Regulation of inflammatory T Cells and Neuroprotection by Calpain Inhibitor in MS

描述(由申请人提供)： 多发性硬化症(MS)是一种与中枢神经系统神经变性相关的脱髓鞘疾病，被认为是由自身反应性T辅助细胞(Th)对髓鞘蛋白的攻击所致。复发缓解型多发性硬化症以急性发作为特征，发作间歇期部分或完全恢复。目前的研究结果表明，在复发期间，促炎症的Th1和Th17细胞占主导地位，而抗炎的Th2和T调节细胞(Tregs)推动缓解。了解这些变化的机制对于开发多发性硬化的治疗方法至关重要。此前，我们的数据显示，与缓解期相比，复发期多发性硬化症患者脑、脊髓和外周血单个核细胞(PBMC)中钙激活的蛋白水解酶Calain的表达和活性增加。Calain激活T细胞并参与Th1细胞因子的产生，而它的抑制则促进Th2细胞因子的上调。Calain还调节与Th谱(STATS、NFAT、NFkappaB)相关的信号蛋白的活性。我们的目标是确定多发性硬化症患者复发和缓解期间钙蛋白酶活性的变化是否通过转录因子的变化参与Th1/Th2的失调。初步数据发现，从RR-MS患者血液中分离出的PBMCs中IL-17水平升高，用CalPeptin处理这些细胞后，MBP激活的PBMCs中Th17细胞的数量减少，并抑制MBP特异性T细胞的增殖。抑制钙蛋白酶还可增加MS PBMCs中髓系抑制细胞(MDSC)的数量。有趣的是，特异性诱导Th2细胞因子基因表达的STAT6在MS患者活化的PBMCs中被降解，与Calain抑制剂孵育逆转了这种降解，这表明Calain抑制可能通过减少STAT6的降解来促进Th2谱。从患者分离的活化的PBMC的上清液与原代神经元培养物孵育可增加神经元死亡，这一作用可被Calain抑制剂治疗所阻断。基于这些数据，我们假设Calain抑制通过改变转录因子和MS中髓系来源的抑制细胞来调节Th细胞，并减少释放到Th1/Th17细胞上清液中的炎性介质的产生，从而导致细胞死亡。三个特定的目标将被用来检验这一假说：(1)检测Calain抑制对MS患者Th和Treg细胞因子/趋化因子谱的影响以及MDSCs的变化；(2)确定在Calain抑制后导致这些变化的信号机制；以及(3)确定由激活的Th细胞分泌的哪些细胞因子/趋化因子在体外负责细胞死亡或增加对神经元的保护，并检测Calain抑制对这些因子分泌的影响。这些研究对于确定钙蛋白酶抑制剂治疗多发性硬化的潜在机制至关重要。

DESCRIPTION (provided by applicant): Multiple sclerosis (MS), a demyelinating disease associated with neurodegeneration of the CNS, is thought to result from an attack on myelin proteins by autoreactive T helper (Th) cells. Relapsing-remitting (RR) MS is characterized by acute attacks with periods of partial or full recovery between episodes. Current findings suggest that pro-inflammatory Th1 and Th17 cells predominate during relapse and that anti-inflammatory Th2 and T regulatory cells (Tregs) drive remission. Understanding the mechanisms of these shifts is crucial to developing therapies for MS. Previously, our data showed increased expression and activity of the calcium- activated protease calpain in brain, spinal cord, and peripheral blood mononuclear cells (PBMCs) of MS patients during relapse compared to remission. Calpain activates T cells and is involved in the production of Th1 cytokines, while its inhibition promotes upregulation of Th2 cytokines. Calpain also modulates the activity of signaling proteins associated with Th profiles (STATs, NFAT, NFkappaB). Our goal is to determine whether alterations in calpain activity during relapse and remission in MS patients are involved in Th1/Th2 dysregulation via alterations in transcription factors. Preliminary data found increased IL-17 levels in PBMCs isolated from the blood of RR-MS patients, and treating these cells with calpeptin reduced the number of Th17 cells in MBP-activated PBMCs, and inhibited the proliferation of MBP-specific T cells as well. Calpain inhibition also increased the number of myeloid-derived suppressor cells (MDSC) in MS PBMCs. Interestingly, STAT6, which specifically induces Th2 cytokine gene expression, was degraded in activated PBMCs from MS patients, and incubation with calpain inhibitor reversed the degradation, suggesting that calpain inhibition may promote a Th2 profile by reducing STAT6 degradation. Incubating primary neuron cultures with supernatant from activated PBMCs isolated from patients increased neuronal death, which was blocked by calpain inhibitor treatment. Based on these data, we hypothesize that calpain inhibition dysregulates Th cells via alterations of transcription factors and myeloid-derived suppressor cells in MS, and reduces production of inflammatory mediators released into the supernatant of Th1/Th17 cells that induce cell death. Three specific aims will be used to examine the hypothesis: (1) examine the effects of calpain inhibition on Th and Treg cytokine/chemokine profiles and alteration of MDSCs in MS patients; (2) determine the signaling mechanisms responsible for these shifts following calpain inhibition; and (3) identify which cytokines/chemokines secreted by activated Th cells are responsible for cell death or increase protection of neurons in vitro and examine the effects of calpain inhibition on the secretion of these factors. These studies are crucial to identfy the mechanisms by which calpain inhibitor therapy may potentially treat MS.