaling is not only necessary to clear potential pathogens, but it also has other advantages, such as maintenance of IEC barrier integrity, production of antimicrobial proteins, IgA production and secretion to the lumen (28), which will be discussed in detail in later sections. Intestinal dendritic cells (DC) In 1973, Ralph M. Steinman and Zanvil A. Cohn were the first to describe dendritic cells (DCs) (30). For his notable contribution Steinman received the Nobel Prize in Medicine 2011 “for his discovery of the DC and its role in adaptive immunity”. A lot has progressed since then and DCs are now identified as the most crucial antigen presenting cells (APCs) that can migrate in afferent lymphatics and prime naïve T cells (7) (31). The function of DC is not limited to their interaction with T cells; they have a multifunctional duty in orchestrating efficient immune responses. The DC in the intestine are identified by their expression of CD11c. They are further divided into two major subsets the CD103+ DC and CD103- DC. CD103 (αE integrin) is expressed by the majority of lamina propria (LP) DC. These DC are efficient in migrating to the MLN via the afferent lymph (32-34). In the steady state, the CD103+ DC migrate constitutively from the LP to MLN to establish appropriate T cell responses (35) (34). The other hallmark of CD103+ DC is its ability to synthesize retinoic acid (RA) (36-38), which is responsible for the generation of gut-homing Treg (35) (39). CD103+ DC also express indoleamine 2,3-dioxygenase (IDO), an enzyme that is involved in driving Treg development in the gut (40). These are the key factors that make CD103+ DC forefront runners in mediating immune tolerance in the gut. The CD103+ DC lack the receptor CX3CR1, which hampers their ability to sample luminal antigen in the intestine. Therefore, they are dependent on the cells in their vicinity. For instance, CD103+ LP DC receives antigens absorbed by goblet cells (9) and IEL (32). Intestinal CD103+ DC can be further subdivided into CD103+CD11b+ and CD103+CD11b- DC (41). CD103+ CD11b+ DC drive Th17 and Th1 cell differentiation (42) 6 (43), whereas CD103+ CD11b- DC facilitate Th1 polarization and IFNγ-production from CD8+ T-cells in the gut (43) (44). However, both subsets are efficient in inducing Treg responses (45). CD103- intestinal DC can trigger both Th17 and Th1 at steady state (44). The expression of CX3CR1 enables these DC to sample luminal antigens. For a long time, it was believed that this population cannot migrate to the MLN. That is one of the reasons why they are considered to be macrophage (MФ). However, it was possible to collect these populations along with CD103+ DC from the thoracic duct, which was directly connected to the lamina propria of the intestine via ``pseudo afferent lymph´´ that was re-anatomized by removing the MLN (32). This indicated that there are CD1013- DC that can migrate from the lamina propria to reach the MLN. Different factors in the gut such as the microbes, dietary factors, both soluble and membrane bound molecules from intestinal epithelial cells and other cells in the gut can shape the properties and functions of DC (7). Intestinal macrophages (MФ) In mucosal immunology, intestinal MФ are given less attention, which is partly due to the general consensuses that they do not migrate to the MLN. However, MФ are involved in various essential immune functions that enable them to contribute in balancing immune tolerance and responding to appropriate immune responses depending on the circumstances (46) (47). In mice, intestinal MФ resemble DC in the expression of MHC Class II, CD11c, and CD11b, but differ in expressing F4/80, CD68 and CD64, which can be used to distinguish them in the gut. It is also now evident that majority of MФ in the gut highly express CX3CR1, which implicates that they indeed can sample luminal antigens (14). The main function of residential MФ in the intestine is to keep the local site inflammatory response at bay. They express low levels of co-stimulatory molecules including CD80, CD86 and CD40 (43) (45). Under steady conditions, residential MФ, just like intestinal DC, are less responsive to stimulation with TLR ligands (35) (48). In addition to maintaining intestinal tolerance, MФ in the gut constitutively produce the anti-inflammatory cytokine interleukin IL10 (49) (50). IL10 secreted by MФ plays a critical role in maintaining FOXP3 expression on Treg cells (51). Furthermore, CX3CR1+ MФ in the mucosal LP is crucial for the expansion and differentiation of FOXP3+ Treg cells, which is important to develop oral tolerance to food antigens (52). Intestinal MФ also play a major role in maintaining epithelial barrier integrity by secreting prostaglandin E2, which favors the proliferation and survival of epithelial progenitors (53). 7 Based on their level of CX3CR1 expression and distinct function, intestinal MФ are classified as either CX3CR1hi MΦ or CX3CR1int MΦ. CX3CR1hi MΦ function as regulatory MΦ (35) and they constitutively produce IL10 and are less responsive to TLR stimulation (46). Unlike the CX3CR1hi MΦ, the CX3CR1int MΦ is more TLR-responsive and show more of a pro-inflammatory phenotype, and it has been shown that they accumulate during experimental colitis (54). Both CX3CR1hi MΦ and CX3CR1int MΦ arise from a common progenitor, Ly6C+CCR2+ monocytes (55). Ly6C+ CCR2+ monocytes first partially differentiate to CX3CR1int MΦ, which later, depending on the surrounding micro-environment, mature into CX3CR1hi MΦ. Ly6Chi monocytes were originally considered as ‘inflammatory’ monocytes due to their efficient migration into inflamed tissue and their vigorous response to TLR ligands in vitro (46). Therefore, it was surprising to find that they are also the source of the antiinflammatory pool of MΦ. Intestinal Monocytes Monocytes are important in initiating immune responses by secreting cytokines and phagocytosis. They are also considered to be precursors of MФ and DC (56). In mice, monocytes develop from the common MФ and DC progenitor. There are two major populations of monocytes in mice: the Ly6c+ (or Gr1+) monocyte and the Ly6clo (or Gr1lo) monocytes (57). There is a clear indication of a developmental link between Ly6clo monocytes and Ly6c+ monocytes, with the latter developing into Ly6clo monocytes driven by CSF1Rdependent signal (58) (59). The Ly6c+ monocytes are known to express high levels of CCR2, but low levels of CX3CR1 (60) (61). Ly6c+ (or Gr1+) monocyte migration from the bone marrow to the intestine is CCR2 ligand dependent, as mice lacking CCL2 (CCR2 ligand) or CCR2 have reduced circulatory monocyte- and macrophage pools in the intestine (62) (63). The cells that secrete CCL2 to attract the monocytes into the intestine are unknown. It has been shown that monocytes can be recruited to the intestine upon the interaction of bacteria and IEC (64), possibly suggesting (although not studied) that IEC could be the cellular source of CCL2 in the intestine. Due to their abundance in inflamed tissue, Ly6c+ monocytes were originally considered as ‘‘inflammatory’’ monocytes, but are now termed ‘classical’ monocytes. Ly6clo monocytes, on the other hand, are positive for CX3CR1, but lack CCR2. They were originally considered as the precursors of steady state MФ (61). However, recent findings clearly indicate that their primary functions are phagocytosis and maintenance of the 8 vasculature in the blood stream. Ly6clo monocytes are commonly known as the non-classical monocytes (57) (65). The human equivalent of the Ly6c+ monocytes and Ly6clo monocytes are the CD14hi CD16- monocytes and the CD14lo CD16+ monocytes, respectively (66). Similar to the mice monocytes, their human homologous subsets are developmentally related. In addition, like the Ly6c+ monocytes, the CD14hi CD16- monocytes express CCR2, while the CD14lo CD16+ monocytes lack CCR2 expression in the same manner as Ly6clo monocytes. Moreover, there are similarities in gene expression profiles between the murine and human homologous subsets (67). Monocytes recruited to the intestine can have deleterious effect, depending on the local microenvironment. During Crohn’s disease associated intestinal inflammation, CD14hi monocytes and their progeny have increased production of TNFα, IL1β (68) as well as increased respiratory burst activity (69), exacerbating the disease condition. On the other hand, monocytes play a regulatory role in the intestine, as they produce IL10 and arginase - a molecule associated with immune suppression (70). In addition, monocyte derived prostaglandin E2 has been shown to inhibit neutrophil-induced inflammation. Moreover, CCR2-deficient mice, which lost the recruitment potential of the Ly6c+ monocytes into the intestine, have increased accumulation of neutrophils in the intestine, during Toxoplasma infection (71). Intestinal eosinophils Eosinophils are granulocytes with an active role in inflammatory conditions such as allergy, inflammatory bowel disease and worm infection. Eosinophils were considered to be present in the gut only in pro-inflammatory conditions and absent in steady state; however it has now been shown that eosinophils substantially contribute to the myeloid population isolated from the healthy murine intestine (30%) (46). Eosinophils, like DC and MФ, can express CD11c, CD11b, and F4/80 (72) (73). Therefore, they might have gone unnoticed and even been considered as DC and MФ in the past. However, due to the advancement of multi-color flow cytometry, it is possible to include more markers to clearly distinguish eosinophils among gut myeloid cells. In intestinal cell preparations, eosinophils can be identified by their high sidescatter profile, and their expression of CCR3 and Siglec-F (72) (74). Apparently gut eosinophils contribute to gut homeostasis as they influence epithelial cell-renewal and intestinal barrier integrity (6). They can also produce cytokine and soluble factors that can modulate and 9 maintain resident DC and MФ. In addition, the mucosal eosinophils can also contribute to the regulation of the IgA class switching in PP and the survival of the IgA+ plasma cells in the LP. These effects of eosinophils are partly mediated by their ability to produce transforming growth factor-β (TGFβ)-activating metalloproteinases that convert latent TGFβ into its active form (75). Intestinal neutrophils Neutrophils are also granulocytes, abundantly found in the circulation where they constitute 50 – 60 % of the circulating leukocytes (76). They are mainly recruited to the intestine in response to inflammation by the production of CXCL8 and chemokines by epithelial cells and resident monocytes/macrophages (76-78). The main function of neutrophils in the gut is to eliminate luminal microbes that invade the underlying mucosa. They have the capability to produce antimicrobial peptides and to phagocytose microbes (79), and can also produce neutrophils extracellular traps, which sequester the microbe inside and thereafter kill the microbe with toxic molecules from intracellular granules (80). In addition, neutrophils produce significant amounts of cytokines such as CXCL8 and IL10 and metalloprotinases that can modulate the activity of the cytokines (76) (81) (82). Intestinal Mast cells Mast cells are also bone marrow derived leukocytes. They are found in abundance in healthy gastrointestinal tracts, mainly in the LP and submucosa but also a few in the epithelium (6). They do express TLR that enables them to detect microorganisms (83), but they are also important in maintaining the intestinal barrier integrity. In addition, mast cells seem to have distinct functions at different intestinal sites. In the SI, mast cells, in response to TGFβ, produce proteases that are involved in tissue remodeling (84), while in the colon, mast cells exhibit a more pro-inflammatory phenotype, which shows that the local micro-environment have an impact on the response of the mast cells (6) (85). Adaptive immune system The adaptive immune system is known for its ability to provide antigen specific response. Also, the ability to develop immunological memory enables adaptive immune cells to identify and respond quickly and efficiently to a microbe that they encountered in the past. T10 and B cells are the primary cellular component of the adaptive immunity. They express a highly diverse repertoire of receptors that enable them to recognize virtually any specific antigen and play a significant role in the mucosal immune response of the intestine. Conventional T cells in the intestine T cells are lymphocytes that develop from hematopoietic progenitor cells in the bone marrow and mature in the thymus. After a series of selection and maturation processes in the thymus, two major subsets of conventional T cells arise, namely the T (Th) helper cells and the T (Tc) cytotoxic cells (86). Both types T cells have αβ-T cell receptors on their surface, which enables Tc cells and Th cells to recognize antigen presented in the context of major histocompatibility complex (MHCI or MHCII) on the surface of either infected cells or APC. Generally, Th- and Tc cells can be distinguished by their expression of expression of CD4 and CD8, respectively. In addition, they differ in their main functions; the Th cells enable the initiation of both humoral and cell-mediated immunity by assisting the activation of immune cells such as B cells, Tc cells and MФ via cell-cell interaction and/or through release of cytokines, while Tc cells are involved in killing infected cells (particularly virus infected) and transformed and/or cancer cells. CD4+T cells in the intestine In the intestine, circulating naïve Th cells that express CD62L (L-selectin) and CCR7 migrate to the MLN and interact with APC, such as CD103+ DC and CD103- DC from the LP (87) (88). Upon interaction, the naïve Th cells differentiate into effector CD4+ T cells, which include, but are not limited to, Th1, Th2, and Th17 cells or Tregulatory cells (Tregs), depending on the cytokine milieu. IL12, produced by DC and MФ, will favor Th1 differentiation and the expression of the Th1 signature transcription factor, T-bet (31); while, IL4 promotes Th2 cell differentiation and expression of Th2 lineage transcription factor, GATA3 (89). The sources of IL4 in the intestine are the γδ T cells, innate lymphoid cells (ILCs) and eosinophils (87). For Th17 development, driven by the transcription factor retinoic-acid-receptor γt (RORγt), the driving forces are TGFβ along with IL1β, IL6 and IL23 produced by innate cells (90) (91). TGFβ also plays a vital role in driving FOXP3+ Treg development. In addition, RA from DC can promote Treg 11 differentiation (92) (36). Once activated, each subset of Th cells upregulate CCR7 and α4β7 to migrate to the effector site of the intestine (88). Th cell subsets are suggested to produce their specific cytokines and to have distinct functions. Th1 cells produce IFN-γ and are vital in clearing intracellular bacteria in the intestine (31). Th2 cells are characterized by the production of cytokines IL4, IL5 and IL13, and are necessary to clear extracellular pathogens, such as helminths (93). Th17 cells are characterized by secretion of cytokines IL17A, IL17F, and IL22. Th17 cells are vital for host defense against extracellular bacteria, viruses and fungi and, in barrier surface maintenance. IL17A can also promote epithelial cell secretion of G-CSF that drives neutrophil recruitment (94). Moreover, Th17-cell derived cytokines can drive the production of antimicrobial peptides (AMP) (βdefensin and Reg family) by epithelial cells (95-97). If not properly regulated, the different Th cell populations can also be involved in pathology. For example exaggerated Th2 responses are seen in allergic conditions and Th17 cells are also involved in inflammation associated with many autoimmune diseases, including IBD (98). Effector T cells in the intestine can overtly react towards antigens derived from the intestinal flora or the diet. However, Treg regulate the activity of effector T cells by expressing CTLA-4 receptor that could dampen the expression of co-stimulatory molecules, such as CD80 and CD86 on APC. In addition, Treg cells produce regulatory cytokines, such as IL10 and TGFβ. In addition to Th1, Th2, Th17 and Treg cells, there are additional Th cell subsets; namely Th9, Th22 and T follicular helper (Tfh) cells (99-102). Th9 cells secrete IL9 and are linked to protection against helminth infection, but also associated with the etiology of immune-mediated diseases, such as IBD, experimental autoimmune encephalomyelitis (EAE), and asthma (101). Th22 cells secrete IL22 and are important for the repair of the intestinal barrier and to protect the host against Gram-negative bacteria (100), while Tfh cells promote activation, differentiation, affinity maturation and survival of B cells by providing CD40 signal and secreting cytokines, including IL21 (102) (103). Furthermore, recent findings are challenging the rigid classification of Th cells subsets, based on their signature cytokines and function. For instance, Th17 cells have been shown to replace IL17- with IFNγ production in an EAE model (104). In our group, Sophia Björkander has shown that CD25+ FOXP3+ T cells that express the CLR CD161 can readily produce both IL17 and IFNγ upon activation (105). Moreover, FOXP3+ Tregs can co-express surface markers and transcription factors associated with the respective subsets of Th cells (106-109). Although, it still needs to be further demonstrated, these recent findings collectively indicate the plasticity in the Th cell lineage. 12 CD8+ T cell in the intestine In the effector site of the GALT, Tc cells constitute a minority of the conventional T cells (6). The main function of Tc cells is to eliminate virus infected cells or tumor cells using different mechanisms, which include degranulation and the release of toxic substances or by activating cell-surface death receptors on the target cell (110) (111). In addition, Tc cells can produce pro inflammatory cytokines like IFN-γ and TNF. In order to get activated, naïve Tc cells need TCR interaction with peptide loaded MHC I molecule presented by APC. Normally, Tc cells encounter endogenously processed peptides in the context of MHC I molecules. However Tc cells can also be activated by a process termed ‘cross-presentation’, which involves the presentation of exogenous antigens on MHC I molecules by DCs to CD8+ T cells (111). Emerging evidence suggests that there are Treg-like CD8+ suppressor T cells (112-115). As their CD4+ counterparts, they express CD25 and FOXP3 and have been shown to impose suppression in vivo in graft versus host disease model (GVHD), influenza infection and colitis. In vitro, they are as efficient as CD4+ Tregs in suppressing effector T cell proliferation (116). Intestinal B cells Unlike T cells, B cells finish their maturation in the bone marrow. In the secondary lymphoid tissues, B cells get activated and differentiate into antibody secreting plasma cells and memory cells upon antigen encounter. Naïve B cells express surface IgM and IgD, but after activation they can undergo a class switch to IgA, IgG or IgE, depending on the cytokine signal. For instance, in the gut, cytokines such as IL10 and TGFβ initiate IgA class switching and secretion of IgA into the lumen of the intestine (117). In addition, RA produced by CD103+ DC not only imprints gut homing markers on B cells, but also promote B cell secretion of IgA (118) (119). The intestinal LP harbors large numbers of plasma cells, with the number ascending from the most proximal to the distal end of the intestinal tract (120). In addition, IgA-producing plasma cells are the predominant population in the intestine (121) (6) and IgA secretion dominates the humoral immune response in the intestine. IgG and IgM are also produced in the intestine to a lesser extent (121). In the gut lumen, secretory IgA (sIgA) protects the epithelium from pathogen invasion by binding and clearing the pathogen (122). In a similar manner, sIgA prevents the evasion of the intestinal antigens such as intestinal microbiota and 13 food antigens into the circulation (35); and sIgA also helps to control the density and the composition of the microbes in the intestine (117) (123). B cells can also contribute as APC as well as produce cytokines. Regulatory B cells (Breg cells), produce IL10 and play a role in suppressing experimental colitis (124-127). Certain types of B-reg cells express the integrin αvβ6 and CX3CR1. These cells have the ability to produce high levels of TGFβ, convert latent TGFβ into its active form, maintain T-reg cell development and suppress T cell activation (128), which further supports the broad contribution of B cells in keeping the homeostasis of the gut. Unconventional T cells and innate lymphoid cells in the intestine Unconventional T cells and ILC cannot be categorized as innate or adaptive cells, as they share features with both innate and adaptive immune cells. Unconventional T cells are further sub-classified into mucosal associated invariant T (MAIT) cells, γδ T cells and NKT cells (129). Unconventional T cells undergo V(D)J recombination, similar to the conventional T cells, but unlike conventional T cells, they lack TCR diversity. MAIT cells are evolutionary conserved T cells expressing CD8 and V7.2 and recognize microbial non-peptidic antigens presented by the non-classical MHC MR1. They are primarily found in the circulation and in the liver, but can also be found in the gut, where they have been described to recognize vitamin metabolites (130). Activation of tissue resident γδ T cells differs from that of conventional T cells as well as from circulating γδ-T cells. They readily detect and respond to MAMPs, such as conserved phosphoantigens of bacterial metabolites and cell damage products. Also, they are not required to interact with MHC and they use other costimulatory molecule than conventional T cells (131). NKT cells express the NK cell marker CD56 and use their invariant TCR to recognize glycolipids when presented by CD1d (MHC-like molecule) on APC (132). Similar to Tc cells and NK cells, NKT cells primary functions are cytotoxicity, mainly via the FasL-Fas pathway, and production of cytokines (133). Recently, ILCs have been in focus of intense research interest in the field of mucosal immunology. They are believed to play a major role in GALT development, epithelial barrier repair and intestinal immunity (134) (135). Although the ILCs are derived from the same 14 lymphoid progenitor as T- and B cells, they lack TCR (6). ILC are heterogeneous populations that constitute lymphoid tissue inducer cells (LTi), NK cells, and other ILC members that are further classified based on their respective transcription factors and their cytokine profile (136). ILC1, which include NK cells, express T-bet and produce IFNγ. Most of the ILC1 cells, including NK cells, are capable of causing granule-mediated cytotoxicity by producing perforin and granzymes (137) (138). ILC1 are also associated with the pathogenesis of Crohn’s disease, as they are abundant in LP of the intestine from patients (139) (138). ILC2 express RORα and GATA3 and, produce Th2 associated cytokines, such as IL4 IL5 and IL13. Like Th2, they are associated with helminth infection and allergy. ILC3 cells, which also include LTi, express the transcription factor RORγt and, secrete IL17 and IL22. Most ILC3 cells reside in the mucosa of the intestine where they play a key role in keeping epithelial barrier integrity and immunity against extracellular bacteria (138) (140). COLONIZATION OF THE GUT Tightly protected by the amniotic membrane and the presence of the antimicrobial factors in the amniotic fluid, the fetus has been considered to be in a sterile environment in utero. However, this has been challenged by recent findings, which suggest microbial transfer from mother to fetus during gestation, as bacterial DNA has been detected in the placenta (141) and viable bacteria have been found in the meconium (142). Nevertheless, the main microbial colonization starts during delivery (143) (144) and continues to develop until it reaches a relative stability in adulthood. The early infant microbiota composition is highly dynamic and relatively low in diversity. It is dominated by facultative anaerobe species, such as Staphylococcus, Streptococcus, Enterobacteriaceae, Enterococcus, Bifiodobacterium and Lactobacillus. However, once the oxygen in the gut is consumed by these facultative anaerobes, the environment is more favorable for strict anaerobes, such as Bacteroides and Clostridia, to dominate in the first year of life (145). Eventually, the microbiota composition becomes successively more diversified (146); in adults the number of bacteria in the human microbiota is estimated to be 1014 consisting of 500-1000 different species (147) (148). The bacterial diversity and density along the gut vary, with the highest number of bacterial colonies residing in the colon. Also, the composition varies along the intestinal sites. In the colon, the Bacteroidetes and Firmicutes are the main colonizers, while in SI the Firmicutes, Bacteroidetes, 15 Proteobacteria and Actinobacteria phyla dominate (147) (148). The SI also contains an adherent microbiota, such as Streptococcus and Neisseria (147). Major factors that influence the microbiota composition The gut microbiota in humans seems to be plastic and different factors can affect its composition, particularly at the early age. Mode of delivery, diet, environmental factors, antibiotics use and host genetics are the main factors. 1. Mode of delivery The mode of delivery is crucial for the establishment of the postnatal intestinal microbiota. The early gut microbiota composition of vaginally born children resembles that of the maternal vagina microbiota, which is best typified by the presence of Lactobacillus and Prevotella (144) (149). On the contrary, those born with Caesarean section acquire maternal skin flora or environmental bacteria at an early age, which are represented by the dominance of Streptococcus, Corynebacterium, and Propionibacterium (144) (149). In addition, a recent study has also shown that Cesarean section-delivered infants lack Bacteroidetes in their gut, and the total microbial diversity is lower compared that of vaginally delivered infants (150). It has also been shown that the gestational age of the neonate can influence the composition of the infant microbiota. For instance, Proteobacteria is the main colonizer in the gut of the preterm infants that lack Bifidobacterium and Lactobacillus which are common genera in the gut of healthy term infants (151). 2. Diet Members of the microbiota have particular nutritional requirements that could determine their survival in the niche, making the diet important in shaping the early infant gut nicrobiota. For instance, there is a substantial difference in the gut microbial composition between breast-fed and formula-fed infants. The gut of breastfed infants contains staphylococci, streptococci, and lactic acid bacteria (152) (153). Moreover, oligosaccharides from breast milk favor the growth of Bifidobacterium species. Eventually with weaning, the gut microbiota is more diverse and relatively established throughout life and more related to the choice of diet (154). Globally, differences in dietary habits between different populations can cause variation in the intestinal microbiota composition. For example, the intestinal microbiota of children in 16 rural village in Burkina Faso was dominated by Bacteroidetes and lower in Firmicutes compared to age matched Italian children. Due to the agrarian life style in that area, the Burkina Faso children consume a plant-based diet, which is rich in cellulose and xylans. Members of the Bacteroidetes can utilize these fibers as a nutrient and generate higher levels of short-chain fatty acids (SCFA) (155). Acetate, butyrate, and propionate are the main SCFAs that result from fermentation of carbohydrates and amino acids in the diet (156). In general, intake of diet that includes fruits, vegetables and fibers are associated with dense and diverse gut microbiota. 3. Environmental factors In addition environmental factors, such as standard of living, exposure to pets, number of siblings and being raised in a rural area, can affect the exposure to different microbes, which could further shape the gut flora composition (157-159). Standard of living can contribute to striking differences in colonization patterns of infants from different countries. For instance, the gut microbiota diversity of infants from USA was lower than that of both Malawian infants and Venezuelan Amazonian infants. The differences were also evident at the species level and functional gene repertoires (160). Although general socio-economic differences can explain such differences, also genetic variation and dietary preferences might contribute as well. However, studies on genetically similar populations, such as Swedish and Estonian children also revealed variation in colonization patterns, supporting a strong environmental impact (161). The importance of environment was further strengthened with a study on genetically similar piglets that were kept in a natural outdoor environment, or housed in very hygienic indoor facility (162). Although there were no differences in microbial diversity, piglets reared outside had higher Firmicutes, particularly Lactobacillus strains in their gut compared to hygienic indoor piglets. 4. Antibiotics Significant changes in the gut microbiota in response to antibiotics include a significant reduction of indigenous commensal bacteria and diminishing the taxonomic diversity. The altered microbiota composition persists, even after ceasing to use antibiotics. For instance, it has been shown that short-term use (7 d) of broad-spectrum antibiotics (e.g., Clindamycin) can affect the diversity of Bacteroides in the gut, up to 2 years post-treatment (163) (164). In addition to the alteration of the normal gut microbial diversity, the persistent use of antibiotics paves the way for the dominance of antibiotic-resistant pathobionts (potential pathogens) in the gut (165). The other major concern is the likelihood of horizontal transfer of antibiotic 17 resistance gene to normal gut microbes. Therefore, it is important to take caution in the use of broad-spectrum antibiotics. 5. Host genetics In humans, a common method to investigate the correlation between host genetics and microbiota composition is by comparing the gut microbiota of monozygotic and dizygotic twins (166). A recent study from UK, showed that the abundance of many microbial taxa were influenced by host genetics (167). On the contrary, other studies involving metagenomics and 16S ribosomal RNA gene sequencing of the gut microbiota DNA from siblings, monozygotic and dizygotic twins in the US, Venezuela and Malawi showed that the genetic contribution was much less than expected and rather gave support for a strong environmental influence on microbiota composition (160). However, under the control of the non-genetic factors (for example diet), host genes associated with metabolism can play a role in shaping the microbiota as leptin-deficient obese mice have been shown to have altered gut microbiota (168) (169). Leptin-deficient mice have the tendency to over-eat, which may contribute to the change in their microbial community. It has also been shown that fasting in mice changed the microbial composition in the gut (170). In addition, host immune system, both innate and adaptive, can impose pressure by secreting AMPs and releasing IgA into the lumen (171). However, one should recall that this is the result of a cross-talk as some of the factors that trigger the immune system have been shown to be the microbes themselves (117) (123). Methods for studying microbial composition in the gut Most of our previous knowledge of the gut microbiota composition is derived from basic microbial culture techniques in the laboratory. Microbial culture is essential for the isolation of a pure culture of a bacterium to further characterize the individual phenotypes and functions, to study the interaction between the individual bacteria with the host in an in vitro or in vivo model and to isolate the biologically active molecules produced by the individual microbe. The shortcoming of this technique is that, due to their selective growth requirements, it is hard to cultivate the majority of the gut microbes in the laboratory. The use of culture-independent approaches has helped enormously to characterize microbial diversity. One of the efficient and affordable culture-independent methods is the use of 16S rRNA gene, 18 which is highly conserved among bacterial species. 16S rRNA genes contain bacteria conserved regions but also species-variable regions that allow identifying bacteria up to the species level. The technique involves extraction of DNA from samples, amplification of 16S rRNA gene using primers, followed by quantitative real-time PCR detection and quantification to reveal bacterial identity (172). Still, 16S rRNA gene-based analysis also has its shortcomings. Some bacteria may have several copies of 16S rRNA genes, which makes it difficult during sequence annotation (173). Another limitation of 16S rRNA gene based analysis is the variability in extraction of DNA from different bacteria. For example, DNA can be extracted easily from Gram-negative bacteria, such as Bacteroides and Enterobacteriaceae, while some Gram-positive bacteria, such as Enterococcus and Staphylococcus, have very thick and strong cell walls, which makes it difficult to access the DNA of these bacteria (163). Therefore, sequencing of the 16S rRNA gene is being replaced by new high-throughput sequencing methods that allow sequencing the entire nucleotide pool (165). These techniques have enabled scientists in the field to have a broad view on the diversity of the microbiota community in the gut (174). Understanding the composition of the microbial community alone does not necessarily give a full picture of its function. Using a high throughput next generation sequencing, it is now possible to sequence the total microbial community DNA, and even compare the sequences to already identified functional genes. However, unless it is supported by a proteomic or metabolic study, evidence on functional capabilities from metagenomic studies will remain to be a prediction. Therefore, the way forward is to include functional profiling, which encompasses protein and metabolite profiling (175). MICROBIOTA-HOST INTERACTION In the interlinked mutualistic relationship of the gut microbiota and the host, the host provides niches and nutrients, which are vital for the existence of the microbes in the gut. On the other hand, the microbes contribute to the digestion and fermentation of indigestible carbohydrates, production of vitamins, organogenesis and protection against pathogens. In addition, commensal microbes are believed to influence the development and response of the host immune system (176). The interaction between the microbiota and the host is complex. It involves the individual microbes, the epithelium and the mucosal and systemic immune system (Figure 2) 19 (177). The microbe can bind to the epithelium and directly interact with the epithelium, leading to the response of the epithelium, which will further impact the underlying immune cells (178). It can also directly interact with members of the immune cells, such as DC and MΦ, which can protrude through the epithelium and sample the luminal antigen (23) (14). In addition, molecules produced by the microbes make their way through the tight junction of the epithelium, especially in a leaky-gut situation, or taken up through transcytosis, as observed by M cells and goblet cells (6) (9). The microbiota can also impose its impact in a diet-dependent manner, i.e., the microbe acts on the nutrient present in the intestine, leading to the formation of metabolites, such as, indole-3-aldehyde (IDO) and SCFA, which have the potential to modulate the immune system. The next sections will focus on the impact of these interactions (179-181). Figure 2. Examples of gut microbiota-IEC-immune cells interaction. Clostridium clusters IV, XIVa, and XVIII promote Treg cell differentiation in the colon by inducing TGFβ production in IEC. SCFA derived from microbes may also facilitate Treg cell generation either direct interaction through GPCR 43 or indirectly via IEC. Bacteroides fragilis derived Polysaccharide A (PSA) directly induce Treg cell differentiation via TLR2 or indirectly by modulating DC. SFB promote Th17 differentiation either via serum amyloid A (SAA) or via IL1β produced by mononuclear phagocytes. Commensal bacteria use ATP to induce Th17 differentiation. Commensal induce BAFF, APRIL and TGFβ production by IEC, which in turn activate B cell differentiation into IgA producing plasma cells. Reprinted with the permission from the Trends in Immunology (177) Microbiota-epithelial cell interaction Commensal bacteria influence the function and development of IEC in a multitude of ways. They use bioactive molecules, which are produced in a diet-independent or dietdependent manner. The main diet-independent products are the bacterial cell wall components, such as LPS, polysaccharide A (PSA) and peptidoglycans, which are ligands for TLR4, TLR2, and NOD, respectively (182). They play a role in protecting the barrier function of the intestinal 20 epithelium. For instance, reduced mucous layer thickness in GF mice can be corrected by treatment with TLR ligands, showing that commensal bacteria can regulate mucus production by goblet cells through TLR signaling (183). In addition, even though some AMP e.g defensins are constitutively expressed by IEC, secretion of others such as RegIIIγ is mediated by sensing of commensal-derived LPS (177), as oral administration of LPS induced expression of RegIII only when TLR4 was expressed in IEC (184). In the colon, there are two mucus layers, in which the inner layer is devoid of microbes. RegIIIγ is crucial in keeping the bacteria free zone in the inner mucus layer (185). In addition to bactericidal activity, AMP can influence chemotaxis, TLR signalling and wound repair (177). For example, RegIIIγ is upregulated in damaged mucosa and participate in the rapid recovery of the epithelial layer by inducing IEC proliferation (186). Another example of commensal microbes enabling antimicrobial protection of epithelial cells, is the capability of Lactobacillus-produced lactic acid to disrupt the outer membrane of bacterial cell walls to pave the way for lysozyme-mediated antimicrobial activity (187). Diet-dependent microbial products such as SCFAs have also been shown to induce the expression of the antimicrobial peptide cathelicidin (188-190). Butyrate, a short chain fatty acid metabolite derived from commensal microbes, enhances the integrity of intestinal epithelium barrier, by facilitating rapid repair and tight junction assembly (191). Similarly Bifidobacterium-derived acetate promotes antiapoptotic responses in IEC and thereby protects the IEC from damage, following challenge with enterohaemorrhagic Escherichia coli (E. coli) infection (192). Furthermore, both dietdependent and independent commensal products influence IEC cytokine production. For instance commensal microbes uses TLR signalling to induce cytokine production by IEC, such as CCL20, CCL28, APRIL, TGF-β1 and thymic stromal lymphoietin (TSLP), which in turn influence the responses of the underlying immune cells, which will be discussed in detail later on (193-196). Similarly, diet derived microbiota products, such as butyrate triggers IL-18 production in IEC through G protein-coupled receptor (GPR) 109A, which in turn inhibits colitis-associated colon cancer (177) (197) (198). Moreover, Clostridium species were shown to enhance expression of matrix metalloproteinases (MMPs) by IEC, which converts latent TGFβ into its active form (199). In this complex interaction between the gut microbe and the host, the conundrum facing the intestinal epithelium is to distinguish the commensal microbe from the pathogenic microbe. Both the commensal microbe and the host epithelium have developed a mechanism to avoid 21 inappropriate responses. The intestinal epithelium regulates the expression and distribution of its MAMPs on the apical and basolateral side of its epithelial cells (200). In concordance with controlling TLR signaling, during the postnatal period epithelial cells increase the expression of the NFκB inhibitor IκBα, to prevent the expression of pro-inflammatory cytokines regulated by NFκB (4). Similarly, there are other molecules, such as SIGIRR, TOLLIP, PPARγ and A20, which implement a different mechanism to regulate the excessive proinflammatory response of IEC via TLR signaling (28). For example A20 that is quickly activated following TLR stimulation of IEC, interferes with a TLR-dependent response by ubiquitin editing. A20deficient mice exhibit excessive inflammation in different organs including the intestine. Commensal microbes have the capacity to use these negative regulators to their advantage. For examples, Bacteroides thetaiotaomicron dampens CXCL8 and TNF production by epithelial cells by inducing the expression of peroxisome-proliferation-activated receptor γ (PPARγ), which prevent the translocation of NFκB to the nucleus (201). Similarly, IEC from SIGIRR deficient mice exhibit excessive cytokine production in response to the commensal microbiota (202). It has also been shown commensal microbes use SCFA to down-regulate IL-1β induced IL-8 production by Caco-2 cells, an intestinal epithelial cell line that is frequently used to study microbe-IEC interaction, in vitro (203). Epithelial cell-regulation of immune cell function It is clear by now that the IEC are not passive barriers, and they can interact with the luminal content and respond in appropriate manner. As already mentioned, IEC produce cytokines, such as TSLP, TGFβ, APRIL, BAFF, CCL20 and CCL28 in response to gut microbe-derived signals (193-196). These cytokines are vital in regulating the development and the function of the underlying immune cells. For instance, IEC-derived TSLP, TGFβ, and retinoic acid (RA) promote the development of tolerogenic DCs and MФ, which are educated to produce IL10, TGFβ and retinoic acid and subsequently regulate the generation of Treg cells and IgA producing B cells (204) (193). IEC can also indirectly regulate ILC3 in response to commensal bacteria-derived signals. For example, IEC-derived IL25 dampens IL23 production by MФ, which in turn decrease IL22 production by ILC3 (205). On the contrary, commensal bacteria induce IL7 by IEC (206), and have been shown to enhance the production of IL22 by ILC3 through stabilization of RORγt (206) (207). IEC can also directly influence B cell recruitment and differentiation by secreting CCL20, CCL28, APRIL and BAFF (193-196). 22 Expression of polymeric Ig receptor (pIgR) – a receptor that is responsible for the shuffling of the IgA into the lumen - is also regulated by commensal microbes (196). Further, M cells and goblet cells transfer luminal antigens to the local DC and MФ, which subsequently present the antigen to the adaptive immune cells in the GALT (6) (9). Although IEC express MHC molecules to present processed antigen, they lack the co-stimulatory molecules for MHC-TCR complex (208). Antigen presentation by IEC in the absence of co-stimulation may promote anergy, which might aid in local and systemic T cell tolerance. Alternatively, memory T cells are less in need for co-stimulatory molecules, thus, the ability of IEC to present antigen may be more important for memory T cell function. The underlying immune cells also protect the epithelium. For instance, luminal IgA prevents microbes from attacking the IEC. Similarly, IL22 from ILC3 has been shown to improve the epithelial barrier. At steady state, mice lacking ILC3 are more vulnerable to microbial translocation from the intestinal lumen to peripheral organs (209) (210), and this can be reversed by the administration of IL-22 (209). Microbiota-immune system interaction The microbiota plays a vital role in orchestrating the structural and functional development of the immune system in the gut. Studies with germ-free (GF) mice revealed that the gut microbiota is required for the proper development of GALT (211) (212). Although, GF mice do not lack functional PP, the number and the size of the PP are smaller in GF mice than those of specific-pathogen-free (SPF) mice, and their MLN contain fewer cells and are smaller in size. Notably, the development and maturation of ILF and CP are impaired in GF mice (120). These ILF are restored following the introduction of gut bacteria into GF mice, which indicates a dynamic relationship between the immune system and the microbiota (212). In addition to its defect in its inductive site, GF mice have smaller numbers of IEL than conventionally reared mice and again, restoring the intestinal microbiota was enough to correct the defect (213) (214). In the next sections, I will discuss the effect of the gut microbes at the cellular level on both mucosal and systemic immunity. Effects of microbiota on mucosal immune cells The benefits of microbiota have been indicated by their involvement in the recruitment and development of immune cells in the mucosal surfaces. GF mice have a lower number of 23 resident DC and MФ. In addition, GF mice have decreased numbers of Treg cells in the LP (199) (213) and the numbers of IgA secreting plasma cells are also affected resulting in reduced amounts of secreted IgA (215). The microbiota uses different mechanisms to modulate the response of the mucosal immune cells. For instance, PSA from commensal B. fragilis facilitates the development of IL10-producing Foxp3+ Treg cells (216) through direct interaction with TLR2 on T lymphocytes (217), or indirectly through TLR2-dependent conditioning of DC (218) (219). In addition, oral inoculation of mice with Clostridium clusters IV, XIVa and XVIII can initiate the differentiation of Treg cells in the colon (199) (220). Some evidence suggests that Clostridia enhance TGFβ production within the colon to promote FOXP3+ Treg cell differentiation (199). In addition, SCFA produced by colonic bacteria can regulate the generation of Treg cells in the colon. For instance, butyrate can induce histone H3 acetylation of the FOXP3 promoter and the conserved non-coding sequence 1 in the Foxp3 enhancer to promote Treg cell differentiation in the colon (221) (222). Similarly the SCFAs propionate, butyrate, and acetate can also drive the expansion of IL10-producing FOXP3+ Treg cells in the colon, by binding to SCFA receptor GPCR43 (223). Segmented filamentous bacteria (SFB) on the other hand, promote Th17 cells differentiation in the SI (224). It has been proposed SFB colonization increases serum amyloid A protein (SAA) in the SI, which facilitates IL6 and IL23 production by CD11c+ cells to drive Th17 cell development (225). Similarly, ATP secreted from commensal bacteria activates CX3CR1+ cells in a TLR-independent manner to promote Th17 cell generation in the gut (226). Other cell types, including DC, have also emerged as targets of SCFA from commensal bacteria. In DC, treatment with butyrate decreased the expression of IL12 and increased expression of IL10 and IL23 (227) (228). It has also been shown that butyrate can regulate the expression of the antigen-presentation machinery of the DC, including CD40, CD80, CD86 and major histocompatibility complex class II (227) (228). However, treatment with butyrate did not limit DC to elicit IL17 and IL10 production by T cells (227). In addition to SCFA, other metabolites of commensal bacteria, such as tryptophan catabolites have also been shown to modulate the function of immune cells. Lactobacillus-produced indole-3 aldehyde from tryptophan promotes IL22 production by ILC3, which confers protection against Candida albicans (229). Gut microbiota species can also use their glycolipids to modulate the response of the NKT cells in a CD1d-dependent manner (132). 24 Effects of microbiota on systemic immune function The influence of the gut microbes extends beyond the intestinal tract and also affects the systemic immune system. Under GF conditions, the Th1- Th2 balance is shifted towards a Th2-dominated response including IgE production that was normalized by exposure to a diverse microbiota during early life. However, colonizing GF mice in adulthood fails to decrease the serum IgE level (230), which also shows the ability for the microbiota to educate the naïve neonatal immune system, an education that could last for life. It has also been shown that administration of PSA from B. fragilis into GF mice is enough to induce T-cell expansion and also corrects the Th1-Th2 imbalance in the periphery (231). In addition, higher intensity of B. fragilis at an early age was found to inversely relate to TLR4 mRNA expression in peripheral blood mononuclear cells (PBMC) from 12-month old infants. As a consequence, LPS induced CXCL8 and IL17 levels were also negatively correlated with B. fragilis colonization for a week after birth (232), which could indicate an influence of bacterial colonization in the induction of tolerance. Similarly, the feeding of a diet rich in fermentable carbohydrates increased the Bacteroidetes phylum in the gut and subsequently enhanced serum level of SCFA, acetate and propionate. The SCFA, in turn, increase the hematopoiesis of DC precursors in the bone marrow (233). The generated DC were inept to initiate allergy-prone Th2 responses in the lung. On the other hand, colonization with SFB has been linked to exacerbated autoimmune arthritis through the induction of auto-antigen specific Th17 cells in a mouse model for arthritis (234). Lactobacilli and Staphylococcus aureus in the gut Both lactobacilli and S. aureus are Gram-positive bacteria. Lactobacilli, which are members of the phylum Firmicutes, are commonly detected in the human gut, where L. salivarius, L. rhamnosus, and L. paracasei are the dominant species (235). S. aureus is commonly found on the skin and in the nasal mucosa. However, S. aureus is nowadays frequently detected in fecal samples from children in Westernized countries (236) (237). Breast milk contains nutrients for these bacteria to grow and favors early colonization and stability of both lactobacilli and S. aureus in the gut of infants (238-240). It is already well known that lactobacilli are common in the gut of vaginally delivered infants. However, recent finding has shown that S. aureus is also more abundant in vaginally born infants (241). S. aureus is known to produce staphylococcal exotoxins (SE) and toxic shock syndrome toxin (TSST) (242). SE and TSST from S. aureus can act as superantigens and can cause non specific binding of the 25 MHC complex on APC with TCR on T cells, leading to uncontrolled T cell activation and cytokine production, which can have pathological ramification of causing septic shock. Although S. aureus is considered as pathobiont in the gut, several strains colonize the infants gut without causing gastrointestinal complication (243). However, early-life colonization with S. aureus has previously been associated with development of non-infectious diseases, such as allergy (244), while early colonization with lactobacilli seemed to decrease the risk of allergy later in life (237). In addition, peripheral immune cells from infants co-colonized with lactobacilli and S. aureus at an early age have a regulated cytokine response ex-vivo, when compared with cells from infants colonized with S. aureus alone (245). Although the intestine harbors many genera and species, repeated studies clearly show that S. aureus and different lactobacilli have strong immunomodulatory properties and that their presence in the early gut associate with immune-mediated disease later in life (232) (237) (244-246). How these bacteria influence the immune system is not clear. That is why in paper I, we investigated the effect of S. aureus and lactobacilli in modulating the response of IEC and how these indirectly affect the immune cells response in vitro. Similarly in paper II, we studied the effects of L. reuteri and S. aureus on the DC differentiation and its subsequent effect on T cells. The therapeutic use of lactobacilli as probiotics, prebiotics or postbiotics An altered microbial composition, also known as dysbiosis, destroys the beneficial influence of gut microbes on host immune physiology, a condition associated with susceptibility to immune-associated diseases, such as necrotizing enterocolitis (NEC), IBD, allergies, type 2 diabetes, diarrhea and infection (247) (248). Probiotics have been shown to correct some of these disorders. Probiotics are defined as “live microorganisms, which when administered in adequate amounts confer a health benefit on the host” (249). Lactobacilli are the most widely used probiotic strain. They have been shown to modulate the immune response both in vitro and in vivo (250). Although, there is a strain difference in function, collectively, lactobacilli contribute to increased intestinal barrier integrity, regulated IEC and DC responses, enhanced IgA responses, and induction of Treg cells (251). The dilemma of using probiotics is that they are live-bacteria, which might be a bit hazardous to give to an individual whose gut barrier is already compromised. The other major challenge is that some of the probiotics are antibiotic resistant, which might be transferred to more pathogenic bacteria through horizontal gene transfer (252) (253). An alternative means of treatment with more or less similar effects as probiotics is to use prebiotics. Prebiotics are defined as ”selectively fermented ingredients that 26 allow specific changes both in the composition and in the activity of the gastrointestinal microflora, which confer benefits upon host well being and health” (251). For instance, oral administration of prebiotics such as inulin and fructo-oligosaccharides has been shown to facilitate the growth of lactobacilli (251) (254). Despite this, there are still contentions in the use of prebiotic as a treatment, since the oral administration of these substrates could favor the growth of unwanted microbes that could induce a pro-inflammatory response. A relatively new paradigm is to use postbiotics, for treatment. Postbiotics are nonviable bacterial products or metabolic byproducts from probiotic microorganisms that have biologic activity in the host. In a strain different manner, lactobacilli-derived postbiotics have already been shown to have potential in modulating epithelial tight junctions and in attenuating tissue inflammation by regulating immune cells (255). However, the way forward is to encourage more research in developing means to deliver the postbiotics to the target site in the host. Since most of these soluble factors are sensitive to host enzymatic activity, they might be degraded before reaching the target site. In addition, identifying optimal postbiotic substances is vital. That is why in paper III, we focused on studying the postbiotic potential of a wellknown and well-tolerated probiotic strain, L. reuteri 17938, in modulating the phenotype and response of DC. 27 PRESENT STUDY General aim: To investigate how common gut microbes - lactobacilli and S. aureus - influence the immune development and responses in vitro Specific aims: To investigate how different strains of lactobacilli and S. aureus influence the response of gut epithelial cells and peripheral immune cells in vitro (paper I). To investigate the influence of L. reuteri and S. aureus on the differentiation of monocyte to DC in vitro, as well as how the generated DC function in regulating T cell production of cytokines, in co-cultures (paper II). To investigate the postbiotic effect of L. reuteri on modulating RA-generated-mucosallike-DC response and their effect on Treg cells in vitro (paper III). 28 Material and Methods Detailed descriptions of the material and methods are found in the specified sections of papers I, II and III, respectively. Paper I In paper I, we used seven Lactobacillus (L.) strains (L. reuteri DSM 17938, L. reuteri ATCC PTA 4659, L. rhamnosus kx151A1, L. rhamnosus GG, L. casei LMG 6904, L. casei Shirota, L. paracasei F19) and three Staphylococcus (S.) aureus strains (S. aureus 139:3, S. aureus 151:2 and S. aureus 161:2), which were grown in their respective growth media (MRS broth and BHI broth). Sterile cell free supernatants (CFS) were separated by pelleting the bacteria by centrifugation, followed by sterile filtration of the CFS. Intestinal epithelial cell lines (IEC) (HT29 and SW480) were treated with different lactobacilli-CFS and S. aureus-CFS. In addition, IEC were stimulated with UV-killed bacteria (L. reuteri DSM 17938 and/or S. aureus 161:2) alone or with their respective bacteria-CFS. To assess the involvement of TLR signaling in the IEC response to S. aureus, the MyD88 expression in the IEC was knockdown by using siRNA. Further, to assess the effect of the bacteria-CFS on immune cells, PBMC and cord blood mononuclear cells (CBMC) from healthy donors were treated directly with bacteria-CFS or with bacteria-conditioned IEC-CFS. The level of cytokines and chemokines in the IEC and PBMC/CBMC-CFS were analyzed using human proteomic array (36 cytokines/chemokines) and ELISA. In addition, PBMC were stimulated with bacteria-CFS in vitro and intracellular staining of T cells for IFN-γ and IL-17 was performed and analyzed by flow cytometry. To investigate the role of histamine in lactobacilli-mediated immune modulation, histamine levels were measured in bacteria-CFS using ELISA. Further, the histamine receptors on PBMC were blocked using ranitidine (the histamine receptor blocking agent) before treating the cells with the bacteria-CFS. To investigate the proteolytic effect of Lactobacillus-CFS on cytokines, recombinant (r) IL-17 and rIFN-γ were pre-incubated with L. reuteri 17938-CFS and then the respective proteins levels were measured with ELISA. Paper II In paper II, we used L. reuteri DSM 17938-CFS and S. aureus 161:2-CFS, which were obtained in a similar manner as in paper I. 29 Using magnetic beads, monocytes were negatively purified from PBMC from healthy donors. The monocytes were kept in complete media with GM-CSF and IL-4 for 3 days, before replacing it with a new complete media, which contained either L. reuteri-CFS, S. aureus-CFS, a combination of L. reuteri-CFS +S. aureus-CFS. As a control the differentiating DC were just kept in culture medium. On day 6, the generated DC were characterized in terms of surface marker expression (flow cytometry) and cytokine production (ELISA). We further assessed the phagocytic ability of the bacteria-conditioned DC by incubating them with enzyme-labeled Escherichia (E.) coli particles, followed by permeabilization of the cells and incubation with substrate. Then, the reaction was stopped and the optical density (OD) value was measured using a spectrophotometer. The generated DC were further stimulated with LPS followed by an analysis of gene expression of 84 DC-related genes. In addition, the LPS stimulated bacteria-CFS generated DC were co-cultured with autologous T cells, negatively isolated from thawed PBMC using magnetic beads. In addition, Mo-DC were pre-treated with bacteria-CFS, before co-cultured with T cells. The cytokine production of the T cells was analyzed using both ELISA and intracellular staining and T cell proliferation was evaluated using flow cytometry. In paper III In paper III, we used L. reuteri DSM 17938-CFS, which was obtained in a similar manner as in paper I. Monocytes were isolated as described above for Paper II. The monocytes were kept in complete media with GM-CSF and IL-4 with or without RA. On day 6, the generated DC (MoDC or RA-DC) were characterized in terms of surface marker expression (flow cytometry) and cytokine production (ELISA and proteome array). Moreover, the Mo-DC and RA-DC were then treated with L. reuteri-CFS, LPS or kept in culture media followed by an analysis of gene expression of 84 DC-related genes as above. In addition, the L. reuteri treated RA-DC and MoDC were co-cultured with autologous T cells, isolated as above, followed by intracellular staining of the T cells for FOXP3 and IL10 expression. 30 Results and Discussion Previous work in our group has shown that an early colonization with lactobacilli is associated with a regulated immunological profile and a reduced allergy risk at five years of age (232) (237) (246) (245). On the contrary, early colonization with S. aureus, in the absence of co-colonization with lactobacilli, is associated with excessive in vitro responses of the immune cells from these children (245). To understand the mechanism how these microbes influence the immune response and development, our group is working on three parallel tracks. First we perform in vitro studies of immune cells from children participating in a prospective allergy cohort and relate these findings to their early-life gut colonization. Here we also perform more mechanistic studies of interactions between bacterial factors and immune- and epithelial cells. Second, we work with an in vivo animal model, where GF mice are colonized with a defined flora from the infants mentioned above and their immune development is characterized. Finally we participate in a randomized placebo controlled probiotic study of preterm neonates where we follow the immune development in relation to probiotic supplementation, gut colonization etc ex vivo. Although I was lucky enough to be involved in all three approaches, the focus of my thesis is on the in vitro studies of interactions between bacterial factors and immune- and epithelial cells, in order to better understand how early cocolonization with lactobacilli and S. aureus affects the host immune system. The initial microbe-host interaction in the gut takes place between IEC and bacteria. In paper I, we aimed to assess the effect of lactobacilli and S. aureus on IEC responses. We initially exposed IEC-lines (HT29 and SW480) to CFS from two specific strains, S. aureus 161:2 and L. reuteri DSM 17938, using human cytokine proteome array (including 36 different cytokines and chemokines). Both HT29 and SW480 produced relatively few factors upon stimulation, but distinctively the IEC-lines stimulated with S. aureus produced the proinflammatory chemokines CXCL8 and CXCL1. To confirm these findings, we further studied the effect of CFS from seven different Lactobacillus strains and three different S. aureus strains in inducing CXCL8 production in HT29 cells. It was only S. aureus 161:2 (having the genes for enterotoxin A and H) that induced CXCL8 production by IEC (as measured by ELISA). The other S. aureus strains (expressing the toxic shock syndrome toxin or lacking enterotoxin genes respectively) did not induce a response in the IEC-lines. We also measured the levels of TSLP, APRIL and TGFβ1 with ELISA as these factors are considered to be produced by IEC upon interaction with bacteria and have vital immune regulatory function. However, none of these cytokines were upregulated in IEC-lines upon stimulation with any of the bacteria-CFS tested. 31 Both HT29 and SW480 are non-polarized IEC-line obtained from adults, and the generality of these findings could be questioned. However, we have further tested the response of the fetal IEC-line (FHS-74 int) and the polarized adult IEC-line (Caco2). Irrespective of the type of IEC-line used, S. aureus-CFS was able to stimulate CXCL8 production (Haileselassie, Holmlund, Nordlander et al unpublished observations). To elucidate whether the bacteria themselves also could induce and/or potentiate this pro-inflammatory response by IEC, we stimulated HT29 with UV-killed bacteria, bacteria-CFS or a combination of both. Our results showed that the IEC-response towards S. aureus was driven by products present in the CFS and not by the UV-killed bacteria. In addition, a combination of both UV-killed bacteria and the bacteria-CFS did not alter the effect of the supernatant alone on IEC. TLR-signaling in IEC contribute to the maintenance of intestinal barrier and production of soluble factors by IEC (256) (200). To investigate whether the S. aureus-induced IEC response was TLR dependent we performed MyD88 silencing of the IEC, which partially dampened the S. aureus induced IEC-response. Although this indicates that S. aureus-CFS induce CXCL8 production by IEC via TLR-signaling, stimulating the IEC with representative TLR2 ligands, such as peptidoglycan (PGN) and PAM3CSK4, did not induce CXCL8 production. This could be a matter of kinetics and dosage, but might also indicate that the bioactive molecule in the S. aureus-CFS inducing CXCL8 is not a TLR ligand. Microbes in the gut can further influence the response of immune cells; either indirectly via IEC or via a direct bacteria-immune cell interactions e.g. through immune cell sampling of luminal antigens and bacterial soluble factors passing the IEC barrier. To investigate the effect of secreted factors from lactobacilli and S. aureus on the responses of the immune cells, and whether the presence of IEC-secreted factors could alter these responses, PBMC were stimulated with L. reuteri 17938-CFS and S. aureus 161:2-CFS directly or with supernatant from IEC conditioned with the same bacteria. In general, the bacteria-CFS treated PBMC were able to produce a wide range of cytokines. However, only S. aureus treated PBMC with or with IEC-CFS produced the T-cell associated cytokines IL-2, IL17 and IFNγ. The presence of IECderived factors did not modify the response of PBMC. The restricted production of different cytokine by the IEC-lines and their inability to influence the PBMC response is somewhat in contrast with the other reports (193) (257) (258). Although we have confirmed our findings using both polarized and non-polarized cells and in cells of different degrees of maturity (adult versus fetal), this could be attributed to the fact that the IEC-lines used in this experiment are not primary cells. We have recently carried out a set 32 of experiments in primary murine cells, where we have stimulated colonic crypts. Also here, we see a relatively restricted epithelial response to microbial ligands (Petursdottir, Nordlander, Haileselssie et al unpublished observations). To investigate whether the effect is bacteria strain specific or general, we measured IL6, IL17, IFNγ, IL2 and TNFα in PBMC-CFS stimulated each Lactobacillus- and S. aureus strain included in this study. Both S. aureus and lactobacilli were able to induce IL6 production by PBMC, showing the potential of lactobacilli to activate the immune system, but in other ways, primarily via innate cells. However, it was only S. aureus that was capable to induce IL17 (S. aureus 161:2 and 139:3), IFNγ, IL2 and TNFα production (S. aureus 161:2). In similar manner, S. aureus readily induced a vigorous IFNγ response by CBMC, which indicates that the response to S. aureus is not a secondary response from a memory T cells pool found in adult donors. Previously, we have observed that early co-colonization with lactobacilli and S. aureus strains are associated with regulated cytokine production in vitro (245). Here we stimulated the PBMC with S. aureus 161:2-CFS alone or S. aureus 161:2-CFS in combination with each Lactobacillus-CFS included in this study. While the presence of lactobacilli did not mitigate the response of IEC to S. aureus 161:2-CFS, it actually dampened S. aureus161:2-CFS induced IL17, IFNγ, IL2 and TNFα production by PBMC. Flow cytometry analysis of intracellular staining of T cells further confirmed that IFNγ and IL17 production following S. aureus stimulation was mainly from CD4+ Tcells and co-stimulation with L. reuteri DSM 17938-CFS and S. aureus 161:2-CFS decreased the percentage of IFNγ positive cells. In addition, S. aureus upregulated CTLA-4 and induced the production of IL10 in Treg cells. Recent findings by our group concur with this finding on the influence of S. aureus on the Treg cell responses (105). It is vital to identify the biologically active molecules present in the CFS of S. aureus 161:2 and the different lactobacilli, which are responsible in modulating the response of the IEC and PBMC. SE from S. aureus strains can act as superantigens and strongly activate T cells, which results in a strong polyclonal activation of T cells. However, its effect on the IEClines and observed differences between S. aureus-CFS and pure SE in some settings made us believe that S. aureus 161:2-CFS also contains other immune-modulatory factors. Therefore we looked at adenosine triphosphate (ATP), since it is known that extracellular ATP from microbes can induce Th17 in the gut (226) and S. aureus can produce extracellular ATP (259). By treating the S. aureus 161:2-CFS with apyrase (ATP hydrolyzing agent), we tried to ablate S. aureus161:2-CFS induced IL17 production. However, treatment with apyrase did not affect the efficiency of S. aureus161:2-CFS to induce IL17 production. 33 Understanding how lactobacilli regulated the S. aureus induced cytokine production by immune cell is also vital. Previous work has shown that histamine secreted by a Lactobacillus strain tune down inflammatory responses by binding to H2 receptors expressed on monocytes (260). However, most of our lactobacilli strains did not produce histamine and pre-incubating PBMC with ranitidine (H2 receptor blocking agent) before exposure to bacteria-CFS did not alter the effect of lactobacilli on the PBMC response to S. aureus 161:2. More recent work in our group have identified that lactate might be responsible for some of the dampening, at least in some cell types (Johansson, Björkander et al submitted). Based on heat-inactivation and fractionation studies we have further shown that it might be different factors that are involved in dampening IFN and IL17 (Mata Forsberg et al, unpublished observations). The dampening effect observed, was not due to a reduced cell-viability in PBMC stimulated with lactobacilli-CFS. To confirm that the modulatory effect of the lactobacilli is at cellular level, and not just a mere degradation of the cytokines in the supernatant by the lactobacilli-CFS, we also investigated the proteolytic potential of the lactobacilli-CFS against secreted cytokine by pre-incubating rIL17 and rIFNγ with L. reuteri-CFS before measuring the cytokine with their respective ELISA. Pretreatment did not hinder their detection by ELISA. Moreover, our flow cytomtery intracellular assays of the cytokine production indicate that the regulation is at cellular level. In paper II, we aimed to further understand how these microbes influence the phenotype and function of immune cells in early life. For obvious reasons, this is difficult to do in vivo in humans and we therefore designed an in vitro experimental system to study the effects L. reuteri, and S. aureus, on the differentiation of monocytes to DC, as well as how the generated DC influenced T cell cytokine production in the absence of antigen. Different signals drive the monocyte migration into the gut (64). However, it is not fully clear what drives the differentiation of monocytes into DC and how the microenvironment modulates the generation of the DC and their phenotype. We generated DC from peripheral blood-derived monocytes according to standard procedures. However on day 3 of DC generation, the respective bacteria-CFS were added. Generated DC were investigated at baseline on day 6, and they expressed CD11c but lacked CD14, as expected of monocyte-derived DC. Although it was a general phenotype of the bacteria conditioned DC, DC generated in the presence of L. reuteri-CFS had a more profound upregulation of the maturation markers (HLADR, CD86, and CD83), characteristic of intestinal DC (261) (262). This suggests that the gut DC phenotype is (partly) driven by gut microbes, which might be very important for gut 34 homeostasis as the absence of some of these markers on DC worsens pathology and disease in a colitis model (262). The increase in expression of these markers indicate that the bacteria conditioned-DC have a mature DC phenotype. Interestingly, DC generated in the presence of L. reuteri-CFS have lower DC-SIGN expression. DC-SIGN has been associated with different pathological conditions. HIV uses this receptor for invasion (263) (264). In addition, DC isolated from ulcerative colitis patients have enhanced expression of DC-SIGN and altered gut DC phenotype, which was reverted by conditioning with serine-threonine peptide from L. plantarum (265). Moreover, Ara h1, one of the major peanut allergen, can bind to DC-SIGN to initiate allergy associated Th2 response (264). Our result might again indicate the potential of L. reuteri-CFS to modulate the DC-SIGN expression on DC and avert a disease condition, such as allergy, which might be a contributing mechanism behind our previous finding where early life colonization with lactobacilli is associated with a decreased risk of developing allergy later in life (244) (246) (237). Similar to gut DC (48), the L. reuteri-CFS treated DC have a reduced phagocytic ability, which might be linked to the decrease in DC-SIGN expression that has a role in antigen capture (27) (266). The gene expression data of the LPS-stimulated DC revealed a profound L. reuteri induced re-programming of the DC population. In general we saw a profound suppression of gene expression for many different genes associated with activation, maturation and T cell interaction. Among other things we observed that L. reuteri-CFS-conditioned DC had an altered secretory profile compared with LPS stimulated control Mo-DC, but also in relation to S. aureus-CFS exposed DC. Further, several chemokines were significantly downregulated while most chemokine receptors showed an opposite pattern. This could suggest that the L. reuteri-treated DC are more sensitive to its environment with their increased receptor expression and thereby more readily sense changes in chemokine gradients. They would in contrast be less likely to attract other cells due to a decreased chemokine production, perhaps in line with a more tolerogenic phenotype. We also observed an increase in TGFβ gene expression for L. reuteri-CFS-treated DC, something that also speaks in favor of L. reuteri-CFS being capable of inducing regulatory DC. However, the enhanced TGFβ was not observed at the protein level, promoting further investigations with different kinetics here. In order to see how these bacteria-exposed DC populations would influence adjacent T cells in the absence of any antigenic insult (i.e. as a model of a steady state situation in the intestine) we investigated T cell cytokines following co-cultures of these DC with autologous T cells. In order to prevent carryover of bacterial factors, we washed our DC extensively before 35 adding the T cells. The L. reuteri-CFS treated DC equalled conventional Mo-DC in T cell activation; while the S. aureus-CFS treated DC induced IL2, IFNγ and a tendency also for IL17. This was a similar pattern to what we observed in paper I, where S. aureus-CFS induced these three cytokines in PBMC cultures. Whether this immunostimulatory capacity of S. aureus is beneficial for the neonatal immune system or not is debated. Early-life colonization with bacteria promoting a T cell pool prone to be hyperresponsive could therefore contribute to allergy development. Indeed, early-life colonization with S. aureus has been linked to an increased risk of allergy (244) and we also observed a tendency of increased S. aureus colonization in early life in children who later developed allergy (245). However, we also noted that S. aureus colonization was very common during the first months of life also in non-allergic children, suggesting that it cannot be a risk factor for allergy per se. Indeed it has rather been suggested to be beneficial for immune development and protecting against allergy development in other studies (267) (268). We think that the presence of S. aureus in the gut does not confer a risk for allergy development as long as other bacteria like lactobacilli are there to modulate the S. aureus-induced immune stimulation. An overall increase in cytokine production is associated with allergy (269) and we could further demonstrate exaggerated immune responses in children that were S. aureus colonized but lacked lactobacilli in early life. Although superantigens produced by S. aureus drive uncontrolled T cell activation, they can also have other effects on the immune system. We have recently demonstrated that the S. aureus-CFS used in our study potently induces FOXP3+ cells and promotes a diverse phenotype with production of regulatory (IL10) and pro-inflammatory (IFN and IL17) cytokines in a partly monocyte-dependent manner (105). Also, others have shown that DC isolated from SE treated mice tend to be more efficient in priming FoxP3 expression and gut homing phenotype (α4β7) on T cells compared to DC from sham-fed mice (270). In our model it is likely that S. aureus is activating the T cells via SE bound to MHC during DC generation and that we have not been able to remove them despite extensive washing. But SE also have other mechanisms to activate cells. For example, SE from S. aureus can bind to other surface proteins, such as gp130 that leads to downstream STAT3 phosphorylation (271). STAT3 phosphorylation in turn can regulate cytokine production by the immune cells. Moreover, epithelial cells do express gp130 (272). It is possible that the S. aureus 161:2 might have used this pathway to activate both epithelial cells and immune cells. We are at present trying to understand how S. aureus and purified enterotoxins activate the 36 immune system more in detail (Björkander, Mata-Forsberg et al, unpublished observations) as we have noted that S. aureus-CFS readily activates both conventional and unconventional T cells as well as NK cells (Johansson, Björkander et al submitted). There are conflicting findings on the generation of DC from monocytes in the gut at steady condition (273) (55) (48). However, there are well established data that monocytes give rise to DC depending on the nature of the environmental signal, for instance inflammatory conditions (274) (54). Therefore one can argue in neonate, where the mucosal barrier are not tight, luminal antigens (microbial factors and dietary factors) can have access to modulate monocytes and further differentiate them to DC. In the context of gut microbe-immune cell interaction, our data might stress the importance of signals from different microbes in shaping the development and response of immune cells, such as DC. It is important for gut DC to develop a tolerogenic phenotype, but yet be responsive to keep the homeostasis of the gut. In addition to gut microbes, diet contributes to the development and response of the immune system. Breast milk takes the lion share by indirectly and directly influencing the immune system development (275). Breast milk inoculates the infant gut with commensal microbes, such as lactobacilli, bifidobacteria and staphylococci (241). Oligosaccharides from breast milk maintain the microbes in the gut (276). In addition, breast milk contains soluble factors, such as cytokines, antimicrobial peptides and antibodies that can provide protection at early age (277). It contains both anti-inflammatory and pro-inflammatory cytokines and we have previously shown that there is a substantial individual variation in breast milk cytokine and chemokine content that is further influenced environmental factors and number of children (278). These differences in breast milk composition influence the cellular response of both immune cells and intestinal epithelial cells (279), and should be considered when neonatal immune responses are evaluated. In addition to breast milk, vitamins also regulate the immune system. In particular, vitamin A plays a multifaceted role, through its bioactive metabolite, RA (280). RA influences proliferation and gut homing phenotype of both B- and T cells. In addition, RA directs the intestinal DC in to the MLN and subsequently promotes the generation of Treg cells (281). In vitro, RA induces CD103 expression and enhances α4β7 and CCR9 expression on Mo-DC (37), promoting mucosal-like DC phenotype. In paper III, we investigated the influence of soluble factors produced by L. reuteri 17938 in modulating the response of the RA-derived mucosal-like DC. L. reuteri 17938 is one of the most commercially and clinically recognized probiotics, which is derived from L. reuteri ATCC 55730, first isolated from human breast milk (282). The probiotic potential of L. reuteri 17938 has been shown in several studies. For this 37 thesis, its influence on NEC is of particular interest. In an experimental study, treatment with L. reuteri 17938 mitigated the inflammatory response in the intestine (283-285). L. reuteri 17938 also reduced the severity of experimental necrotizing enterocolitis disease in mice by modulation of the balance between T effector/memory (Tem) cells and regulatory T (Treg) cells (286). Due to its potential to support the gastrointestinal tract, we have initiated a study where the premature, extremely low birth weight infants are treated with L. reuteri 17938, and these children are monitored for e.g. microbiota establishment and immune development. Normally, probiotics do not cause disease. However, introducing live bacteria to individuals where the gut barrier is compromised or under-developed, as in the extreme prematures mentioned above, might confer a risk. An alternative mechanism is to use secreted factors from the probiotics, i.e. postbiotics, to modulate the immune response. However, the knowledge about whether bacteria-derived products can mimic the probiotic effects is very limited. In paper III we generated DC from monocytes in the presence of RA. We combined investigations of gene- and protein expressions to get a detailed characterization of RA-DC and Mo-DC. RA-DC showed downregulation of a majority of the genes, when compared with MoDC. For example, RA-DC exhibited a reduced expression of TNF and NFκB1. The regulated expression of these genes in RA-DC supports the tolerogenic phenotype of these cells. Among the few genes that were significantly increased in the RA-DC were IL6 and IL10, which was also confirmed at protein level for IL10. In line with previous work (287) the RA-DC also had up-regulated CCR7 surface protein, a molecule that drives CD103+ DC to the MLN in vivo (288). In general, RA-DC expressed less chemokine mRNA compared to Mo-DC. However, both at gene and protein level, CCL2 and CCL7, which are CCR2 ligands, were expressed higher in RA-DC. CCL2 seems important in recruiting CCR2 positive mononuclear phagocytes into the intestine (57). RA might play a role in upregulating CCL2 on DC, which in turn attracts more phagocytic cells in the gut. In addition, CCL2 and CCL7 have been shown to regulate IL4 production and protect against infection (289), mechanisms that might be used by DC under certain circumstances to maintain tissue integrity. Stimulation of the RA-DC and Mo-DC with LPS revealed that more genes are up-regulated in the Mo-DC than in RA-DC, showing that RA-DC are more refractory to activation, further supporting its tolerogenic profile. We were then interested to see whether postbiotic L. reuteri treatment modulated these two types of DC in different ways. Treatment with L. reuteri-CFS induced IL6, IL23 and IL10 38 by both RA-DC and Mo-DC, while TGFβ1 levels did not change. In the gut, the production of IL6 and IL23 is not always detrimental. IL23 can trigger the production of IL22 that is important in maintaining the epithelial barrier (290). IL6, on the other hand, plays a role in IgA production (291) (292). L. reuteri-CFS also induced the production of several chemokines, including CXCL1, CXCL5, CCL3, CCL15 and CCL20 in both RA-DC and Mo-DC, which are involved in gut homeostasis. In the intestine, local CXCL5 production control the migration of neutrophils, at steady state. CXCL5-/- mice are devoid of neutrophils in the gut (293). CCL15 functions as an antimicrobial peptide in steady state conditions in the gut (257). CXCL1 is vital in maintaining the intestinal barrier (294) and CCL3 and CCL20 appear to be involved in the recruitment of mononuclear phagocytes (295). Although epithelial cells are the main CCL3 and CCL20 producers in the gut, DC might serve as an additional source of CCL3 and CCL20. As we used the whole bacteria-CFS, we do not know what factor(s) that are responsible for the stimulatory effects of L. reuteri-CFS on DC cytokine and chemokine gene- and protein expressions. It could be argued that LPS derived from L. reuteri is present in the supernatant and can mediate the observed effects. However, we do not think this is very likely. The gene expression profile induced by L. reuteri-CFS is distinctly different from that following LPS exposure, for both RA-DC and Mo-DC. Further, although LPS readily induced IEC CXCL8response in paper I, we did not observe any CXCL8 production by IEC following lactobacilliexposure. L. reuteri-CFS exposure made a notable impact on the surface marker expression on RA-DC. Although RA-DC had a higher proportion of CD14+ cells to start with, L. reuteri treatment further enhanced the percentage of CD14+ cells in the RA-DC, a phenotype characteristic of tolerogenic DC (296), which suggests that L. reuteri is able to promote this phenotype. The driving factor in L. reuteri-CFS that enhanced the CD14 expression is unknown. However, histamine has been shown to induce CD14 expression in DC (297). Indeed, in the histamine measurement we performed on the lactobacilli-CFS in paper I, we found low but detectable histamine production by L. reuteri DSM 17938. In paper II we see a decreased expression of DC-SIGN on DC generated in the presence of L. reuteri-CFS, and in paper III we further observe that L. reuteri-CFS down-regulates DC-SIGN expression on RADC. This might be important for the tolerogenic phenotype that L. reuteri conferred to the DC in our studies, in line with previous observations where lactobacilli have been implicated to induce a regulatory phenotype in DC via DC-SIGN (298) (299). In the gut, DC up-regulate CCR7 in order to migrate from LP to the MLN and interact 39 with T cells. Here, L. reuteri-CFS increased CCR7 expression on the surface of RA-DC, which shows its migratory potential to get into contact with T cells. By co-culturing the RA-DC with autologous T cells, we observed that the RA-DC were superior in inducing FOXP3- and IL-10 expression in Treg cells compared with Mo-DC. Although our data fit with previously published findings, we observe less notable effects than others (288). This might be due to variations in the experimental setups. However, we have repeated our experiments using both longer co-cultures as well as allogeneic instead of autologous T cells, without notable difference in our results. A previous study where NEC was induced experimentally in mice showed that L. reuteri DSM 17938 treatment increases the frequency of FOXP3+ Treg cells and mitigate the disease (284). In our hands, pretreatment of RA-DC with L. reuteri-CFS did not further improve their ability to induce Treg cells. This shows that the in vitro setting has its limitations and that probiotic L. reuteri-CFS probably needs to act on other cells in the gut, such as IEC to reinforce the DC ability to induce T cells. To further answer this, we would have to establish an in vivo model in mice. It is also possible that live probiotic L. reuteri DSM 17938 are required to induce the Treg cell responses reported in vivo. It has previously been shown that feeding probiotic lactic acid bacteria to mice induces endogenous RA activity in intestinal DC, which in turn drive T reg cells (300). Very recent data from our group indicates that feeding mice with live probiotic bacteria impacts the “IL-17-Tfh cell-IgA” axis in the gut (Petursdottir, Nordlander, Carvalho-Queiroz, Qazi et al, unpublished observations). General conclusion In general, these papers shed light on the influence of the two common gut microbes, S. aureus and lactobacilli on host immune homeostasis. In paper I, S. aureus-CFS induced an inflammatory response by IEC and a diverse cytokine response by PBMC. Interestingly, costimulation with CFS from Lactobacillus strains down-regulated the responses induced by S. aureus-CFS in immune cells but not in IEC. Subsequently, in paper II, the simultaneous presence of both L. reuteri-CFS and S. aureus-CFS during DC-differentiation generated a DC, which were less responsive to LPS stimulation than conventional Mo-DC yet very efficient in inducing T cell responses. Today, S. aureus is commonly found in the infant gut. For long, it was considered as an enemy, however, paper II, along with other recent papers suggests that in the presence of other bacteria, such as lactobacilli that can balance the stimulatory effects, S aureus might contribute in the development and response of host 40 immune system. Paper III indicates the importance of diet in the interaction of microbes and immune cells and broadens our previous knowledge on the impact of RA on the DC phenotype. Further it indicates the potential of L. reuteri-CFS to be used as a postbiotic, with its ability to regulate the response and phenotype of DC. Its ability to dampen excessive immune responses and influence the generation of tolerogenic DC suggests that is a good candidate to explore further for maintaining gut homeostasis and regulating excessive inflammation in diseases, such as NEC. It is undeniable that the gut microbiota is complex and these two species are definitely not the only species that could influence the immune response in the gut. Also, isolated bacteria might behave differently when they are together with other species in their natural habitat. However, our studies in humans and in mice, in vitro and in vivo, together with the work of many others cited here, strongly suggest that these two bacteria have a profound influence on the developing immune system. FUTURE PERSPECTIVES Our investigations on how lactobacilli and S. aureus are interacting with the immune system both in vivo and in vitro are continuing. We are aiming to understand the interactions between these bacteria and immune cells more in detail together with their relevance for the in vivo situation, as indicated at several places in the Result and Discussion section. My own work has generated several pieces of information that would be interesting to pursue further. The transcriptome data will serve as a platform for future studies. We have identified molecules, such as Fc epsilon RI (FCεRI) and thrombospondin 1 (THBS1) that were regulated by L. reuteri-CFS. FCεRI expression on DC has previously been associated to initiating allergic responses (301). However, recent paper claims FCεR+ DC helps to contain the IgE allergic inflammation at mucosal site (302). On the other hand, THBS1 production by DC has been shown to have anti-inflammatory effect by negatively regulating IL12 and TNFα production (303). We will study whether the impact of L. reuteri-CFS on these molecules is evident also at protein level, and whether it influences DC functions. Manipulation of monocytes in vitro to generate DC and retransferring the generated DC to recipient host is the frontline in future individualized immunotherapy for several diseases (304). However in vivo, the DC experience a tissue-specific micromillieu during differentiation that shape their phenotype and function. With the profound differences of RA-DC and Mo-DC depicted here in mind, including microbial signals and vital diet metabolites during the 41 generation of DC from monocytes might be very important to consider in future approaches when aiming at treating gut related ailments, such as colonic cancer. 42 ACKNOWLEDGEMENT What a wonderful journey! In my route, a lot of you have contributed (even sacrificed) for me to reach this level. I thank God for each and every one of you. You were my blessings. Words do not describe how much I am grateful to my supervisor, Eva Sverremark-Ekström. First and foremost, thank you for giving me the opportunity to do a PhD. I learned a lot and was involved in different projects. Thank you for believing in me. I always enjoyed when we sit and discuss the results. You have provided me the platform to think freely and express my idea. You pushed me forward in the right direction in the implementation of the ideas into practice. Thank you for your moral support and for providing me conducive-work environment. All in all, you are the best handledare (in its literal definition). Ulrika, my former co-supervisor, thanks for your patience and guidance during my first two years. Thank you for being a good friend. The Fantastic Four (the former immunology departments’ seniors): Carmen Fernández, Eva Severinson, Klavs Berzins and Marita Troye-Blomberg. Thank you for your exemplified mentorship. I always enjoyed discussing science with you. P.S future PhD student at MBW, if you have questions or need help, don’t hesitate to knock on their door. Most of the times, they have the answer, and they are eager to help. For the luck of our North-South corridor, we have very talented and amazing seniors: AnnBeth Jonsson, Anna-Lenna Spetz, Antonio Barragan, Klas Udekwu, and Sushil Pathak. Thank you for being an inspiration. The future is bright for North-South corridor. I am sure the tradition of generating amazing works will continue. I know I am preaching to the choir, but collaboration between groups will further strengthen your group. For me, it was nice to see in my eyes the transition from WGI to MBW. To be honest, I was a bit skeptical in the beginning. Now I can see it was the right move to go forward. For anyone who reads this acknowledgement, you might be asking yourself why I am so passionate about the development of MBW and the departments. Honestly, the sense of belongingness and familyship is high in MBW. I do not think I am the only one who feels the same. I partly would like to give the credit to Per Ljungdahl for inspiring us and made us feel proud of MBW. 43 Personally, I would also like to thank you for the moral support and inspiration. You and Roger Karlsson were two non-immunologists, who have a great impact on my development as a scientist. I am so grateful for the administration at MBW. You are always helpful and make things easy for us. Special thanks to Gelana Yadeta. It is always easy to ask for your helping hand. Thank you for making be part of your special celebration. I have not forgotten your generosity when I was doing my masters. Beatriz Campos “Bea”, I have never seen a person so eager to help. You go extra miles to help. Special thanks to the unforgettable Margareta Hagstedt “Maggan”, our former lab technician. You were always helpful. Special thanks to our current group members: Clô, Dag, Mano, Mukti, Sofia N and Sophia B. It was always nice to work or discuss science with you all. In my view, you all have one particular character that I like about you. I will just randomly list them out: easy going but talented, the most caring one, the talented one, the funny one, the perfectionist one and the gifted scientist. Dag, whenever I work on isolating GALT, I will always remember you. Thank you for teaching me those techniques. Themis, thank you for being an awesome friend. Anna, you joined the group, when I started writing my thesis. I am sure you will have a wonderful time working in our group. Former colleagues at the department, we shared a lot of fun. The most notable ones: Charles (my walking immunology-book), Ebba, Shanie (the caring one), Maria, Jubayer, Pablo, Ming Hui (c-course teaching partner), Halima, Nancy, Natalije, Stephie and Ali. North-South corridor colleagues, thank you for being so helpful. Special thanks to Ioana, Sara S, Hanna, Gabi, Sunil, Jacob, Pilar, Eva H, Spyridon, Aleksandra, Candice, Lucille, Peter, Raquel, Xiao, Sachie K, Einar and Linda for being so helpful when I needed your help. My heartfelt thanks to co-authors and collaborators: your diligent contribution has helped a lot for the betterment of our published and soon to be published paper. I would like to send my 44 special gratitude to Stefan Roos: tack för hjälpen! Bence, thank you for introducing me to the world of Dendritic cells. A number of students worked besides me in different projects. Christine (Tine), my first student, it was fun working with you. Isabelle, you are talented and patient. Nam, the future is bright (you are talented individual). Simon, I enjoyed your short time in our lab (most of all, I used to like your data presentation). Marit, it is hard for me to call you my student. You were my colleague. I actually learned a lot from you. It was wonderful having you in the lab. You are energetic and a good friend and not to forget: a good international conference companion. To my family: you know I love you all. I am indebted to you for all the sacrifices you have made on behalf of the family. My mom, my first supervisor, you did a great job. I am so proud to be your son. Your life turn of event is inspirational. I am blessed with wonderful siblings. Ephiti, thank you for all the advice and for always being there to help me out. Dani, I have no words to express my gratitude. If it was not for your sacrifice, I might not have reached this level. Your advices (hard talk) were always valuable. My beautiful sister (Rahel), my easy going, yet focused brother (Yonas), my life mentor (Jonny), my sister (Hiwi) who was always there in all my childhood fun and menace. My young brother (Dave), I am very proud of you. The pressure is on my beautiful nephew and nieces: Naomi, Betty, Joni and Mehret. We have set the standards of ‘team Adi’, we do not expect less. My new family in Sweden: my peeps at JEC and Hillsong. My Swedish mom-Kiye: your moral support means a lot to me. Tigi, you made my transition from Ethiopia to Sweden so easy. Keberes’ family, thank you for always inviting me in all the Ethiopian holidays (Thanks to you I did not have home sickness). Johny Asrat and Peter Paul, thank you for the moral support and your care. Gulle, I thank you for being a spiritual big brother. Beresh and Sami, you made my weekend filled with fun. Girume, thank you for always being there for me. Bini, my old roommate, we had some memorable times. Neba, my bro, I have never encountered a knowledgeable person like you. I am really proud of you. The passionate host team members at Hillsong, I love my Sunday with you guys. Ghazal and Maryam, my Persian sisters, thank you for helping with obtaining journals from KI library. Miki, it is so nice to have a friend for life. Thank you for always making my US 45 journey memorable. Wuhibo, Johny and Ela, my high school friends end up in Sweden. It was always nice having you around Lappis. Lord, you are my shepherd and redeemer. Your love is my strength. Stor är Din trofasthet! This work was supported by Stockholm University, The Swedish Research Council (grant 57X-15160-10-4), The Swedish Heart-and Lung Foundation, The Ragnar Söderberg Foundation, The Torsten Söderberg Foundation, The Cancer- and Allergy Foundation, The Engqvist Foundations, The Mjölkdroppen-, The Hesselman-, The Golden Jubilee Memorialand The Crownprincess Lovisa/Axel Tielman Foundations. 46 REFERENCES (1) Davies JM, Abreu MT. Host-microbe interactions in the small bowel. Curr Opin Gastroenterol 2015 Mar;31(2):118-123. (2) Mowat AM. Anatomical basis of tolerance and immunity to intestinal antigens. Nat Rev Immunol 2003 Apr;3(4):331-341. (3) Caballero S, Pamer EG. Microbiota-Mediated Inflammation and Antimicrobial Defense in the Intestine. Annu Rev Immunol 2015;33(1):227-256. (4) Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol 2014 Mar;14(3):141-153. (5) Gonzalez-Mariscal L, Betanzos A, Nava P, Jaramillo BE. Tight junction proteins. Prog Biophys Mol Biol 2003 Jan;81(1):1-44. (6) Mowat AM, Agace WW. Regional specialization within the intestinal immune system. Nat Rev Immunol 2014 Oct;14(10):667-685. (7) Rescigno M. Dendritic cell-epithelial cell crosstalk in the gut. Immunol Rev 2014;260(1):118-128. (8) Birchenough GM, Johansson ME, Gustafsson JK, Bergstrom JH, Hansson GC. New developments in goblet cell mucus secretion and function. Mucosal Immunol 2015 Jul;8(4):712-719. (9) McDole JR, Wheeler LW, McDonald KG, Wang B, Konjufca V, Knoop KA, et al. Goblet cells deliver luminal antigen to CD103+ dendritic cells in the small intestine. Nature 2012 Mar 14;483(7389):345-349. (10) Howe SE, Lickteig DJ, Plunkett KN, Ryerse JS, Konjufca V. The uptake of soluble and particulate antigens by epithelial cells in the mouse small intestine. PLoS One 2014 Jan 27;9(1):e86656. (11) Rindi G, Ratineau C, Ronco A, Candusso ME, Tsai M, Leiter AB. Targeted ablation of secretin-producing cells in transgenic mice reveals a common differentiation pathway with multiple enteroendocrine cell lineages in the small intestine. Development 1999 Sep;126(18):4149-4156. (12) Olszak T, Neves JF, Dowds CM, Baker K, Glickman J, Davidson NO, et al. Protective mucosal immunity mediated by epithelial CD1d and IL-10. Nature 2014 May 22;509(7501):497-502. (13) Jabri B, Ebert E. Human CD8+ intraepithelial lymphocytes: a unique model to study the regulation of effector cytotoxic T lymphocytes in tissue. Immunol Rev 2007 Feb;215:202214. 47 (14) Niess JH, Brand S, Gu X, Landsman L, Jung S, McCormick BA, et al. CX3CR1mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science 2005 Jan 14;307(5707):254-258. (15) Rescigno M. Mucosal immunology and bacterial handling in the intestine. Best Pract Res Clin Gastroenterol 2013 Feb;27(1):17-24. (16) Makala LH, Suzuki N, Nagasawa H. Peyer's patches: organized lymphoid structures for the induction of mucosal immune responses in the intestine. Pathobiology 2002 2003;70(2):55-68. (17) Kanamori Y, Ishimaru K, Nanno M, Maki K, Ikuta K, Nariuchi H, et al. Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors develop. J Exp Med 1996 Oct 1;184(4):1449-1459. (18) Pabst O, Herbrand H, Worbs T, Friedrichsen M, Yan S, Hoffmann MW, et al. Cryptopatches and isolated lymphoid follicles: dynamic lymphoid tissues dispensable for the generation of intraepithelial lymphocytes. Eur J Immunol 2005 Jan;35(1):98-107. (19) Lugering A, Ross M, Sieker M, Heidemann J, Williams IR, Domschke W, et al. CCR6 identifies lymphoid tissue inducer cells within cryptopatches. Clin Exp Immunol 2010 Jun;160(3):440-449. (20) Saito H, Kanamori Y, Takemori T, Nariuchi H, Kubota E, Takahashi-Iwanaga H, et al. Generation of intestinal T cells from progenitors residing in gut cryptopatches. Science 1998 Apr 10;280(5361):275-278. (21) Eberl G, Sawa S. Opening the crypt: current facts and hypotheses on the function of cryptopatches. Trends Immunol 2010 Feb;31(2):50-55. (22) Hamada H, Hiroi T, Nishiyama Y, Takahashi H, Masunaga Y, Hachimura S, et al. Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine. J Immunol 2002 Jan 1;168(1):57-64. (23) Rescigno M, Urbano M, Valzasina B, Francolini M, Rotta G, Bonasio R, et al. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol 2001 Apr;2(4):361-367. (24) Sixt M, Kanazawa N, Selg M, Samson T, Roos G, Reinhardt DP, et al. The conduit system transports soluble antigens from the afferent lymph to resident dendritic cells in the T cell area of the lymph node. Immunity 2005 Jan;22(1):19-29. (25) Kumagai Y, Takeuchi O, Akira S. Pathogen recognition by innate receptors. J Infect Chemother 2008 Apr;14(2):86-92. (26) Kawai T, Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 2011 May 27;34(5):637-650. (27) Geijtenbeek TB, Gringhuis SI. Signalling through C-type lectin receptors: shaping immune responses. Nat Rev Immunol 2009 Jul;9(7):465-479. 48 (28) Honda K, Littman DR. The microbiome in infectious disease and inflammation. Annu Rev Immunol 2012;30:759-795. (29) Pulendran B. The varieties of immunological experience: of pathogens, stress, and dendritic cells. Annu Rev Immunol 2015;33:563-606. (30) Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med 1973 May 1;137(5):1142-1162. (31) Moser M, Murphy KM. Dendritic cell regulation of TH1-TH2 development. Nat Immunol 2000 Sep;1(3):199-205. (32) Cerovic V, Houston SA, Scott CL, Aumeunier A, Yrlid U, Mowat AM, et al. Intestinal CD103(-) dendritic cells migrate in lymph and prime effector T cells. Mucosal Immunol 2013 Jan;6(1):104-113. (33) Schulz O, Jaensson E, Persson EK, Liu X, Worbs T, Agace WW, et al. Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions. J Exp Med 2009;206(13):3101-3114. (34) Jaensson E, Uronen-Hansson H, Pabst O, Eksteen B, Tian J, Coombes JL, et al. Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans. J Exp Med 2008 Sep 1;205(9):2139-2149. (35) Mann ER, Li X. Intestinal antigen-presenting cells in mucosal immune homeostasis: crosstalk between dendritic cells, macrophages and B-cells. World J Gastroenterol 2014 Aug 7;20(29):9653-9664. (36) Coombes JL, Siddiqui KRR, Arancibia-Carcamo C, Hall J, Sun C-, Belkaid Y, et al. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF- and retinoic acid dependent mechanism. J Exp Med 2007;204(8):17571764. (37) Bakdash G, Vogelpoel LT, van Capel ,T.M.M., Kapsenberg ML, de Jong ,E.C. Retinoic acid primes human dendritic cells to induce gut-homing, IL-10-producing regulatory T cells. Mucosal Immunology 2015;8(2):265-278. (38) Sun CM, Hall JA, Blank RB, Bouladoux N, Oukka M, Mora JR, et al. Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid. J Exp Med 2007 Aug 6;204(8):1775-1785. (39) Benson MJ, Pino-Lagos K, Rosemblatt M, Noelle RJ. All-trans retinoic acid mediates enhanced T reg cell growth, differentiation, and gut homing in the face of high levels of costimulation. J Exp Med 2007 Aug 6;204(8):1765-1774. (40) Matteoli G, Mazzini E, Iliev ID, Mileti E, Fallarino F, Puccetti P, et al. Gut CD103+ dendritic cells express indoleamine 2,3-dioxygenase which influences T regulatory/T effector cell balance and oral tolerance induction. Gut 2010 May;59(5):595-604. 49 (41) Persson EK, Jaensson E, Agace WW. The diverse ontogeny and function of murine small intestinal dendritic cell/macrophage subsets. Immunobiology 2010 Sep-Oct;215(910):692-697. (42) Persson EK, Uronen-Hansson H, Semmrich M, Rivollier A, Hagerbrand K, Marsal J, et al. IRF4 transcription-factor-dependent CD103(+)CD11b(+) dendritic cells drive mucosal T helper 17 cell differentiation. Immunity 2013 May 23;38(5):958-969. (43) Rogler G, Hausmann M, Vogl D, Aschenbrenner E, Andus T, Falk W, et al. Isolation and phenotypic characterization of colonic macrophages. Clin Exp Immunol 1998 May;112(2):205-215. (44) Smith PD, Smythies LE, Shen R, Greenwell-Wild T, Gliozzi M, Wahl SM. Intestinal macrophages and response to microbial encroachment. Mucosal Immunol 2011 Jan;4(1):3142. (45) Uematsu S, Jang MH, Chevrier N, Guo Z, Kumagai Y, Yamamoto M, et al. Detection of pathogenic intestinal bacteria by Toll-like receptor 5 on intestinal CD11c+ lamina propria cells. Nat Immunol 2006 Aug;7(8):868-874. (46) Bain CC, Scott CL, Uronen-Hansson H, Gudjonsson S, Jansson O, Grip O, et al. Resident and pro-inflammatory macrophages in the colon represent alternative contextdependent fates of the same Ly6Chi monocyte precursors. Mucosal Immunol 2013 May;6(3):498-510. (47) Zigmond E, Jung S. Intestinal macrophages: well educated exceptions from the rule. Trends Immunol 2013 Apr;34(4):162-168. (48) Scott CL, Bain CC, Wright PB, Sichien D, Kotarsky K, Persson EK, et al. CCR2(+)CD103(-) intestinal dendritic cells develop from DC-committed precursors and induce interleukin-17 production by T cells. Mucosal Immunol 2015 Mar;8(2):327-339. (49) Denning TL, Wang YC, Patel SR, Williams IR, Pulendran B. Lamina propria macrophages and dendritic cells differentially induce regulatory and interleukin 17-producing T cell responses. Nat Immunol 2007 Oct;8(10):1086-1094. (50) Platt AM, Bain CC, Bordon Y, Sester DP, Mowat AM. An independent subset of TLR expressing CCR2-dependent macrophages promotes colonic inflammation. J Immunol 2010 Jun 15;184(12):6843-6854. (51) Murai M, Turovskaya O, Kim G, Madan R, Karp CL, Cheroutre H, et al. Interleukin 10 acts on regulatory T cells to maintain expression of the transcription factor Foxp3 and suppressive function in mice with colitis. Nat Immunol 2009 Nov;10(11):1178-1184. (52) Hadis U, Wahl B, Schulz O, Hardtke-Wolenski M, Schippers A, Wagner N, et al. Intestinal tolerance requires gut homing and expansion of FoxP3+ regulatory T cells in the lamina propria. Immunity 2011 Feb 25;34(2):237-246. 50 (53) Pull SL, Doherty JM, Mills JC, Gordon JI, Stappenbeck TS. Activated macrophages are an adaptive element of the colonic epithelial progenitor niche necessary for regenerative responses to injury. Proc Natl Acad Sci U S A 2005 Jan 4;102(1):99-104. (54) Rivollier A, He J, Kole A, Valatas V, Kelsall BL. Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon. J Exp Med 2012 Jan 16;209(1):139-155. (55) Tamoutounour S, Henri S, Lelouard H, de Bovis B, de Haar C, van der Woude CJ, et al. CD64 distinguishes macrophages from dendritic cells in the gut and reveals the Th1-inducing role of mesenteric lymph node macrophages during colitis. Eur J Immunol 2012 Dec;42(12):3150-3166. (56) Varol C, Mildner A, Jung S. Macrophages: development and tissue specialization. Annu Rev Immunol 2015;33:643-675. (57) Bain CC, Mowat AM. The monocyte-macrophage axis in the intestine. Cell Immunol 2014;291(1-2):41-48. (58) Yona S, Kim KW, Wolf Y, Mildner A, Varol D, Breker M, et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 2013 Jan 24;38(1):79-91. (59) Sunderkotter C, Nikolic T, Dillon MJ, Van Rooijen N, Stehling M, Drevets DA, et al. Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response. J Immunol 2004 Apr 1;172(7):4410-4417. (60) Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 2003 Jul;19(1):71-82. (61) Bain CC, Mowat AM. Macrophages in intestinal homeostasis and inflammation. Immunol Rev 2014 Jul;260(1):102-117. (62) Kurihara T, Warr G, Loy J, Bravo R. Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor. J Exp Med 1997 Nov 17;186(10):1757-1762. (63) Serbina NV, Pamer EG. Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2. Nat Immunol 2006 Mar;7(3):311-317. (64) Skoczek DA, Walczysko P, Horn N, Parris A, Clare S, Williams MR, et al. Luminal microbes promote monocyte-stem cell interactions across a healthy colonic epithelium. J Immunol 2014 Jul 1;193(1):439-451. (65) Carlin LM, Stamatiades EG, Auffray C, Hanna RN, Glover L, Vizcay-Barrena G, et al. Nr4a1-dependent Ly6C(low) monocytes monitor endothelial cells and orchestrate their disposal. Cell 2013 Apr 11;153(2):362-375. 51 (66) Ziegler-Heitbrock L, Ancuta P, Crowe S, Dalod M, Grau V, Hart DN, et al. Nomenclature of monocytes and dendritic cells in blood. Blood 2010 Oct 21;116(16):e74-80. (67) Ingersoll MA, Spanbroek R, Lottaz C, Gautier EL, Frankenberger M, Hoffmann R, et al. Comparison of gene expression profiles between human and mouse monocyte subsets. Blood 2010 Jan 21;115(3):e10-9. (68) Rugtveit J, Nilsen EM, Bakka A, Carlsen H, Brandtzaeg P, Scott H. Cytokine profiles differ in newly recruited and resident subsets of mucosal macrophages from inflammatory bowel disease. Gastroenterology 1997 May;112(5):1493-1505. (69) Rugtveit J, Haraldsen G, Hogasen AK, Bakka A, Brandtzaeg P, Scott H. Respiratory burst of intestinal macrophages in inflammatory bowel disease is mainly caused by CD14+L1+ monocyte derived cells. Gut 1995 Sep;37(3):367-373. (70) Grainger JR, Wohlfert EA, Fuss IJ, Bouladoux N, Askenase MH, Legrand F, et al. Inflammatory monocytes regulate pathologic responses to commensals during acute gastrointestinal infection. Nat Med 2013 Jun;19(6):713-721. (71) Dunay IR, Damatta RA, Fux B, Presti R, Greco S, Colonna M, et al. Gr1(+) inflammatory monocytes are required for mucosal resistance to the pathogen Toxoplasma gondii. Immunity 2008 Aug 15;29(2):306-317. (72) Carlens J, Wahl B, Ballmaier M, Bulfone-Paus S, Forster R, Pabst O. Common gammachain-dependent signals confer selective survival of eosinophils in the murine small intestine. J Immunol 2009 Nov 1;183(9):5600-5607. (73) Shang L, Thirunarayanan N, Viejo-Borbolla A, Martin AP, Bogunovic M, Marchesi F, et al. Expression of the chemokine binding protein M3 promotes marked changes in the accumulation of specific leukocytes subsets within the intestine. Gastroenterology 2009 Sep;137(3):1006-18, 1018.e1-3. (74) Straumann A, Kristl J, Conus S, Vassina E, Spichtin HP, Beglinger C, et al. Cytokine expression in healthy and inflamed mucosa: probing the role of eosinophils in the digestive tract. Inflamm Bowel Dis 2005 Aug;11(8):720-726. (75) Chu VT, Beller A, Rausch S, Strandmark J, Zanker M, Arbach O, et al. Eosinophils promote generation and maintenance of immunoglobulin-A-expressing plasma cells and contribute to gut immune homeostasis. Immunity 2014 Apr 17;40(4):582-593. (76) Fournier BM, Parkos CA. The role of neutrophils during intestinal inflammation. Mucosal Immunol 2012 Jul;5(4):354-366. (77) Ajuebor MN, Das AM, Virag L, Flower RJ, Szabo C, Perretti M. Role of resident peritoneal macrophages and mast cells in chemokine production and neutrophil migration in acute inflammation: evidence for an inhibitory loop involving endogenous IL-10. J Immunol 1999 Feb 1;162(3):1685-1691. (78) Soehnlein O, Lindbom L. Phagocyte partnership during the onset and resolution of inflammation. Nat Rev Immunol 2010 Jun;10(6):427-439. 52 (79) Kuhl AA, Kakirman H, Janotta M, Dreher S, Cremer P, Pawlowski NN, et al. Aggravation of different types of experimental colitis by depletion or adhesion blockade of neutrophils. Gastroenterology 2007 Dec;133(6):1882-1892. (80) Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science 2004 Mar 5;303(5663):1532-1535. (81) Mantovani A, Cassatella MA, Costantini C, Jaillon S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol 2011 Jul 25;11(8):519-531. (82) Raab Y, Gerdin B, Ahlstedt S, Hallgren R. Neutrophil mucosal involvement is accompanied by enhanced local production of interleukin-8 in ulcerative colitis. Gut 1993 Sep;34(9):1203-1206. (83) Lloyd G, Green FH, Fox H, Mani V, Turnberg LA. Mast cells and immunoglobulin E in inflammatory bowel disease. Gut 1975 Nov;16(11):861-865. (84) Miller HR, Pemberton AD. Tissue-specific expression of mast cell granule serine proteinases and their role in inflammation in the lung and gut. Immunology 2002 Apr;105(4):375-390. (85) Miller HR, Huntley JF, Newlands GF, Mackellar A, Lammas DA, Wakelin D. Granule proteinases define mast cell heterogeneity in the serosa and the gastrointestinal mucosa of the mouse. Immunology 1988 Dec;65(4):559-566. (86) Hogquist KA, Baldwin TA, Jameson SC. Central tolerance: learning self-control in the thymus. Nat Rev Immunol 2005 Oct;5(10):772-782. (87) Holmkvist P. Regulation of T cell effector functions in the intestinal mucosa. Lund: Lund University; 2014. (88) Agace WW. Tissue-tropic effector T cells: generation and targeting opportunities. Nat Rev Immunol 2006 Sep;6(9):682-692. (89) Zhu J, Paul WE. CD4 T cells: fates, functions, and faults. Blood 2008 Sep 1;112(5):1557-1569. (90) Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ, Konkel JE, et al. Generation of pathogenic T(H)17 cells in the absence of TGF-beta signalling. Nature 2010 Oct 21;467(7318):967-971. (91) Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annu Rev Immunol 2009;27:485-517. (92) Moore C, Fuentes C, Sauma D, Morales J, Bono MR, Rosemblatt M, et al. Retinoic acid generates regulatory T cells in experimental transplantation. Transplant Proc 2011 JulAug;43(6):2334-2337. 53 (93) Maizels RM, Pearce EJ, Artis D, Yazdanbakhsh M, Wynn TA. Regulation of pathogenesis and immunity in helminth infections. J Exp Med 2009 Sep 28;206(10):20592066. (94) Fossiez F, Djossou O, Chomarat P, Flores-Romo L, Ait-Yahia S, Maat C, et al. T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. J Exp Med 1996 Jun 1;183(6):2593-2603. (95) Ishigame H, Kakuta S, Nagai T, Kadoki M, Nambu A, Komiyama Y, et al. Differential roles of interleukin-17A and -17F in host defense against mucoepithelial bacterial infection and allergic responses. Immunity 2009 Jan 16;30(1):108-119. (96) Liang SC, Tan XY, Luxenberg DP, Karim R, Dunussi-Joannopoulos K, Collins M, et al. Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J Exp Med 2006 Oct 2;203(10):2271-2279. (97) Zheng Y, Valdez Pa, Danilenko DM, Hu Y, Sa SM, Gong Q, et al. Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat Med 2008;14(3):282-289. (98) Maynard CL, Weaver CT. Intestinal effector T cells in health and disease. Immunity 2009 Sep 18;31(3):389-400. (99) Cosmi L, Maggi L, Santarlasci V, Liotta F, Annunziato F. T helper cells plasticity in inflammation. Cytometry A 2013 Sep 5. (100) Trifari S, Kaplan CD, Tran EH, Crellin NK, Spits H. Identification of a human helper T cell population that has abundant production of interleukin 22 and is distinct from T(H)-17, T(H)1 and T(H)2 cells. Nat Immunol 2009 Aug;10(8):864-871. (101) Veldhoen M, Uyttenhove C, van Snick J, Helmby H, Westendorf A, Buer J, et al. Transforming growth factor-beta 'reprograms' the differentiation of T helper 2 cells and promotes an interleukin 9-producing subset. Nat Immunol 2008 Dec;9(12):1341-1346. (102) Kato LM, Kawamoto S, Maruya M, Fagarasan S. Gut TFH and IgA: key players for regulation of bacterial communities and immune homeostasis. Immunol Cell Biol 2014 Jan;92(1):49-56. (103) Cannons JL, Lu KT, Schwartzberg PL. T follicular helper cell diversity and plasticity. Trends Immunol 2013 May;34(5):200-207. (104) Hirota K, Duarte JH, Veldhoen M, Hornsby E, Li Y, Cua DJ, et al. Fate mapping of IL17-producing T cells in inflammatory responses. Nat Immunol 2011 Mar;12(3):255-263. (105) Björkander S, Hell L, Johansson MA, Forsberg MM, Lasaviciute G, Roos S, et al. Staphylococcus aureus-derived factors induce IL-10, IFN-gamma and IL-17A-expressing FOXP3(+)CD161(+) T-helper cells in a partly monocyte-dependent manner. Sci Rep 2016 Feb 26;6:22083. 54 (106) Duhen T, Duhen R, Lanzavecchia A, Sallusto F, Campbell DJ. Functionally distinct subsets of human FOXP3+ Treg cells that phenotypically mirror effector Th cells. Blood 2012 May 10;119(19):4430-4440. (107) Chaudhry A, Rudra D, Treuting P, Samstein RM, Liang Y, Kas A, et al. CD4+ regulatory T cells control TH17 responses in a Stat3-dependent manner. Science 2009 Nov 13;326(5955):986-991. (108) Koch MA, Tucker-Heard G, Perdue NR, Killebrew JR, Urdahl KB, Campbell DJ. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat Immunol 2009 Jun;10(6):595-602. (109) Zheng Y, Chaudhry A, Kas A, deRoos P, Kim JM, Chu TT, et al. Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control T(H)2 responses. Nature 2009 Mar 19;458(7236):351-356. (110) Lieberman J. The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal. Nat Rev Immunol 2003 May;3(5):361-370. (111) Joffre OP, Segura E, Savina A, Amigorena S. Cross-presentation by dendritic cells. Nat Rev Immunol 2012 Jul 13;12(8):557-569. (112) Liu Y, Lan Q, Lu L, Chen M, Xia Z, Ma J, et al. Phenotypic and functional characteristic of a newly identified CD8+ Foxp3- CD103+ regulatory T cells. J Mol Cell Biol 2014 Feb;6(1):81-92. (113) Nakagawa T, Tsuruoka M, Ogura H, Okuyama Y, Arima Y, Hirano T, et al. IL-6 positively regulates Foxp3+CD8+ T cells in vivo. Int Immunol 2010 Feb;22(2):129-139. (114) Horwitz DA, Pan S, Ou JN, Wang J, Chen M, Gray JD, et al. Therapeutic polyclonal human CD8+ CD25+ Fox3+ TNFR2+ PD-L1+ regulatory cells induced ex-vivo. Clin Immunol 2013 Dec;149(3):450-463. (115) Kishi M, Yasuda H, Abe Y, Sasaki H, Shimizu M, Arai T, et al. Regulatory CD8+ T cells induced by exposure to all-trans retinoic acid and TGF-beta suppress autoimmune diabetes. Biochem Biophys Res Commun 2010 Mar 26;394(1):228-232. (116) Churlaud G, Pitoiset F, Jebbawi F, Lorenzon R, Bellier B, Rosenzwajg M, et al. Human and Mouse CD8(+)CD25(+)FOXP3(+) Regulatory T Cells at Steady State and during Interleukin-2 Therapy. Front Immunol 2015 Apr 15;6:171. (117) Macpherson AJ, Koller Y, McCoy KD. The bilateral responsiveness between intestinal microbes and IgA. Trends Immunol 2015 Aug;36(8):460-470. (118) Mora JR, Iwata M, Eksteen B, Song SY, Junt T, Senman B, et al. Generation of guthoming IgA-secreting B cells by intestinal dendritic cells. Science 2006 Nov 17;314(5802):1157-1160. 55 (119) Cong Y, Feng T, Fujihashi K, Schoeb TR, Elson CO. A dominant, coordinated T regulatory cell-IgA response to the intestinal microbiota. Proc Natl Acad Sci U S A 2009 Nov 17;106(46):19256-19261. (120) Brandtzaeg P. Function of mucosa-associated lymphoid tissue in antibody formation. Immunol Invest 2010;39(4-5):303-355. (121) Brandtzaeg P. Secretory IgA: Designed for Anti-Microbial Defense. Front Immunol 2013 Aug 6;4:222. (122) Cerutti A, Chen K, Chorny A. Immunoglobulin responses at the mucosal interface. Annu Rev Immunol 2011;29:273-293. (123) Kubinak JL, Petersen C, Stephens WZ, Soto R, Bake E, O'Connell RM, et al. MyD88 signaling in T cells directs IgA-mediated control of the microbiota to promote health. Cell Host Microbe 2015 Feb 11;17(2):153-163. (124) Mizoguchi A, Mizoguchi E, Takedatsu H, Blumberg RS, Bhan AK. Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation. Immunity 2002 Feb;16(2):219-230. (125) Pabst O. New concepts in the generation and functions of IgA. Nat Rev Immunol 2012 Dec;12(12):821-832. (126) Fillatreau S, Sweenie CH, McGeachy MJ, Gray D, Anderton SM. B cells regulate autoimmunity by provision of IL-10. Nat Immunol 2002 Oct;3(10):944-950. (127) Mauri C, Gray D, Mushtaq N, Londei M. Prevention of arthritis by interleukin 10producing B cells. J Exp Med 2003 Feb 17;197(4):489-501. (128) Liu ZQ, Wu Y, Song JP, Liu X, Liu Z, Zheng PY, et al. Tolerogenic CX3CR1+ B cells suppress food allergy-induced intestinal inflammation in mice. Allergy 2013 Oct;68(10):1241-1248. (129) Roberts S, Girardi M. Conventional and Unconventional T Cells. 2008:85-104. (130) Kjer-Nielsen L, Patel O, Corbett AJ, Le Nours J, Meehan B, Liu L, et al. MR1 presents microbial vitamin B metabolites to MAIT cells. Nature 2012 Nov 29;491(7426):717-723. (131) Witherden DA, Havran WL. Molecular aspects of epithelial gammadelta T cell regulation. Trends Immunol 2011 Jun;32(6):265-271. (132) An D, Oh SF, Olszak T, Neves JF, Avci FY, Erturk-Hasdemir D, et al. Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells. Cell 2014 Jan 16;156(1-2):123-133. (133) Loh L, Ivarsson MA, Michaelsson J, Sandberg JK, Nixon DF. Invariant natural killer T cells developing in the human fetus accumulate and mature in the small intestine. Mucosal Immunol 2014 Sep;7(5):1233-1243. 56 (134) Sonnenberg GF, Artis D. Innate lymphoid cells in the initiation, regulation and resolution of inflammation. Nat Med 2015 Jul;21(7):698-708. (135) Tait Wojno ED, Artis D. Innate lymphoid cells: balancing immunity, inflammation, and tissue repair in the intestine. Cell Host Microbe 2012 Oct 18;12(4):445-457. (136) Serafini N, Vosshenrich CA, Di Santo JP. Transcriptional regulation of innate lymphoid cell fate. Nat Rev Immunol 2015 Jul;15(7):415-428. (137) Artis D, Spits H. The biology of innate lymphoid cells. Nature 2015 Jan 15;517(7534):293-301. (138) Bernink JH, Peters CP, Munneke M, te Velde AA, Meijer SL, Weijer K, et al. Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues. Nat Immunol 2013 Mar;14(3):221-229. (139) Bernink J, Mjosberg J, Spits H. Th1- and Th2-like subsets of innate lymphoid cells. Immunol Rev 2013 Mar;252(1):133-138. (140) Walker JA, Barlow JL, McKenzie AN. Innate lymphoid cells--how did we miss them? Nat Rev Immunol 2013 Feb;13(2):75-87. (141) Satokari R, Gronroos T, Laitinen K, Salminen S, Isolauri E. Bifidobacterium and Lactobacillus DNA in the human placenta. Lett Appl Microbiol 2009 Jan;48(1):8-12. (142) Gosalbes MJ, Llop S, Valles Y, Moya A, Ballester F, Francino MP. Meconium microbiota types dominated by lactic acid or enteric bacteria are differentially associated with maternal eczema and respiratory problems in infants. Clin Exp Allergy 2013 Feb;43(2):198211. (143) Palmer, Chana AND Bik, Elisabeth M AND DiGiulio, Daniel B AND Relman, David A AND Brown,Patrick O. Development of the Human Infant Intestinal Microbiota. PLoS Biol 2007 06;5(7):e177. (144) Dominguez-Bello MG, Costello EK, Contreras M, Magris M, Hidalgo G, Fierer N, et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci U S A 2010 Jun 29;107(26):1197111975. (145) Matamoros S, Gras-Leguen C, Le Vacon F, Potel G, de La Cochetiere MF. Development of intestinal microbiota in infants and its impact on health. Trends Microbiol 2013 Apr;21(4):167-173. (146) Hernell O, West CE. Clinical effects of probiotics: scientific evidence from a paediatric perspective. Br J Nutr 2013 1;109(Supplement S2):S70-S75. (147) Ou G, Hedberg M, Horstedt P, Baranov V, Forsberg G, Drobni M, et al. Proximal small intestinal microbiota and identification of rod-shaped bacteria associated with childhood celiac disease. Am J Gastroenterol 2009 Dec;104(12):3058-3067. 57 (148) Sekirov I, Russell SL, Antunes LC, Finlay BB. Gut microbiota in health and disease. Physiol Rev 2010 Jul;90(3):859-904. (149) Mackie RI, Sghir A, Gaskins HR. Developmental microbial ecology of the neonatal gastrointestinal tract. Am J Clin Nutr 1999 May;69(5):1035S-1045S. (150) Jakobsson HE, Abrahamsson TR, Jenmalm MC, Harris K, Quince C, Jernberg C, et al. Decreased gut microbiota diversity, delayed Bacteroidetes colonisation and reduced Th1 responses in infants delivered by Caesarean section. Gut 2013 Aug 7. (151) Barrett E, Kerr C, Murphy K, O'Sullivan O, Ryan CA, Dempsey EM, et al. The individual-specific and diverse nature of the preterm infant microbiota. Arch Dis Child Fetal Neonatal Ed 2013 Jul;98(4):F334-40. (152) Gronlund MM, Gueimonde M, Laitinen K, Kociubinski G, Gronroos T, Salminen S, et al. Maternal breast-milk and intestinal bifidobacteria guide the compositional development of the Bifidobacterium microbiota in infants at risk of allergic disease. Clin Exp Allergy 2007 Dec;37(12):1764-1772. (153) Collado MC, Delgado S, Maldonado A, Rodriguez JM. Assessment of the bacterial diversity of breast milk of healthy women by quantitative real-time PCR. Lett Appl Microbiol 2009 May;48(5):523-528. (154) Koenig JE, Spor A, Scalfone N, Fricker AD, Stombaugh J, Knight R, et al. Succession of microbial consortia in the developing infant gut microbiome. Proc Natl Acad Sci U S A 2011 Mar 15;108 Suppl 1:4578-4585. (155) De Filippo C, Cavalieri D, Di Paola M, Ramazzotti M, Poullet JB, Massart S, et al. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc Natl Acad Sci U S A 2010 Aug 17;107(33):14691-14696. (156) Macfarlane S, Macfarlane GT. Regulation of short-chain fatty acid production. Proc Nutr Soc 2003 Feb;62(1):67-72. (157) Azad MB, Konya T, Maughan H, Guttman DS, Field CJ, Sears MR, et al. Infant gut microbiota and the hygiene hypothesis of allergic disease: impact of household pets and siblings on microbiota composition and diversity. Allergy Asthma Clin Immunol 2013 Apr 22;9(1):15. (158) von Mutius E, Vercelli D. Farm living: effects on childhood asthma and allergy. Nat Rev Immunol 2010 Dec;10(12):861-868. (159) Rook GA, Lowry CA, Raison CL. Hygiene and other early childhood influences on the subsequent function of the immune system. Brain Res 2015 Aug 18;1617:47-62. (160) Yatsunenko T, Rey FE, Manary MJ, Trehan I, Dominguez-Bello MG, Contreras M, et al. Human gut microbiome viewed across age and geography. Nature 2012 May 9;486(7402):222-227. 58 (161) Sepp E, Julge K, Vasar M, Naaber P, Bjorksten B, Mikelsaar M. Intestinal microflora of Estonian and Swedish infants. Acta Paediatr 1997 Sep;86(9):956-961. (162) Mulder IE, Schmidt B, Stokes CR, Lewis M, Bailey M, Aminov RI, et al. Environmentally-acquired bacteria influence microbial diversity and natural innate immune responses at gut surfaces. BMC Biol 2009 Nov 20;7:79-7007-7-79. (163) Sjöberg F. Microbiota of the alimentary tract of children: implications for allergy and inflammatory bowel disease. Göteborg: Institute of Biomedicine, Department of Microbiology and Immunology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg; 2014. (164) Jernberg C, Lofmark S, Edlund C, Jansson JK. Long-term ecological impacts of antibiotic administration on the human intestinal microbiota. ISME J 2007 May;1(1):56-66. (165) Hill DA, Hoffmann C, Abt MC, Du Y, Kobuley D, Kirn TJ, et al. Metagenomic analyses reveal antibiotic-induced temporal and spatial changes in intestinal microbiota with associated alterations in immune cell homeostasis. Mucosal Immunol 2010 Mar;3(2):148158. (166) Marietta E, Rishi A, Taneja V. Immunogenetic control of the intestinal microbiota. Immunology 2015 Jul;145(3):313-322. (167) Goodrich JK, Waters JL, Poole AC, Sutter JL, Koren O, Blekhman R, et al. Human genetics shape the gut microbiome. Cell 2014 Nov 6;159(4):789-799. (168) Ley RE, Backhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI. Obesity alters gut microbial ecology. Proc Natl Acad Sci U S A 2005 Aug 2;102(31):11070-11075. (169) Spor A, Koren O, Ley R. Unravelling the effects of the environment and host genotype on the gut microbiome. Nat Rev Microbiol 2011 Apr;9(4):279-290. (170) Crawford PA, Crowley JR, Sambandam N, Muegge BD, Costello EK, Hamady M, et al. Regulation of myocardial ketone body metabolism by the gut microbiota during nutrient deprivation. Proc Natl Acad Sci U S A 2009 Jul 7;106(27):11276-11281. (171) Donaldson GP, Lee SM, Mazmanian SK. Gut biogeography of the bacterial microbiota. Nat Rev Microbiol 2016 Jan;14(1):20-32. (172) Lagier JC, Armougom F, Million M, Hugon P, Pagnier I, Robert C, et al. Microbial culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect 2012 Dec;18(12):1185-1193. (173) Ueda K, Seki T, Kudo T, Yoshida T, Kataoka M. Two distinct mechanisms cause heterogeneity of 16S rRNA. J Bacteriol 1999 Jan;181(1):78-82. (174) West CE, Renz H, Jenmalm MC, Kozyrskyj AL, Allen KJ, Vuillermin P, et al. The gut microbiota and inflammatory noncommunicable diseases: associations and potentials for gut microbiota therapies. J Allergy Clin Immunol 2015 Jan;135(1):3-13; quiz 14. 59 (175) Lozupone CA, Stombaugh JI, Gordon JI, Jansson JK, Knight R. Diversity, stability and resilience of the human gut microbiota. Nature 2012 Sep 13;489(7415):220-230. (176) Hill DA, Artis D. Intestinal bacteria and the regulation of immune cell homeostasis. Annu Rev Immunol 2010 Mar;28:623-667. (177) Kabat AM, Srinivasan N, Maloy KJ. Modulation of immune development and function by intestinal microbiota. Trends Immunol 2014 Nov;35(11):507-517. (178) Pott J, Hornef M. Innate immune signalling at the intestinal epithelium in homeostasis and disease. EMBO Rep 2012 Aug;13(8):684-698. (179) Maslowski KM, Mackay CR. Diet, gut microbiota and immune responses. Nat Immunol 2011 Jan;12(1):5-9. (180) Shimada Y, Kinoshita M, Harada K, Mizutani M, Masahata K, Kayama H, et al. Commensal bacteria-dependent indole production enhances epithelial barrier function in the colon. PLoS One 2013 Nov 20;8(11):e80604. (181) Lee WJ, Hase K. Gut microbiota-generated metabolites in animal health and disease. Nat Chem Biol 2014 Jun;10(6):416-424. (182) Kamada N, Chen GY, Inohara N, Nunez G. Control of pathogens and pathobionts by the gut microbiota. Nat Immunol 2013 Jul;14(7):685-690. (183) Wrzosek L, Miquel S, Noordine ML, Bouet S, Joncquel Chevalier-Curt M, Robert V, et al. Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii influence the production of mucus glycans and the development of goblet cells in the colonic epithelium of a gnotobiotic model rodent. BMC Biol 2013 May 21;11:61-7007-11-61. (184) Brandl K, Plitas G, Mihu CN, Ubeda C, Jia T, Fleisher M, et al. Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits. Nature 2008 Oct 9;455(7214):804-807. (185) Vaishnava S, Yamamoto M, Severson KM, Ruhn KA, Yu X, Koren O, et al. The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine. Science 2011 Oct 14;334(6053):255-258. (186) Miyaoka Y, Kadowaki Y, Ishihara S, Ose T, Fukuhara H, Kazumori H, et al. Transgenic overexpression of Reg protein caused gastric cell proliferation and differentiation along parietal cell and chief cell lineages. Oncogene 2004 Apr 29;23(20):3572-3579. (187) Alakomi HL, Skytta E, Saarela M, Mattila-Sandholm T, Latva-Kala K, Helander IM. Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Appl Environ Microbiol 2000 May;66(5):2001-2005. (188) Kida Y, Shimizu T, Kuwano K. Sodium butyrate up-regulates cathelicidin gene expression via activator protein-1 and histone acetylation at the promoter region in a human lung epithelial cell line, EBC-1. Mol Immunol 2006 May;43(12):1972-1981. 60 (189) Schauber J, Svanholm C, Termen S, Iffland K, Menzel T, Scheppach W, et al. Expression of the cathelicidin LL-37 is modulated by short chain fatty acids in colonocytes: relevance of signalling pathways. Gut 2003 May;52(5):735-741. (190) Termen S, Tollin M, Rodriguez E, Sveinsdottir SH, Johannesson B, Cederlund A, et al. PU.1 and bacterial metabolites regulate the human gene CAMP encoding antimicrobial peptide LL-37 in colon epithelial cells. Mol Immunol 2008 Sep;45(15):3947-3955. (191) Tremaroli V, Backhed F. Functional interactions between the gut microbiota and host metabolism. Nature 2012 Sep 13;489(7415):242-249. (192) Fukuda S, Toh H, Hase K, Oshima K, Nakanishi Y, Yoshimura K, et al. Bifidobacteria can protect from enteropathogenic infection through production of acetate. Nature 2011 Jan 27;469(7331):543-547. (193) Zeuthen LH, Fink LN, Frokiaer H. Epithelial cells prime the immune response to an array of gut-derived commensals towards a tolerogenic phenotype through distinct actions of thymic stromal lymphopoietin and transforming growth factor-beta. Immunology 2008 Feb;123(2):197-208. (194) Fukata M, Shang L, Santaolalla R, Sotolongo J, Pastorini C, Espana C, et al. Constitutive activation of epithelial TLR4 augments inflammatory responses to mucosal injury and drives colitis-associated tumorigenesis. Inflamm Bowel Dis 2011 Jul;17(7):14641473. (195) Shang L, Fukata M, Thirunarayanan N, Martin AP, Arnaboldi P, Maussang D, et al. Toll-like receptor signaling in small intestinal epithelium promotes B-cell recruitment and IgA production in lamina propria. Gastroenterology 2008 Aug;135(2):529-538. (196) Swamy M, Jamora C, Havran W, Hayday A. Epithelial decision makers: in search of the 'epimmunome'. Nat Immunol 2010 Aug;11(8):656-665. (197) Kalina U, Koyama N, Hosoda T, Nuernberger H, Sato K, Hoelzer D, et al. Enhanced production of IL-18 in butyrate-treated intestinal epithelium by stimulation of the proximal promoter region. Eur J Immunol 2002 Sep;32(9):2635-2643. (198) Singh N, Gurav A, Sivaprakasam S, Brady E, Padia R, Shi H, et al. Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunity 2014 Jan 16;40(1):128-139. (199) Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara T, Momose Y, et al. Induction of colonic regulatory T cells by indigenous Clostridium species. Science 2011 Jan 21;331(6015):337-341. (200) Abreu MT. Toll-like receptor signalling in the intestinal epithelium: how bacterial recognition shapes intestinal function. Nat Rev Immunol 2010 Feb;10(2):131-144. (201) Kelly D, Campbell JI, King TP, Grant G, Jansson EA, Coutts AG, et al. Commensal anaerobic gut bacteria attenuate inflammation by regulating nuclear-cytoplasmic shuttling of PPAR-gamma and RelA. Nat Immunol 2004 Jan;5(1):104-112. 61 (202) Xiao H, Gulen MF, Qin J, Yao J, Bulek K, Kish D, et al. The Toll-interleukin-1 receptor member SIGIRR regulates colonic epithelial homeostasis, inflammation, and tumorigenesis. Immunity 2007 Apr;26(4):461-475. (203) Huang N, Katz JP, Martin DR, Wu GD. Inhibition of IL-8 gene expression in Caco-2 cells by compounds which induce histone hyperacetylation. Cytokine 1997 Jan;9(1):27-36. (204) Rimoldi M, Chieppa M, Salucci V, Avogadri F, Sonzogni A, Sampietro GM, et al. Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nat Immunol 2005 May;6(5):507-514. (205) Sawa S, Lochner M, Satoh-Takayama N, Dulauroy S, Berard M, Kleinschek M, et al. RORgammat+ innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota. Nat Immunol 2011 Apr;12(4):320-326. (206) Vonarbourg C, Mortha A, Bui VL, Hernandez PP, Kiss EA, Hoyler T, et al. Regulated expression of nuclear receptor RORgammat confers distinct functional fates to NK cell receptor-expressing RORgammat(+) innate lymphocytes. Immunity 2010 Nov 24;33(5):736751. (207) Satoh-Takayama N, Vosshenrich CA, Lesjean-Pottier S, Sawa S, Lochner M, Rattis F, et al. Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense. Immunity 2008 Dec 19;29(6):958-970. (208) Sanderson IR, Ouellette AJ, Carter EA, Walker WA, Harmatz PR. Differential regulation of B7 mRNA in enterocytes and lymphoid cells. Immunology 1993 Jul;79(3):434438. (209) Sonnenberg GF, Monticelli LA, Alenghat T, Fung TC, Hutnick NA, Kunisawa J, et al. Innate lymphoid cells promote anatomical containment of lymphoid-resident commensal bacteria. Science 2012 Jun 8;336(6086):1321-1325. (210) Lochner M, Ohnmacht C, Presley L, Bruhns P, Si-Tahar M, Sawa S, et al. Microbiotainduced tertiary lymphoid tissues aggravate inflammatory disease in the absence of RORgamma t and LTi cells. J Exp Med 2011 Jan 17;208(1):125-134. (211) Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 2005 Jul 15;122(1):107-118. (212) Bouskra D, Brezillon C, Berard M, Werts C, Varona R, Boneca IG, et al. Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. Nature 2008 Nov 27;456(7221):507-510. (213) Min YW, Rhee PL. The Role of Microbiota on the Gut Immunology. Clin Ther 2015 May 1;37(5):968-975. (214) Hooper LV. Bacterial contributions to mammalian gut development. Trends Microbiol 2004 Mar;12(3):129-134. 62 (215) Hapfelmeier S, Lawson MA, Slack E, Kirundi JK, Stoel M, Heikenwalder M, et al. Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses. Science 2010 Jun 25;328(5986):1705-1709. (216) Round JL, Mazmanian SK. Inducible Foxp3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota. Proc Natl Acad Sci U S A 2010 Jul 6;107(27):12204-12209. (217) Round JL, Lee SM, Li J, Tran G, Jabri B, Chatila TA, et al. The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota. Science 2011 May 20;332(6032):974-977. (218) Dasgupta S, Erturk-Hasdemir D, Ochoa-Reparaz J, Reinecker HC, Kasper DL. Plasmacytoid dendritic cells mediate anti-inflammatory responses to a gut commensal molecule via both innate and adaptive mechanisms. Cell Host Microbe 2014 Apr 9;15(4):413-423. (219) Shen Y, Giardino Torchia ML, Lawson GW, Karp CL, Ashwell JD, Mazmanian SK. Outer membrane vesicles of a human commensal mediate immune regulation and disease protection. Cell Host Microbe 2012 Oct 18;12(4):509-520. (220) Geuking MB, Cahenzli J, Lawson MA, Ng DC, Slack E, Hapfelmeier S, et al. Intestinal bacterial colonization induces mutualistic regulatory T cell responses. Immunity 2011 May 27;34(5):794-806. (221) Arpaia N, Campbell C, Fan X, Dikiy S, van der Veeken J, deRoos P, et al. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature 2013 Dec 19;504(7480):451-455. (222) Furusawa Y, Obata Y, Fukuda S, Endo TA, Nakato G, Takahashi D, et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 2013 Dec 19;504(7480):446-450. (223) Smith PM, Howitt MR, Panikov N, Michaud M, Gallini CA, Bohlooly-Y M, et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science 2013 Aug 2;341(6145):569-573. (224) Ivanov II, Manel N. Induction of gut mucosal Th17 cells by segmented filamentous bacteria. Med Sci (Paris) 2010 Apr;26(4):352-355. (225) Shaw MH, Kamada N, Kim YG, Nunez G. Microbiota-induced IL-1beta, but not IL-6, is critical for the development of steady-state TH17 cells in the intestine. J Exp Med 2012 Feb 13;209(2):251-258. (226) Atarashi K, Nishimura J, Shima T, Umesaki Y, Yamamoto M, Onoue M, et al. ATP drives lamina propria T(H)17 cell differentiation. Nature 2008 Oct 9;455(7214):808-812. (227) Berndt BE, Zhang M, Owyang SY, Cole TS, Wang TW, Luther J, et al. Butyrate increases IL-23 production by stimulated dendritic cells. Am J Physiol Gastrointest Liver Physiol 2012 Dec 15;303(12):G1384-92. 63 (228) Liu L, Li L, Min J, Wang J, Wu H, Zeng Y, et al. Butyrate interferes with the differentiation and function of human monocyte-derived dendritic cells. Cell Immunol 2012 May-Jun;277(1-2):66-73. (229) Zelante T, Iannitti RG, Cunha C, De Luca A, Giovannini G, Pieraccini G, et al. Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22. Immunity 2013 Aug 22;39(2):372-385. (230) Cahenzli J, Koller Y, Wyss M, Geuking MB, McCoy KD. Intestinal microbial diversity during early-life colonization shapes long-term IgE levels. Cell Host Microbe 2013 Nov 13;14(5):559-570. (231) Bjursell M, Admyre T, Goransson M, Marley AE, Smith DM, Oscarsson J, et al. Improved glucose control and reduced body fat mass in free fatty acid receptor 2-deficient mice fed a high-fat diet. Am J Physiol Endocrinol Metab 2011 Jan;300(1):E211-20. (232) Sjögren YM, Tomicic S, Lundberg A, Bottcher MF, Björkstén B, Sverremark-Ekström E, et al. Influence of early gut microbiota on the maturation of childhood mucosal and systemic immune responses. Clin Exp Allergy 2009 Dec;39(12):1842-1851. (233) Trompette A, Gollwitzer ES, Yadava K, Sichelstiel AK, Sprenger N, Ngom-Bru C, et al. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nat Med 2014 Feb;20(2):159-166. (234) Wu HJ, Ivanov II, Darce J, Hattori K, Shima T, Umesaki Y, et al. Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity 2010 Jun 25;32(6):815-827. (235) Wells JM. Immunomodulatory mechanisms of lactobacilli. Microb Cell Fact 2011 Aug 30;10 Suppl 1:S17-2859-10-S1-S17. Epub 2011 Aug 30. (236) Nowrouzian FL, Dauwalder O, Meugnier H, Bes M, Etienne J, Vandenesch F, et al. Adhesin and superantigen genes and the capacity of Staphylococcus aureus to colonize the infantile gut. J Infect Dis 2011 Sep 1;204(5):714-721. (237) Johansson MA, Sjögren YM, Persson JO, Nilsson C, Sverremark-Ekström E. Early colonization with a group of Lactobacilli decreases the risk for allergy at five years of age despite allergic heredity. PLoS One 2011;6(8):e23031. (238) Balmer SE, Wharton BA. Diet and faecal flora in the newborn: breast milk and infant formula. Arch Dis Child 1989 Dec;64(12):1672-1677. (239) Adlerberth I, Lindberg E, Aberg N, Hesselmar B, Saalman R, Strannegard IL, et al. Reduced enterobacterial and increased staphylococcal colonization of the infantile bowel: an effect of hygienic lifestyle? Pediatr Res 2006 Jan;59(1):96-101. (240) Jost T, Lacroix C, Braegger C, Chassard C. Impact of human milk bacteria and oligosaccharides on neonatal gut microbiota establishment and gut health. Nutr Rev 2015 Jul;73(7):426-437. 64 (241) Salminen S, Endo A, Isolauri E, Scalabrin D. Early Gut Colonization With Lactobacilli and Staphylococcus in Infants: The Hygiene Hypothesis Extended. J Pediatr Gastroenterol Nutr 2016 Jan;62(1):80-86. (242) Pinchuk IV, Beswick EJ, Reyes VE. Staphylococcal enterotoxins. Toxins (Basel) 2010 Aug;2(8):2177-2197. (243) Lindberg E, Nowrouzian F, Adlerberth I, Wold AE. Long-time persistence of superantigen-producing Staphylococcus aureus strains in the intestinal microflora of healthy infants. Pediatr Res 2000 Dec;48(6):741-747. (244) Björkstén B, Sepp E, Julge K, Voor T, Mikelsaar M. Allergy development and the intestinal microflora during the first year of life. J Allergy Clin Immunol 2001 Oct;108(4):516-520. (245) Johansson MA, Saghafian-Hedengren S, Haileselassie Y, Roos S, Troye-Blomberg M, Nilsson C, et al. Early-life gut bacteria associate with IL-4-, IL-10- and IFN-gamma production at two years of age. PLoS One 2012;7(11):e49315. (246) Sjögren YM, Jenmalm MC, Bottcher MF, Björkstén B, Sverremark-Ekström E. Altered early infant gut microbiota in children developing allergy up to 5 years of age. Clin Exp Allergy 2009 Apr;39(4):518-526. (247) Walsh CJ, Guinane CM, O'Toole PW, Cotter PD. Beneficial modulation of the gut microbiota. FEBS Lett 2014 Nov 17;588(22):4120-4130. (248) Belkaid Y, Hand TW. Role of the microbiota in immunity and inflammation. Cell 2014 Mar 27;157(1):121-141. (249) Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, et al. Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 2014 Aug;11(8):506-514. (250) Vitaliti G, Pavone P, Guglielmo F, Spataro G, Falsaperla R. The immunomodulatory effect of probiotics beyond atopy: an update. The Journal of asthma : official journal of the Association for the Care of Asthma 2013;0903(3):1-13. (251) Frei R, Akdis M, O'Mahony L. Prebiotics, probiotics, synbiotics, and the immune system: experimental data and clinical evidence. Curr Opin Gastroenterol 2015 Mar;31(2):153-158. (252) Wong A, Ngu DY, Dan LA, Ooi A, Lim RL. Detection of antibiotic resistance in probiotics of dietary supplements. Nutr J 2015 Sep 14;14:95-015-0084-2. (253) Sharma P, Tomar SK, Goswami P, Sangwan V, Singh R. Antibiotic resistance among commercially available probiotics. Food Res Int 2014 3;57:176-195. 65 (254) Flint HJ, Duncan SH, Scott KP, Louis P. Interactions and competition within the microbial community of the human colon: links between diet and health. Environ Microbiol 2007 May;9(5):1101-1111. (255) Cicenia A, Scirocco A, Carabotti M, Pallotta L, Marignani M, Severi C. Postbiotic activities of lactobacilli-derived factors. J Clin Gastroenterol 2014 Nov-Dec;48 Suppl 1:S1822. (256) Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 2004 Jul 23;118(2):229-241. (257) Kotarsky K, Sitnik KM, Stenstad H, Kotarsky H, Schmidtchen a, Koslowski M, et al. A novel role for constitutively expressed epithelial-derived chemokines as antibacterial peptides in the intestinal mucosa. Mucosal immunology 2010;3(1):40-8. (258) He B, Xu W, Santini PA, Polydorides AD, Chiu A, Estrella J, et al. Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL. Immunity 2007 Jun;26(6):812-826. (259) Hironaka I, Iwase T, Sugimoto S, Okuda K, Tajima A, Yanaga K, et al. Glucose triggers ATP secretion from bacteria in a growth-phase-dependent manner. Appl Environ Microbiol 2013 Apr;79(7):2328-2335. (260) Thomas CM, Hong T, van Pijkeren JP, Hemarajata P, Trinh DV, Hu W, et al. Histamine derived from probiotic Lactobacillus reuteri suppresses TNF via modulation of PKA and ERK signaling. PLoS One 2012;7(2):e31951. (261) Bogunovic M, Ginhoux F, Helft J, Shang L, Hashimoto D, Greter M, et al. Origin of the lamina propria dendritic cell network. Immunity 2009 Sep 18;31(3):513-525. (262) Bates JM, Flanagan K, Mo L, Ota N, Ding J, Ho S, et al. Dendritic cell CD83 homotypic interactions regulate inflammation and promote mucosal homeostasis. Mucosal Immunol 2015 Mar;8(2):414-428. (263) Hodges A, Sharrocks K, Edelmann M, Baban D, Moris A, Schwartz O, et al. Activation of the lectin DC-SIGN induces an immature dendritic cell phenotype triggering Rho-GTPase activity required for HIV-1 replication. Nat Immunol 2007 Jun;8(6):569-577. (264) Shreffler WG, Castro RR, Kucuk ZY, Charlop-Powers Z, Grishina G, Yoo S, et al. The major glycoprotein allergen from Arachis hypogaea, Ara h 1, is a ligand of dendritic cellspecific ICAM-grabbing nonintegrin and acts as a Th2 adjuvant in vitro. J Immunol 2006 Sep 15;177(6):3677-3685. (265) Al-Hassi HO, Mann ER, Sanchez B, English NR, Peake ST, Landy J, et al. Altered human gut dendritic cell properties in ulcerative colitis are reversed by Lactobacillus plantarum extracellular encrypted peptide STp. Mol Nutr Food Res 2014 May;58(5):11321143. 66 (266) Koppel EA, van Gisbergen KP, Geijtenbeek TB, van Kooyk Y. Distinct functions of DC-SIGN and its homologues L-SIGN (DC-SIGNR) and mSIGNR1 in pathogen recognition and immune regulation. Cell Microbiol 2005 Feb;7(2):157-165. (267) Lundell AC, Adlerberth I, Lindberg E, Karlsson H, Ekberg S, Aberg N, et al. Increased levels of circulating soluble CD14 but not CD83 in infants are associated with early intestinal colonization with Staphylococcus aureus. 2007;37(1):71. (268) Lundell AC, Hesselmar B, Nordstrom I, Saalman R, Karlsson H, Lindberg E, et al. High circulating immunoglobulin A levels in infants are associated with intestinal toxigenic Staphylococcus aureus and a lower frequency of eczema. Clin Exp Allergy 2009 May;39(5):662-670. (269) Holgate ST, Polosa R. Treatment strategies for allergy and asthma. Nat Rev Immunol 2008 Mar;8(3):218-230. (270) Stern A, Wold AE, Ostman S. Neonatal mucosal immune stimulation by microbial superantigen improves the tolerogenic capacity of CD103(+) dendritic cells. PLoS One 2013 Sep 27;8(9):e75594. (271) Banke E, Rodstrom K, Ekelund M, Dalla-Riva J, Lagerstedt JO, Nilsson S, et al. Superantigen activates the gp130 receptor on adipocytes resulting in altered adipocyte metabolism. Metabolism 2014 Jun;63(6):831-840. (272) Ernst M, Thiem S, Nguyen PM, Eissmann M, Putoczki TL. Epithelial gp130/Stat3 functions: an intestinal signaling node in health and disease. Semin Immunol 2014 Feb;26(1):29-37. (273) Varol C, Vallon-Eberhard A, Elinav E, Aychek T, Shapira Y, Luche H, et al. Intestinal lamina propria dendritic cell subsets have different origin and functions. Immunity 2009 Sep 18;31(3):502-512. (274) Zigmond E, Varol C, Farache J, Elmaliah E, Satpathy AT, Friedlander G, et al. Ly6C hi monocytes in the inflamed colon give rise to proinflammatory effector cells and migratory antigen-presenting cells. Immunity 2012 Dec 14;37(6):1076-1090. (275) Walker WA, Iyengar RS. Breast milk, microbiota, and intestinal immune homeostasis. Pediatr Res 2015 Jan;77(1-2):220-228. (276) Bode L. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology 2012 Sep;22(9):1147-1162. (277) Ballard O, Morrow AL. Human milk composition: nutrients and bioactive factors. Pediatr Clin North Am 2013 Feb;60(1):49-74. (278) Amoudruz P, Holmlund U, Schollin J, Sverremark-Ekström E, Montgomery SM. Maternal country of birth and previous pregnancies are associated with breast milk characteristics. Pediatr Allergy Immunol 2009 Feb;20(1):19-29. 67 (279) Holmlund U, Amoudruz P, Johansson MA, Haileselassie Y, Ongoiba A, Kayentao K, et al. Maternal country of origin, breast milk characteristics and potential influences on immunity in offspring. Clin Exp Immunol 2010 Dec;162(3):500-509. (280) Mora JR, Iwata M, von Andrian UH. Vitamin effects on the immune system: vitamins A and D take centre stage. Nat Rev Immunol 2008 Sep;8(9):685-698. (281) Coombes JL, Maloy KJ. Control of intestinal homeostasis by regulatory T cells and dendritic cells. Semin Immunol 2007 Apr;19(2):116-126. (282) Rosander A, Connolly E, Roos S. Removal of antibiotic resistance gene-carrying plasmids from Lactobacillus reuteri ATCC 55730 and characterization of the resulting daughter strain, L. reuteri DSM 17938. Appl Environ Microbiol 2008 Oct;74(19):6032-6040. (283) Liu Y, Fatheree NY, Mangalat N, Rhoads JM. Lactobacillus reuteri strains reduce incidence and severity of experimental necrotizing enterocolitis via modulation of TLR4 and NF-κ B signaling in the intestine. AJP: Gastrointestinal and Liver Physiology 2012;302(6):G608-G617. (284) Liu Y, Fatheree NY, Dingle BM, Tran DQ, Rhoads JM. Lactobacillus reuteri DSM 17938 changes the frequency of Foxp3+ regulatory T cells in the intestine and mesenteric lymph node in experimental necrotizing enterocolitis. PLoS One 2013;8(2):e56547. (285) Liu Y, Fatheree NY, Mangalat N, Rhoads JM. Human-derived probiotic Lactobacillus reuteri strains differentially reduce intestinal inflammation. American Journal of Physiology Gastrointestinal and Liver Physiology 2010;299(5):1087-1096. (286) Liu Y, Tran DQ, Fatheree NY, Rhoads JM. Lactobacillus reuteri DSM 17938 differentially modulates effector memory T cells and Foxp3+ regulatory T cells in a mouse model of necrotizing enterocolitis. American journal of physiology.Gastrointestinal and liver physiology 2014;307(2):177-186. (287) den Hartog G, Van Altena C, Savelkoul HFJ, Van Neerven ,R.J.Joost. The mucosal factors retinoic acid and TGF-β1 induce phenotypically and functionally distinct dendritic cell types. Int Arch Allergy Immunol 2013;162(3):225-236. (288) Iliev ID, Spadoni I, Mileti E, Matteoli G, Sonzogni a, Sampietro GM, et al. Human intestinal epithelial cells promote the differentiation of tolerogenic dendritic cells. Gut 2009;58(11):1481-1489. (289) Szymczak WA, Deepe GS. The CCL7-CCL2-CCR2 axis regulates IL-4 production in lungs and fungal immunity. Journal of Immunology 2009;183(3):1964-1974. (290) Aychek T, Mildner A, Yona S, Kim K, Lampl N, Reich-Zeliger S, et al. IL-23mediated mononuclear phagocyte crosstalk protects mice from Citrobacter rodentiuminduced colon immunopathology. Nature communications 2015;6:6525-6525. (291) Goodrich ME, McGee DW. Effect of intestinal epithelial cell cytokines on mucosal Bcell IgA secretion: enhancing effect of epithelial-derived IL-6 but not TGF-beta on IgA+ B cells. Immunol Lett 1999;67(1):11-14. 68 (292) Ramsay AJ, Husband AJ, Ramshaw IA, Bao S, Matthaei KI, Koehler G, et al. The role of interleukin-6 in mucosal IgA antibody responses in vivo. Science 1994 Apr 22;264(5158):561-563. (293) Mei J, Liu Y, Dai N, Hoffmann C, Hudock KM, Zhang P, et al. Cxcr2 and Cxcl5 regulate the IL-17/G-CSF axis and neutrophil homeostasis in mice. J Clin Invest 2012;122(3):974-986. (294) Shea-Donohue T, Thomas K, Cody MJ, Zhao A, Detolla LJ, Kopydlowski KM, et al. Mice deficient in the CXCR2 ligand, CXCL1 (KC/GRO-alpha), exhibit increased susceptibility to dextran sodium sulfate (DSS)-induced colitis. Innate immunity 2008;14(2):117-24. (295) Cruickshank SM, Deschoolmeester ML, Svensson M, Howell G, Bazakou A, Logunova L, et al. Rapid dendritic cell mobilization to the large intestinal epithelium is associated with resistance to Trichuris muris infection. Journal of immunology (Baltimore, Md.: 1950) 2009;182(5):3055-3062. (296) Torres-Aguilar H, Aguilar-Ruiz S, González - Pérez G,, Munguía R, Bajaña S, MerazRíos MA, et al. Tolerogenic dendritic cells generated with different immunosuppressive cytokines induce antigen-specific anergy and regulatory properties in memory CD4+ T cells. Journal of immunology (Baltimore, Md.: 1950) 2010;184(4):1765-1775. (297) van der Does A,M., Kenne E, Koppelaar E, Agerberth B, Lindbom L. Vitamin D3 and phenylbutyrate promote development of a human dendritic cell subset displaying enhanced antimicrobial properties. J Leukoc Biol 2014;95(6):883-91. (298) Smits HH, Engering A, van dK, de Jong ,E.C., Schipper K, van Capel ,T.M., et al. Selective probiotic bacteria induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3grabbing nonintegrin. J Allergy Clin Immunol 2005;115(6):1260-1267. (299) Cerovic V, Houston SA, Scott CL, Aumeunier A, Yrlid U, Mowat AM, et al. Intestinal CD103(-) dendritic cells migrate in lymph and prime effector T cells. Mucosal Immunol 2013 Jan;6(1):104-113. (300) Konieczna P, Ferstl R, Ziegler M, Frei R, Nehrbass D, Lauener RP, et al. Immunomodulation by Bifidobacterium infantis 35624 in the murine lamina propria requires retinoic acid-dependent and independent mechanisms. PLoS One 2013 May 21;8(5):e62617. (301) Sallmann E, Reininger B, Brandt S, Duschek N, Hoflehner E, Garner-Spitzer E, et al. High-affinity IgE receptors on dendritic cells exacerbate Th2-dependent inflammation. J Immunol 2011 Jul 1;187(1):164-171. (302) Platzer B, Baker K, Vera MP, Singer K, Panduro M, Lexmond WS, et al. Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites. Mucosal Immunol 2015 May;8(3):516-532. 69 (303) Doyen V, Rubio M, Braun D, Nakajima T, Abe J, Saito H, et al. Thrombospondin 1 is an autocrine negative regulator of human dendritic cell activation. J Exp Med 2003;198(8):1277-83. (304) Bernardo D, Mann ER, Al-Hassi HO, English NR, Man R, Lee GH, et al. Lost therapeutic potential of monocyte-derived dendritic cells through lost tissue homing: stable restoration of gut specificity with retinoic acid. Clin Exp Immunol 2013 Oct;174(1):109-119. 70
