Immune Regulation of Neuronal Injury and Repair

描述(申请人提供)：我们已经建立了涉及切断轴突的野生型(WT)小鼠面部运动神经元(FMN)的CD4+T细胞介导的神经保护的生理相关机制，该机制可能对治疗肌萎缩侧索硬化症(ALS)具有重要意义，ALS是一种致命的运动神经元疾病。在ALS转基因小鼠模型(SOD1G93A)上叠加面神经切断(FNA)在症状前阶段表明SOD1小鼠在FMN丢失增加和功能恢复降低方面表现得像免疫缺陷(ID)小鼠，其机制矛盾的是，这种机制并不是运动神经元本身固有的，而是涉及外周免疫的缺陷：中枢神经胶质细胞的相互作用。该项目的长期目标是利用我们的WT小鼠FNA后免疫介导的神经保护模型作为模板，以阐明外周免疫系统故障如何导致ALS SOD1小鼠模型中运动神经元的丢失。我们的中心假设是，CD4+效应T细胞通过调节中枢小胶质细胞和星形胶质细胞对周围神经损伤的反应来为运动神经元提供神经保护。该研究有4个特定目的旨在：1)阐明IL-10在FNA后WT面核中的功能作用；2)确定CD4+T细胞是否通过激活面核内IL-10级联通路，使FNA诱导的RAG-2 KO小鼠FMN免于细胞死亡；3)确定阻止无症状SOD1小鼠FNA后CD4+T细胞介导的神经保护的机制；以及4)测试体外产生的具有神经保护作用的SOD1 CD4+T细胞改变SOD1小鼠疾病发生和/或进展的能力。将使用IL-10报告基因和条件性IL-10基因敲除小鼠，以及WT、ID和SOD1小鼠的过继转移实验，以及激光捕获显微切割。根据这次修订产生的新数据，SOD1T细胞是正常的，但抗原提呈细胞(APC)是缺陷的，SOD1T细胞和APC将在体外使用相关和无关的抗原进行表型和功能检测，并在体内使用过继转移进行检测。最后，将在疾病发生前对SOD1小鼠进行神经保护性FNA特异性T细胞或APC过继免疫治疗，并评估对疾病发生、进展和寿命的影响。就预期结果而言，这项工作有望确定FNA后CD4+T细胞介导的神经保护的关键机制元素，揭示FNA后ALS SOD1小鼠模型中阻止有效CD4+T细胞激活/分化的外周免疫系统缺陷，并确定FNA抗原体外产生的神经保护性SOD1T细胞过继免疫治疗的治疗潜力。每年有30,000名美国人罹患肌萎缩侧索硬化症(ALS)(每天15例)，通常是在他们生活中的生产高峰期，受影响的个人、他们的家庭和医疗保健系统付出了巨大的代价。识别神经缺陷：免疫相互作用将产生重要的积极影响，因为它们可以成为ALS治疗中治疗干预的目标。

DESCRIPTION (provided by applicant): We have established a physiologically relevant mechanism of CD4+ T cell-mediated neuroprotection involving axotomized wildtype (WT) mouse facial motoneurons (FMN) that may have significance in the treatment of amyotrophic lateral sclerosis (ALS), a fatal motoneuron disease. Superimposition of facial nerve axotomy (FNA) on the transgenic mouse model of ALS (SOD1G93A) during pre-symptomatic stages indicates that SOD1 mice behave like immunodeficient (ID) mice in terms of increased FMN loss and decreased functional recovery, through a mechanism that, paradoxically, is not inherent within the motoneuron itself, but, instead, involves a defect in peripheral immune: CNS glial cell interactions. The long-term goal of this project is to utilize our WT mouse model of immune-mediated neuroprotection after FNA as a template to elucidate how a malfunctioning peripheral immune system contributes to motoneuron cell loss in the SOD1 mouse model of ALS. Our central hypothesis is that CD4+ effector T cells provide neuroprotection to motoneurons through regulation of the central microglial and astrocytic response to peripheral nerve damage. There are 4 specific aims designed to: 1) elucidate the functional role of IL-10 in the WT facial nucleus following FNA, 2) determine if CD4+ T cells rescue FMN from FNA-induced cell death in RAG-2 KO mice through activation of the IL-10 cascade within the facial nucleus, 3) determine the mechanism that prevents CD4+T cell-mediated neuroprotection following FNA in the pre-symptomatic SOD1 mouse, and 4) test the ability of in vitro-generated, neuroprotective SOD1 CD4+ T cells to alter disease onset and/or progression in SOD1 mice. IL-10 reporter and conditional IL-10 knockout mice, along with adoptive transfer experiments with WT, ID and SOD1 mice, and laser capture microdissection will be used. Based on new data generated for this revision suggesting that SOD1 T cells are normal, but antigen-presenting cells (APC) are defective, SOD1 T cells and APC will be examined phenotypically and functionally both in vitro using relevant and irrelevant antigens and in vivo using adoptive transfer. Finally, adoptive immunotherapy with either neuroprotective FNA-specific T cells or APC into SOD1 mice will be done prior to disease onset, and the effects on disease onset, progression, and lifespan assessed. With respect to expected outcomes, the work is expected to identify key mechanistic elements in CD4+ T-mediated neuroprotection following FNA, reveal peripheral immune system defects following FNA in the SOD1 mouse model of ALS that prevent effective CD4+ T cell activation/differentiation and determine the therapeutic potential of adoptive immunotherapy with neuroprotective SOD1 T cells generated in vitro by FNA antigens. ALS strikes 30,000 Americans each year (15 cases a day), often at peak productive points in their lives and with enormous cost to affected individuals, their families, and the health care system. Identification of defects in neural:immune interactions will have an important positive impact because they can then be the target of therapeutic intervention in ALS treatment.