Gemfibrozil Ameliorates Relapsing-Remitting Experimental Autoimmune Encephalomyelitis Independent of Peroxisome Proliferator-Activated Receptor-

The present study underlines the importance of gemfibrozil, a lipid-lowering drug and an activator of peroxisome proliferatoractivated receptor(PPAR), in inhibiting the disease process of adoptively transferred experimental allergic encephalomyelitis (EAE), an animal model of relapsing-remitting multiple sclerosis. Clinical symptoms of EAE, infiltration of mononuclear cells, and demyelination were significantly lower in SJL/J female mice receiving gemfibrozil through food chow than those without gemfibrozil. It is noteworthy that the drug was equally effective in treating EAE in PPARwild-type as well as knockout mice. Gemfibrozil also inhibited the encephalitogenicity of MBP-primed T cells and switched the immune response from a Th1 to a Th2 profile independent of PPAR. Gemfibrozil consistently inhibited the expression and DNA-binding activity of T-bet, a key regulator of interferon(IFN) expression and stimulated the expression and DNA-binding activity of GATA3, a key regulator of IL-4. Gemfibrozil treatment decreased the number of T-bet–positive T cells and increased the number of GATA3-positive T cells in spleen of donor mice. The histological and immunohistochemical analyses also demonstrate the inhibitory effect of gemfibrozil on the invasion of T-bet–positive T cells into the spinal cord of EAE mice. Furthermore, we demonstrate that the differential effect of gemfibrozil on the expression of T-bet and GATA3 was due to its inhibitory effect on NO production. Although excess NO favored the expression of T-bet, scavenging of NO stimulated the expression of GATA-3. Taken together, our results suggest gemfibrozil, an approved drug for hyperlipidemia in humans, may find further therapeutic use in multiple sclerosis. Multiple sclerosis (MS) is one of the most common neurological diseases affecting young adults. Although the etiology of MS is not completely understood, studies of patients with MS suggest that the observed demyelination in the CNS is a result of a T-cell-mediated autoimmune response (Martin et al., 1992). Experimental allergic encephalomyelitis (EAE) serves as an animal model for MS (Tuohy et al., 1988; Benveniste, 1997; Du et al., 2001). Studies using the adoptively transferred EAE model strongly support the view that activated neuroantigen-specific T cells cross the blood-brain barrier, infiltrate the CNS parenchyma, and initiate an inflammatory response (Tuohy et al., 1988; Benveniste, 1997). The identification of wide range of proinflammatory cytokines, cell adhesion molecules, chemokines, proinflammatory enzymes such as inducible nitric-oxide synthase (iNOS) and cyclooxygenase in CNS lesions of patients with MS and animals with EAE (Martin et al., 1992; Benveniste, 1997) suggests that the inflammatory process initiated by activated T cells eventually becomes a broad-spectrum one. Therefore, analysis of molecular mechanisms for the regulation of this broad-spectrum inflammatory process in EAE should help decipher the mechanisms of the disease process in MS/EAE and further the possibility of developing effective therapeutic strategies for patients with MS. Peroxisome proliferator-activated receptors (PPARs), members of the nuclear hormone receptor superfamily, have been implicated in a variety of human disorders. Three isoThis study was supported by National Multiple Sclerosis Society grant RG3422A1/1 and National Institutes of Health grants NS39940 and NS48923. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.033787. ABBREVIATIONS: MS, multiple sclerosis; CNS, central nervous system; EAE, experimental allergic encephalomyelitis; iNOS, inducible nitricoxide synthase; PPAR, peroxisome proliferator-activated receptor; HDL, high-density lipoprotein; Th, T-helper; RR-EAE, relapsing-remitting EAE; FBS, fetal bovine serum; carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, potassium salt; PCR, polymerase chain reaction; IFA, incomplete Freund’s adjuvant; MBP, myelin basic protein; LNC, lymph node cells; dpt, days post-transfer; HPLC, highperformance liquid chromatography; H&E, hematoxylin & eosin; PBST, PBS containing Tween 20; LFB, Luxol fast blue; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; IL, interleukin; WY14643, pirinixic acid; GSNO, S-nitrosoglutathione; IFN, interferon; dpt, days post-transfer. 0026-895X/07/7204-934–946$20.00 MOLECULAR PHARMACOLOGY Vol. 72, No. 4 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 33787/3255539 Mol Pharmacol 72:934–946, 2007 Printed in U.S.A. 934 at A PE T Jornals on M ay 9, 2017 m oharm .aspeurnals.org D ow nladed from types have been described: PPAR, PPAR, and PPAR(Lemberger et al., 1996). Activation of PPARmainly leads to the induction of a variety of genes, such as those coding for the enzymes for and -oxidation of fatty acids (Dreyer et al., 1992). Gemfibrozil, an activator of PPAR, has often been prescribed in patients to lower the level of triglycerides (Bloomfield Rubins et al., 2001; Hsu et al., 2001). This drug decreases the risk of coronary heart disease by increasing the level of high-density lipoprotein (HDL) cholesterol and decreasing the level of low-density lipoprotein cholesterol (Bloomfield Rubins et al., 2001; Hsu et al., 2001). We (Pahan et al., 2002) have shown that gemfibrozil inhibits the expression of iNOS in human astrocytes, suggesting that this drug may ameliorate neuroinflammatory disorders. Racke and colleagues (Lovett-Racke et al., 2004; Xu et al., 2005) have found consistently that gemfibrozil and other fibrate drugs suppress the induction of NO production in microglia and attenuate the disease process in an actively immunized primary progressive EAE model in B10.PL mice, one of the animal models of primary progressive MS. Considering that the majority (85%) of patients with MS at the time of original diagnosis suffer from the relapsing-remitting disease, that the B10.PL mouse does not have the relevant locus for relapsingremitting EAE (RR-EAE), and that gemfibrozil is a known agonist of PPAR, it is important to determine whether gemfibrozil is capable of suppressing the disease process of RR-EAE and whether antineuroimmune effect of gemfibrozil depends on PPAR. Here, we demonstrate that gemfibrozil inhibited the disease process of RR-EAE in an adoptively transferred EAE model in female SJL/J mice by shifting the encephalitogenic immune response from Th1 to Th2 mode without the involvement of PPAR. We also present interesting evidence that gemfibrozil increases the expression of GATA-3, a key regulator of Th2 cytokines, and decreases the expression of T-bet, a key regulator of Th1 cytokines, through the inhibition of NO production. Materials and Methods Reagents. Fetal bovine serum (FBS) and RPMI 1640 medium were from Invitrogen (Carlsbad, CA). Gemfibrozil was purchased from Sigma (St. Louis, MO). L-N-(1-Iminoethyl)-lysine and carboxyPTIO were obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). Oligonucleotide probes for T-bet and GATA and antibodies against T-bet and GATA3 were purchased from SantaCruz Biotechnology (Santa Cruz, CA). Rat anti-mouse pan macrophage marker (moma-2) was purchased from Biosource International (Camarillo, CA). Rabbit anti-mouse iNOS antibody was obtained from Calbiochem (San Diego, CA). [ -P]ATP was purchased from PerkinElmer Life and Analytical Sciences (Waltham,