Peroxisome Proliferator-Activated Receptor-Gamma Agonists Inhibit

Home , Ciglitazone

Genes and Immunity (2002) 3, 59–70  2002 Nature Publishing Group All rights reserved 1466-4879/02 $25.00 www.nature.com/gene Peroxisome proliferator-activated receptor-gamma agonists inhibit experimental allergic encephalomyelitis by blocking IL-12 production, IL-12 signaling and Th1 differentiation

C Natarajan and JJ Bright Division of Neuroimmunology, Department of Neurology, Vanderbilt University School of Medicine, Nashville, TN 37212, USA

Peroxisome proliferator-activated receptor-gamma (PPAR␥) is a nuclear receptor transcription factor that regulates adipocyte differentiation and glucose homeostasis. PPAR␥ agonists are potent therapeutic agents for the treatment of type 2 diabetes and obesity. PPAR␥ agonists also prevent inflammation in animal models, suggesting their use for the treatment of human inflammatory diseases. Experimental allergic encephalomyelitis (EAE) is a Th1 cell-mediated inflammatory demyelinating disease model of multiple sclerosis (MS) and IL-12 plays a crucial role in the pathogenesis of EAE and MS. In this study we have examined the effect of PPAR␥ agonists on the pathogenesis of EAE. In vivo ␥ ⌬12,14 treatment of SJL/J mice with PPAR agonists, 15-deoxy prostaglandin J2 or Ciglitazone, decreased the duration and clinical severity of active immunization and adoptive transfer models of EAE. PPAR␥ agonists inhibited EAE in association with a decrease in IL-12 production and differentiation of neural antigen-specific Th1 cells. In vitro treatment of activated T cells with PPAR␥ agonists inhibited IL-12-induced activation of JAK-STAT signaling pathway and Th1 differentiation. These findings highlight the fact that PPAR␥ agonists regulate central nervous system inflammation and demyelination by inhibiting IL-12 production, IL-12 signaling and Th1 differentiation in EAE. Genes and Immunity (2002) 3, 59–70. DOI: 10.1038/sj/gene/6363832

Keywords: cytokine; signal transduction; Th1 cells; inﬂammation; immunomodulation

Introduction cardiac, and adipose tissue.6 Several fatty acids and ecosanoids including 9-Hydroxyoctadecadienoic acid Peroxisome proliferator activated receptors (PPARs) are (HODE) and 13-HODE function as physiological ligands members of the nuclear hormone receptor superfamily of ␥ 7,8 ⌬12,14 for PPAR . The 15-deoxy prostaglandin J2 ligand-activated transcription factors.1,2 PPAR␣, ␦ and ␥ ␥ (15d-PGJ2) is a natural ligand for PPAR that binds and are the three known members of the PPAR family, en- activates PPAR␥ at micromolar concentrations.9 Thiazo- 2 ␣ coded by distinct genes. In mammals, the PPAR is lidinediones (TZD) or glitazones are a class of synthetic expressed abundantly in tissues with high capacity for compounds that function as high affinity agonists for lipid-metabolism such as liver, kidney, heart and adrenal PPAR␥.10 Upon activation with specific ligands, PPAR␥ ␦ gland, whereas, PPAR is expressed ubiquitously in all heterodimerizes with 9-cis-retinoic acid receptor (RXR) 3 ␥ tissues. PPAR has been cloned from many species and and binds to direct repeats of AGGTCA hexanucleotide ␥ ␥ the human PPAR is homologous to murine PPAR with sequence on the promoter region of target genes.11–13 4 ␥ ␥ 95% identity at the amino acid level. PPAR 1, PPAR PPAR␥ is an important regulator of adipogenesis and ␥ ␥ 2 and PPAR 3 are the three isoforms of PPAR , which treatment with PPAR␥ agonists prevent obesity.10,14 arise as products of different promoter usage and alterna- PPAR␥ is a potent regulator of glucose metabolism and ␥ tive splicing. While PPAR 2 is expressed predominantly PPAR␥ agonists enhance the sensitivity of tissues to insu- ␥ ␥ in adipose tissue, PPAR 1 and PPAR 3 are expressed in lin, thereby reducing the plasma glucose and insulin adipose tissue, heart, kidney, pancreas, spleen, intestine, levels in human and animal models of type 2 diabetes.15,16 5 colon epithelial cells and skeletal muscle. The targeted Troglitazone (Rezulin), pioglitazone (ACTOS) and rosig- ␥ disruption of PPAR is embryonically lethal and dies by litazone (Avandia) have been approved by the FDA and day E10 due to defects in the development of placental, are currently marketed as therapeutic agents for the treatment of type 2 diabetes. PPAR␥ agonists also inhibit malignant growth of liposarcoma,17 breast cancer,18 Correspondence: JJ Bright, PhD, Department of Neurology, Vanderbilt myeloid leukemia,19 lung cancer,20 and colon cancer,21,22 University Medical Center, 1222F VSRH, 2201 Capers Avenue, Nash- Ȱ suggesting their use for the treatment of cancer. Interest- ville, TN37212, USA. E-mail: john.brightj mcmail.vanderbilt.edu ingly, recent studies also showed the importance of This work was supported in part by National Multiple Sclerosis ␥ Society Grant RG 3069A2/1 and National Institutes of Health Grant PPAR in the regulation of immune and inflammatory 23,24 R01 NS42257-01A1 (to JJB). responses. In vivo treatment with PPAR␥ agonists pre- Received 23 August 2001; revised and accepted 30 October 2001 vent the pathogenesis of rheumatoid arthritis,25 athero- PPAR␥ regulation of IL-12 in EAE C Natarajan and JJ Bright 60 sclerosis,26,27 colitis,28 and psoriasis29 in animal models, with PPAR␥ agonists following induction of active EAE suggesting their potential use for the treatment of human by immunization with mouse spinal cord homogenate inflammatory diseases. Macrophage has well-established (MSCH). All 10 mice in the DMSO treated control group involvement in the inflammatory processes and the nat- developed clinical paralysis for a mean duration of 17 ural and synthetic PPAR␥ ligands block macrophage acti- days with a mean maximum clinical severity (MMCS) of vation and expression of inflammatory cytokines, 3.55 on day 13 (Figure 1). Conversely, treatment with inducible nitric oxide synthase, gelatinase B and scaven- PPAR␥ agonists decreased the clinical severity and dur- ger receptor A gene.23,24,30,31 Recent studies have also ation of active EAE (Figure 1). The mice treated with shown that activated T cells express PPAR␥ and treat- 50 ␮g Ciglitazone developed paralysis only for a mean ment with PPAR␥ agonists inhibit antigen-specific T cell duration of 13 days (23.5% reduction) with an MMCS of proliferation, suggesting the role of PPAR␥ in the regu- 1.95 (23.5% reduction). Treatment of mice with 100 ␮g lation of T cell-mediated immune and inflammatory Ciglitazone decreased the mean duration of disease to 8 responses.32,33 days (53% reduction) with an MMCS of 0.65 (82% inhi- Multiple sclerosis (MS) is an inflammatory demyelin- bition, P Ͻ 0.025%). Similarly, mice treated with 50 ␮g ating disease of the central nervous system (CNS) that 15d-PGJ2 developed paralysis only for a mean duration of afflicts around 1 million people worldwide.34 About 30% 12 days (29.5% reduction) with an MMCS of 1.62 (43.7% ␮ of the MS patients develop clinical paralysis and become inhibition). Treatment of mice with 100 g 15d-PGJ2 wheel chair bound for the rest of their lives. There is no decreased the duration of 7 days (59% inhibition) and medical treatment available so far that can cure MS.35 The MMCS to 0.25 (93% inhibition, P Ͻ 0.001). These results destruction of oligodendrocyte and myelin sheath in the suggest that PPAR␥ agonists inhibit the severity and dur- CNS is the pathological hall mark of MS.36 Although the ation of clinical paralysis in active EAE. etiology of MS is not known, it is generally viewed as an inflammatory autoimmune disease of the CNS, resulting PPAR␥ agonists inhibit passive EAE from activation, expansion and homing of myelin anti- To further study the role of PPAR␥ in the regulation of gen-sensitized T cells in the CNS.37–39 Experimental CNS inflammation and demyelination, we examined the allergic encephalomyelitis (EAE) is a CD4+ Th1 cell- in vivo effect of PPAR␥ agonists on adoptive transfer mediated inflammatory autoimmune demyelinating EAE. SJL/J mice were treated with PPAR␥ agonists fol- disease of the CNS.40 EAE can be induced in many sus- lowing induction of passive EAE by adoptive transfer of ceptible strains of rodents and primates by immunizing MBP specific T cells. All the 10 mice in the DMSO treated with whole brain homogenate or purified neural antigens control group developed clinical paralysis for a duration such as myelin basic protein (MBP), proteolipid protein of 16 days with a mean maximum clinical severity of 2.6 (PLP) and myelin oligodendrocyte glycoprotein on day 10 (Figure 2). Treatment of mice with PPAR␥ (MOG).40–42 The antigenic epitopes of neural antigens agonists decreased the clinical severity of adoptive trans- have been mapped and adoptive transfer of T cells fer EAE. Treatment of mice with 50 ␮g Ciglitazone reactive to these epitopes is sufficient to induce the dis- decreased the clinical paralysis for a duration of 12 days ease.41,42 Many clinical and pathological features of EAE (25% reduction) with an MMCS of 1.2 (53.8% inhibition) show close similarity to MS and therefore EAE has been and that decreased to a duration of 7 days (56% commonly used as a model system to study the mech- inhibition) with an MMCS of 0.15 (94.5% inhibition, anism of MS pathogenesis and to test the efficacy of P Ͻ 0.001%) following treatment with 100 ␮g Ciglitazone potential therapeutic agents for the treatment of MS.40–42 (Figure 2). Similarly, treatment of mice with 50 ␮g 15d- The pathogenesis of EAE/MS is a complex process that involves activation of macrophage/microglial cells, differentiation of encephalitogenic Th1 cells and secretion of inflammatory cytokines in the CNS.43 Interleukin-12 is a proinflammatory cytokine produced mainly by macrophage/microglia that plays a critical role in the differentiation of encephalitogenic Th1 cells and pathogenesis of EAE and MS.44–46 We and others have shown earlier that the inhibition of IL-12 production or IL-12 signaling through JAK-STAT pathway prevents Th1 differentiation and pathogenesis of EAE.45–50 In this study, we have examined the protective effects and mechanism of action of PPAR␥ agonists in EAE. Our results show that PPAR␥ agonists prevent CNS demyelination by inhibiting IL-12 production, IL-12 signaling and differentiation of neural antigen-specific Th1 cells in EAE.

Results

␥ Figure 1 Inhibition of active EAE by PPAR␥ agonists. SJL/J mice PPAR agonists inhibit active EAE ␮ To define the role of PPAR␥ in the regulation of CNS immunized with MSCH were treated (i.p) with 50 and 100 g Cigli- tazone or 15d-PGJ2 on every other day. The clinical symptoms were inflammation and demyelination, we first examine the scored every day and the mean clinical score of 10 mice from two protective effect of PPAR␥ agonists on the pathogenesis independent experiments is shown. The figure is a representative of active immunization EAE. SJL/J mice were treated of four experiments.

Genes and Immunity PPAR␥ regulation of IL-12 in EAE C Natarajan and JJ Bright 61

Figure 3 Pathological scores of spinal cord sections from EAE mice treated with PPAR␥ agonists. The spinal cord sections from cervical, upper thoracic, lower thoracic and lumbar regions were prepared (four sections per mouse) and each quadrant of every spinal cord section from each mouse was scored for the presence or Figure 2 Inhibition of adoptive transfer EAE by PPAR␥ agonists. absence of inflammation or demyelination. The pathological score SJL/J mice induced to develop passive EAE by adoptive transfer for each treatment group was expressed as the percentage over the of MBP-sensitized T cells were treated (i.p) with 50 and 100 ␮g Cig- total number of quadrants examined. The average number of quadrants examined per mouse was 16, therefore, this study included litazone or 15d-PGJ2 on every other day. The clinical symptoms = were scored every day and the mean clinical score of 10 mice from the analysis of 240 spinal cord quadrants (n 15 mice). two independent experiments is shown. The figure is a representative of four experiments. of PPAR␥ agonists on the neural antigen-specific T cell responses in vitro. As shown in Figure 4a, MBP-immune

PGJ2 inhibited the clinical paralysis for a duration of 12 T cells displayed a strong proliferative response to anti- days with an MMCS of 1.1 (57.7% inhibition) and treat- gen in vitro and treatment with PPAR␥ agonists resulted ␮ ment with 100 g 15d-PGJ2 inhibited the clinical paralysis a dose-dependent decrease in proliferation. Stimulation to a duration of 6 days with an MMCS of 0.12 (95.5% of cells with 100 ␮g/ml MBP increased the 3H thymidine inhibition, P Ͻ 0.001). These results suggest that PPAR␥ uptake from 742 ± 89 in the control background to ± agonists inhibit the clinical severity and duration of 12213 595 cpm. Treatment with Ciglitazone or 15d-PGJ2 adoptive transfer EAE. resulted a dose-dependent decrease in T cell proliferation with a maximum of 80.1% or 84.1% inhibition at PPAR␥ agonists decrease CNS inflammation and 20 ␮g/ml levels respectively (Figure 4a). We have also demyelination in EAE examined the antigen-induced secretion of IFN␥ from We have further examined the effect of PPAR␥ agonists MBP-immune spleen cells in vitro. The spleen cells cul- on the pathogenesis of inflammation and demyelination tured with 100 ␮g/ml MBP produced 6.24 ± 0.15 ng/ml in the CNS of mice with EAE. Spinal cord sections from of IFN␥ in 48 h. Treatment of cells with Ciglitazone or ␥ ␥ mice treated with PPAR agonists following induction of 15d-PGJ2 resulted in a dose-dependent decrease in IFN active EAE were analyzed for the infiltration of mono- production with a maximum of 84.6% or 90.1% inhibition nuclear cells (inflammation) and myelin loss (de- at 20 ␮g/ml levels respectively (Figure 4b). These results myelination). As shown in Figure 3, the DMSO treated suggest that PPAR␥ agonists inhibit proliferation and control EAE mice showed profound inflammation and secretion of IFN␥ from encephalitogenic Th1 cells in EAE. demyelination in the CNS that decreased following treatment with PPAR␥ agonists. In the control group of EAE PPAR␥ agonists inhibit IL-12 production in mice, 51.25% of spinal cord quadrants were positive for macrophage and microglial cells infiltration of mononuclear cells (inflammation) and To determine the mechanisms involved in the PPAR␥ 46.25% of spinal cord quadrants positive for demyelin- regulation of Th1 responses in EAE, we examined the ation. Conversely, treatment with Ciglitazone resulted in effect of PPAR␥ agonists on IL-12 production in 17.53% (65.8% inhibition) and 13.74% (70.3% inhibition) macrophage/microglial cells. As shown in Figure 5a, spinal cord quadrants positive for inflammation and stimulation of MBP immune spleen cells with 100 ␮g/ml demyelination respectively. Similarly, treatment with MBP increased the production of IL-12 in vitro. Treatment ␥ 15d-PGJ2 resulted in 14.97% (70.8% inhibition) and with PPAR agonists, Ciglitazone or 15d-PGJ2 resulted in 12.49% (73.0% inhibition) spinal cord quadrants positive a dose-dependent decrease in neural antigen-induced IL- for inflammation and demyelination respectively 12 production with a maximum of 86.5% or 90.8% inhi- (Figure 3). These results suggest that PPAR␥ agonists bition at 20 ␮g/ml levels respectively (Figure 5a). Since inhibit CNS inflammation and demyelination in EAE. macrophages are the major producers of IL-12 in spleen, we isolated splenic macrophages from SJL/J mice and PPAR␥ agonists inhibit neural antigen-specific T cell stimulated with 1 ␮g/ml LPS and 50 ng/ml of IFN␥ in responses the absence or presence of PPAR␥ agonists in vitro.As To define the mechanisms involved in the PPAR␥ regu- shown in Figure 5b, treatment of macrophages with 5 and lation of CNS demyelination, we first examined the effect 20 ␮g/ml Ciglitazone respectively resulted in 40% and

Genes and Immunity PPAR␥ regulation of IL-12 in EAE C Natarajan and JJ Bright 62

Figure 5 Inhibition of IL-12 production by PPAR␥ agonists. (a) MBP-immune spleen cells were stimulated with 25 ␮g/ml MBP in

the presence of Ciglitazone or 15d-PGJ2 for 48 h. (b) Splenic macrophage or EOC-20 microglial cells were stimulated with LPS in the presence of Ciglitazone or 15d-PGJ for 48 h. The secretion of IL- ␥ 2 Figure 4 Inhibition of MBP-speciﬁc T cell responses by PPAR 12 in the culture supernatant was measured by ELISA. The values agonists in vitro.(a) MBP-immune spleen cells were stimulated in are mean of triplicates at each point and the error bars represent vitro with MBP in the presence of Ciglitazone or 15d-PGJ2.[3H] standard deviation. The ﬁgure is a representative of four differ- thymidine was added at 72 h and the uptake of radioactivity meas- ent experiments. ured after 96 h. (b) MBP-immune spleen cells were stimulated with

MBP in the presence of Ciglitazone or 15d-PGJ2 for 48 h. The levels of IFN␥ secreted in the culture supernatants measured by ELISA. The values are mean of triplicates at each point and the error bars IL-12 production from spleen cells, macrophage and represent standard deviation. The ﬁgures are representatives of microglia in EAE. three independent experiments. PPAR␥ agonists inhibit IL-12-induced T cell responses To further understand the effect of PPAR␥ agonists on 74.2% inhibition of IL-12 production induced by IFN␥ IL-12-induced responses in T cells, we have examined the + ␮ LPS. A similar treatment with 5 and 20 g/ml 15d-PGJ2 proliferative response of MBP immune T cells in the pres- respectively resulted in 45.2% and 84.8% inhibition of ence of exogenous IL-12 in culture. As shown in IFN␥ + LPS-induced IL-12 production in macrophage Figure 6a, PPAR␥ agonists inhibited the proliferation of cells. Since the microglial cells are the major producers T cells in response to MBP in vitro. Addition of rIL-12 of IL-12 in the CNS, we have further examined the effect failed to restore the MBP-induced proliferation of T cells of PPAR␥ agonists on IL-12 production in microglia. from inhibition by PPAR␥ agonists, suggesting that Treatment of EOC-20 microglial cells with 5 and PPAR␥ agonists may also regulate IL-12-induced 20 ␮g/ml Ciglitazone respectively induced 37.2% and responses in T cells. To determine this possibility, we 69.7% inhibition of IL-12 production induced by have further tested the effect of PPAR␥ agonists on IL- IFN␥ + LPS in microglia (Figure 5b). A similar treatment 12-induced proliferation of activated T cells. Stimulation ␮ with 5 and 20 g/ml 15d-PGJ2 also respectively induced of ConA-activated T cells with IL-12 resulted in a dose- 52.8% and 80.8% inhibition of IL-12 production in dependent increase in proliferation as measured by [3H] microglia. These results suggest that PPAR␥ agonists thymidine uptake assay. The cells cultured in medium inhibit CNS inﬂammation and demyelination by blocking alone showed a background count of 930 ± 207 CPM and

Genes and Immunity PPAR␥ regulation of IL-12 in EAE C Natarajan and JJ Bright 63 proliferation of activated T cells in vitro. Finally, we have also examined the effect of PPAR␥ agonists on IL-12- induced differentiation of Th1 cells in vitro. When com- pared with T cells stimulated with anti-CD3 alone, splenic T cells activated in the presence of IL-12 displayed a dramatic increase in Th1 differentiation, as measured by IFN␥ production upon secondary stimulation with anti-CD3 mAb. Strikingly, the addition of PPAR␥

agonists, Ciglitazone or 15d-PGJ2 resulted in a dose- dependent decrease in Th1 differentiation with a maximum of 84.8% and 92.1% inhibition respectively at 20 ␮g/ml levels (Figure 6c), suggesting the inhibition of IL-12-induced Th1 differentiation by PPAR␥ agonists in vitro.

PPAR␥ agonists inhibit IL-12-induced tyrosine phosphorylation of STAT 3 and STAT 4 in T cells To deﬁne the mechanisms involved in the PPAR␥ regulation of IL-12-induced T cell responses, we have examined the effect of PPAR␥ agonists on the activation of STAT proteins in IL-12 signaling. Stimulation of ConA- activated T cells with 2 ng/ml of IL-12 induced the tyrosine phosphorylation of STAT3 and STAT4 in 15 min (Figure 7a, b). Pretreatment of T cells with PPAR␥ agon-

ists, Ciglitazone or 15d-PGJ2 for 15 min inhibited the IL- 12-induced tyrosine phosphorylation of STAT3 and STAT4. While 5 ␮g/ml PPAR␥ agonists induced only partial inhibition, addition of 20 ␮g/ml Ciglitazone or

15d-PGJ2 resulted in a signiﬁcant decrease in IL-12- induced tyrosine phosphorylation of STAT3 in T cells (Figure 7a). Similarly, treatment with 5 ␮g/ml Ciglita-

zone or 15d-PGJ2 induced only partial inhibition of IL- 12-induced STAT4 phosphorylation and that was further inhibited by the addition of 20 ␮g/ml Ciglitazone or 15d- ␥ PGJ2 (Figure 7b). These results suggest that PPAR agonists inhibit IL-12-induced T cell responses by blocking the tyrosine phosphorylation of STAT3 and STAT4 in T cells.

PPAR␥ agonists inhibit IL-12-induced tyrosine Figure 6 Inhibition of IL-12-induced responses by PPAR␥ agonists phosphorylation of JAK2 and TYK2 in T cells in T cells. (a) MBP immune spleen cells were stimulated with MBP To determine whether the inhibition of STAT proteins by ␥ ␥ and PPAR agonists, Ciglitazone or 15d-PGJ2 in the presence of PPAR agonists was a direct effect or consequent to the 2 ng/ml of rIL-12. [3H] thymidine was added at 72 h and the uptake inhibition of upstream JAK kinases, we examined the of radioactivity measured after 96 h. (b) Con A-activated T cells effect of PPAR␥ agonists on IL-12-induced tyrosine phos- were stimulated with 2 ng/ml IL-12 in the presence of 0, 1, 5, 10, ␮ ␥ 3 phorylation of JAK2 and TYK2 kinases in T cells. 15 and 20 g PPAR agonists, Ciglitazone or 15d-PGJ2.[H] thymidine was added at 36 h and the radioactivity was measured after Immunoprecipitation and Western blot analyses showed 48 h. The values (CPM) are mean of triplicates at each point and that the stimulation of ConA-activated T cells with the error bars represent standard deviation. The figure is a rep- 2 ng/ml IL-12 induced the tyrosine phosphorylation of resentative of five experiments. (c) Naive T cells were stimulated JAK2 and TYK2 kinases in 15 min (Figure 8a, b). Pretreat- with anti-CD3 mAb in the presence of 2 ng/ml IL-12 and 0, 1, 5, ␥ ␮ ␥ ment of cells with PPAR agonists for 15 min signifi- 10 and 20 g PPAR agonists, Ciglitazone or 15d-PGJ2. After 5 days, the cells were restimulated with anti-CD3 for 24 h and the cantly reduced the IL-12-induced tyrosine phosphoryl- IFN␥ in the culture supernatant measured by ELISA. ation of both JAK2 and TYK2 kinases. While the addition ␮ of 5 g/ml Ciglitazone or 15d-PGJ2 induced partial inhi- ␮ bition, treatment with 20 g/ml Ciglitazone or 15d-PGJ2 that increased to 11751 ± 879 following addition of resulted in a significant inhibition of JAK2 phosphoryl- 2 ng/ml rIL-12. Treatment of cells with PPAR␥ agonists, ation (Figure 8a). Similarly, treatment with 5 ␮g/ml Cig-

Ciglitazone or 15d-PGJ2, resulted a dose-dependant litazone or 15d-PGJ2 also induced partial inhibition of decrease in IL-12-induced proliferation of T cells TYK2 phosphorylation and that was further inhibited ␮ (Figure 6b). While the cells treated with 20 ␮g/ml Ciglita- following addition of 20 g/ml Ciglitazone or 15d-PGJ2 zone showed 83.4% inhibition, treatment with 20 ␮g/ml (Figure 8b). These results suggest that PPAR␥ agonists 15d-PGJ2 resulted in 90.1% inhibition of proliferation, inhibit IL-12-induced tyrosine phosphorylation and acti- suggesting that PPAR␥ agonists inhibit IL-12-induced vation of JAK2 and TYK2 kinases in T cells.

Genes and Immunity PPAR␥ regulation of IL-12 in EAE C Natarajan and JJ Bright 64

Figure 7 Inhibition of IL-12-induced tyrosine phosphorylation of STAT transcription factors by PPAR␥ agonists in T cells. Western Figure 8 Inhibition of IL-12-induced tyrosine phosphorylation of blot analysis of STAT3 (a) and STAT4 (b) proteins immunoprecipit- JAK kinases by PPAR␥ agonists in T cells. Western blot analysis of ated from Con A-activated T cells after incubation in medium alone, ConA-activated T cells incubated in medium alone, 2 ng/l IL-2, IL- 2 ng/ml IL-12, IL-12 + 5 and 20 ␮g/ml Ciglitazone or 5 and 2 + 5 and 20 ␮g/ml Ciglitazone (Cg), or IL-12 + 5and20␮g/ml ␮ ° ° 20 g/ml 15d-PGJ2 at 37 C for 15 min. The cells were pretreated 15d-PGJ2 at 37 C for 15 min. The cells were pretreated for 10 min for 10 min prior to induction of IL-12 signaling. The STAT3 (a) and prior to induction of IL-12 signaling. The JAK2 (a) and TYK2 (b) STAT4 (b) immune complexes were resolved on 7.5% SDS-PAGE, immunoprecipitates were resolved on 7.5% SDS-PAGE, transferred transferred onto nylon membrane and probed with anti-phospho- onto nylon membrane and probed with anti-phosphotyrosine mAb, tyrosine mAb, 4G10. The blots were striped and reprobed with anti- 4G10. The blots were striped and reprobed with anti-JAK2 (a)or STAT3 (a) or STAT4 (b) antibody. Arrows point to 92 kDa STAT3 anti-TYK2 (b) antibody. Arrows point to 130 kDa JAK2 and 135 kDa (a) and 89 kDa STAT4 (b) protein bands. The ﬁgure is a representa- TYK2 protein band. The ﬁgure is a representative of six experi- tive of six experiments. ments.

PPAR␥ agonists induce a speciﬁc and reversible induced JAK-STAT pathway was speciﬁc to PPAR␥ inhibition of IL-12 signaling through JAK-STAT agonists, we have examined the effect of PGF2␣, a nega-

pathway in T cells tive control for 15d-PGJ2 and Wy14,643, a synthetic To determine whether the blockade of IL-12 signaling by PPAR␣ specific agonist, on IL-12-induced phosphoryl- PPAR␥ agonists resulted the inhibition of IL-12 responses ation of JAK2, TYK2, STAT3 and STAT4 in T cells. As in T cells, we examined the reversibility of JAK and STAT shown in Figure 10, treatment with PGF2␣ or Wy14,643, phosphorylation inhibited by PPAR␥ agonists in T cells. resulted in no decrease in IL-12-induced phosphorylation Con A activated T cells were pretreated with 20 ␮g/ml of JAK2, TYK2, STAT3 or STAT4, suggesting that the ° ␥ 15d-PGJ2 or Ciglitazone for 30 or 120 min at 37 C. The PPAR agonists induced specific inhibitory effect on IL- cells were washed three times with PBS, cultured in fresh 12 signaling and IL-12 mediated responses in T cell. medium for an additional 3 h and then stimulated with IL-12 for 15 min. As shown in Figure 9, the cells washed Discussion free from PPAR␥ agonists after treatment for 30 min and 120 min showed no inhibition of IL-12-induced phos- In this study, we have shown that PPAR␥ agonists are phorylation of JAK2, TYK2, STAT3 or STAT4 in T cells, potent regulators of IL-12 production, IL-12 signaling, suggesting that the blockade of IL-12-induced activation Th1 differentiation and pathogenesis of CNS inflam- of JAK-STAT pathway by PPAR␥ agonists be reversible. mation and demyelination in EAE. In vivo treatment with ␥ To further determine whether the inhibition of IL-12- PPAR agonists, 15d-PGJ2 and Ciglitazone decreased the

Genes and Immunity PPAR␥ regulation of IL-12 in EAE C Natarajan and JJ Bright 65

Figure 9 Reversible inhibition of IL-12 signaling by PPAR␥ agonists in T cells. Western blot analysis of ConA-activated T cells incubated + ␮ + ␮ in medium alone, 2 ng/ml IL-12, IL-12 20 g/ml Ciglitazone (Cg, 30 and 120 min) or IL-12 20 g/ml 15d-PGJ2 (Pg 30 and 120 min) at ° ␮ 37 C for 15 min. The cells were pretreated with 20 g/ml Ciglitazone or 15d-PGJ2 for 30 and 120 min, washed off, cultured in fresh medium for 3 h and then stimulated with medium or IL-12 for 15 min at 37°C. The JAK2 (a) and TYK2 (b) STAT3 (c) and STAT4 (d) immunoprecipitates were resolved on 7.5% SDS-PAGE, transferred onto nylon membrane and probed with anti-phosphotyrosine mAb, 4G10. The blots were striped and reprobed with specific JAK or STAT antibody. Arrows point to 135 kDa JAK2, 135 kDa TYK2, 92 kDa STAT3 (c)or89kDa STAT4 (d) protein band. The figure is a representative of six experiments. clinical and pathological symptoms of CNS demyelin- severity of active immunization and adoptive transfer ation in EAE. The inhibition of EAE by PPAR␥ agonists EAE. The inhibition of clinical paralysis by PPAR␥ agon- was associated with a decrease in IL-12 production and ists was associated with a decrease in inflammation and differentiation of neural antigen-specific Th1 cells in vivo. demyelination in the CNS. Earlier studies showed that In vitro treatment of activated T cells with PPAR␥ agon- PPAR␥ agonists inhibit inflammatory disease models of ists inhibited IL-12-induced activation of JAK-STAT path- arthritis, atherosclerosis, psoriasis, and inflammatory way, suggesting the molecular mechanism in the PPAR␥ bowl disease.25–29 Those studies attributed the blockade regulation of EAE. of macrophage activation and secretion of proinflamma- The pathogenesis of EAE/MS is a complex process tory cytokines as the mechanism of regulation of involving activation of macrophage/microglial cells and inflammatory diseases by PPAR␥ agonists.23–31 differentiation of neural antigen-specific Th1 cells in IL-12 is a macrophage/microglia derived proinflam- which the proinflammatory cytokines determine the out- matory cytokine that plays a crucial role in the pathogen- come of the disease.35,36 Using the EAE model of MS, we esis of CNS demyelination in EAE/MS. Earlier studies have shown earlier that in vivo treatment with lisofyline showed that the inhibition of IL-12 production or IL-12 or tyrphostin prevents the pathogenesis of CNS inflam- signaling was effective in preventing the clinical and mation and demyelination.49,50 In this study, we have pathological symptoms of EAE.49,50 In consistent with a used EAE model to test the potential use of PPAR␥ agon- recent report we have also observed that the PPAR␥ ists for the treatment of MS. The treatment of mice with agonists inhibit LPS-induced production of IL-12 in 51 15d-PGJ2 or Ciglitazone reduced the duration and clinical macrophage cells in vitro. However, the inhibition of

Genes and Immunity PPAR␥ regulation of IL-12 in EAE C Natarajan and JJ Bright 66

Figure 10 Lack of inhibition of IL-12 signaling by PPAR␥ agonists in T cells. Western blot analysis of JAK2 (a) and TYK2 (b) STAT3 (c) and STAT4 (d) proteins immunoprecipitated from Con A-activated T cells after incubation in medium alone, 2 ng/ml IL-12, IL- 12 + 20 ␮g/ml PGF2␣ or WY14643 at 37°C for 15 min. The cells were pretreated for 10 min prior to induction of IL-12 signaling. The JAK2 (a), TYK2 (b) STAT3 (c) and STAT4 (d) immune complexes were resolved on 7.5% SDS-PAGE, transferred onto nylon membrane and probed with anti-phosphotyrosine mAb, 4G10. The blots were striped and reprobed with speciﬁc JAK or STAT antibody. Arrows point to 130 kDa JAK2, 135 kDa TYK2, 92 kDa STAT3 (c) or 89 kDa STAT4 (d) protein band. The ﬁgure is a representative of six experiments.

IL-12 production in microglial cells and MBP-immune to T cell proliferation and Th1 differentiation. Signaling spleen cells in response to LPS and neural antigens through its receptor, IL-12 induces tyrosine phosphoryl- respectively by PPAR␥ agonists observed in this study ation and activation of JAK2 and TYK2 kinases that leads suggests that the inhibition of IL-12 be a mechanism of to activation of STAT3 and STAT4 transcription fac- regulation of EAE by PPAR␥ agonists. Although the exact tors.54,55 Interestingly, treatment with PPAR␥ agonists,

mechanism involved in the regulation of IL-12 gene 15d-PGJ2 or Ciglitazone blocked the IL-12-induced tyro- expression by PPAR␥ agonists in EAE is not known, in sine phosphorylation of STAT3 and STAT4 transcription view of the pivotal role played by NF-kB in IL-12 gene factors and the upstream JAK2 and TYK2 kinases in T expression,51,52 the blockade of this pathway may be a cells. This is consistent with our earlier report showing mechanism of inhibition of IL-12 in EAE. the blockade of STAT3 and STAT4 following inhibition To further define the mechanisms involved in the of IL-12-induced tyrosine phosphorylation of JAK2 and PPAR␥ regulation of CNS demyelination, we examined TYK2 by TGF-␤ in T cells.56 The blockade of JAK-STAT the effect of PPAR␥ agonists on neural antigen-specificT pathway by PPAR␥ agonists (signal 2) resulted a cells. In vitro treatment of MBP-immune spleen cells with decrease in IL-12-induced proliferation and Th1 differen- PPAR␥ agonists resulted in a significant decrease in the tiation in activated T cells. The reversibility of IL-12 sig- neural antigen-specific T cell proliferation and IFN␥ pro- naling inhibited by PPAR␥ agonists suggests that the duction. The T cell proliferation is a multistep process inhibition of IL-12-mediated responses in T cells was initiated through T cell antigen receptor (signal 1) that consequent to the blockade of IL-12 signaling by PPAR␥ ␥ drives resting naive T cells from G0 to G1 phase of the agonists but not the cytotoxic action of PPAR agonists cell cycle. The signals delivered through IL-12 receptor resulted in the blockade of IL-12 signaling in T cells. The (signal 2) is required for the transition of activated T cells lack of inhibition of IL-12-induced activation of JAK- from G1 to S/G2/M phase of the cell cycle and differen- STAT pathway by PGF2␣ or Wy14,643 suggests that the tiation of Th1 cells.53 Earlier studies showed that PPAR␥ PPAR␥ agonists induced a specific inhibitory effect to IL- agonists inhibit antigen (signal 1)-induced T cell prolifer- 12 signaling and IL-12-mediated responses in T cell. The ation.32 We have further examined the effect of PPAR␥ effect of PPAR␥ agonists on the activation of JAK-STAT agonists on IL-12 signaling (signal 2) and its consequence pathways induced by other pro- and anti-inflammatory

Genes and Immunity PPAR␥ regulation of IL-12 in EAE C Natarajan and JJ Bright 67 cytokines including IFN␥ and IL-4 in EAE is yet to be the regulation of IL-12 signaling by PPAR␥ agonists can- determined. However, the inhibition of JAK kinases and not be completely ruled out. STAT transcription factors by PPAR␥ agonists observed Our results highlight the fact that PPAR␥ agonists in this study suggests that the IL-12 signaling pathway inhibit the pathogenesis of EAE and suggests the use of be the molecular target for PPAR␥ regulation of Th1 dif- PPAR␥ agonists for the treatment of MS and other ferentiation and pathogenesis of CNS inflammation and inflammatory demyelinating disease of the CNS. PPAR␥ demyelination in EAE (Figure 11). agonists also inhibit IL-12 production, IL-12 signaling The identification of intracellular targets involved in and Th1 differentiation in EAE, suggesting the use of the anti-inflammatory actions of PPAR␥ agonists is con- PPAR␥ agonists for the treatment of Th1 cell-mediated troversial. While some of the effects have been attributed inflammatory and autoimmune diseases. Further investi- to PPAR␥, recent studies suggested the involvement of gations will define the molecular mechanisms in the PPAR␥ independent mechanisms in the induction of PPAR␥ regulation of IL-12 gene expression and IL-12- 57 anti-inflammatory effects by 15d-PGJ2. However, the induced Th1 differentiation in EAE and MS. inhibition of IL-12 signaling and Th1 differentiation by 15d-PGJ2 as well as Ciglitazone observed in this study, implies the involvement of PPAR␥ dependent mech- Materials and methods anisms in the regulation of JAK-STAT pathway in T cells. Although the exact mechanisms involved in the PPAR␥ Animals and cells regulation of JAK-STAT pathway in EAE ia not known, SJL/J mice were purchased from Clarence Reader it is likely that the ligand activated PPAR␥ may directly (National Institute of Health, Bethesda, MD, USA) and interact with and block the tyrosine phosphorylation and maintained in the animal care facility at Vanderbilt Uni- activation of JAKs and STATs and interfere with the versity Medical Center. Activated T cells were prepared nuclear translocation and DNA binding activity of STAT by stimulation of spleen cells from SJL/J mice proteins. Alternatively, PPAR␥ may activate cofactors, (2 × 106/ml) with 5 ␮g/ml of Concanavalin A (ConA, phosphatases or other unknown proteins that in turn Pharmacia Biotech, Uppsala, Sweden) in RPMI medium inhibit the activation of JAK-STAT pathway. However, ␥ supplemented with 10% FBS (Hyclone, Logan Utah, the involvement of PPAR independent mechanisms in ° USA) at 37 C and 5% CO2. After 3 days of culture, cells were harvested and cultured in medium containing 0.5% FBS for an additional 24 h to synchronize to G1 phase of the cell cycle. The T cell blasts were isolated by centrifug- ation over Histopaque (Sigma MO, USA) at 1200 g for 15 min and used for experiments.50,56,58 This population of cells normally contains Ͼ98% T cell blasts as measured by flow cytometry. The peritoneal macrophages were isolated from thioglycollate-stimulated SJL/mice as described elsewhere.59 The EOC-20 mouse microglial cell line60 was a kind gift of W Walker (St Jude Children’s Research Hospital, Memphis, TN, USA).

Reagents ␥ ⌬12,14 The PPAR agonist, 15-Deoxy- -Prostaglandin J2, ␣ Prostaglandin F2 alpha (PGF2 ) and [4-chloro-6-(2,3- xylidine)-2-pirimidinylthio] acetic acid (Wy14,643) were purchased from Calbiochem (La Jolla, CA, USA). Ciglita- zone was purchased from Alexis Corporation (San Diego, CA, USA). Recombinant murine IL-12 and IFN␥ were purchased from R&D Systems (Minneapolis, MN, USA). The anti-IFN␥ mAb, R4-6A2 was purified from ascitic fluid collected from nude mice following transplantation of R4-6A2 hybridoma cells (American Type Culture Col- lection # HB 170, Rockville, MD, USA). The anti-IFN␥ mAb, MM700 was obtained from Endogen (Woburn, MA, USA) and conjugated with biotin according to standard protocol. Anti-IL-12 mAb C17.8 (anti-p40) was prepared from hybridoma cells kindly provided by G Trinchieri (Wistar Institute, Philadelphia, PA, USA). Mouse spinal cord homogenate (MSCH) and Guinea pig Figure 11 Model on PPAR␥ regulation of IL-12 signaling in CNS MBP were prepared according to standard proto- demyelination. IL-12 induces tyrosine phosphorylation and acti- cols.45,49,50 Anti-JAK2 Ab and anti-phosphotyrosine mAb vation of JAK2, TYK2, STAT3 and STAT4 that are critical in the 4G10 were purchased from Upstate Biotechnology (Lake differentiation of Th1 cells and pathogenesis of CNS inflammation placid, NY, USA). Anti-TYK2, anti-STAT3 and anti- and demyelination. Treatment of cells with PPAR␥ agonists, Ciglit- ␥ STAT4 Abs were from Santa Cruz Biotechnology (Santa azone or 15d-PGJ2 induces the activation of PPAR that in turn blocks the IL-12-induced activation of JAK-STAT pathway and Cruz, CA, USA). Anti-CD40 Ab was obtained from Bio- inhibits Th1 differentiation, CNS inflammation and demyelination. Science (San Diego, CA, USA).

Genes and Immunity PPAR␥ regulation of IL-12 in EAE C Natarajan and JJ Bright

68 3 Induction and treatment of EAE PGJ2 or Ciglitazone, for 48 h. [ H] thymidine was added To induce active EAE, 4 to 6-week-old female SJL/J mice for the last 12 h and the radiolabel measured as above.49,50 were immunized (s.c) with 800 ␮g of MSCH in 150 ␮l emulsion of incomplete Freund’s adjuvant containing 50 ␮g/ml H37Ra in the lower dorsum on days 0 and 7. Immunoprecipitation and Western blot analyses To induce adoptive transfer EAE, 4 to 6 weeks old, female The immunoprecipitation and Western blot analyses of SJL/J mice (donor) were immunized with 350 ␮g MBP in JAK and STAT proteins were performed as described 50,56,58 × CFA on days 0 and 7. On day 14, the lymph node and earlier. Briefly, ConA-activated T cells (2.5 7 ␮ ␥ spleen cells were isolated and cultured in RPMI medium 10 /lane) were pretreated with 5 or 20 g/ml PPAR × 6 × 6 ␮ agonists, 15d-PGJ2 or Ciglitazone for 15 min, and then (5 10 or 2.5 10 cells/ml respectively) with 25 g/ml ° MBP. After 4 days of culture, the T cell blasts were har- stimulated with 2 ng/ml IL-12 at 37 C for 15 min. Cell vested and 1 × 107 cells injected (i.p) into naive female lysates were prepared and JAK2, TYK2, STAT3 and SJL/J mice. Mice in the test groups were treated (i.p) with STAT4 proteins immunoprecipitated using specific anti- ␮ ␮ bodies and protein A Sepharose. The phosphoproteins in 15d-PGJ2 or Ciglitazone (50 and 100 g) in 25 l DMSO on every other day from 0 to 25 days following induction the immune complex were analyzed by 7.5% SDS-PAGE of active or passive EAE. Mice in the control group and Western blot using anti-phosphotyrosine mAb 4G10 received 25 ␮l DMSO. The clinical paralysis in active and and visualized by ECL detection system. The blots were passive EAE was graded as follows; 0, normal; 0.5, stiff stripped and reprobed with specific Ab to ensure equal tail; 1, limp tail; 1.5, limp tail with inability to right; 2, protein loading. paralysis of one limb; 2.5, paralysis of one limb and weakness of one other limb; 3, complete paralysis of both ␥ 45,49,50 Culture for IL-12 and IFN assay hind limbs; 4, moribund; 5, death. MBP immune spleen cells were cultured in 24 well plates in RPMI medium (5 × 105/ml) with 25 ␮g/ml MBP in the Histological analysis presence of different concentrations of 15d-PGJ2 or Ciglit- To assess the degree of CNS inflammation and demyelin- azone and the culture supernatants collected after 24 h. ation, mice induced to develop active EAE were euthan- Peritoneal macrophage and EOC-20 microglial cells were

ized on day 15 (at the peak of the disease) by CO2 cultured in DMEM containing 10% FBS with 50 ng/ml asphyxiation and perfused by intracardiac injection of 4% IFN␥ + 1 ␮g/ml LPS or 2 ␮g/ml anti-CD40 Ab + 2nd Ab paraformaldehyde and 1% glutaraldehyde in PBS. Trans- in the absence or presence of Ciglitazone or 15d-PGJ2 and verse sections (five each) taken from different levels of the culture supernatants collected after 48 h. Naive spleen cervical, upper thoracic, lower thoracic, and lumbar T cells from SJL/J mice were enriched (Ͼ95% purity) by regions of the spinal cord were stained with Luxol Fast passing through nylon wool column and plastic adher- Blue or hematoxylin and eosin. Each spinal cord section ence. Anti-CD3 mAb (2C11, 5 ␮g/ml) was immobilized was further subdivided into anterior, posterior and two onto six-well tissue culture plates by incubation for 1 h lateral columns (4 quadrants). Each quadrant displaying at 37°C. After washing the plates with PBS, T cells were the infiltration of mononuclear cells or loss of myelin added into the wells (1 × 106 cells/ml) and cultured in staining were assigned for a score of one inflammation the presence or absence of 2 ng/ml rIL-12 and different ␥ or demyelination respectively. Thus, each animal had a concentrations of PPAR agonists, 15d-PGJ2 or Ciglita- potential maximum score of 80 and this study represents zone. After 5 days of culture, equal number of viable cells the analysis of spinal cord from five representative mice (5 × 105 cells/ml) were restimulated with soluble anti- per group (n = 15). The pathologic score for each group CD3 mAb (2C11, 5 ␮g/ml) and the supernatants were was expressed as percent positive over the total number collected after 36 h.49 of quadrants examined.49 ELISA for IL-12 and IFN␥ T cell proliferation assay The levels of IL-12 and IFN␥ in the culture supernatants The effect of PPAR␥ agonists on neural antigen- or IL- were measured by ELISA as described earlier.45,49,50 12-induced T cell proliferation was measured by [3H] thy- Briefly, ELISA plates were coated with 2 ␮g/ml of anti- midine incorporation assay. MBP immune spleen cells IL-12 mAb, C17.15 or anti-IFN␥ mAb, R4–6A2 capture Ab were cultured in 96 well tissue culture plates in RPMI in 100 ␮l/well of bicarbonate buffer, pH 9.3. After over- medium (2 × 105/200 ␮l/well) in the presence of 0 or night incubation at 4°C, excess Ab was washed off and 100 ␮g/ml MBP with different concentrations (0 to the residual binding sites blocked by the addition of 3% ␮ ␥ 20 g/ml) of PPAR agonists, 15d-PGJ2 or Ciglitazone. BSA in PBS for 1 h. The test samples (culture [3H] thymidine (0.5 ␮Ci/ml) was added at 72 h and the supernatants) and standards (rIL-12 or rIFN␥) were uptake of radiolabel measured after 96 h by Wallac beta added and incubated overnight at 4°C. Plates were plate scintillation counter.45,49,50 MBP immune spleen cells washed with PBS containing 0.05% Tween-20 and were also cultured with 0 or 25 ␮g/ml MBP + 2 ng/ml 0.2 ␮g/ml of biotin conjugated anti-IL-12 mAb, C15.6 or rIL-12 with different concentrations (0 to 20 ␮g/ml) of anti-IFN␥ mAb, MM700 added as detection Ab. After ␥ 3 PPAR agonists, 15d-PGJ2 or Ciglitazone. [ H] thymidine incubation at room temperature for 1 h, the plates were (0.5 ␮Ci/ml) was added at 72 h and the uptake of radio- washed three times and avidin-alkaline phosphatase label measured after 96 h by Wallac beta plate scintil- added followed by 1 mg/ml of p-nitrophenyl phosphate. lation counter. ConA-activated T cells were cultured in After 30 min incubation at room temperature, the RPMI medium in 96 well tissue culture plate absorbance was read at 405 nm and the concentration of (1 × 105/well) with 2 ng/ml rIL-12 in the absence or pres- IL-12 and IFN␥ in the culture supernatants calculated ence of different concentrations of PPAR␥ agonists, 15d- from standard curve.

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