Control of Th17 differentiation by PPARγ
We first investigated the influence of PPARγ on the Th17 responses during MOG-induced EAE. Pharmacological activation of PPARγ with PIO in vivo ameliorated the disease course over the entire observation period (), as previously reported (Niino et al., 2001
; Diab et al., 2002
; Feinstein et al., 2002
). Importantly, CD4+
T cells isolated from the central nervous system (CNS) of PIO-treated EAE mice at day 17 after disease induction produced significantly less IL-17A after PMA/ionomycin restimulation (). This prompted us to investigate in more detail the influence of PPARγ on the function of autoreactive MOG-specific T cells. We therefore determined the frequency of antigen-specific Th17 cells in the CNS by ELISpot. After MOG35-55
-peptide specific stimulation of equal numbers of CNS-derived T cells, we observed a strong reduction in antigen-specific IL-17 producing, but interestingly not IFN-γ producing, CD4+
T cells (). In light of these results, we next investigated the influence of PPARγ on CD4+
Th differentiation. To focus exclusively on the effect of PPARγ in T cells, we used stimulation with αCD3/αCD28 in the absence of antigen-presenting cells. Interestingly, PPARγ activation by PIO selectively inhibited Th17 differentiation induced by TGF-β and IL-6, whereas IL-12–induced Th1 differentiation was completely unaffected (; Fig. S1
). To obtain unequivocal evidence for the role of PPARγ for Th17 differentiation, we generated T cell–specific PPARγ knockout mice by crossing CD4-Cre mice with mice carrying loxP sites within the PPARγ gene (CD4-PPARγKO
; Fig. S2
). In the absence of PPARγ, Th17 differentiation was strongly increased when compared with wild-type CD4+
T cells (), indicating that PPARγ serves as a T cell–intrinsic brake of Th17 differentiation under physiological conditions. Accordingly, Th1 differentiation was not altered in CD4-PPARγKO
T cells (). Interestingly, the endogenous PPARγ agonist 13s-HODE, a linoleic acid derivative (Huang et al., 1999
), equally suppressed Th17, but not Th1, differentiation (), further indicating that PPARγ activity limits Th17 differentiation under physiological conditions. Given the fact that 12/15-lipoxygenase is expressed in T cells (Vanderhoek, 1988
), it is reasonable to assume that endogenous ligands produced by T cells themselves serve as a brake for Th17 differentiation in an autocrine fashion. Also, PPARγ ligand production by antigen-presenting cells may contribute to local control of Th17 differentiation (Huang et al., 1999
Figure 1. Control of Th17 differentiation by PPARγ. (a) MOG-EAE was induced in PIO or vehicle-treated wild-type mice (n = 6 per group, 3 experiments), and the clinical disease score was assessed daily. (b) In a separate experiment, mice were sacrificed (more ...)
We further substantiated the inhibitory effect of PPARγ on Th17 differentiation by investigating other classical markers of Th17 cells. In addition to IL-17A, we found that PPARγ activation by PIO suppressed expression of TNF and IL-22 (), as well as IL-17F, IL-21, and IL-23R, in T cells (). Likewise, expression of the chemokine receptor CCR6 and its ligand CCL20 were also strongly controlled by PPARγ activation (). This demonstrates that PPARγ, indeed, influenced differentiation of Th17 cells rather than merely suppressing IL-17A production.
Selectivity of PPARγ for Th17 differentiation
To further characterize the specificity of PPARγ on the differentiation of Th17 cells, we evaluated the effect of PIO on cytokine-induced CD4+
T cell differentiation into Th1, Th2, or regulatory T cells. Importantly, PIO did not modulate TGF-β–mediated induction of Foxp3+
regulatory T cells, IL-4–mediated induction of Th2 cells, or IL-12–mediated induction of Th1 cells (). This is in contrast to the effect of RA, which is a natural ligand of the nuclear RA receptor (Chambon, 1994
) that has been shown to reciprocally regulate Th17 and regulatory T cell differentiation (Mucida et al., 2007
). In direct comparison, RA and PIO both efficiently suppressed Th17 differentiation, whereas RA but not PIO induced TGF-β–mediated expression of Foxp3 (). Accordingly, CD4-PPARγKO
T cells did not show altered TGF-β–mediated Foxp3-induction (unpublished data). A further distinction between RA and PIO was observed on Th1 differentiation, as RA slightly but significantly impeded IL-12–mediated induction of IFN-γ expression in T cells (), as has been previously reported (Iwata et al., 2003
). Collectively, these data indicate that distinct molecular mechanisms were involved in PPARγ-mediated, as compared with RA-mediated, control of T cell differentiation.
Figure 2. Selectivity of PPARγ for Th17 differentiation. (a) CD4+ T cells were subjected to Th1, Th2, Th17, and regulatory T cell differentiation protocols, as described in the Materials and methods section, and the influence of RA and PIO on the induction (more ...)
We next investigated whether PPARγ also affected expression of the key transcription factors determining CD4+
T cell differentiation. PPARγ-activation selectively suppressed TGF-β/IL-6–mediated expression of RORγt, the transcription factor required for Th17 induction, whereas the expression of the transcription factors determining Th1, Th2, and regulatory T cell differentiation, i.e., T-bet, GATA-3, and FoxP3, was not influenced by PIO (), again confirming that PPARγ acted specifically on the differentiation of Th17 cells. Other transcriptional regulators have been reported to influence Th17 differentiation. Foxp3 has been shown to directly antagonize RORγt activity, and thus prevent Th17 differentiation (Zhou et al., 2008
). Furthermore, several groups have reported that the aryl hydrocarbon receptor elicits either regulatory T cell or Th17 responses when activated by distinct ligands; however, the underlying mechanisms do not seem to involve RORγt regulation (Quintana et al., 2008
; Veldhoen et al., 2008
). Additionally, the nuclear orphan receptor NR2F6 seems to regulate Th17-dependent autoimmunity, but with no apparent involvement of RORγt (Hermann-Kleiter et al., 2008
). It can therefore be concluded that several receptors are involved in the T cell–intrinsic control of Th17-responses, but that the molecular pathways involved in these processes are distinct. Even among the family of PPARs, the regulatory effect on Th17 differentiation is not a general feature, as lack of PPARα in T cells did not result in altered IL-17 expression levels (Dunn et al., 2007
PPARγ inhibits Th17 differentiation by controlling RORγt induction
We next evaluated whether PPARγ influenced RORγt expression in T cells. In PPARγKO
T cells, we observed enhanced cytokine-induced RORγt induction compared with PPARγWT
T cells (). The suppressive effect of PPARγ activation by PIO on the one hand and the increased expression of RORγt in PPARγKO
T cells on the other hand illustrate the dynamic range of PPARγ-mediated control of Th17 differentiation. We substantiated the influence of PPARγ activation on RORγt expression using reporter mice, which express GFP under control of the RORc(γt) promoter (Lochner et al., 2008
). In such T cells, we observed that PIO strongly reduced TGF-β/IL-6–mediated GFP-expression (). Importantly, both the frequency of GFPpos
T cells and the mean fluorescence intensity of GFP-expressing T cells were reduced by PIO (). These results indicated that most CD4+
T cells failed to express RORγt under the influence of PPARγ activation, thus giving rise to less Th17 cells. Furthermore, the decreased mean fluorescence intensity of GFP in PIO-treated RORc(γt) reporter T cells () revealed that upon PPARγ activation there was less GFP, i.e., RORγt, on a per cell basis, suggesting that PPARγ reduced RORγt transcription on a single-cell level.
Figure 3. PPARγ inhibits Th17 differentiation by controlling RORγt induction. (a) Th17 differentiation from PPARγKO and wild-type T cells was induced as described in Materials and methods; RORγt expression was determined by quantitative (more ...)
The control of PPARγ over RORγt transcription led us to examine whether the RORγt promoter contained a bona fide PPARγ-binding site (PPRE), which might permit direct interaction of PPARγ with the RORγt promoter. Bioinformatic analysis did not reveal any known PPRE sequence within the mouse RORγt promoter (unpublished data). In addition, we excluded direct interaction of PPARγ with the RORγt promoter by examining the binding of recombinant PPARγ to the full-length RORγt promoter using surface plasmon resonance analysis. In contrast to strong and specific binding of PPARγ to the AP2 promoter, which contains a PPRE site (Frohnert et al., 1999
), we did not observe significant binding to the RORγt promoter ().
The lack of a high-affinity PPARγ binding site in the RORγt promoter raised the possibility that PPARγ might negatively regulate RORγt transcription through a transrepression mechanism that does not require direct DNA binding. One such mechanism involves the ability of ligand-activated PPARγ to inhibit signal-dependent clearance of NCoR or SMRT corepressor complexes from promoters of regulated genes (Pascual et al., 2005
; Ghisletti et al., 2009
). To investigate this possibility, we used chromatin immunoprecipitation (ChIP) to screen of genomic sequences surrounding the RORγt promoter (unpublished data) for corepressor binding. These studies revealed the binding of the corepressor SMRT, but not NCoR, at the RORγt promoter in unstimulated mouse CD4+
T cells ( and not depicted). Importantly, stimulation of CD4+
T cells with TGF-β and IL-6 resulted in rapid and nearly complete loss of SMRT from the RORγt promoter (), indicating that SMRT clearance precedes RORγt activation. Interestingly, this cytokine-induced clearance of SMRT from the RORγt promoter was prevented by the PPARγ agonist PIO (). These data suggest that the retention of SMRT results in persistent repression of RORγt in the presence of activating cytokines, and are consistent with prior studies demonstrating that PPARγ suppresses activation of inflammatory response genes in macrophages by preventing NCoR/SMRT turnover (Ghisletti et al., 2009
). Interference of SMRT clearance from the RORγt promoter thus provides a previously unrecognized mechanism by which ligand-activated PPARγ may control Th17 differentiation in T cells. However, these findings do not exclude other mechanisms, such as modulation of STAT3 or IRF4 signaling (Nurieva et al., 2007
; Huber et al., 2008
PPARγ in T cells controls CNS autoimmunity and restricts Th17 differentiation in vivo
To analyze whether PPARγ is involved in T cell–intrinsic control of CNS autoimmunity, we induced EAE in CD4-PPARγKO
mice and wild-type littermates. CD4-PPARγKO
mice showed a significantly earlier onset and aggravated disease course during the initial T cell–dependent phase of disease until d15 (). However, this difference was not observed in the effector phase, when disease activity is mainly determined by a local inflammatory response within the CNS governed by microglial cells (Heppner et al., 2005
). Disease activity in CD4-PPARγKO
mice directly correlated with the total numbers of infiltrating CD4+
T cells in the CNS (). Both at the beginning of clinical disease activity (day 8), and at the peak of disease in CD4-PPARγKO
mice (day 13), we found significantly increased total CD4+
T cell numbers in the CNS. Later (day 18) disease score and T cell influx were not different from wild-type littermates. As expected, PIO-treated wild-type mice exhibited decreased T cell numbers within the CNS at all time points investigated (). Importantly, at the peak of disease in CD4-PPARγKO
mice, the frequency of MOG35-55
peptide-specific, IL-17–producing CD4+
T cells in the CNS was increased by threefold, which, together with the increase in T cell influx, enhanced the numbers of IL-17–producing autoreactive T cells within the target organ by nearly fivefold (). In contrast, there was no alteration in antigen-specific IFN-γ–producing CD4+
T cells in these mice ().
Figure 4. PPARγ in T cells controls CNS autoimmunity and restricts Th17 differentiation in vivo. (a) MOG-EAE was induced in CD4-PPARγKO mice and CD4-PPARγWT littermates (n = 8 per group, 3 experiments), and the clinical disease score was (more ...)
The clinical symptoms and antigen-specific Th17 responses in CD4-PPARγKO
mice both revealed that the kinetics of CNS autoimmunity in vivo were modulated by PPARγ in a T cell–intrinsic fashion. There was pronounced accumulation of IL-17–producing T cells in the CNS compared with the spleen in CD4-PPARγKO
mice (), which may be caused by guided entry of Th17 cells into the CNS. A recent study demonstrated that CCR6-expressing Th17 cells function as “pioneer” cells, enabling immune cell entry into the CNS at the beginning of CNS autoimmunity (Reboldi et al., 2009
). In this regard, the control of expression of both CCR6 and its ligand CCL20 by PPARγ activation () may explain the decreased influx of T cells and the reduced disease activity in the CNS of PIO-treated wild-type mice. The protective effect of PIO on disease activity was greatly diminished in CD4-PPARγKO
mice (Fig. S4 b
), thus excluding off-target effects that had been reported previously (Chawla et al., 2001
) and further demonstrating that PPARγ expression in T cells was required for full protective effect of PIO on CNS autoimmunity. The observation that PPARγ activation in vivo did not entirely protect from EAE development, despite its profound effect on Th17 differentiation, lends support for a key but not exclusive role of Th17 cells in CNS inflammation, as previously reported (Yang et al., 2009
). The persistent Th1 responses, which were not altered by PPARγ activation, may explain persistent disease activity, despite diminished Th17 responses.
As we also observed a significant increase in antigen-specific Th17 cell numbers in the spleen in CD4-PPARγKO mice at the peak of disease (), we next asked whether PPARγ influenced Th17 differentiation in vivo at early time points. To this end, we adoptively transferred CD90.2+ CD4+ T cells from OT-II mice, followed by immunization with OVA in CFA. Importantly, PIO treatment of these mice strongly interfered with the expression of activation markers () and IL-17 production () by the adoptively transferred T cells 4 d after immunization; this persisted for longer than 4 d (day 7; not depicted), demonstrating that PPARγ controls antigen-specific Th17 differentiation in vivo.
Collectively, the entire range of PPARγ-sensitive control of Th17 differentiation in vivo and CNS autoimmunity is reflected by the combination of pharmacological PPARγ activation on the one hand and by the absence of PPARγ-activity in CD4-PPARγKO mice on the other hand.
PPARγ selectively controls Th17 differentiation in T cells from healthy controls (HCs) and MS patients
The protective effects of PPARγ on both clinical manifestation and Th17-responses during EAE prompted us to investigate whether T cells from HCs and MS patients were susceptible to treatment with PPARγ agonists. Again, we focused on the effect of PPARγ activation on T cells by using direct stimulation with TGF-β/IL-21 in the absence of antigen-presenting cells. Pharmacologic PPARγ activation reduced the frequency of IL-17A–producing CD45RA+ CD4+ T cells both in HC and MS patients (). Although in our experiments there was no apparent difference in Th17 differentiation between HC and MS patients in vitro, it is important to note that PIO-treatment was equally effective in potent suppression of IL-17A release from T cells (). Moreover, no influence of PIO was observed during IFN-γ production (). Pharmacologic PPARγ activation prevented Th17 differentiation, as demonstrated by diminished expression of the Th17 markers IL-17F, IL-21, IL-22, and IL-23R upon PIO treatment (). Importantly, the specific effect of PPARγ activation on Th17 induction in human CD4+ T cells was further illustrated by selective regulation of RORγt expression, whereas T-bet and GATA-3 expression were not altered by PIO ().
Figure 5. PPARγ selectively controls Th17 differentiation in T cells from HCs and MS patients. CD45RA+ CD4+ T cells from HC (n = 5) and from relapsing-remitting MS patient (n = 7) were treated with PIO and stimulated as described in Materials and methods. (more ...)
In summary, we identify PPARγ as a defined molecular target to selectively modulate Th17 differentiation in a T cell–intrinsic fashion, which opens up new possibilities for specific immunointervention in Th17-mediated autoimmune diseases such as MS.