Disease Agonists As Therapy for Autoimmune Α Peroxisome

Peroxisome Proliferator-Activated Receptor α Agonists as Therapy for Autoimmune Disease

This information is current as Amy E. Lovett-Racke, Rehana Z. Hussain, Sara Northrop, of September 29, 2021. Judy Choy, Anne Rocchini, Lela Matthes, Janet A. Chavis, Asim Diab, Paul D. Drew and Michael K. Racke J Immunol 2004; 172:5790-5798; ; doi: 10.4049/jimmunol.172.9.5790

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Peroxisome Proliferator-Activated Receptor ␣ Agonists as Therapy for Autoimmune Disease1

Amy E. Lovett-Racke,* Rehana Z. Hussain,* Sara Northrop,* Judy Choy,* Anne Rocchini,* Lela Matthes,* Janet A. Chavis,‡ Asim Diab,* Paul D. Drew,‡ and Michael K. Racke2*†

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. PPAR␥ ligands, ⌬12,14 which include the naturally occurring PG metabolite 15-deoxy- -PGJ2 (15d-PGJ2), as well as thiazolidinediones, have been shown to have anti-inflammatory activity. The PPAR␣ agonists, gemfibrozil, ciprofibrate, and fenofibrate, have an excellent track history as oral agents used to treat hypertriglyceridemia. In the present study, we demonstrate that these PPAR␣ agonists can increase the production of the Th2 cytokine, IL-4, and suppress proliferation by TCR transgenic T cells specific for the myelin basic protein Ac1–11, as well as reduce NO production by microglia. Oral administration of gemfibrozil and fenofibrate inhibited clinical signs of experimental autoimmune encephalomyelitis. More importantly, gemfibrozil was shown to shift the cytokine Downloaded from secretion of human T cell lines by inhibiting IFN-␥ and promoting IL-4 secretion. These results suggest that PPAR␣ agonists such as gemfibrozil and fenofibrate, may be attractive candidates for use in human inflammatory conditions such as multiple sclerosis. The Journal of Immunology, 2004, 172: 5790–5798.

eroxisome proliferator-activated receptors (PPARs)3 are affect T cell function indirectly by inhibiting the production of

members of the nuclear hormone receptor superfamily that chemokines by endothelial cells (9). The evidence that PPAR ago- http://www.jimmunol.org/ P also include steroid, retinoid, and thyroid hormone recep- nists can regulate inflammation is supported by several animal tors (1). There are three PPAR subtypes (␣, ␦, and ␥) and they studies. These include the suppression of adjuvant arthritis in rats exhibit different tissue distribution as well as different ligand spec- (10), inhibition of atherosclerosis in mice (11), and amelioration of ificities. PPARs have been most extensively studied in the regu- inflammatory bowel disease in mice (12). Our recent work using lation of genes involved in glucose and lipid metabolism (2). In ␥ 15d-PGJ2, as well as that of others using other PPAR agonists, addition to adipocytes, it has also been recently shown that cells of demonstrated inhibition of the clinical signs of experimental au- the monocyte/macrophage lineage express both PPAR␣ and toimmune encephalomyelitis (EAE) (13Ð16). PPAR␥, suggesting a possible role for these receptors in immune PPAR␣ ligands have also been shown to regulate inflammatory by guest on September 29, 2021 function (3, 4). responses, although there is clearly less evidence along this line ␥ PPAR ligands, which include the naturally occurring PG me- compared with PPAR␥ agonists. Both PPAR␣ and PPAR␥ have ⌬12,14 tabolite 15-deoxy- -PGJ2 (15d-PGJ2), as well as thiazo- been shown to be expressed on T cells and their ligands can inhibit lidinediones, have been shown to have anti-inflammatory activity. IL-2 production and T cell proliferation (7, 8). PPAR␣-deficient 15d-PGJ2 was shown to inhibit inducible nitric oxide synthase, mice have abnormally prolonged responses to inflammatory stim- ␤ ␣ matrix-metalloproteinase-9, IL-1 , IL-6, and TNF- production uli such as arachidonic acid and leukotrienes (17). The expression ␥ by monocytes/macrophages (5, 6). PPAR ligands can inhibit IL-2 of IL-6, VCAM, and cyclooxygenase-2 in response to cytokine production and T cell proliferation (7, 8). PPAR␥ ligands can also activation can be inhibited by PPAR␣ ligands (18). PPAR␣ ligands have been shown to decrease NF-␬B activation, IL-12, and *Department of Neurology, and †Center for Immunology, University of Texas South- IL-6 production in aged mice (19). PPAR␣ ligands may inhibit western Medical Center, Dallas, TX 75390; and ‡Department of Neurobiology and ␬ Developmental Sciences, University of Arkansas for the Medical Sciences, Little functional expression of NF- B, in part by augmenting the expres- Rock, AR 72205 sion of I␬B␣ (20). Received for publication December 2, 2003. Accepted for publication February Recently, the PPAR␣ ligand, WY14,643, was shown to inhibit 26, 2004. IgG responses in myelin oligodendrocyte glycoprotein 35Ð55/ The costs of publication of this article were defrayed in part by the payment of page CFA-immunized mice (21). When mice were fed this agonist, charges. This article must therefore be hereby marked advertisement in accordance ␥ with 18 U.S.C. Section 1734 solely to indicate this fact. splenocytes demonstrated impaired production of IFN- , IL-6, and ␣ 1 These studies were supported by the following research grants: U.S. Public Health TNF- . Interestingly, the authors had hoped to examine the effect Service Grant K24 NS044250 (to M.K.R.), U.S. Public Health Service Grant RO1 of fibrates on EAE; however, the combination of immunization NS37513 (to M.K.R.), U.S. Public Health Service Grant RO1 NS42860 (to P.D.D.), with myelin oligodendrocyte glycoprotein 35Ð55/CFA, pertussis National Multiple Sclerosis Society Grant RG 3427-A-8 (to M.K.R.), and grants from the Yellow Rose Foundation to M.K.R. and A.E.L.-R. This study was also supported toxin, and WY14,643 treatment consistently induced mortality by an advanced postdoctoral fellowship from the National Multiple Sclerosis Society 5Ð10 days after immunization. (to A.D.). Organ-specific autoimmune diseases, such as EAE, are medi- 2 Address correspondence and reprint requests to Dr. Michael K. Racke, Department ated by IFN-␥-producing Th1 cells (22). Evidence in the EAE of Neurology and the Center for Immunology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9036. E-mail ad- model suggests that the production of Th2 cytokines such as IL-4 dress: [email protected] or IL-10 may play an important role in the remissions observed in 3 Abbreviations used in this paper: PPAR, peroxisome proliferator-activated receptor; this disease (23, 24, 25). Interestingly, it appears that proteolipid ⌬12,14 15d-PGJ2, 15-deoxy- -PGJ2; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; TCL, T cell line; PLP, proteolipid protein; MS, mul- protein (PLP)-specific T cell clones isolated from multiple sclero- tiple sclerosis. sis (MS) patients have different cytokine phenotypes depending on

when they are isolated (26). T cell clones derived during MS at- When mice were fed by gavage, stock solution was diluted in PBS so that tacks are more likely to have a Th1 phenotype, while those derived indicated dose of PPAR␣ agonist was delivered in a total of 200 ␮l. Mouse during remission were more variable but included several T cells chow was supplemented with gemfibrozil or fenofibrate by adding the stock solution to the chow (0.25% w/w), allowing the ethanol to evaporate, of a Th2 phenotype, suggesting that shifting the phenotype of T and then using it as the source of food. Control diet was given the same cells from Th1 to Th2 may be beneficial in MS. amount of ethanol without the PPAR␣ agonist. In the present studies, we have examined the effects of PPAR␣ Pathology and immunohistochemistry agonists on the differentiation of autoreactive T cells and their effects on clinical signs of disease in the murine model of EAE. In Mice were deeply anesthetized with sodium pentobarbital and transcardi- addition, we have also examined the effects of gemfibrozil, a syn- ally perfused with sterile PBS, followed by 4% paraformaldehyde in phos- ␣ phate buffer. The brain and spinal cord were removed, fixed in 4% para- thetic PPAR agonist with a long history of oral use in humans, on formaldehyde for 6 h, and cryoprotected in 30% sucrose overnight at 4¡C the secretion of cytokines by human myelin-reactive T cells. Our before being embedded in Tissue-Tek (T. Pella, Redding, CA) and quick- results suggest that PPAR␣ agonists may be a useful therapeutic frozen in isopentane. Immunohistochemistry was performed on adjacent agent for human inflammatory diseases such as MS. sections. After pretreatment with 0.3% hydrogen peroxide in absolute methanol, sections were blocked with 1% BSA for2hatroom temperature and then incubated with primary Ab overnight at 4¡C. Primary Abs used Materials and Methods were MOMA-2 (1/20) mAb, a marker for macrophages (BioSource Inter- Mice national) and KT174 (1/20), which recognizes CD4ϩ T cells (BioSource ␣ ␤ International). Transgenic mice bearing the rearranged V 2.3,V 8.2 gene encoding the The binding of the primary Abs was detected using a biotinylated sec- TCR specific for the myelin basic protein (MBP) Ac1Ð11 peptide on ondary Ab and an avidin-biotin-peroxidase method under humidified con- Downloaded from the B10.PL background were obtained by crossing transgenic mice bearing ditions (ABC Elite kit; Vector Laboratories, Burlingame, CA). Peroxidase the individual rearranged genes (27). The V␣2.3 TCR-transgenic mice Ј ␤ activity was visualized with 3,3 -diaminobenzidine (DAB kit; Vector Lab- and the V 8.2 TCR transgenic mice were kindly provided by Dr. J. Gov- oratories) as a substrate. Isotype IgG2c Ab served as a negative control. erman (University of Washington, Seattle, WA). These mice were bred and The tissue area was measured by a Scion image analysis system (Scion, maintained in a federally approved animal facility at the University of Frederick, MD). Positive cells were counted by automatic video scanning Texas Southwestern Medical Center (Dallas, TX) in accordance with the using a Leica Q500 MC (Zeiss, Oberkochen, Germany), and the numbers Institutional Animal Care and Use Committee. All mice were 7Ð10 wk old of stained cells per 104 square pixels tissue area were calculated. when experiments were performed. B10.PL mice were purchased from The http://www.jimmunol.org/ Jackson Laboratory (Bar Harbor, ME) and then bred in our animal facility. Cytokine ELISA Murine cell culture Mouse IFN-␥, IL-4, and IL-10 were quantified using a sandwich ELISA based on noncompeting pairs of Abs as previously described (13). Murine ␣ ␤ Spleens from naive V 2.3, V 8.2 TCR transgenic mice were harvested splenocytes from B10.PL mice with a transgenic TCR specific for MBP and single-cell suspensions were obtained by pressing the tissue through a Ac1Ð11 were cultured at 8 ϫ 106 cells/well in 24-well plates with 2 ␮g/ml wire mesh screen, as previously described (27). Proliferative responses of Ac1Ð11. Supernatants were collected at 24, 48, and 72 h and cytokine ϫ 5 splenocytes (4 10 cells/well) were determined using MBPAc1Ð11 (2 levels determined by ELISA. Human IFN-␥ and IL-4 were determined ␮ g/ml) in the presence of various concentrations of gemfibrozil, ciprofi- similarly, using the same basic protocol with human-specific Abs. In brief, brate, and fenofibrate (Sigma-Aldrich, St. Louis, MO) as indicated, or Immulon 2 plates (Dynex Technologies, Chantilly, VA) were coated with equivalent volumes of solvent. Gemfibrozil, ciprofibrate, and fenofibrate ␥ purified monoclonal anti-IFN- (Code: M-700A; Endogen, Woburn, MA) by guest on September 29, 2021 were prepared as recommended by the manufacturer. Cultures were main- or anti-IL-4 (catalog no. 554515; BD PharMingen, San Diego, CA) at 2 tained in 96-well flat-bottom plates for 96 h at 37¡C in humidified 5% ␮g/ml in 0.1 M NaHCO at 4¡C overnight. The plates were washed two ␮ 3 3 CO2/air. The wells were pulsed with 0.5 Ci/well [methyl- H]thymidine times with PBS/1% Tween, blocked with 1% BSA/PBS, and 100 ␮lof for the final 16 h of culture. Cells were harvested on glass filters and supernatant was added to the plates in duplicate. The plates were placed at 3 incorporated [methyl- H]thymidine was measured with a Betaplate counter 4¡C overnight and then washed four times with PBS/Tween. Biotin-labeled (PerkinElmer Wallac, Gaithersburg, MD). Results were determined as anti-IFN-␥ (code: M-701B; Endogen) or anti-IL-4 (catalog no. 554483; BD means from quadruplicate cultures and are shown with SEM. PharMingen) were added at 1 ␮g/ml and incubated at room temperature for The N9 microglial cell line is derived from myc-immortalized mouse 1.5 h. An avidin-peroxidase substrate was used to complete the assay. microglia (28), and was graciously provided by P. Ricciardi-Castagnoli Concentrations of IFN-␥ and IL-4 were determined using a standard curve (University of Milan, Milan, Italy). Cells were cultured in MEM medium generated using known concentrations of human recombinant IFN-␥ (BD ␮ containing 10% FBS, 1.4 mM glutamine, and 20 M 2-ME. Where indi- PharMingen) and IL-4 (Calbiochem, San Diego, CA) in the assay. cated, cells were treated with IFN-␥ (PBL Laboratories, Piscataway, NJ) plus TNF-␣ (R&D Systems, Minneapolis, MN) and/or the PPAR␣ ago- Generation of human T cell lines (TCL) nists, gemfibrozil, ciprofibrate, or fenofibrate (Sigma-Aldrich). Human lymphocytes were collected via leukapheresis from MS patients NO production assay and healthy individuals under an Institutional Review Board approved protocol. Human TCL specific for MBP and PLP were generated as previously N9 microglial cell production of the NO derivative nitrite was assessed in described (30). Bovine MBP (Sigma-Aldrich) or a mix of five PLP peptides culture medium by Greiss reaction as we have described previously (29). (31Ð60, 91Ð120, 111Ð140, 131Ð160, and 177Ð206) were used at 10 ␮g/ml Cells were grown in 96-well plates. Where indicated, cells were pretreated to generate the TCL. Lines designated with the letter M were derived from for 1 h with gemfibrozil, ciprofibrate, or fenofibrate. Cells were then treated MS patients, while those designated with an H were derived from healthy as indicated with IFN-␥ (100 U/ml) plus TNF-␣ (500 U/ml), and nitrite individuals. levels were determined following incubation for 24 h. Human T cell proliferation assay Induction and clinical evaluation of EAE For Con A proliferation assays, 2 ϫ 105 PBMC were placed in each well EAE was induced in B10.PL mice by s.c. injection over four sites in the of a 96-well plate. Con A was added at 1 ␮g/ml and various concentrations flank with 200 ␮g of MBP Ac1Ð11 (BioSource International, Camarillo, of gemfibrozil (or the same volume of ethanol which was the diluent for CA) in an emulsion with CFA (Difco, Detroit, MI). Pertussis toxin (200 gemfibrozil) was added to quadruplicate wells. The wells were pulsed with ng/mouse) was injected i.p. at the time of immunization and 48 h later. 0.5 ␮Ci/well [methyl-3H]thymidine for 18 h before harvesting at 96 h. Mice were scored on a scale of 0Ð6 as previously described (13): 0, no Cells were harvested on glass filters using a Tomtec harvester (Tomtec, clinical disease; 1, limp/flaccid tail; 2, moderate hind limb weakness; 3, Hamden, CT), and incorporated [methyl-3H]thymidine was measured with severe hind limb weakness; 4, complete hind limb paralysis; 5, quadriple- a Betaplate counter (PerkinElmer Wallac). gia or premoribund state; 6, death. T cell viability assay Administration of PPAR␣ agonists to mice with EAE Human PBMC or murine splenocytes were placed in 24-well plates at 5 ϫ For administration of gemfibrozil and fenofibrate to mice with EAE, stock 106 cells per well. Gemfibrozil, ciprofibrate, or fenofibrate (or an equal solutions were made by dissolving these agents in ethanol (50 mg/ml). volume of solvent) were added to the wells at a final concentration of 5792 PPAR␣ AGONISTS IN AUTOIMMUNE DISEASE

0Ð500 ␮M. At various time points, the cells were collected, resuspended in trypan blue, and the number of viable and nonviable cells were counted. Results PPAR␣ agonists suppress lymphocyte proliferation in a dose-dependent manner Because EAE is caused by CD4ϩ T cells specific for myelin Ags, we first examined whether various PPAR␣ agonists have any effect on CD4ϩ Ag-specific proliferation. Splenocytes from naive V␣2.3,V␤8.2 TCR transgenic mice were cultured with MBP Ac1Ð11 (2 ␮g/ml) in the presence of various concentrations of gemfibrozil, ciprofibrate, and fenofibrate. MBP Ac1Ð11-induced proliferation was inhibited by high doses of gemfibrozil (100Ð400 ␮M); however, lower doses did not affect T cell proliferation (20Ð50 ␮M) (Fig. 1A). Proliferation was decreased by 79.2% at the highest concentration tested. Similarly, lymphocyte proliferation was suppressed at higher concentrations of ciprofibrate (200Ð 400 ␮M), with some minor fluctuations in proliferation at the lower doses (Fig. 1B). Fenofibrate completely inhibited lympho- Downloaded from cyte proliferation at 50Ð100 ␮M concentrations, but had no effect at doses of 10 ␮M or less (Fig. 1C). To determine whether the suppression of lymphocyte proliferation was a reflection of drug toxicity, a trypan blue exclusion assay was performed. TCR transgenic splenocytes were cultured in

the presence of various concentrations of gemfibrozil, ciprofibrate, http://www.jimmunol.org/ and fenofibrate. The cells were collected, washed, and counted at 1, 3, and 5 days. Both the viable and nonviable cells were counted by trypan blue exclusion. Gemfibrozil and ciprofibrate appeared to have no effect on cell viability except at 400 ␮M (Fig. 2, A and B). Fenofibrate appeared to have only a minimal effect on cell viabiltiy at the higher concentrations (Fig. 2C). For all three PPAR␣ agonists, there is a suppression of proliferation at higher concentrations that is not due solely to lymphocyte death. by guest on September 29, 2021 Stimulation of CD4ϩ T cells in the presence of PPAR␣ agonists induces the expression of Th2 cytokines Prior work had suggested that stimulation of lymphocytes with mitogen in the presence of PPAR␣ agonists induced IL-4 production (7). We examined whether the presence of gemfibrozil, cip- FIGURE 1. PPAR␣ agonists inhibit lymphocyte proliferation in a dose- rofibrate, and fenofibrate affected cytokine secretion of MBPAc1Ð dependent manner. MBP Ac1Ð11-specific murine TCR transgenic splenocytes 11-specific T cells. Using ELISA, we examined the expression of were diluted 1/3 with irradiated wild-type B10.PL splenocytes. A total of 4 ϫ IFN-␥, IL-4 and IL-10 in the supernatant of MBPAc1Ð11-specific 105 splenocytes were placed in each well of a 96-well plate. MBP Ac1Ð11 was T cells stimulated in the presence of 50Ð400 ␮M gemfibrozil and added to the wells at a final concentration of 2 ␮g/ml. Gemfibrozil (A), cip- ciprofibrate. A single stimulation of naive TCR transgenic spleno- rofibrate (B), or fenofibrate (C), were added to four replicate wells at various cytes in the presence of gemfibrozil or ciprofibrate induced IL-4 concentrations to wells with Ag. Equivalent volumes of solvent were used in 3 expression (Fig. 3, A and C). For gemfibrozil, IL-4 levels were control wells. Proliferation was determined by adding [ H]thymidine during the final 16 h of incubation. Solvent alone had no effect on lymphocyte pro- highest at 50 ␮M, a concentration that had no effect on prolifera- liferation at any of the concentrations tested. tion or cell viability. For ciprofibrate, IL-4 levels were highest at 200 ␮M, a concentration that suppressed proliferation by 58.6%, but had no effect on lymphocyte viability. Fenofibrate did not induce IL-4 production after a single stimulation, but IL-4 produc- lectively, these studies suggest that 15d-PGJ2 may modulate EAE, tion was doubled in the second stimulation in the presence of 10 at least in part, via effects on microglial cells, resident CNS cells ␮M fenofibrate (Fig. 3E). Although there was a modest decrease in that function in Ag presentation and phagocytosis. In the present the levels of IFN-␥ at higher concentrations of the PPAR␣ ago- study, we demonstrate that the PPAR␣ agonists, gemfibrozil, cip- nists, IFN-␥ production were not significantly affected by the pres- rofibrate, and fenofibrate, inhibited NO production by microglial ence of any of the three PPAR␣ agonists (Fig. 3B, D, and F). cells (Fig. 4). Gemfibrozil shows a dose-dependent decrease in NO Similarly, there was no significant affect on IL-10 production by production. Ciprofibrate and fenofibrate show a significant effect at the PPAR␣ agonists (data not shown). doses as low as 1 ␮M. Fenofibrate showed a complete inhibition of NO by microglia at 50 ␮M which was not due to toxicity because NO production by N9 microglial cells MTT analyses indicated that these PPAR␣ agonists did not de- ␥ Previously, we demonstrated that the PPAR agonist, 15d-PGJ2, crease microglial cell viability (data not shown). These studies inhibited the activation of microglial cells (29), repressed micro- suggest that these PPAR␣ agonists inhibit microglial cell activa- glial expression of CD40, a molecule which plays a critical role in tion, providing a second potential mechanism by which PPAR␣ T cell costimulation, and repressed development of EAE (13). Col- may exert its anti-inflammatory properties. The Journal of Immunology 5793

fibrate had to be removed from their diet due to toxicity. In the mice that had received fenofibrate through day 10, signs of disease were suppressed for ϳ25 days after feeding had stopped, although by day 50 postimmunization, treated animals could not be distin- guished from those fed the control diet (Fig. 5C). Having shown that PPAR␣ agonists could inhibit the development of EAE in a prevention setting, we next examined whether these agents could be effective in the setting of administration after the onset of signs of EAE. EAE was induced as before and mice were monitored for signs of disease. When mice began showing signs of disease at day 10, mice were fed either gemfibrozil, fenofibrate, or PBS by gavage for 21 days. The incidence of clinical disease was dramatically reduced in the mice that received the PPAR␣ agonists, demonstrating a therapeutic benefit after disease induction (Fig. 5D). Administration of fenofibrate beginning 10 days postimmunization had no apparent toxicity as seen in the previous experiment, suggesting that the fenofibrate toxicity may be associated with the immunization. Downloaded from Histologic analysis of mice induced to develop EAE with or without gemfibrozil Having determined that PPAR␣ agonists protected against the clinical signs of EAE, we next examined the effects of gemfibrozil on the pathology of EAE. Mice that had been induced to develop

EAE were fed gemfibrozil for 21 days and then followed another http://www.jimmunol.org/ three weeks before being sacrificed and having their spinal cords removed for histologic analysis (mice from Fig. 5A). Infiltrating macrophages and CD4ϩ T cells were detected by immunohistochemistry. The difference in the number of infiltrating cells between the control-treated animals and the gemfibrozil-treated animals were quantified by a Scion image analysis system (Scion). As shown in Fig. 6, the number of infiltrating immune cells in the lumbosacral spinal cord of gemfibrozil-treated mice was much less

␣ by guest on September 29, 2021 FIGURE 2. Effect of PPAR agonists on lymphocyte viability. Ac1Ð than those present in the placebo-treated mice. The actual number ϫ 6 11-specific splenocytes were cultured at 4 10 cells per well in 24-well of infiltrating macrophages and CD4ϩ T cells quantified by the plates in the presence of gemfibrozil (A), ciprofibrate (B), or fenofibrate Scion image analysis system (Scion) is shown in Fig. 7. These (C). Cells were collected and both the viable and nonviable cells were ␣ counted trypan blue exclusion at days 1, 3, and 5. The percentage of viable results suggest that the administration of the PPAR agonist, gem- cells compared with total number of cells was calculated. fibrozil, reduces the inflammation in the CNS of mice induced to develop EAE. Effect of gemfibrozil on human lymphocyte viability and PPAR␣ agonists protect mice from developing EAE proliferation Since gemfibrozil altered the phenotype of MBP Ac1Ð11-specific Having demonstrated that gemfibrozil was effective in altering the T cells with regard to their cytokine secretion, we next determined cytokine secretion phenotype of murine myelin-specific T cells whether administration of gemfibrozil altered the pathogenesis of and effective in prevention and treatment of EAE, we next exam- EAE. Gemfibrozil was chosen as the first drug to evaluate in EAE, ined the effects of this agent on human lymphocyte function. Gem- because it has been extensively studied in both mice and humans fibrozil was chosen because it is an FDA-approved drug with an with few side effects and little toxicity. In our initial experiment, established safety record. Concentrations of gemfibrozil at 50 ␮M B10.PL mice were fed gemfibrozil (500 ␮g) daily for 21 days had no increase in toxicity over the carrier alone (Fig. 8A). Lym- starting on the day of immunization with MBP Ac1Ð11. Control phocytes exposed to gemfibrozil at higher doses experienced in- mice either received PBS by gavage or were not fed. Although creased toxicity when exposed to the drug for longer than 24 h. both groups of control mice developed severe signs of EAE, only Similar to what was observed with murine T cells, gemfibrozil also one mouse that received gemfibrozil developed signs of disease inhibited T cell proliferation in response to Con A in a dose-de- (Fig. 5A). pendent manner (Fig. 8B). Again, lower doses of gemfibrozil had We next determined whether a diet supplemented with gemfi- only minimal effects on T cell proliferation when Con A was used brozil had a similar effect to daily feeding by gavage. B10.PL mice as the stimulating agent. were started on a diet continuously supplemented with 0.25% w/w gemfibrozil three days before immunization with MBP Ac1Ð11. In Effects of gemfibrozil on cytokine production by human T cells this experiment, the majority of mice developed clinical signs of Because gemfibrozil had important effects on altering cytokine EAE, but the mice that received gemfibrozil in their diet had very production of murine lymphocytes, we examined whether it had mild disease (Fig. 5B). similar effects on human T cell cytokine production. Because 50 Using another PPAR␣ agonist, fenofibrate, which was shown to ␮M gemfibrozil did not affect T cell proliferation or cell viability, dramatically suppress lymphocyte proliferation and NO produc- this concentration was chosen to examine effects on cytokine pro- tion by microglia, a similar experiment was performed. The feno- duction. Human PLP-specific Th1 cell lines were stimulated with 5794 PPAR␣ AGONISTS IN AUTOIMMUNE DISEASE

FIGURE 3. In vitro stimulation of lymphocytes in the presence of gemfibrozil or ciprofibrate results in IL-4 cytokine expression. MBP Ac1Ð11-specific murine TCR␣␤ splenocytes were stimulated with 2 ␮g/ml Ac1Ð11 in the presence of various concentrations of gemfibrozil (A and B), ciprofibrate (C and D), or fenofibrate (E and F). Supernatants were collected at 24, 48, and 72 h. IL-4 and IFN-␥ expressions were determined

by ELISA. Since fenoﬁbrate did not show an effect after Downloaded from the primary stimulation, the TCR transgenic cells were restimulated in the presence of fenoﬁbrate and supernatants were collected at 24, 48, and 72 h. http://www.jimmunol.org/ by guest on September 29, 2021

Ag in the presence of 50 ␮M gemfibrozil. IFN-␥ production was of human myelin-specific TCLs, suggesting that this agent might inhibited at both 48 and 72 h after stimulation when measuring the be a potential therapeutic agent for MS. presence of the cytokine in the supernatant by ELISA (Fig. 9A). This phenomenon was not Ag-specific, since IFN-␥ production was inhibited in both MBP- and PLP-specific human Th1-like Discussion lines (Fig. 9B). IL-4 and IL-10 were occasionally secreted by Th1 In the present study, we have shown that the PPAR␣ agonists, cells following gemfibrozil treatment (data not shown). Human T gemfibrozil and fenofibrate, are able to prevent and treat the organ- cells lines specific for myelin Ags were also generated in the pres- specific autoimmune disorder EAE. We investigated several po- ence of gemfibrozil (50 ␮M). TCLs generated from MS patients tential mechanisms by which this may be occurring, including al- and healthy subjects in this manner secreted significant amounts of tering cytokine production, inhibiting T cell proliferation, and IL-4 as determined by ELISA (Fig. 9C). Thus, gemfibrozil was suppressing microglia activation. We have demonstrated that gem- able to both promote IL-4 production and inhibit IFN-␥ production fibrozil can induce the secretion of the prototypic Th2 cytokine,

FIGURE 4. PPAR␣ agonists inhibit NO production by microglial cells. N9 mouse microglial cells were pretreated for 1 h with various concentrations of gemfibrozil, ciprofibrate, or fenofibrate, and then treated with IFN-␥ plus TNF-␣. Only the control N9 cells were not treated with IFN-␥ and TNF-␣ (negative control on left side of graph). All other samples were treated with the concentration of PPAR␣ agonists as indicated (G, gemfibrozil; C, ciprofibrate; F, fenofibrate). The black bar represents nitrite production in the absence of PPAR␣ agonists (positive control). After an additional 24 h of incubation, the pro- p Ͻ 0.001 vs ,ء .duction of nitrite was determined IFN-␥/TNF-␣-treated cultures. The Journal of Immunology 5795 Downloaded from http://www.jimmunol.org/

FIGURE 5. Oral administration of PPAR␣ agonists has therapeutic benefit in EAE. A, B10.PL mice were fed 500 ␮gofgemfibrozil daily by gavage for 21 days, beginning the day before immunization with 200 ␮g of MBP Ac1Ð11/CFA. Control mice were immunized only and “PBS” mice were fed PBS by gavage daily for 21 days. Disease incidence is shown in parentheses. PBS vs gemfibrozil (GEM), p Ͻ 0.001; control vs GEM, p Ͻ 0.001. B, B10.PL mice were given a diet supplemented with gemfibrozil (0.25% w/w) beginning 3 days before immunization with 200 ␮g of Ac1Ð11/CFA. Control vs GEM, p Ͼ 0.001. C, B10.PL mice were given a diet supplemented with fenofibrate (0.25% w/w) beginning 3 days before immunization with Ac1Ð11/CFA. Half of the mice in the fenofibrate group died within the first 10 days, and the fenofibrate-supplemented diet was stopped. Control vs fenofibrate (FEN), p Ͻ

0.001. D, B10.PL mice were immunized with Ac1Ð11/CFA. At the first signs of disease, the mice were fed 500 ␮gofgemfibrozil, fenofibrate, or PBS by by guest on September 29, 2021 gavage for 21 days. PBS vs GEM, p Ͻ 0.001, PBS vs FEN, p Ͻ 0.001.

IL-4, in both murine and human T cells. The role that Th2 cyto- used to treat patients with diabetes, the activation of PPAR␥ by kines play in EAE has been studied extensively by us and others thiazolidinediones may potentially augment the immune response. (23, 24, 31). The Th2 cytokines, IL-4 and IL-10, have been linked The ability of PPAR␣ agonists to regulate the immune response to both remissions and recovery in EAE and MS (23Ð26). Shifting is an area of active investigation. Because of our observations and the balance of cytokine production by encephalitogenic, Th1-like those of others that PPAR␣ agonists such as gemfibrozil and cip- response to the protective Th2-like response has been a focus of rofibrate can induce IL-4 production, it is likely that transcription several therapeutic strategies in EAE and has been termed “im- factors involved in T cell differentiation are affected by PPAR␣ mune deviation.” (7). Differentiation of T cells to a Th2 phenotype involves activa- We previously demonstrated that administration of IL-4 could tion of STAT-6, which is translocated to the nucleus, resulting in alter the cytokine secreting phenotype of MBP-reactive T cells and expression of GATA-3 (35). Although the mechanism of STAT-6 reduce their encephalitogenic potential (23). Several agents have induction of GATA-3 is not entirely clear, GATA-3 is thought to been used to induce immune deviation in MS and EAE, including be the master regulator of Th2 differentiation (36). There is an retinoids, glatiramer acetate, and cyclophosphamide (24, 32, 33). increasing body of evidence that there is cross-talk between PPAR PPAR␣ agonists would represent another class of drugs with a signaling pathways with the STAT and GATA transcription fac- favorable safety profile that could potentially be used in the treat- tors (37). Studies are in progress evaluating whether the production ment of human autoimmune disorders such as MS. of IL-4 is responsible for the alteration of the T cell phenotype or The precise mechanism of how PPAR agonists exert their ther- whether this is occurring at the level of activation of PPAR/reti- apeutic effects remain unclear. Our work and that of others using noid X receptor heterodimers. Prior studies by our group with reti- PPAR␥ agonists suggested that Th1 cytokine secretion could be noids would suggest that even if the effects of IL-4 are neutralized, suppressed and that apoptosis of autoreactive T cells would be the activation of Th2-inducing transcription factors would result in promoted (13Ð16). Interestingly, one study examining the effect of an alteration in encephalitogenic potential (24). How PPAR␣ ago- thiazolidinediones on T cell death suggested apoptosis was in- nists induce Th2 differentiation is an area of active investigation by duced in a dose-dependent manner (34). The doses required to our group at the present time. induce programmed cell death were well above the doses required Interestingly, a recent study suggests that PPAR␣ also plays a to induce optimal transcriptional activation. Surprisingly, at those physiologic role in regulating T-bet, an inducible transcription fac- doses, thiazolidinedione activation of PPAR␥-protected cells from tor important in the initiation of cytokine gene transcription, par- apoptosis following growth factor withdrawal. Thus, at the doses ticularly Th1 cytokines (38). This study demonstrated that PPAR␣ 5796 PPAR␣ AGONISTS IN AUTOIMMUNE DISEASE Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. Pathology and immunohistochemistry of the CNS of gemfibrozil-treated mice. Immunohistochemistry for macrophages (A and C) and CD4ϩ T cells (B and D) was performed on frozen sections (10 ␮m) from EAE-affected mice perfused on day 42 postimmunization. Mice receiving gemfibrozil from day Ϫ1 until day 20 are shown in C and D, while sections in A and B are from a PBS-treated control. Magnification, ϫ100. present in the cytoplasm of T cells was able to negatively regulate entiation of autoreactive lymphocytes, as well as the activation of the transcription of T-bet, which indirectly influenced the amount microglia in the CNS. of IFN-␥ produced by the T cell. This regulation occurred inde- Although the ability of PPAR␥ agonists to regulate inflamma- pendent of DNA-binding, suggesting that there may be several tory processes in vivo appears to be true in a number of models mechanisms of how PPAR␣ can influence T cell activation and (10Ð16), limited data is available on PPAR␣ agonists in such sit- cytokine production. uations. In a model of colitis induced by administration of dextran In addition to the effects of PPAR␣ on lymphocytes, we have sodium sulfate, gastric gavage of the PPAR␣ ligand, bezafibrate, investigated the effects of gemfibrozil, ciprofibrate, and fenofibrate significantly inhibited the expression of colitis, and reduced pro- on microglia, resident CNS cells that function in Ag presentation liferation of colonic mucosa (41). Use of fenofibrate in experimen- and phagocytosis. A variety of neuroinflammatory disorders in- tal autoimmune myocarditis reduced the number of inflammatory cluding MS are characterized by the presence of activated micro- lesions in the heart and reduced the ventricular size (42). Cardiac glia (39). Upon activation, microglia produce a variety of mole- expression of IL-10 also appeared to be increased in the rats that cules including NO and TNF-␣, which are important for the received fenofibrate. Thus, this suggests that PPAR␣ agonists may elimination of invading pathogens, but may also be toxic to host also have the potential to regulate inflammatory processes in vivo. cells including oligodendrocytes and neurons, which are compro- In summary, we have demonstrated that administration of the mised in MS. In addition, fenofibrate has been shown to have PPAR␣ agonists, gemfibrozil and fenofibrate, are able to treat on- protective effects in models of cerebral ischemia, suggesting that it going signs of EAE. Exposure of both murine and human T cells may have neuroprotective, as well as anti-inflammatory effects to these agents in vitro results in a shift in cytokine secretion pat- (40). This suggests that agents which block microglial cell activa- tern from a Th1-like response to that of a Th2-like response, a tion may also be protective in diseases such as MS. process known as immune deviation. In addition, PPAR␣ agonists Gemfibrozil, ciprofibrate, and fenofibrate were also capable of appear to inhibit microglial activation which may minimize CNS suppressing lymphocyte proliferation at doses that had miminal inflammation. Gemfibrozil has an excellent track record as a safe, effects on cells viability. This observation was most profound at beneficial, and cost-effective drug for the treatment of hyperlipid- the higher concentrations of fenofibrate. Together, this data sug- emia. Although fenofibrate has shown toxicity in mice, it has been gests that PPAR␣ can potentially affect the activation and differ- shown to be safe and well-tolerated in humans (43). These results The Journal of Immunology 5797

FIGURE 7. Gemfibrozil-treated mice have decreased numbers of infiltrating CD4ϩ T cells and macrophages. Number of CD4ϩ T cells and macrophages expressed in infiltrates per 104 square pixels from spinal cord sections of mice induced to develop EAE. Mice received either gemfibrozil (500 ␮g) or PBS by gavage from day Ϫ1 until day 20 postimmunization. Spinal cords were harvested on day 42 postimmunization. Infiltrating cells were quantified as described in Materials and Methods. The mean number of infiltrating cells and SD are depicted. Value of p Ͻ 0.05 for both mac- Downloaded from rophages and CD4ϩ T cells. http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 9. Gemfibrozil suppresses IFN-␥ and enhances IL-4 production by human lymphocytes. A, A human PLP-specific Th1 cell line was stimulated with 10 ␮g/ml PLP peptide mix in the presence of 50 ␮M gemfibrozil. IFN-␥ production was measured by ELISA at 48 h. B, Human Th1 cell lines specific for MBP or PLP were stimulated with Ag in the presence of 50 ␮M gemfibrozil. IFN-␥ production was determined at 48 h by ELISA. C, Human MBP-specific (M876-B) and PLP-specific (H145-E) TCLs were generated by weekly stimulation with Ag in the presence of 50 mM gemfibrozil. IL-4 production was determined by stimulation of the cell lines in the absence of gemfibrozil by ELISA. A and B, TCLs were generated from MS patients. C, TCLs were generated from both MS (M876-B) and healthy individuals (H145-E).

FIGURE 8. Effect of gemfibrozil on the viability and proliferation of human lymphocytes. A, Human PBL (5 ϫ 106 cells per well of 24-well plate) were cultured in the presence of various concentrations of gemfi- raise the possibility that PPAR␣ agonists might have benefitasa brozil and the viability of the lymphocytes was checked by a trypan blue therapy in inflammatory human diseases such as MS. exclusion assay. B, Human PBL were placed in 96-well plates at 2 ϫ 105 cells per well and stimulated with 1 ␮g/ml Con A. Various concentrations References of gemfibrozil were added to quadruplicate wells. Proliferation was mea- 1. Desvergne, B., and W. Wali. 1999. Peroxisome proliferator-activated receptors: sured by addition of [3H]thymidine during the final 16 h of incubation. nuclear control of metabolism. Endocr. Rev. 20:649. 5798 PPAR␣ AGONISTS IN AUTOIMMUNE DISEASE

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