Induction of Treg cells in the mouse colonic mucosa: A central mechanism to maintain host–microbiota homeostasis

Seminars in Immunology 24 (2012) 50–57

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Review

Induction of Treg cells in the mouse colonic mucosa: A central mechanism to maintain host–microbiota homeostasis Takeshi Tanoue, Kenya Honda ∗ Department of Immunology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan

a r t i c l e

i n f o

a b s t r a c t

Keywords: Intestinal microbiota Treg Germ-free Clostridium

CD4+ regulatory T (Treg) cells expressing the transcription factor forkhead box P3 (Foxp3) play a critical role in maintaining immunological homeostasis. Treg cells are highly abundant in the mouse intestinal lamina propria, particularly in the colon. Recent studies using germ-free and gnotobiotic mice have revealed that specific components of the intestinal microbiota influence the number and function of Treg cells. Substantial changes in the composition of microbiota have been associated with inflammatory bowel disease. In this review, we will discuss recent findings that associate intestinal microbiota in mice with Treg responses and with the maintenance of intestinal immune homeostasis. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction

intestinal epithelium and induce disease in certain genetic or environmental contexts [3]. There have been a number of reports describing a link between dysbiosis and immune disorders. In particular, dysbiosis has been implicated in chronic inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, where colitogenic bacteria disrupt gut epithelial barrier function and activate the intestinal immune system [4–6]. The activated immune system may in turn aberrantly react to commensal microbes. Recent studies using gene-targeted mice, such as T-bet−/− Rag2−/− mice [7] or Nlrp6−/− mice [8], have revealed that colitis or susceptibility to colitis is transmissible horizontally and vertically via the fecal–oral transmission of gut microbiota. In these mice, intestinal inflammation might select for colitogenic species, which could further promote colitis. In addition to their influence on the local immune status, accumulating evidence suggests that the components of gut microbiota may be instrumental for the development of systemic immune diseases, such as rheumatoid arthritis, autoimmune encephalomyelitis, and type-1 diabetes [9–12]. Intestinal microbiota constitutively and profoundly affects the development of the mucosal immune system. The effects of microbiota include organization of Peyer’s patches and isolated lymphoid follicles [13], induction of antimicrobial peptide secretion by epithelial cells, and accumulation of various lymphocytes at mucosal sites. IgA-producing plasma cells, intraepithelial lymphocytes (IELs) and ␥␦T cell receptor (TCR)-expressing T cells (␥␦T cells) are lymphocytes that are uniquely present in the mucosa. In addition, recent studies have shown the presence of various innate immune leukocytes in the mucosa, such as CD4+ CD3− lymphoid tissue inducer cells (LTi cells) and interleukin (IL)-22-producing natural killer (NK)-like cells [13–15]. Furthermore, of the CD4+ T cells in the intestinal lamina propria (LP), there are significant

The gastrointestinal tract is a very unique tissue, particularly with regard to the presence of a large number of bacteria in this site. The number of bacteria resident in the intestine can be up to 1011 or 1012 cells/g of luminal contents. This concentration is similar to or even higher than those found in colonies growing under optimum conditions on a laboratory plate, indicating that the intestinal lumen represents an extremely efficient natural bioreactor for bacteria. These communities of bacteria are referred to as the intestinal ‘microbiota’ or ‘microbial flora’; under normal conditions, the gut microbiota is not pathogenic, but actually confers health benefits to the host. The microbiota aids in the digestion and absorption of nutrients, and stimulates fat storage. Furthermore, the microbiota contributes to the construction of the intestinal epithelial barrier and also competes with pathogenic microorganisms to prevent their harmful propagation [1,2]. The collective genome of intestinal microbes is referred to as the ‘microbiome’, which is estimated to contain at least 100-times as many genes as the human genome. Unlike the human genome, which is rarely manipulated by xenobiotic intervention, the microbiome is readily changeable by diet, ingestion of antibiotics, infection by pathogens and other life events. The plasticity of the microbiome has been implicated in numerous disease conditions, and an unfavorable alteration of the commensal structure of gut microbiota is referred to as ‘dysbiosis’; this includes a reduction in the number of tolerogenic bacteria and an outgrowth of potentially pathogenic bacteria (‘pathobionts’) that can penetrate the

∗ Corresponding author. Tel.: +81 3 5841 3373; fax: +81 3 5841 3450. E-mail address: [email protected] (K. Honda). 1044-5323/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2011.11.009

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numbers of IL-17-producing T (Th17) cells [16] and Foxp3expressing regulatory T (Treg) [17] cells. The accumulation and function of these mucosal leukocytes are regulated by the presence of intestinal microbiota. By regulating these immune cells, intestinal microbiota enhances the mucosal barrier function and allows the host to mount robust immune responses against invading pathogens, and simultaneously maintains immune homeostasis. Indeed, when properly guided by the microbiota, the mucosal immune system maintains a state of non-responsiveness to dietary antigens and harmless commensal microbes [18]. Recent studies using germ-free (GF) and gnotobiotic mice have revealed the role of several specific gut microbiota components in the differentiation and activation of particular immune cell populations. For example, monocolonization by segmented filamentous bacterium (SFB) induces Th17 cell responses [16,19]. The presence of SFB and consequent induction of Th17 cells influences the development of disease in several rodent autoimmune disease models [16]. The presence of SFB exacerbates disease symptoms in the K/BxN arthritis model [10] and in an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis [11], whereas SFB inhibits disease development in NOD mice [20], which is a spontaneous model of type-1 diabetes. A number of reports have also shown that Treg cells are regulated by intestinal microbiota, including Bacteroides species and Clostridium species [17,21,22]. Depending on the bacterial origin, SFB also induces Treg cell responses in the mouse small intestine (SI) [19]. This review will discuss recent advances in this specific research field, particularly focusing on microbiota-mediated induction of Treg cells and how Treg cells may contribute to maintaining homeostasis in the colonic mucosa. 2. Treg cells: a brief overview Multiple immune cells subsets, including CD4+ Foxp3+ Treg cells, tolerogenic dendritic cells (DCs), myeloid suppressor cells, IL10producing CD4+ T cells (Tr1 cells), and IL10-producing B cells (Breg cells), and their products have been implicated in the suppression of immune response-associated inflammation. Among these instigators, CD4+ Foxp3+ Treg cells play an outstanding and indispensable role in maintaining immunological unresponsiveness to self-antigens and in suppressing excessive immune responses. Indeed, mice lacking the Foxp3 gene develop fatal multi-organ inflammation that can be suppressed by the adoptive transfer of Foxp3+ Treg cells [23]. In humans, mutations in the FOXP3 gene locus are responsible for the IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome [24]. Foxp3 is critical to the initiation and maintenance of the genetic program for the differentiation and function of Treg cells. Retroviral gene expression of Foxp3 converts naïve CD4+ T cells into Treg cells that can suppress proliferation of CD4+ effector T cells in vitro and inhibit the development of autoimmune disease in vivo [25]. Foxp3expressing Treg cells regulate immune responses through multiple mechanisms, which include their expression of cytotoxic T cellassociated antigen-4 (CTLA-4) [26] and also depletion of local IL2 [27]. In addition, Treg cells are more mobile than naïve T cells and can out-compete the latter by aggregating around DCs, thereby inhibiting the differentiation and proliferation of effector T cells [28]. Treg cells migrate to inflamed tissues and selectively regulate specific T cell lineages. For example, under conditions of Th1 inflammation, Treg cells upregulate T-bet expression, which promotes the expression of CXCR3 and allows for the accumulation of Treg cells at Th1 cell-inflamed sites [29]. Similarly, the expression of interferon regulatory factor 4 (IRF4) in Treg cells is required for the expression of ICOS and CCR8, and subsequent suppression of Th2 responses [30]. Likewise, the expression of STAT3 in Treg cells is required

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for suppression of Th17 responses. STAT3 activation results in the expression of characteristic Th17 cell markers by Treg cells, including CCR6, which allows Treg cells to migrate to inflamed tissues in which a Th17 cell response is taking place [31]. Therefore, the inflammatory environment may induce the functional maturation of Treg cells. 3. Treg cells in the intestine Foxp3+ Treg cells are known to play an important role in intestinal immune homeostasis in mice. Foxp3+ Treg cells efficiently inhibit experimental colitis induced by the adoptive transfer of naïve CD4+ T cells into Rag2−/− or scid mice [32,33]. Treg cells also suppress innate immunity-mediated colitis triggered by Helicobacter hepaticus. Adoptive transfer of Treg cells before oral inoculation with H. hepaticus prevents the accumulation of innate immune cells and intestinal inflammation [34]. Furthermore, Foxp3-deficient mice exhibit intestinal inflammation, although less severe than that in the skin and liver [35]. In humans, some IPEX patients show intestinal inflammation, but the IPEX enteropathy mainly occurs in the SI, and the lesions in the colon are less frequent and less severe [36–38]. Given that individuals with FOXP3 mutations do not always develop colitis and that intestinal inflammation is not generally associated with a decrease in the number of Treg cells, compensatory mechanisms for Tregs may be operative in the human colon. In the intestine, one of the most important effectors produced by Treg cells is IL-10. More than half of all Foxp3+ Treg cells in the intestine express a high level of IL-10 [17]. IL-10 is an indispensable immunoregulatory cytokine, and it is particularly important for intestinal immune homeostasis. Recent reports have identified IL-10 as a susceptibility locus for the development of IBD in humans [39]. The injection of mice with recombinant IL10 can inhibit the development of colitis in the T-cell-transfer model [40]. Il10-deficient mice spontaneously develop severe colitis [41], particularly in the presence of H. hepaticus [42]. CD4+ T cell- or Treg-specific disruption of IL-10 in mice (CD4-Cre or Foxp3-Cre × Il10flox/flox ) also results in spontaneous colitis [43,44]. Therefore, IL-10 produced by Treg cells is indispensable for the maintenance of intestinal immune homeostasis. Definitely, other cells, including B cells, macrophages, and DCs, all have the potential to produce IL-10. Indeed, patients lacking functional IL10 receptor (IL10R) develop colitis with an earlier onset and higher penetrance compared with IPEX patients [45]. Therefore, it is highly likely that IL-10 secretion by both Treg cells and non-Treg cells play a role in the control of intestinal immune homeostasis. IL-10 receptor signaling in macrophages requires the transcription factor STAT3, and the targeted deletion of STAT3 in myeloid cells (LysM-Cre x Stat3flox/flox mice) leads to the development of spontaneous intestinal inflammation [46]. Therefore, IL-10 suppresses unnecessary inflammation created by myeloid cells through activation of STAT3. It is interesting to note that STAT3-deficiency in T cells (Lck-Cre × Stat3flox/flox mice) results in different phenotypes, including a defect in IL-6 signaling and a failure of CD4 T cells to differentiate into Th17 cells. Furthermore, IL-10 from Treg cell is necessary for suppressing ␥␦T cell proliferation. Deletion of phosphoinositide-dependent protein kinase 1 (Pdk1) in CD4+ T cells (Cd4-Cre × Pdk1flox/flox ) induces chronic inflammation in the intestine, where colonic CD8␣+ TCR␥␦+ IELs expressing IL-17 markedly expand in the absence of IL-10 from Treg cells [47]. It has also been shown that IL-10 production by Treg cells is required for the autocrine activation of Treg cells. IL10 binds to the IL-10R on Treg cells and activates STAT3, which induces Treg regulatory function. Mice in which IL-10R or STAT3 has been selectively deleted in Treg cells (Foxp3-Cre × Il10rflox/flox , or Foxp3-Cre × Stat3flox/flox ) develop spontaneous colitis [48].

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Deficiency of STAT3 or IL-10R in Treg cells interferes with their ability to suppress Th17 cell responses but does not affect regulation of Th1- or Th2-cell-mediated inflammation [48]. Furthermore, it has been reported that IL-10R is also expressed on Th17 cells. Foxp3+ Treg cells suppress colitis caused by the transfer of Th17 cells into lymphopenic hosts in an IL-10-dependent manner [49]. Several molecules that are important for IL-10 induction in Treg cells have been identified. TGF-␤ contributes to IL-10 production by Foxp3+ Treg cells in the gut [50]. Likewise, IL-27 has been suggested to induce IL-10 expression. IL-27 induces the gene expression of cMaf, IL-21, and ICOS, which coordinately act together to promote the production of IL-10 [51]. In addition, IL-27 upregulates the aryl hydrocarbon receptor (AhR), which acts in synergy with c-Maf to induce IL-10 production [52]. The transcription factors Blimp-1 and IRF4 are also required for IL-10 production by mucosal Treg cells. IL-2 and proinflammatory cytokines, such as IL-12, IL-6 and IL-4, induce Blimp1 expression, which then induces the expression of IL-10 in Treg cells and provides a negative-feedback regulation [53]. TGF-␤ is another effector produced by intestinal Treg cells. Injection of blocking anti-TGF-␤ monoclonal antibody abrogates the Treg cell-dependent prevention of wasting disease and colitis [54,55]. Mice lacking TGF-␤1 specifically in T cells spontaneously develop wasting colitis [56]. Treg cells lacking TGF-␤1 fail to inhibit colitis caused by adoptive transfer of naïve T cell into Rag1−/− mice [56]. Mice overexpressing a dominant-negative mutant of TGF-␤RII under the CD4 promoter show inhibited TGF-␤ signaling in T cells (CD4-DNRII Tg mice). The transfer of naïve CD4+ T cells isolated from CD4-DNRII Tg mice induce colitis are refractory to Treg cells from wild-type mice [57]. IL-35 also contributes to the suppressive function of Treg cells in the intestine. IL-35 is a heterodimer of Ebi3 and IL-12a, and is constitutively secreted by Foxp3+ Treg cells. Ebi3−/− and IL-12a−/− Treg cells fail to cure experimental colitis induced by the adoptive transfer of CD4+ CD25− CD45RBhigh effector T cells into Rag1−/− mice [58]. 4. nTreg and iTreg cells The thymus is the primary site for the generation of Treg cells. Thymus-derived Treg cells are called naturally occurring Treg (nTreg) cells. Neonatally thymectomized mice lack Treg cells and show severe inflammation and fatal autoimmune lesions similar to those observed in Foxp3-deficient mice [59]. The expression of Foxp3 is detectable among immature CD4+ CD8+ double positive cells, but mainly among CD4 single positive cells [60]. In the MHC class-I- and -II-deficient mice, very few Foxp3+ thymocytes are detected [60]. Therefore, positive selection is required for the development of nTreg cells. Thymic development of Treg cells requires strong TCR signaling. In mice harboring the mutated gene encoding linker of activated T cell (LAT), which is a transmembrane adaptor protein involved in TCR signal transduction, the generation of Foxp3+ Treg cells in the thymus is abolished, whereas the development of other T cells is normal [61]. IL-2 is a critical regulator for the development of nTreg cells. The number of Treg cells is markedly reduced in mice lacking IL-2, IL-2R␣ (CD25) or IL2R␤ (CD122), and these mice spontaneously develop autoimmune diseases [62–64]. Neutralization of IL-2 by anti-IL-2 monoclonal antibody during the neonatal period of development substantially reduces the number of Treg cells and elicits autoimmune diseases [65]. T cell-specific deletion of STAT5, which mediates signaling through IL-2R, prevents Treg cell development, resulting in autoimmune and inflammatory disease [66]. STAT5 binds to an enhancer region of the Foxp3 gene, called conserved non-coding DNA sequence 2 (CNS2), and thereby induces Treg cell differentiation [66]. Activation of the NF-␬B pathway, which is down stream of TCR signaling, is also important for Treg cells development. Conditional knockout mice of several molecules that link the TCR to

NF-␬B, such as PKC-␪, CARMA1, IKK2, and TAK1, have defective nTreg cell development [67–73]. Among the components of NF␬B, c-Rel binds to the promoter region of the Foxp3 gene, which is called CNS3 [74]. The promoter binding by c-Rel after TCR-induced activation is likely to facilitate opening of the Foxp3 locus [74]. Aside from nTreg cells that develop in the thymus, there is mounting evidence which shows that Foxp3+ Treg cells can develop extra-thymically under certain conditions. Treg cells induced in the periphery are referred to as induced Treg (iTreg) cells. The peripheral conversion of Foxp3− CD4+ T cells to Foxp3+ Treg cells can be observed experimentally in the intestinal LP and gut-associated lymphoid tissues (GALT) after oral exposure to antigen [75–77]. In the presence of TGF-␤, Foxp3 expression is induced in TCRstimulated naïve CD4+ T cells, which confers Treg function in vitro [78]. TGF-␤R signaling leads to Smad recruitment to an enhancer region of the Foxp3 gene, which is called CNS1 [79]. The ablation of the CNS1 sequence in mice impairs iTreg induction, particularly in the GALT, whereas thymic nTreg differentiation remains normal [74]. Moreover, TGF-␤ restrains recruitment of a silencer for the Foxp3 gene, DNA methyltransferase 1 (Dnmt1), thereby promoting iTreg cell differentiation [80]. Although TGF-␤ alone promotes iTreg differentiation, a combination of IL-6 and TGF-␤ facilitates the differentiation of Th17 cells. IL-6 stimulation of naïve CD4+ T cells results in increased expression of the transcription factor ROR␥t, which effectively shuts down TGF-␤-induced Foxp3 expression [81,82]. In contrast, IL-2 promotes the generation of iTreg cells but inhibits the differentiation of Th17 cells from naïve T cells [83]. In addition, retinoic acid, which is produced by DCs expressing retinal dehydrogenase (RALDH) in the intestinal LP and GALT [84], can inhibit Th17 induction and promote iTreg induction [75]. Indeed, the conversion of Foxp3− CD4+ T cells to Foxp3+ iTreg cells preferentially occurs in the intestinal LP and GALT, perhaps because the gut is a TGF-␤- and retinoic-acid-rich environment [75–77]. The gut is also rich in IL-6 and TGF-␤, which preferentially induce Th17 cells. Although iTreg cells suppress immune responses through cytokine production and contact-dependent mechanisms, and express cell surface markers which are similar to those expressed by nTreg cells [85], it has been proposed that iTreg cells are different from nTreg cells in certain regards. For example, the evolutionarily conserved region within the Foxp3 locus upstream of exon1 is widely demethylated in nTreg cells, but not in TGF-␤-induced iTreg cells [86]. The TCR repertoires of iTreg cells are similar to those of effector or naïve T cells, but are totally different from those of nTreg cells [85]. Helios is a member of the ikaros transcription factor family that is expressed in Treg cells in the thymus, but not in Foxp3+ Treg cells induced in vitro by TGF-␤, suggesting that Helios-negative Foxp3+ cells may be iTreg cells [87]. As discussed later, Heliosnegative Foxp3+ Treg cells are highly abundant in the intestinal LP [17,88,89]. Several intestinal LP DC subsets have been implicated in the differentiation and activity of iTreg cells. In particular, a CD103 (also known as ␣E-integrin)-expressing DC population in the intestinal LP preferentially promotes the generation and homing of iTreg cells to the intestinal mucosa [76,77]. CD103+ DCs express retinal dehydrogenase (RALDH) and produce retinoic acid. Retinoic acid mediates the expression of gut-homing receptors on T cells [84,90] and enhances iTreg differentiation by co-operating with TGF-␤ [75]. In addition to CD103+ DCs, CD11bhigh CD11c− LP macrophages have been shown to preferentially promote iTreg cell development. These LP macrophages express high levels of IL-10 and TGF-␤, and preferentially induce the differentiation of naïve CD4+ T cells into Treg cells [91]. It has also been shown that the constitutive activation of ␤-catenin in DCs and macrophages in the intestinal LP is required to maintain immune homeostasis and tolerance. Intestinal DCs from mice selectively lacking ␤-catenin expression in DCs

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(␤-catDC−/− mice) express low levels of RALDH genes, IL-10 and TGF-␤, but express high levels of IL-23 and IL-6 [92]. ␤-catDC−/− mice show a decrease in the number of Treg cells and an increase in the number of Th1 and Th17 cells in the intestine, indicating that ␤-catenin expression by intestinal DCs is important for maintaining the balance between regulatory and effector T cell populations in the gut [92]. Collectively, the preferential induction of iTreg cells at mucosal sites is at least in part attributed to the gut-specific presence of CD103+ DCs, CD11bhigh CD11c− macrophages, and DCs with active ␤-catenin.

5. Induction of Treg cells by the microbiota Foxp3+ Treg cells distribute to essentially all organs, and the frequencies of Foxp3+ cells are approximately 10% within the CD4+ cell subset. In contrast, the frequency of Foxp3+ Treg cells in the gut LP is notably higher (>30%) than in other organs [17]. It is highly likely that the intestinal microbiota plays a critical role in the accumulation and functional maturation of intestinal Treg cells. Indeed, we have shown that in the colonic LP, Treg cell numbers decreased in GF mice and antibiotic-treated mice [17]. In the LP of the SI, however, the proportion and absolute number of Foxp3+ Treg cells were unchanged or increased in GF mice compared with specificpathogen-free (SPF) mice, whereas the proportion of Th17 cells was greatly reduced [17,93,94]. Therefore, the number of colonic Treg cells is likely to be affected by the presence of intestinal microbiota, whereas different mechanisms are active in the induction of SI Treg cells. It should be noted that analyses of the number of colonic Treg cells in various studies have provided discordant results, with some studies, including those from our lab, showing a decrease in GF mice [17,95–97] and other studies showing normal numbers of Treg cells in GF mice [22,88,89,98] relative to SPF mice. These different results might stem from the methods that were used to purify lymphocytes from the gut LP. The discrepancies could also be attributed to differences in the components of the intestinal microbiota in mice among various SPF animal facilities. Furthermore, there might be differences in the stringency of cleanliness among GF facilities. Notably, in SPF mice, colonic LP Treg cells contain a high number of Helios-negative cells (Helios-negative and -positive cells are at an about 1:1 ratio among colonic Treg cells) [17,88]. As described above, Helios is a putative marker for thymically derived nTreg cells [87]. Thus, Helios− Treg cells in the colonic LP may be iTreg cells. Importantly, many reports have consistently shown that the percentage and number of Helios-negative Tregs was markedly reduced in GF mice [17,88,89]. Therefore, it is likely that the presence of microbiota does affect this population of Treg cells, particularly by promoting the peripheral conversion of naïve CD4+ T cells to iTreg cells. Alternatively, loss of Helios expression could represent a further activation of nTreg cells by the microbiota. More detailed studies are required to fully characterize Helios-negative Treg cells in the intestine. Recent analyses using high-throughput sequencing have revealed that the amino-acid sequences of TCRs on Treg cells from the mouse colon were different from those of receptors on T cells from other organs [89]. Many of these TCRs on colonic Treg cells recognize antigens derived from the intestinal microbiota, such as Clostridiales species and species within the Parabacteroides/Bacteroides genuses [89]. Furthermore, if the TCRs, which are normally expressed on Treg cells in the colon, were instead present on effector T cells, such T cells induced markedly severe colitis when transferred into Rag1−/− mice [89]. Therefore, if Foxp3 expression does not adequately occur in T cells expressing commensal-specific TCRs, a severe inflammatory response against commensal microbes could be induced by these T cells.

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It has been postulated that certain components of the microbiota specifically affect the accumulation and activity of Treg cells in the intestine. Species of lactobacilli and bifidobacteria have been implicated in the induction of Treg cells. Treatment of mice with the probiotic mixture VSL#3 (a mixture of bifidobacteria, lactobacilli, and Streptococcus salivarius) or the probiotic strain Lactobacillus reuteri increased the intestinal concentration of IL-10, likely due to Treg activation [99–101]. Furthermore, daily ingestion of either Lactobacillus acidophilus [102], Lactobacillus rhamnosus [103], Lactobacillus reuteri [104], or Bifidobacterium infantis [105] resulted in the modification of inflammatory status or autoimmune responses in mice, and these effects might be mediated in part by the induction of Treg cells. However, these probiotic strains are not necessarily part of the indigenous microbiota. Indeed, some Lactobacillus species are only transiently present (allochthonous) and do not usually exist in the intestines of certain mice or humans [106]. Furthermore, the effects of colonization of these probiotic strains, including the possible induction of Treg cells, could be a secondary effect, because these bacterial preparations may affect the microbial ecology within the gut and thus may not directly induce Treg cells. A human commensal Bacteroids fragilis species was shown to facilitate the functional maturation of Treg cells in mice. Colonization with B. fragilis boosted IL-10 production in Treg cells from the colon, although the effect on Foxp3+ Treg proportions was marginal [107]. Furthermore, the colonization with B. fragilis prevented intestinal inflammation caused by H. hepaticus or trinitrobenzene sulfonic acid (TNBS) [107]. It was shown that the induction of IL-10 expression from Treg cells by B. fragilis was mediated by polysaccharide A (PSA), a unique surface polysaccharide of B. fragilis [22]. Injection of or feeding mice with PSA was sufficient to replicate the immune effects of B. fragilis. PSA was shown to directly bind to TLR2 on CD4+ T cells and induce IL-10 production [21] (Fig. 1). Importantly, PSA suppressed Th17 cell responses and facilitated the intimate colonization of B. fragilis on the intestinal epithelium. Indeed, animals colonized with mutant B. fragilis lacking PSA displayed increases in the number of Th17 cells in the colon and profoundly reduced numbers of tissue-associated bacteria when compared to animals colonized with wild-type B. fragilis [21]. It was also shown that PSA from B. fragilis induced a Th1 response and corrected the Th2 cell skew of germ-free mice [108]. Although the mechanisms by which PSA carries out such versatile functions are still poorly understood, PSA may be a critical factor for colonization with B. fragilis that permits beneficial host–bacterial mutualism.

6. Induction of Treg cells by Clostridium species Because B. fragilis are human commensals, their effects observed in mice may or may not reflect evolutionarily selected commensal functions. Among the microbiota indigenous to the murine colon, the spore-forming components, particularly those belonging to the genus Clostridium clusters IV and XIVa (also known as Clostridium leptum and coccoides groups, respectively), are outstanding inducers of colonic Treg cells. The colonization of GF mice with a defined mixture of 46 strains of Clostridium, which were originally isolated from chloroform-treated fecal material (sporulated fraction) from conventionally reared mice [109], sufficiently induced Treg cells [17]. These Clostridium spp. preferentially colonize and form a thick layer on the cecum and colon. In the epithelial cells from Clostridium colonized mice, the increased expression of matrix metalloproteinases (MMPs) and production of the active form of TGF-␤ was observed [17]. MMPs are known to convert TGF-␤ from the latent to the active form [110]. In addition, the colonization of Clostridium induced the expression of indoleamine 2,3-dioxygenase (IDO) in

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Fig. 1. The complexity of the Treg regulation by intestinal bacteria. Colonization with SFB, Bacteroids fragilis, Clostridium spp. or altered Schaedler flora (ASF), in mice has been shown to increase the number of intestinal Foxp3+ Treg cells (particularly cells negative for Helios) and/or enhance the expression of IL-10 and CTLA-4 in Treg cells. The molecular and cellular mechanisms underlying the influence of intestinal bacteria on Treg cells are largely unknown. See text for details.

epithelial cells [17]. The metabolites of IDO have been implicated in the induction of iTreg cells. Similar to mice reared in SPF condition, Treg cells in the colon of mice colonized with Clostridium spp. consisted of a large number of Helios− cells [17]. Therefore, it is possible that Clostridium spp. induce the iTreg cell conversion in the colon by producing the active form of TGF-␤ and metabolic products of IDO. Interestingly, a large subset of Treg cells induced by the colonization of Clostridium spp. expressed high levels of IL-10 and CTLA-4 [17], which are required for Treg-induced suppression of immune responses. Therefore, Clostridium spp. may influence the qualitative properties of colonic Treg cells, as well as their quantity (Fig. 1). Oral inoculation of Clostridium spp. early in life of conventionally reared mice resulted in an increased frequency of Clostridium spp. in their intestinal microbiota and also of Treg cells in the CD4+ cell subset. Such mice were more resistant to experimental models of colitis and systemic immunoglobulin E responses in the adult stage [17]. Perhaps consistent with the importance of Clostridium spp., the colonization of mice with the altered Schaedler flora, which is a precisely defined mixture of commensal bacterial strains including Clostridium clostridioforme, induced Helios− Treg cell accumulation in the colonic LP [88] (Fig. 1). It was also shown that a reduction of Faecalibacterium prausnitzii, which belongs to the Clostridium cluster IV, was associated with a higher risk of postoperative recurrence of Crohn’s disease [111]. Supernatants from F. prausnitzii cultures increased the production of IL-10 by peripheral blood mononuclear cells in vitro [112]. In addition, SFB are functionally related to members of the genus Clostridium and were shown to simultaneously stimulate pro-inflammatory and Treg cell responses that balanced each others in the mouse intestine [19] (Fig. 1). It was shown that Clostridium clusters IV and XIVa formed a smaller proportion of the fecal community in patients with IBD than in healthy controls [113]. Although it still remains unknown whether defects in Treg cells contribute to human IBD, these reports raise the possibility that Clostridium- and its related species-dependent constitutive induction of Treg cells may contribute to the suppression of autoimmunity and deleterious inflammation.

7. Context-dependent effects by the microbiota The findings described above support the notion that specific components of commensal microbiota, Bacteroides and Clostridium in particular, preferentially induce Treg cells, which then contribute to the maintenance of immune homeostasis in the gut. However, accumulating evidence suggests that even beneficial members of the microbiota can have detrimental effects on the host, depending on the context. For instance, animals colonized with a mutant B. fragilis lacking PSA display increased numbers of Th17 cells, rather than IL-10-producing Treg cells, in the colon [21]. A wellcharacterized symbiotic species, Bacteroides thetaiotaomicron, can induce colitis in mice with T cells that have a deficiency in IL-10R2 and that express a dominant-negative TGF-␤R2 [114]. The colonization of GF mice with altered Schaedler flora, which typically only activates the immunosuppressive axis including T cell IL-10 expression and Helios− Treg accumulation, may induce Th1 and Th17 cell responses and inflammation in the colon when the mice are simultaneously treated with a blocking antibody to IL-10R [88], a situation reminiscent of that observed in early onset colitis in a subset of children lacking a functional IL-10R [45]. Therefore, the intestinal commensal bacteria are not always benign towards their host. The cells and factors which are typically immunoregulatory in the gut can also become inflammatory, depending on the local conditions. Retinoic acid is a well-known mediator of iTreg differentiation; however, in the presence of IL-15, retinoic acid activates LP DCs to release IL-12p70 and IL-23. As a result, under these conditions, retinoic acid acts as an adjuvant that promotes Th1 and Th17 responses rather than Treg induction [115]. IL-15 is a cytokine greatly upregulated during inflammatory disease conditions, such as celiac disease [116,117]. This functional conversion of retinoic acid is highly reminiscent of the variable effects of TGF-␤, which is otherwise a critical inducer of iTreg cells differentiation, but can lead to the generation of Th17 cells in the presence of IL-6 [118]. Therefore, the functional outcomes of regulatory factors and cells may be context-dependent.

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Importantly, the microbiota of individuals with chronic inflammation exhibit lower bacterial diversity as compared to healthy individuals. The MetaHIT consortium reported that IBD patients harbored, on average, 25% fewer bacterial genes compared to individuals not suffering from IBD [119]. Although it still remains unclear whether the decrease in microbial diversity is a causative factor or a consequence of chronic inflammation in IBD patients, it is likely that maintaining the diversity of the gut microbial community is a prerequisite for immune homeostasis. Furthermore, there may be a positive feedback mechanism in which intestinal inflammation caused by infection or genetic predisposition can render the nature of the microbiome more prone to induce inflammation. Further detailed investigation will be required to elucidate the conditions and environments that determine whether the microbial community residing in the individual gut as a whole contributes to the induction of Treg cells or to the promotion of inflammation. 8. Concluding remarks We have discussed the role of Treg cells in the intestine, and the effects of intestinal microbiota on the differentiation and function of these cells. Specific components of the gut microbiota, such as B. fragilis and Clostridium spp., affect the numbers and activities of Treg cells; however, it is still unclear whether these commensalinduced Treg cells contribute to the organism’s overall tolerance for commensal bacteria. The community membership of the microbiota certainly affects the immune status of the host. Several diseases, including IBD, have been associated with an imbalanced microbial population and/or with host genotype. However, the reason why a specific community structure of the microbiota has a deleterious effect on the host remains to be resolved. Further dissection of the individual members of the microbiota by examination of monocolonized and gene-manipulated mice, as well as more extensive systems approaches using metagenomic and metabolomic analyses, will unravel key molecular and cellular pathways that govern host and microbiota interactions. Acknowledgements The work was supported by the Japan Science and Technology Agency for Precursory Research for Embryonic Science and Technology (PRESTO) and the Japan Society for the Promotion of Science NEXT program. References [1] Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host–bacterial mutualism in the human intestine. Science 2005;307:1915–20. [2] Garrett WS, Gordon JI, Glimcher LH. Homeostasis and inflammation in the intestine. Cell 2010;140:859–70. [3] Chow J, Mazmanian SK. A pathobiont of the microbiota balances host colonization and intestinal inflammation. Cell Host Microbe 2010;7:265–76. [4] Tamboli CP, Neut C, Desreumaux P, Colombel JF. Dysbiosis in inflammatory bowel disease. Gut 2004;53:1–4. [5] Hill DA, Artis D. Intestinal bacteria and the regulation of immune cell homeostasis. Annu Rev Immunol 2010;28:623–67. [6] Lee YK, Mazmanian SK. Has the microbiota played a critical role in the evolution of the adaptive immune system? Science 2010;330:1768–73. [7] Garrett WS, Lord GM, Punit S, Lugo-Villarino G, Mazmanian SK, Ito S, et al. Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system. Cell 2007;131:33–45. [8] Elinav E, Strowig T, Kau AL, Henao-Mejia J, Thaiss CA, Booth CJ, et al. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell 2011;145:745–57. [9] Bach JF. The effect of infections on susceptibility to autoimmune and allergic diseases. N Engl J Med 2002;347:911–20. [10] Wu HJ, Ivanov II, Darce J, Hattori K, Shima T, Umesaki Y, et al. Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity 2010;32:815–27. [11] Lee YK, Menezes JS, Umesaki Y, Mazmanian SK. Proinflammatory Tcell responses to gut microbiota promote experimental autoimmune encephalomyelitis. Proc Natl Acad Sci U S A 2011;108(Suppl. 1):4615–22.

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