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The Role of Microbiota on the Gut Immunology Yang Won Min, MD; and Poong-Lyul Rhee, MD, PhD Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea ABSTRACT Purpose: The human gut contains 4100 trillion microbes. This microbiota plays a crucial role in the gut homeostasis. Importantly, the microbiota contributes to the development and regulation of the gut immune system. Dysbiosis of the gut microbiota could also cause several intestinal and extraintestinal diseases. Many experimental studies help us to understand the complex interplay between the host and microbiota. Methods: This review presents our current understanding of the mucosal immune system and the role of gut microbiota for the development and functionality of the mucosal immunity, with a particular focus on gutassociated lymphoid tissues, mucosal barrier, TH17 cells, regulatory T cells, innate lymphoid cells, dendritic cells, and IgA-producing B cells and plasma cells. Findings: Comparative studies using germ-free and conventionally-raised animals reveal that the presence of microbiota is important for the development and regulation of innate and adaptive immune systems. The host-microbial symbiosis seems necessary for gut homeostasis. However, the precise mechanisms by which microbiota contributes to development and functionality of the immune system remain to be elucidated. Implications: Understanding the complex interplay between the host and microbiota and further investigation of the host-microbiota relationship could provide us the insight into the therapeutic and/or preventive strategy for the disorders related to dysbiosis of the gut microbiota. (Clin Ther. 2015;]:]]]– ]]]) & 2015 Elsevier HS Journals, Inc. All rights reserved. Key words: gut, immune system, microbiota, mucosal.
INTRODUCTION The human gut harbors 4100 trillion microbes referred to as the gut microbiota, and most of these microbes reside in the colon, where densities approach
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1011 to 1012 cells/mL.1,2 The gut microbiota plays an important role in health and disease in humans.3 Although the gut provides a rich environment for supporting microbial survival, the microbiota also contributes to the well-being of the host. The main benefits of the microbiota include metabolic, trophic, and immunologic functions.4–7 The microbiota degrades undigested carbohydrates to produce important energy sources (eg, short-chain fatty acids [SCFAs])8 and synthesizes essential vitamins.9 Perhaps even more importantly, the microbiota contributes to the development and regulation of the gut immune system.10–16 Studies with germ-free (GF) animals reveal that the microbiota is necessary for the development of the gut mucosal immunity. In addition, microbiota-driven immune response can prevent the development of inappropriate inflammation, which in turn allows the microbiota to survive in the absence of unnecessary inflammation. Therefore, the host-microbial symbiosis is necessary for the gut homeostasis. On the contrary, dysbiosis of the gut microbiota could cause immune-related disorders,17 diabetes,18 allergies,19 and even obesity.20 In this review, we discuss the mucosal immune system and the role of gut microbiota for the development and functionality of the mucosal immunity.
THE MICROBIOTA INFLUENCES THE DEVELOPMENT OF THE GUT MUCOSAL IMMUNE SYSTEM Gut-Associated Lymphoid Tissues and Mucosal Barrier Gut-associated lymphoid tissues (GALTs) are lymphoid structures and aggregates that line the gut, such as the tonsils, Peyer patches (PPs), isolated lymphoid Accepted for publication March 9, 2015. http://dx.doi.org/10.1016/j.clinthera.2015.03.009 0149-2918/$ - see front matter & 2015 Elsevier HS Journals, Inc. All rights reserved.
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Clinical Therapeutics follicles (ILFs), and mesenteric lymph nodes (MLNs).21 PPs are the most recognizable immune structure in the small intestine and appear as clusters of Z3 large lymphoid aggregates with an overlying follicleassociated epithelium, a T-cell zone, and a subepithelial dome containing dendritic cells (DCs).22,23 The follicleassociated epithelium contains M cells, which are specialized epithelial cells that facilitate the uptake of antigen and microbes from the gut lumen and its delivery to underlying lymphoid tissue.24,25 Although ILFs are structurally and functionally similar to PPs, they are smaller, lack a T-cell zone, and are also present in the large intestine.26,27 DCs within PPs promote the production of IgA from B cells,28 and IgAþ B cells are prevalent in the germinal centers of PPs.29 The intestinal epithelium is a single layer of cells derived from the epithelial stem cells within the crypt.30 Epithelial cells are responsible for the mucosal barrier function, participating in immunologic surveillance and direction of the host responses in the gut. Epithelial cells produce mucus to inhibit pathogen invasion by separating the gut lumen from the surface of the intestinal epithelium31,32 and also produce antimicrobial peptides, such as regenerating islet-derived protein 3β (REGIIIβ) and REGIIIγ in response to stimulation with interleukin (IL) 22.33,34 Epithelial cells can express numerous pattern recognition receptors (PRRs), including Toll-like receptor (TLRs) and nucleotide oligomerization domain-like receptors.35–37 PRRs are germline-encoded receptors in the epithelial and innate immune cells to recognize microbial particles, such as DNA, lipopolysaccharides, peptidoglycans, flagellin, and metabolites.21,38,39 PRRs have crucial roles in innate immunity because they can sense pathogen-associated molecular patterns and initiate signaling cascades that lead to innate immune response.21 Intraepithelial lymphocytes (IELs) are composed of CD8þ T cells and reside within the epithelium of the intestine.40 IELs have both protective and pathogenic roles during inflammation. IELs help preserve the integrity of damaged epithelial surfaces by providing the localized delivery of an epithelial cell growth factor, such as keratinocyte growth factor.41,42 On the other hand, IELs capable of interferon γ production have been associated with the development of inflammatory bowel disease.43
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Studies with GF animals reveal that the microbiota contributes to the development of GALT and promotes mucosal barrier function. PPs in GF mice are less active and contain small germinal zones than those in conventionally raised mice.44 In mice that are deficient in PRRs, maturation of ileal and colonic ILFs is incomplete.45 These observations indicate that the generation of the intestinal lymphoid tissues is induced by the microbiota through an innate detection system. GF animals also have lower levels of antimicrobial peptides46 and smaller numbers of IELs than conventional animals.47 However, intestinal microbial stimulation in GF animals can restore the proper organization of the intestinal immune system.46,48 Likewise, the colonic adherent mucous layer in GF mice is significantly thinner than that in conventional mice, but when exposed to bacterial products, such as lipopolysaccharide and peptidoglycan, the thickness of the adherent mucous layer is restored to levels observed in conventional mice.49 Production of antimicrobial peptides REGIIIγ and REGIIIβ is impaired in mice that lack myeloid differentiation factor 88, a signaling adaptor for several TLRs, resulting in the increased susceptibility of mice to infection by enteric pathogens.34,50,51 Thus, microbiota can contribute to enhance the innate immunity through the regulation of mucous secretion and production of antimicrobial peptides. In addition, the microbiota can enhance mucosal barrier function through the production of metabolic by-products. SCFAs, such as acetate, propionate, and butyrate, are by-products of fermentation of dietary fiber by colonic bacteria.8 Bifidobacteria inhibit the translocation of the Escherichia coli O157:H7 Shiga toxin from the gut lumen to the blood through the production of acetate.52 Butyrate also reduces T-cell–mediated immune reaction via modulating antigen-presenting cell function.53 SCFAs bind the G-protein–coupled receptor 43 (GPR43) and SCFAGPR43 interactions profoundly affect inflammatory responses.54 GPR43-deficient mice have severe inflammation in the models of colitis. In addition, GF mice, which express little or no SCFAs, have a similar dysregulation of inflammatory responses.
TH17 Cells IL-17–producing TH17 cells are presented in high numbers in the lamina propria (LP) of the small intestine. They play a role in host defense and
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Y.W. Min and P.-L. Rhee development of autoimmune disease by producing the proinflammatory cytokines IL-17 and IL-22.55,56 Because the number of intestinal TH17 cells is greatly reduced in antibiotic-treated or GF mice,56–60 microbiota seems to have an important role in TH17 cell development. For instance, specific members of the microbiota as segmented filamentous bacteria (SFB), Clostridium arthromitus, promote TH17 cell development in mice.56,58,59 SFB colonization up-regulates serum amyloid A production, which in turn enhances IL-6 and IL-23 production by LP DCs to stimulate a TH17 cell-inducing environment.58 Luminal adenosine triphosphate, which is provided by microbiota, also promotes TH17 cell development through the activation of LP DCs similar to the mechanism by SFB.57 In addition, SFB colonization enhances resistance to the intestinal pathogen, such as Citrobacter rodentium.58 The aryl hydrocarbon receptor (AhR) participates in TH17 cell differentiation and is required for the secretion of IL-22 by TH17 cells.61–63 Tryptophan metabolites from microbiota in mice produce an AhR ligand indole-3aldehyde that can drive IL-22 expression.64 Collectively, microbiota seems to influence the development of TH17 cell in the gut.
Regulatory T Cells Regulatory T (Treg) cells are T cells that express the transcription factor Foxp3 and accumulate in the intestine, where they have anti-inflammatory activity, including secretion of transforming growth factor β and/or IL-10.65 Treg cells are required to prevent the abnormal expansion of CD4þ TH cells to commensal bacteria.66 In addition, the development of colonic Treg cells is induced in response to microbiota because they are significantly decreased in the colonic LP of GF mice.67–69 Furthermore, certain species populations of commensal bacteria, such as Clostridium clusters IV and XIVa, induce Treg cells in the colon,68 which is facilitated by high amounts of transforming growth factor β.65 Thus, the microbiota can prevent the development of inappropriate inflammation, which in turn allows the microbiota to survive in the absence of unnecessary inflammation.
DCs and IgA-Producing B Cells and Plasma Cells Mucosal DCs play a role in determining whether to mount tolerant or protective immune responses.70 They are composed of 2 distinct subpopulations,
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conventional DCs and plasmacytoid DCs, both of which are present in the GALT.71 Conventional DCs have a critical role in the maintenance of gut homeostasis, immune responses against gut infection, and inflammatory bowel diseases. CD103þ DCs (major conventional DC population in the LP) migrate from the LP to the MLNs and promote the generation of Treg cells by means of retinoic acid.72 Their subsets also generate T-cell–producing IL-17,73 have high expression of TLR5, and induce inflammatory responses when stimulated with the TLR5 ligand bacterial flagellin.74 However, DCs carrying their commensal load do not stray beyond lymphoid tissues to prevent a systemic infection, and live bacteria transported to the MLNs by DCs never gain access to systemic circulation.75,76 Unlike conventional DCs, plasmacytoid DCs, found in the PPs and LP, play a critical role in T-cell–independent IgA production by B cells in the GALT.71 IgA has an irreplaceable role in the mucosal defense against infectious microbes. After sampling luminal bacteria via endocytosis of the resident microbes,77,78 DCs induce the activation and differentiation of naive B cells to yield plasma cells that produce commensalspecific IgA in the LP.79,80 Antimicrobial immunoglobulin is transported via the immunoglobulin receptor, which is expressed on the epithelial cells to their apical surface and into the gut lumen as secreted IgA (SIgA).81 SIgA binds to commensal bacteria and soluble antigens, thereby inhibiting their binding to the host epithelium and penetration the epithelial barrier.82 The commensal specific IgA response remains restricted to the intestinal tissue, keeping the microbiota at bay.75,76 IgA also regulates the composition and function of the gut microbiota. Activationinduced cytidine deaminase is an enzyme that plays an essential role in class switch recombination and somatic hypermutation of immunoglobulin genes.83 Deficiency in activation-induced cytidine deaminase results in the development of hyperplasia of ILFs associated with a 100-fold expansion of anaerobic flora in the small intestine.84 IgA also reduces the intestinal innate immune response by affecting bacterial gene expression in a gnotobiotic mouse model.85 In GF mice lacking B and T cells, which is monoassociated with Bacteroides thetaiotaomicron, bacteria elicit fewer proinflammatory signals in the presence of specific IgA for a capsular polysaccharide than in the absence of that. In addition, bacteria
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Clinical Therapeutics Microbiota
Clostridium cluster IV and XIVa
Microbiota REG III γ/β
SIgA SCFA TLR
Tryptophan
Mucus
SFB
Mucous Layer
ATP
Integrity
Goblet cell
Indole-3 -aldehyde
M cell
IL-22 IL-22
pDC
IEL
Lamina propria
Epithelial cell
SAA
TGF-β
AhR IgA CD103+ DC
ROR γ t+ ILC IL-23 IL-6 B cell
Bacterial flagella TH17 cell
IgA+ Plasma cell
RA Treg cell DC
TLR5+ CD11c+ LPC
Figure. The interplay between the gut microbiota and the immune system. Epithelial cells produce antimicrobial peptides, such as regenerating islet-derived protein 3β (REGIIIβ) and REGIIIγ, in response to interleukin (IL) 22. They can express pattern recognition receptors, such as the Toll-like receptor (TLR). The microbiota regulates mucous secretion and antimicrobial peptides production. In addition, the microbiota can enhance the epithelial integrity through the production of short chain fatty acids (SCFAs), which are by-products of fermentation of dietary fiber by colonic bacteria. Goblet cells produce mucus to inhibit pathogen invasion. TH17 cells play a role in host defense by producing the proinflammatory cytokine IL-22. Segmented filamentous bacteria (SFB) colonization up-regulates serum amyloid A (SAA) production, which in turn promotes IL-6 and IL-23 production by dendritic cells (DCs) to promote TH17 cell development. Adenosine triphosphate (ATP), which is provided by microbiota, also promotes TH17 cell development similar to the mechanism by SFB. Clostridium clusters IV and XIVa induce regulatory T (Treg) cells, which are facilitated by transforming growth factor β (TGF-β). Treg cells are required to prevent the abnormal expansion of CD4þ TH cells to commensal bacteria. CD103þ DCs promote the generation of Treg cells by means of retinoic acid (RA). On the other hand, TLR5þ CD11cþ LP cells generate TH17 cells and induce inflammatory responses when stimulated with the TLR5 ligand bacterial flagellin. Plasmacytoid DCs (pDCs) induce the activation and differentiation of naive B cells to yield plasma cells that produce commensalspecific IgA in the LP. IgA is transported into the gut lumen as secreted IgA (SIgA). SIgA binds to commensal bacteria and soluble antigens, thereby inhibiting their binding to the host epithelium and penetration the epithelial barrier. Tryptophan metabolites from microbiota enhance the differentiation of RORγtþ innate lymphoid cells (ILCs) and their production of IL-22. IEL ¼ intraepithelial lymphocyte.
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Y.W. Min and P.-L. Rhee adapts to the presence of specific IgA by switching the expression of the target epitope to another capsular polysaccharide and by decreasing the expression of genes involved in nitric oxide metabolism. Thus, the complex interplay among microbiota and DCs and IgAþ B cells contributes to the gut immune homeostasis.
Innate Lymphoid Cells ILCs are innate immune cells that share functional characteristics with CD4þ TH cells in the LP.86,87 They arise from a common lymphoid progenitor but differentiate into multiple lineages on the basis of the expression of specific transcriptional factors.88–90 ILC can be categorized into 3 broad groups according to the expression of distinct transcription factors and effector molecules.88–90 Group 1 ILCs, such as natural killer cells, are characterized by the expression of the transcription factors T-bet and the production of interferon γ. Group 2 ILCs are characterized by the expression of GATA3 and RORα and the production of IL-5 and IL-13. Finally, group 3 ILCs, such as lymphoid tissue inducer cells, NKp46þ, and NKp46 cells, are characterized by the expression of RORγt and the production of IL-22 and/or IL-17. Group 3 ILCs are particularly relevant to the gut. Microbiota is necessary for the differentiation of group 3 ILCs and for their IL-22 production.86 IL-22 promotes the production of the antimicrobial peptides REGIIIβ and REGIIIγ by intestinal epithelial cells.33,34 However, GF mice have impaired IL-22 production.60 In addition, AhR-deficient mice were highly susceptible to infection with C rodentium, a mouse model for attaching and effacing infections.62 AhR has a crucial role in promoting innate gut immunity by regulating RORγtþ ILCs.62,63 In turn, administration of IL-22 to Ahr-deficient mice with impaired production of IL-22 provides protection against C rodentium infection.91 In addition, depletion of ILCs results in dissemination of commensal bacteria and systemic inflammation, which could be prevented by administration of IL22.92 These observations indicate that the microbiotadriven IL-22 by stimulating ILCs might enhance the innate immunity Figure.
CONCLUSION Humans have coevolved with microscopic organisms in a mutualistic relationship. Many comparative studies in GF and conventionally raised animals indicate
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that the presence of microbiota is important for the development and regulation of innate and adaptive immune systems. Thus, the host-microbial symbiosis is necessary for gut homeostasis. Dysbiosis of the gut microbiota could be related to several intestinal and extraintestinal diseases, although that is not covered in this review. Understanding the complex interplay between the host and microbiota might provide us the insights into the genesis of those diseases (Figure). However, we still have a long way to understand the precise mechanisms by which microbiota contributes to development and functionality of the immune system. Thus, further investigation of the hostmicrobiota relationship is warranted to establish the therapeutic and/or preventive strategy for the disorders related with dysbiosis of the gut microbiota.
ACKNOWLEDGMENTS Yang Won Min searched literature and drafted the manuscript and Poong-Lyul Rhee edited the manuscript. All authors approved the final version of the manuscript. We thanks to Sung-Kab Kim for figure creation.
CONFLICTS OF INTEREST The authors have indicated that they have no conflicts of interest regarding the content of this article.
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79. Hooper LV, Littman DR, Macpherson AJ. Interactions between the microbiota and the immune system. Science. 2012;336: 1268–1273. 80. Mowat AM. Anatomical basis of tolerance and immunity to intestinal antigens. Nat Rev Immunol. 2003;3:331–341. 81. Strugnell RA, Wijburg OL. The role of secretory antibodies in infection immunity. Nat Rev Microbiol. 2010; 8:656–667. 82. Fagarasan S, Kawamoto S, Kanagawa O, et al. Adaptive immune regulation in the gut: T celldependent and T cell-independent IgA synthesis. Annu Rev Immunol. 2010;28:243–273. 83. Muramatsu M, Kinoshita K, Fagarasan S, et al. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell. 2000;102:553– 563. 84. Fagarasan S, Muramatsu M, Suzuki K, et al. Critical roles of activationinduced cytidine deaminase in the homeostasis of gut flora. Science. 2002;298:1424–1427. 85. Peterson DA, McNulty NP, Guruge JL, et al. IgA response to symbiotic bacteria as a mediator of gut homeostasis. Cell Host Microbe. 2007;2:328–339.
86. Satoh-Takayama N, Vosshenrich CA, Lesjean-Pottier S, et al. Microbial flora drives interleukin 22 production in intestinal NKp46þ cells that provide innate mucosal immune defense. Immunity. 2008; 29:958–970. 87. Buonocore S, Ahern PP, Uhlig HH, et al. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology. Nature. 2010;464: 1371–1375. 88. Spits H, Artis D, Colonna M, et al. Innate lymphoid cells: a proposal for uniform nomenclature. Nat Rev Immunol. 2013;13:145–149. 89. Spits H, Cupedo T. Innate lymphoid cells: emerging insights in development, lineage relationships, and function. Annu Rev Immunol. 2012;30:647–675. 90. Spits H, Di Santo JP. The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling. Nat Immunol. 2011;12:21–27. 91. Qiu J, Heller JJ, Guo X, et al. The aryl hydrocarbon receptor regulates gut immunity through modulation of innate lymphoid cells. Immunity. 2012;36:92–104. 92. Sonnenberg GF, Monticelli LA, Alenghat T, et al. Innate lymphoid cells promote anatomical containment of lymphoid-resident commensal bacteria. Science. 2012;336:1321–1325.
Address correspondence to: Poong-Lyul Rhee, MD, PhD, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul, 135-710, South Korea. E-mail: [email protected]
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The Role of Microbiota on the Gut Immunology Yang Won Min, MD; and Poong-Lyul Rhee, MD, PhD Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea ABSTRACT Purpose: The human gut contains 4100 trillion microbes. This microbiota plays a crucial role in the gut homeostasis. Importantly, the microbiota contributes to the development and regulation of the gut immune system. Dysbiosis of the gut microbiota could also cause several intestinal and extraintestinal diseases. Many experimental studies help us to understand the complex interplay between the host and microbiota. Methods: This review presents our current understanding of the mucosal immune system and the role of gut microbiota for the development and functionality of the mucosal immunity, with a particular focus on gutassociated lymphoid tissues, mucosal barrier, TH17 cells, regulatory T cells, innate lymphoid cells, dendritic cells, and IgA-producing B cells and plasma cells. Findings: Comparative studies using germ-free and conventionally-raised animals reveal that the presence of microbiota is important for the development and regulation of innate and adaptive immune systems. The host-microbial symbiosis seems necessary for gut homeostasis. However, the precise mechanisms by which microbiota contributes to development and functionality of the immune system remain to be elucidated. Implications: Understanding the complex interplay between the host and microbiota and further investigation of the host-microbiota relationship could provide us the insight into the therapeutic and/or preventive strategy for the disorders related to dysbiosis of the gut microbiota. (Clin Ther. 2015;]:]]]– ]]]) & 2015 Elsevier HS Journals, Inc. All rights reserved. Key words: gut, immune system, microbiota, mucosal.
INTRODUCTION The human gut harbors 4100 trillion microbes referred to as the gut microbiota, and most of these microbes reside in the colon, where densities approach
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1011 to 1012 cells/mL.1,2 The gut microbiota plays an important role in health and disease in humans.3 Although the gut provides a rich environment for supporting microbial survival, the microbiota also contributes to the well-being of the host. The main benefits of the microbiota include metabolic, trophic, and immunologic functions.4–7 The microbiota degrades undigested carbohydrates to produce important energy sources (eg, short-chain fatty acids [SCFAs])8 and synthesizes essential vitamins.9 Perhaps even more importantly, the microbiota contributes to the development and regulation of the gut immune system.10–16 Studies with germ-free (GF) animals reveal that the microbiota is necessary for the development of the gut mucosal immunity. In addition, microbiota-driven immune response can prevent the development of inappropriate inflammation, which in turn allows the microbiota to survive in the absence of unnecessary inflammation. Therefore, the host-microbial symbiosis is necessary for the gut homeostasis. On the contrary, dysbiosis of the gut microbiota could cause immune-related disorders,17 diabetes,18 allergies,19 and even obesity.20 In this review, we discuss the mucosal immune system and the role of gut microbiota for the development and functionality of the mucosal immunity.
THE MICROBIOTA INFLUENCES THE DEVELOPMENT OF THE GUT MUCOSAL IMMUNE SYSTEM Gut-Associated Lymphoid Tissues and Mucosal Barrier Gut-associated lymphoid tissues (GALTs) are lymphoid structures and aggregates that line the gut, such as the tonsils, Peyer patches (PPs), isolated lymphoid Accepted for publication March 9, 2015. http://dx.doi.org/10.1016/j.clinthera.2015.03.009 0149-2918/$ - see front matter & 2015 Elsevier HS Journals, Inc. All rights reserved.
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Clinical Therapeutics follicles (ILFs), and mesenteric lymph nodes (MLNs).21 PPs are the most recognizable immune structure in the small intestine and appear as clusters of Z3 large lymphoid aggregates with an overlying follicleassociated epithelium, a T-cell zone, and a subepithelial dome containing dendritic cells (DCs).22,23 The follicleassociated epithelium contains M cells, which are specialized epithelial cells that facilitate the uptake of antigen and microbes from the gut lumen and its delivery to underlying lymphoid tissue.24,25 Although ILFs are structurally and functionally similar to PPs, they are smaller, lack a T-cell zone, and are also present in the large intestine.26,27 DCs within PPs promote the production of IgA from B cells,28 and IgAþ B cells are prevalent in the germinal centers of PPs.29 The intestinal epithelium is a single layer of cells derived from the epithelial stem cells within the crypt.30 Epithelial cells are responsible for the mucosal barrier function, participating in immunologic surveillance and direction of the host responses in the gut. Epithelial cells produce mucus to inhibit pathogen invasion by separating the gut lumen from the surface of the intestinal epithelium31,32 and also produce antimicrobial peptides, such as regenerating islet-derived protein 3β (REGIIIβ) and REGIIIγ in response to stimulation with interleukin (IL) 22.33,34 Epithelial cells can express numerous pattern recognition receptors (PRRs), including Toll-like receptor (TLRs) and nucleotide oligomerization domain-like receptors.35–37 PRRs are germline-encoded receptors in the epithelial and innate immune cells to recognize microbial particles, such as DNA, lipopolysaccharides, peptidoglycans, flagellin, and metabolites.21,38,39 PRRs have crucial roles in innate immunity because they can sense pathogen-associated molecular patterns and initiate signaling cascades that lead to innate immune response.21 Intraepithelial lymphocytes (IELs) are composed of CD8þ T cells and reside within the epithelium of the intestine.40 IELs have both protective and pathogenic roles during inflammation. IELs help preserve the integrity of damaged epithelial surfaces by providing the localized delivery of an epithelial cell growth factor, such as keratinocyte growth factor.41,42 On the other hand, IELs capable of interferon γ production have been associated with the development of inflammatory bowel disease.43
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Studies with GF animals reveal that the microbiota contributes to the development of GALT and promotes mucosal barrier function. PPs in GF mice are less active and contain small germinal zones than those in conventionally raised mice.44 In mice that are deficient in PRRs, maturation of ileal and colonic ILFs is incomplete.45 These observations indicate that the generation of the intestinal lymphoid tissues is induced by the microbiota through an innate detection system. GF animals also have lower levels of antimicrobial peptides46 and smaller numbers of IELs than conventional animals.47 However, intestinal microbial stimulation in GF animals can restore the proper organization of the intestinal immune system.46,48 Likewise, the colonic adherent mucous layer in GF mice is significantly thinner than that in conventional mice, but when exposed to bacterial products, such as lipopolysaccharide and peptidoglycan, the thickness of the adherent mucous layer is restored to levels observed in conventional mice.49 Production of antimicrobial peptides REGIIIγ and REGIIIβ is impaired in mice that lack myeloid differentiation factor 88, a signaling adaptor for several TLRs, resulting in the increased susceptibility of mice to infection by enteric pathogens.34,50,51 Thus, microbiota can contribute to enhance the innate immunity through the regulation of mucous secretion and production of antimicrobial peptides. In addition, the microbiota can enhance mucosal barrier function through the production of metabolic by-products. SCFAs, such as acetate, propionate, and butyrate, are by-products of fermentation of dietary fiber by colonic bacteria.8 Bifidobacteria inhibit the translocation of the Escherichia coli O157:H7 Shiga toxin from the gut lumen to the blood through the production of acetate.52 Butyrate also reduces T-cell–mediated immune reaction via modulating antigen-presenting cell function.53 SCFAs bind the G-protein–coupled receptor 43 (GPR43) and SCFAGPR43 interactions profoundly affect inflammatory responses.54 GPR43-deficient mice have severe inflammation in the models of colitis. In addition, GF mice, which express little or no SCFAs, have a similar dysregulation of inflammatory responses.
TH17 Cells IL-17–producing TH17 cells are presented in high numbers in the lamina propria (LP) of the small intestine. They play a role in host defense and
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Y.W. Min and P.-L. Rhee development of autoimmune disease by producing the proinflammatory cytokines IL-17 and IL-22.55,56 Because the number of intestinal TH17 cells is greatly reduced in antibiotic-treated or GF mice,56–60 microbiota seems to have an important role in TH17 cell development. For instance, specific members of the microbiota as segmented filamentous bacteria (SFB), Clostridium arthromitus, promote TH17 cell development in mice.56,58,59 SFB colonization up-regulates serum amyloid A production, which in turn enhances IL-6 and IL-23 production by LP DCs to stimulate a TH17 cell-inducing environment.58 Luminal adenosine triphosphate, which is provided by microbiota, also promotes TH17 cell development through the activation of LP DCs similar to the mechanism by SFB.57 In addition, SFB colonization enhances resistance to the intestinal pathogen, such as Citrobacter rodentium.58 The aryl hydrocarbon receptor (AhR) participates in TH17 cell differentiation and is required for the secretion of IL-22 by TH17 cells.61–63 Tryptophan metabolites from microbiota in mice produce an AhR ligand indole-3aldehyde that can drive IL-22 expression.64 Collectively, microbiota seems to influence the development of TH17 cell in the gut.
Regulatory T Cells Regulatory T (Treg) cells are T cells that express the transcription factor Foxp3 and accumulate in the intestine, where they have anti-inflammatory activity, including secretion of transforming growth factor β and/or IL-10.65 Treg cells are required to prevent the abnormal expansion of CD4þ TH cells to commensal bacteria.66 In addition, the development of colonic Treg cells is induced in response to microbiota because they are significantly decreased in the colonic LP of GF mice.67–69 Furthermore, certain species populations of commensal bacteria, such as Clostridium clusters IV and XIVa, induce Treg cells in the colon,68 which is facilitated by high amounts of transforming growth factor β.65 Thus, the microbiota can prevent the development of inappropriate inflammation, which in turn allows the microbiota to survive in the absence of unnecessary inflammation.
DCs and IgA-Producing B Cells and Plasma Cells Mucosal DCs play a role in determining whether to mount tolerant or protective immune responses.70 They are composed of 2 distinct subpopulations,
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conventional DCs and plasmacytoid DCs, both of which are present in the GALT.71 Conventional DCs have a critical role in the maintenance of gut homeostasis, immune responses against gut infection, and inflammatory bowel diseases. CD103þ DCs (major conventional DC population in the LP) migrate from the LP to the MLNs and promote the generation of Treg cells by means of retinoic acid.72 Their subsets also generate T-cell–producing IL-17,73 have high expression of TLR5, and induce inflammatory responses when stimulated with the TLR5 ligand bacterial flagellin.74 However, DCs carrying their commensal load do not stray beyond lymphoid tissues to prevent a systemic infection, and live bacteria transported to the MLNs by DCs never gain access to systemic circulation.75,76 Unlike conventional DCs, plasmacytoid DCs, found in the PPs and LP, play a critical role in T-cell–independent IgA production by B cells in the GALT.71 IgA has an irreplaceable role in the mucosal defense against infectious microbes. After sampling luminal bacteria via endocytosis of the resident microbes,77,78 DCs induce the activation and differentiation of naive B cells to yield plasma cells that produce commensalspecific IgA in the LP.79,80 Antimicrobial immunoglobulin is transported via the immunoglobulin receptor, which is expressed on the epithelial cells to their apical surface and into the gut lumen as secreted IgA (SIgA).81 SIgA binds to commensal bacteria and soluble antigens, thereby inhibiting their binding to the host epithelium and penetration the epithelial barrier.82 The commensal specific IgA response remains restricted to the intestinal tissue, keeping the microbiota at bay.75,76 IgA also regulates the composition and function of the gut microbiota. Activationinduced cytidine deaminase is an enzyme that plays an essential role in class switch recombination and somatic hypermutation of immunoglobulin genes.83 Deficiency in activation-induced cytidine deaminase results in the development of hyperplasia of ILFs associated with a 100-fold expansion of anaerobic flora in the small intestine.84 IgA also reduces the intestinal innate immune response by affecting bacterial gene expression in a gnotobiotic mouse model.85 In GF mice lacking B and T cells, which is monoassociated with Bacteroides thetaiotaomicron, bacteria elicit fewer proinflammatory signals in the presence of specific IgA for a capsular polysaccharide than in the absence of that. In addition, bacteria
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Clinical Therapeutics Microbiota
Clostridium cluster IV and XIVa
Microbiota REG III γ/β
SIgA SCFA TLR
Tryptophan
Mucus
SFB
Mucous Layer
ATP
Integrity
Goblet cell
Indole-3 -aldehyde
M cell
IL-22 IL-22
pDC
IEL
Lamina propria
Epithelial cell
SAA
TGF-β
AhR IgA CD103+ DC
ROR γ t+ ILC IL-23 IL-6 B cell
Bacterial flagella TH17 cell
IgA+ Plasma cell
RA Treg cell DC
TLR5+ CD11c+ LPC
Figure. The interplay between the gut microbiota and the immune system. Epithelial cells produce antimicrobial peptides, such as regenerating islet-derived protein 3β (REGIIIβ) and REGIIIγ, in response to interleukin (IL) 22. They can express pattern recognition receptors, such as the Toll-like receptor (TLR). The microbiota regulates mucous secretion and antimicrobial peptides production. In addition, the microbiota can enhance the epithelial integrity through the production of short chain fatty acids (SCFAs), which are by-products of fermentation of dietary fiber by colonic bacteria. Goblet cells produce mucus to inhibit pathogen invasion. TH17 cells play a role in host defense by producing the proinflammatory cytokine IL-22. Segmented filamentous bacteria (SFB) colonization up-regulates serum amyloid A (SAA) production, which in turn promotes IL-6 and IL-23 production by dendritic cells (DCs) to promote TH17 cell development. Adenosine triphosphate (ATP), which is provided by microbiota, also promotes TH17 cell development similar to the mechanism by SFB. Clostridium clusters IV and XIVa induce regulatory T (Treg) cells, which are facilitated by transforming growth factor β (TGF-β). Treg cells are required to prevent the abnormal expansion of CD4þ TH cells to commensal bacteria. CD103þ DCs promote the generation of Treg cells by means of retinoic acid (RA). On the other hand, TLR5þ CD11cþ LP cells generate TH17 cells and induce inflammatory responses when stimulated with the TLR5 ligand bacterial flagellin. Plasmacytoid DCs (pDCs) induce the activation and differentiation of naive B cells to yield plasma cells that produce commensalspecific IgA in the LP. IgA is transported into the gut lumen as secreted IgA (SIgA). SIgA binds to commensal bacteria and soluble antigens, thereby inhibiting their binding to the host epithelium and penetration the epithelial barrier. Tryptophan metabolites from microbiota enhance the differentiation of RORγtþ innate lymphoid cells (ILCs) and their production of IL-22. IEL ¼ intraepithelial lymphocyte.
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Y.W. Min and P.-L. Rhee adapts to the presence of specific IgA by switching the expression of the target epitope to another capsular polysaccharide and by decreasing the expression of genes involved in nitric oxide metabolism. Thus, the complex interplay among microbiota and DCs and IgAþ B cells contributes to the gut immune homeostasis.
Innate Lymphoid Cells ILCs are innate immune cells that share functional characteristics with CD4þ TH cells in the LP.86,87 They arise from a common lymphoid progenitor but differentiate into multiple lineages on the basis of the expression of specific transcriptional factors.88–90 ILC can be categorized into 3 broad groups according to the expression of distinct transcription factors and effector molecules.88–90 Group 1 ILCs, such as natural killer cells, are characterized by the expression of the transcription factors T-bet and the production of interferon γ. Group 2 ILCs are characterized by the expression of GATA3 and RORα and the production of IL-5 and IL-13. Finally, group 3 ILCs, such as lymphoid tissue inducer cells, NKp46þ, and NKp46 cells, are characterized by the expression of RORγt and the production of IL-22 and/or IL-17. Group 3 ILCs are particularly relevant to the gut. Microbiota is necessary for the differentiation of group 3 ILCs and for their IL-22 production.86 IL-22 promotes the production of the antimicrobial peptides REGIIIβ and REGIIIγ by intestinal epithelial cells.33,34 However, GF mice have impaired IL-22 production.60 In addition, AhR-deficient mice were highly susceptible to infection with C rodentium, a mouse model for attaching and effacing infections.62 AhR has a crucial role in promoting innate gut immunity by regulating RORγtþ ILCs.62,63 In turn, administration of IL-22 to Ahr-deficient mice with impaired production of IL-22 provides protection against C rodentium infection.91 In addition, depletion of ILCs results in dissemination of commensal bacteria and systemic inflammation, which could be prevented by administration of IL22.92 These observations indicate that the microbiotadriven IL-22 by stimulating ILCs might enhance the innate immunity Figure.
CONCLUSION Humans have coevolved with microscopic organisms in a mutualistic relationship. Many comparative studies in GF and conventionally raised animals indicate
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that the presence of microbiota is important for the development and regulation of innate and adaptive immune systems. Thus, the host-microbial symbiosis is necessary for gut homeostasis. Dysbiosis of the gut microbiota could be related to several intestinal and extraintestinal diseases, although that is not covered in this review. Understanding the complex interplay between the host and microbiota might provide us the insights into the genesis of those diseases (Figure). However, we still have a long way to understand the precise mechanisms by which microbiota contributes to development and functionality of the immune system. Thus, further investigation of the hostmicrobiota relationship is warranted to establish the therapeutic and/or preventive strategy for the disorders related with dysbiosis of the gut microbiota.
ACKNOWLEDGMENTS Yang Won Min searched literature and drafted the manuscript and Poong-Lyul Rhee edited the manuscript. All authors approved the final version of the manuscript. We thanks to Sung-Kab Kim for figure creation.
CONFLICTS OF INTEREST The authors have indicated that they have no conflicts of interest regarding the content of this article.
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Address correspondence to: Poong-Lyul Rhee, MD, PhD, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul, 135-710, South Korea. E-mail: [email protected]
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