The biology of Th17 cells has elicited considerable excitement in recent years due to the recognition that these cells are involved in a wide variety of inflammatory conditions and autoimmune diseases. There has been extensive investigation of the cytokine requirements and genetic regulation in Th17 cell differentiation both under well-defined conditions in vitro and in the context of inflammatory stimuli in vivo (Bettelli et al., 2007
; Ivanov et al., 2007; Stockinger and Veldhoen, 2007
). The role of Th17 cells in normal physiology and the requirements for their differentiation at sites where they are most prevalent has received comparatively little attention. We previously reported that Th17 cells are abundant in the small intestinal lamina propria at steady state (Ivanov et al., 2006
). In this report, we have shown that the small intestine provides an environment that uniquely favors the differentiation of Th17 cells, which are scarce in other organs and at other mucosal sites. The composition of the commensal intestinal bacteria was found to have a crucial role in the differentiation of SI LP Th17 cells and in their balance with Treg cells, which also make up a large proportion of CD4+
T cells in the intestinal mucosa. Our results indicate that only a subset of bacterial species can induce Th17 cell differentiation, and suggest that unique innate immune signaling pathways, distinct from the TLR-mediated signals that can be initiated by numerous microorganisms, are required for this process.
Commensal bacteria have been shown to have a critical role in intestinal epithelial cell repair and homeostasis, acting through TLR-dependent mechanisms (Rakoff-Nahoum et al., 2004
). It is not clear how different members of the microbiota specifically affect different branches of innate or acquired mucosal immunity. However, it has been reported that certain bacterial species are more potent immune stimulators than others (Talham et al., 1999
); (Umesaki et al., 1999
). In support of this notion, recent studies have identified Bacteroides fragilis
as capable of inducing Th1 systemic immunity as well as IL-10 producing regulatory T cells in the colonic lamina propria (Mazmanian et al., 2005
; Mazmanian et al., 2008
). We found that simply the presence of commensal bacteria, even with a diverse composition, as in C57BL/6 mice from the Jackson Laboratories, is not sufficient to induce Th17 cells in the lamina propria. Instead, specific vancomycin-sensitive species in the commensal microbiota appear to be required. In our experiments, the presence of Th17 cells in the mucosa correlated with the presence of members of the CFB phylum, implicating these bacteria as Th17 cell inducers. In contrast, the abundance of γ-Proteobacteria and Firmicutes did not correlate with presence of Th17 cells. γ-Proteobacteria were present in high numbers in the cecum only after vancomycin treatment, but were scarce in Jackson B6 mice () and total numbers of Firmicutes were actually increased in the ileum of Th17 cell-deficient Jackson B6 mice (). However, the species composition of the Firmicutes phylum was not investigated in this study and thus we cannot rule out the involvement of individual species from this phylum. The exact nature of components of the microbiota that induce Th17 cells awaits a more detailed species-specific comparison of the microbiome of Th17 cell-sufficient and Th17 cell-deficient mice.
Induction of SI LP Th17 cells by specific microbiota is likely to involve activation of distinct innate signaling pathways in epithelial cells or different subsets of antigen presenting cells in the lamina propria (Denning et al., 2007
; Fujiwara et al., 2008
; He et al., 2007
; Mucida et al., 2007
; Uematsu et al., 2008
). Our results with TLR signaling-deficient mice rule out an absolute requirement for this pathway in Th17 cell induction. Instead, combined with the results with mice from different sources, they suggest that activation of other microbial pattern recognition pathways or production of a metabolite by subsets of commensal organisms in the gut direct the differentiation of IL-17 producing T-helper cells. While this paper was under review, another study reported the absence of Th17 cells in large intestine of germ-free mice and showed that TLR signaling is not required for their accumulation in the lamina propria (Atarashi et al., 2008
). That study showed that luminal ATP can drive the differentiation of colonic Th17 cells, most likely by activating a specific population of intestinal DCs (Atarashi et al., 2008
). Combined, the two studies suggest that similar signals from the intestinal microbiota drive Th17 cell differentiation in both small and large intestine. Our study demonstrates that the presence of even very diverse populations of intestinal bacteria is not sufficient to induce Th17 cell differentiation, but that a specific subset of the intestinal microbiota is required. Although ATP can clearly induce Th17 cell differentiation as demonstrated by Atarashi et al., it is unclear at this point if this is the main mechanism of Th17 cell induction in vivo and whether it involves bacterial-derived ATP. It is formally possible that Th17 cell-inducing bacteria represent a small intestinal subpopulation that specifically produces ATP. On the other hand, ATP may be produced by many types of bacteria, in which case there may be bacterial species-specific sequestration of ATP accessibility to dendritic cells or engagement of ATP-independent mechanisms for induction of Th17 cell differentiation in the intestine.
An unexpected finding was that neither the IL-21 nor the IL-23 ligand-receptor interaction was required for the differentiation of Th17 cells in the SI LP. We and others showed that IL-21 is induced by IL-6 in vitro in naïve CD4+
T cells and contributes significantly towards production of IL-17 in response to the combination of IL-6 and TGF-β(Korn et al., 2007
; Nurieva et al., 2007
; Zhou et al., 2007
). Normal or even enhanced differentiation of mucosal Th17 cells in IL-21R-deficient mice suggests that cytokines other than IL-21 can mediate the inductive effect of IL-6 in vivo in the intestinal lamina propria. Consistent with our data, two recent studies have demonstrated that IL-21 is dispensable for Th17 cell differentiation in vivo in a model of autoimmunity (Coquet et al., 2008
; Sonderegger et al., 2008
). In contrast to studies on IL-21, there is a consensus that IL-23 is required in vivo for Th17 cells to mediate inflammatory and protective immune responses (McKenzie et al., 2006
; Weaver et al., 2007
). In vitro, IL-23 can enhance production of IL-17, particularly at low TGF-β concentrations (Zhou et al., 2008
). It is therefore difficult to explain why Th17 cells were present in the lamina propria of Il23a-/-
mice. We noted that lack of IL-23 selectively depleted IL-22-producing Th17 cells in the SI LP at steady state. Thus, IL-23 may selectively influence the differentiation of distinct Th17 cell subpopulations. Mice lacking IL-23 have been shown to succumb to intestinal infection with Citrobacter rodentium
even though there was little effect on IL-17 levels in the colon (Mangan et al., 2006
). Absence of IL-22 results in similarly increased susceptibility to C. rodentium
infection, and the IL-22 response was shown to require IL-23 and to be independent of adaptive immunity (Zheng et al., 2008
). Therefore, IL-22 may execute an innate effector function that has been appropriated by Th17 cells, and its expression may be more dependent than that of IL-17 on IL-23R signaling.
We also show in this report that appropriate activation of TGF-β by the extracellular matrix is necessary for steady-state Th17 cell differentiation. TGF-β, depending on its concentration and the presence of other cytokines, controls the balance between Foxp3+
Treg and Th17 cell differentiation in vitro (Bettelli et al., 2006
; Zhou et al., 2008
). Of note, the mutation that we have generated in TGF-β leads to abnormal targeting of TGF-β to the extracellular matrix. It does not decrease the amount of TGF-β precursor, nor does it decrease the level of activated TGF-β in the serum (data not shown). In addition, all mechanisms of TGF-β activation are still in place. Thus, this mutation may lead to activation of TGF-β at a different location or by different mechanisms, which may result in changes in the local concentration of active TGF-β, and thus affect Th17 as well as Foxp3+
Treg cell production. Accordingly, Th17 cells were not present in the lamina propria in these mice. Another study recently reported that lack of αvβ8, one of the major TGF-β activators, on DCs led to loss of Foxp3+
Tregs in the lamina propria (Travis et al., 2007
). However Th17 cell differentiation was not investigated in that study. The mechanism of αvβ8 activation is not expected to be affected in our model. Nevertheless we also found that Foxp3+
cell proportions in the lamina propria were greatly reduced in our mice. Combined, these data suggest that the concentration of TGF-β established by the appropriate activation mechanism at the appropriate location is crucial for Th17 and Foxp3+
Treg cell differentiation in the lamina propria.
Our findings demonstrate the importance of the composition of the intestinal bacteria in regulating the balance and homeostasis of different helper T cell populations in the lamina propria and further emphasize the critical role that the microbiota play in the development of the immune system. In addition to controlling the Th17:Treg balance, the composition of the commensal microbiota may also have a role in regulating levels of different Th17 cell subpopulations (IL-22+, IL-17F+, IL-10+, and IFNγ +) whose functions in disease and host defense have yet to be defined. The representation of different effector T cell populations generated by a particular commensal repertoire will dictate the nature and robustness of the immune responses in the intestine. Elucidation of intestinal microbial species and products that influence the differentiation of the various effector CD4+ T cell subsets promises to provide a better understanding of how the intestinal host-microbial relationship regulates host defense and inflammation.