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. Author manuscript; available in PMC: 2008 Apr 7.
Published in final edited form as: J Immunol. 2007 Feb 1;178(3):1904–1913. doi: 10.4049/jimmunol.178.3.1904

Peroxisome Proliferator-Activated Receptor-γ Agonists Suppress the Production of IL-12 Family Cytokines by Activated Glia1

Jihong Xu 1, Paul D Drew 1,2
PMCID: PMC2288778  NIHMSID: NIHMS42829  PMID: 17237441

Abstract

The IL-12 family of cytokines, which include IL-12, IL-23, and IL-27, play critical roles in the differentiation of Th1 cells and are believed to contribute to the development of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Relatively little is known concerning the expression of IL-12 family cytokines by cells of the CNS, the affected tissue in MS. Previously, we and others demonstrated that peroxisome proliferator-activated receptor (PPAR)-γ agonists suppress the development of EAE, alter T cell proliferation and phenotype, and suppress the activation of APCs. The present studies demonstrated that PPAR-γ agonists, including the naturally occurring 15-deoxy-Δ12,14-PGJ2 and the synthetic thiazoladinedione rosiglitazone, inhibited the induction of IL-12p40, IL-12p70 (p35/p40), IL-23 (p19/p40), and IL-27p28 proteins by LPS-stimulated primary microglia. In primary astrocytes, LPS induced the production of IL-12p40, IL-23, and IL-27p28 proteins. However, IL-12p70 production was not detected in these cells. The 15-deoxy-Δ12,14-PGJ2 potently suppressed IL-12p40, IL-23, and IL-27p28 production by primary astrocytes, whereas rosiglitazone suppressed IL-23 and IL-27p28, but not IL-12p40 in these cells. These novel observations suggest that PPAR-γ agonists modulate the development of EAE, at least in part, by inhibiting the production of IL-12 family cytokines by CNS glia. In addition, we demonstrate that PPAR-γ agonists inhibit TLR2, MyD88, and CD14 expression in glia, suggesting a possible mechanism by which these agonists modulate IL-12 family cytokine expression. Collectively, these studies suggest that PPAR-γ agonists may be beneficial in the treatment of MS.

Multiple sclerosis (MS)3 is believed to be an autoimmune disorder initiated by T cells reactive against self CNS Ags (1, 2). The disease is characterized by CNS inflammation and associated demyelination and axonal pathology (3–5). The differentiation of CD4+ autoreactive T cells into a Th1 phenotype plays a critical role in the development of MS and other autoimmune diseases (6).

IL-12 is the prototypic member of the IL-12 family of proteins, which also includes the more recently identified IL-23 and IL-27. These cytokines are heterodimeric proteins. IL-12 is composed of p40 and p35 subunits. IL-23 is composed of the p40 subunit shared with IL-12 and the unique p19 subunit, which is related to the IL-12p35 subunit (7). IL-27 is composed of EBV-induced molecule 3 (EBI3), which is related to IL-12p40, and p28, which is an IL-12p35-related protein (8). These cytokines are expressed by a variety of cells, including APCs, such as macrophages and dendritic cells in peripheral tissues. These proteins are also expressed by microglia, which are monocytic cells resident to the CNS. Controversy exists as to whether CNS astrocytes express IL-12 family cytokines. This family of proteins displays a diverse array of functions, but each is believed to play an important role in the generation of Th1 cells (9–11).

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of proteins. Three subtypes of PPAR-γ exist (α, β, and γ), each exhibiting distinct tissue distribution and ligand specificities (12). The role of PPAR-γ in glucose metabolism and adipogenesis is well established. In fact, PPAR-γ agonists of the thiazolidinedione class are commonly used in the treatment of type II diabetes. More recently, the role of PPAR-γ in modulating inflammatory response has begun to be appreciated. For example, we (13) and others (14–16) have demonstrated that administration of PPAR-γ agonists inhibits the clinical signs of experimental autoimmune encephalomyelitis (EAE), suggesting that these agonists may be effective in the treatment of MS. In addition, we have demonstrated that PPAR-γ agonists also suppress the development of EAE and suppress the expression of proinflammatory cytokines and chemokines by glia (17–19).

TLRs are a family of pattern recognition receptors expressed by cells of the innate immune system that recognize specific pathogen-associated molecular patterns (PAMPs), including those present on bacteria, viruses, and fungi. In both mice and humans, 11 TLRs have been identified. Like TLR, CD14 is a pattern recognition receptor. This receptor is expressed on cells on the monocyte lineage and recognizes PAMPS, including LPS on the surface of Gram-negative bacteria as well as peptidoglycan found on the surface of Gram-positive bacteria. The activation of most TLRs (with the exception of TLR3) stimulates MyD88-dependent signaling pathways. In MyD88-dependent signaling pathways, ligand activation of TLR results in the recruitment of the adaptor molecule MyD88, which triggers a signaling cascade that ultimately results in the phosphorylation and subsequent ubiquitination and proteasome-mediated degradation of I-κB. Degradation of I-κB unmasks the nuclear localization signal of NF-κB, resulting in the nuclear localization of this transcription factor and subsequent activation of NF-κB-responsive genes (20). Previously, PPAR-γ agonists were demonstrated to suppress NF-κB activity (21). This suggests that PPAR-γ agonists may inhibit NF-κB activity through effects on TLR/MyD88 signaling pathways.

IL-12 family cytokines are important modulators of EAE and MS. The current studies demonstrate that PPAR-γ agonists suppress the production of IL-12 family cytokines by microglia and astrocytes, CNS glial cells that contribute to the pathology of MS and EAE when chronically activated. This suggests that PPAR-γ agonists modulate EAE, at least in part, by suppressing glial production of IL-12 family cytokines, molecules known to stimulate the differentiation of Th1 cells that exacerbate disease. In addition, these agonists suppressed TLR2, MyD88, and CD14 expression in glia, suggesting a molecular mechanism by which these agonists alter the expression of IL-12 family proteins. These studies further suggest that PPAR-γ agonists may be effective in the treatment of MS.

Materials and Methods

Reagents

The 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) and rosiglitazone were obtained from Cayman Chemical. LPS, the lectin Griffonia simplicifolia, and l-leucine methyl ester hydrochloride were obtained from Sigma-Aldrich. DMEM medium, glutamine, trypsin, and antibiotics used for tissue culture were obtained from BioWhittaker. Oxaloacetate pyruvate insulin medium supplement was obtained from Sigma-Aldrich. FBS was obtained from HyClone. GM-CSF was obtained from BD Pharmingen. The cytokines IFN-γ or TNF-α were obtained from R&D Systems. Glial fibrillary acidic protein was obtained from DakoCytomation. C57BL/6 mice were obtained from Harlan, and bred in house.

Cell culture

Primary mouse microglia cultures were obtained through a modification of the McCarthy and de Vellis (22) protocol. Briefly, cerebral cortices from 1- to 3-day-old C57BL/6 mice were excised, meninges were removed, and cortices were minced into small pieces. Cells were separated by trypsinization, followed by trituration of cortical tissue. The cell suspension was filtered through a 70-µm cell strainer to remove debris. Cells were centrifuged at 153 × g for 5 min at 4°C; resuspended in DMEM medium containing 10% FBS, 1.4 mm l-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, OPI medium supplement, and 0.5 ng/ml mouse rGM-CSF; and plated into tissue culture flasks. Cells were allowed to grow to confluence (7–10 days) at 37°C/5% CO2. Flasks were then shaken overnight (200 rpm at 37°C) in a temperature-controlled shaker to loosen microglia and oligodendrocytes from the more adherent astrocytes. These less adherent cells were plated for 2–3 h and then lightly shaken to separate oligodendrocytes from the more adherent microglia. Microglia were seeded in 24- or 6-well plates and incubated overnight at 37°C/5% CO2. Astrocyte medium contained all the substances described in the microglia medium above except GM-CSF. Astrocyte medium was also supplemented with 0.1 mm l-leucine methyl ester to eliminate any remaining microglia. After shaking to remove microglia and oligodendrocytes, astrocytes were recovered by trypsinization, seeded in 24- or 6-well plates, and incubated overnight at 37°C/5% CO2. After overnight incubation, microglia and astrocytes were treated with 15d-PGJ2 and rosiglitazone for 1 h, and then stimulated with LPS or IFN-γ plus TNF-α, as indicated. The purity of microglia and astrocyte cultures was >95% determined by immunohistochemical staining with the lectin Griffonia simplicifolia or glial fibrillary acidic protein, respectively.

Cell viability assay

Cell viability was determined by MTT reduction assay, as described previously (23). ODs were determined using a Spectromax 190 microplate reader (Molecular Devices) at 570 nm. Results were reported as percentage of viability relative to untreated cultures.

ELISA

Cytokine (IL-12p40, IL-12p70) levels in tissue culture medium were determined by ELISA, as described by the manufacturer (OptEIA Sets; BD Pharmingen). Cytokine IL-23 (p19/p40) levels in tissue culture medium were determined by ELISA, as described by the manufacturer (eBioscience). The use of a p19-specific capture Ab and a p40-specific detection Ab renders this assay specific for mouse IL-23. Cytokine IL-27p28 levels in tissue culture medium were determined by ELISA, as described by the manufacturer (R&D Systems). ODs were determined using a Spectromax 190 microplate reader (Molecular Devices) at 450 nm. Cytokine concentrations in medium were determined from standards containing known concentrations of the proteins.

RNA isolation and cDNA synthesis

Total RNA was isolated from microglia and astrocytes using the TRIzol reagent (Invitrogen Life Technologies). The integrity and amount of RNA were tested on the Agilent Bioanalyzer. Only samples with lack of degradation were further analyzed. RNA samples were treated with DNase I (Invitrogen Life Technologies) to remove any traces of contaminating DNA. The reverse-transcription reactions were conducted using an iScript cDNA synthesis kit (Bio-Rad), according to the manufacturer’s instructions.

Real-time quantitative RT-PCR assay

IL-12p40, IL-12p35, IL-23p19, IL-27EBI3, IL-27p28, TLR2, TLR4, MyD88, and CD14 mRNA were quantified by real-time PCR using an iCycler IQ multicolor real-time PCR detection system (Bio-Rad). All primers and TaqMan MGB probes (FAM dye labeled) were designed and synthesized by Applied Biosystems. The real-time PCR were performed in a total volume of 25 µl using an iCycler kit (Bio-Rad). The levels of IL-12p40, IL-12p35, IL-23p19, IL-27EBI3, IL-27p28, TLR2, TLR4, MyD88, and CD14 mRNA expression in primary microglia and astrocyte were calculated after normalizing cycle thresholds against the housekeeping gene GAPDH and are presented as the fold change value (2−ΔΔCt) relative to LPS or IFN-γ plus TNF-α-stimulated microglia or astrocytes.

Protein extraction and Western blotting

Protein extracts were prepared from primary microglia or astrocytes cultured in 6-well plates by lysing cells in radioimmunoprecipitation assay buffer (1% Triton X-100, 0.1% SDS in PBS (pH 7.4)) supplemented with a Complete protease inhibitor mixture tablet (Roche). Lysates were incubated on ice for 30 min, followed by centrifugation at 21,000 × g for 20 min at 4°C to pellet debris. The resulting supernatants represent total cell protein lysates. The amount of total protein was quantitated by using a standard protein assay (bicinchoninic acid protein assay reagent; Pierce). Cellular proteins (25 µg) were separated electrophoretically on 4–15% Tris-HCl Ready Gels (Bio-Rad). Proteins were transferred electrophoretically to nitrocellulose membranes (NitroBind; GE Osmonics) and then incubated with goat anti-mouse TLR2 Ab (R&D Systems), rabbit antihuman/ mouse MyD88 Ab (eBioscience), or rat anti-mouse CD14 Ab (BD Pharmingen), overnight at 4°C. Blots were then incubated with HRP-conjugated donkey anti-goat IgG for TLR2, goat anti-rabbit IgG for MyD88, or goat anti-rat IgG for CD14 (Santa Cruz Biotechnology) secondary Abs for 1 h at room temperature. TLR2, MyD88, or CD14 protein was detected by ChemoGlow West substrate, as described by the manufacturer (Alpha Innotech), and visualized using a ChemiDoc XRS (Bio-Rad) image analysis system. Actin protein was detected in a similar manner using a rabbit anti-actin polyclonal Ab (Sigma-Aldrich; catalogue A-5060) to verify uniformity in gel loading.

Statistics

Data were analyzed by one-way ANOVA, followed by a Bonferroni post hoc test to determine the significance of difference.

Results

Effects of PPAR-γ agonists on IL-12 family subunit mRNA expression by primary mouse microglia

Previously, we (13) and others (14–16) demonstrated that PPAR-γ agonists suppress the development of EAE. We have also demonstrated that these agonists potently suppress the production of proinflammatory mediators, including NO, various cytokines, and chemokines by primary mouse microglia and astrocytes (23–25). These studies suggest that PPAR-γ agonists may modulate EAE in part through effects on resident CNS glia. The IL-12 family of cytokines is believed to be important modulators of EAE and MS. The expression of these cytokines in peripheral APCs is well characterized. However, relatively little is known concerning the expression of IL-12 family cytokines in CNS glia. In the present studies, real-time quantitative RT-PCR analysis indicated that primary mouse microglia constitutively express very low levels of IL-12 family subunit RNAs, including IL-12p40 (Fig. 1A), IL-12p35 (Fig. 1B), IL-23p19 (Fig. 1C), and IL-27p28 (Fig. 1E). However, these cells constitutively express EBI3 mRNA (Fig. 1D). LPS dramatically induced the mRNA expression of IL-12p40 and IL-12p35 mRNAs that encode subunits that comprise the IL-12p70 holoprotein. In addition, LPS induced the expression of IL-23p19 mRNA, which encodes IL-23p19 that heterodimerizes with IL-12p40 to form IL-23. Finally, LPS induced the expression of EBI3 and IL-27p28, which encode subunits that comprise IL-27. Interestingly, we demonstrate for the first time that the PPAR-γ agonists 15d-PGJ2 and rosiglitazone significantly repressed the expression of each of these IL-12 family subunit mRNAs.

FIGURE 1.

FIGURE 1

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PPAR-γ agonists inhibit LPS induction of IL-12 family subunit mRNA expression in primary mouse microglia. Cells were pretreated for 1 h with the indicated concentrations of 15d-PGJ2 (10 µM) and rosiglitazone (300 µM). LPS (0.1 µg/ml) was added as indicated, and 6 h later, total RNA was isolated. IL-12p40 (A), IL-12p35 (B), IL-23p19 (C), IL-27EBI3 (D), and IL-27p28 (E) mRNA levels were quantified by real-time quantitative RT-PCR. Results are expressed as fold changes relative to LPS-treated cells, and all values are normalized against GAPDH. Values are mean ± SEM from four independent experiments, and duplicate reactions were performed on each sample in each experiment. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 vs LPS-treated cultures.

Effects of PPAR-γ agonists on production of IL-12 family cytokines by primary mouse microglia

ELISA analysis was performed to determine whether PPAR-γ agonists suppress microglial production of IL-12 family proteins, as observed for IL-12 family subunit mRNAs. Microglia constitutively expressed little or no detectable IL-12p40 (Fig. 2A), IL-12p70 (p35/p40) (Fig. 2B), IL-23 (p19/p40) (Fig. 2C), and IL-27p28 (Fig. 2D) protein. However, LPS potently induced production of these cytokines. Furthermore, 15d-PGJ2 and rosiglitazone suppressed LPS induction of IL-12 family cytokines by microglia (Fig. 2). Control studies demonstrated that the doses of PPAR-γ agonists used in these studies did not alter cell viability as determined by MTT analysis (data not shown) (24). Interestingly, the effective dose required to inhibit IL-12 family proteins and RNAs was much higher for rosiglitazone than 15d-PGJ2. Because rosiglitazone binds PPAR-γ with higher affinity than 15d-PGJ2, this suggests that 15d-PGJ2 may act at least in part through receptor-independent mechanisms. Collectively, these studies demonstrate for the first time that PPAR-γ agonists inhibit the production of IL-12 family cytokines by microglia. The studies further suggest that these agonists inhibit transcription of the genes encoding IL-12 family subunit mRNAs. However, the effects of PPAR-γ agonists on the stability of these mRNAs and other posttranscriptional activities have not been evaluated.

FIGURE 2.

FIGURE 2

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PPAR-γ agonists inhibit LPS induction of IL-12 family cytokine production in primary mouse microglia. Cells were pretreated for 1 h with the indicated concentrations (µM) of 15d-PGJ2 and rosiglitazone. LPS (0.1 µg/ml) was added as indicated, and 24 h later, the concentration of IL-12p40 (A), IL-12p70 (p35/p40) (B), IL-23 (p19/p40) (C), and IL-27p28 (D) in the culture medium was determined. Values represent the mean ± SEM for a representative experiment run in triplicate. Three independent experiments were conducted. *, p < 0.05, and ***, p < 0.001 vs LPS-treated cultures.

Effects of PPAR-γ agonists on IL-12 family subunit mRNA expression by IFN-γ plus TNF-α-stimulated primary mouse microglia

Like LPS, the proinflammatory cytokines IFN-γ and TNF-α stimulate the activation of CNS glia. Importantly, these cytokines are believed to contribute to the pathogenesis associated with MS. Therefore, we wished to determine whether a combination of IFN-γ plus TNF-α induced the expression of IL-12 family cytokines in microglia, and whether the PPAR-γ agonists altered the expression of IL-12 family cytokines in these cells. The cytokines IFN-γ plus TNF-α induced the expression of IL-12p40 (Fig. 3A), IL-12p35 (Fig. 3B), and IL-27p28 (Fig. 3D) mRNAs in primary microglia. As opposed to LPS (Fig. 1), these cytokines did not significantly induce the expression of IL-23p19 (data not shown) and IL-27 EBI3 mRNA (Fig. 3C). Importantly, the PPAR-γ agonists 15d-PGJ2 and rosiglitazone potently suppressed IFN-γ plus TNF-α induction of IL-12p40, IL-12p35, and IL-27p28 mRNA expression in primary microglia.

FIGURE 3.

FIGURE 3

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PPAR-γ agonists inhibit IFN-γ plus TNF-α (IT) induction of IL-12 family subunit mRNA expression in primary mouse microglia. Cells were pretreated for 1 h with 15d-PGJ2 (10 µM) or rosiglitazone (300 µM). IFN-α (250 U/ml) and TNF-α (500 U/ml) were added as indicated, and 6 h later, total RNA was isolated. IL-12p40 (A), IL-12p35 (B), IL-27EBI3 (C), and IL-27p28 (D) mRNA levels were quantified by real-time quantitative RT-PCR. Results are expressed as fold changes relative to IFN-γ plus TNF-α-treated cells, and all values are normalized against GAPDH. Values are mean ± SEM from three independent experiments, and duplicate reactions were performed on each sample in each experiment. **, p < 0.01, and ***, p < 0.001 vs IFN-γ plus TNF-α-treated cultures.

Effects of PPAR-γ agonists on production of IL-12 family cytokines by IFN-γ plus TNF-α-stimulated primary mouse microglia

The cytokines IFN-γ and TNF-α induced the expression of IL-12p40 (Fig. 4A) and IL-27p28 (Fig. 4B) proteins by primary microglia, as determined by ELISA analysis. However, these cytokines did not induce the expression of detectable levels of IL-12p70 and IL-23 proteins in these cells (data not shown). Because IFN-γ plus TNF-α induced the expression of both IL-12p40 and IL-12p35 mRNAs (Fig. 3), it is surprising that these cytokines do not induce IL-12p70. However, it is possible that the sensitivity of this assay will not allow detection of very low levels of this cytokine. It is likely that the lack of induction of IL-23p19 by IFN-γ plus TNF-α (Fig. 3) is responsible for the absence of microglial production of IL-23 protein. Importantly, the PPAR-γ agonists 15d-PGJ2 and rosiglitazone potently suppressed IFN-γ plus TNF-α induction of IL-12p40 and IL-27p28 protein expression in primary microglia. In summary, LPS is a more potent stimulus of IL-12 family cytokine expression in primary microglia than a combination of IFN-γ plus TNF-α. However, PPAR-γ agonists appear to suppress the production of IL-12 family cytokines in a similar manner in response to each of these stimuli.

FIGURE 4.

FIGURE 4

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PPAR-γ agonists inhibit IFN-γ plus TNF-α (IT) induction of IL-12 family proteins in primary mouse microglia. Cells were pretreated for 1 h with 15d-PGJ2 (10 µM) or rosiglitazone (300 µM). IFN-γ (250 U/ml) and TNF-α (500 U/ml) were added as indicated, and 24 h later, the concentration of IL-12p40 (A) and IL-27p28 (B) in the culture medium was determined. Values represent the mean ± SEM for a representative experiment run in triplicate. Three independent experiments were conducted. **, p < 0.01, and ***, p < 0.001 vs IFN-γ plus TNF-α-treated cultures.

Effects of PPAR-γ agonists on IL-12 family subunit mRNA expression by primary mouse astrocytes

Controversy exists as to whether or not activated astrocytes express IL-12. Some studies indicated that these cells express IL-12p40, but not the p70 holoprotein (26). Other studies indicated that astrocytes are capable of producing both IL-12p40 and IL-12p70 (27). The present studies indicate that astrocytes constitutively express little or no IL-12p40 (Fig. 5A), IL-12p35 (data not shown), IL-23p19 (Fig. 5B), and IL-27p28 (Fig. 5D), but do constitutively express EBI3 (Fig. 5C) mRNA. LPS potently induced the expression of each of these subunit mRNAs with the exception of IL-12p35 mRNA, which was not significantly induced by LPS stimulation of astrocytes. Furthermore, the PPAR-γ agonist 15d-PGJ2 suppressed the LPS induction of IL-12p40, IL-23p19, EBI3, and IL-27p28 mRNAs (Fig. 5). In addition, the PPAR-γ agonist rosiglitazone inhibited LPS induction of IL-23p19, EBI3, and IL-27p28 mRNA, but did not significantly inhibit IL-12p40 mRNA (Fig. 5).

FIGURE 5.

FIGURE 5

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Effects of PPAR-γ agonists on LPS induction of IL-12 family subunit mRNA expression in primary mouse astrocytes. Cells were pretreated for 1 h with the indicated concentrations of 15d-PGJ2 (25 µM) and rosiglitazone (300 µM). LPS (2.5 µg/ml) was added as indicated, and 6 h later, total RNA was isolated. IL-12p40 (A), IL-23p19 (B), IL-27EBI3 (C), and IL-27p28 (D) mRNA levels were quantified by real-time quantitative RT-PCR. Results are expressed as fold changes relative to LPS-treated cells, and all values are normalized against GAPDH. Values are mean ± SEM from four independent experiments, and duplicate reactions were performed on each sample in each experiment. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 vs LPS-treated cultures.

Effects of PPAR-γ agonists on production of IL-12 family cytokines by primary mouse astrocytes

Primary astrocytes express low or undetectable levels of IL-12p40 (Fig. 6A), IL-12p70 (data not shown), IL-23 (Fig. 6B), and IL-27p28 (Fig. 6C) proteins. LPS induced the expression of IL-12p40, IL-23, and IL-27p28, but IL-12p70 was not detected in these same cultures. The PPAR-γ agonist 15d-PGJ2 potently inhibited LPS induction of IL-12p40, IL-23, and IL-27p28, whereas rosiglitazone inhibited LPS induction of IL-23 and IL-27p28, but did not significantly inhibit IL-12p40 (Fig. 6). In addition, as observed with microglia, 15d-PGJ2 more potently suppressed LPS induction of IL-12 family cytokines than rosiglitazone. The PPAR-γ agonists did not significantly alter astrocyte cell viability relative to LPS alone in these studies (data not shown) (24). To the best of our knowledge, this is the first time that primary astrocytes have been demonstrated to express IL-23 and IL-27p28. In addition, the effects of PPAR-γ agonists on IL-12 family cytokines had not been previously investigated.

FIGURE 6.

FIGURE 6

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PPAR-γ agonists inhibit LPS induction of IL-12 family cytokine production in primary mouse astrocytes. Cells were pretreated for 1 h with the indicated concentrations (µM) of 15d-PGJ2 and rosiglitazone. LPS (2.5 µg/ml) was added as indicated, and 24 h later, the concentration of IL-12p40 (A), IL-23 (p19/p40) (B), and IL-27p28 (C) in the culture medium was determined. Values represent the mean ± SEM for a representative experiment run in triplicate. Three independent experiments were conducted. *, p < 0.05, and ***, p < 0.001 vs LPS-treated cultures.

Effects of PPAR-γ agonists on TLR signaling by primary mouse glia

The NF-κB family of transcription factors plays a critical role in regulating the expression of a variety of genes that encode proinflammatory molecules. Previously, PPAR-γ agonists were demonstrated to suppress NF-κB activity. MyD88-dependent TLR signaling results in the activation of NF-κB. Therefore, in the present study, we investigated the effects of LPS and PPAR-γ agonists on TLR2, TLR4, MyD88, and CD14 expression in primary glia. As expected, TLR4 mRNA was constitutively expressed in primary microglia (Fig. 7A) and astrocytes (Fig. 8A), and the levels of TLR4 mRNA were not induced by LPS, as has been demonstrated previously (20). In addition, PPAR-γ agonists did not significantly alter TLR4 mRNA expression in these cells. However, LPS induced the expression of TLR2, MyD88, and CD14 mRNA in primary microglia (Fig. 7, B–D) and astrocytes (Fig. 8, B–D). Interestingly, the PPAR-γ agonist 15d-PGJ2 suppressed TLR2, MyD88, and CD14 mRNA expression in these cells. However, rosiglitazone only suppressed CD14 mRNA in microglia and MyD88 mRNA expression in primary astrocytes. Similarly, 15d-PGJ2 inhibited LPS induction of TLR2, MyD88, and CD14 protein expression in primary microglia (Fig. 9), whereas rosiglitazone only suppressed CD14 protein production in these cells. The differential regulation of TLR2, MyD88, and CD14 expression by 15d-PGJ2 and rosiglitazone suggests that 15d-PGJ2 may regulate the expression of these molecules at least in part through PPAR-γ-independent mechanisms. The studies also suggest that 15d-PGJ2 suppresses the expression of IL-12 family cytokines in part by suppressing MyD88-dependent TLR signaling pathways in glia.

FIGURE 7.

FIGURE 7

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Effects of PPAR-γ agonists on LPS induction of TLR2, TLR4, MyD88, and CD14 mRNA expression in primary mouse microglia. Cells were pretreated for 1 h with 15d-PGJ2 (10 µM) or rosiglitazone (300 µM). LPS (0.1 µg/ml) was added as indicated, and 6 h later, total RNA was isolated. TLR4 (A), TLR2 (B), MyD88 (C), and CD14 (D) mRNA levels were quantified by real-time quantitative RT-PCR. Results are expressed as fold changes relative to LPS-treated cells, and all values were normalized against GAPDH. Values are mean ± SEM from four independent experiments, and duplicate reactions were performed on each sample in each experiment. **, p < 0.01, and ***, p < 0.001 vs LPS-treated cultures.

FIGURE 8.

FIGURE 8

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Effects of PPAR-γ on LPS induction of TLR2, TLR4, MyD88, and CD14 mRNA expression in primary mouse astrocytes. Cells were pretreated for 1 h with 15d-PGJ2 (25 µM) or rosiglitazone (300 µM). LPS (2.5 µg/ml) was added as indicated, and 6 h later, total RNA was isolated. TLR4 (A), TLR2 (B), MyD88 (C), and CD14 (D) mRNA levels were quantified by real-time quantitative RT-PCR. Results are expressed as fold changes relative to LPS-treated cells, and all values are normalized against GAPDH. Values are mean ± SEM from four independent experiments, and duplicate reactions were performed on each sample in each experiment. **, p < 0.01, and ***, p < 0.001 vs LPS-treated cultures.

FIGURE 9.

FIGURE 9

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Effects of PPAR-γ agonists on LPS induction of TLR2, MyD88, or CD14 protein expression in mouse primary microglia. Cells were pretreated for 1 h with 15d-PGJ2 (10 µM) or rosiglitazone (300 µM). LPS (0.1 µg/ml) was added as indicated to cultures, and 24 h later, protein extracts from whole cell lysates were prepared. Protein expression for TLR2, MyD88, or CD14 was evaluated by Western blotting, as described in Materials and Methods. Western blot analyses are representative of four independent experiments (A). Each TLR2, MyD88, CD14, and actin band was quantified by densitometry. Results are expressed in arbitrary units as the ratio of TLR2, MyD88, or CD14 to actin, and LPS was used as reference and assigned the arbitrary value of 1. Values represent the mean ± SEM from four independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 vs LPS-treated cultures (B).

Discussion

PPAR-γ agonists were previously demonstrated to inhibit the development of EAE (13–16). Chronically activated microglia and astrocytes produce proinflammatory molecules that may contribute to the loss of neurons and oligodendrocytes, cells that are compromised in MS. Previously, we demonstrated that PPAR-γ agonists block the production of proinflammatory molecules by CNS glia (23–25). Collectively, these studies suggest that PPAR-γ agonists may modulate the development of EAE in part through effects on glial cell activation. The IL-12 family of proteins plays a critical role in differentiation of Th1 cells, which are believed to exacerbate EAE and MS. The current studies indicate that the PPAR-γ agonists 15d-PGJ2 and rosiglitazone inhibit the production of IL-12 family cytokines by CNS glia. These studies suggest that PPAR-γ agonists may alter T cell phenotype by limiting IL-12 family cytokine production by glia.

IL-12 plays an important role in eliminating pathogens, including bacteria and viruses (28, 29). However, this cytokine can contribute to the development of a variety of autoimmune diseases, including MS, by triggering the differentiation of naive T cells into CD4+ Th1 cells (30, 31). The fact that IL-12 expression in CNS lesions correlates with disease activity in MS patients supports a role for this cytokine in modulating disease (32–34). A variety of studies indicates that IL-12 plays a role in modulating EAE. For example, IL-12 induces disease in mouse strains that are normally resistant to the development of EAE (35, 36). Furthermore, IL-12 treatment of T cells reactive to CNS Ags ex vivo increases the severity of disease following transfer of these cells into naive recipient mice in adoptive transfer models of EAE (37). Suppression of EAE with IL-12-neutralizing Abs further supports a role for this cytokine in modulating disease (38). Additional studies demonstrated that IL-12p40 knockout mice were resistant to EAE induction. In contrast, IL-12p35 knockout mice showed similar or even more severe clinical and pathological EAE compared with wildtype mice (39). These studies indicated a redundancy within the IL-12 system, and suggested that other p40-containing cytokines, such as the more recently discovered IL-23, may be required for establishment of EAE.

Recent studies suggest that IL-23 plays a critical role in the development of EAE (40). Studies demonstrating that IL-12 p40−/− mice are resistant to EAE were originally interpreted to support a critical role for IL-12 in disease. However, subsequent studies demonstrated that like IL-12 p40−/− mice, IL-12 p19−/− mice do not develop EAE (41), whereas IL-12 p35−/− mice develop severe disease (39, 42). Because IL-23 exists as a heterodimer consisting of p40 and p19 subunits, these studies defined a critical role for IL-23 in the pathogenesis of EAE. IL-23 induces the production of IL-17 by memory T cells termed ThIL-17 cells (43), and IL-17 has been demonstrated to modulate the development of autoimmune diseases, including EAE (44, 45). Collectively, these studies suggest that IL-23 may influence the development of EAE via altering IL-17 expression by memory T cells.

IL-27 was recently added to the IL-12 family of proteins based on sequence and functional similarities to existing family members (8). The transcription factors T-bet and GATA-3 are believed to play critical roles in the differentiation of Th1 and Th2 cells, respectively. Interestingly, IL-27 induces the expression of T-bet and suppresses the expression of GATA-3, suggesting a mechanism by which IL-27 skews T cell differentiation toward a Th1 cell phenotype (46, 47). In addition, IL-27 induces the expression of IL-12Rβ2 in CD4+ and NK cells, thus increasing the response of these cells to IL-12. This suggests a mechanism by which IL-12 and IL-27 act cooperatively in inducing Th1 cell differentiation (46, 47). Recent studies also suggest that IL-27 may modulate the development of EAE. For example, inflammatory cells present in the CNS during EAE express elevated levels of IL-27 subunits mRNAs as well as IL-27R (WSX-1) mRNA (48). In addition, IL-27-specific Abs block the development of EAE (49). It should be noted that IL-27 exhibits a complex array of proand anti-inflammatory properties that may contribute to or alternatively suppress the development of EAE (50). In fact, IL-27 blocks the development of ThIL-17 cells that are believed to exacerbate disease (51, 52). In summary, collectively, our present studies demonstrate for the first time that PPAR-γ agonists suppress the production of IL-12 family cytokines by CNS glia, suggesting that these agonists may be effective in the treatment of MS.

The mechanisms by which PPAR-γ agonists inhibit the production of IL-12-family cytokines in activated glia are unknown. NF-κB is believed to play a critical role in regulating IL-12p40 gene expression and possible genes that encode other IL-12 family subunits (53, 54). Furthermore, NF-κB activity has been demonstrated to be suppressed by PPAR-γ agonists, including 15d-PGJ2 (21). The present studies demonstrate that 15d-PGJ2 inhibits the LPS induction of TLR2, MyD88, and CD14 expression in primary microglia. This suggests that this agonist suppresses NF-κB activity by altering MyD88-dependent TLR signaling pathways. Previous studies demonstrated that primary microglia constitutively express TLR2 and TLR4, but expression of these molecules in astrocytes remains controversial (20). Furthermore, LPS induces microglial and macrophage expression of TLR2, but not TLR4 (55–57). Similarly, our current studies demonstrated that LPS did not significantly alter TLR4 expression by primary microglia. LPS signaling is principally mediated through TLR4. However, at high doses, LPS signaling can also occur through TLR4-independent mechanisms, the details of which have not been elucidated (58). Furthermore, TLR2 and TLR4 have been demonstrated to act cooperatively in responding to bacterial cell wall components (57). The current studies demonstrate for the first time that PPAR-γ agonists modulate the expression of TLR2, MyD88, and CD14, critical molecules in MyD88-dependent signaling pathways.

In summary, we have demonstrated that PPAR-γ agonists suppressed the production of IL-12-family proteins by activated glia. These cytokines are key modulators of the development of EAE and MS. Therefore, these studies suggest that PPAR-γ agonists may be effective in the treatment of MS.

Footnotes

1

This work was supported by grants from the National Institutes of Health (NS42860 and NS 047546), the National Multiple Sclerosis Society (RG 3427-A-8), and the Arkansas Biosciences Institute.

3

Abbreviations used in this paper: MS, multiple sclerosis; 15d-PGJ2, 15-deoxy-Δ12,14-PGJ2; EAE, experimental autoimmune encephalomyelitis; EBI3, EBV-induced molecule 3; PAMP, pathogen-associated molecular pattern; PPAR, peroxisome proliferator-activated receptor.

Disclosures The authors have no financial conflict of interest.

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