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The Journal of Immunology, 2004, 172: 7530-7536.
Copyright © 2004 by The American Association of Immunologists

Repression of IFN-{gamma} Expression by Peroxisome Proliferator-Activated Receptor {gamma}1

Robyn Cunard2,*,{ddagger}, Yoko Eto*, Julie T. Muljadi*, Christopher K. Glass{dagger},{ddagger}, Carolyn J. Kelly*,{ddagger} and Mercedes Ricote{dagger}

* Research Service and Division of Nephrology-Hypertension, Veterans Affairs San Diego Healthcare System, San Diego, CA 92161; and {dagger} Departments of Cellular and Molecular Medicine and {ddagger} Medicine, University of California, San Diego, CA 92093


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in a wide variety of cells. Our studies and others have demonstrated that both human and murine T cells express PPAR{gamma} and that expression can be augmented over time in mitogen-activated splenocytes. PPAR{gamma} ligands decrease proliferation and IL-2 production, and induce apoptosis in both B and T cells. PPAR{gamma} ligands have also been shown to be anti-inflammatory in multiple models of inflammatory disease. In the following study, we demonstrate for the first time that PPAR{gamma} is expressed in both murine CD4 and CD8 cells and that PPAR{gamma} ligands directly decrease IFN-{gamma} expression by murine and transformed T cell lines. Unexpectedly, GW9662, a PPAR{gamma} antagonist, increases lymphocyte IFN-{gamma} expression. Transient transfection studies reveal that PPAR{gamma} ligands, in a PPAR{gamma}-dependent manner, potently repress an IFN-{gamma} promoter construct. Repression localizes to the distal conserved sequence of the IFN-{gamma} promoter. Our studies also demonstrate that PPAR{gamma} acts on the IFN-{gamma} promoter by interfering with c-Jun activation. These studies suggest that many of the observed anti-inflammatory effects of PPAR{gamma} ligands may be related to direct inhibition of IFN-{gamma} by PPAR{gamma}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Peroxisome proliferator-activated receptors (PPARs)3 are ligand-activated transcription factors expressed in a wide variety of cells (1). The PPAR subfamily of receptors is encoded by three genes; {alpha}, {delta} ({beta} and NUC1), and {gamma}, each with distinct ligands and expression patterns (2). Our studies and others have demonstrated that both human and murine T cells express PPAR{gamma} and that expression can be augmented over time in mitogen-activated splenocytes (3, 4, 5, 6, 7, 8). PPAR{gamma} ligands decrease proliferation and IL-2 production, and induce apoptosis in both B and T cells (3, 4, 5, 8).

Ligands for PPAR{gamma} are lipophilic molecules, and putative endogenous ligands include 15-deoxy-{Delta}(12,14)-PGJ2, 13-hydroxyoctadecadienoic acid (13-HODE), and 15-hydroxyeicosatetraenoic acid (9, 10). Thiazolidinediones are synthetic PPAR{gamma} ligands used clinically to treat type 2 diabetes (11). PPAR ligands exert effects in multiple metabolic pathways and adipocyte and macrophage differentiation (12, 13). PPAR{gamma} ligands have also been shown to be anti-inflammatory in models of inflammatory bowel disease (14, 15, 16), adjuvant-induced arthritis (17, 18), experimental autoimmune encephalomyelitis (19, 20, 21), ischemia-reperfusion (22), and atherosclerosis (23, 24, 25). Early studies suggested that these ligands exerted direct effects on macrophages/monocytes (26, 27), and endothelial (28), colonic (14), and smooth muscle cells (29, 30). However, increasing evidence suggests that PPAR{gamma} ligands may mediate their anti-inflammatory actions at least in part through their effects on T cells.

IFN-{gamma} plays a central role in inflammatory reactions and is predominately produced by CD4, CD8, and NK cells (31). IFN-{gamma} drives inflammatory reactions by stimulating the release of NO, TNF-{alpha}, and IL-1{beta} by monocytes/macrophages. IFN-{gamma} is also a major effector cytokine, responsible for driving cell-mediated immunity and mediating organ-specific autoimmunity (32). Recent studies have shown that PPAR{gamma} ligands inhibit IFN-{gamma} production by T lymphocytes; however, the mechanism underlying this observation has not been clarified (4, 6, 16, 21, 33, 34, 35, 36). Based on previous studies, PPAR{gamma} ligands could indirectly decrease IFN-{gamma} by inhibiting activation of T cells, production of IL-2, or induction of apoptosis (3, 5, 8), or inhibiting IL-12 production by APCs (37, 38, 39, 40).

In the following study, we demonstrate for the first time that PPAR{gamma} is expressed in both murine CD4 and CD8 cells and that PPAR{gamma} ligands directly decrease IFN-{gamma} expression by murine and transformed T cell lines. In contrast, GW9662, a PPAR{gamma} antagonist, increases IFN-{gamma} expression. Transient transfection studies reveal that PPAR{gamma} ligands, in a PPAR{gamma}-dependent manner, potently repress an IFN-{gamma} promoter construct. Repression localizes to the distal conserved sequence (DCS) of the minimal IFN-{gamma} promoter. Our studies also demonstrate that PPAR{gamma} acts on the minimal IFN-{gamma} promoter by interfering with c-Jun activation. These studies suggest that many of the observed anti-inflammatory effects of PPAR{gamma} ligands may be related to direct inhibition of IFN-{gamma} by PPAR{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The mice were used between 6 and 24 wk of age. Mice were housed and handled in accordance with Department of Veterans Affairs and National Institutes of Health guidelines under Institutional Animal Care and Use Committee approved protocols.

Reagents and cell lines

Reagents used included Con A, PMA, and ionomycin from Sigma-Aldrich (St. Louis, MO), and BRL49,653, troglitazone, and GW9662 (41) from Cayman Chemicals (Ann Arbor, MI). GW742, a PPAR{delta} ligand, was a kind gift from T. Willson (GlaxoSmithKline, Research Triangle Park, NC). An EL4.IL-2 cell line from American Type Culture Collection (TIB-39; Manassas, VA) and a Jurkat TAg cell line that is stably transfected with the SV40 large T Ag (42) were used.

Cytokine ELISAs

EL4.IL-2 cells were plated in 96-well plates at 0.1 x 106 cells/well in RPMI 1640 (Invitrogen, San Diego, CA) supplemented with 10% heat-inactivated FCS (Atlanta Biologicals, Norcross, GA), 5.5 x 10–5 M 2-ME (Invitrogen), 130 U/ml penicillin, 130 µg/ml streptomycin, and 2.5 mM L-glutamine (Invitrogen) (RPMI-C). Cells were treated with PPAR{gamma} agonists and antagonists, and stimulated with PMA (25 ng/ml) and ionomycin (1 µM). After 72-h incubation, culture supernatant concentrations of murine IFN-{gamma} were determined by sandwich ELISA with the OPTEIA set (BD PharMingen, San Diego, CA).

Intracellular cytokine staining

BALB/c mice spleens were prepared into single cell suspensions, as previously described (4). Ninety hours after Con A stimulation, splenocytes were restimulated with PMA (25 ng/ml) and ionomycin (1 µM). After 2 h, they were treated with Golgi Plug (Brefeldin A; BD PharMingen), harvested 4 h later, fixed, and permeabilized using the Cytofix/Cytoperm kit (BD PharMingen). Cells were then stained with FITC CD3{epsilon}, PE IFN-{gamma}, and PE IFN-{gamma} isotype control (BD PharMingen), and flow cytometry was performed by the Veterans Affairs San Diego Healthcare System Core Flow Cytometry facility. The isotype control was subtracted from all of the PE IFN-{gamma} levels reported.

TaqMan PCR

BALB/c splenocytes stimulated with Con A were harvested at 72 h, stained with FITC anti-CD4 and PE anti-CD8{alpha} (BD PharMingen), and separated by MOFLO (Cytomation, Fort Collins, CO, Veterans Affairs San Diego Healthcare System Core FACS facility). After sorting, the cells were determined to be >99% pure. RNA was prepared with TRIzol (Invitrogen), then cleaned up with RNeasy Mini kit (Qiagen, Valencia, CA) with on-column DNase digestion. DNase I (Invitrogen) was used again, and cDNA was prepared with the Superscript II Preamplification System (Invitrogen). TaqMan PCR were run by the Center for AIDS Research Genomics Core (University of California, San Diego, and Veterans Medical Research Foundation) using an ABO Prism 7700 Sequence Detector (TaqMan; PerkinElmer, Wellesley, MA; Applied Biosystems, Foster City, CA). A control without reverse transcriptase was used to control for efficiency of the DNase reactions. Equal amounts of cDNA were used in duplicate and amplified with the TaqMan Master Mix. Amplification efficiencies were normalized against GAPDH, and fold increases were calculated by generating a standard curve (23, 43). Each TaqMan experiment was also performed at least three times. Amplification of RNA from EL4.IL-2 cells was performed in a similar manner. Primers for murine IFN-{gamma} were: F, 5'-CAA TGA ACG CTA CAC ACT GCA TCT; R, 5'-CGT GGC AGT AAC AGC CAG AA; and T, 5'-TGG CTT TGC AGC TCT TCC TCA TGG C.

Plasmids and transient transfections

Jurkat T lymphocytes cultured in RPMI-C were transfected with 5 µg of reporter vector by electroporation at 250 V/975 µF using a Bio-Rad electroporator with a capacitance extender (Bio-Rad, Hercules, CA). Three hours after transfection, cells were treated with PPAR ligands in the presence or absence of PMA (25 ng/ml) and ionomycin (1 µM). Luciferase activity was assessed, as described (44).

To establish a positive control for the transfections, the first set of studies investigated whether PPAR ligands could activate the (acyl CoA oxidase)3-thymidine kinase-luciferase construct, which contains three copies of a PPAR response element (PPRE). Transfections were performed with or without 250 ng of the pSG5-PPAR{alpha}, pCMX-PPAR{delta}, and pCMX-PPAR{gamma} expression vectors. The pCMX empty vector was used to control for total amounts of DNA transfectants. The IFN-{gamma} promoter construct (pGL3-IFN-{gamma}-Luc, –541/+111) isolated from a C57BL/6 mouse was used. Truncations of the IFN-{gamma} promoter were prepared by designing primers that bound to relevant sequences of the IFN-{gamma} promoter. After PCR and isolation, these sequences were subcloned back into pGL3-Luc (Promega, Madison, WI). Plasmids containing head to tail (5' to 3') dimers of the IFN-{gamma} proximal conserved sequence (PCS) and four copies of the DCS of the IFN-{gamma} promoter were subcloned into a pIFN-39 basal promoter construct (45). pCMX-PPAR{gamma}-{Delta} activation function 2 (AF2) lacks the last 17 aa of the full-length protein; pCMX-PPAR{gamma}-{Delta}DBD was constructed by deleting the DNA binding domain (DBD) and most of the hinge region of PPAR{gamma} (46). His323 and His449 of PPAR{gamma} form hydrogen bonds with the ligand, and therefore are important for ligand binding and ligand-dependent receptor function. In His-Ala323-His-Ala449, these two His residues were mutated to Ala (46). pRC-CMV-c-Jun and pRC-CMV were also used.

Statistics

Comparisons were performed using Student’s t test for independent samples. However, when the comparison was performed with data that had been normalized, then a one-sample t test was used. All studies were performed at least three times on 3 different days to ensure reproducibility. Analysis was accomplished with Statview v5.0.1 (SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD4 and CD8 T cells express PPAR{gamma}

We have previously shown that at 72 h, Con A-stimulated T cells express PPAR{gamma}. In this study, 72 h after Con A stimulation of BALB/c splenocytes, CD4 and CD8 cells were separated by MOFLO. TaqMan PCR studies demonstrate that PPAR{gamma} is expressed in both the CD4 and CD8 subpopulations (Fig. 1).



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  		FIGURE 1. CD4 and CD8 T cells express PPAR{gamma}. RNA was isolated from CD4, CD8, and total T cell populations (separated by MOFLO). cDNA was prepared, and TaqMan PCR studies were performed. Amplification efficiencies were normalized against GAPDH. GAPDH cDNA was detected at ~22 cycles, whereas the PPAR{gamma} transcript was detected at ~35 cycles. Data are compared as relative fold increase in mRNA compared with CD4 cell mRNA and represent the average ± SEM of three separate experiments.

 
PPAR{gamma} ligands decrease and PPAR{gamma} antagonists increase IFN-{gamma} protein expression by T lymphocytes

Our previous studies demonstrated that a PPAR{gamma} ligand, ciglitazone, inhibits IFN-{gamma} production in mitogen-stimulated splenocytes (4). In the current studies, intracellular cytokine staining was used to confirm that troglitazone, another PPAR{gamma} ligand, decreases the number of IFN-{gamma}-producing cells (Fig. 2, A and B). Surprisingly, GW9662, an antagonist of PPAR{gamma} ligands (41), increases the number of IFN-{gamma}-producing cells (Fig. 2C). This novel finding suggests that IFN-{gamma} expression is inhibited in this culture system by an endogenous PPAR{gamma} ligand. Thus, GW9662 is able to relieve repression, thereby increasing the number of cells producing IFN-{gamma}.



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  		FIGURE 2. PPAR{gamma} agonists decrease, while PPAR{gamma} antagonists increase IFN-{gamma} protein expression. A, BALB/c splenocytes were treated with vehicle (DMSO); B, 10 µM troglitazone (a PPAR{gamma} ligand); or C, 10 µM GW9662 (a PPAR{gamma} antagonist) and stimulated with Con A. Ninety-six hours after stimulation, intracellular cytokine staining was performed using PE IFN-{gamma} and FITC CD3{epsilon}. The numbers in the upper right quadrants depict the percentage of CD3{epsilon}+, IFN-{gamma}+-producing cells. Results are representative of at least three experiments. D, EL4.IL-2 T cells were treated with the PPAR{gamma} ligand BRL49,653 (BRL, rosiglitazone), vehicle control (Veh), or GW9662 (GW), and stimulated with PMA (25 ng/ml) and ionomycin (1 µM). Seventy-two hours after stimulation, cytokine ELISA studies for IFN-{gamma} were performed on culture supernatants. Data represent the average ± SEM of five studies. *, p < 0.05 vs control (Veh 10 µM); **, p < 0.01 vs control.

 
EL-4.IL-2 cells are a T cell line transgenically altered to produce IL-2. These cells produce IFN-{gamma} and IL-4 in response to stimulation (PMA/ionomycin). PPAR{gamma} expression is also up-regulated with PMA and ionomycin (our unpublished data); therefore, they serve as a useful model system to study the immunological effects of PPAR{gamma} ligands. ELISA studies performed demonstrate that treatment of EL-4.IL-2 cells with the PPAR{gamma} ligand, BRL49,653, and PMA and ionomycin decreases supernatant IFN-{gamma} levels, whereas the antagonist, GW9662, increases by >2-fold supernatant IFN-{gamma} levels (Fig. 2D). Again, these results suggest that there is an endogenous PPAR{gamma} ligand present in the T cell culture system. It also helps to confirm that the effect of PPAR{gamma} ligands on IFN-{gamma} expression occurs in a PPAR{gamma}-dependent manner.

IFN-{gamma} mRNA expression is enhanced by PPAR{gamma} antagonists and diminished by PPAR{gamma} agonists

In concert with the previous findings, TaqMan PCR studies performed on RNA isolated from EL4.IL-2 cells demonstrate that the PPAR{gamma} ligand, BRL49,653 (rosiglitazone), decreases, while the PPAR{gamma} antagonist augments IFN-{gamma} mRNA expression (Fig. 3). These studies suggest that PPAR{gamma} may inhibit IFN-{gamma} expression at the transcriptional level.



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  		FIGURE 3. IFN-{gamma} mRNA expression is enhanced by PPAR{gamma} antagonists and diminished by PPAR{gamma} agonists. EL4.IL-2 T cells were treated with vehicle, 10 µM BRL49,653 (BRL, rosiglitazone), or 10 µM GW9662 (GW), and stimulated with PMA (25 ng/ml) and ionomycin (1 µM). RNA was isolated at 24 and 36 h after stimulation, and cDNA was prepared. TaqMan PCR studies were performed with primers corresponding to murine IFN-{gamma}, and normalization was conducted by comparing transcripts with GAPDH. Results are representative of at least four experiments. Error bars represent SDs of duplicate PCR samples.

 
PPAR{gamma} ligands inhibit activation of the IFN-{gamma} promoter

We next determined whether Jurkat T cells could support PPAR-dependent transactivation. The (acyl CoA oxidase)3-thymidine kinase-luciferase plasmid, which contains three copies of a PPRE (26), was transfected into Jurkat cells with either PPAR{alpha}, PPAR{delta}, or PPAR{gamma}. All PPAR isoforms with their specific ligand could activate the PPRE reporter (our unpublished data). Transfection of PPAR{gamma} was required for transactivation, which suggests that these Jurkat T cells do not express sufficient PPAR{gamma} to support trans- activation or repression. Fig. 4 depicts the effect of each PPAR isoform and its specific ligand on the IFN-{gamma} promoter construct (pGL3-IFN-{gamma}, –541/+111). PPAR{gamma} potently inhibits activation of the promoter, and BRL49,653 further suppresses activation. Troglitazone had a similar effect on the IFN-{gamma} promoter construct (our unpublished data). PPAR{alpha} and its ligand WY14,643 had no effect on the IFN-{gamma} promoter construct. PPAR{delta} had a repressive effect on the IFN-{gamma} promoter; however, this did not appear to occur in a ligand-dependent manner.



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  		FIGURE 4. PPAR{gamma} inhibits transcription of the IFN-{gamma} promoter. Jurkat T cells were cotransfected with 5 µg of the IFN-{gamma} promoter construct (pGL3-IFN-{gamma}-Luc) and 250 ng of pCMX-PPAR{gamma} (A), pSG5-PPAR{alpha} (B), and pCMX-PPAR{delta} (C). Cells were stimulated (25 ng/ml PMA and 1 µM ionomycin, P/I) and treated with their respective PPAR ligands: PPAR{gamma} ligand (BRL, BRL49,653), PPAR{alpha} ligand (WY, WY14,643), and PPAR{delta} ligand (GW, GW742). PPAR{gamma} inhibits transcription of the IFN-{gamma} promoter construct in a PPAR{gamma}-dependent manner. Data represent the average ± SEM of at least three studies. *, p < 0.05 vs control (no PPAR{delta}); **, p < 0.01 vs control (no PPAR{gamma}).

 
PPAR{gamma}’s ligand binding and DBD are required for PPAR{gamma}-dependent repression of the IFN-{gamma} promoter

PPAR{gamma} shares similar structural and functional features with other nuclear receptor family members. There is a C-terminal domain that mediates ligand binding, dimerization, and transactivation functions (AF2) and a central DBD (47). To further study which domains are necessary for mediating IFN-{gamma}-dependent repression, we used mutants of PPAR{gamma} in which these domains have been deleted or altered. First, we used pCMX-PPAR{gamma}-{Delta}AF2, in which the ligand binding domain of PPAR{gamma} has been deleted. Fig. 5 demonstrates that this construct was not able to repress promoter activity in the presence or absence of ligand. His323 and His449 of PPAR{gamma} form hydrogen bonds with the ligand, and therefore are important for ligand binding and ligand-dependent receptor function. In His-Ala323-His-Ala449, these two His residues were mutated to Ala (46). Again, this construct was not able to repress stimulated IFN-{gamma} promoter activity. We have previously shown by Western blot that after transfection there is approximately equivalent expression of each PPAR{gamma} mutant (46). Therefore, these studies suggest that PPAR{gamma} must bind ligand to repress IFN-{gamma} promoter activity. PPAR{gamma} was able to inhibit activation of the IFN-{gamma} promoter without addition of exogenous ligand. This again suggests the presence of endogenous ligand in our culture system. Deletion of the DBD of PPAR{gamma} (pCMX-PPAR{gamma}-{Delta}DBD) also abrogated the ability of PPAR{gamma} to repress the activated IFN-{gamma} promoter. However, EMSAs did not show evidence of PPAR{gamma} binding to the IFN-{gamma} promoter (our unpublished data).



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  		FIGURE 5. Both ligand and DBD of PPAR{gamma} are required for repression of the IFN-{gamma} promoter. Jurkat T cells were cotransfected with 5 µg of the IFN-{gamma} promoter construct (pGL3-IFN-{gamma}-Luc) and 250 ng of pCMX-PPAR{gamma}-{Delta}AF2 (PPAR{gamma} lacking the ligand binding domain), pCMX-His-Ala323/His-Ala449 (PPAR{gamma} with mutations in His323 and His449, residues important for ligand binding and ligand-dependent receptor function), and pCMX-PPAR{gamma}-{Delta}DBD (PPAR{gamma} lacking the DBD). The cells were stimulated with PMA (25 ng/ml) and ionomycin (1 µM): P/I. Data represent the average ± SEM of at least three studies.

 
PPAR{gamma}-dependent repression localizes to the minimal IFN-{gamma} promoter

The next set of studies depicted in Fig. 6 was performed to determine which segments in the IFN-{gamma} promoter were critical for PPAR{gamma}-dependent repression. Truncation constructs were designed to successively remove sequences important for IFN-{gamma} gene regulation. All truncation constructs were inhibited by BRL49,653 when PPAR{gamma} was transfected into the system. To maintain inducibility with PMA and ionomycin, the minimal promoter p124 cannot be further truncated. There are two highly conserved regions in the minimal promoter, the PCS and DCS (48). PPAR{gamma} was able to inhibit activity of the construct containing the multimerized DCS, but not the PCS, suggesting that PPAR{gamma}-dependent repression localizes to this area (Fig. 7).



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  		FIGURE 6. PPAR{gamma} inhibits transcription of the minimal IFN-{gamma} promoter. A, T cells were cotransfected with 5 µg of each IFN-{gamma} promoter truncation construct and pCMX-PPAR{gamma} or pCMX. The cells were stimulated (PMA and ionomycin: P/I) and treated with 500 nM BRL49,653 (BRL) or vehicle control. BRL 49,653 inhibits activity of all of the truncation constructs in a PPAR{gamma}-dependent manner. Data represent the average ± SEM of at least three studies. *, p < 0.05 vs control (no PPAR{gamma}); **, p < 0.01 vs control (no PPAR{gamma}). B, Each truncation construct is represented in this figure. Progressive NF-AT and AP-1 sites were deleted to determine which sites are important for PPAR{gamma}-dependent transcriptional regulation of the IFN-{gamma} promoter. p124 is the minimal IFN-{gamma} promoter; YY-1, ying-yang-1 site.

 


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  		FIGURE 7. PPAR{gamma} inhibits the DCS of the minimal IFN-{gamma} promoter. Jurkat T cells were cotransfected with 2.5 µg of the multimerized PCS and the multimerized DCS of the IFN-{gamma} promoter. PPAR{gamma} or the empty vector (pCMX) was also cotransfected. Cells were stimulated with PMA (25 ng/ml) and ionomycin (1 µM): P/I, and treated with BRL49,653 (500 nM). Data represent the average ± SEM of at least three studies. *, p < 0.05 vs control.

 
PPAR{gamma} inhibits c-Jun-mediated induction of the IFN-{gamma} promoter

The DCS of the minimal promoter has been shown to bind CREB/activating transcription factor 2 (ATF2) and AP-1 family members (45, 48, 49, 50). Both CREB and c-Jun activated the DCS and the minimal IFN-{gamma} promoter (our unpublished data). However, PPAR{gamma} was only able to interefere with c-Jun-induced activation of the IFN-{gamma} promoter (Fig. 8). Therefore, PPAR{gamma} inhibits the IFN-{gamma} promoter by inhibiting c-Jun-mediated activation.



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  		FIGURE 8. PPAR{gamma} inhibits c-Jun-mediated activation of IFN-{gamma} promoter. Jurkat T cells were cotransfected with 2.5 µg of the multimerized DCS of the IFN-{gamma} promoter and 5 µg of pCMX-PPAR{gamma}, pCMX, and 2.5 µg of pRC-CMV (empty vector) or pRC-CMV-c-Jun (cJun). Cells were treated with BRL49,653 (500 nM) or vehicle control. Data represent the average ± SEM of at least three studies. *, p < 0.05 vs control (no PPAR{gamma}); **, p < 0.01 vs control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IFN-{gamma} plays a central role in Th1 inflammatory and autoimmune reactions, and modulation of IFN-{gamma} expression can alter the outcome of inflammatory diseases. Our studies demonstrate that agents used extensively to treat type 2 diabetes, PPAR{gamma} ligands, decrease T cell IFN-{gamma} production. Moreover, for the first time, we have shown that a PPAR{gamma} antagonist, GW9662, augments IFN-{gamma} production.

Given its central importance in inflammatory reactions, IFN-{gamma} expression has multiple levels of lineage-specific transcriptional control, including chromatin remodeling (51, 52), DNA methylation (53), and interaction of cis- and trans-acting transcription factors. T-bet, NF-AT, NF-{kappa}B, ying-yang-1, STAT4, AP-1, and members of the CREB/ATF family of transcription factors play important roles in IFN-{gamma} gene transcription (45, 48, 49, 50, 54, 55, 56, 57, 58, 59). Transient transfection studies demonstrate that the region between –108 and –40 (–124/–40 in the mouse, relative to the transcription initiation site; the minimal IFN-{gamma} promoter) is required for full transcriptional activity of the IFN-{gamma} promoter (48).

In our studies, we have shown by deletion analysis that the minimal IFN-{gamma} promoter is the site responsive to PPAR{gamma}-dependent repression. TCR engagement activates two highly conserved regions in the minimal promoter, the PCS and DCS (48). The distal sequence binds AP-1 and CREB/ATF family members and may bind GATA and Oct-1 (48, 49), whereas the PCS binds CREB, ATF-1, ATF-2, and Jun family members (45). Investigators using reporter transgenic mice expressing the luciferase gene under control of a multimerized DCS of the IFN-{gamma} promoter showed that this element is active in memory and not naive T cells. In CD4+ T cells, IL-4 priming partially inhibits distal IFN-{gamma} transcriptional activity (49, 59). It is tempting to postulate that IL-4 could up-regulate the expression of PPAR{gamma} (4), thereby repressing IFN-{gamma} gene transcription. In contrast, Murphy and colleagues (52), using transgenic mice with 3.4-kb retroviral IFN-{gamma} promoter inserts, showed that the IFN-{gamma} promoter was constitutively active in both Th1 and Th2 cells. Interestingly, the site of constitutive activity resided within the DCS. EMSAs and transfection studies suggest that CREB/ATF family members inhibit, whereas c-Jun activates transcription of the DCS (59). Our studies show that repression of PPAR{gamma} is localized to the DCS. These findings are in concert with studies performed on CD4 T cells, in which ciglitazone, a PPAR{gamma} ligand, interferes with AP-1 and NF-{kappa}B activation of the IFN-{gamma} promoter (35).

Multiple stimuli, including growth factors, cytokines, and PMA, induce AP-1 activity and activate mitogen-activated protein kinase (MAPK) cascades (60). Interestingly, several proteins with different abilities to activate transcription can form complexes at AP-1 sites; thus, AP-1 activation may not be associated with alterations in DNA binding (60). Furthermore, transcriptional activation of c-Jun is enhanced when it is phosphorylated in its N-terminal region by c-Jun N-terminal kinase and p38 MAPK cascades (60).

In our studies, we have shown that PPAR{gamma} inhibits c-Jun activation of the DCS. Also, repression of the IFN-{gamma} promoter was abrogated by deletion of the DBD of PPAR{gamma} (Fig. 5). However, using oligonucleotides corresponding to the distal and proximal regulatory sequences, EMSAs have not shown alterations in DNA binding associated with PPAR{gamma} agonist or antagonist treatment (our unpublished data). EMSAs also did not show evidence of PPAR{gamma} binding to the DCS or the PCS. This is similar to glucocorticoid receptor-mediated repression of NF-{kappa}B activation of the ICAM promoter. Both DNA and ligand binding domains are required for repression, even though the glucocorticoid receptor does not bind to the ICAM NF-{kappa}B promoter element (61). Competition for shared transcriptional coactivators (62, 63), alterations in MAPK activity (64), or increases in the recruitment of corepressor complexes (65) could be alternative mechanisms whereby nuclear receptors inhibit AP-1 activity.

In macrophages, PPAR{gamma} blocks both AP-1- and NF-{kappa}B-mediated gene expression (26). Activation of AP-1 requires CREB-binding protein (CBP)/p300 (62) and PPAR{gamma} ligands enhance the interaction of CBP with PPAR{gamma} (66, 67), thereby blocking AP-1 activation. In human epithelial cells, PPAR{gamma} ligands inhibit cyclooxygenase-2 (COX-2) transcription by blocking the induction of c-Jun and competing for CBP (68). In an elegant study, Delerive et al. (69) demonstrated that PPAR{alpha} represses c-Jun-induced transcription of the IL-6 promoter. In this study, CBP did not relieve the PPAR{alpha}-mediated transcriptional repression; however, the authors proposed that inhibition may be associated with the ability of unliganded PPAR{alpha} and c-Jun to form a physical complex. In a similar manner, in our studies, cotransfection of CBP was not able to mitigate the ability of PPAR{gamma} to interfere with c-Jun activation of the IFN-{gamma} promoter. Nor did we observe a PPAR{gamma}-dependent alteration in the induction or phosphorylation of c-Jun (our unpublished data).

If PPAR{gamma} plays a physiological role in IFN-{gamma} expression, then endogenous PPAR{gamma} ligands should be present in the T cell milieu. In macrophages, IL-4 induces expression of PPAR{gamma} and 12,15 lipoxygenase, which favors the generation of 13-HODE and 15-hydroxyeicosatetraenoic acid, potential PPAR{gamma} ligands (70). Likewise, stimulated human monocytes (71) treated with IL-4 produce 13-HODE, which inhibits IL-2 production by human peripheral T lymphocytes (71). In our system, treatment of either BALB/c splenocytes or EL4.IL-2 cells with a PPAR{gamma} antagonist increases the expression of IFN-{gamma}. This suggests the existence of endogenous ligand in our culture system. In the Jurkat transfection studies, independent of ligand, PPAR{gamma} inhibits an IFN-{gamma} promoter construct. Moreover, studies performed with mutations in PPAR{gamma} that delete the ligand binding domain (pCMX-PPAR{gamma}-{Delta}AF2) or alter 2 aa critical for ligand binding suggest that PPAR{gamma} must bind ligand to repress IFN-{gamma} gene transcription. Therefore, we propose that EL4.IL-2 and Jurkat T cells may synthesize endogenous PPAR{gamma} ligands.

There is increasing evidence that T cells may produce PGs (72). Work by Tanaka et al. (73) demonstrates that PGD synthase and COX-2 are up-regulated in activated Th2 cells compared with Th1 cells. This is associated with the preferential synthesis of PGD2 by Th2 cells. In vitro, PGD2 can spontaneously dehydrate to 15-deoxy-{Delta}12, 14-PGJ2, a putative endogenous PPAR{gamma} ligand (74, 75). Also in T cells, IL-4 augments the expression of PPAR{gamma} (4). Thus, Th2 cells may preferentially express PPAR{gamma} and its ligands. This could lead to diminished IFN-{gamma} production and represent a positive feedback loop driving further IL-4 production, PPAR{gamma} expression, ligand production, and Th2 differentiation. Correspondingly, we have shown that in EL4.IL-2 cells, COX-2 protein and RNA are up-regulated (our unpublished data). We are currently assaying for the molecule or molecules in our T cell culture systems that endogenously activate PPAR{gamma}.

Other nuclear receptors negatively regulate IFN-{gamma} gene transcription. Transfection assays demonstrate that dexamethasone, in the presence of the glucocorticoid receptor, inhibits the minimal IFN-{gamma} promoter (50). In contrast, retinoic acid receptor and vitamin D receptor-mediated inhibition of IFN-{gamma} transcription occur in regions upstream of the minimal promoter (76). Interestingly, 17-{beta}-estradiol in the presence of the estrogen receptor markedly increases expression of an IFN-{gamma} promoter construct (77).

Multiple studies have demonstrated that in vivo administration of PPAR{gamma} ligands decreases IFN-{gamma} expression. Our studies show that the effect of these ligands may be a direct effect on T cells. Moreover, we propose that these agents may be used to treat inflammatory reactions dominated by IFN-{gamma} expression. Patients using thiazolidinediones for glucose control could exhibit impaired protective immune responses. This is highly significant as diabetic patients already display impaired infection-fighting ability.

In conclusion, PPAR{gamma} ligands, in a PPAR{gamma}-dependent manner, decrease IFN-{gamma} production by T lymphocytes by a mechanism of transcriptional transrepression. This effect appears to be related to the ability of PPAR{gamma} to interfere with c-Jun-induced activation of the proximal IFN-{gamma} promoter.


   Acknowledgments
 
We thank Judy Nordberg and the rest of the staff at the Veterans Affairs San Diego Healthcare System Core Flow Cytometry facility. We thank Christine Plotkin and the Center for AIDS Research Genomics Core facility (University of California, San Diego and Veterans Medical Research Foundation) for performing the TaqMan PCR studies. We also thank Dr. Tim Willson from GlaxoSmithKline for providing GW742, Dr. Stephan Ho and Dr. Jason Trama for pGL3-IFN-{gamma}-Luc, and Dr. Thomas Aune for providing the multimerized PCS and DCS luciferase constructs. Finally, we thank Paul Clopton for his assistance with the statistical analysis.


   Footnotes
 
1 The studies were performed with the support of National Institutes of Health Grants DK45346 (to C.J.K.) and P42ES10337 (to C.K.G.) and Department of Veterans Affairs Career Development Grant for R.C., and the support of the Veterans Affairs Merit Review Grant for C.J.K. The National Kidney Foundation Young Investigator Award and Medicine Education and Research Foundation scholarship awards also provided support (for R.C.). M.R. was supported by American Heart Association, Western Affiliate Beginning Grant-In-Aid. Y.E. was supported by the National Kidney Foundation Fellowship Award. Back

2 Address correspondence and reprint requests to Dr. Robyn Cunard, FRCPC, Research and Medicine Services, Division of Nephrology, University of California and Veterans Affairs San Diego Healthcare System 151, 3350 La Jolla Village Drive, San Diego, CA 92161. E-mail address: rcunard{at}ucsd.edu Back

3 Abbreviations used in this paper: PPAR, peroxisome proliferator-activated receptor; AF2, activation function 2; ATF, activating transcription factor; CBP, CREB-binding protein; COX-2, cyclooxygenase-2; DBD, DNA binding domain; DCS, distal conserved sequence; 13-HODE, 13-hydroxyoctadecadienoic acid; MAPK, mitogen-activated protein kinase; PCS, proximal conserved sequence; PPRE, PPAR response element. Back

Received for publication January 7, 2004. Accepted for publication April 13, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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C. Fionda, F. Nappi, M. Piccoli, L. Frati, A. Santoni, and M. Cippitelli
Inhibition of Trail Gene Expression by Cyclopentenonic Prostaglandin 15-Deoxy-{Delta}12,14-Prostaglandin J2 in T Lymphocytes
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F. Zapata-Gonzalez, F. Rueda, J. Petriz, P. Domingo, F. Villarroya, A. de Madariaga, and J. C. Domingo
9-cis-Retinoic Acid (9cRA), a Retinoid X Receptor (RXR) Ligand, Exerts Immunosuppressive Effects on Dendritic Cells by RXR-Dependent Activation: Inhibition of Peroxisome Proliferator-Activated Receptor {gamma} Blocks Some of the 9cRA Activities, and Precludes Them to Mature Phenotype Development
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R. Hontecillas and J. Bassaganya-Riera
Peroxisome Proliferator-Activated Receptor {gamma} Is Required for Regulatory CD4+ T Cell-Mediated Protection against Colitis
J. Immunol., March 1, 2007; 178(5): 2940 - 2949.
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L. Klotz, I. Dani, F. Edenhofer, L. Nolden, B. Evert, B. Paul, W. Kolanus, T. Klockgether, P. Knolle, and L. Diehl
Peroxisome Proliferator-Activated Receptor {gamma} Control of Dendritic Cell Function Contributes to Development of CD4+ T Cell Anergy
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S.-H. Jo, C. Yang, Q. Miao, M. Marzec, M. A. Wasik, P. Lu, and Y. L. Wang
Peroxisome Proliferator-Activated Receptor {gamma} Promotes Lymphocyte Survival through Its Actions on Cellular Metabolic Activities
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M. Soller, A. Tautenhahn, B. Brune, K. Zacharowski, S. John, H. Link, and A. von Knethen
Peroxisome proliferator-activated receptor {gamma} contributes to T lymphocyte apoptosis during sepsis
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L. Klotz, M. Schmidt, T. Giese, M. Sastre, P. Knolle, T. Klockgether, and M. T. Heneka
Proinflammatory Stimulation and Pioglitazone Treatment Regulate Peroxisome Proliferator-Activated Receptor {gamma} Levels in Peripheral Blood Mononuclear Cells from Healthy Controls and Multiple Sclerosis Patients
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