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Agonists as Therapy for Autoimmune Disease1
* Department of Neurology, and
Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390; and
Department of Neurobiology and Developmental Sciences, University of Arkansas for the Medical Sciences, Little Rock, AR 72205
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Abstract |
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ligands, which include the naturally occurring PG metabolite 15-deoxy-
12,14-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 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. |
Introduction |
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, 
, and ) and they exhibit different tissue distribution as well as different ligand specificities. PPARs have been most extensively studied in the regulation of genes involved in glucose and lipid metabolism (2). In addition to adipocytes, it has also been recently shown that cells of the monocyte/macrophage lineage express both PPAR and PPAR, suggesting a possible role for these receptors in immune function (3, 4).
PPAR ligands, which include the naturally occurring PG metabolite 15-deoxy-12,14-PGJ2 (15d-PGJ2), as well as thiazolidinediones, have been shown to have anti-inflammatory activity. 15d-PGJ2 was shown to inhibit inducible nitric oxide synthase, matrix-metalloproteinase-9, IL-1
, IL-6, and TNF- production by monocytes/macrophages (5, 6). PPAR ligands can inhibit IL-2 production and T cell proliferation (7, 8). PPAR ligands can also affect T cell function indirectly by inhibiting the production of chemokines by endothelial cells (9). The evidence that PPAR agonists can regulate inflammation is supported by several animal studies. These include the suppression of adjuvant arthritis in rats (10), inhibition of atherosclerosis in mice (11), and amelioration of inflammatory bowel disease in mice (12). Our recent work using 15d-PGJ2, as well as that of others using other PPAR agonists, demonstrated inhibition of the clinical signs of experimental autoimmune encephalomyelitis (EAE) (13, 14, 15, 16).
PPAR ligands have also been shown to regulate inflammatory responses, although there is clearly less evidence along this line compared with PPAR agonists. Both PPAR and PPAR have been shown to be expressed on T cells and their ligands can inhibit IL-2 production and T cell proliferation (7, 8). PPAR-deficient mice have abnormally prolonged responses to inflammatory stimuli such as arachidonic acid and leukotrienes (17). The expression of IL-6, VCAM, and cyclooxygenase-2 in response to cytokine activation can be inhibited by PPAR ligands (18). PPAR ligands have been shown to decrease NF-
B activation, IL-12, and IL-6 production in aged mice (19). PPAR ligands may inhibit functional expression of NF-B, in part by augmenting the expression of IB (20).
Recently, the PPAR ligand, WY14,643, was shown to inhibit IgG responses in myelin oligodendrocyte glycoprotein 35–55/CFA-immunized mice (21). When mice were fed this agonist, splenocytes demonstrated impaired production of IFN-, IL-6, and TNF-. Interestingly, the authors had hoped to examine the effect of fibrates on EAE; however, the combination of immunization with myelin oligodendrocyte glycoprotein 35–55/CFA, pertussis toxin, and WY14,643 treatment consistently induced mortality 5–10 days after immunization.
Organ-specific autoimmune diseases, such as EAE, are mediated by IFN--producing Th1 cells (22). Evidence in the EAE model suggests that the production of Th2 cytokines such as IL-4 or IL-10 may play an important role in the remissions observed in this disease (23, 24, 25). Interestingly, it appears that proteolipid protein (PLP)-specific T cell clones isolated from multiple sclerosis (MS) patients have different cytokine phenotypes depending on when they are isolated (26). T cell clones derived during MS attacks are more likely to have a Th1 phenotype, while those derived during remission were more variable but included several T cells of a Th2 phenotype, suggesting that shifting the phenotype of T cells from Th1 to Th2 may be beneficial in MS.
In the present studies, we have examined the effects of PPAR agonists on the differentiation of autoreactive T cells and their effects on clinical signs of disease in the murine model of EAE. In addition, we have also examined the effects of gemfibrozil, a synthetic PPAR agonist with a long history of oral use in humans, on the secretion of cytokines by human myelin-reactive T cells. Our results suggest that PPAR agonists may be a useful therapeutic agent for human inflammatory diseases such as MS.
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Materials and Methods |
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Transgenic mice bearing the rearranged V2.3,V8.2 gene encoding the TCR specific for the myelin basic protein (MBP) Ac1–11 peptide on the B10.PL background were obtained by crossing transgenic mice bearing the individual rearranged genes (27). The V2.3 TCR-transgenic mice and the V8.2 TCR transgenic mice were kindly provided by Dr. J. Goverman (University of Washington, Seattle, WA). These mice were bred and maintained in a federally approved animal facility at the University of Texas Southwestern Medical Center (Dallas, TX) in accordance with the Institutional Animal Care and Use Committee. All mice were 7–10 wk old when experiments were performed. B10.PL mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and then bred in our animal facility.
Murine cell culture
Spleens from naive V2.3, V8.2 TCR transgenic mice were harvested and single-cell suspensions were obtained by pressing the tissue through a wire mesh screen, as previously described (27). Proliferative responses of splenocytes (4 x 105 cells/well) were determined using MBPAc1–11 (2 µg/ml) in the presence of various concentrations of gemfibrozil, ciprofibrate, and fenofibrate (Sigma-Aldrich, St. Louis, MO) as indicated, or equivalent volumes of solvent. Gemfibrozil, ciprofibrate, and fenofibrate were prepared as recommended by the manufacturer. Cultures were maintained in 96-well flat-bottom plates for 96 h at 37°C in humidified 5% CO2/air. The wells were pulsed with 0.5 µCi/well [methyl-3H]thymidine for the final 16 h of culture. Cells were harvested on glass filters and incorporated [methyl-3H]thymidine was measured with a Betaplate counter (PerkinElmer Wallac, Gaithersburg, MD). Results were determined as means from quadruplicate cultures and are shown with SEM.
The N9 microglial cell line is derived from myc-immortalized mouse microglia (28), and was graciously provided by P. Ricciardi-Castagnoli (University of Milan, Milan, Italy). Cells were cultured in MEM medium containing 10% FBS, 1.4 mM glutamine, and 20 µM 2-ME. Where indicated, cells were treated with IFN- (PBL Laboratories, Piscataway, NJ) plus TNF- (R&D Systems, Minneapolis, MN) and/or the PPAR agonists, gemfibrozil, ciprofibrate, or fenofibrate (Sigma-Aldrich).
NO production assay
N9 microglial cell production of the NO derivative nitrite was assessed in culture medium by Greiss reaction as we have described previously (29). Cells were grown in 96-well plates. Where indicated, cells were pretreated for 1 h with gemfibrozil, ciprofibrate, or fenofibrate. Cells were then treated as indicated with IFN- (100 U/ml) plus TNF- (500 U/ml), and nitrite levels were determined following incubation for 24 h.
Induction and clinical evaluation of EAE
EAE was induced in B10.PL mice by s.c. injection over four sites in the flank with 200 µg of MBP Ac1–11 (BioSource International, Camarillo, CA) in an emulsion with CFA (Difco, Detroit, MI). Pertussis toxin (200 ng/mouse) was injected i.p. at the time of immunization and 48 h later. Mice were scored on a scale of 0–6 as previously described (13): 0, no clinical disease; 1, limp/flaccid tail; 2, moderate hind limb weakness; 3, severe hind limb weakness; 4, complete hind limb paralysis; 5, quadriplegia or premoribund state; 6, death.
Administration of PPAR agonists to mice with EAE
For administration of gemfibrozil and fenofibrate to mice with EAE, stock solutions were made by dissolving these agents in ethanol (50 mg/ml). When mice were fed by gavage, stock solution was diluted in PBS so that indicated dose of PPAR agonist was delivered in a total of 200 µl. Mouse chow was supplemented with gemfibrozil or fenofibrate by adding the stock solution to the chow (0.25% w/w), allowing the ethanol to evaporate, and then using it as the source of food. Control diet was given the same amount of ethanol without the PPAR agonist.
Pathology and immunohistochemistry
Mice were deeply anesthetized with sodium pentobarbital and transcardially perfused with sterile PBS, followed by 4% paraformaldehyde in phosphate buffer. The brain and spinal cord were removed, fixed in 4% paraformaldehyde for 6 h, and cryoprotected in 30% sucrose overnight at 4°C before being embedded in Tissue-Tek (T. Pella, Redding, CA) and quick-frozen in isopentane. Immunohistochemistry was performed on adjacent sections. After pretreatment with 0.3% hydrogen peroxide in absolute methanol, sections were blocked with 1% BSA for 2 h at room temperature and then incubated with primary Ab overnight at 4°C. Primary Abs used were MOMA-2 (1/20) mAb, a marker for macrophages (BioSource International) and KT174 (1/20), which recognizes CD4+ T cells (BioSource International).
The binding of the primary Abs was detected using a biotinylated secondary Ab and an avidin-biotin-peroxidase method under humidified conditions (ABC Elite kit; Vector Laboratories, Burlingame, CA). Peroxidase activity was visualized with 3,3'-diaminobenzidine (DAB kit; Vector Laboratories) as a substrate. Isotype IgG2c Ab served as a negative control. The tissue area was measured by a Scion image analysis system (Scion, Frederick, MD). Positive cells were counted by automatic video scanning using a Leica Q500 MC (Zeiss, Oberkochen, Germany), and the numbers of stained cells per 104 square pixels tissue area were calculated.
Cytokine ELISA
Mouse IFN-, IL-4, and IL-10 were quantified using a sandwich ELISA based on noncompeting pairs of Abs as previously described (13). Murine splenocytes from B10.PL mice with a transgenic TCR specific for MBP Ac1–11 were cultured at 8 x 106 cells/well in 24-well plates with 2 µg/ml Ac1–11. Supernatants were collected at 24, 48, and 72 h and cytokine levels determined by ELISA. Human IFN- and IL-4 were determined similarly, using the same basic protocol with human-specific Abs. In brief, Immulon 2 plates (Dynex Technologies, Chantilly, VA) were coated with purified monoclonal anti-IFN- (Code: M-700A; Endogen, Woburn, MA) or anti-IL-4 (catalog no. 554515; BD PharMingen, San Diego, CA) at 2 µg/ml in 0.1 M NaHCO3 at 4°C overnight. The plates were washed two times with PBS/1% Tween, blocked with 1% BSA/PBS, and 100 µl of supernatant was added to the plates in duplicate. The plates were placed at 4°C overnight and then washed four times with PBS/Tween. Biotin-labeled anti-IFN- (code: M-701B; Endogen) or anti-IL-4 (catalog no. 554483; BD PharMingen) were added at 1 µg/ml and incubated at room temperature for 1.5 h. An avidin-peroxidase substrate was used to complete the assay. Concentrations of IFN- and IL-4 were determined using a standard curve generated using known concentrations of human recombinant IFN- (BD PharMingen) and IL-4 (Calbiochem, San Diego, CA) in the assay.
Generation of human T cell lines (TCL)
Human lymphocytes were collected via leukapheresis from MS patients and healthy individuals under an Institutional Review Board approved protocol. Human TCL specific for MBP and PLP were generated as previously described (30). Bovine MBP (Sigma-Aldrich) or a mix of five PLP peptides (31–60, 91–120, 111–140, 131–160, and 177–206) were used at 10 µg/ml to generate the TCL. Lines designated with the letter M were derived from MS patients, while those designated with an H were derived from healthy individuals.
Human T cell proliferation assay
For Con A proliferation assays, 2 x 105 PBMC were placed in each well of a 96-well plate. Con A was added at 1 µg/ml and various concentrations of gemfibrozil (or the same volume of ethanol which was the diluent for gemfibrozil) was added to quadruplicate wells. The wells were pulsed with 0.5 µCi/well [methyl-3H]thymidine for 18 h before harvesting at 96 h. Cells were harvested on glass filters using a Tomtec harvester (Tomtec, Hamden, CT), and incorporated [methyl-3H]thymidine was measured with a Betaplate counter (PerkinElmer Wallac).
T cell viability assay
Human PBMC or murine splenocytes were placed in 24-well plates at 5 x 106 cells per well. Gemfibrozil, ciprofibrate, or fenofibrate (or an equal volume of solvent) were added to the wells at a final concentration of 0–500 µM. At various time points, the cells were collected, resuspended in trypan blue, and the number of viable and nonviable cells were counted.
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Results |
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agonists suppress lymphocyte proliferation in a dose-dependent manner
Because EAE is caused by CD4+ T cells specific for myelin Ags, we first examined whether various PPAR agonists have any effect on CD4+ Ag-specific proliferation. Splenocytes from naive V2.3,V8.2 TCR transgenic mice were cultured with MBP Ac1–11 (2 µg/ml) in the presence of various concentrations of gemfibrozil, ciprofibrate, and fenofibrate. MBP Ac1–11-induced proliferation was inhibited by high doses of gemfibrozil (100–400 µM); however, lower doses did not affect T cell proliferation (20–50 µM) (Fig. 1A). Proliferation was decreased by 79.2% at the highest concentration tested. Similarly, lymphocyte proliferation was suppressed at higher concentrations of ciprofibrate (200–400 µM), with some minor fluctuations in proliferation at the lower doses (Fig. 1B). Fenofibrate completely inhibited lymphocyte proliferation at 50–100 µM concentrations, but had no effect at doses of 10 µM or less (Fig. 1C).
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agonists, there is a suppression of proliferation at higher concentrations that is not due solely to lymphocyte death.
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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, ciprofibrate, and fenofibrate affected cytokine secretion of MBPAc1–11-specific T cells. Using ELISA, we examined the expression of IFN-, IL-4 and IL-10 in the supernatant of MBPAc1–11-specific T cells stimulated in the presence of 50–400 µM gemfibrozil and ciprofibrate. A single stimulation of naive TCR transgenic splenocytes in the presence of gemfibrozil or ciprofibrate induced IL-4 expression (Fig. 3, A and C). For gemfibrozil, IL-4 levels were highest at 50 µM, a concentration that had no effect on proliferation or cell viability. For ciprofibrate, IL-4 levels were highest at 200 µM, 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 production was doubled in the second stimulation in the presence of 10 µM fenofibrate (Fig. 3E). Although there was a modest decrease in the levels of IFN- at higher concentrations of the PPAR agonists, IFN- production were not significantly affected by the presence of any of the three PPAR agonists (Fig. 3B, D, and F). Similarly, there was no significant affect on IL-10 production by the PPAR agonists (data not shown).
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Previously, we demonstrated that the PPAR agonist, 15d-PGJ2, inhibited the activation of microglial cells (29), repressed microglial expression of CD40, a molecule which plays a critical role in T cell costimulation, and repressed development of EAE (13). Collectively, these studies suggest that 15d-PGJ2 may modulate EAE, at least in part, via effects on microglial cells, resident CNS cells that function in Ag presentation and phagocytosis. In the present study, we demonstrate that the PPAR agonists, gemfibrozil, ciprofibrate, and fenofibrate, inhibited NO production by microglial cells (Fig. 4). Gemfibrozil shows a dose-dependent decrease in NO production. Ciprofibrate and fenofibrate show a significant effect at doses as low as 1 µM. Fenofibrate showed a complete inhibition of NO by microglia at 50 µM which was not due to toxicity because MTT analyses indicated that these PPAR agonists did not decrease microglial cell viability (data not shown). These studies suggest that these PPAR agonists inhibit microglial cell activation, providing a second potential mechanism by which PPAR may exert its anti-inflammatory properties.
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agonists protect mice from developing EAESince gemfibrozil altered the phenotype of MBP Ac1–11-specific T cells with regard to their cytokine secretion, we next determined whether administration of gemfibrozil altered the pathogenesis of EAE. Gemfibrozil was chosen as the first drug to evaluate in EAE, because it has been extensively studied in both mice and humans with few side effects and little toxicity. In our initial experiment, B10.PL mice were fed gemfibrozil (500 µg) daily for 21 days starting on the day of immunization with MBP Ac1–11. Control mice either received PBS by gavage or were not fed. Although both groups of control mice developed severe signs of EAE, only one mouse that received gemfibrozil developed signs of disease (Fig. 5A).
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Using another PPAR agonist, fenofibrate, which was shown to dramatically suppress lymphocyte proliferation and NO production by microglia, a similar experiment was performed. The fenofibrate 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 
25 days after feeding had stopped, although by day 50 postimmunization, treated animals could not be distinguished 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.
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 three weeks before being sacrificed and having their spinal cords removed for histologic analysis (mice from Fig. 5A). Infiltrating macrophages and CD4+ 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 than those present in the placebo-treated mice. The actual number of infiltrating macrophages and CD4+ T cells quantified by the Scion image analysis system (Scion) is shown in Fig. 7. These results suggest that the administration of the PPAR agonist, gemfibrozil, reduces the inflammation in the CNS of mice induced to develop EAE.
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Having demonstrated that gemfibrozil was effective in altering the cytokine secretion phenotype of murine myelin-specific T cells and effective in prevention and treatment of EAE, we next examined the effects of this agent on human lymphocyte function. Gemfibrozil was chosen because it is an FDA-approved drug with an established safety record. Concentrations of gemfibrozil at 50 µM had no increase in toxicity over the carrier alone (Fig. 8A). Lymphocytes exposed to gemfibrozil at higher doses experienced increased toxicity when exposed to the drug for longer than 24 h. Similar to what was observed with murine T cells, gemfibrozil also inhibited T cell proliferation in response to Con A in a dose-dependent manner (Fig. 8B). Again, lower doses of gemfibrozil had only minimal effects on T cell proliferation when Con A was used as the stimulating agent.
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Because gemfibrozil had important effects on altering cytokine production of murine lymphocytes, we examined whether it had similar effects on human T cell cytokine production. Because 50 µM gemfibrozil did not affect T cell proliferation or cell viability, this concentration was chosen to examine effects on cytokine production. Human PLP-specific Th1 cell lines were stimulated with Ag in the presence of 50 µM gemfibrozil. IFN- production was inhibited at both 48 and 72 h after stimulation when measuring the 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 lines (Fig. 9B). IL-4 and IL-10 were occasionally secreted by Th1 cells following gemfibrozil treatment (data not shown). Human T cells lines specific for myelin Ags were also generated in the presence of gemfibrozil (50 µM). TCLs generated from MS patients and healthy subjects in this manner secreted significant amounts of IL-4 as determined by ELISA (Fig. 9C). Thus, gemfibrozil was able to both promote IL-4 production and inhibit IFN- production of human myelin-specific TCLs, suggesting that this agent might be a potential therapeutic agent for MS.
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Discussion |
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agonists, gemfibrozil and fenofibrate, are able to prevent and treat the organ-specific autoimmune disorder EAE. We investigated several potential mechanisms by which this may be occurring, including altering cytokine production, inhibiting T cell proliferation, and suppressing microglia activation. We have demonstrated that gemfibrozil can induce the secretion of the prototypic Th2 cytokine, IL-4, in both murine and human T cells. The role that Th2 cytokines play in EAE has been studied extensively by us and others (23, 24, 31). The Th2 cytokines, IL-4 and IL-10, have been linked to both remissions and recovery in EAE and MS (23, 24, 25, 26). Shifting the balance of cytokine production by encephalitogenic, Th1-like response to the protective Th2-like response has been a focus of several therapeutic strategies in EAE and has been termed "immune deviation."
We previously demonstrated that administration of IL-4 could alter the cytokine secreting phenotype of MBP-reactive T cells and reduce their encephalitogenic potential (23). Several agents have been used to induce immune deviation in MS and EAE, including retinoids, glatiramer acetate, and cyclophosphamide (24, 32, 33). PPAR agonists would represent another class of drugs with a favorable safety profile that could potentially be used in the treatment of human autoimmune disorders such as MS.
The precise mechanism of how PPAR agonists exert their therapeutic effects remain unclear. Our work and that of others using PPAR agonists suggested that Th1 cytokine secretion could be suppressed and that apoptosis of autoreactive T cells would be promoted (13, 14, 15, 16). Interestingly, one study examining the effect of thiazolidinediones on T cell death suggested apoptosis was induced in a dose-dependent manner (34). The doses required to induce programmed cell death were well above the doses required to induce optimal transcriptional activation. Surprisingly, at those doses, thiazolidinedione activation of PPAR-protected cells from apoptosis following growth factor withdrawal. Thus, at the doses used to treat patients with diabetes, the activation of PPAR by thiazolidinediones may potentially augment the immune response.
The ability of PPAR agonists to regulate the immune response is an area of active investigation. Because of our observations and those of others that PPAR agonists such as gemfibrozil and ciprofibrate can induce IL-4 production, it is likely that transcription factors involved in T cell differentiation are affected by PPAR (7). Differentiation of T cells to a Th2 phenotype involves activation of STAT-6, which is translocated to the nucleus, resulting in expression of GATA-3 (35). Although the mechanism of STAT-6 induction of GATA-3 is not entirely clear, GATA-3 is thought to be the master regulator of Th2 differentiation (36). There is an increasing body of evidence that there is cross-talk between PPAR signaling pathways with the STAT and GATA transcription factors (37). Studies are in progress evaluating whether the production of IL-4 is responsible for the alteration of the T cell phenotype or whether this is occurring at the level of activation of PPAR/retinoid X receptor heterodimers. Prior studies by our group with retinoids would suggest that even if the effects of IL-4 are neutralized, the activation of Th2-inducing transcription factors would result in an alteration in encephalitogenic potential (24). How PPAR agonists induce Th2 differentiation is an area of active investigation by our group at the present time.
Interestingly, a recent study suggests that PPAR also plays a physiologic role in regulating T-bet, an inducible transcription factor important in the initiation of cytokine gene transcription, particularly Th1 cytokines (38). This study demonstrated that PPAR present in the cytoplasm of T cells was able to negatively regulate the transcription of T-bet, which indirectly influenced the amount of IFN- produced by the T cell. This regulation occurred independent of DNA-binding, suggesting that there may be several mechanisms of how PPAR can influence T cell activation and cytokine production.
In addition to the effects of PPAR on lymphocytes, we have investigated the effects of gemfibrozil, ciprofibrate, and fenofibrate on microglia, resident CNS cells that function in Ag presentation and phagocytosis. A variety of neuroinflammatory disorders including MS are characterized by the presence of activated microglia (39). Upon activation, microglia produce a variety of molecules including NO and TNF-, which are important for the elimination of invading pathogens, but may also be toxic to host cells including oligodendrocytes and neurons, which are compromised in MS. In addition, fenofibrate has been shown to have protective effects in models of cerebral ischemia, suggesting that it may have neuroprotective, as well as anti-inflammatory effects (40). This suggests that agents which block microglial cell activation may also be protective in diseases such as MS.
Gemfibrozil, ciprofibrate, and fenofibrate were also capable of suppressing lymphocyte proliferation at doses that had miminal effects on cells viability. This observation was most profound at the higher concentrations of fenofibrate. Together, this data suggests that PPAR can potentially affect the activation and differentiation of autoreactive lymphocytes, as well as the activation of microglia in the CNS.
Although the ability of PPAR agonists to regulate inflammatory processes in vivo appears to be true in a number of models (10, 11, 12, 13, 14, 15, 16), limited data is available on PPAR agonists in such situations. In a model of colitis induced by administration of dextran sodium sulfate, gastric gavage of the PPAR ligand, bezafibrate, significantly inhibited the expression of colitis, and reduced proliferation of colonic mucosa (41). Use of fenofibrate in experimental autoimmune myocarditis reduced the number of inflammatory lesions in the heart and reduced the ventricular size (42). Cardiac expression of IL-10 also appeared to be increased in the rats that received fenofibrate. Thus, this suggests that PPAR agonists may also have the potential to regulate inflammatory processes in vivo.
In summary, we have demonstrated that administration of the PPAR agonists, gemfibrozil and fenofibrate, are able to treat ongoing signs of EAE. Exposure of both murine and human T cells to these agents in vitro results in a shift in cytokine secretion pattern from a Th1-like response to that of a Th2-like response, a process known as immune deviation. In addition, PPAR agonists appear to inhibit microglial activation which may minimize CNS inflammation. Gemfibrozil has an excellent track record as a safe, beneficial, and cost-effective drug for the treatment of hyperlipidemia. Although fenofibrate has shown toxicity in mice, it has been shown to be safe and well-tolerated in humans (43). These results raise the possibility that PPAR agonists might have benefit as a therapy in inflammatory human diseases such as MS.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael K. Racke, Department of Neurology and the Center for Immunology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9036. E-mail address: Michael.Racke{at}utsouthwestern.edu
3 Abbreviations used in this paper: PPAR, peroxisome proliferator-activated receptor; 15d-PGJ2, 15-deoxy-12,14-PGJ2; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; TCL, T cell line; PLP, proteolipid protein; MS, multiple sclerosis.
Received for publication December 2, 2003. Accepted for publication February 26, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and are activated by indomethacin and other non-steroidal anti-inflammatory drugs. J. Biol. Chem. 272:3406. is a negative regulator of macrophage activation. Nature 391:79.[Medline]
agonists inhibit production of monocyte inflammatory cytokines. Nature 391:82.[Medline]
dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation. Nat. Med. 7:48.[Medline]
activators inhibit IFN--induced expression of the T cell-active CXC chemokines IP-10, Mig, and I-TAC in human endothelial cells. J. Immunol. 164:6503.12,14-PGJ2 induces synoviocyte apoptosis and suppresses adjuvant-induced arthritis in rats. J. Clin. Invest. 106:189.[Medline]
ligands inhibit development of atherosclerosis in LDL receptor-deficient mice. J. Clin. Invest. 106:523.[Medline]
ligands to inhibit the epithelial inflammatory response. J. Clin. Invest. 104:383.[Medline]
agonist 15-deoxy-12,14-PGJ2 ameliorates experimental autoimmune encephalomyelitis. J. Immunol. 168:2508.. J. Neuroimmunol. 116:40.[Medline]
agonists inhibit experimental allergic encephalomyelitis by blocking IL-12 production, IL-12 signaling, and Th1 differentiation. Genes Immun. 3:59.[Medline]
agonists prevent experimental auotimmune encephalomyelitis. Ann. Neurol. 51:694.[Medline]
-leukotriene B4 pathway to inflammation control. Nature 384:39.[Medline]
B and AP-1. J. Biol. Chem. 274:32048.B in an animal model of aging. Int. Immunol. 9:1581.B expression as a mechanism contributing to the anti-inflammatory activities of peroxisome proliferator-activated receptor- activators. J. Biol. Chem. 275:36703. ligand, has profound effects on immune responses in vivo. J. Immunol. 169:6806.12,14 prostaglandin J2 represses nitric oxide, TNF-, and IL-12 production by microglial cells. J. Neuroimmunol. 115:28.[Medline]
can enhance mitochondrial potential and promote cell survival. J. Biol. Chem. 277:31781. (PPAR) agonists. PPAR co-association with transcription factor NFAT. J. Biol. Chem. 275:4541. activation as a mechanism of preventive neuroprotection induced by chronic fenofibrate treatment. J. Neurosci. 23:6264. and inhibit chemically induced colitis and formation of aberrant crypt foci in rats. Cancer Res. 61:2424. activator, suppresses experimental autoimmune myocarditis by stimulating the interleukin-10 pathway in rats. J. Atheroscler. Thromb. 9:87.[Medline]
. J. Natl. Cancer Inst. 90:1702.This article has been cited by other articles:
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 M. B. Schaefer, A. Pose, J. Ott, M. Hecker, A. Behnk, R. Schulz, N. Weissmann, A. Gunther, W. Seeger, and K. Mayer Peroxisome proliferator-activated receptor-{alpha} reduces inflammation and vascular leakage in a murine model of acute lung injury Eur. Respir. J., November 1, 2008; 32(5): 1344 - 1353. [Abstract] [Full Text] [PDF]
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X. Fang, L. Zhang, Y. Feng, Y. Zhao, and J. Dai Immunotoxic Effects of Perfluorononanoic Acid on BALB/c Mice Toxicol. Sci., October 1, 2008; 105(2): 312 - 321. [Abstract] [Full Text] [PDF]
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 H. P. Carroll, V. Paunovic, and M. Gadina Signalling, inflammation and arthritis: Crossed signals: the role of interleukin-15 and -18 in autoimmunity Rheumatology, September 1, 2008; 47(9): 1269 - 1277. [Abstract] [Full Text] [PDF]
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 S. Cuzzocrea, S. Bruscoli, E. Mazzon, C. Crisafulli, V. Donato, R. Di Paola, E. Velardi, E. Esposito, G. Nocentini, and C. Riccardi Peroxisome Proliferator-Activated Receptor-{alpha} Contributes to the Anti-Inflammatory Activity of Glucocorticoids Mol. Pharmacol., February 1, 2008; 73(2): 323 - 337. [Abstract] [Full Text] [PDF]
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 A. Roy, X. Liu, and K. Pahan Myelin Basic Protein-primed T Cells Induce Neurotrophins in Glial Cells via {alpha}5beta3 Integrin J. Biol. Chem., November 2, 2007; 282(44): 32222 - 32232. [Abstract] [Full Text] [PDF]
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S. Dasgupta, A. Roy, M. Jana, D. M. Hartley, and K. Pahan Gemfibrozil Ameliorates Relapsing-Remitting Experimental Autoimmune Encephalomyelitis Independent of Peroxisome Proliferator-Activated Receptor-{alpha} Mol. Pharmacol., October 1, 2007; 72(4): 934 - 946. [Abstract] [Full Text] [PDF]
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M. Jana, A. Jana, X. Liu, S. Ghosh, and K. Pahan Involvement of Phosphatidylinositol 3-Kinase-Mediated Up-Regulation of I{kappa}B{alpha} in Anti-Inflammatory Effect of Gemfibrozil in Microglia J. Immunol., September 15, 2007; 179(6): 4142 - 4152. [Abstract] [Full Text] [PDF]
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 M. Rotondi, L. Chiovato, S. Romagnani, M. Serio, and P. Romagnani Role of Chemokines in Endocrine Autoimmune Diseases Endocr. Rev., August 1, 2007; 28(5): 492 - 520. [Abstract] [Full Text] [PDF]
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 A. Budd, L. Alleva, M. Alsharifi, A. Koskinen, V. Smythe, A. Mullbacher, J. Wood, and I. Clark Increased Survival after Gemfibrozil Treatment of Severe Mouse Influenza Antimicrob. Agents Chemother., August 1, 2007; 51(8): 2965 - 2968. [Abstract] [Full Text] [PDF]
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J. M. Reynolds, Q. Liu, K. C. Brittingham, Y. Liu, M. Gruenthal, C. Z. Gorgun, G. S. Hotamisligil, R. D. Stout, and J. Suttles Deficiency of Fatty Acid-Binding Proteins in Mice Confers Protection from Development of Experimental Autoimmune Encephalomyelitis J. Immunol., July 1, 2007; 179(1): 313 - 321. [Abstract] [Full Text] [PDF]
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K. J. Fairley, R. Purdy, S. Kearns, S. E. Anderson, and B. Meade Exposure to the Immunosuppresant, Perfluorooctanoic Acid, Enhances the Murine IgE and Airway Hyperreactivity Response to Ovalbumin Toxicol. Sci., June 1, 2007; 97(2): 375 - 383. [Abstract] [Full Text] [PDF]
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 S. E. Dunn, S. S. Ousman, R. A. Sobel, L. Zuniga, S. E. Baranzini, S. Youssef, A. Crowell, J. Loh, J. Oksenberg, and L. Steinman Peroxisome proliferator-activated receptor (PPAR){alpha} expression in T cells mediates gender differences in development of T cell-mediated autoimmunity J. Exp. Med., February 19, 2007; 204(2): 321 - 330. [Abstract] [Full Text] [PDF]
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A. R. Gocke, P. D. Cravens, L.-H. Ben, R. Z. Hussain, S. C. Northrop, M. K. Racke, and A. E. Lovett-Racke T-bet Regulates the Fate of Th1 and Th17 Lymphocytes in Autoimmunity J. Immunol., February 1, 2007; 178(3): 1341 - 1348. [Abstract] [Full Text] [PDF]
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J. Xu and P. D. Drew Peroxisome Proliferator-Activated Receptor-{gamma} Agonists Suppress the Production of IL-12 Family Cytokines by Activated Glia J. Immunol., February 1, 2007; 178(3): 1904 - 1913. [Abstract] [Full Text] [PDF]
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 A. Yessoufou, A. Hichami, P. Besnard, K. Moutairou, and N. A. Khan Peroxisome Proliferator-Activated Receptor {alpha} Deficiency Increases the Risk of Maternal Abortion and Neonatal Mortality in Murine Pregnancy with or without Diabetes Mellitus: Modulation of T Cell Differentiation Endocrinology, September 1, 2006; 147(9): 4410 - 4418. [Abstract] [Full Text] [PDF]
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 S. Cuzzocrea, E. Mazzon, R. Di Paola, A. Peli, A. Bonato, D. Britti, T. Genovese, C. Muia, C. Crisafulli, and A. P. Caputi The role of the peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) in the regulation of acute inflammation J. Leukoc. Biol., May 1, 2006; 79(5): 999 - 1010. [Abstract] [Full Text] [PDF]
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 R Bordet, P Gele, P Duriez, and J-C Fruchart PPARs: a new target for neuroprotection. J. Neurol. Neurosurg. Psychiatry, March 1, 2006; 77(3): 285 - 287. [Full Text] [PDF]
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 M. K. Racke, A. R. Gocke, M. Muir, A. Diab, P. D. Drew, and A. E. Lovett-Racke Nuclear Receptors and Autoimmune Disease: The Potential of PPAR Agonists to Treat Multiple Sclerosis J. Nutr., March 1, 2006; 136(3): 700 - 703. [Abstract] [Full Text] [PDF]
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 B. Staels and J.-C. Fruchart Therapeutic Roles of Peroxisome Proliferator-Activated Receptor Agonists Diabetes, August 1, 2005; 54(8): 2460 - 2470. [Abstract] [Full Text] [PDF]
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 L. Steinman Immune Therapy for Autoimmune Diseases Science, July 9, 2004; 305(5681): 212 - 216. [Abstract] [Full Text] [PDF]
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