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The Journal of Immunology, 2003, 171: 196-203.
Copyright © 2003 by The American Association of Immunologists

Peroxisome Proliferator-Activated Receptor {alpha} Negatively Regulates T-bet Transcription Through Suppression of p38 Mitogen-Activated Protein Kinase Activation1

Dallas C. Jones*, Xiaohong Ding*, Tian Y. Zhang* and Raymond A. Daynes2,*,{dagger}

* Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132; and {dagger} Geriatric Research, Education and Clinical Center, Veterans Affairs Medical Center, Salt Lake City, UT 84112


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of the nuclear hormone receptor peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) in resting lymphocytes was recently established, although the physiologic role(s) played by this nuclear hormone receptor in these cell types remains unresolved. In this study, we used CD4+ T cells isolated from PPAR{alpha}-/- and wild-type mice, as well as cell lines that constitutively express PPAR{alpha}, in experiments designed to evaluate the role of this hormone receptor in the regulation of T cell function. We report that activated CD4+ T cells lacking PPAR{alpha} produce increased levels of IFN-{gamma}, but significantly lower levels of IL-2 when compared with activated wild-type CD4+ T cells. Furthermore, we demonstrate that PPAR{alpha} regulates the expression of these cytokines by CD4+ T cells in part, through its ability to negatively regulate the transcription of T-bet. The induction of T-bet expression in CD4+ T cells was determined to be positively influenced by p38 mitogen-activated protein (MAP) kinase activation, and the presence of unliganded PPAR{alpha} effectively suppressed the phosphorylation of p38 MAP kinase. The activation of PPAR{alpha} with highly specific ligands relaxed its capacity to suppress p38 MAP kinase phosphorylation and promoted T-bet expression. These results demonstrate a novel DNA-binding independent and agonist-controlled regulatory influence by the nuclear hormone receptor PPAR{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})3 is a member of the nuclear hormone receptor superfamily that was initially described for its ability to induce peroxisome proliferation in rodent hepatocytes in response to certain xenobiotic compounds (1). PPAR{alpha} has now been demonstrated to exist within numerous tissues and to be positively involved in important physiologic and developmental processes, including the control of inflammatory responses (2, 3).

The influence of PPAR{alpha} on numerous biological activities arises through its ability to carry out both positive and negative gene regulation, a characteristic shared by other nuclear hormone receptors (3). Upon activation, PPAR{alpha} binds to DNA as a heterodimer with the 9-cis retinoic acid receptor and transcriptionally activates a subset of genes possessing a peroxisome proliferator response element in their promoter region (4). PPAR{alpha} can also negatively regulate gene expression by antagonizing various transcription factors involved in an array of important signaling pathways. Such antagonism can be achieved through different mechanisms, including physical interaction with the transcription factor itself or through the ability of PPAR{alpha} to sequester essential transcriptional coactivators (5). This transrepression capability of PPAR{alpha} appears critical for controlling the expression of certain proinflammatory genes within vascular endothelial cells and macrophages (6, 7).

It has recently been reported that PPAR{alpha} exists in T and B lymphocytes (8, 9, 10). Although demonstrated to be both transactivation and transrepression competent within lymphocytes, the role(s) of PPAR{alpha} in lymphocyte biology remains largely unknown. To gain further insight into the physiological function of PPAR{alpha} within lymphocytes, we investigated physiological responses by T cells isolated from PPAR{alpha}-/- mice as well as responses elicited by T cell lines that overexpress PPAR{alpha}. In this study, we present experiments that describe a unique role for PPAR{alpha} in T cell activation. We show that unliganded PPAR{alpha} has the ability to negatively regulate the transcription of T-bet, an inducible transcription factor in lymphocytes that is important in the initiation and termination of activation-induced cytokine gene transcription (11). By controlling the initiation of T-bet transcription, PPAR{alpha} was able to indirectly influence the level of activation-induced IFN-{gamma} produced by CD4+ T cells. Furthermore, we report that the control of PPAR{alpha} over T-bet expression occurs via a DNA-binding independent mechanism, mediated through the ability of PPAR{alpha} to repress the phosphorylation of p38 mitogen-activated protein (MAP) kinase following T cell activation. These data suggest a novel and important function for PPAR{alpha} within T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals

Colonies of PPAR{alpha} wild-type (PPAR{alpha}+/+) and homozygous knockout (PPAR{alpha}-/-) mice on the 129sv background were expanded from breeding pairs originally obtained from F. Gonzalez (Metabolism Branch, National Institutes of Health, Bethesda, MD). The derivation and phenotypic characteristics of these animals have previously been reported (12). PPAR{alpha}-/- mice fail to express a functional PPAR{alpha} protein in all tissues, including CD4+ T cells. An analysis was made, using quantitative real-time PCR, of PPAR{beta} and PPAR{gamma} mRNA levels in CD4+ T cells from PPAR{alpha}-/- and wild-type (WT) donors. The mRNA levels of PPAR{gamma} were found to be similar in PPAR{alpha}-/- and WT CD4+ T cells, before and following activation. Similarly, PPAR{beta} mRNA levels were equivalent in PPAR{alpha}-/- and WT CD4+ T cells before and following activation (data not shown). Female mice were used for all of the experiments reported in this work. All mice were housed in the University of Utah Animal Resource Center, which routinely monitors for the most prevalent murine pathogens, employs sentinel animals as a means for early detection of murine hepatitis virus and parvovirus, and guarantees strict compliance with regulations established by the Animal Welfare Act. Mice were anesthetized with Halothane and sacrificed by cervical dislocation.

Cell lines and transfection

Jurkat T cells were purchased from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (HyClone Laboratories, Logan, UT), 200 mM L-glutamine, antibiotics, and 5 x 10-5 M 2-ME. Where indicated, cells were resuspended at a concentration of 5 x 106 cells/ml and were activated in multiwell plates with a solution of 50 ng/ml PMA with or without 1 µM ionomycin (Sigma-Aldrich, St. Louis, MO) for the times indicated. In certain experiments, cells were pretreated for 2 h before stimulation with the PPAR{alpha}-specific agonist GW9578 or GW7647 (generous gifts from P. Brown, Glaxo Wellcome, Research Triangle Park, NC) or vehicle (0.1% DMSO). These ligands have been reported to have high specificity for the PPAR{alpha} isoform and are able to activate PPAR{alpha} at nanomolar concentrations (13, 14).

Transfections were performed, as previously described (15). Briefly, 5 x 106 cells were resuspended in 0.65 ml of growth medium, and 10 µg of plasmid was then added. Cells were incubated with plasmids for 5 min in 0.4-cm electrode gap cuvettes (Invitrogen, Carlsbad, CA) and electroporated at room temperature using the Gene Pulser (Bio-Rad, Hercules, CA) set at 260 V and 960 µF. Cells were then incubated for 5 min at room temperature, transferred to 100 x 20-mm tissue culture dishes containing 10 ml of growth medium, and incubated at 37°C for 48 h before use. Stable transfectants were generated in Clona-cell TCS following the manufacturer’s protocol (Stem Cell Technologies, Vancouver, Canada).

Dynabead cell enrichment

For the preparation of CD4+ T lymphocytes, freshly isolated splenic lymphoid cells were suspended at a concentration of 2 x 107 cells/ml in RPMI 1640 containing 5% FBS. The erythrocytes present in the cell suspension were lysed by brief treatment with sterile aqueous 0.83% (w/v) ammonium chloride. The single cell suspension was incubated with 2 µg/ml each of biotinylated anti-CD45R/B220, anti-CD11b, and anti-CD8 Abs (BD PharMingen, San Diego, CA) for 20 min on ice. Following washing with PBS, the cells were resuspended with M-280 magnetic Dynabeads coated with streptavidin (Dynal, New York, NY), and incubated at a bead:cell ratio of 1:1 for 20 min with agitation at 4°C. The residual cells were collected, washed, and separated for use in culture or for mRNA analysis. The level of purity of the cell preparations was assessed by staining cells with FITC anti-mouse CD4, FITC anti-mouse CD8, and FITC anti-mouse B220. The level of cell purity was routinely >90%.

ELISA

Freshly isolated CD4+ T cells were activated on multiwell plates treated with 2 µg/ml immobilized anti-CD3 with the addition of 1 µg/ml soluble anti-CD28 for various times at 37°C in an atmosphere of 5% CO2 in air. Cell culture supernatants were collected for quantitative evaluation of immunoactive IL-2, IFN-{gamma}, or IL-4 by ELISA, as described previously (16). Rat anti-murine cytokine mAbs and murine rIL-2 and rIFN-{gamma} cytokine standards were purchased from BD PharMingen.

Quantitative real-time PCR

Reverse transcription was performed, as previously described (17). mRNA was isolated by the method of Chomczynski and Sacchi (18), and PCR was performed in a fluorescence temperature cycler (Light Cycler; Idaho Technology, Salt Lake City, UT), as fully described elsewhere (19). The Light Cycler monitors the cycle-by-cycle accumulation of fluorescently labeled products. The cycle at which the product is first detected is used as an indicator of relative starting copy. Melting curves were acquired to determine specificity of the PCR (19). PCR products for each of the primer sets were confirmed by running samples on agarose gels. The PCR was conducted in 10 µl final volume containing 3 mM MgCl2, 0.2 mM dNTPs, 1/30,000 dilution of SYBR Green I, 5 µM (each) primer, 0.05 U Taq polymerase, and 11 ng TaqStart Ab. Oligonucleotides used for these analyses are as follows: murine GAPDH, 5'-AGT ATG TCG TGG AGT CTA C-3' and 5'-CAT ACT TGG CAG GTT TCT C-3'; murine T-bet, 5'-GGA TTC TGG GGT TTA CTT CTT-3' and 5'-TTC TCT GTT TGG CTG GCT GTT-3'; murine IFN-{gamma}, 5'-CTT CCT CAT GGC TGT TTC TGG-3' and 5'-CGA CTC CTT TTC CGC TTC CTG-3'; and murine IL-2, 5'-GTC ACA TTG ACA CTT GTG CTC C-3' and 5'-AGT CAA ATC CAG AAC ATG CCG-3'. GAPDH transcript levels were used to normalize the amount of cDNA in each sample, and T-bet, IFN-{gamma}, and IL-2 transcript levels were reported relative to levels found in the control sample.

Preparation of nuclear extracts and immunoblot analysis

Nuclear extracts were prepared from T cells following treatment for various times with immobilized anti-CD3 and soluble anti-CD28, as described previously (20). Briefly, cells were washed twice with ice-cold PBS containing 1 mM PMSF, resuspended in 250 µl buffer A (10 mM HEPES, pH 7.8, 0.1 mM EDTA, 10 mM NaCl, 3 mM MgCl2, 300 mM sucrose, 10 µg/ml aprotinin, 100 µM leupeptin, 1 mM DTT, and 1 mM PMSF), and incubated on ice for 10 min. Next, 25 µl of 1% Nonidet P-40 was added and mixed carefully. Cells were then collected by centrifugation at 800 x g for 1 min at 4°C and washed with 200 µl buffer A. Nuclei were then resuspended in 50 µl buffer B (20 mM HEPES, pH 7.8, 3 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 10 µg/ml aprotinin, 100 µM leupeptin, 1 mM DTT, and 1 mM PMSF) and incubated for 15 min on ice. Nuclear debris was removed by centrifugation at 16,000 x g for 1 min. In certain experiments, cells were pretreated with the extracellular signal-regulated kinase (ERK) inhibitor PD98059 (Sigma-Aldrich) or the p38 MAP kinase inhibitor PD169,316 (Alexis Biochemicals, San Diego, CA) before activation. Whole cell extracts used in the analysis of the MAP kinases were generated, as described previously (21).

The supernatant was then removed, and protein content was determined by Bradford Assay (17). Equal amounts of nuclear protein were subjected to 10% SDS-PAGE and polyvinylidene difluoride membrane (Millipore, Bedford, MA), as previously described (20). After blocking with 5% nonfat milk TBS, blots were incubated with either anti-T-bet Ab (kindly provided by L. Glimcher, Harvard University, Boston, MA) or Abs against the double-phosphorylated forms of p38, c-Jun N-terminal kinase (JNK), and ERK (Promega, Madison, WI) for 1 h at room temperature or overnight at 4°C for anti-p38-Ab, anti-JNK, and anti-ERK1/2 Ab (Cell Signaling, Beverly, MA). Membranes were then washed and incubated with goat anti-rabbit HRP conjugate (1/2000 dilution in TBS-Tween) for 45 min at room temperature. After washing, bands were visualized using a chemiluminescence kit, according to the manufacturer’s instructions (Santa Cruz Biotechnology, Santa Cruz, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{gamma} and IL-2 production is dysregulated in activated PPAR{alpha}-/- T cells

We and other investigators have previously reported that the nuclear hormone receptor PPAR{alpha} is expressed in resting T lymphocytes (8, 9, 10). The transcription and protein levels of PPAR{alpha} were found to decline following T cell activation (8, 9, 10). We have now questioned whether the expression of PPAR{alpha} in resting T lymphocytes is involved in the regulation of T cell responses that occur early postactivation. An analysis of the cytokines produced following the activation of splenic CD4+ T cells from WT and PPAR{alpha}-/- mice revealed consistent differences. As shown in Fig. 1a, CD4+ T cells from PPAR{alpha}-/- mice produce greater amounts of IFN-{gamma} over the 24-h period postactivation with immobilized anti-CD3 and soluble anti-CD28, when compared with CD4+ T cells isolated from age-matched WT mice. However, under the same activating conditions, CD4+ T cells from PPAR{alpha}-/- mice produced lower amounts of IL-2 than T cells from the WT animals (Fig. 1b). Cell types from both sources produced similar low levels of IL-4. The addition of neutralizing anti-IL-4 and/or anti-IL-12 Abs at the initiation of the cell cultures period did not alter the consistent differences in cytokine production observed between PPAR{alpha}-/- and WT T cells (data not shown). The observed differences in cytokine production were due to kinetic differences in the transcription of the IFN-{gamma} and IL-2 genes postactivation, as determined by quantitative real-time PCR. As shown in Fig. 1c, transcription of the IFN-{gamma} gene was initiated much earlier in PPAR{alpha}-/- T cells postactivation, and the level of IFN-{gamma} transcripts was significantly higher at each of the time points analyzed compared with WT T cells. When IL-2 transcripts were compared kinetically under the same conditions, the level of IL-2 mRNA from PPAR{alpha}-/- T cells optimized before 3 h postactivation and decreased markedly at all the later time points tested. IL-2 mRNA levels in the WT T cells, however, continued to increase until 12 h postactivation (Fig. 1d), similar to what has been reported previously (22). From these results it is apparent that the presence of PPAR{alpha} in CD4+ T cells contributes to the regulation of IFN-{gamma} and IL-2 expression in response to activation.



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  		FIGURE 1. Dysregulated production of IFN-{gamma} and IL-2 in PPAR{alpha}-/- T cells. Splenic CD4+ T cells, isolated from PPAR{alpha}-/- and WT mice, were activated with 2 µg/ml of immobilized anti-CD3 and 1 µg/ml soluble anti-CD28. Following a 24-h activation period, the levels of IFN-{gamma} (a) and IL-2 (b) in the culture supernatants were measured by ELISA. The levels of IFN-{gamma} mRNA (c) and IL-2 mRNA (d) were measured, using quantitative real-time PCR, under these same activating conditions in PPAR{alpha}-/- and WT T cells at the various time points. Experiments were repeated three times. The results of a representative experiment are shown.

 
T-bet is expressed earlier in PPAR{alpha}-/- T cells following activation

The lineage-specific transcription factor T-bet is now appreciated to be critical for the activation-induced progression of T cells down a Th1 pathway (11, 23, 24). Interestingly, T-bet was originally isolated based on its ability to bind to the IL-2 promoter and was later demonstrated to actually repress IL-2 expression by T cells in in vitro experiments. Based on the reported ability of T-bet to repress IL-2 expression as well as to transactivate the IFN-{gamma} gene (11, 23, 24), we questioned whether the activation-induced expression of T-bet is altered in PPAR{alpha}-/- T cells. T-bet mRNA levels were analyzed using quantitative real-time PCR before T cell activation and at 3, 6, 12, and 24 h postactivation with immobilized anti-CD3 and anti-CD28. As shown in Fig. 2a, the initiation of T-bet transcription postactivation was kinetically accelerated in PPAR{alpha}-/- CD4+ T cells. T-bet mRNA levels were maximal by 3 h postactivation in PPAR{alpha}-/- T cells, while the levels of T-bet mRNA in WT CD4+ T cells did not peak until 6 h postactivation. Similarly, Western blot analysis determined that T-bet protein synthesis postactivation in PPAR{alpha}-/- T cells was accelerated when compared with T cells isolated from WT animals (Fig. 2b). Thus, differences in the kinetics of T-bet expression might contribute to the differences observed in cytokine production in WT and PPAR{alpha}-/- T cells.



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  		FIGURE 2. Kinetic induction of T-bet mRNA and protein is accelerated in PPAR{alpha}-/- T cells. a, T-bet mRNA was analyzed by quantitative real-time PCR in PPAR{alpha}-/- and WT CD4+ T cells at the time points indicated, following activation with 2 µg/ml immobilized anti-CD3 and 1 µg/ml soluble anti-CD28. b, T-bet protein was analyzed by Western blot on nuclear extracts generated from freshly isolated PPAR{alpha}-/- and WT CD4+ T cells and T cells from these two groups that were activated for 6 and 24 h under the same conditions, as stated above. Data are the results of one of three experiments that gave similar results.

 
T-bet expression in PPAR{alpha}-/- CD4+ T cells is independent of signaling through the IFN-{gamma} receptor

It has recently been reported that IFN-{gamma} exposure rapidly up-regulates the expression of T-bet following activation of CD4+ T cells (25, 26). We therefore questioned whether the accelerated expression of T-bet in PPAR{alpha}-/- T cells was due to the influences caused by an increased IFN-{gamma} expression in these same cells. PPAR{alpha}-/- and WT CD4+ T cells were activated with immobilized anti-CD3 and anti-CD28 in the presence or absence of neutralizing Ab against IFN-{gamma}. T-bet and IFN-{gamma} mRNA levels, as well as T-bet protein levels, were measured 48 h postactivation. Similar to what has been reported previously (25), WT T cells were severely compromised in their ability to up-regulate expression of T-bet or IFN-{gamma} mRNA when activated in the presence of anti-IFN-{gamma}, but were able to up-regulate expression of both these genes when activated in the absence of anti-IFN-{gamma} (Fig. 3, a and b). Surprisingly, when PPAR{alpha}-/- CD4+ T cells were activated in the presence of anti-IFN-{gamma}, they were induced to express both T-bet mRNA and protein. This correlated with a retention in their ability to express mRNA for IFN-{gamma} (Fig. 3, a–c). Fig. 3d demonstrates that PPAR{alpha}-/- CD4+ T cells, activated with immobilized anti-CD3 plus anti-CD28 for 48 h in the presence of anti-IFN-{gamma}, retain their ability to reinitiate synthesis of IFN-{gamma} protein when restimulated with anti-CD3. WT T cells treated in a similar manner, as expected, failed to up-regulate IFN-{gamma} production following restimulation. The accelerated IFN-{gamma} production in the PPAR{alpha}-/- T cells upon restimulation most likely arises through a retained ability to express T-bet, as WT T cells that failed to express T-bet under the same conditions also failed to produce IFN-{gamma} upon restimulation (Fig. 3d). These results suggest PPAR{alpha} regulates the expression of T-bet by antagonizing a signaling pathway that is independent of IFN-{gamma} signaling.



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  		FIGURE 3. T-bet is expressed and IFN-{gamma} produced in PPAR{alpha}-/- T cells activated in the presence of anti-IFN-{gamma}. CD4+ T cells, isolated from PPAR{alpha}-/- and WT mice, were activated over a 48-h period with immobilized anti-CD3 (2 µg/ml) plus 1 µg/ml anti-CD28 in the absence or presence of 10 µg/ml anti-IFN-{gamma}. a, T-bet mRNA levels along with T-bet protein levels (b) were then measured by quantitative real-time PCR and Western blot, respectively. c, IFN-{gamma} mRNA levels were also measured in these same samples by quantitative real-time PCR. d, The culture supernatants of WT and PPAR{alpha}-/- T cells reactivated with immobilized anti-CD3 (2 µg/ml) in the absence of anti-IFN-{gamma} were assayed for the presence of IFN-{gamma} protein by ELISA. Experiments were repeated three times. The results of a representative experiment are shown.

 
Inhibition of T-bet expression does not require ligand activation of PPAR{alpha}

It is well recognized that activated PPARs can suppress the expression of many distinct genes using a variety of molecular mechanisms (3). In almost all cases, agonist activation of the PPAR is required for effective transrepression to occur. Interestingly, the differences in T-bet expression in WT and PPAR{alpha}-/- T cells, following activation, were observed without adding exogenous ligand. In an attempt to define the mechanism through which PPAR{alpha} regulates T-bet expression, we analyzed the influences that ligand-activated PPAR{alpha} would have on T-bet expression by activated T cells. CD4+ T cells isolated from WT mice were treated with increasing doses of the highly specific PPAR{alpha} ligand GW9578 (13) or vehicle 2 h before cellular activation with immobilized anti-CD3 and anti-CD28. T-bet protein levels were then analyzed at 24 h postactivation by Western blot analysis. As shown in Fig. 4a, treatment of WT CD4+ T cells with GW9578 enhanced the activation-induced expression of T-bet in a dose-dependent manner. However, GW9578 treatment of T cells isolated from PPAR{alpha}-/- donors did not show an increase in T-bet expression compared with vehicle-treated T cells following activation (Fig. 4b). These data suggest that the effects being elicited by the GW9578 compound were mediated through a PPAR{alpha}-dependent mechanism. Similar results have been obtained using GW7647, another highly specific PPAR{alpha} ligand (data not shown). Collectively, these results suggest that the ability of PPAR{alpha} to suppress T-bet expression is independent of PPAR{alpha} activation and that ligand activation of PPAR{alpha} abrogates its normally suppressive effects.



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  		FIGURE 4. PPAR{alpha}-mediated inhibition of T-bet expression does not require PPAR{alpha} ligand activation. a, CD4+ T cells isolated from PPAR{alpha}+/+ donors were activated with anti-CD3 and anti-CD28 following a 2-h pretreatment with 1, 10, or100 nM GW9578 or vehicle (0.1% DMSO). T-bet protein levels were analyzed by Western blot 24 h after activation. b, CD+ T cells isolated from PPAR{alpha}-/- and WT donors were pretreated for 2 h with 10 nM GW9578 or vehicle (0.1% DMSO). The levels of T-bet protein were analyzed by Western blot 24 h after activation with immobilized anti-CD3 (2 µg/ml) and soluble anti-CD28 (1 µg/ml). Experiments were repeated three times. The results of a representative experiment are shown.

 
Inhibition of the p38 MAP kinase represses activation-induced T-bet mRNA and protein expression

The PPAR{alpha} isoform, unlike PPAR{beta}, is unable to negatively regulate gene expression through a DNA-binding dependent mechanism (27). This results from the ability of unliganded PPAR{alpha} to maintain an association with corepressor complexes when bound to DNA (27). We therefore reasoned that the negative regulation of T-bet expression by PPAR{alpha} was not due to a DNA-binding dependent repression of the T-bet gene. This led us to question whether PPAR{alpha} was inhibiting T-bet expression through the regulation of an upstream signal cascade. Because the activity of the p38 MAP kinase has been positively linked with IFN-{gamma} production in activated T cells, as well as Th1 T cell development (28, 29, 30), we questioned whether activation of p38 MAP kinase might be involved in the regulation of T-bet expression. To address this, CD4+ T cells were pretreated with PD169,316, a specific chemical inhibitor of p38 MAP kinase, or with the chemical ERK MAP kinase inhibitor, PD98059 (31). Treated and control T cells were then activated with immobilized anti-CD3 plus soluble anti-CD28. The level of T-bet mRNA was quantitated at 6 h postactivation, and T-bet protein was analyzed by Western blot 24 h postactivation. As shown in Fig. 5a, activation-induced expression of T-bet mRNA was inhibited in a dose-dependent manner in T cells treated with PD169,316, while inhibition of the ERK MAP kinase did not influence the induced expression of T-bet mRNA. Likewise, the inhibition of p38 MAP kinase activation correlated with an inhibition of T-bet protein expression in both PPAR{alpha}-/- and WT T cells (Fig. 5b). These data suggest that activation of p38 MAP kinase positively contributes to the activation-induced expression of T-bet in T cells.



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  		FIGURE 5. Inhibition of the p38 MAP kinase inhibits activation-induced expression of T-bet in T cells. a, Splenic CD4+ T cells, isolated from WT mice, were pretreated with various concentrations of PD169,316, PD98059, or vehicle (0.1% DMSO) for 1 h before activation with anti-CD3 and anti-CD28. T-bet mRNA levels were then analyzed by quantitative real-time PCR in each of these samples following a 6-h activation period. b, T-bet protein levels were analyzed in PPAR{alpha}-/- and WT T cells that were treated for 1 h with 25 µM of PD169,316, 25 µM PD98059, or vehicle (0.1% DMSO) before activation for 24 h with anti-CD3 and anti-CD28. Data are the results of one of three experiments that gave similar results.

 
PPAR{alpha}-/- T cells express elevated levels of phosphorylated p38 MAP kinase following activation

It has previously been reported that certain members of the nuclear hormone receptor superfamily possess the ability to inhibit activation of MAP kinases, including the p38 MAP kinase (32, 33, 34, 35). This led us to question whether the ability of PPAR{alpha} to regulate the transcription of T-bet might arise through its ability to somehow regulate the activation of the p38 MAP kinase. To address this, splenic CD4+ T cells from PPAR{alpha}-/- and WT mice were activated with PMA for 5, 15, and 30 min. The induced levels of phosphorylated p38 MAP kinase and phosphorylated MAP kinase kinase (MKK)3/6 within the activated T cells were then analyzed by Western blot and compared with resting cells. As shown in Fig. 6a, the levels of phosphorylated p38 MAP kinase were greater in activated PPAR{alpha}-/- T cells when compared with WT T cells activated under the same conditions. Interestingly, the induced levels of phosphorylated MKK3/6 were similar between the activated WT and PPAR{alpha}-/- T cells (Fig. 6a).



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  		FIGURE 6. Phosphorylation of p38 MAP kinase in activated T cells is suppressed by PPAR{alpha}. a, Following activation with 50 ng/ml PMA, the levels of phosphorylated MKK3/6 and phosphorylated p38 MAP kinase were measured in cellular extracts generated from CD4+ T cells isolated from PPAR{alpha}-/- and WT mice. b, Jurkat cells stably transfected with PPAR{alpha} were activated with 50 ng/ml PMA for 5, 15, and 30 min. Following activation, the levels of phosphorylated p38 MAP kinase and ERK were measured in cellular extracts by Western blot analysis. c, Jurkat T cells stably expressing PPAR{alpha} were treated with 10 nM GW9578 or vehicle (0.1% DMSO) for 2 h before activation with 50 ng/ml PMA. Phosphorylated p38 MAP kinase levels were then assayed by Western blot analysis on cell extracts generated from cells before or following activation with PMA for 5, 15, and 30 min. Experiments were repeated three times. The results of a representative experiment are shown.

 
To further support a role for PPAR{alpha} in the regulation of p38 MAP kinase activation, PPAR{alpha} was stably expressed in Jurkat T cells using a constitutively active expression construct. As shown in Fig. 6b, the level of phospho-p38 MAP kinase was much lower in activated Jurkat cells transfected with PPAR{alpha} when compared with the levels observed in activated Jurkat cells that were transfected with an empty vector. Overexpression of PPAR{alpha} in Jurkat T cells had no effect on the level of activation-induced phosphorylated ERK. Similar to what was observed with T-bet expression, ligand activation of PPAR{alpha} attenuated the ability of this receptor to inhibit p38 MAP kinase activation (Fig. 6c). These results suggest that the ability of PPAR{alpha} to regulate the transcription of T-bet within T cells postactivation arises through the ability of the unliganded receptor to repress the activation of p38 MAP kinase at a site distal to MKK3/6 phosphorylation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During the differentiation of naive CD4+ T cells into effector Th1 and Th2 subsets, activated T cells shift from producing predominantly IL-2 into producing the specific effector cytokines that are important for the generation of cellular or humoral immunity (36). This important differentiation process is now appreciated to be tightly regulated through the activities of specific signaling pathways and transcription factors (37). The T-box transcription factor, T-bet, represents a key regulator of Th1 CD4+ T cell development through its ability to transactivate the IFN-{gamma} gene while concomitantly repressing IL-4 gene expression (11). If T-bet expression becomes dysregulated, however, it can contribute in the development of certain pathological disease states, ranging from asthma to inflammatory bowel disease (38, 39). Consequently, a better understanding of the cellular signaling pathways that regulate the transcription and cellular expression of T-bet subsequent to T cell activation could be of great importance in the design of therapeutic interventions for many distinct immunopathological conditions.

In this study, we have experimentally demonstrated in CD4+ T cells that unliganded PPAR{alpha} negatively regulates the activation-induced expression of T-bet. CD4+ T cells lacking PPAR{alpha} undergo an early termination of induced IL-2 gene expression and protein production, and a concomitant overexpression of IFN-{gamma}. PPAR{alpha} exerts its regulatory influences over T-bet by suppressing the activation-induced phosphorylation of p38 MAP kinase, a signaling molecule whose activity is associated with the expression of IFN-{gamma} (28, 29, 30). We demonstrate in this study that p38 MAP kinase activation also contributes to inducing the expression of T-bet following TCR-mediated activation of CD4+ T cells. The data we present, linking regulation of p38 MAP kinase activation to transcription of T-bet, are consistent with previous reports demonstrating that activity of the p38 MAP kinase is associated with Th1 T cell differentiation and IFN-{gamma} production (28, 29, 30).

IFN-{gamma} signaling in CD4+ T cells rapidly induces the expression of T-bet (25). It has also been determined that T-bet expression becomes compromised without sufficient IFN-{gamma} signaling. Consequently, the enhanced p38 MAP kinase activity seen in stimulated PPAR{alpha}-/- CD4+ T cells could be accelerating T-bet expression indirectly, with the early activation of p38 MAP kinase resulting in increased IFN-{gamma} expression and signaling. However, our present studies show that CD4+ T cells lacking a functional PPAR{alpha} retain their ability to express T-bet in the complete absence of IFN-{gamma} signaling. This ability by activated PPAR{alpha}-/- T cells to express T-bet without IFN-{gamma} signaling results in a rapid secretion of newly synthesized IFN-{gamma} protein following restimulation. Wild-type CD4+ T cells that are initially activated under conditions in which IFN-{gamma} signaling is inhibited fail to express T-bet or IFN-{gamma} following restimulation. These results indicate that the ability of PPAR{alpha} to suppress the activation-induced expression of T-bet is mediated through an ability to antagonize aspects of TCR signaling, and is independent of regulation through IFN-{gamma} signaling.

The novel regulation of T-bet by PPAR{alpha} would allow CD4+ T cells to control the expression of T-bet based on the presence of IFN-{gamma} in the microenvironment. Recently, it was reported that dendritic cells and other types of APCs produce IFN-{gamma} following appropriate stimulation (40, 41). It was proposed that the IFN-{gamma} produced by APCs might play an important role in regulating the development of Th1 cells through the known ability of this cytokine to induce T-bet expression in T cells (42). However, the ability of APCs to produce IFN-{gamma} is dictated by the specific conditions under which these cells are activated (42). Therefore, under conditions in which the APCs are not producing IFN-{gamma}, the PPAR{alpha} within CD4+ T lymphocytes might help to stabilize the initiation of Th2 cell development. Conversely, CD4+ T cells that are stimulated by IFN-{gamma}-producing APCs should be induced to express T-bet, thereby circumventing the TCR-dependent PPAR{alpha}-mediated repression of T-bet.

The ability of PPAR{alpha} to repress T-bet expression appears to require unactivated PPAR{alpha}, because ligand activation of the receptor relaxes its repression over T-bet expression by allowing p38 MAP kinase to be phosphorylated. It is presently unclear how unliganded PPAR{alpha} mediates its suppressive effects on p38 MAP kinase activation. However, we have previously reported that PPAR{alpha} is expressed almost exclusively in the cytoplasmic compartment of resting T cells, suggesting that unliganded PPAR{alpha} must be repressing p38 MAP kinase activation through a DNA-binding independent mechanism (8). In support of this finding is a recent report demonstrating that glucocorticoid receptors (GR) are able to inhibit JNK activation through a DNA-binding independent mechanism that has yet to be mechanistically defined (33). In this report, the authors stated that they were unable to detect a physical association between GR and JNK, possibly due to a transient association between these two proteins. It was also stated that the GR might be part of a larger complex that associates with JNK, making it difficult to detect by the methods used. Further support of this concept comes from a recent report describing that JNK activation can be inhibited by the nuclear hormone receptor corepressor protein N-CoR that is associated with a number of other bridge proteins (43). Interestingly, we have found that the PPAR{alpha} within resting lymphocytes is associated with N-CoR, and that following ligand addition, the receptor dissociates from this corepressor (unpublished data). However, we have not yet been able to establish a physical association between PPAR{alpha} and p38 MAP kinase in resting T cells. Therefore, the ability of unliganded PPAR{alpha} to inhibit p38 MAP kinase activation may not require a direct interaction between these two proteins, but may possibly interact with proteins that are upstream of p38 MAP kinase. It was recently established that p38 MAP kinase is activated in Th1 CD4+ T cells through a signaling cascade that involves GADD45{gamma}, a protein that can physically interact with PPAR{alpha} (44). Although this represents an intriguing mechanism to describe how PPAR{alpha} might functionally inhibit p38 MAP kinase activation, the activation of the CD4+ T cells in our culture systems was conducted under conditions in which GADD45{gamma} is not expressed (44). Alternatively, PPAR{alpha} may suppress the activation of p38 MAP kinase through an association with some secondary complex of proteins, making it difficult to detect with the methods used.

Because the constitutive expression of PPAR{alpha} inhibits the phosphorylation of p38 MAP kinase, we questioned whether the constitutive expression of PPAR{alpha} could also inhibit T-bet expression. Because Jurkat T cells are unable to express IFN-{gamma} and T-bet, we transfected PPAR{alpha} into FS7-20.6.18, a cell line that can be induced to express IFN-{gamma} (45) and T-bet. We found that the constitutive expression of PPAR{alpha} repressed the ability of the FS7-20.6.18 cells to express T-bet and IFN-{gamma} subsequent to their activation (data not shown). However, after several passages in vitro, the FS7-20.6.18 cell line lost its ability to be induced to express T-bet and IFN-{gamma}. We are therefore hesitant to conclude that an overexpression of PPAR{alpha} can suppress T-bet and IFN-{gamma} expression until a more stable cell line can be analyzed.

It will be of interest to establish whether PPAR{alpha}’s tonic suppression of p38 MAP kinase activation and its downstream regulation of T-bet expression result in altered T cell function in vivo. The accelerated and increased expression of IFN-{gamma} expression in activated PPAR{alpha}-/- CD4+ T cells, which results from a dysregulation in the p38 MAP kinase signaling pathway, could result in a number of detrimental effects. It has recently been reported that IFN-{gamma} signaling in dendritic cells induces the expression of the enzyme indeolamine 2,3-dioxygenase (IDO) (41). The expression of IDO within dendritic cells was established to inhibit T cell proliferation and induce T cell apoptosis through IDO catalyzing the breakdown of tryptophan, decreasing the availability of this essential amino acid. The rapid production of IFN-{gamma} by PPAR{alpha}-/- CD4+ T cells following their stimulation might cause an accelerated expression of IDO within dendritic cells that are presenting Ag to these T cells. An increase in IDO expression by peptide-presenting dendritic cells, coupled with the decreased IL-2 production by PPAR{alpha}-/- CD4+ T cells subsequent to activation, could severely compromise the ability of Ag-specific T cells to undergo adequate clonal expansion.

In this study, we provide data indicating that PPAR{alpha} regulates specific aspects of T cell function in the absence of ligand. Contrary to the more global suppression that has been reported by agonist-activated PPAR{alpha}, unliganded PPAR{alpha} appears to be specific in the signaling cascades it regulates. It has now been demonstrated that a number of the ligand-induced effects can be achieved in the absence of PPAR{alpha} (10). Consequently, it is not yet possible to conclude whether all of the reported effects by these compounds are mediated by a PPAR{alpha}-dependent process. It is still intriguing to suggest that the function of PPAR{alpha} within T cells is dictated by the microenvironments in which the cell resides. Interestingly, we have found that treatment of CD4+ T cells with GW9578 inhibits the production of IFN-{gamma} following T cell activation through a PPAR{alpha}-dependent process (data not shown). The mechanism through which ligand-activated PPAR{alpha} influences IFN-{gamma} gene expression has yet to be uncovered. However, it is known that ligand-activated PPAR{alpha} can negatively regulate the transcriptional activities of NF-{kappa}B, a transcription factor that is important in the up-regulation of IFN-{gamma} gene expression (6, 8, 17).

Collectively, our studies demonstrate that the nuclear hormone receptor PPAR{alpha} plays a novel role in the transcriptional regulation of T-bet gene expression within T cells. The ability of PPAR{alpha} to negatively regulate the activation-induced expression of T-bet in naive T cells influences the timing of the switch from transcription of the IL-2 gene to the transcription of the IFN-{gamma} gene in these cells. This process, which controls CD4+ T cell production of IFN-{gamma}, appears to be facilitated by the capacity of unliganded PPAR{alpha} to transiently suppress the phosphorylation of p38 MAP kinase subsequent to T cell activation. The ability of PPAR{alpha} to transiently suppress activation of the p38 MAP kinase and to subsequently delay the expression of T-bet and IFN-{gamma} production may allow activated CD4+ T cells to sense and respond to unique environmental influences that serve to influence these cells’ actions following stimulation.


   Acknowledgments
 
We thank Dr. Laurie Glimcher for providing the T-bet Ab, Dr. Ron Evans for the PPAR{alpha} construct, and Dr. Peter Brown for the GW9578 and GW7647.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants CA25917 and DK55491, a Browning Foundation grant, and by DVA Medical Research Funds. D.C.J. was supported by National Institutes of Health, Department of Health and Human Services, National Institute of Diabetes and Digestive and Kidney Diseases, Hematology Research Training Grant T32 DK07115. Back

2 Address correspondence and reprint requests to: Dr. Raymond A. Daynes, Pathology Department, University of Utah, 30 North 1900 East, Salt Lake City, UT 84132-2501. E-mail address: daynes.office{at}path.utah.edu Back

3 Abbreviations used in this paper: PPAR{alpha}, peroxisome proliferator-activated receptor {alpha}; ERK, extracellular signal-regulated kinase; GR, glucocorticoid receptor; IDO, indeolamine 2,3-dioxygenase; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MKK, MAP kinase kinase; WT, wild type. Back

Received for publication January 13, 2003. Accepted for publication April 23, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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