Peroxisome proliferator-activated receptor (PPAR)α and -γ regulate IFNγ and IL-17A production by human T cells in a sex-specific way

Sex Bias in CD4⁺ T-Cell Expansion and Th Cytokine Production.

SJL is one of the few mouse strains that exhibits a female bias in EAE development (8, 9). We, thus, used this strain as a model system to explore the basis of the sex differences in Th cytokine production. We immunized male and female mice of this strain with proteolipid protein (PLP) p139-151 in complete Freund’s adjuvant (CFA) and examined the recall proliferation and cytokine responses of splenic and draining lymph node cells to PLP p139-151 ex vivo. We detected PLP p139-151-reactive cells primarily in the spleens of males and in both the spleen and draining lymph nodes of females at 8 d postimmunization, indicating possible sex differences in the trafficking of these cells in vivo (Fig. 1 A and B). However, when considered together, splenocytes and lymph node cells cultured from female mice clearly expanded more than male T cells (Fig. 1 A and B). T regulatory cells (T regs) (CD25⁺FoxP3⁺CD4⁺) also tended to expand more in female mice (Fig. S1A).

Consistent with previous reports (8, 9), we observed that female SJL PLP p139-151-reactive T cells produced higher levels of IFNγ compared with male cells (Fig. 1 A and B). However, instead of exhibiting a Th2 bias (no IL-4 was detected), male splenic PLP p139-151-reactive T cells tended to produce more IL-17A than female cells (Fig. 1B). We also followed several male and female mice to the acute stage of EAE and examined the cytokine production by spinal cord-infiltrating CD4⁺ cells. We observed a higher frequency CD4⁺ cells that produced IFNγ (mainly IFNγ- and IL-17A-coproducing CD4⁺ cells) in the central nervous system of female as compared with male mice (Fig. S1C). Thus, these results indicate that Th cells from female SJL mice have a higher propensity to expand in general and produce more IFNγ and less IL-17A than male T cells. The discrepancy between our results and past studies that indicated a Th2 bias in male SJL mice may relate to the fact that: (i) past studies were conducted before the discovery of Th17 cells; and (ii) the levels of IL-4 reported in these studies were measured in cultures of whole lymph nodes or spleens (8) or by IL-2 expanded T-cell lines (9).

Androgen Dependence of Sex Difference in Th Cytokine Production.

Castration of male mice worsens EAE in males (17), whereas ovariectomy does not have a major impact on this disease in female mice (18, 19). Such observations suggest that it is the higher level of androgens in males that is protecting this sex from EAE development. We, thus, explored the effect of castration on Th proliferation and cytokine production. Male SJL mice (4–5 wk of age) were castrated or provided a sham surgery. When these mice reached adulthood, they were immunized with PLP p139-151 and CFA, and, as before, the recall responses to antigen were measured. We found that castration was associated with a “feminization” of the Th profile in male mice, accompanied by an enhanced expansion of PLP p139-151-reactive T cells over sham-operated counterparts (Fig. 2A) and a selective outgrowth of IFNγ- vs. IL-17A-producing cells (Fig. 2 B vs. C). Castrated mice that were followed to the development of EAE symptoms were also found to exhibit a higher ratio of IFNγ- to IL-17-producing T cells in the central nervous system as compared with sham mice (Fig. 2D). These observations suggest that androgens shift Th cytokines from Th1 to Th17.

Cellular Basis of T-Cell Expansion and Cytokine Production.

To investigate the cellular basis of the sex difference in Th proliferation and cytokine production, we took advantage of the fact that female SJL T cells do not react against the male Y-linked histocompatibility (HY) antigen (10) and conducted coculture experiments of male or female antigen-presenting cells (APCs) with naïve male or female PLP p139-151-specific T-cell receptor transgenic (TCR Tg) T cells. We found that cultures that contained female APCs and female naïve CD4⁺ T cells exhibited the highest proliferation to PLP p139-151 (Fig. S2A). On the other hand, the production of Th cytokines more closely mapped with the sex of the T cells in these assays (Fig. S2A). Cultures that contained female T cells produced higher levels of IFNγ and IL-12p40, whereas the cultures that contained male T cells instead exhibited higher levels of IL-17A at the lowest PLP p139-151 concentration tested (Fig. S2A). Again, the levels of IL-4 and IL-10 were low and not found to be different between groups (Fig. S2A). Similar results were observed when we cultured purified CD4⁺ T cells in isolation with anti-CD3 and anti-CD28 (Fig. 1C). Furthermore, the Th17 bias by male CD4⁺ T cells was evident regardless of the tissue source of cells (Fig. S2B). Collectively, these results suggest that two mechanisms are operating to regulate the sex differences in Th proliferation and cytokine production: (i) a mechanism that regulates the expansion of T cells that that is determined by the sex of the APCs and the T cell; and (ii) a mechanism that regulates Th cytokine production that is determined primarily by the sex of the CD4⁺ T cell. Herein, we focused on the T-cell-intrinsic mechanisms that regulate sex differences in the production of IFNγ and IL-17A.

PPARα Is an Androgen-Sensitive Regulator of Th Cytokine Production.

We found previously that PPARα has a male-specific role in dampening Th1 cytokine production in mice (20). To address whether PPARα is an intermediary of androgen effects in shifting the Th cytokine production from Th1 → Th17, we “re-programmed” female WT or PPARα^−/− mouse T cells in vivo with the androgen receptor agonist 5α-dihydrotestosterone (DHT). Three weeks later, we isolated splenic CD4⁺ T cells from mice and, as before, stimulated them in vitro with anti-CD3 and anti-CD28. We observed that DHT-treated WT T cells displayed higher expression of PPARα mRNAs compared with placebo control cells (Fig. 2E). This was associated with lowered IFNγ and higher IL-17A production by WT T cells (Fig. 2 F and G). Interestingly, the decrease in the potential to make IFNγ in response to androgen treatment did not occur in PPARα^−/− T cells, whereas the increased potential to make IL-17A did (Fig. 2 F and G). These results indicate that PPARα may be a molecular intermediary of androgen effects on IFNγ, but not IL-17A.

Human Female CD4⁺ T Cells Are Th1-Prone.

We next set out to investigate whether human CD4⁺ T cells exhibit a sex difference in Th cytokine production. The variability associated with past reports of sex differences in human Th cytokine production (12–16) may be attributed to the fact that cytokine production was assayed in whole blood or in cultures of peripheral blood mononuclear cells (PBMCs), which are comprised of mixtures of cells that are present at varying frequencies in individuals. We, thus, investigated cytokine production by naive CD4⁺CD45RA⁺ T cells in response to anti-CD3 and anti-CD28 stimulation. This stimulus was titrated to produce cytokine levels and proliferative rates that were within the dynamic range of these assays (Fig. S3A). Healthy men and women were recruited in a pair-wise fashion with one woman and one man donating a blood sample each day (n = 25 pairs) to control for the day-to-day variability. Furthermore, to further reduce noise in the system, we controlled for the time of the menstrual cycle in women (follicular phase) (Fig. S3 B and C).

Consistent with our findings in SJL mice, we observed that naïve CD4⁺ T cells of women secreted significantly higher levels of IFNγ compared with men in response to anti-CD3 and anti-CD28 stimulation (Fig. 3A). In fact, 20 of the 25 women sampled exhibited higher production of IFNγ than their male pair (Fig. 3F). In females, we also detected a higher frequency of IFNγ-producing T cells and significantly higher T-cell expression of IFNγ mRNAs than male counterparts (Fig. 3 B and C and Table S1). A trend for higher proliferative rates by female T cells was also observed (Table S1). Although the level of IL-17A detected in these cultures under Th0 conditions was low and not different between the sexes (Fig. 3D), when IL-17A production was examined in individual pairs, we observed a tendency for higher production of this cytokine in cultures of male vs. female T cells (Fig. 3G). A trend for higher IL-17A mRNA expression by male T cells was also observed (Fig. 3E). As in the SJL mouse, the productions of Th2 cytokines were low and not found to be different between the sexes (Table S1).

To further investigate the male Th17 bias, we explored the production of IL-17A in two T culture systems associated with enhanced production of this cytokine: (i) naïve CD4⁺ T cells activated with anti-CD3 plus peptidoglycan-matured autologous monocytes (21); and (ii) memory CD4⁺ T cells that were cultured with anti-CD3 plus autologous monocytes before expansion with IL-23 (6). In both of these systems, we observed a higher propensity for IL-17A production when donor cells were derived from men compared with women (Fig. S4). Thus, the presence of male monocytes amplified the sex difference in IL-17A that was specified by TCR- and CD28-elicited signals. Altogether, these findings in human T cells align with our results in the murine system and suggest a Th1 bias in females and a Th17 bias in males.

Regulation of Human PPARα by Androgens and TCR Stimulation.

Next, we focused on a possible role for PPARα in the Th1 bias in women. First, we investigated whether PPARα exhibits sexual dimorphic expression in human CD4⁺ T cells and then followed with experiments to address whether this gene is sensitive to androgen levels. We found that this gene was expressed at low levels in naïve CD4⁺ T cells of both sexes. However, PPARα mRNAs were strikingly up-regulated in T cells upon TCR and CD28 stimulation, particularly in males (Fig. 4 A and B). Next, we stimulated CD4⁺ T cells from men and women in the presence of either 100 nM DHT or vehicle, as well as examined the relationship of PPARα mRNAs with circulating testosterone and 17-β-estradiol levels in men and women. We found that in vitro DHT treatment enhanced PPARα expression in T cells of women but did not alter the expression of this gene in T cells of men (Fig. 4C). In addition, we observed that PPARα mRNA expression in freshly isolated male T cells tended to associate with circulating levels of testosterone (P = 0.07) but not 17-β-estradiol levels (Fig. S5). The lack of sensitivity of male T cells to in vitro DHT treatment may relate to the fact that these cells were taken from an environment that already contained high levels of androgens.

To address the molecular basis of androgen-sensitivity human PPARα, we used Alibaba2 prediction software (www.gene-regulation.com/pub/programs.html) to search for hormone-responsive elements in promoter region of this gene. This search revealed the presence of an androgen receptor (AR)/glucocorticoid receptor-binding site in close proximity to an estrogen-binding site in the human and mouse promoters (Fig. 4D). Chromatin immunoprecipitation (ChIP) experiments revealed that DHT and anti-CD3 and -CD28 signals act in synergy to induce the recruitment of the AR to the human PPARα promoter in primary T cells (Fig. 4 E and F). These studies suggest a direct effect of androgens in increasing PPARα mRNAs.

PPARα Represses IFNγ Production by Human T Cells.

To address whether PPARα functions in human T cells to repress IFNγ, we transfected primary T cells of men and women (n = 10/group) with PPARα-specific siRNAs before activating these cells with anti-CD3 and anti-CD28. Approximately half of T cells (52.5 ± 3.0% in men and 56.1 ± 3.8% in women) were transfected using our approach, resulting in a ∼70–75% reduction of T-cell PPARα mRNAs in T cells of both sexes (Fig. 4G). When stimulated with anti-CD3 and anti-CD28, male T cells transfected with PPARα siRNAs produced higher levels of IFNγ compared with those transfected with the nontargeting siRNAs (Fig. 4 H and I). This effect was not observed in women (Fig. 4H), and appeared to be specific to IFNγ (Table S2). Similar results were obtained when siRNA transfection studies were conducted in SJL mice (Fig. S6).

We also investigated whether the PPARα ligand fenofibrate would have sex-specific effects in limiting IFNγ production. We found that fenofibrate reduced IFNγ levels by WT but not by PPARα^−/− T cells when administered at concentrations from 1 to 5 μM (Fig. S7A). This same drug also had a sex-specific effect in decreasing IFNγ production by male but not female T cells both in mice (Fig. S7B) and humans (Fig. 4J) when provided at a 5 μM dose. Together with siRNA findings, these results validate that PPARα has a sex-specific role in the negative regulation of T-cell IFNγ production.

Mechanism of Repression of Human IFNγ by PPARα.

The induction of expression of the IFNγ gene is regulated by both TCR- and cytokine-dependent signals acting at multiple genomic elements, located far upstream and downstream of this gene (22, 23). These genomic elements comprise a number of highly conserved noncoding sequences (CNSs) that contain binding sites for transcription factors including T-box expressed in T cells (T-bet) and signal transducer and activator of transcription (STAT)4 (22, 23). Under Th1 conditions, T-bet and STAT4 play key roles in the establishment of histone marks at these regions, thus permitting chromatin opening, transcription factor binding, and recruitment of the transcriptional activation machinery (22, 23). To gain insight into how PPARα may factor into this process, we used ChIP to assess the abundance of the activating histone mark acetylated-histone H4 at two well-characterized CNS regions, CNS-22 and CNS-6, in male T cells that were transfected with PPARα or nontargeting siRNAs (Fig. S8A). We found that T cells transfected with PPARα siRNAs displayed an enhanced abundance of acetylated-histone H4 at both of these CNS regions after anti-CD3 and anti-CD28 stimulation (Fig. S8 B and C). Thus, one way that PPARα may operate to repress IFNγ production is through epigenetic changes in the IFNγ locus that make it less poised for IFNγ transcription.

In light of our previous findings of a higher abundance of RelA (reticuloendotheliosis viral oncogene homolog A) in the nucleus of PPARα^−/− T cells (20), we further investigated whether this transcription factor is recruited in increased amounts to these CNS regions. Using ChIP, we detected an enhanced abundance of RelA at these sites in T cells that were transfected with the PPARα siRNAs relative to those transfected with the nontargeting siRNAs (Fig. S8D). This occurred even in quiescent T cells (Fig. S8D). Taken together, our findings suggest that PPARα actions on the IFNγ gene are very complex and involve effects on upstream TCR-signaling intermediates in addition to epigenetic changes at the IFNG locus.

PPARγ Is a Potential Sex-Dependent Regulator of Th17.

Our findings in mice indicated that PPARα expression is not responsible for the higher IL-17A production by male T cells. Indeed, recent reports have implicated roles for a number of other nuclear receptors in the control of IL-17A production [i.e., retinoic acid-related orphan receptor (ROR)γT, vitamin D receptor, retinoic acid receptor, liver X receptor, and PPARγ1] (24). With the knowledge that PPARγ exhibits a female preponderance in certain adipose stores (25, 26), we investigated whether the immune-expressed PPARγ variant, PPARγ1 is expressed by human CD4⁺ T cells in sex-specific way or is sensitive to androgen levels. In contrast to our findings for PPARα, we observed that PPARγ1 mRNAs were down-regulated more strikingly in T cells of men upon anti-CD3 and anti-CD28 stimulation (Fig. S9A). Furthermore, in vitro DHT treatment inhibited the T-cell expression of this gene in mice and humans, suggesting that PPARγ1 is tuned down in T cells by antigen and androgen signals (Fig. S9 B and G).

Using the approach of siRNA transfection and ligand activation, we next explored whether the control of T-cell IL-17A production by PPARγ1 is sex-dependent. As for our studies for PPARα, we isolated T cells from males and females of both species, transfected these cells with either PPARγ or nontargeting siRNAs and then stimulated cells with anti-CD3 and anti-CD28, this time in the presence of Th17-skewing cytokines. Although we did not detect a sex difference in IL-17A production by the human CD4⁺ T cells transfected with nontargeting siRNAs (Fig. S9E), we did observe this effect in mice (Fig. S9H). Furthermore, in both the murine and human systems, knockdown of PPARγ1 mRNAs had a more profound effect in increasing IL-17A production by T cells of females vs. males (Fig. S9 E and H). Finally, we observed that the synthetic PPARγ ligand rosiglitazone reduced IL-17A production by CD4⁺ T cells of women but not men (Fig. S9F). Altogether, these data suggest that PPARγ1 may be one molecule that may have sex-specific effects in limiting the production of IL-17A by female T cells.