T cell functional differentiation is regulated not only by cytokines from the environment but also their intrinsic programs. TH17 as a novel lineage of T cells has now been shown to be positively and negatively regulated by many cytokines. However, the genetic programming of their lineage differentiation in response to these cytokines has not been well understood. In the current study, we found that RORα, downstream of STAT3, functions together with RORγ in directing TH17 differentiation and cytokine expression.
RORα, regulated by STAT3, is selectively expressed in TH17 cells. RORα overexpression promotes IL-17 and IL-17F but not IL-22 expression. However, RORα deficiency only selectively impairs IL-17 but not IL-17F expression. Only in the absence of RORγ, RORα accounts for the remaining IL-17F production. RORα thus appears only to be important for IL-17 expression among all TH17 cytokines. The past work has indicated that TH cell cytokine expression is not only determined by master regulators but also regulated by other lineage-specific transcription factors that fine tune specific gene expression. For example, in TH1 cells, Hlx genetically interacts with T-bet to promote TH1 response (Mullen et al., 2002
; Zheng et al., 2004
). In TH2 cells, c-MAF and JunB selectively regulate IL-4 expression (Kim et al., 1999
; Li et al., 1999
). We previously found ICOS-c-Maf pathway only controls IL-4 but not IL-5 or IL-10 expression in effector TH2 cells and ICOS deficiency selectively abrogates IL-4- dependent IgE production but not IL-5-mediated airway eosinophilia (Dong et al., 2001
; Nurieva et al., 2003
). Interestingly, recently we observed different ratios of IL-17 and IL-17F expression in different T cell populations in vitro
and in vivo
(unpublished data). This suggests differential cytokine expression in differentiated TH17 cells. Whether RORα regulates this differential regulation is unknown at this point. Nor was the biological or pathologic significance of this regulation. In addition to IL-17 regulation, RORα also appears to upregulate IL-23R and downregulate IFNγ and T-bet expression. The molecular basis of this regulation also requires further investigation.
Since RORα or RORγ on their own is sufficient to induce TH17 differentiation under neutral conditions, it is curious why both are co-expressed in TH17 cells. Single ROR over-expression or deficiency did not substantially alter the expression of the other (data not shown), suggesting that these are two parallel but not sequential or inter-dependent pathways induced by STAT3. In support of this idea, RORα expression in RORγ-deficient cells drove TH17 differentiation, indicating that RORα could function independent of RORγ. Our retroviral co-expression has revealed a synergistic function by RORα and RORγ during TH differentiation. The molecular basis for this synergy is unclear at this point. ROR factors do not typically form heterodimeric complexes, which we confirmed via over-expression and co-immunoprecipitation of RORα and RORγt (data not shown). Moreover, we did not observe any synergy of the two factors in activating CNS2-IL-17
reporter expression. Although it is possible that RORα and RORγ synergistically activate some other elements in the locus, our data also strongly suggest a dose-dependent function of RORs in activation of IL-17 production. Low expression levels of RORα or RORγt did not effectively induce IL-17 expression (Fig. S1). Moreover, RORα enhanced IL-17 production in a dose-dependent manner in cells expressing variable concentrations of RORγt (Fig. S3A). These observations from retrovirus overexpression experiments suggest that when RORαt is expressed at physiological concentration, it requires the presence of RORα to induce TH17 differentiation effectively. Alternatively, both receptors are activated by similar but different ligands which can be regulated differentially to control the amount of pro-inflammatory TH17 cells
. Co-expression and co-activation of both factors is necessary to counteract the inhibitory mechanisms to reach the threshold levels for TH17 differentiation. In support of this idea, we observe that during polarized differentiation of T cells into TH1, TH2 or iTreg cells, RORs had different capacity of supporting TH17 development-TH2 was the easiest while iTreg was the most difficult. Consistent with these results, we found that TH17 differentiation was strongly inhibited by T-bet and Foxp3 but not by GATA-3 (unpublished results). RORα and RORγ can both be induced to some extent by TGFβ and IL-6; however, this level of expression may not be sufficient to drive TH17 differentiation.
In addition, our results also indicate redundancy of ROR factors. Although RORγ deficiency substantially inhibits TH17 differentiation, TH17 development was not completely abrogated (Ivanov et al., 2006
). The combination with RORα mutation further diminished the expression of TH17-specific genes by at least several folds. The redundancy is best seen in terms of IL-21 expression. While single mutation did not affect, the compound mutation greatly inhibited IL-21 production. This redundancy may have a biological significance, as in our EAE model involving two times of immunization, whereas RORγ−/−
cell-reconstituted mice only exhibited moderately reduced EAE, in contrast to an earlier report using RORγt knockout mice (Ivanov et al., 2006
). Importantly, RORα and RORγ double deficiencies led to complete protection. Since IFNγ production is not diminished in the absence of both ROR factors, our current results, in agreement with our data on IL-21 −/−
(Nurieva et al., 2007
) and IL-17−/−
(our unpublished results) mice, support TH17 cells as pathogenic mediators in this disease. Our data also suggest that simultaneous inhibition of RORα and RORγ would be most beneficial for treatment of autoimmune diseases, although it is not clear at this stage whether they are required for maintaining TH17 programs.
In summary, our study has elucidated the function of RORα, another TH17 lineage-specific transcription factor and its synergy and redundancy with RORγ. This work not only demonstrates genetic interaction between two nuclear receptors in TH17 differentiation but also reveals the complexity in the regulation of TH17 program and function.