The Toll-Like Receptor 2 Pathway Establishes Colonization by a Commensal of the Human Microbiota

Throughout our lives, we continuously encounter microorganisms that range from those essential for health to those causing death (1). Consequently, our immune system is charged with the critical task of distinguishing between beneficial and pathogenic microbes. Toll-like receptors (TLRs) are evolutionarily conserved molecules that promote immune responses, and TLR signaling by innate immune cells is indispensable for proper activation of the immune system during infections. T cells also express functional TLRs (2–4), and TLR signaling has furthermore been shown to restrain immune responses (5). As symbionts and pathogens produce similar molecular patterns that are sensed by TLRs, the mechanisms by which our immune system differentiates between the microbiota and enteric infections remain unknown.

Whereas the intestinal microbiota contains hundreds of bacterial species and is integral to human health (6), the mucosal immune system employs an arsenal of responses to control enteric pathogens. Germ-free mice lack proinflammatory T helper 17 (T_H17) cells in the gut (7, 8) (Fig. 1A), and only select symbiotic bacteria can induce T_H17 cells (9, 10). Most microbes express common TLR ligands (e.g., peptidoglycan, unmethylated CpG, and lipoproteins); therefore, how do symbionts avoid triggering intestinal immunity in their mammalian hosts? We examined the hypothesis that the human gut commensal Bacteroides fragilis evolved molecular mechanisms to suppress T_H17 responses during homeostatic colonization. As predicted previously (7, 10, 11), we found that B. fragilis mono-associated animals did not induce T_H17 cell development in the colon compared to germ-free controls (Fig. 1A). The beneficial contributions of B. fragilis require a single immunomodulatory molecule named polysaccharide A (PSA), which prevents and cures inflammatory disease (12–14). Colonization with B. fragilis in the absence of PSA (B. fragilisΔPSA), however, resulted in significant T_H17 cell responses in the gut (Fig. 1, A and B). Colonic lamina propria (LP) lymphocytes from B. fragilisΔPSA mono-associated animals displayed increased secretion of interleukin-17A (IL-17A) in vitro (fig. S1) and elevated transcription of Il17a and the T_H17-specific lineage differentiation factor RORγt (Rorc) (Fig. 1, C and D) (15). Differences in T_H1 responses were not observed during B. fragilisΔPSA colonization (fig. S2). Cells from B. fragilis mono-associated animals produced low amounts of IL-17A during in vitro T_H17 skewing assays, whereas CD4⁺ T cells from B. fragilisΔPSA animals display elevated IL-17A production (fig. S3). Administration of purified PSA to B. fragilisΔPSA mono-associated animals suppresses T_H17 immunity (Fig. 1E). Thus, B. fragilis actively restrains T_H17 cell responses during colonization.

• Download high-res image
• Open in new tab
• Download Powerpoint

Recent studies have shown that certain gut bacteria can promote regulatory T cell (T_reg) induction (11, 14). T_regs expressing the transcription factor Foxp3 (forkhead box P3) suppress proinflammatory T_H17 cell reactions. To test whether T_regs prevent immune responses during B. fragilis colonization, we reconstituted germ-free Rag1^−/− mice with bone marrow from Foxp3-DTR (diphtheria toxin receptor) donors, which allowed for specific ablation of T_regs by administration of diphtheria toxin (DT) (16). Mice were mono-associated with B. fragilis to induce T_reg development (Fig. 1F). DT treatment of mice resulted in depletion of Foxp3⁺ T cells (Fig. 1F), with a concomitant increase in T_H17 responses (Fig. 1G and fig. S4), which suggests that Foxp3⁺ T_regs are required for suppression of T_H17 cells during B. fragilis colonization.

PSA is an immunomodulatory bacterial molecule that shapes host immune responses (17). Induction of IL-10 and interferon-γ (IFN-γ) from CD4⁺ T cells by PSA requires TLR2 signaling (14, 18). We sought to determine the mechanism whereby B. fragilis suppresses T_H17 cell responses by testing whether PSA functions through TLR2 signaling by dendritic cells (DCs) and/or CD4⁺ T cells. PSA elicited a significant increase in IL-10 and IFN-γ production from mixed cultures of wild-type DCs and wild-type CD4⁺ T cells in vitro (Fig. 2A and fig. S5). When Tlr2^−/− T cells were cocultured with wild-type DCs, however, PSA-induced IL-10 production was reduced, whereas IFN-γ expression was not affected (Fig. 2A and fig. S5), which indicated that PSA required TLR2 expression on T cells to promote IL-10 production. IL-10 responses to PSA were specific to T cells (fig. S6). Consistent with previous findings (18), proinflammatory IFN-γ production was dependent on TLR2 signaling by DCs (fig. S5); however, IL-10 production was unaffected in cultures containing wild-type CD4⁺ T cells and Tlr2^−/− DCs (Fig. 2A). Therefore, TLR2 expression by T lymphocytes is necessary for IL-10 production by PSA.

• Download high-res image
• Open in new tab
• Download Powerpoint

CD4⁺ T cells produce IL-10 in response to PSA in the absence of antigen-presenting cells (APCs) (Fig. 2B). Moreover, PSA induces IL-10 expression from purified T cells in a dose-dependent manner, whereas other TLR2 ligands do not (fig. S7). TLR2 can function as either a homodimer or a heterodimer with TLR1 or TLR6 (19). PSA could induce high amounts of IL-10 from wild-type, Tlr1^−/−, and Tlr6^−/− CD4⁺ T cells; however, IL-10 production was lost only from Tlr2^−/− CD4⁺ T cells and T cells deleted in the TLR adapter molecule MyD88 (Fig. 2B). To determine T_reg suppression as a function of cell-intrinsic TLR2 signaling, we measured proliferation of responder T cells after coculture with T_regs stimulated in vitro with PSA, or classical TLR2 ligands Pam3CysK or Pam2CysK. The proportion of Foxp3⁺ T cells was equivalent under all conditions (fig. S8). PSA-treated T_regs displayed increased suppressive capacity compared to media or other TLR2 ligand–treated T_regs (Fig. 2C). IL-2 was not produced by T_regs during treatment with any of the TLR2 ligands, and IL-2 neutralization had no effect on in vitro suppression (fig. S9). Notably, the suppressive capacity of PSA-treated T_regs was lost when Foxp3⁺ T cells were deficient in TLR2 (Fig. 2C). PSA likely directed the development of inducible T_regs by promoting the expression of IL-10, transforming growth factor–β2 (TGF-β2), and Foxp3 from purified Foxp3⁺ T cells in a TLR2-dependent fashion (fig. S10). Collectively, these studies show that unlike other TLR2 ligands, PSA enhances T_reg function and gene expression in the absence of APCs through TLR2 signaling directly on CD4⁺Foxp3⁺ Treg cells.

We next determined whether PSA signals through TLR2 on Foxp3⁺ T_regs during B. fragilis colonization of animals to suppress T_H17 cell responses. Germ-free Rag1^−/− mice were reconstituted with purified CD4⁺ T cells from wild-type or Tlr2^−/− animals. Animals were subsequently mono-associated with either B. fragilis or B. fragilisΔPSA. Unlike wild-type animals, IL-10–producing Foxp3⁺ T_regs were not induced in the gut of B. fragilis mono-colonized animals that were reconstituted with Tlr2^−/− CD4⁺ T cells (fig. S11). Although we observed minimal T_H17 cell development in wild-type mice mono-associated with B. fragilis, T_H17 cell responses were significantly increased during colonization of animals that were reconstituted with Tlr2^−/− CD4⁺ T cells (Fig. 2, D and E). Furthermore, TLR2-deficient CD4⁺ T cells from B. fragilis mono-associated mice produced more IL-17A compared to mice reconstituted with wild-type CD4⁺ T cells (fig. S12). Collectively, these data demonstrate that B. fragilis requires TLR2 to induce Foxp3⁺ T_regs during intestinal colonization and actively suppresses T_H17 responses through engagement of TLR2 specifically on T cells.

The intestinal microbiota occupies both mucosal and luminal niches during normal colonization; however, the biogeographic distributions of specific microbial species are poorly characterized. We reasoned that the functions of PSA were driven by an evolutionary impetus to prevent deleterious mucosal immune reactions to B. fragilis, enabling bacteria to associate with host tissue. Intriguingly, we discovered a population of bacteria that intimately associates with the intestinal epithelium (Fig. 3A). Whole-mount preparations of medial colons were probed for bacteria after labeling with B. fragilis–specific antisera (fig. S13). Three-dimensional reconstruction of confocal microscopic images revealed microcolonies of B. fragilis residing within colonic crypts (Fig. 3A). The amounts of B. fragilis associated with host tissue represented a fraction of total bacteria (Fig. 3, B and C), but likely are an important population that are in close proximity to the host immune system. We speculated that the amounts of tissue-associated bacteria would be sensitive to host immune responses such as T_H17 cell induction. Notably, animals colonized with B. fragilisΔPSA displayed profoundly reduced numbers of tissue-associated bacteria when compared to animals colonized with wild-type B. fragilis (Fig. 3D). Treatment of B. fragilisΔPSA–colonized animals with purified PSA corrected this defect and increased colonization of B. fragilisΔPSA to wild-type bacterial levels (Fig. 3D). Only tissue-associated bacteria were affected, because no differences were observed in the amounts of bacteria in the gut lumen by either strain (fig. S14) (17). Collectively, these data identify a previously unappreciated mucosal niche for B. fragilis and reveal that PSA is required for maintaining host-bacterial symbiosis at the epithelial surface of the gut.

• Download high-res image
• Open in new tab
• Download Powerpoint

Our findings suggest that PSA induces T_regs through TLR2 signaling to suppress T_H17 cell responses and promote mucosal colonization by B. fragilis. To test this model, we measured colonization levels of B. fragilis in Rag1^−/− mice reconstituted with TLR2-deficient CD4⁺ T cells. Tissue association by wild-type B. fragilis in the colon was reduced to the levels of B. fragilisΔPSA in these mice (Fig. 3E and fig. S15). Moreover, Foxp3⁺ T_reg ablation in B. fragilis mono-associated animals resulted in significantly reduced amounts of tissue-associated B. fragilis (Fig. 3F), without affecting bacterial numbers in the lumen of the gut (fig. S16). Finally, to functionally determine the role of IL-17 responses in mucosal association, we treated B. fragilisΔPSA mono-associated animals with a neutralizing antibody to IL-17A. Whereas the amounts of B. fragilisΔPSA in isotype control–treated animals remained low, neutralization of IL-17A resulted in a 1000-fold increase in tissue-associated bacteria (Fig. 3, G and H). These data indicate that IL-17 suppression by PSA is required by B. fragilis during association with its host. Therefore, unlike pathogens that trigger inflammatory responses through TLRs to clear infections, symbiotic colonization by B. fragilis is actually enhanced via the TLR pathway. We conclude that PSA evolved to engender host-bacterial mutualism by inducing mucosal tolerance through TLR2 activation of T_reg cells.

The gastrointestinal tract represents a primary portal for entry by numerous pathogens. Toll-like receptors recognize MAMPs (microbial-associated molecular patterns) expressed by bacteria and coordinate a cascade of innate and adaptive immune responses that control infections (20). Although TLRs have classically been studied on innate immune cells, recent reports have demonstrated their expression by T cells in both mice and humans (4, 21–23). As bacteria contain universally conserved MAMPs, how do commensal microbes, unlike pathogens, avoid triggering TLR activation? It is historically believed that the microbiota is excluded from the mucosal surface (24). However, certain symbiotic bacteria tightly adhere to the intestinal mucosa (9–11), and thus immunologic ignorance may not explain why inflammation is averted by the microbiota. Our study provides new insight into the mechanisms by which the immune system distinguishes between pathogens and symbionts. The functional activity of PSA on T_regs contrasts with the role of TLR2 ligands of pathogens, which elicit inflammation, and thus reveals an unexpected function for TLR signaling during homeostatic intestinal colonization by the microbiota. Although engagement of TLR2 by previously identified ligands is known to stimulate microbial clearance of pathogens, TLR signaling by PSA paradoxically allows B. fragilis persistence on mucosal surfaces. These results identify PSA as the incipient member of a new class of TLR ligands termed “symbiont-associated molecular patterns (SAMPs)” that function to orchestrate immune responses to establish host-commensal symbiosis. On the basis of the importance of the microbiota to mammalian health (25), evolution appears to have created molecular interactions that engender host-bacterial mutualism. In conclusion, our findings suggest that animals are not “hard-wired” to intrinsically distinguish pathogens from symbionts, and that microbial-derived mechanisms have evolved to actively promote immunologic tolerance to symbiotic bacteria. This concept suggests a reconsideration of how we define self versus nonself.