Immunomodulation and Neuroprotection in Multiple Sclerosis

 描述（由申请人提供）： 多发性硬化症（MS）及其动物模型（实验性自身免疫性脑脊髓炎; EAE）是CNS的自身免疫性疾病，并且被认为是由髓鞘反应性T辅助细胞（TH 1、TH 2、Treg和TH 17）介导的。本研究的目的是探讨S-亚硝基谷胱甘肽（GSNO）在EAE疾病模型中的免疫调节和神经保护/神经修复机制，其基础是我们的新发现：1）GSNO在PLP 139 -151免疫的SJL小鼠、MOG 35 -55免疫的C57 BL/6小鼠和MBP免疫的刘易斯大鼠三种不同的EAE疾病模型中减轻疾病;（2）GSNO抑制TH 17极化/效应子功能，诱导Treg极化/效应子功能，对TH 1和TH 2无明显影响; 3）GSN 0介导的STAT 3的翻译后修饰（Cys 259处的S-亚硝基化）抑制STAT 3活化，所述STAT 3活化在TH 17/Treg极化/效应器功能和EAE/MS的疾病进展中起关键作用; 4）GSNO抑制NF κ B活性和iNOS表达，NF κ B活性和iNOS表达在炎症条件下CNS的氧化/亚硝化损伤中起关键作用; 5）GSNO诱导星形胶质细胞表达在神经修复过程中起关键作用的神经营养因子。 基于这些新的发现，我们假设GSNO介导的机制通过S-亚硝基化和抑制STAT 3来减轻EAE/MS疾病，并且还通过减弱NF κ B/iNOS介导的亚硝化/氧化应激来促进EAE中的神经保护和神经修复过程，同时通过补充GSNO仍然支持细胞对GSNO/NO的需求。所提出的研究将通过研究GSNO在TH 17/Treg极化和神经保护/神经修复过程中的作用以及在EAE疾病过程中的作用来测试该假设，所述EAE疾病过程使用遗传方法（iNOS敲除和GSNO还原酶敲除小鼠）以及药理学方法，通过使用GSNO还原酶抑制剂（N6022）来增加内源性GSNO和通过补充外源性GSNO。GSNO的细胞内稳态通过其由GSH和NO的基于氧化还原的合成以及其主要通过GSNO还原酶（GSNOR）的酶促失活/降解来维持。拟议的研究包括：具体目标1：评价外源性与内源性GSNO对活动性EAE疾病模型中Treg/TH 17特异性免疫调节的功效。具体目标二：评价外源性与内源性GSNO对过继转移被动EAE疾病模型中Treg/TH 17特异性免疫调节的功效。具体目的3：评估GSNO对EAE疾病模型中的神经保护和神经修复的功效。该研究的新奇是为TH 17介导的免疫应答和神经保护（保护血脑屏障和CNS）和促进神经修复提供新的治疗靶点，而不影响由其他TH细胞（例如TH 1和TH 2）介导的天然免疫监视机制。我们期望对EAE疾病中基于S-亚硝基化的TH 17/Treg极化/效应器功能和神经保护/神经修复的调节机制的研究不仅将描绘自身免疫性疾病中NO/GSNO介导的机制，而且其作为单独或与批准的MS药物组合的新型基于NO的治疗剂的临床相关性。GSNO是人体的天然组分没有任何副作用因此，GSNO本身或与目前的药物组合作为MS的转化药物的潜在用途是有希望的，并且与退伍军人的健康和VA使命高度相关。

 DESCRIPTION (provided by applicant): Multiple Sclerosis (MS) and its animal model (experimental autoimmune encephalomyelitis; EAE) are autoimmune disorders of the CNS, and are considered to be mediated by myelin reactive T helper cells (TH1, TH2, Treg and TH17). The present proposal is to investigate S-nitrosoglutathione (GSNO)-mediated immuno- modulation and neuroprotection/neurorepair mechanisms in EAE disease models based on our novel finding that: 1) GSNO attenuated disease in three different EAE disease models; SJL mice immunized with PLP139-151, C57BL/6 mice immunized with MOG35-55, and Lewis rats immunized with MBP; 2) GSNO inhibited TH17 polarization/effector functions and induced Treg polarization/effector functions without obvious effects on TH1 and TH2; 3) GSNO-mediated post-translational modification of STAT3 (S-nitrosylation at Cys259) inhibited STAT3 activation which plays a critical role in TH17/Treg polarization/effector functions and disease progression of EAE/MS; 4) GSNO inhibited NFkappaB activity and iNOS expression which play a critical role in oxidative/nitrosative damage of the CNS under inflammatory conditions;.5) GSNO induced astroglial expression of neurotrophic factors that play a critical role in the neurorepair process. Based on these novel findings, we hypothesize that GSNO-mediated mechanisms attenuate the EAE/MS disease by S-nitrosylation and inhibition of STAT3 and also promote neuroprotection and neurorepair processes in EAE by attenuation of NFkappaB/iNOS-mediated nitrosative/oxidative stress while still supporting cellular requirements for GSNO/NO with supplementation of GSNO. The proposed studies will test the hypothesis by investigating the role of GSNO in TH17/Treg polarization and neuroprotection/neurorepair processes and in EAE disease processes using genetic (iNOS knockout and GSNO reductase knock mice) as well as pharmacological approaches by using inhibitor of GSNO reductase (N6022) to increase endogenous GSNO and by supplementation of exogenous GSNO. Cellular homeostasis of GSNO is maintained by its redox based synthesis from GSH and NO and its enzymatic inactivation/degradation primarily by GSNO reductase (GSNOR). The proposed studies are: Specific Aim 1: To evaluate the efficacy of exogenous vs. endogenous GSNO on Treg/TH17 specific immunomodulation in an active EAE disease model. Specific Aim 2: To evaluate the efficacy of exogenous vs. endogenous GSNO on Treg/TH17 specific immunomodulation in the adoptive transfer passive EAE disease model. Specific Aim 3: To evaluate the efficacy of GSNO on neuroprotection and neurorepair in the EAE disease model. The novelty of the study is the promise of new therapeutic targets for TH17-mediated immune response and neuroprotection (protection of blood brain barrier and CNS) and promotion of neurorepair without affecting natural immuno-surveillance mechanisms mediated by other TH cells (e.g. TH1 and TH2). We expect that study of the S-nitrosylation-based regulatory mechanisms of TH17/Treg polarization/effector functions and neuroprotection/neurorepair in EAE disease will not only delineate the NO/GSNO mediated mechanisms in autoimmune disease, but also its clinical relevance as novel NO based therapeutics either alone or in combination with approved drug for MS. GSNO is a natural component of the human body and its exogenous administration in humans was not associated with any adverse effects. Therefore, potential use of GSNO by itself or in combination with present day medications as a translational drug for MS is promising and highly relevant to Veterans' health and the VA mission.