Immunomodulation and Neuroprotection in Multiple Sclerosis

多发性硬化症(MS)，一种致残的自身免疫性疾病，影响着全球近250万人 在世界上，这种疾病影响到个人最具生产力的年龄段，并在美国退伍军人中流行。是由MS引起的 通过髓鞘特异性自身反应性淋巴细胞的异常激活及其中枢神经系统在血液中的渗透- 脑屏障(BBB)。目前FDA批准的针对免疫系统的多发性硬化症药物疗效有限 随着疾病的继续发展。为了改进多发性硬化症的治疗，这项提案的目标是评估 一氧化氮合酶和一氧化氮代谢产物作为潜在治疗靶点的可行性(S) 对于免疫和血脑屏障的发病机制，MS的两个关键疾病机制。 我们实验室和其他实验室最近的研究证明了一氧化氮合酶和一氧化氮代谢产物的作用(S- 亚硝基谷胱甘肽；GSNO与过氧亚硝酸根；ONOO)在实验动物生理和病理调节中的作用 自身免疫性脑脊髓炎(EAE；多发性硬化症模型)。不对称二甲基精氨酸(ADMA)，一种细胞 L的代谢产物-精氨酸，将一氧化氮合酶的活性从一氧化氮的产生转移到超氧阴离子(O2·)的产生，从而导致 以增加有毒的ONOO形成。因此，EAE和MS中ADMA水平的升高可能会发生变化 一氧化氮合酶活性的增加导致ONOO的产生，从而导致相关的严重病理。 ADMA在多发性硬化症中的病理重要性首先被报道的血液中ADMA的升高所认识 多发性硬化患者。接下来，我们的研究记录了EAE患者血液中ADMA的升高。我们进一步观察到，治疗 外源性ADMA可通过增加TH1/TH17介导的促肾上腺皮质激素释放激素而加重EAE动物的病情 炎性免疫反应。此外，ADMA还可诱导脑微血管中ONOO的合成 内皮细胞和引起的BBB功能障碍/破坏的中枢神经系统的免疫细胞即使在 没有百日咳毒素。这些发现第一次证明了ADMA中介的参与 EAE免疫发病机制及血脑屏障功能障碍。我们的实验室之前报告了 NO及其生理载体分子GSNO在自身免疫反应中起关键作用 EAE患者内皮屏障完整性的维持。这些观察结果使我们假设地球的高度 ADMA由于其代谢缺陷，在EAE过程中导致一氧化氮合酶功能障碍，导致 抑制NO/GSNO介导的抗炎和血管保护活性，同时诱导O2·‘’/ONOO- 在EAE中介导的促炎和血管破坏活性。因此，ADMA介导的机制有 为MS/EAE的免疫和血脑屏障发病机制提供新的和潜在的治疗靶点。基于这一假设， 建议的研究是调查功能障碍的ADMA和NO之间的时间关系 (ONOO与GSNO)代谢和免疫学、血管、神经和临床病理学 EAE(AIM 1)，DDAH-1/2损害EAE ADMA分解代谢的机制(AIM 2) 评估针对EAE中ADMA和NO代谢的潜在治疗方法(AIM 3)。 建议的研究是基于我们最初关于ADMA在NOS/NO-1中的病理意义的发现。 EAE介导的免疫和血管疾病机制。这些研究的结果有望提供 对疾病机制的新见解，并确定治疗MS的新的潜在靶点和治疗方法。 这项研究的翻译潜力很高，因为GSNO介导的机制针对的是免疫调节 (Th1/TH17&lt；TH2/Treg)，而不是全球免疫抑制，以及EAE的血脑屏障病理。

Multiple sclerosis (MS), a disabling autoimmune disease affecting almost 2.5 million people around the world, affects individuals in their most productive years and is prevalent among US Veterans. MS is caused by abnormal activation of myelin-specific autoreactive lymphocytes and their CNS infiltration across the blood- brain barrier (BBB). Present day FDA approved MS drugs, targeting immune system, have limited efficacies as disease progression continues. In an effort to improve MS therapy, the goal of this proposal is to evaluate the feasibility of nitric oxide synthase (NOS) and nitric oxide (NO) metabolites as potential therapeutic target(s) for immune as well as BBB pathogenesis, two critical disease mechanisms of MS. Recent studies from our laboratory and others have documented the roles of NOS and NO metabolites (S- nitrosoglutathione; GSNO vs. peroxynitrite; ONOO¯) in physiological vs. pathological regulation of experimental autoimmune encephalomyelitis (EAE; a model for MS). Asymmetric dimethylarginine (ADMA), a cellular metabolite of L-arginine, shifts the NOS activity from NO production to superoxide (O2·¯) production, thus leading to increased toxic ONOO¯ formation. Therefore, elevation of ADMA levels in EAE and MS could potentially shift the NOS activity for increased production of ONOO¯ and thus cause related severe pathologies. The pathological importance of ADMA in MS was recognized first by the reported elevation of blood ADMA in MS patients. Next, our studies document the elevated blood ADMA in EAE. We further observed that treatment of EAE animals with exogenous ADMA aggravates the disease severity by increasing TH1/TH17 mediated pro- inflammatory immune responses. In addition, ADMA induced ONOO¯ synthesis in the brain microvascular endothelial cells and caused BBB dysfunction/disruption for CNS infiltration of immunocytes even in the absence of pertussis toxin. These findings, for the first time, document the participation of ADMA-mediated mechanisms in immune pathogenesis as well as BBB dysfunction in EAE. Our laboratory previously reported that NO and its physiological carrier molecule GSNO play critical roles in autoimmune responses as well as in maintenance of endothelial barrier integrity in EAE. These observations lead us to hypothesize that elevation of ADMA as a result of its defective metabolism, during the course of EAE, induces NOS dysfunction leading to inhibition of NO/GSNO-mediated anti-inflammatory and vaso-protective activity, while inducing the O2·¯/ONOO¯- mediated pro-inflammatory and vaso-disruptive activity in EAE. Therefore, ADMA-mediated mechanisms are novel and a potential therapeutic target for immune and BBB pathogenesis of MS/EAE. Based on this hypothesis, the proposed studies are to investigate the temporal relationship between dysfunctional ADMA and NO (ONOO¯ vs. GSNO) metabolism and immunological, vascular, neurological, and clinical pathologies of EAE (AIM 1), mechanism underlying impaired ADMA catabolism by DDAH-1/2 in EAE (AIM 2) and to evaluate potential therapeutics targeting ADMA and NO metabolism in EAE (AIM 3). The proposed studies are based on our original findings of pathological significance of ADMA in NOS/NO- mediated immune and vascular disease mechanisms of EAE. Results from these studies are expected to provide novel insights into disease mechanisms and to identify novel potential targets and therapeutics for MS. Translational potential of this study is high as GSNO mediated mechanisms target immune modulation (TH1/TH17 < TH2/Treg), rather than global immune suppression, as well as BBB pathologies of EAE.