In the present study, we addressed the role of IL-1 in the Th17 lineage differentiation pathway. We found that Th17 cells expressed higher levels of IL-1R1 mRNA, which was dependent on STAT3, RORα and RORγt. IL-1R1-deficient CD4+ T cells failed to induce EAE, which was associated with a selective defect in IL-17-producing T cells and an accumulation of Treg cells in the inflamed tissue. IL-1R1 signal was required for the early differentiation of Th17 cells and conversion of Foxp3+ T cells into IL-17-producing cells. After polarization, IL-1 also allowed Th17 cells to maintain their cytokine secretion profile. The IL-1R1 signaling in T cells functioned by upregulating IRF4 and RORγt.
IL-1 is a pleiotropic cytokine with many target cells. Although IL-1 was shown previously to be important in Th17 generation in vivo
, its exact action has not been understood. Higher IL-1R1 expression on Th17 cells has been observed in the SKG mouse strain that spontaneously develops arthritis (Hirota et al., 2007
). On the other hand, IL-1R1 expression on DC has been reported to be critical for onset of autoimmune myocarditis (Eriksson et al., 2003
), a Th17-mediated autoimmune disorder (Rangachari et al., 2006
). In our study, we clearly demonstrated that IL-1R1 expression in CD4 T cells but not on DC is critically required for Th17 generation in vitro
. Moreover, Rag1−/−
mice receiving IL-1R1 KO CD4 T cells displayed significantly attenuated EAE compared with recipients of WT T cells. Taken together, these observations indicate that IL-1 responsiveness in T cells is required for Th17 cell development and Th17-mediated autoimmunity.
The regulation of IL-1R1 expression has not been well understood. In the current study, we show that although TcR/costimulation activation resulted in elevated IL-1R1 mRNA expression, IL-6, but not IL-23 or TGFβ, serves a unique role to further enhance its expression. Consistent with this observation, IL-1 only functioned in the presence of IL-6 in upregulating Th17-specific genes. IL-6 is an essential Th17 priming cytokine- it can not only initiate Th17 differentiation and downregulate Foxp3 expression, but also has been shown to allow T cells to produce IL-21 (Nurieva et al., 2007
; Zhou et al., 2007
) and respond to IL-1 and IL-23 (Yang et al., 2007
; Zhou et al., 2007
). Interestingly, naïve T cells stimulated with IL-6, IL-23 and IL-1 can be differentiated into Th17 cells in the absence of exogenous TGF-β. Although this type of Th17 differentiation is still dependent on TGF-β, most likely from T cell itself or contained in serum, it led to greater IL-22 expression than those polarized by only TGF-β and IL-6. This finding suggests flexibility of Th17 differentiation. As previously shown in the literature, Th17 cells can be generated in the presence of IL-6 and relatively high concentrations of TGF-β which could be found at sites such as the intestine or tumor microenvironment On the other hand, with low TGF-β concentrations but with strong innate responses, IL-1, along with the other proinflammatory cytokines, can also drive Th17 polarization. This idea is supported by the early defect of Th17 differentiation in vivo
in the absence of IL-1 signaling. However, the function and regulation of Th17 generated by two different conditions remain to be understood. Interestingly, IL-1 induces IL-22 expression in Th17 cells differentiated in the presence of exogenous TGFβ suggesting the plasticity of IL-22 expression. Most importantly, our results also indicate that similar to human Th17 cells, mouse Th17 cells can be developed in the presence of pro-inflammatory cytokines IL-6, IL-1 and IL-23 in the absence of exogenous TGF-β.
IL-23 has been suggested to support Th17 expansion and maintenance (Veldhoen et al., 2006
). In the present study, we observed that IL-1 regulates early Th17 cell differentiation and maintenance of polarized effector Th17 cells. For the latter, IL-1 may have an overlapping function with IL-23. However, IL-23 alone had little effect on Th17 cells in the absence of TCR stimulation whereas the combination of IL-1 and IL-23 greatly synergized to expand Th17 cells and maintain their cytokine profiles. As IL-1R1 KO T cells expressed lower levels of IL-23R, IL-1 may function to enhance the IL-23R expression on Th17. Nevertheless, the expansion and cytokine production of Th17 cells by IL-1 in the absence of TcR stimulation suggests an important role of IL-1 in the homeostatic maintenance of Th17 cells. Since tissue inflammation induces abundant IL-1 and IL-23 expression, it would be interesting to surmise that Th17 cells that are not antigen-specific in inflamed tissue might expand and produce Th17 cytokines in a TCR-independent manner, and thus aggravates tissue damage. Further in vivo
study will be needed to clarify the role of inflammatory cytokines on Th17 cells.
A recent study demonstrates that IRF4 is necessary for Th17 differentiation; IRF4-deficient T cells fail to produce IL-17 in Th17 conditions but produces IFNγ in Th1 conditions (Brustle et al., 2007
). Moreover, overexpression of RORγt in IRF4 KO T cells only partially restores IL-17 production (Brustle et al., 2007
). The physiological factor(s) regulating IRF4 induction during Th17 polarization has been unclear. In the present study, we utilized DC/T cell coculture system and observed a severe defect in IRF4 expression in IL-1R1 KO T cells compared to WT T cells. Overexpression of IRF4 or RORγt alone partially overcame Th17 polarization in IL-1R1 KO. Interestingly, coexpression of these two transcription factors enhanced Th17 polarization in IL-1R1 KO but not in WT T cells. Therefore IL-1 from activated antigen-presenting cells delivers signals to T cells to upregulate IRF4 and RORγt during their early lineage programming and to sustain their differentiation. The present study unveiled IL-1 as a critical factor for inducing IRF4 in CD4+ T cells during Th17 polarization. Lack of an effect after IRF4 overexpression in WT cells is probably because IRF4 levels in WT T cells after stimulation by TcR and DC was sufficient for Th17 polarization.
The balance between Treg and Th17 is important for the generation of immunity against extracellular bacteria and regulating autoimmunity. Recent studies by our own group and others showed physical interaction between Foxp3 and RORγt or RORα(Yamazaki et al., 2008
; Yang et al., 2008b
; Zhou et al., 2008
). Of note, Foxp3+ cells can produce IL-17 in response to inflammatory stimuli. In the mixed bone-marrow chimera study, we observed higher percentage of Foxp3+ T cells in the IL-1R1 KO population compared with the WT population in the inflamed CNS. More importantly, about 10 % of Foxp3+ WT T cells in CNS and spleen expressed IL-17 whereas nearly no Foxp3+ KO T cells expressed IL-17. It has been reported that Foxp3+ IL-17F+ cells are much less efficient in suppressing the activation of naïve T cells (Yang et al., 2008b
). Therefore the IL-1R1 signal is critically required for Foxp3+ T cells to produce IL-17. However, IL-1 did not affect the downregulation of Foxp3 or the TGF-β-induction of Foxp3, indicating that the mechanism of IL-1-mediated IL-17 expression is distinct from that of IL-6. IL-1R1 delivers signal through the recruitment of MyD88 and TRAF6 leading to the activation of NF-κ B and MAPK pathways, which is distinct from IL-6 and IL-23 signaling. This may explain the synergism of IL-1 with the other Th17-inducing factors and their different effects on Foxp3, RORα and RORγt. Ahr is recently discovered as a critical transcription factor for both Treg and Th17 cells. Of note, we observed higher Ahr expression in the IL-1R1 KO T cells, suggesting inhibitory role of IL-1 on Ahr. The precise regulation by IL-1 signaling on Ahr expression remains to be determined.
The present study unveiled the critical roles of IL-1 during early Th17 differentiation and expansion/maintenance. Our results may explain the molecular mechanism of the spontaneous arthritis onset in IL-1Ra-deficient mice, which is a Th17-mediated autoimmune disorder (Nakae et al., 2003
). Of interest, a recent study showed that administration of ATP, a well known inflammasome activator, drives Th17 differentiation in the gut (Atarashi et al., 2008
), suggesting a role of IL-1 for the generation of Th17 in the gut. Our data thus corroborate these findings. The identification of IL-1 as a key regulator of Th17 biology may provide a support for therapeutic targeting of IL-1 in autoimmune disorders.