REVIEWS

Role of the gut microbiota in
immunity and inflammatory disease
Nobuhiko Kamada1, Sang‑Uk Seo1, Grace Y. Chen2 an...
REVIEWS
Box 1 | Pattern recognition receptors
Pattern recognition receptors (PRRs) are receptor molecules expressed in imm...
REVIEWS
a

• SFB
• Other microbiota

Gut lumen

• Clostridium cluster IV
and XIVa strains
• PSA+ Bacteroides fragilis
• Ot...
REVIEWS
inhibits innate immune responses by affecting bacterial
gene expression27. The gut microbiota also regulates IgA
p...
REVIEWS
Indirect

Direct

Pathogen strategies

Microbiota strategies

Pathogen

Pathogen

Commensal
microbiota

Competitio...
REVIEWS

Inflammasome
A molecular complex of several
proteins, including a NOD-like
receptor (NLR), the adaptor
protein AS...
REVIEWS

Inflammatory bowel disease
(IBD). Immune-mediated
inflammation of the bowel.
There are two main forms:
Crohn’s di...
REVIEWS
Table 1 | Role of the gut microbiota in the protection or induction of IBD
Commensal bacterium Host

Genotype

Dis...
REVIEWS
Homeostasis

Inflammatory bowel disease
Genetic factors (for
example, mutations in
Nod2, Atg16I1 and Il23r)

Symbio...
REVIEWS
There is increasing evidence that defects in the NODlike receptor (NLR) family of proteins are associated with
dys...
REVIEWS
Experimental autoimmune
encephalomyelitis
(EAE). An experimental model
of multiple sclerosis that is
induced by im...
REVIEWS
excessive IL‑1 signalling, enhanced IL‑17 expression
and increased arthritic symptoms154. Similar to K/BxN
mice, a...
REVIEWS
factors are important in triggering dysbiosis. Obviously,
both genetic and environmental factors are important in
...
REVIEWS
53.	Leatham, M. P. et al. Precolonized human commensal
Escherichia coli strains serve as a barrier to E. coli
O157...
1 (2013) role of the gut microbiota in immunity and inflammatory disease
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1 (2013) role of the gut microbiota in immunity and inflammatory disease

  1. 1. REVIEWS Role of the gut microbiota in immunity and inflammatory disease Nobuhiko Kamada1, Sang‑Uk Seo1, Grace Y. Chen2 and Gabriel Núñez1 Abstract | The mammalian intestine is colonized by trillions of microorganisms, most of which are bacteria that have co‑evolved with the host in a symbiotic relationship. The collection of microbial populations that reside on and in the host is commonly referred to as the microbiota. A principal function of the microbiota is to protect the intestine against colonization by exogenous pathogens and potentially harmful indigenous microorganisms via several mechanisms, which include direct competition for limited nutrients and the modulation of host immune responses. Conversely, pathogens have developed strategies to promote their replication in the presence of competing microbiota. Breakdown of the normal microbial community increases the risk of pathogen infection, the overgrowth of harmful pathobionts and inflammatory disease. Understanding the interaction of the microbiota with pathogens and the host might provide new insights into the pathogenesis of disease, as well as novel avenues for preventing and treating intestinal and systemic disorders. Gut-associated lymphoid tissues (GALTs). Lymphoid structures and aggregates that are associated with the intestinal mucosa, specifically the tonsils, Peyer’s patches, lymphoid follicles, appendix or cecal patch, and mesenteric lymph nodes. They are enriched in conventional and unconventional lymphocytes, and in specialized dendritic cell and macrophage subsets. Department of Pathology and Comprehensive Cancer Center, University of Michigan, Ann Arbor 48109, Michigan, USA. 2 Department of Internal Medicine and Comprehensive Cancer Center, University of Michigan, Ann Arbor 48109, Michigan, USA. Correspondence to G.N. e-mail: gabriel.nunez@umich.edu doi:10.1038/nri3430 1 The skin and mucosal surfaces of vertebrates are colo‑ nized by large numbers of microorganisms, includ‑ ing bacteria, fungi, parasites and viruses, commonly referred to as the microbiota. In humans, more than 100 trillion microorganisms, mostly bacteria, colonize the oral–gastrointestinal tract, and most of these micro‑ organisms reside in the distal intestine. Millions of years of co‑evolution between the host and micro­ rganisms o have led to a mutualistic relationship in which the micro‑ biota contributes to many host physiological processes and, in turn, the host provides niches and nutrients for microbial survival1. The main contributions of the microbiota to the host include the digestion and fermen‑ tation of carbohydrates, the production of vitamins, the develop­ment of gut-associated lymphoid tissues (GALTs), the polarization of gut-specific immune responses and the prevention of colonization by pathogens1–3. In turn, gut immune responses that are induced by commensal populations regulate the composition of the micro‑ biota. Thus, a complex interplay between the host immune system and the microbiota is necessary for gut homeostasis. However, when the mutualistic rela‑ tionship between the host and microbiota is disrupted, the gut microbiota can cause or contribute to disease4,5. In this Review, we provide an overview of the current understanding of the dual role of the gut microbiota in health and disease. Microbiota-dependent lymphoid development GALTs. The study of germ-free mice led to the discov‑ ery that the gut microbiota is required for the normal generation and/or maturation of GALTs. GALTs are immune structures in which antigen can be taken up and presented by antigen-presenting cells, and there‑ fore these structures have important roles in lympho‑ cyte functions that lead to inflammation or tolerance. GALTs include the Peyer’s patches, crypt patches and isolated lymphoid follicles (ILFs)6–8. In the fetus, lym‑ phoid tissue inducer (LTi) cells promote the devel‑ opment of Peyer’s patches in the absence of resident bacteria, although Peyer’s patches in germ-free mice are smaller in size than those in specific-pathogen-free mice9. Unlike Peyer’s patches, the maturation of ILFs and crypt patches requires stimulation by the gut microbiota7,10. Specifically, incomplete maturation of ileal and colonic ILFs is observed in mice that are deficient in various pattern recognition receptors (PRRs) (BOX 1) that are acti‑ vated by bacterial stimuli, such as Toll-like receptor 2 (TLR2), TLR4, and nucleotide-binding oligomerization domain 2 (NOD2), and in their adaptor molecules, such as myeloid differentiation primary-response protein 88 (MYD88) and TIR domain-containing adaptor protein inducing IFNβ (TRIF; also known as TICAM1)8. The development of ILFs is also tightly regulated by NOD1, which is a bacterial sensor that responds to bacterial NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | MAY 2013 | 321 © 2013 Macmillan Publishers Limited. All rights reserved
  2. 2. REVIEWS Box 1 | Pattern recognition receptors Pattern recognition receptors (PRRs) are receptor molecules expressed in immune cells. PRRs recognize pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide, flagellin, bacterial DNA and RNA, and they have crucial roles in innate immunity and protection against pathogens. In addition to pathogens, the gut-resident microbiota contains PAMPs that are recognized by PRRs. Most PRRs fall into three families: the Toll-like receptors (TLRs), NOD-like receptors (NLRs) and retinoic acid-inducible gene I (RIG‑I)‑like receptors (RLRs). TLR family members are expressed on the cell surface and in endosomes, and stimulation of TLRs by bacterial components activates downstream adaptor molecules such as myeloid differentiation primary response 88 protein (MYD88) and/or TIR domaincontaining adaptor protein inducing IFNβ (TRIF). TLR activation results in the upregulation of pro-inflammatory mediators that facilitate host immune responses. NLRs are cytoplasmic proteins that regulate inflammatory and apoptotic responses. The NLRs nucleotide-binding oligomerization domain 1 (NOD1) and NOD2 share the downstream adaptor molecule receptor-interacting serine/threonine kinase (RICK), which activates innate immunity through nuclear factor‑κB (NF‑κB) and mitogen-activated protein kinases (MAPKs). A specific subset of NLRs is involved in the formation of the inflammasome, which activates caspase 1. These NLRs include NLRP3 (NOD-, LRR- and pyrin domain-containing 3), NLRP1 and NLRC4 (NOD-, LRR- and CARD-containing 4), and they contain specific domains (a pyrin domain or a CARD) that assist in the recruitment of the adaptor protein ASC, which forms an oligomeric complex with caspase 1. Activation of the inflammasome and caspase 1 leads to the secretion of mature interleukin‑1β (IL‑1β) and IL‑18, which are involved in host defence and in the pathogenesis of multiple autoimmune diseases. RLRs are cytoplasmic proteins that are involved in intracellular virus recognition but have been demonstrated to also be important in gut immunity and in disease pathogenesis166,167. The RLRs RIG‑I and melanoma differentiation-associated protein 5 (MDA5) recognize double-stranded RNA. Another RLR member, LGP2 (also known DHX58), lacks amino‑terminal caspase-recruitment domains and functions as a regulator of RIG‑I and MDA5 signalling. Germ-free mice Mice that are born and raised in isolators, without exposure to microorganisms. Peyer’s patches Collections of lymphoid tissue that are located in the mucosa of the small intestine. They have an outer epithelial layer consisting of specialized epithelial cells called M cells. Peyer’s patches consist of a dome area, B cell follicles and interfollicular T cell areas. High endothelial venules are present mainly in the interfollicular areas. Specific-pathogen-free mice Mice raised in conditions whereby an increasing number of pathogens are excluded or eradicated from the colony. These animals are maintained in the absence of most of the known chronic and latent persistent pathogens. Although this enables better control of experimental conditions related to immunity and infection, it also sets apart such animal models from pathogen-exposed humans or non-human primates, whose immune systems are in constant contact with pathogens. peptidoglycan molecules8. However, although it is clear from studies using germ-free mice that GALT develop‑ ment requires the presence of microbiota, the require‑ ment for specific PRRs is less clear, as these studies have been confounded by differences between mouse strains that can result in environmental alterations in the microbiota, which, in turn, might affect lymphoid development independently of a specific gene defect. TH17 cells. It is now well known that the resident micro‑ biota regulates the development of specific lymphocyte subsets in the gut. T helper 17 (TH17) cells are a specific lineage of CD4+ TH cells that are crucial for host defence and that have a role in the development of autoimmune disease by producing the pro-inflammatory cytokines interleukin‑17A (IL‑17A), IL‑17F and IL‑22 (REF. 11). Unlike other subsets of CD4+ TH cells, such as TH1 and TH2 cells, TH17 cells preferentially accumulate in the intestine, which indicates that the development of TH17 cells might be regulated by gut-intrinsic mechanisms. Consistent with this hypothesis, the presence of intes‑ tinal TH17 cells is greatly reduced in antibiotic-treated or germ-free mice12–16, which shows that the microbiota has a crucial role in TH17 cell development. In fact, a particular species of Clostridia-related bacteria, called segmented filamentous bacteria (SFB), promotes the generation of TH17 cells in mice13–15 (FIG. 1a). The precise mechanism by which SFB promote TH17 cell develop‑ ment remains to be fully elucidated. The adhesion of SFB to the host epithelium upregulates serum amyloid A protein (SAA) production, which, in turn, promotes IL‑6 and IL‑23 production by CD11c+ lamina propria dendritic cells (DCs) and the subsequent induction of TH17 cell differentiation14 (FIG. 1a). Likewise, luminal ATP, which is provided by commensal bacteria but not by pathogenic bacteria, promotes TH17 cell development through a mechanism that is distinct to that elicited by SFB12. However, whether SAA and ATP are required for TH17 cell development in vivo remains unclear. IL‑1β is also crucial for TH17 cell differentiation and is specifi‑ cally induced by the gut microbiota16 (FIG. 1a). Although reconstitution of germ-free mice with the microbiota of conventionally raised mice rescues the development of TH17 cells in the gut, colonization with rat or human microbiota does not17. This indicates that the microbiota might regulate the development of TH17 cells in the gut in a species-specific manner. TReg cells. Forkhead box P3 (FOXP3)+ regulatory T (TReg) cells comprise a subset of CD4+ TH cells that accumu‑ late in the intestine, where they help to maintain gut homeostasis. The depletion of TReg cells results in the abnormal expansion of CD4+ TH cells expressing com‑ mensal bacteria-specific T cell receptors (TCRs); conse‑ quently, intestinal inflammation ensues18. Importantly, the development of peripherally derived TReg cells is partly dependent on the gut microbiota, as the number of TReg cells is greatly decreased in the colonic lamina propria of germ-free mice19,20 (FIG. 1a). The induction of TReg cells is triggered by specific populations of commensal bac‑ teria. The accumulation of IL-10-producing TReg cells in the colon is promoted by colonization of germ-free mice with one of the following bacterial populations: a mix‑ ture of 46 Clostridium spp. cluster IV and XIVa strains; altered Schaedler flora (ASF), which consists of a cock‑ tail of eight defined commensal bacteria; or the human commensal bacterium Bacteroides fragilis19–21 (FIG. 1a). However, the mechanism by which specific bacterial populations induce the development of TReg cells remains poorly understood. IgA-producing B cells and plasma cells. A close relation‑ ship also exists between the microbiota and gut-specific B cell responses. IgA is a major class of immunoglobu‑ lin that is produced in mucosal tissues, including in the intestine. In the intestinal lumen, IgA is produced as pol‑ ymeric IgA at high concentrations22 (FIG. 1b). Polymeric IgA is transported via the polymeric immunoglobulin receptor (pIgR) that is expressed on intestinal epithelial cells and is released into the intestinal lumen as secreted IgA (SIgA)23 (FIG. 1b). SIgA coats commensal bacteria and soluble antigens to inhibit their binding to the host epi‑ thelium and their penetration into the lamina propria22. Thus, SIgA promotes intestinal barrier function and helps to maintain host–commensal mutualism (FIG. 1b). In addition, IgA has been shown to regulate the com‑ position and the function of the gut microbiota24. For example, deficiency or dysfunction of IgA as a result of deficiency or mutation of activation-induced cytidine deaminase (AID), respectively, leads to alterations in the composition of the gut microbiota25,26. Moreover, bind‑ ing of IgA to the commensal Bacteroides thetaiotaomicron 322 | MAY 2013 | VOLUME 13 www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved
  3. 3. REVIEWS a • SFB • Other microbiota Gut lumen • Clostridium cluster IV and XIVa strains • PSA+ Bacteroides fragilis • Other microbiota REGIIIγ Epithelial cell ATP Lamina propria IL-1β, IL-6 and IL-23 SAA • PSA • Other factors? IL-22 TGFβ CD103+ DC and TLR5+ DC TH17 cell CD70+CD11clow DC and CX3CR1+CD11c+ DC CD11b+CD11c– macrophage IL-12(?) TH1 cell b Retinoic acid and IL-10 FOXP3+ TReg cell c SIgA CD11b+CD11c– macrophage REGIIIγ TLR TSLP Pattern recognition receptors (PRRs). Germline-encoded receptors (such as the Toll-like receptors (TLRs) and the NOD-like receptors (NLRs)) that can sense pathogenassociated molecular patterns and initiate signalling cascades that lead to an innate immune response. They can be membrane-bound (such as TLRs and C-type lectin receptors (CLRs)) or soluble cytoplasmic receptors (such as retinoic acid-inducible gene I (RIG‑I), melanoma differentiationassociated protein 5 (MDA5) and NLRs). Peripherally derived TReg cells A subset of forkhead box P3 (FOXP3)+ regulatory T (TReg) cells that are induced in the periphery and mediate their suppressive effects through the secretion of cytokines such as interleukin‑10 and transforming growth factor‑β, which inhibit other T cells. Their role is to maintain self-tolerance. Activation-induced cytidine deaminase (AID). An enzyme that is required for two crucial events in the germinal centre: somatic hypermutation and class-switch recombination. BAFF and APRIL pDC, TIP DC and TLR5+ DC APRIL, BAFF, NO, retinoic acid and TNF FDC IL-22 IL-25 BAFF, APRIL IgA IgA+ B cell B cell • ↑ AID • Class switching RORγt+ ILC3 IL-17RB+ DC IgA+ plasma cell Figure 1 | The gut microbiota-mediated development of the intestinal immune system.  a | Segmented filamentous bacteria (SFB) and other commensal microorganisms activate lamina propria dendritic cells (DCs) and macrophages to induce T helper 17 (TH17) cells and TH1 cells through the production of interleukin‑1β (IL‑1β), Nature Reviews |the case IL‑6 and IL‑23 in Immunology of TH17 cells, and possibly IL‑12 in the case of TH1 cells (although the role of IL-12 in TH1 development in vivo in the gut remains to be confirmed). TH17 cells regulate the gut microbiota community in an IL‑22- and regenerating islet-derived protein 3γ (REGIIIγ)-dependent manner. Clostridium spp. clusters IV and XIVa, polysaccharide A (PSA)+ Bacteroides fragilis and other microbiota stimulate intestinal epithelial cells, T cells, and lamina propria DCs and macrophages to promote the development and/or the activation of forkhead box P3 (FOXP3)+ regulatory T (TReg) cells. b | The microbiota stimulates intestinal epithelial cells and DCs to promote IgA-producing B cell and plasma cell differentiation in the lamina propria. Toll-like receptor (TLR) activation on intestinal epithelial cells induces the secretion of B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL), which promote the differentiation of IgA-producing plasma cells. Intestinal epithelial cells also produce thymic stromal lymphoprotein (TSLP) to promote BAFF and APRIL expression by DCs. Various types of DCs, such as plasmacytoid DCs (pDCs), TIP DCs (TNF and inducible nitric oxide synthase (iNOS)-producing DCs) and TLR5+ DCs secrete BAFF, APRIL, nitric oxide (NO), retinoic acid and tumour necrosis factor (TNF) to facilitate the expression of activation-induced cytidine deaminase (AID) and IgA class-switching in B cells. Follicular DCs (FDCs) also induce the differentiation of IgA-producing plasma cells in Peyer’s patches and isolated lymphoid follicles. IgA that is produced by lamina propria B cells is secreted into the intestinal lumen (SIgA), where it alters microbiota composition and function. c | Innate lymphoid cells (ILCs) that express retinoic acid receptor-related orphan receptor-γt (RORγt) and produce IL-22 (termed ILC3s) regulate the gut microbiome through the induction of REGIIIγ in intestinal epithelial cells. The microbiota positively regulates the production of IL‑22 by RORγt+ ILC3s via an unknown mechanism. In addition, the microbiota induces IL‑25 secretion by endothelial cells, which acts on lamina propria IL‑17 receptor B (IL‑17RB)+ DCs, and the IL‑25‑activated DC subset suppresses IL‑22 production by RORγt+ ILC3s. CX3CR1, CX3C-chemokine receptor 1; SAA, serum amyloid A protein; TGFβ, transforming growth factor‑β. NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | MAY 2013 | 323 © 2013 Macmillan Publishers Limited. All rights reserved
  4. 4. REVIEWS inhibits innate immune responses by affecting bacterial gene expression27. The gut microbiota also regulates IgA production, as the number of IgA-producing cells in the intestine is markedly decreased in germ-free mice22. Although the precise mechanisms by which commensal bacteria promote the development of IgA-producing cells remain poorly understood, bacterial recognition through MYD88 in follicular DCs (FDCs) is known to be important for the generation of IgA28 (FIG. 1b). Notably, for a subset of lamina propria DCs, commensal bacteriaderived flagellin promotes the synthesis of retinoic acid, which is an important molecule that facilitates the differ‑ entiation of IgA-producing B cells29,30 (FIG. 1b). Likewise, commensal bacteria promote the expression of factors that are involved in the induction of IgA+ B cells, such as tumour necrosis factor (TNF), inducible nitric oxide synthase (iNOS; also known as NOS2), B cell activating factor (BAFF; also known as TNFSF13B) and a prolifer‑ ation-inducing ligand (APRIL; also known as TNFSF13) in lamina propria DCs31,32. In addition to TNF- and iNOSproducing (TIP) DCs, intestinal plasma cells express TNF and iNOS following microbial exposure, which further promotes the IgA secretory function of intestinal B cells33 (FIG. 1b). Thus, the microbiota instructs lamina propria DCs and/or FDCs to induce the differentiation of IgAproducing B cells, and in turn, IgA regulates the func‑ tion and composition of the gut microbiota to maintain mutualism between the host and the microbiota. Follicular DCs (FDCs). Specialized non-haematopoietic stromal cells that reside in the follicles and germinal centres. These cells possess long dendrites, but are not related to dendritic cells, and carry intact antigen on their surface. ILCs. Innate lymphoid cells (ILCs) are increasingly recog­ nized as innate immune cells that share functional charac‑ teristics with T cells (reviewed in REFS 34,35). ILCs arise from a common lymphoid precursor cell but differenti‑ ate into multiple lineages on the basis of the expression of specific transcriptional factors. Currently, ILCs have been categorized into three main groups: T‑bet+ ILCs (termed ILC1s) GATA-binding protein 3 (GATA3)+ ILCs (termed ILC2s), and retinoic acid receptor-related orphan receptor-γt (RORγt)+ ILCs (termed ILC3s)34–37. The role of the microbiota in the development and the function of ILCs has been controversial. Although some studies report that the microbiota is required for the differentiation of ILCs and for their production of IL‑22 (REFS 38,39) (FIG. 1c), another study suggested that the microbiota suppressed IL‑22 production by RORγt+ ILCs40 (FIG. 1c). A crucial function of IL‑22 is to promote antimicrobial peptide production by intestinal epithe‑ lial cells. Specifically, IL‑22 induces the expression of the C‑type lectin antimicrobial peptides regenerating isletderived protein 3β (REGIIIβ) and REGIIIγ, which can affect the gut microbiota41,42. Depletion of either IL‑22 or IL‑22‑producing ILCs leads to overgrowth and/or dis‑ semination of potentially pathogenic bacteria, such as Alcaligenes xylosoxidans, which might increase the risk of subsequent intestinal damage and systemic inflamma‑ tion43. Thus, the regulation of gut-specific immune cells by commensal bacteria has far-reaching effects that are not limited to immune homeostasis, but also include the regulation of the balance between beneficial and poten‑ tially pathogenic commensals in the microbiota, and resistance to intestinal pathology. Microbiota and resistance to pathogens It has been known for many years that germ-free mice are more susceptible to infection than conventionally raised mice44,45. Furthermore, treatment with antibiot‑ ics is associated with increased colonization of patho‑ gens in both mice and humans46–49. These observations indicate that an important function of the indigenous microbiota might be to protect the host from infection. The mechanisms by which commensal bacteria achieve this remain poorly understood, but there is evidence to indicate that both direct and indirect mechanisms might be involved. Direct competition for nutrients. Several studies have shown that commensal bacteria can inhibit pathogen colonization by successfully competing for the limited supply of nutrients within the intestine. For example, Escherichia coli competes with enterohaemorrhagic E. coli (EHEC; an enteric pathogen that causes substan‑ tial morbidity and mortality worldwide) for organic acids, amino acids and other nutrients50–53 (FIG. 2). As different commensal E. coli strains have distinct meta‑ bolic profiles, each strain can differentially compete with pathogens53. Although our understanding of how commensal bacteria outcompete pathogens is still poor, studies thus far indicate that pathogen eradica‑ tion might be most effective with bacteria that are meta‑ bolically related to the pathogen. For example, E. coli, but not Bacteroides spp., can effectively outcompete the metabolically related pathogen Citrobacter rodentium, which is a mouse bacterium that models infection by enteropathogenic E. coli (EPEC) and EHEC 54. The ability of E. coli to outcompete C. rodentium is partly mediated by competition for simple sugars that are used by both bacteria in the intestine54 (FIG. 2). Likewise, the enteric pathogen Salmonella enteria subsp. enterica serovar Typhimurium is capable of colo‑ nizing the intestine of mice that have been harbouring a limited microbiota for several weeks, but it is rapidly eradicated after co‑housing with conventionally raised mice, which indicates that not all commensal bacteria have the ability to out-compete the pathogen55. The metabolic activity of the microbiota can also affect pathogen colonization by a mechanism that is distinct from nutritional competition. The commensal bacterial strain B. thetaiotaomicron produces multiple fucosidases that generate fucose from host-derived glycans, such as mucin, and this modulates the expression of EHEC viru‑ lence genes through the activation of the fucose sensor FusKR56 (FIG. 2). Pathogens, in turn, have also evolved strategies to over‑ come competition by commensal bacteria. One strategy is to use nutrients that are not primarily metabolized by commensal bacteria. Carbohydrate usage can differ substantially between pathogenic and commensal E. coli strains52,57. For example, EHEC, but not certain commen‑ sal E. coli strains, can use galactose, hexuronates, man‑ nose, ribose and ethanolamine, which is released into the intestine during epithelial cell turnover, as a carbon or nitrogen source52,58. Use of ethanolamine can be explained by the presence of the ethanolamine utilization (eut) 324 | MAY 2013 | VOLUME 13 www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved
  5. 5. REVIEWS Indirect Direct Pathogen strategies Microbiota strategies Pathogen Pathogen Commensal microbiota Competition for nutrients Fucose Virulence factor expression Killing (T6SS) Pathogen colonization Gut lumen Nutrients specifically used by pathogens Epithelial cell SCFAs REGIIIγ • Pathogen colonization • Systemic translocation REGIIIγ IgA Mucus ↑ Barrier function Lamina propria Inflamed epithelial cell IL-22 IL-22 Plasma cell Induce pro-IL-1β ILC3 Neutrophil IL-1β and IL-23 IL-1β B cell TH1 or TH17 cell Macrophage Figure 2 | Direct and indirect resistance of the microbiota to colonization by enteric pathogens.  Direct competition Nature Reviews | inhibit between pathogens and commensal bacteria is shown on the left-hand side. Resident microorganisms directlyImmunology the colonization and/or the proliferation of incoming enteric pathogens. Commensal microorganisms can outcompete pathogens for shared nutrients, such as carbohydrates, amino acids and organic acids. In addition, commensal bacterial strains such as Bacteroides thetaiotaomicron catabolize mucin to produce fucose, which inhibits virulence factor expression by pathogenic Escherichia coli. Enteric pathogens have evolved strategies to overcome competition by commensal bacteria. Some pathogens can directly kill their commensal competitors through their type VI secretion system (T6SS). Pathogen-induced inflammation, which leads to increased epithelial cell turnover, provides nutrients that selectively promote the growth of pathogens. Moreover, pathogens can localize to epithelium-associated niches that are devoid of commensal bacteria and use nutrients near the epithelium to escape direct competition with resident microorganisms. Indirect mechanisms of competition between commensal bacteria and pathogens are shown on the right-hand side. Commensal bacteria catabolize polysaccharides to generate short-chain fatty acid (SCFAs), such as acetate, which enhances intestinal epithelial cell barrier function. In addition, commensal microbiota promotes the production of mucus and the release of antimicrobial peptides such as regenerating islet-derived protein 3γ (REGIIIγ) from epithelial cells to limit pathogen colonization and proliferation. Innate immune cells, such as intestinal resident macrophages, neutrophils and some group 3 innate lymphoid cells (namely ILC3s), as well as T helper 1 (TH1) cells, TH17 cells and IgA-producing B cells and plasma cells, are also activated by the microbiota to limit pathogen colonization. IL, interleukin. Type IV secretion system (T6SS). An export system that is used by Gram-negative bacteria to deliver effector proteins to other cells in a cell contact-dependent manner. operon in the genome of the EHEC strain and not in the commensal E. coli strain58. Thus, pathogens have evolved to use nutritional resources that are not consumed by commensal bacteria to acquire a growth advantage. Another strategy used by pathogens is the induction of inflammation by their virulence factors. For example, infection by S. Typhimurium results in the production of reactive oxygen species by neutrophils, which facili‑ tates the conversion of endogenous thio­ ulphate into s tetrathionate, thereby selectively promoting the growth of S. Typhimurium59,60 (FIG. 2). Moreover, virulence factors such as intimin allow C. rodentium to attach to the host epithelium where commensal bacteria do not normally reside54. Localization to epithelium-associated niches might also allow certain pathogens such as EPEC and EHEC to use nutrients that are available at or near the epithelium and to escape from direct competition with resident microorganisms (FIG. 2). Furthermore, certain Gram-negative enteric pathogens, such as Serratia marce‑ scens and C. rodentium, can directly kill their commensal competitors through the expression of the type VI secretion system (T6SS)61,62 (FIG. 2). Thus, the outcome of infection by pathogens is ultimately determined by both host and commensal bacterial factors. NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | MAY 2013 | 325 © 2013 Macmillan Publishers Limited. All rights reserved
  6. 6. REVIEWS Inflammasome A molecular complex of several proteins, including a NOD-like receptor (NLR), the adaptor protein ASC and a caspase, that, upon assembly, cleaves pro‑interleukin‑1β (pro-IL‑1β) and pro‑IL‑18, thereby producing mature IL‑1β and IL‑18. Aryl hydrocarbon receptor (AHR). A cytosolic, ligand-dependent transcription factor that translocates to the nucleus following the binding of specific ligands, which include dietary and microbial metabolites. AHR participates in the differentiation of regulatory T cells, T helper 17 (TH17) cells and intraepithelial intestinal T cells, and it is required for the secretion of interleukin‑22 by TH17 cells. More recently, AHR has been shown to have crucial roles in the development and function of group 3 innate lymphoid cells, including lymphoid tissue inducer (LTi) cells and ILC3s. Commensal bacteria promote mucosal barrier function. The attachment of pathogens to the surface of the intes‑ tinal epithelium is a crucial initial step for infection to occur. As a defence mechanism, the epithelium produces mucus and antimicrobial molecules to inhibit pathogen invasion. In the colon, the mucus layer forms a strong bar‑ rier against both pathogens and commensal bacteria63,64. In the small and large intestines, the inner mucus layer near the epithelium is devoid of commensal bacteria63,64. However, the mucus layer in germ-free mice is much thinner than that in conventionally raised mice, which indicates that the microbiota might contribute to mucus production65 (FIG. 2). Consistent with this hypothesis, the thickness of the mucus layer of germ-free mice can be restored to normal by oral administration of the bacterial products lipopolysaccharide (LPS) and peptidoglycan65. The microbiota also contributes to the production of antimicrobial molecules by epithelial cells of the small and large intestines (FIG. 2). For example, REGIIIγ and REGIIIβ that are contained in Paneth cell granules are released into the lumen to regulate host–bacte‑ ria inter­ ctions through their antimicrobial activity 66. a Epithelium-intrinsic deletion of MYD88 impairs the production of REGIIIγ and REGIIIβ; therefore, it seems that recognition of the microbiota by TLRs medi‑ ates the expression of these antimicrobial peptides42,66. Importantly, the impaired production of REGIIIγ and REGIIIβ increases the susceptibility of mice to infection by enteric pathogens including Listeria mono­cytogenes, Yersinia pseudotuberculosis and S. enterica subsp. enterica serovar Enteritidis67–69. The microbiota can also enhance epithelial barrier function through the production of metabolites. For instance, short-chain fatty acids (SCFAs), especially acetate, produced by Bifidobacterium spp. act on the epi‑ thelium to inhibit the translocation of Shiga toxin that is produced by E. coli O157:H7 (REF. 45) (FIG. 2). The precise signalling mechanism by which bacterial products and metabolites enhance epithelial defence, and how this mutually affects the microbiota and the host, remains to be elucidated. The microbiota enhances innate immunity to pathogens. Mononuclear phagocytes, such as macrophages and DCs, are located in the lamina propria and promote immunological unresponsiveness to commensal bacteria, which is important for maintaining gut homeostasis70,71. Specifically, gut-resident phagocytes are hyporespon‑ sive to microbial ligands and commensal bacteria, and they do not produce biologically significant levels of pro-inflammatory molecules upon stimulation70–72. However, the microbiota is essential for upregulating the production of pro‑IL‑1β, the precursor to IL‑1β, in resident mononuclear phagocytes71. Under steady-state conditions when the epithelial barrier is intact, resident commensal bacteria cannot also induce the processing of pro‑IL‑1β into biologically active mature IL‑1β and thus a state of hyporesponsiveness is maintained71. By contrast, infection by enteric pathogens such as S. Typhimurium and Pseudomonas aeruginosa can induce the processing of pro‑IL‑1β by promoting the activation of caspase 1 via the NLRC4 (NOD-, LRR- and CARD-containing 4) inflammasome71. Unlike commensal bacteria, these patho‑ gens are capable of activating the NLRC4 inflammasome to produce IL‑1β because they express a type III secretion system, which allows the transfer of the NLRC4 agonist flagellin into the host cytosol71. Consequently, BALB/c mice that are deficient in NLRC4 or in the IL‑1 receptor (IL‑1R) are highly susceptible to orogastric infection with S. Typhimurium71. The protective role of IL‑1β in intesti‑ nal immunity is, at least partly, mediated by its ability to induce the expression of endothelial adhesion molecules that contribute to neutrophil recruitment and pathogen clearance in the intestine. Thus, NLRC4‑dependent IL‑1β production by resident phagocytes represents a specific response that can discriminate between pathogenic and commensal bacteria. The intestinal microbiota also promotes immunity through the production of IL‑22 by ILC3s38. Germ-free mice show impaired intestinal production of IL‑22, which indicates that there might be a requirement for commen‑ sal bacteria or their metabolites16. Mice with defects in IL‑22‑expressing cells exhibit increased susceptibility to C. rodentium infection, which indicates that commen‑ sal bacterial-driven IL‑22 that is produced by ILC3s is important for protection against infectious pathogens38,73,74. Consistent with this idea, administration of IL‑22 to aryl hydrocarbon receptor (Ahr)−/− mice, which exhibit impaired IL‑22 production, provides protection against C. rodentium infection74. Taken together, these results indicate that commensal bacteria might also promote host defence by stimulating ILCs to produce IL‑22 (FIG. 2). The microbiota promotes adaptive immunity. As men‑ tioned above, specific commensal bacteria promote the generation of different T cell subsets in the intes‑ tine12,14,19–21,75 that have unique roles during pathogen infection (FIG. 2). For instance, TH17 cell differentiation that is induced by SFB colonization facilitates protec‑ tion against C. rodentium infection14. Likewise, micro‑ biota-induced TReg cells attenuate intestinal damage that is caused by exaggerated immune responses against infectious pathogens. Specifically, B. fragilis promotes IL‑10‑producing TReg cells that protect against Helicobacter hepaticus infection21,76, and Bifidobacterium infantis increases the number of TReg cells that attenuate intes‑ tinal disease severity following S. Typhimurium infec‑ tion77. Commensal bacteria also induce specific immune responses, including IgA production and the generation of CD4+ T cells, that are directed against their own anti‑ gens78–80. Although the precise role of commensal bacte‑ ria-specific adaptive immunity against invasive pathogens remains poorly understood, there is evidence indicating that it is important for limiting the systemic dissemination of commensal bacteria81. The induction of this ‘firewall’ function of the adaptive immune system by the micro‑ biota might prevent collateral damage — which could be caused by the translocation of indigenous bacteria — that is often associated with pathogen infection and epithelial barrier disruption. In addition, it might contribute to the elimination of pathogens through opsonization or other immune mechanisms. 326 | MAY 2013 | VOLUME 13 www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved
  7. 7. REVIEWS Inflammatory bowel disease (IBD). Immune-mediated inflammation of the bowel. There are two main forms: Crohn’s disease, which is a granulomatous segmental inflammation affecting any part of the intestine; and ulcerative colitis, which is a mucosal inflammation involving the rectum and extending for a variable distance along the colon. In developed countries, the incidence of inflammatory bowel disease is approximately one in 50,000. It usually starts in early adult life and continues afterwards with a relapsing, remitting course. SAMP1/yit mice A mutant mouse strain that spontaneously develops a chronic intestinal inflammation that is mainly localized in the terminal ileum. Autophagy An evolutionarily conserved process in which acidic double-membrane vacuoles sequester intracellular contents (such as damaged organelles and macromolecules) and target them for degradation through fusion to secondary lysosomes. α-defensins One subset of defensin proteins. Defensins and cathelicidins are members of a family of small antimicrobial polypeptides that are abundant in neutrophils and epithelial cells. They contribute to host defence by disrupting the cytoplasmic membrane of microorganisms. Protective role of the commensal microbiota against systemic infection. The mechanism by which the micro‑ biota promotes systemic pathogen eradication is poorly understood. Pretreatment of germ-free mice with LPS induces pro-inflammatory cytokine production and neutrophil recruitment, and can prevent systemic bac‑ terial infection82. This observation indicates that com‑ mensal bacteria might, at least partly, promote host defence at distant sites through the release of microbial molecules. Consistent with this hypothesis, peptido­ glycan molecules that are derived from the microbiota are found in the periphery and can prime peripheral blood neutrophils to facilitate their bactericidal capacity via NOD1 signalling 83. This neutrophil priming effect, which is presumably induced by intestinal bacteria, enhances host defence against systemic infection with Streptococcus pneumoniae83. The microbiota might also have an important role in systemic antiviral immune responses. Germ-free mice and antibiotic-treated mice show reduced innate and adaptive immune responses to influenza virus, which results in increased viral loads in their tissues84,85. The microbiota might enhance antiviral immunity through the inflammasome and the production of innate cytokines that are required for optimal immune responses against viruses84. For example, the microbiota promotes type I interferon (IFN) production by macrophages and the subsequent IFN-priming of natural killer (NK) cells, which is important for protection against infection with mouse cytomegalovirus and lymphocytic chorio­ meningitis virus86. Moreover, the gut microbiota induces intestinal TLR-dependent DC activation and inhibits systemic parasitic infection87. Recent studies also indicate that commensal bacteria might provide tissue-specific defence mechanisms, as has been demonstrated by the protection provided by resident skin bacteria against local pathogen infection88. Taken together, these studies using different infection models show a protective effect of the microbiota against systemic infection; however, additional studies are nec‑ essary to identify the underlying mechanisms as well as the relative contributions of host defence mechanisms that are promoted by the microbiota of specific tissues such as the skin or the respiratory tract. It is also impor‑ tant to note that commensal bacteria do not always pro‑ tect against pathogenic infection and in certain contexts they can facilitate it (as discussed below)89–91. The microbiota and intestinal disease The microbiota contributes to IBD. The gut micro‑ biota is essential for triggering or enhancing chronic intestinal inflammation in various inflammatory bowel disease (IBD)-prone mouse strains (TABLE  1). Under germ-free conditions, Il10−/− or Tcra−/− mice, as well as transgenic rats that have been engineered to overexpress HLA‑B27 and human β2 microglobulin, do not develop colitis92–95. In Il2−/− or SAMP1/yit mice, although sponta‑ neous intestinal inflammation occurs even under germfree conditions ,symptoms are attenuated compared with conventionally raised mutant mice96,97. In many spontaneous colitis models, intestinal inflammation is abrogated if mice also have a deficiency in individual TLRs or in MYD88. For example, spontaneous coli‑ tis that develops in Il10−/− mice, in conditional signal transducer and activator of transcription 3 (Stat3)−/− mice or in mice with a deletion in inhibitor of nuclear factor-κB kinase-γ (IKKγ; also known as NEMO) spe‑ cifically in intestinal epithelial cells (NEMOΔIEC mice) is reversed when these mice also lack TLRs or MYD88 (REFS 98–101). However, Il2−/− mice that are deficient in MYD88 still develop spontaneous colitis99. In this model, the microbiota might drive colitis through the promo‑ tion of inflammatory TH17 cell responses12 that occur independently of MYD88. Similar to mice, human intestinal mononuclear phagocytes show hyporesponsiveness to microbial stim‑ ulation under steady-state conditions102,103. However, in patients with IBD, intestinal mononuclear phagocytes robustly respond to microbial products and to the resi‑ dent bacteria, which results in the production of large amounts of pro-inflammatory cytokines such as TNF and IL‑23, as occurs in IBD-prone mice103. Thus, the abnormal activation of resident intestinal mononuclear phagocytes by commensal bacteria might facilitate the development or persistence of intestinal inflammation in IBD. Consistent with this idea, intestinal macrophages isolated from Il10−/− mice robustly respond to gut bac‑ teria, whereas wild-type intestinal macrophages are hyporesponsive70–72. However, the increased production of pro-inflammatory molecules by intestinal phagocytes might also reflect the activity of recruited monocytes in areas of infection or inflammation. There is genetic evidence showing that the impaired recognition and killing of commensal bacteria also con‑ tributes to IBD development, as has been suggested by the fact that many of the identified IBD-susceptibility genes regulate host–microbial interactions104. NOD2, which is an intracellular sensor of bacterial peptidog‑ lycan, was identified as a susceptibility gene for Crohn’s disease, and Crohn’s disease-associated NOD2 muta‑ tions are associated with a loss of function of the pro‑ tein105–107. Mutations in the autophagy regulatory genes autophagy related gene 16‑like 1 (ATG16L1) and immu‑ nity-related GTPase family M (IRGM) are also linked to Crohn’s disease108,109, and autophagy dysfunction is associated with defects in bacterial killing 110–112. In addi‑ tion, NOD2 and autophagy proteins regulate the func‑ tion of Paneth cells, which release granules containing antimicrobial peptides in response to bacteria113. NOD2 is highly expressed in Paneth cells, and some studies report diminished expression of Paneth cell α‑defensins in individuals with Crohn’s disease-associated NOD2 mutations114,115. Nod2−/− mice have impaired antimicro‑ bial functions in Paneth cells, resulting in the accumu‑ lation of ileal bacteria, which might contribute to IBD pathogenesis116. Atg16l1‑mutant (Atg16l1HM) mice show abnormal granule formation in Paneth cells, which is also observed in patients with Crohn’s disease who have homozygous mutations in ATG16L1 (REF. 117). In Atg16l1HM mice this abnormality can be triggered by enteric viral infection (for example, by murine noro‑ virus) and is associated with increased susceptibility NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | MAY 2013 | 327 © 2013 Macmillan Publishers Limited. All rights reserved
  8. 8. REVIEWS Table 1 | Role of the gut microbiota in the protection or induction of IBD Commensal bacterium Host Genotype Disease Receptors Possible mechanism Refs Commensal microbiota that protect against IBD Clostridium spp. clusters IV and XIVa Mouse Wild-type DSS colitis Unknown Induction of TReg cells 19 Altered Schaedler flora Mouse Wild-type DSS colitis MYD88 and TRIF Induction of TReg cells 20 Bacteroides fragilis Mouse Wild-type TNBS colitis TLR2, MYD88 Induction of TReg cells via polysaccharide A 21 Bacteroides vulgatus Mouse Il2−/− Spontaneous colitis Unknown Suppression of Escherichia colitriggered colitis in Il2−/− mice 120 Faecalibacterium prausnitzii Human NA IBD Unknown Induction of IL‑10 by PBMCs 148 Commensal microbiota that promote the development of IBD E. coli Mouse Il10−/− Spontaneous colitis Unknown Monocolonization of germ-free mice induces colitis 119 E. coli Mouse Il2−/− Spontaneous colitis Unknown Monocolonization of germ-free mice induces colitis 96, 120 Enterococcus faecalis Mouse Il10−/− Spontaneous colitis Unknown Monocolonization of germ-free mice induces colitis 119 B. vulgatus Rat HLA‑B27–B2m transgenic Spontaneous colitis Unknown Monocolonization of germ-free rats induces colitis 96 B. vulgatus Mouse Il10r2−/−Tgfbr2−/− Spontaneous colitis Unknown Colonization of antibiotictreated mice triggers colitis 122 Bacteroides thetaiotaomicron Mouse Il10r2−/–Tgfbr2−/− Spontaneous colitis Unknown Colonization of antibiotic-treated mice triggers colitis 122 B. thetaiotaomicron Rat HLA‑B27–B2m transgenic Spontaneous colitis Unknown Monocolonization of germ-free rats induces colitis Bacteroides unifirmatis Mouse Il10r2−/− Tgfbr2−/− Spontaneous colitis Unknown Colonization of antibiotic-treated mice triggers colitis Klebsiella pneumoniae*‡ Mouse Tbx21−/−Rag2−/− Spontaneous colitis Unknown Other commensal bacteria are required for the induction of colitis 123,124 Proteus mirabilis*‡ Mouse Tbx21−/−Rag2−/− Spontaneous colitis Unknown Other commensal bacteria are required for the induction of colitis 123,124 Helicobacter typhlonius* Mouse Tbx21−/−Rag2−/− Spontaneous colitis Unknown Transmissible to non-colitogenic TRUC mice 125 Prevotellaceae*‡ Mouse Nlrp6−/−, Asc−/− or Casp1−/− DSS colitis Unknown Impaired IL‑18 signalling promotes pathobiont expansion 126 TM7*‡ Mouse Nlrp6−/−, Asc−/− or Casp1−/− DSS colitis Unknown Impaired IL‑18 signalling promotes pathobiont expansion 126 Bilophila wadsworthia Mouse Il10−/− Spontaneous colitis Unknown Consumption of a diet composed of milk-derived fat induces pathobiont expansion 128 95 122 B2m, gene encoding β2‑microglobulin; Casp1, caspase 1; DSS, dextran-sulphate sodium; IBD, inflammatory bowel disease; IL, interleukin; MYD88, myeloid differentiation primary-response protein 88; NA, not applicable; Nlrp6, NOD-, LRR- and pyrin domain-containing 6; PBMCs, peripheral blood mononuclear cells; Rag2, recombination-activating gene 2; Tbx21, T-box 21 (encodes T‑bet); Tgfbr2, transforming growth factor‑β receptor 2; TLR, Toll-like receptor; TNBS, 2,4,6‑trinitrobenzene sulphonic acid; TReg cell, regulatory T cell; TRIF, TIR domain-containing adaptor protein inducing IFNβ; TRUC, Tbx21−/−Rag2−/− mice. *Transmissable to mutant hosts. ‡Transmissable to wild-type hosts. to chemically induced colitis118. Thus, defects in host mechanisms that recognize and clear bacteria are associated with the development of human IBD. How genetic defects lead to chronic colitis in patients with IBD remains unknown, but it is possible that impaired NOD2 or autophagy function might result in the accu‑ mulation of intestinal commensal bacteria that have the capacity to locally invade the intestinal mucosa and to trigger an abnormal inflammatory response. The role of the pathobiont in IBD. As non-pathogenic symbionts, the gut microbiota derives nutritional ben‑ efits from the host and help to maintain gut homeostasis. However, under certain conditions, particular bacterial populations that are typically found in very low abun‑ dance can acquire pathogenic properties. These condi‑ tions include inherent immune defects as well as changes in diet and/or acute inflammation, and can result in the disruption of the normal balanced state of the gut 328 | MAY 2013 | VOLUME 13 www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved
  9. 9. REVIEWS Homeostasis Inflammatory bowel disease Genetic factors (for example, mutations in Nod2, Atg16I1 and Il23r) Symbiont Unclassified bacteria Beneficial bacteria Environmental factors (diet, stress and infection) Dysbiosis Transmissable to healthy host Pathobionts Gut lumen Lamina propria • ↑↑↑ TH17 cells • ↑↑↑ TH1 cells • GALT development • ↑ TH17 and TH1 cells • ↑ Barrier function • ↑ REGIIIγ • ↑ TReg cell • ↑ IL-10 • ↑ REGIIIγ Unknown contribution • ↓ TReg cells • ↓ IL-10 • ↓ REGIIIγ Chronic inflammation Homeostasis Figure 3 | Protective and pathogenic role of the gut microbiota in IBD.  During homeostasis (left-hand side), the gut microbiota has important roles in the development of intestinal immunity. Beneficial subsets of commensal bacteria tend to have anti-inflammatory activities. Pathobionts that are colitogenic are directly suppressed by beneficial commensal Nature Reviews | Immunology bacteria partly through the induction of regulatory immune responses, involving regulatory T (TReg) cells, interleukin‑10 (IL‑10) and regenerating islet-derived protein 3γ (REGIIIγ). In inflammatory bowel disease (IBD) (right-hand side), a combination of genetic factors (for example, mutations in nucleotide-binding oligomerization domain 2 (Nod2), autophagy-related gene 16‑like 1 (Atg16l1) and interleukin‑23 receptor (Il23r)) and environmental factors (such as infection, stress and diet) result in disruption of the microbial community structure, a process termed dysbiosis. Dysbiosis results in a loss of protective bacteria and/or in the accumulation of colitogenic pathobionts, which leads to chronic inflammation involving hyperactivation of T helper 1 (TH1) and TH17 cells. Dashed line shows that that the suppression of pathobionts by beneficial bacteria is diminished. In certain contexts, pathobionts can be transferred to the host and can cause disease without the host having a predisposing genetic susceptibility. It is unknown whether unclassified bacteria have a role in the pathogenesis of IBD. GALT, gut-associated lymphoid tissue. Rag Recombinase activating genes; Rag1and Rag2 are expressed in developing lymphocytes. Mice that are deficient in either of these genes fail to produce B or T cells owing to a developmental block in the gene rearrangement that is necessary for receptor expression. microbiota, which is referred to as dysbiosis5. Dysbiosis involves the abnormal accumulation or increased viru‑ lence of certain commensal populations of bacteria, thereby transforming former symbionts into ‘patho‑ bionts’ (FIG. 3). Pathobionts are typically colitogenic in that they can trigger intestinal inflammation; therefore, there has been considerable interest in the identification of pathobionts to understand the pathogenesis of IBD. Colitogenic pathobionts have been identified using rodent models of spontaneous colitis (TABLE 1). For exam‑ ple, colonization of germ-free Il10−/− or Il2−/− mice with one of several particular E. coli or Enterococcus faecalis strains, but not with Bacteroides vulgatus, induces colitis119–121. By contrast, B. vulgatus, but not E. coli, triggers colitis in HLA‑B27‑transgenic rats94,95. The differential colitogenic capacity of these bacteria cannot be merely explained by differences in the host species (mouse versus rat), as B. vulgatus, but not E. coli, induces intestinal inflamma‑ tion in mice that are deficient in both IL‑10R2 and TGFβ receptor 2 (REF. 122). These findings indicate that specific immune defects in the host might determine the ability of individual commensal bacteria to trigger colitis (FIG. 3). However, the physiological relevance of these findings remains unclear because monocolonization of germ-free mice does not mimic the normal complex gut ecosystem. Mice that have only innate immune cells with a defi‑ ciency in T‑bet (Tbx21−/− (gene encoding T-bet) Rag−/− mice; also known as TRUC mice) develop spontaneous ulcerative colitis-like inflammation123. This colitis is trans‑ missible to T‑bet-sufficient Rag−/− mice and even to wild-type mice through co‑housing, which indicates that impaired T‑bet signalling in innate immune cells leads to the overgrowth of pathobionts that cause disease123. Indeed, the analysis of gut microbial communities in TRUC mice showed that Klebsiella pneumoniae and Proteus mirabilis, both of which are found in very low abundance in healthy mice, accumulate in the guts of TRUC mice124. These bac‑ teria can trigger colitis in T‑bet-sufficient Rag−/− mice and in wild-type mice, which indicates that they can function as pathobionts. Interestingly, although K. pneumoniae or P. mirabilis can induce colitis in healthy mice, monoor dual-colonization of germ-free TRUC mice with these bacteria does not, which indicates that interactions with additional commensal bacteria are required to actually trigger inflammation124. The proteobacterium Helicobacter typhlonius also accumulates in colitic TRUC mice, and oral administration of the bacterium is sufficient to induce colitis in non-colitic TRUC mice, but not in T‑betsufficient Rag−/− mice125. Similarly, the colitis induced by H. typhlonius is not transmissible to wild-type mice125. NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | MAY 2013 | 329 © 2013 Macmillan Publishers Limited. All rights reserved
  10. 10. REVIEWS There is increasing evidence that defects in the NODlike receptor (NLR) family of proteins are associated with dysbiosis. For example, Nlrp6−/− mice have impaired IL‑18 production, which is associated with an abnormal expan‑ sion of the commensal bacterial species Prevotellaceae and the candidate division TM7 (REF. 126). Although Nlrp6−/− mice do not develop spontaneous colitis, these mice have an increased susceptibility to dextran sulphate sodium (DSS)-induced colitis, and this phenotype is transmis‑ sible to wild-type mice by co-housing 126. This study demonstrates a correlation between the accumulation of Prevotellaceae and TM7, as well as DSS-induced colitis susceptibility, but whether these bacteria are the direct cause of this susceptibility and whether their accumu‑ lation is indeed NLRP6‑dependent remain to be deter‑ mined. Similarly, bacterial genomic sequencing shows that Nod2−/− mice, which have an increased susceptibility to DSS-induced colitis, have an altered microbiota that can be transferred to wild-type mice to increase dis‑ ease susceptibility 127. However, the specific pathobionts involved have not yet been identified. Diet has a considerable effect on the composition of microbial communities and can lead to the expansion of pathobionts. For instance, a diet rich in saturated milk fats, but not in polyunsaturated vegetable oil fats, induces dysbiosis and expansion of Bilophila wadsworthia, which is a hydrogen-consuming, sulphite-reducing commen‑ sal bacterium. This intestinal bacterium promotes proinflammatory TH1 immunity and exacerbates colitis in IBD-prone Il10−/− mice but not in wild-type mice128. Although further studies are necessary, the increase in taurine conjugation of hepatic bile acids might stimulate the growth of sulphite-reducing microorganisms such as B. wadsworthia, which promote harmful inflammation via mechanisms that remain to be elucidated128. Taken together, these observations indicate that defects in the immune system and/or changes in diet can trigger dysbiosis, which results in the accumulation of pathobionts that promote transmissible or non-trans‑ missible colitis (FIG. 3). There is increasing evidence that dysbiosis affects susceptibility to developing not only IBD but also colitis-associated colon cancer in mouse models, as well as colon cancer in humans127,129–133. The role of commensal bacteria in intestinal cancer has been recently reviewed134 and is not discussed here. iNKT cells Invariant natural killer (iNKT) cells; a population of cells that are thought to be particularly important in bridging the innate and adaptive immune responses. iNKT cells are typified by a capacity for self-recognition and rapid release of cytokines such as interferon‑γ. Protective effect of the microbiota in IBD. Although colitogenic pathobionts promote the development of IBD, commensal bacteria are also crucial for reducing IBD susceptibility, and this has generated considerable interest in the development of probiotic approaches to prevent IBD (TABLE 1). The protective effect of com‑ mensal bacteria is evident from studies using germ-free mice, which are more susceptible to DSS-induced colitis than conventionally housed mice135–137. Consistent with these observations, deficiency in TLR2, TLR4, TLR9 or MYD88 are all associated with increased susceptibility to DSS-induced colitis138–140. Tlr5−/− mice also develop spon‑ taneous colitis in certain mouse housing facilities, which strongly indicates that the microbiota might affect disease susceptibility 141,142. However, the precise mechanisms by which the microbiota mediates resistance against the development of colitis through TLR signalling remain unclear. Administration of LPS (a TLR4 ligand) or CpG (a TLR9 ligand) protects mice from colitis by promoting epithelial barrier function following the upregulation of cytoprotective heat shock proteins or by the induction of type I IFN production, respectively 138,139,143. In addi‑ tion, the production of protective IgA and IgM suppresses the harmful dissemination of commensal bacteria after colonic damage through MYD88 signalling in B cells140. The human commensal bacterium B. fragilis also pro‑ tects mice from colitis by promoting the accumulation of IL‑10‑producing TReg cells in the colon76,144. This protective effect is mediated by the outer membrane polysaccharide A (PSA) of B. fragilis76,144. TLR2‑mediated internalization of PSA into DCs, followed by its surface presentation, activates IL‑10‑producing TReg cells145,146. Commensal bacterial stimulation through MYD88 also limits the trafficking of CX3CR1+ mononuclear phagocytes that capture luminal non-invasive bacteria to the mesenteric lymph nodes (MLNs)147. Consequently, disruption of the commensal microbiota and/or MYD88 signalling results in excessive B and T cell stimulation in the MLNs, which might contribute to the development of IBD. It has also been reported that the microbiota protects against colitis through TLR-independent mechanisms. For example, the colonization of neonatal germ-free mice with whole microbiota results in decreased hyper‑ methylation of the CXC-chemokine ligand 16 (Cxcl16) gene75. This epigenetic modification of the Cxcl16 gene reduces CXCL16 expression and CXCL16‑mediated accumulation of iNKT cells that can contribute to colitis development 75. Clostridium spp. (clusters IV and XIVa) and ASF also promote the development of FOXP3+ TReg cells, which can protect against chemically induced colitis19,20 (FIG. 3). Differentiation of TReg cells that are induced by Clostridium spp. (clusters IV and XIV) is independent of NOD1, NOD2, caspase-recruitment domain-containing protein 9 (CARD9; a signalling molecule downstream of Dectin-1) and MYD88 sig‑ nalling 19; however, the induction of TReg cells by ASF requires MYD88–TRIF signalling20. Intestinal commen‑ sal bacteria can also exert protective anti-inflammatory effects through their production of metabolites, such as SCFAs136. Likewise, numbers of Faecalibacterium praus‑ nitzii, a principal member of the phylum Firmicutes, are reduced in patients with Crohn’s disease. Culture super‑ natants of F. prausnitzii containing secreted metabolites exhibit anti-inflammatory effects on human epithelial cell lines in vitro, and they decrease the susceptibility of mice to chemically induced colitis in vivo, although it is unclear whether these effects are TLR-independent 148. Thus, commensal bacteria and their products have activ‑ ities that can protect the host against inflammation by different mechanisms (FIG. 3). Microbiota and extra-intestinal diseases Multiple sclerosis. The gut microbiota has the capacity to affect the development of autoimmune central nerv‑ ous system (CNS) disorders. Broad-spectrum antibi‑ otics are given orally to mice reduce the symptoms of 330 | MAY 2013 | VOLUME 13 www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved
  11. 11. REVIEWS Experimental autoimmune encephalomyelitis (EAE). An experimental model of multiple sclerosis that is induced by immunization of susceptible animals with myelin-derived antigens, such as myelin basic protein, proteolipid protein or myelin oligodendrocyte glycoprotein. K/BxN transgenic mice Mice formed by crossing non-obese diabetic (NOD)/Lt mice with KRN T cell receptortransgenic mice on the C57BL/6 background. As the KRN receptor on T cells recognizes a peptide from the autoantigen glucose-6‑phosphate isomerase, these mice develop an arthritis that is mediated, and transferable, by circulating antibodies against glucose-6‑ phosphate isomerase. (EAE)149. In one model of multiple sclerosis, mice are immunized with the self antigen myelin oligodendrocyte glyco‑ protein (MOG) in complete Freund’s adjuvant (CFA). Disease symptoms in either MOG–CFA-induced EAE or in a spontaneous EAE mouse model are reduced when the mice are housed under germ-free conditions150,151. Monocolonization of germ-free mice with SFB results in an increase in the number of TH17 cells in both the intestinal lamina propria and the CNS, which results in severe EAE150 (FIG. 4). Thus, SFB-enhanced TH17 cellmediated inflammation might contribute to EAE exac‑ erbation. However, it is unclear whether the disease is caused by the migration of SFB-specific TH17 cells into the CNS or by the expansion of pathogenic autoantigenspecific T cells that are promoted by intestinal TH17 cell responses (FIG. 4). By contrast, certain populations of commensal bacteria are capable of attenuating CNS inflammation. For example, PSA+ B. fragilis, which experimental autoimmune encephalomyelitis CNS induces FOXP3+ TReg cell differentiation, can prevent EAE symptoms152 (FIG. 4). Thus, the pathogenesis of CNS disorders might ultimately depend on the balance of different community members in the gut microbiota. Arthritis. Autoimmune arthritis, such as rheumatoid arthritis, is a systemic inflammatory disease that pri‑ marily affects the joints but can also affect other parts of the body. The events that trigger the development of autoimmune arthritis remains unknown. However, in mouse models, the gut microbiota contributes to disease symptoms. Arthritis symptoms in K/BxN transgenic mice are reduced in a germ-free environment136,153. As in EAE, TH17 cell responses are implicated in promoting disease, and SFB-mediated enhancement of TH17 cell immunity stimulates autoantibody production by B cells, which leads to arthritic symptoms153 (FIG. 4). TH17 cell immunity is also a key factor in spontaneous rheumatoid arthri‑ tis in IL‑1R antagonist (Il1rn)−/− mice, which exhibit Joint Multiple sclerosis EAE TH17 cell TH17 cell Arthritis IL-1R antagonist TReg cell IL-1β TH17 cell Beneficial commensal bacteria Gut lumen SFB Lamina propria IgE B cell ↓ Firmicutes ↑ Bacteroidetes Peripheral blood Basophil iNKT cell Type 1 diabetes Pancreatitis Pancreas Allergic inflammation Lungs Figure 4 | Gut microbiota affects extra-intestinal autoimmune diseases.  Segmented filamentous bacteria (SFB) colonization induces T helper 17 (TH17) cell development in the intestine. These TH17 cells might migrate to the periphery to affect systemic and central nervous system (CNS) immunity; increased intestinal TH17 cells enhance the expansion Nature Reviews | Immunology of pathogenic autoantigen-specific T cells in the intestine and cause inflammation in the CNS. By contrast, ‘beneficial’ commensal bacteria can attenuate CNS inflammation through the induction of forkhead box P3 (FOXP3)+ regulatory T (TReg) cells. Induced TH17 cells can also promote autoimmune arthritis by facilitating autoantibody production by B cells (not shown). In addition, microbiota-induced interleukin‑1β (IL‑1β) signalling participates in the development of rheumatoid arthritis through the induction of TH17 cells. The IL‑1 receptor (IL‑1R) antagonist blocks IL‑1β signalling and abrogates joint inflammation. Balance in the microbial community also determines susceptibility to type 1 diabetes. A decreased Firmicutes/Bacteroidetes ratio as a result of a deficiency in myeloid differentiation primary-response protein 88 (MYD88) in non-obese diabetic mice is associated with an attenuated risk of type 1 diabetes. SFB-induced TH17 cells protect the host against type 1 diabetes development by an unknown mechanism. Finally, exposure to microorganisms in neonatal, but not adult, life decreases the accumulation of invariant natural killer T (iNKT) cells in the gut, which results in protection against allergic inflammation in the lungs. In addition, microbial compounds stimulate peripheral B cells through B cell-intrinsic MYD88 signalling and inhibit IgE production. Decreased levels of peripheral IgE result in decreased numbers of basophils, and attenuate the risk of allergic airway inflammation. EAE, experimental autoimmune encephalomyelitis. NATURE REVIEWS | IMMUNOLOGY VOLUME 13 | MAY 2013 | 331 © 2013 Macmillan Publishers Limited. All rights reserved
  12. 12. REVIEWS excessive IL‑1 signalling, enhanced IL‑17 expression and increased arthritic symptoms154. Similar to K/BxN mice, arthritic symptoms in Il1rn−/− mice are diminished when the mice are housed in germ-free conditions, which indicates that the microbiota might be essential for the progression of arthritis in this model, although whether SFB are involved is unclear 155 (FIG. 4). Together with the observation that IL‑1β signalling is essential for TH17 cell differentiation16, these results indicate that the microbiota is involved in the pathogenesis of rheumatoid arthritis partly through the promotion of TH17 cell responses. NOD mice Non-obese diabetic (NOD) mice spontaneously develop type 1 diabetes as a result of autoreactive T cell-mediated destruction of pancreatic β‑islet cells. Hygiene hypothesis This hypothesis originally stated that the increased incidence of atopic diseases in westernized countries was a consequence of living in an overly clean environment, which resulted in an understimulated immune system that responded inappropriately to harmless antigens. More recently it has been proposed that an absence of exposure to pathogens, in particular helminths, might predispose individuals to both increased allergy and autoimmune disease. T1D. Type 1 diabetes (T1D) is an autoimmune disorder that results in the destruction of insulin-producing cells in the pancreas. Recent studies with NOD mice, which have been used to model this disease, have provided some insights into how the gut microbiota can affect the devel‑ opment of T1D. Diabetes in NOD mice is ameliorated in the absence of MYD88 (REF. 156). However, protection against diabetes in Myd88−/− NOD mice is abrogated by antibiotic treatment and in germ-free conditions156. These data indicate that commensal bacteria might be impor‑ tant for reducing disease susceptibility in this model. Indeed, 16S ribosomal RNA sequencing showed altera‑ tions in the composition of the microbiota in Myd88−/− NOD mice compared with their Myd88‑sufficient NOD littermates. These alterations were characterized by a lower Firmicutes/Bacteroides ratio on a phylum level and indicate that certain bacterial populations might contribute to protection against T1D (FIG. 4). As it has been recently demonstrated that deficiencies in MYD88 or individual TLRs alone do not affect the composition of the gut microbiota157, it is possible that the genetic background of the NOD mice contributes to the effect of MYD88 deficiency on microbiota composition. Interestingly, SFB has also been implicated in pro‑ tection against T1D, which indicates that intestinal TH17 cells in certain contexts might be protective, despite their link to autoimmunity 158 (FIG. 4). SFB-mediated pro‑ tection against T1D seems to be gender-specific: male NOD mice with SFB have a lower incidence of T1D than their female counterparts158. Furthermore, the microbiota in male NOD mice is distinct from that in females and it contributes to increased testosterone levels, which are associated with protection against T1D159. Indeed, trans‑ ferring the microbiota from male into female NOD mice results in increased levels of testosterone and reduced susceptibility to T1D in female NOD mice159. Much work remains to be done to understand the complexities of TH17 cell responses and the mechanisms by which the gut microbiota contributes to the development of T1D. Allergic inflammation. Under germ-free conditions, host immune responses are TH2‑biased17. Restoration of the gut microbiota in germ-free mice results in an increase in TH1 and TH17 cells and a reduction of TH2‑type responses to levels that are observed in conven‑ tionally housed mice17. This indicates that the microbi‑ ota might be important in maintaining balance in TH cell responses. The mechanism by which the microbiota achieves this is still unclear. Germ-free mice or mice treated with antibiotics have an expansion of basophils in the peripheral blood as well as increased serum IgE levels160 (FIG. 4). The expansion of basophils as a result of microbiota depletion exacerbates the allergic airway inflammation that is triggered by exposure to house dust mite allergen160. The microbiota is directly sensed by B cells and activates MYD88 signalling to limit IgE class-switching in B cells. As CpG stimulation alone can prevent IgE production by B cells, the TLR9–MYD88 axis might have a crucial role in IgE responses that can lead to allergic inflammation160. Consistent with this idea, allergic responses to food antigens are aggravated in antibiotic-treated mice or in Tlr4−/− mice, but are ameliorated by CpG treatment 161. Thus, exposure to microorganisms that stimulate TLR signalling might be important in reducing the develop‑ ment of allergic inflammation by the promotion of TH1 rather than TH2 cell polarization. A recent study dem‑ onstrated that neonatal colonization of the gut micro‑ biota is essential for reducing numbers of iNKT cells in the intestine, and iNKT cells have been implicated in mediating allergic responses in the lungs75 (FIG. 4). Taken together, the essential role of the gut microbiota in regu‑ lating immune balance in the intestine might explain its ability to influence susceptibility to allergies. These find‑ ings support the hygiene hypothesis, which arose from the observation that early exposure to bacteria during infancy is associated with a reduced incidence of allergic diseases. Other extra-intestinal diseases. Systemic dissemination of the gut microbiota has a crucial role in the develop‑ ment of severe acute pancreatitis162. Cerulein-induced pancreatitis is dramatically reduced in Nod1−/− mice, which indicates that there might be an important role for commensal bacteria-mediated NOD1 activation in the development of pancreatitis163. NOD1 stimulation induces CC-chemokine ligand 2 (CCL2) production, thereby promoting the recruitment of CCR2+ monocytes that facilitate pancreatitis163. Intestinal dysbiosis might also be linked to the pathogenesis of cystic fibrosis, as mice with cystic fibrosis-like disease exhibit an increased abundance of Mycobacteriaceae spp., Enterobacteriaceae spp., Clostridaceae spp., and B. fragilis 164. However, the pre‑ cise mechanisms by which a dysbiotic microbiota affects the pathophysiology of cystic fibrosis still remains to be elucidated. Perspectives The gut microbiota has been studied for more than a century; however, recent studies have shown everexpanding roles for these microscopic organisms in health and disease. Despite the complexity of the gut microbiota, there is a delicate balance in bacterial popu‑ lations such that any disruption in this balance leads to dysbiosis and, consequently, to decreased resistance to pathogen colonization, to the favoured growth of patho‑ bionts and to pathological immune responses. Although the association of dysbiosis with disease pathogenesis has become evident, it is still largely unknown which 332 | MAY 2013 | VOLUME 13 www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved
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