Blockade of cytosolic phospholipase A2α prevents experimental autoimmune encephalomyelitis and diminishes development of Th1 and Th17 responses

https://doi.org/10.1016/j.jneuroim.2008.08.012Get rights and content

Abstract

Cytosolic phospholipase A2α (cPLA2α) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to prostaglandins via the cyclooxygenase (COX) 1/2 pathways, and leukotrienes via the 5-lipoxygenase (LO) pathway. We utilized inhibitors of cPLA2α, COX-1/2 and 5-LO to determine the potential roles of these enzymes in development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Blocking cPLA2α prevented EAE development and greatly reduced antigen-induced production of Th1-type cytokines and IL-17. Blocking COX-1/2 delayed onset and reduced severity of EAE, and reduced production of Th1-type cytokines, but not IL-17. Blocking 5-LO delayed onset and reduced cumulative severity of EAE, but did not reduce production of Th1-type cytokines or IL-17. Finally, blockade of cPLA2α from the onset of clinical EAE reduced duration of EAE relapses. Therefore, cPLA2α represents a potential therapeutic target for treatment of MS.

Introduction

Multiple sclerosis (MS) is the most prevalent autoimmune disease of the central nervous system in people and a better understanding of its pathogenesis is needed in order to develop more effective therapies. Experimental autoimmune encephalomyelitis (EAE) is a commonly used animal model of MS (Zamvil and Steinman, 1990, Martin and McFarland, 1995, Steinman, 1999) and clinically different EAE develops in different mouse strains. To induce EAE, different susceptible mouse strains need to be immunized with different myelin-derived proteins or peptides. For example, chronic-progressive EAE develops in C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein (MOG)35–55 peptide, while a remitting–relapsing form of EAE develops in SJL mice immunized with proteolipid protein (PLP)139–151 peptide (Gold et al., 2006) and in NOD mice immunized with MOG35–55 (Maron et al., 1999). Agents efficacious in preventing EAE have had variable success in treatment of MS (Gold et al., 2006, Steinman and Zamvil, 2005, Steinman and Zamvil, 2006, Sriram and Steiner, 2005). However, the agents successful in treating MS, generally showed efficacy in multiple EAE models (Gold et al., 2006, Maron et al., 1999).

Both MS and EAE are believed to be Th1-induced autoimmune diseases because of the increased expression of Th1 cytokines in the affected CNS, and because injection of myelin specific Th1 but not Th2 cells into immunocompetent mice is sufficient to induce EAE (Steinman et al., 2002, Kuchroo et al., 1993, Kennedy et al., 1992, Baron et al., 1993, Miller and Karpus, 1994, Steinman, 1996, Lafaille et al., 1997). More recently, Th17 cells producing IL-17 have also been implicated in pathogenesis of EAE (Thakker et al., 2007, Langrish et al., 2005, Bettelli et al., 2007). In addition to Th1-type cytokines and IL-17, many other mediators of inflammation are increased during EAE and MS development, and it is likely that a subset of these is also critical in the pathogenesis of EAE and MS.

We have previously demonstrated that mice deficient in cytosolic phospholipase A2α (cPLA2α−/−), which are deficient in production of prostaglandins (PGs), leukotrienes (LTs) and platelet activating factor (PAF), are resistant to EAE induction (Marusic et al., 2005, Uozumi et al., 1997). However, it was possible that cPLA2α is essential for development of the immune system but not critical for mounting immune responses when adult animals are challenged. Multiple molecules, which play a role in mounting immune responses, also play roles in development of immune system. For example, lymphotoxin plays a critical role in generation of efficient humoral immune response as well as in development of lymph nodes, Peyer's patches and spleen architecture (Eugster et al., 1996, Tumanov et al., 2007). Therefore, studies using a selective inhibitor, with dosing limited to the duration of disease induction and development, complement findings from gene-deficient mice and provide further confirmation of the role of a specific gene product in disease development. Kalyvas and David (2004) have demonstrated that AACOCF3, which inactivates cPLA2α can reduce EAE severity in C57BL/6 mice. However, as was noted by the authors, AACOCF3 also inhibits calcium independent PLA2, thromboxane synthase, and fatty acid amide hydrolase, all of which may play a role in EAE development (Riendeau et al., 1994, Ghomashchi et al., 1999, Koutek et al., 1994, Croxford and Miller, 2003). It was therefore critical to test if a highly selective inhibitor of cPLA2α would prevent disease if administered during the induction phase of EAE. We have recently developed a highly specific cPLA2α inhibitor, WAY-196025, which is selective for cPLA2α over the closely related β and γ isoforms, and blocks production of PGs, LTs and PAF in whole blood assays (McKew et al., 2008). We have used WAY-196025 in the present study to block cPLA2α during the induction and development of EAE. To further characterize which mediators downstream of arachidonic acid (AA) may play a role in the pathogenesis of EAE, we used small molecule inhibitors of cyclooxygenase (COX) 1 and 2 and 5-lipoxygenase (LO).

cPLA2α selectively cleaves arachidonyl phospholipids to release free AA, which is converted primarily to PGs via the COX-1/2 pathway, and LTs [such as leukotriene B4 (LTB4) and cysteinyl leukotrienes (cysLTs)] via the 5-LO pathway. Concurrent with the generation of AA, cPLA2α also releases lysophosphatidylcholine, which serves as the precursor for PAF (Kudo and Murakami, 2002, Funk, 2001). All three classes of lipid mediators are increased in cerebrospinal fluid of MS patients (Dore-Duffy and Donovan, 1991, Neu et al., 2002, Callea et al., 1999) and there is evidence suggesting that they play a pathogenic role in EAE development (Marusic et al., 2005, Kihara et al., 2005, Gladue et al., 1996, Muthian et al., 2006, Ni et al., 2007). Thus, there is ample evidence to suggest blocking cPLA2α in these diseases would be beneficial because it would block production of these proinflammatory mediators. However, in addition to being converted into proinflammatory mediators, AA is also a precursor of anti-inflammatory lipid mediators such as lipoxins (LXs) and agonists of peroxisome proliferator-activated receptor γ (PPAR γ). These anti-inflammatory mediators have been shown to enhance resolution of inflammation and reduce severity of EAE (McMahon and Godson, 2004, Serhan, 2005, Huang et al., 1999) by negatively regulating both macrophage and T cell functions (Niino et al., 2001). In addition, conflicting data on the pathogenic roles of AA-derived lipid mediators in EAE have been obtained using small molecule inhibitors compared with genetically modified mice. For example, LTB4 appears to play a proinflammatory role in EAE because a LTB4 receptor antagonist reduced the severity of EAE (Gladue et al., 1996). However, 5-LO−/− mice, which are deficient in LTB4 production, developed more severe EAE than wild-type mice (Emerson and LeVine, 2004). Similarly, small molecule COX-2 inhibitors were reported to prevent development of EAE, but COX-2−/− mice developed EAE similar to wild-type controls (Muthian et al., 2006, Ni et al., 2007, Miyamoto et al., 2006).

In the present study, we demonstrate that blockade of cPLA2α from the time of immunization, using a highly specific small molecule inhibitor, WAY-196025 blocked EAE development, and blockade of cPLA2α starting at disease onset reduced the duration of EAE relapses. In addition, blockade of cPLA2α from the time of immunization led to a reduction in antigen-specific Th1-type cytokine and IL-17 production. Treatment of mice with a COX-1/2 inhibitor delayed onset and significantly reduced severity of EAE, accompanied by reduced production of Th1-type cytokines, but not IL-17. In contrast to an earlier report that 5-LO−/− mice develop more severe EAE than wild-type controls, we report here that treatment of mice with a 5-LO-inhibitor delayed onset of EAE and reduced cumulative severity of EAE, but did not decrease maximum disease severity. These results indicate that both 5-LO and COX-1/2 pathways play a pathophysiological role in EAE development and that cPLA2α, which provides the AA substrate for both pathways is a potential therapeutic target for treatment of MS.

Section snippets

Mice

Female C57BL/6, SJL and NOD mice were purchased from Jackson Laboratories or Taconic Farms and used at 8–10 weeks of age. All in vivo experiments were performed in accordance with Wyeth's Institutional Animal Care and Use Committee.

EAE induction and drug treatment

EAE was induced in C57BL/6 and NOD mice using immunization with MOG35–55. The mice were injected subcutaneously at two sites with a total of 200 μg of MOG35–55 emulsified in complete Freund's adjuvant (CFA) containing 6mg/mL killed M. tuberculosis. On the same day,

Treatment with cPLA2α inhibitor, WAY-196025 from the time of immunization prevents EAE development in C57BL/6 mice

We demonstrated previously that cPLA2α−/− mice are resistant to EAE (Marusic et al., 2005). To exclude the possibility that the effects observed in cPLA2α−/− mice were related to altered immune system development resulting from the absence of cPLA2α expression during embryonic, fetal and postnatal development, we tested the effects of cPLA2α-specific small molecule inhibitor, WAY-196025 (McKew et al., 2008) on EAE development in wild-type (WT) mice. We immunized C57BL/6 mice with MOG35–55 and

Discussion

Understanding the roles of proinflammatory mediators in the progress of EAE may help develop more effective therapies for MS. In the present study we showed that a small molecule inhibitor of cPLA2α, WAY-196025 prevented EAE development and diminished production of both Th1-type cytokines and IL-17 in C57BL/6 mice. Because AA, released by cPLA2α, is converted primarily to PGs via COX-1/2 and LTs via 5-LO pathways, we also evaluated the contribution of each of these classes of the AA-derived

Acknowledgments

Statistics on the microscopic changes were done by Dr. Youping Huang.

The research described in this publication was fully funded by Wyeth Research.

The authors declare no conflicting interest.

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