Role of the Gut Microbiota in the Development and Function of Lymphoid Cells

The gut microbiota

Vertebrates are colonized by a large number of commensal microorganisms including bacteria, fungi and viruses that are referred collectively as the microbiota. In humans, more than 100 trillion bacteria colonize the skin and mucosal surfaces including the oral cavity and the intestine. By far, the majority of indigenous bacteria reside in the distal intestine. Millions of years of co-evolution between the host and microbes have led to a symbiotic relationship in which the microbiota contribute to many host physiological processes and the host, in turn, provides hospitable and nutritious niches for microbial survival (1). In the intestine, the microbiota contributes to the development of specific organ structures, induction of immunity, and metabolism of nutrients, such as carbohydrates and certain vitamins. On the other hand, the gut microbiota can also cause disease or increase the risk for disease development in genetically-susceptible individuals (2). In this review, we provide an overview of the current understanding of the role of the microbiota in the development of gut-specific immunity with an emphasis in the regulation of lymphoid function.

Microbiota-dependent development of intestinal steady-state Th17 cells

T helper 17 (Th17) cells are a selective lineage of CD4⁺ T-helper cells that are critical for host defense and play a role in the development of autoimmune disease by producing the pro-inflammatory cytokines IL-17A, IL-17F, and IL-22 (3). Recent studies have revealed that the presence of intestinal Th17 cells is dramatically reduced in antibiotic-treated or germ-free animals (4, 5). In addition, a specific species of Clostridia-related bacteria, called segmented filamentous bacteria (SFB), has been found to promote the generation of Th17 cells (4, 6, 7). Mounting evidence indicates that lamina propria (LP) mononuclear phagocytes including dendritic cells (DCs) and macrophages sense the gut microbiota and promote Th17 cell development in the intestine (Fig. 1). Colonic CD70^highCD11c⁺ LP DCs express purinergic P2X and P2Y receptors and promote Th17 development in the presence of commensal microbes-generated ATP (5). This subset of DCs express Th17-drivers such as IL-6 and the integrin molecules, integrin-αV and β8, which contribute to the activation of the latent form of TGF-β in response to the ATP (5). However, SFB mono-colonization does not increase luminal ATP levels following activation of the CD70^highCD11c⁺ LP DC subset (7). SFB adhesion to the epithelium induces SAA production, which promotes IL-6 and IL-23 production by CD11c⁺ LP DCs in the ileum (7). Thus, SFB and other commensals appear to promote Th17 cell development through different mechanisms (Fig. 1). Another key molecule that is induced by the gut microbiota is the cytokine IL-1β (8). Impaired IL-1β signaling leads to blunted development of Th17 cells, but not of Th1 cells in the small intestine (8). The gut microbiota constitutively primes CD11b⁺CD11c⁻ LP macrophages to induce pro-IL-1β, though the specific commensals that regulate pro-IL-1β in LP phagocytes and the mechanism involved remain to be identified (8, 9). Because resident macrophages and DCs are hyporesponsive to microbial stimulation (9), it is possible that commensal microbes induce pro-IL-1β in LP macrophages/DCs by an indirect mechanism which may involve epithelial or stromal cells. Since MyD88-deficient mice exhibit reduced pro-IL-1β expression in CD11b⁺CD11c⁻ LP macrophages and reduced Th17 cell development (8), it is likely that commensals induce pro-IL-1β via stimulation of Toll-like receptors or members of the IL-1/IL-18 receptor family which are upstream of MyD88. Another aspect that remains unclear is the signal that leads to the processing of pro-IL-1β into the mature IL-1β. One possibility is that ATP and SAA, which are known to activate the NLRP3 inflammasome, may regulate the processing of pro-IL1β into mature IL-1β via Caspase-1 (10). However, there is no conclusive evidence as yet that Caspase-1 or the inflammasome regulates the development of intestinal Th17 cells.

Although the microbiota promote the development of steady-state Th17 cells in the gut, it is unclear whether resident Th17 cells exhibit specificity for microbial antigens. So far, the body of evidence does not support an antigen-specific role for steady-state Th17 cells in the intestine. For example, development of the gut Th17 cells is normally observed in TCR transgenic mice in the absence of cognate antigen (11). Furthermore, intraperitoneal administration of recombinant IL-1β injection into GF mice promotes the development of intestinal Th17 development in the absence of commensal microbes (8). These results suggest that the induction of Th17 cells in response to commensal stimulation is not likely due to the expansion of commensal-specific Th17 cells. Instead, microbiota-driven Th17 cells may merely reflect bystander expansion of multiple repertoires of Th17 cells or alternatively of populations of Th17 cells with skewed TCRs that home to the intestine. Because SFB-induced Th17 cells contribute to the pathogenesis of autoimmune disease outside of the gut, such as arthritis in K/BxN mice and experimental autoimmune encephalomyelitis (EAE) (12, 13), steady-state Th17 cells appear capable of recognizing self-antigens or promoting the pathogenic activity of autoreactive T cells.

Homeostatic versus pathogenic Th17 cells

Although Th17 cells have been shown to promote pathology in inflammatory disease models (12, 13), they are numerous under steady-state conditions and presumably exert a beneficial role in the host. Indeed, Th17 cells are protective against infection with Citrobacter rodentium, a mouse enteric pathogen that models infection with pathogenic Escherichia coli in humans. Mice reared under specific-pathogen free (SPF) conditions, but lacking SFB exhibit higher susceptibility to C. rodentium than SPF mice colonized with SFB (7). However, the interpretation of these experiments is difficult because the differences could be explained by commensals other than SFB or unrelated host factors. Monocolonization of GF mice with SFB induces Th17 cells in the gut, but does not promote Salmonella clearance (14). Thus, the role of steady-state Th17 cells in host defense in the gut remains unclear. In addition, intestinal Th17 cells can promote the pathogenesis of autoimmune diseases under certain conditions (12, 13). However, the involvement of steady-state Th17 cells in intestinal inflammation is controversial. In humans, the production of Th17-related cytokines is elevated in the intestinal mucosa of patients with inflammatory bowel disease (IBD) (15). However, mono-colonization of mice with SFB did not promote intestinal inflammation in the adaptive T-cell transfer colitis model (16). Notably, co-colonization of mice with SFB and other commensals induced severe colitis in the T-cell transfer model, while inflammation was mild or moderate in mice colonized with commensals other than SFB (16). These results suggest that SFB-induced Th17 cells are not harmful in mice, but they can promote inflammation or be converted into pathogenic T cells under inflammatory conditions or in the presence of other commensals (Fig. 1).

What is the difference between steady-state and pathogenic Th17 cells? There is evidence for plasticity of Th17 cells, which may ultimately influence disease susceptibility. For example, the ability of Th17 cells to produce the anti-inflammatory cytokine IL-10 (17), which is important for suppression of autoimmune diseases such as EAE (17), is context-dependent. The production of IL-10 by Th17 cells is induced when cells are differentiated and maintained with TGF-β1 and IL-6, whereas IL-10 is suppressed when the cells are stimulated with IL-23 (17). Importantly, Th17 cells stimulated by IL-23 and lacking expression of IL-10 are capable of inducing autoimmune disease in mice (17). In addition, TGF-β3-induced Th17 cells are functionally different from TGF-β1-induced Th17 cells (18). Like IL-23-induced Th17 cells, TGF-β3-induced Th17 cells are pathogenic and promote EAE or colitis whereas TGF-β1-induced Th17 cells are not (18). Therefore, the capacity to produce IL-10 may explain the functional difference between steady-state homeostatic Th17 and pathogenic Th17 cells. Another example that reflects the plasticity of Th17 cells is that Th17 cells developed in vitro show a typical Th17 phenotype and produce IL-17 but not IFN-γ; however, when stimulated with IL-12 or IL-23, Th17 cells produce IFN-γ (19). This conversion of Th17 into IFN-γ-producing Th1-like cells is also observed in vivo (20, 21). In the course of colitis, Th17 cells can convert into IL-17/IFN-γ-double positive cells and IFN-γ-producing Th1-like cells (20, 21). In humans, IL-1β regulates the conversion of IL-17⁺IL-10⁺ Th17 cells into pathogenic IL-17⁺IFN-γ⁺ Th17 cells (22). Thus, although commensal-induced steady-state Th17 cells are not harmful and may be homeostatic, the intestinal inflammatory microenvironment, such as that found in the presence of IL-12, IL-23, IL-1β or TGF-β3 promotes conversion of the resident Th17 cells to IFN-γ-producing pathogenic Th17 cells, and may contribute to the progression of intestinal inflammation (Fig. 1).

Microbiota-dependent induction of Treg cells in the gut

Foxp3⁺ regulatory T cells (Tregs) are key suppressive cell types that regulate autoimmune inflammation in the body (23). In the gut, Tregs accumulate under steady-state conditions where they play an important role in the regulation of inflammation against microbial stimuli. Indeed, adoptive transfer of CD4⁺ T cells in the absence of Tregs, but not in their presence, elicits commensal-driven colitis (24). Furthermore, depletion of Tregs induces spontaneous colitis which is abrogated when the mice are reared under GF conditions (25). Thus, Tregs are critical for the prevention of spontaneous inflammation against commensal microbes (Fig. 1). In antibiotic-treated mice or GF mice, Tregs remain detectable, but their numbers are significantly decreased in the intestinal LP, suggesting that the microbiota promotes the differentiation and/or maintenance of Tregs (26, 27). Colonization of GF mice with 46 strains of Clostridium (26), or with a cocktail of 8 defined commensals, called altered Schaedler flora (ASF) is sufficient to induce Tregs in the gut (26, 27). Furthermore, a human commensal bacterium Bacteroides fragilis can also induce Tregs in the mouse intestine (28). In the absence of the microbiota, the number of Foxp3⁺ Tregs is decreased in the colon, but not in the small intestine and lymph nodes (26, 29). Thus, Treg development is largely dependent on the microbiota in the colon, but not in the small intestine. A key mechanism by which commensal microbiota induce Treg differentiation involves TGF-β (Fig. 1). Colonization of specific commensal microbes to the intestinal epithelium may induce activation of the latent form of TGF-β in epithelial cells (2). In addition to epithelial cell-derived TGF-β, certain subsets of LP mononuclear phagocytes are involved in the induction of Treg differentiation in the gut. For example, CD103-expressing DC subsets (including CD103⁺CD11b⁺CD11c⁺ LP DCs and CD103⁺CD11b⁻CD11c⁺ LP DCs) preferentially induce Treg differentiation from naïve CD4⁺ T cells (30, 31). CD103⁺ LP DCs robustly express molecules which drive Treg differentiation, such as TGF-β and retinoic acid (RA) dehydrogenase (RALDH), a key enzyme in the synthesis of retinoic acid (32). RA acts as a potent inducer of Tregs in the presence of TGF-β (32). In addition to DC subsets, CD11b⁺CD11c⁻ LP macrophages also express RALDH and are able to induce the differentiation of intestinal Tregs cells (33). In addition to Treg differentiation, RA acts on T cells to imprint gut-homing properties (34). Although the exact role of the microbiota in the generation of gut-specific DCs remains poorly understood, it is possible that commensals regulate the expression of RALDH in DCs to promote their ability to instruct gut-specific functions in T cells.

The microbiota play a role not only in development of Treg cells, but also in the functional regulation of Treg cells. It is known that the regulatory function of Tregs is mediated, in part, by the production of IL-10. A key role for IL-10 in the regulation of intestinal homeostasis is supported by the observation that IL-10 deficiency can lead to the development of colitis which is triggered by the presence of commensals or enteric pathogens (35). Various cell types in the gut produce IL-10 including Foxp3⁺ Tregs (36), Foxp3⁻ Tr1 cells (36, 37), CD11b⁺CD11c⁻ LP macrophages (33), and regulatory B cells (38). The fact that conditional deletion of IL-10 in Foxp3⁺ Tregs triggers spontaneous colitis in mice (39), suggests that Treg-intrinsic production of IL-10 is critical for the prevention of inflammatory responses to commensals or pathogens present in the intestine. Although the mechanism by which commensals control IL-10 production by Treg cells is largely unknown, certain microbes facilitate the production of IL-10 by Treg cells through direct or indirect mechanisms. As we described earlier, a human commensal bacterium B. fragilis induces Foxp3⁺ Tregs cells in mice (28). A key microbial molecule that is involved in the induction of Tregs by B. fragilis is polysaccharide A (PSA). B. fragilis PSA directly act on Tregs to promote robust production of IL-10 by T cells that is TLR2-dependent (28). Thus, certain commensals have the ability to directly activate Tregs. Unraveling the mechanism by which commensals induce the production of IL-10 by Treg cells may provide further mechanistic insight into the link between the microbiota and Treg function in the gut.

The impact of the microbiota on intestinal B cell immunity

The production of immunoglobulin (Ig) is an adaptive immune response which is critical for the clearance of infectious microorganisms, vaccination and development of autoimmune disease. IgA plays an important role in shaping the composition and function of the gut microbiota (40). Consistently, mice deficient in activation-induced cytidine deaminase (AID) that regulates Ig class switching recombination (CSR) and therefore lack IgA contain an altered microbiota (41). Not only the quantity, but also the quality of IgA, is important in the regulation of the gut microbiota. For example, G23S knock-in mutation in the AID gene that produce IgA with lower affinity for commensal microbes exhibit alteration in the composition of the gut microbiota (42). Mono-colonization of GF mice with the commensal Bacteroides thetaiotaomicron does not induce inflammatory responses in wild-type mice, but triggers innate immune responses in Rag-2 deficient mice (43). Notably, implantation of IgA-producing hybridoma cells reactive with capsular polysaccharide of B. thetaiotaomicron into Rag-2 deficient mice affects gene expression in the bacterium and inhibits the innate immune response in the host (43). Thus, commensals and IgA can regulate each other and this interaction promotes peaceful mutualism between host and the gut microbiota.

In the intestine, T cells in the germinal center of gut-specific secondary lymphoid organs, such as peyer’s patches (PPs), crypt patches (CPs), or isolated lymphoid follicles (ILFs), induce the generation of IgA⁺ B cells via TCR and CD40L-dependent interactions (44). DCs in these lymphoid organs are required for the activation of follicular T cells to promote T cell-dependent and T cell-independent B cell activation (44). During T cell-dependent or independent generation of IgA⁺ B cells, TGF-β and RA signaling are thought to be important for the induction of CSR in B cells (45). It is known that LP DCs are capable of generating RA and activating the latent form of TGF-β through matrix metalloproteinases (MMPs) or integrin αvβ8 (46, 47). In addition, certain subsets of DCs in LP or lymphoid follicles express various factors such as TNF-α, inducible nitric oxide synthetase (iNOS), B cell activating factor (BAFF), and proliferation-inducing ligand (APRIL) that promote the generation of IgA⁺ B cells (48). Various mechanisms have been proposed to explain the induction of IgA⁺ B cells by the intestinal microbiota. One mechanism involves a role for commensals in the formation of gut-specific lymphoid structures, which are essential for the generation of IgA⁺ B cells. Consistently, the generation and/or maturation of gut-specific lymphoid organs, such as PPs and ILFs, are significantly decreased in GF mice or antibiotic-treated mice, (49, 50). Furthermore, bacteria derived molecules including TLR ligands and NOD1 agonist have been shown to be involved in the development of gut-specific lymphoid structures (50). Although experiments in gnotobiotic mice suggest that SFB induces IgA⁺ B cell development (51), the commensal species that stimulate the formation of lymphoid structures in the gut under steady-state conditions remain to be identified. It has become evident that bacteria recognition through MyD88 within follicular DCs is important for the generation of IgA⁺ B cells (52); however the molecular link between the gut microbiota and activation of LP DCs remains poorly understood. Because LP DCs play a critical role in the development of IgA⁺ B cells (48), it is possible that the gut microbiota induces the expression of molecules such as RA, BAFF, APRIL, in LP DCs to trigger the development of IgA⁺ B cells.

Microbiota-dependent regulation of innate lymphocyte development and function

T and B lymphocytes play a major role in antigen-specific adaptive immune responses; however, some subsets of lymphocytes act as ‘innate’ lymphocytes. Intraepithelial lymphocytes (IELs), including TCRγδT cells and TCRαβCD8αα T cells, are considered to be part of the innate lymphocyte family, although some of these cells also play a role in antigen-specific adaptive immunity (53). Although IELs express a skewed TCR repertoire and are activated through pattern recognition receptors (53), the antigen-specificity of these IELs is not fully understood. Given their close proximity to the intestinal lumen, IELs are thought to play a role in the regulation of immune responses against enteric pathogens and the commensal microbiota. However, a role for the gut microbiota in the development of IELs is still controversial. Several lines of studies have shown comparable or minimal differences in the number of IELs in GF mice and conventionally-raised mice (54–56). The number of TCRαβCD8αα IELs and/or TCRγδ IELs are comparable or modestly decreased in GF mice (54–56). In addition to the development of IELs, the gut microbiota may regulate the activation of TCRγδ IELs. It is reported that production of the antimicrobial peptide RegIIIγ by TCRγδ IELs is decreased in GF mice (57). Finally, microbiota-induced IL-1β and IL-23 can regulate the ability of IL-1R⁺ TCRγδ T cells to produce IL-17 and to promote following migration of neutrophils (58). RegIIIγ or IL-17 production by intraepithelial and/or lamina propria TCRγδ innate-like T cells limits the penetration and dissemination of pathogens including S. typhimurium (57, 58). In addition to the protective role against pathogens, microbiota-induced RegIIIγ by γδIELs controls the composition of commensal bacteria (59). Thus, the gut microbiota and host IELs reciprocally regulate each other, and control gut homeostasis.

Invariant NKT (iNKT) cells are another innate lymphocyte population that has been linked to gut homeostasis via the microbiota. Accumulation of iNKT cells in the gut is observed in GF mice, and increased iNKT cells promote susceptibility to colitis through upregulation of IL-4, IL-13 and IL-1β (60). Microbiota colonization can prevent Cxcl16 methylation, thereby reducing CXCL16 expression and preventing iNKT cell accumulation in the intestine. The microbiota-mediated reduction of Cxcl16 methylation is observed neonatal, but not adult GF mice, suggesting that microbial exposure in early life is important for shaping the homeostatic development of the gut immune system (60). In addition to IELs and iNKT cells, NK-related cell types are considered to function as innate lymphocytes. These NK-related cell types are referred as ‘innate lymphoid cells (ILCs)’. Classical NK cells, which selectively produce IFN-γ, are considered as type 1 ILC (ILC1). Moreover, other ILC subsets including IL-13-producing ILCs (ILC2 or nuocytes), IL-17-producing ILCs (ILC17), and IL-22-producing ILCs (ILC22) have been identified (61). Recent studies have revealed an important role for IL-22-producing ILC22 in the regulation of gut immunity (62, 63). CD3⁻NKp46⁺ lymphocytes in mice and CD3⁻NKp44⁺ in humans were originally defined as a subset of ILC22. These cells express the pan-NK cell marker NKp46 in mice (or NKp44 in humans), but lack the expression of the NK cell marker NK1.1 and typical NK cell functions such as cytotoxic activity or IFN-γ production (62). Expression of the transcriptional factor RORγt and production of IL-22 are two key features of the ILC subset (64–66). In addition to RORγt⁺NKp46⁺ ILCs, other RORγt⁺ ILC subsets, which do not express NKp46, are present in the gut and play an important role in intestinal homeostasis (67). In the fetal intestine, RORγt-expressing CD3⁻CD4^+/−CD127⁺ cells, named lymphoid tissue inducer (LTi) cells, play a critical role in the generation of lymph nodes and PPs. After birth, LTi cells are also present in the intestinal LP as cell clusters called CPs, and induce the development of ILFs (68). Recently, several studies have reported the presence of LTi-like cells in the adult intestine as a part of the IL-22-producing ILCs (69). LTi-like ILCs include both CD3⁻CD4⁺RORγt⁺c-kit⁺CD127⁺ cells and CD3⁻CD4⁻RORγt⁺c-kit⁺CD127⁺ cells (68). Thus, ILCs are heterogenous and include multiple cell subsets of cells that need to be better defined.

A critical function of IL-22 is to promote antimicrobial peptide production by intestinal epithelial cells. IL-22 induces the expression of RegIIIβ and RegIIIγ which are important for the regulation of the gut microbiota (59) (Fig. 1). Indeed, depletion of IL-22 or IL-22-producing ILCs causes intra- and extra-intestinal overgrowth and/or dissemination of certain bacterial species, such as Alcaligenes xylosoxidians, that may increase the risk of subsequent intestinal damage and/or systemic inflammation (70).

The relationship between the development and function of ILCs and the gut microbiota is controversial. Some reports showed that the microbiota is required for the differentiation of RORγt⁺NKp46⁺CD127⁺NK1.1⁻ or RORγt⁺NKp46⁺CD127⁺NK1.1^int ILCs (64, 65), whereas other studies concluded that the microbiota is dispensable, but is nonetheless required for the production of IL-22 by NKp46⁺CD127⁺NK1.1⁺ ILCs (64). In contrast, another study found that the microbiota is dispensable for the development of RORγt⁺ ILCs, but suppress the production of IL-22 by RORγt⁺ ILCs (71). In the latter study, GF mice exhibited an accelerated production of IL-22 by RORγt⁺ ILCs. The microbiota-dependent inhibition of IL-22 production by RORγt⁺ ILCs was mediated indirectly by intestinal epithelial cell-derived IL-25, which in turn activates IL-17BR, a receptor for IL-25-expressing DCs in ILFs. Another recent finding has demonstrated that the gut microbiota stabilizes the expression of RORγt within NKp46⁺ ILCs (72). Commensals induce intestinal epithelial IL-7 production, which stabilizes the expression of RORγt and maintains the homeostatic phenotype of NKp46⁺ ILCs, such as IL-22 production (72). In contrast, the absence of the microbiota results in decreased IL-7 production and down-regulation of RORγt gene expression in ILCs (72). As a consequence, ILCs are converted into IFN-γ-producing ILCs, which can promote intestinal inflammation (72). Thus, the gut microbiota can exert a homeostatic and pathogenic role in RORγt⁺ ILCs by regulating their development and function (Fig. 1).

Conclusions

There is clear evidence supporting a critical role for the microbiota in the development and functional regulation of the gut immune system. In addition, intestinal immune cells may regulate, in turn, the composition and function of the microbiota. This flexible interaction between the host and the microbiota may determine, in part, the balance between peaceful mutualism and disease in the intestine. Mounting evidence suggests that different commensals play unique roles in the development and/or maintenance of intestinal immune populations. Furthermore, some members of the microbiota facilitate beneficial immune responses, such as the development of steady-state Th17 cells, induction or activation of Tregs, and the regulation of ILCs. On the other hand, some commensal populations referred as ‘pathobionts’ can promote harmful immune responses such as conversion of steady-state Th17 cells into pathogenic Th17 to elicit intestinal or systemic inflammation. Determining the genetic and functional differences between "beneficial commensals" and "pathobionts" may provide insight into mechanisms of disease. Collectively, the studies to date indicate that the link between the microbiota and the host immune systems is complex. Further work is needed to obtain a more comprehensive understanding of the relationship of host immune cells and microbes in the gut.