RA, the physiological vitamin A metabolite, has recently been shown to enhance TGF-β-induced Foxp3 expression and Treg conversion (23
) and to decrease Th17 differentiation and IL-17 production (26
). In this study, we confirmed these results and determined the mechanism by which RA increased Foxp3 expression but reciprocally decreased IL-17 production. Furthermore, we analyzed whether RA exerted similar effects in vivo
during an autoimmune reaction. We found that RA affected components in the TGF-β, IL-6/IL-21, and TcR/CD3 signaling pathways that are involved in the development of Treg and Th17 cells. Moreover, treatment with RA prevented autoimmune disease mainly by decreasing pathogenic Th17 and Th1 responses, however, under this in vivo
setting RA had no significant effect on Treg cells possibly due to ongoing inflammation.
RA strongly increased TGF-β-induced Foxp3 expression in vitro
and thus enhanced Treg conversion. Under these conditions, RA enhanced TGF-β signaling by increasing the expression and phosphorylation of Smad3, which is one of the key elements responsible for the induction Foxp3
. RA exerts its regulatory effects by binding to its receptor RAR, which is a transcription factor by itself. RA alone in the absence of TGF-β could increase the expression of Smad3, however, the increased Smad3 phosphorylation and Foxp3 expression by RA is completely dependent on TGF-β. On the other hand, it has been reported that the expression of RAR can be increased by TGF-β signaling (33
). Therefore, RA and TGF-β could cooperatively augment their mutual signaling to further enhance Foxp3 expression. Moreover, RA may also enhance Smad3-driven transactivation by promoting a direct RAR-Smad3 interaction since a direct RAR/Smad3 interaction has been demonstrated previously (34
). RA has also been reported to increase histone acetylation of the Foxp3
promoter regions (35
). Since histone acetylation is associated with increased gene activation and expression (36
), this may contribute to the RA-mediated increase in TGF-βinduced Foxp3
In addition to the positive effect on TGF-β-induced Foxp3 expression and Treg conversion, RA also inhibited IL-6/IL-21-mediated Th17 differentiation. Inhibition of Th17 differentiation by RA appears to be achieved by multiple mechanisms: 1) The enhanced TGF-β-induced Foxp3 expression by RA counteracts Th17 differentiation; 2) Inhibition of IL-6 receptor upregulation by RA decreases Th17 differentiation; 3) RA downregulates IRF4 expression and thus impairs IL-6-induced inhibition of Foxp3 expression and finally 4) RA inhibits IL-23R expression and thus impairs the stabilization and further maturation of the Th17 phenotype. The inhibition of IL-6 receptor expression not only decreases IL-6-initiated Th17 differentiation directly but also counteracts the inhibitory effect of IL-6 on Foxp3 induction and results in increased Foxp3 levels that further inhibit the induction of IL-17. RA seems to have only minor effects on proximal IL-6 or IL-21 signaling events. RA did not considerably affect either STAT3 phosphorylation or IL-21 expression induced by IL-6 or IL-21. However, RA reduced the expression of IL-23R induced by either IL-6 or IL-21 alone or in combination with TGF-β. This might be due to the fact that the RA/RAR complex binds to the promoter of the IL-23R
gene and transcriptionally represses IL-23R expression. Alternatively, RA binding to RAR might exert a direct inhibitory effect on IL-6/IL-21 signaling downstream of STAT3 since the expression of IL-23R is a downstream event of RORγt function and inhibition of RORγt expression by RA has recently been reported (26
). Both RAR and RORγt belong to a large superfamily of ligand-inducible nuclear receptors that includes RXR and ROR, steroid, vitamin D, and thyroid hormone receptors, estrogen receptor, peroxisome proliferator-activated receptors (PPAR), and a number of orphan receptors whose ligands are unknown as yet (15
). Direct interactions among the family members (such as RAR/RXR heterodimers and RXR/vitamin D3 receptor heterodimers) and between the transcription factors from other families (such as vitamin D3 receptor/c-Jun and RAR/STAT3 interactions) have been reported (15
). Therefore, it is possible that RA interferes with the transactivation function of STAT3 and/or RORγt by inducing direct RAR/STAT3 and/or RAR/RORγt interactions and thus inhibiting the IL-6/IL-21-induced expression of IL-23R, in addition to RA caused decrease of RORγt expression. Conversely, it will be interesting to determine whether RAR also binds to Foxp3, similar to NFAT (38
), and enhances Foxp3 function.
Although downregulation of IL-23R by RA may not play a role for Th17 differentiation in our in vitro
APC-free systems, in which there is no source of IL-23 cytokine, IL-23/IL-23R signaling is required for stabilizing the development and pathogenic function of Th17 cells in vivo
). Indeed, RA-treated MOG35-55
-immunized mice have a decrease in the generation of pathogenic Th17 responses, which is associated with a decreased expression of IL-23R. In addition to its direct inhibition of pathogenic Th17 and Th1 responses, RA also exerts this effect indirectly by decreasing the ability of APCs to generate Th1 and Th17 cells (data not shown), although RA does not affect the production of IL-6 in these cells (). It has previously been demonstrated that RA impairs the secretion of IL-12 from APCs and therefore decreases Th1 differentiation (39
). Whether RA-treated dendritic cells have an impaired IL-23 production remains to be determined. Taken together, we propose that in vivo
, the inhibitory effects of RA on the generation of Th17 cells are due to direct inhibitory effects on Th17 cells and modulation of APC function.
The decreased ratio of antigen-specific effector versus regulatory T cells upon treatment with RA attenuates autoimmunity. Here, we provide evidence that this change in Teff/Treg ratio is mainly due to a decrease of effector T cells in RA-treated mice. Unlike the in vitro
observation that RA strongly enhances Treg conversion, RA treatment in vivo
only slightly increases the population of Treg cells. Interestingly, Mucida et al
also observed a remarkable reduction of Th17 cells but not measurable enhancement of the differentiation of Foxp3+
Treg cells in mice infected with Listeria monocytogenes
and treated with RA, and they suggested that TGF-β might be a limiting factor in the lack of Treg cell differentiation induced by exogenous RA in vivo
). Here we show that this minor expansion of Treg cells by RA in vivo
appears to be due to the strong induction of IL-6 as well as TNF-α and IL-1 during the inflammatory process induced by MOG35-55
/CFA immunization as addition of all these inflammatory cytokines into Th17 differentiation cultures in vitro
strongly inhibited the enhancement of Foxp3 by RA, but could not abrogate the inhibitory effects of RA on Th17 cells (). This is also consistent with our previous data showing that IL-6 inhibits the generation of iTreg cells and in MOG/CFA immunized mice or under strongly inflammatory conditions, antigen specific Treg cells are not generated de novo but are derived from nTreg cells (32
The significant reduction of EAE by RA is associated with a decrease in Th17 and Th1 responses. However, the observation that IFN-g signaling may not be absolutely required for the induction of EAE (2
) suggests that the decreased Th17 response is most likely responsible for the reduction of EAE by RA. Also, the decreased IL-23R expression by RA impairs IL-23 signaling which further dampens the encephalitogenicity of Th17 cells, since IL-23 signaling has been shown to be crucial for full expression of the pathogenic phenotype in Th17 cells (10
). RA treatment after disease onset still attenuates the severity of EAE, suggesting that RA also inhibits established disease by regulating not just the expansion but most likely the effector functions of pathogenic Th17 and Th1 cells. Accordingly, we observed that addition of RA into in vitro
cultures of LN or spleen cells from mice with EAE also strongly inhibited CD4+
T cell proliferation and their production of IL-17 and IFN-g upon antigenic restimulation (data not shown).
Although under inflammatory conditions in vivo
, RA has minor effects on increasing the frequency of Treg cells, it is likely that the blockade of TGF-β-induced upregulation of IL-6Rα and the decrease of IRF4 expression by RA would reduce IL-6-mediated Treg inhibition (41
) and thus enhance their suppressive function, which may also play a role in the strong inhibition of pathogenic Th1/Th17 responses in RA treated mice during EAE.
In summary, we demonstrated that RA both in vitro and in vivo inhibits the development of Th17 cells by interfering with signaling events in the TGF-β and IL-6/IL-21/IL-23 pathways that are involved in the development and function of these cells. Although RA strongly promotes Treg conversion in vitro, RA treatment in vivo does not considerably increase the population of Treg cells in the face of ongoing inflammation. However, in the absence of strong inflammatory conditions, RA may be able to increase Treg cells in vivo as well. Therefore, RA regulates the balance between pathogenic Th17 and protective Treg cells by enhancing TGF-β signaling and by suppressing IL-6/IL-21/IL-23-driven signaling.