Nuclear Receptors and Autoimmune Disease: The Potential of PPAR Agonists to Treat Multiple Sclerosis^,

Abstract

Experimental autoimmune encephalomyelitis (EAE) is a T-cell–mediated, autoimmune disorder characterized by central nervous system inflammation and demyelination, features reminiscent of the human disease, multiple sclerosis (MS). Prior work in the EAE model has suggested that encephalitogenic T cells are of the T helper (Th)-1 phenotype. Our group has performed several studies in the EAE model that suggest that a strategy for treating autoimmune disorders is to convert the pathogenic cells from the Th1 to Th2 phenotype. Peroxisome proliferator-activated receptors (PPARs) are members of a nuclear hormone receptor superfamily that include receptors for steroids, retinoids, and thyroid hormone, all of which are known to affect the immune response. Recently, we examined the role of PPARγ in EAE and observed that administration of the PPARγ agonist 15-deoxy-Δ^12,14 prostaglandin J2 exerted a significant therapeutic effect predominantly by inhibiting the activation and expansion of encephalitogenic T cells. One potential advantage in studying PPARα agonists is that they have been very well tolerated when used in humans to treat conditions such as elevated triglycerides. Building on prior work in immune deviation and with PPAR agonists, we have demonstrated that PPARα agonists can alter the cytokine phenotype of myelin-reactive T cells, alter their encephalitogenicity, and be useful in the treatment of EAE. The fact that PPARα agonists have been used as therapeutic agents in humans to treat metabolic conditions for over 25 years with little toxicity makes them attractive candidates for use as adjunctive therapies in MS.

Approximately 350,000 people in the United States have physician-diagnosed multiple sclerosis (MS)⁴ (1,2). Following trauma it is the next leading cause of neurologic disability in the United States in young adults, and most patients suffer from the effects of MS for most of their adult life. The cause of MS remains unknown, but because the disease involves perivascular mononuclear cell infiltrates and demyelination [features also characteristic of experimental autoimmune encephalomyelitis (EAE)], an autoimmune process is hypothesized to be involved in disease pathogenesis (3,4). There are now five drugs approved for use in the treatment of MS by the FDA, however, none of these agents are a cure for the disease, and the need for better treatment strategies for MS remains (5–8). In addition, with the withdrawal of drugs such as nataluzimab and its potential for infectious toxicity, it is clear that long-term safety is an important consideration for new MS therapies.

Relevance of EAE to multiple sclerosis

Several animal models have been used to study MS (3,4). Our laboratory has exclusively used the murine model of EAE for a number of reasons. Murine EAE results in a relapsing-remitting disease, similar to the early phase of disease for most MS patients. In chronic murine EAE, the pathology observed in the white matter shows demyelination that is reminiscent of the pathology seen in the central nervous system of humans with MS. With the advent of transgenic and homologous recombination technology, it is increasingly clear that many powerful molecular tools are becoming available for studying the immune response in pathologic processes such as EAE.

T cell phenotypes and autoimmune demyelination

Organ-specific autoimmune diseases such as EAE are mediated by IFN-γ–producing T helper (Th)-1 type cells (9). An abundance of data suggests that inflammatory immune responses or delayed type hypersensitivity responses are primarily mediated by these Th1 cells, which produce cytokines such as lymphotoxin and IFN-γ, but little IL-4 (10). In contrast, CD4+ Th2 populations produce large amounts of IL-4 and IL-5, mediate immune responses characterized by high levels of noncomplement binding IgG, IgE, and eosinophil-mediated cytotoxicity, but produce no organ-specific tissue destruction in normal mice. The production by Th2 cells of macrophage-deactivating cytokines, such as IL-4, IL-10, and IL-13, provides a strong foundation for their anti-inflammatory effects in vivo (11). On the other hand, it is very clear that cytokines such as IL-12 and type-I IFN can activate a transcription factor called signal transducing activator of transcription (STAT)-4 in human T cells and instruct the differentiation of Th1 cells (12). STAT4 is recruited to the human IFN-α or -β receptor through an interaction with the C-terminus of human STAT2, and this portion of STAT2 is not conserved between mice and humans (13). Thus, the most commonly used therapy for MS may be able to potentiate the development of autoreactive T cells.

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 (3,14,15). The ability of Th2 cells to regulate an already initiated immune response in EAE is less clear (16). Interestingly, it appears that proteolipid protein-specific T-cell clones isolated from MS patients have different cytokine phenotypes depending on when they are isolated (17). T-cell clones derived during acute attacks were more likely to be of a Th1 phenotype, whereas during remission, these cytokine responses were much more variable but included T-cell clones of a Th2 phenotype.

In the Th1 differentiation pathway, both IL-12 and IFN-γ are important in the differentiation of naïve T cells (see Fig. 1). IFN-γ is necessary for translocation of the transcription factor STAT1 to the nucleus. T box expressed in T cells (T-bet), which has been proposed to be the master regulator of Th1 differentiation, is a strong transactivator of the IFN-γ gene (18,19). IL-12, which is produced by antigen-presenting cells, is necessary for STAT4 activation and full activation of the IFN-γ gene (20). In addition to IL-12, it is now clear there is a family of heterodimeric cytokines that may play a role in the regulation of T-cell responses (21). IL-12p40 associates not only with IL-12p35, but also with a p19 chain to form IL-23. An essential role is suggested for IL-23 because mice deficient in IL-12p40 could not develop EAE, whereas mice deficient in IL-12p35 developed more severe disease (22). IL-23p19–deficient mice are resistant to EAE development, but can develop a normal Th1 response (23), thus the role of cytokines in producing encephalitogenic T cells may be more complicated than previously appreciated.

FIGURE 1.

Transcriptional regulation of the differentiation of Th1 cells. IL-12 activates the STAT4 pathway while IFN-γ activates the STAT1 or T-bet pathway. It is unclear where PPARs regulate these pathways at a molecular level.

Our own work suggests that T-bet and STAT1 regulate each other in a feedback loop (24). Our studies suggest peroxisome proliferator-activated receptor-α (PPAR)α agonists affect the differentiation of myelin basic protein (MBP) Ac1-11–specific T cells into a Th2 phenotype, although the molecular mechanisms for this effect are still unclear (14). In the differentiation of Th2 cells, IL-4 is the critical cytokine that leads to activation and translocation of STAT6 to the nucleus and subsequent expression of GATA binding protein-3 (GATA-3) (see Fig. 2). GATA-3 is felt to be the master regulator of Th2 cell differentiation (25). Future studies will need to examine whether the increased IL-4 production we have observed in MBP Ac1-11–specific T cells due to PPARα agonists is the result of activating transcription factors of the Th2 differentiation pathway.

FIGURE 2.

Transcriptional regulation of the differentiation of Th2 cells. IL-4 turns on the STAT6 pathway and GATA-3. It is unclear where PPARs such as PPARα turns on Th2 differentiation.

PPARγ: Role in inflammatory diseases

PPARs are members of the nuclear hormone receptor superfamily that also include steroid, retinoid, and thyroid hormone receptors. PPARs have been studied most extensively in the regulation of genes involved in glucose and lipid metabolism (26). Three PPAR subtypes exist (α, δ, and γ) and they exhibit different tissue distribution as well as different ligand specificities. In addition to adipocytes, it was recently shown that cells of the monocyte and/or macrophage lineage express PPARγ and PPARα, suggesting a possible role for these receptors in the function of these cells. Subsequently, PPARγ ligands, which include the naturally occurring prostaglandin metabolite 15-deoxy-Δ^12,14 prostaglandin J2 as well as thiazolidinediones, have been shown to have anti-inflammatory activity (27). 15-Deoxy-Δ^12,14 prostaglandin J2 was shown to inhibit inducible nitric oxide synthase, matrix metalloproteinase-9, IL-1β, IL-6, and tumor necrosis factor (TNF)-α production by monocytes and/or macrophages (28,29). Both PPARγ and PPARα have been shown to be expressed on T cells and their ligands inhibit IL-2 secretion and T-cell proliferation (30,31). PPARγ ligands likely block IL-2 production by a mechanism that inhibits the nuclear factor of activated T cells that bind to the IL-2 promoter (32). PPARγ ligands can also affect T-cell function indirectly by inhibiting endothelial-cell production of chemokines (33). The evidence that PPAR agonists can regulate inflammation is supported by several animal studies. These include suppression of adjuvant arthritis in rats (34), inhibition of atherosclerosis in mice (35), inhibition of the inflammatory response and stroke size following cerebrovascular occlusion in rats, and amelioration of inflammatory bowel disease in mice (36). As mentioned previously, our studies and those of others show that PPARγ agonists can inhibit the clinical signs of EAE (27,37–39).

PPARα: Role in inflammatory diseases

PPARα ligands have also been shown to regulate inflammatory responses, although there is clearly less evidence along this line compared with PPARγ agonists. PPARα-deficient mice have abnormally prolonged responses to inflammatory stimuli such as arachidonic acid and leukotrienes (40). The expression of IL-6, the vascular cell adhesion molecule, and cyclooxygenase-2 in response to cytokine activation can be inhibited by PPARα ligands (41). PPARα ligands were also shown to decrease nuclear factor-κB (NF-κB) activation, IL-12, and IL-6 production in aged mice (42). PPARα ligands may inhibit the functional expression of NF-κB, in part by augmenting the expression of inhibitor of NF-κB (IκBα) (43). Recently, the PPARα ligand WY14,643 was shown to inhibit IgG responses in myelin oligodendrocyte glycoprotein 35–55/complete Freund’s adjuvant FA-immunized mice (44). When mice were fed this agonist, splenocytes demonstrated impaired production of IFN-γ, IL-6, and TNFα. Interestingly, the authors had hoped to examine the effect of fibrates on EAE; however, the combination of immunization with myelin oligodendrocyte glycoprotein 35–55/complete Freund’s adjuvant, pertussis toxin and WY14,643 treatment consistently induced mortality 5–10 d after immunization. Several PPARα agonists have been shown to potentiate TNFα secretion in response to endotoxins; however, this was observed in mice and rats, and not in guinea pigs, monkeys, or humans (45,46).

Regulation of cytokine expression by PPAR

Recently, several studies have examined the effect of PPARs to mediate anti-inflammatory activity. The majority of these studies have focused on PPARγ and have examined their effects on cells of the macrophage and/or monocyte lineage (47). As noted above, the majority of these studies have shown that PPARγ agonists inhibit the expression of inflammatory mediators such as inducible nitric oxide synthase, presumably by antagonizing activation of transcription factors such as NF-κB. PPARα is expressed in human monocytes that differentiate into macrophages, and PPARα agonists have been shown to induce apoptosis in macrophages (48).

Our work and the work of others demonstate that PPARγ ligands can inhibit T-cell proliferation or the production of IL-2 and IFN-γ by T cells stimulated with antigen (27,30,31). Studies that compare the ability of PPARα ligands with those of PPARγ ligands suggest that the effect on the inhibition of T-cell proliferation and IFN-γ secretion is more marked with PPARγ agonists (30). Some fibrates, such as gemfibrozil and ciprofibrate, are able to induce IL-4 secretion by splenocytes stimulated with concanavalin A (Con A). However, another PPARα agonist, GW7647, did not increase IL-4 secretion.

Recently, a study examining the regulation of T-bet by PPARα concluded that it is unliganded PPARα that suppresses T-bet expression and subsequent IFN-γ expression in T cells (49). In that study, CD4+ T cells deficient in PPARα showed that unliganded PPARα was important in regulating the phosphorylation of p38 mitogen-activated protein kinase and T-bet expression. PPARα agonists resulted in suppressed p38 mitogen-activated protein kinase phosphorylation and promoted T-bet expression. Studies by our group are examining the effects of PPARα activation on T cell differentiation and determining the effects on the T-bet or STAT1 pathway as well as on Th2 differentiation pathways.

PPAR activators in human T lymphocytes

Recent work has shown that both PPARα and PPARγ are expressed by human CD4+ T cells at both the mRNA and protein levels (31). Activation of human CD4+ T cells with anti-CD3 increased IFN-γ production, but this was dramatically inhibited in the presence of either PPARα or PPARγ agonists. The PPARα agonist WY14643 was also quite effective in inhibiting TNFα production and IL-2 secretion. It should be noted that these studies examined the effect of the PPARα agonists at clinically relevant concentrations and did not observe any evidence of effects on T-cell viability. However, these studies did not examine the effect of PPARα agonists on Th2 cytokines such as IL-4. Our studies have examined the effect of PPARα agonists gemfibrozil and fenofibrate on promoting Th2 cytokine production by human myelin-specific T cells (14). Human MBP-specific T-cell lines secreted increased IL-4 when generated in the presence of gemfibrozil (Fig. 3).

FIGURE 3.

Gemfibrozil increases IL-4 secretion by a human T-cell line. The MBP-specific T-cell line was generated by weekly stimulation with antigen in the presence or absence of gemfibrozil (100 μM). IL-4 was measured at 48 h time point by ELISA. Means of quadruplicate cultures are shown. SD were <10% of the mean.

Relevance to multiple sclerosis

Although there are presently several medications approved by the FDA for the treatment of MS, none of them are a cure. Similar to the situation that has occurred in oncology, it is likely that improved treatment protocols will eventually arise from combinations of therapies targeting different aspects of the disease process. Future studies will determine whether PPARα agonists, which are presently being used to treat humans, can also be beneficial in autoimmune demyelination. As several groups continue to gather data on various PPAR agonists, it is expected that there will be an attempt to launch an investigator-initiated trial to test one or several of these compounds in patients with MS.