The Journal of Immunology Antagonizing Peroxisome Proliferator–Activated Receptor a Activity Selectively Enhances Th1 Immunity in Male Mice Monan Angela Zhang,* Jeeyoon Jennifer Ahn,* Fei Linda Zhao,* Thirumahal Selvanantham,* Thierry Mallevaey,* Nick Stock,† Lucia Correa,† Ryan Clark,† David Spaner,*,‡,x and Shannon E. Dunn*,{,‖ Females exhibit more robust Th1 responses than males. Our previous work suggested that this sex disparity is a consequence of higher activity of the androgen-induced gene peroxisome proliferator–activated receptor a (PPARa) in male CD4+ T cells. The objective of this study was to elucidate the cellular and molecular mechanism of how PPARa inhibits Th1 responses in male mice. In this study, we found that PPARa functions within CD4+ and CD8+ T lymphocytes and NKT cells to negatively regulate IFN-g responses in male mice and identified Ifng as the gene target of PPARa repression. Treatment of male CD4+ T cells with the PPARa agonist fenofibrate induced the recruitment of PPARa and the nuclear receptor-interacting protein, nuclear receptor corepressor 1, to specific cis-regulatory elements in the Ifng locus. This recruitment associated with reduced histone acetylation at these sites. Knockdown of nuclear receptor corepressor 1 in primary male T cells abolished the effect of fenofibrate in reducing IFN-g production. In contrast, treatment of male T cells with IS001, a novel antagonist of PPARa, increased Ifng gene expression and histone acetylation across the Ifng locus. Finally, we investigated the effects of IS001 on IFN-g responses in mice during infection with the Th1-associated pathogen Listeria monocytogenes and observed that IS001 enhanced IFN-g production by NKT, CD4+, and CD8+ T cells and improved the survival of male, but not female, mice. Our findings provide a novel mechanism of why IFN-g responses are more robust in females and introduce a small-molecule IS001 that can be used to enhance Th1 immunity in males. The Journal of Immunology, 2015, 195: 5189–5202. helper cells are classified into distinct subsets based on the 1) directly transactivating Ifng; 2) mediating epigenetic changes expression of effector cytokines: IFN-g for Th1, IL-4 for that make chromatin more accessible at the Ifng locus; and 3) T Th2, and IL-17A for Th17 cells (1). Naive CD4+ T cells increasing CD4+ T cell responsiveness to IL-12 (1–3). In addition acquire these distinct Th cell fates to orchestrate the appropriate to being the major Th1 effector cytokine, IFN-g serves as an immune response against invading pathogens (1). Th1 cells are amplification signal in the Th1 pathway to further enhance T-bet specialized for the generation of cellular immunity against intra- expression (4). by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. cellular pathogens and are generated upon Ag receptor engage- One intriguing feature of Th1 immune responses is that they are ment in the presence of IL-12 (1). During Th1 differentiation, the more robust in females than in males (5–8). This sex difference is IFN-g receptor–STAT1 signaling pathway converges with the thought to underlie why women generate enhanced antiviral (9) TCR signaling pathway to induce expression of T-bet, the master and antitumor (10) immune responses, but also have a higher regulator of Th1 programming. T-bet promotes Th1 immunity by: propensity to develop certain autoimmune diseases (8). Past studies have suggested that sex differences in Th1 responses arise *Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, because of suppressive effects of androgens at key nodes in the Canada; †Inception Sciences, San Diego, CA 92121; ‡Sunnybrook Health Sciences Th1 differentiation pathway including IL-12 production (11) and Centre, Toronto, Ontario M4N 3M5, Canada; xDepartment of Medical Biophysics, http://classic.jimmunol.org University of Toronto, Toronto, Ontario M5G 1L7, Canada; {Toronto General Re- signaling (12, 13) and IFN-g production downstream of Ag search Institute, University Health Network, Toronto, Ontario M5G 2M1, Canada; stimulation (6, 7). Until recently, the molecular players involved in ‖ and Women’s College Research Institute, Toronto, Ontario M5G 2M1, Canada this androgen-dependent regulation were not known. Received for publication February 23, 2015. Accepted for publication September 22, We previously identified that the nuclear receptor peroxisome 2015. proliferator–activated receptor a (PPARa) is expressed at higher This work was supported by a Canadian Institutes for Health Research grant (MOP- levels by male compared with female T cells both in humans and 97807) and the Arthritis Research Foundation through a generous donation from Downloaded from Janice Wright (both to S.E.D.). M.A.Z., J.J.A., and F.L.Z. were supported by stu- in mice and acts to limit Th1 cytokine production exclusively in dentships and S.E.D. by a Don Paty award from the Multiple Sclerosis Society of the male sex (6, 7). We further showed that expression of PPARa Canada. M.A.Z. was also supported by an Ontario Health Scholar Award. is sensitive to androgen levels (6, 7) and that deficiency of this Address correspondence and reprint requests to Dr. Shannon E. Dunn, Toronto Gen- eral Research Institute, 67 College Street, Room 4-421, Toronto, ON M5G 2M1, gene leads to heightened Th1 responses and more severe acute Canada. E-mail address: [email protected] experimental autoimmune encephalomyelitis in male, but not fe- The online version of this article contains supplemental material. male mice (6). Despite these advances, the precise mechanism of Abbreviations used in this article: ChIP, chromatin immunoprecipitation; CNS, con- how PPARa limits Th1 immune responses is unknown. served noncoding sequence; Eomes, eomesdermin; H4-Ac, acetylated histone 4; Past studies have shown that PPAR family members (PPARa, HDAC, histone deacetylase; H3K27-Me, trimethylated histone 3 lysine 27; Inos, inducible NO synthase; MOG, myelin oligodendrocyte glycoprotein; NCOR, nuclear PPARd, and PPARg) function to activate or repress target genes receptor corepressor 1; PPARa, peroxisome proliferator–activated receptor a; ROR, by regulating gene transcription (14). Positive regulation of target retinoic acid receptor–related orphan receptor; RXR, retinoid X receptor; siRNA, genes occurs through the binding of PPARs and retinoid X re- small interfering RNA; Teff, effector T; Treg, regulatory T; WT, wild-type. ceptor (RXR) heterodimers to PPAR responsive elements in gene Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 promoter regions (14). The binding of lipid ligands activates www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500449 5190 PPARa REGULATES SEX DIFFERENCES IN IFN-g PRODUCTION PPARs by promoting an allosteric change in the ligand-binding In vitro T cell stimulations, cytokine measurements, and flow domain of the receptor that supports an increased association with cytometry coactivator proteins and a decreased association with corepressor CD4+ and CD8+ T cells were isolated from spleens and lymph nodes of proteins such as nuclear receptor corepressor 1 (NCOR) (14). In mice using magnetic beads (Miltenyi Biotec or Stemcell Technologies). contrast, the repressive activities of PPARs feature more in in- The purity of isolated cells was routinely monitored and ranged between flammation control and instead occur through the indirect 94 and 98%. Purified cells were resuspended in complete RPMI 1640 binding of PPARs to gene regulatory regions: a mechanism media or X-VIVO-20 media (Lonza) supplemented with 2 mmol/l L-glu- tamine and were cultured in 96-well flat-bottom plates (2 3 105/well) that that has been termed “transrepression” (14). The molecular de- were precoated with anti-CD3 (clone 145-2C11) and anti-CD28 (clone tails of this transrepression mechanism are less understood, but 37.51) (each at 0.5 mg/ml). Th polarizations were conducted as described have been resolved for certain gene targets of PPARg including previously (7) using the following cytokines and Abs from eBioscience: inducible NO synthase (Inos) and retinoic acid–related orphan 10 ng/ml IL-12 + 10 mg/ml anti–IL-4 (clone 11B11) for Th1-, 10 ng/ml IL-4 + 10 mg/ml anti–IFN-g (clone XMG1.2) for Th2-, or 3 ng/ml TGF-b + receptor (ROR) gt(Rorc) (15, 16). For example, it has been shown 30 ng/ml IL-6 + 10 mg/ml anti–IFN-g +10mg/ml anti–IL-4 (clone 11B11) that treatment of macrophages with the ligand rosiglitazone in- for Th17-polarizing conditions. Cytokine levels in culture supernatants duces the sumoylation and recruitment of PPARg to the Inos were measured using Ready-SET-Go! ELISA kits (eBioscience). PMA/ promoter, where it stabilizes the presence of the histone deacet- ionomycin stimulations and cell surface and intracellular cytokine stain- ings were performed as described previously (19, 20). Intranuclear staining ylase (HDAC) 3/NCOR-containing corepressor complex, thereby for transcription factors was performed using the Foxp3 Transcription inhibiting gene transcription (15). Whether PPARa works via a Factor Staining Buffer Set (eBioscience). The following Abs and tetramers similar mechanism to repress Th1 responses in males is not were used in this study and were purchased from eBioscience unless known. otherwise noted: CD4-PE–Cy5 (GK1.5), CD8-FITC (53-6.7), TCRb-PE– The aim of this study was to dissect the cellular and molecular Cy7 (H56-597), IFN-g–PE (XMG1.2), NKp46-allophycocyanin–eFluor780 (29A1.4), NKp46–Alexa Fluor 700 (29A1.4; BD Biosciences), GATA-3–PE mechanism of how PPARa inhibits Th1 immunity in male mice (TWAJ), T-bet–PE-Cy7 (4B10), RORgt-PE (B2D), and unloaded and and to explore the utility of a novel small-molecule antagonist of PBS57-loaded mCD1d tetramer–allophycocyanin (National Institutes of PPARa to enhance Th1 immune responses. Health Tetramer Core Facility). Data were acquired using an LSRII (BD Biosciences) (Sickkids Flow Cytometry facility) and analyzed using FlowJo Materials and Methods software (V9+) (Tree Star).