STAT5 tetramerization in autoimmune-mediated neuroinflammation

项目摘要/摘要 多发性硬化症(MS)是一种免疫介导的疾病，全球约有230万人受到影响- 很宽。MS是由不同免疫细胞类型之间的激活和复杂的相互作用引起的 导致中枢神经系统(CNS)发炎，导致脱髓鞘和轴突变性。 一种经过验证的多发性硬化症动物模型，实验性自身免疫性脑脊髓炎(EAE)，已经被 用于证明CD4+T细胞和单核细胞衍生细胞(MDCs)之间的同源相互作用， 在此定义了从单核细胞分化而来的树突状细胞和巨噬细胞的混合群体 自身免疫驱动的神经炎症是EAE发病所必需的。这个项目的总体目标是 建议是确定信号通路及其下游调节相互作用的效应介体 中枢神经系统中的CD4+T细胞和MDCs促进了EAE的发病。信号换能器和激活器 转录因子5A和5B(STAT5A和STAT5B)在下列细胞反应中起关键作用 刺激细胞因子、干扰素、生长因子。激活的STAT5蛋白可以形成二聚体和 四聚体。STAT5四聚体的生物学功能尚不完全清楚。使用Stat5a-Stat5b N域 双敲入(DKI)小鼠品系，其中不能形成STAT5四聚体，但形成STAT5二聚体 在未受影响的情况下，我们发现STAT5四聚体促进EAE的发病。DKI患者的轻度EAE表型 小鼠脊髓膜中CD4+T细胞与单核细胞/单核细胞间的相互作用减少 在EAE期间。我们进一步证明了细胞因子GM-CSF激活STAT5四聚体可以促进单核细胞 树突状细胞的分化及趋化因子CCL17的表达。与之对应的是， 在EAE期间，DKI小鼠脊髓中的CCL17显著减少。重要的是，接受STAT5的小鼠 经CCL17治疗的四聚体缺陷Th17细胞发生更严重的EAE，发病时间更早 与接受未经CCL17处理的细胞的小鼠相比。我们的中心假设是GM-CSF介导的 Stat5四聚化对促进致病Th17细胞和单核细胞/MDCs的相互作用至关重要 在脊髓脑膜中，通过CCL17依赖的机制，这些细胞相互作用是必需的 促进EAE发病机制的研究。我们将在三个具体目标上检验我们的假设。在目标1中，我们将利用 不同的EAE诱导策略以确定STAT5四聚体在CD4+T细胞和 单核细胞/巨噬细胞在EAE中的表达。在目标2中，我们将检验我们的假设，即STAT5四聚体信号促进 活体观察EAE过程中脊髓脑膜单核细胞分化及Th17细胞-MDC相互作用 成像技术。在目标3中，我们将探讨CCL17促进糖尿病发病的机制。 EAE。具体地说，我们将检验我们的假设，即CCL17增加Th17细胞上整合素LFA-1的亲和力， 从而促进Th17细胞与MDC的相互作用。以控制相互作用的途径和中介为目标 中枢神经系统内CD4+T细胞与MDCs之间的相互作用可能成为治疗MS的一种新策略。

Project Summary/Abstract Multiple sclerosis (MS) is an immune-mediated disease that impacts approximately 2.3 million people world- wide. MS is caused by the activation and the complex interactions between different immune cell types that cause inflammation in the central nervous system (CNS), which leads to demyelination and axonal degeneration. A validated animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), has been utilized to demonstrate that the cognate interactions between CD4+ T cells and monocyte-derived cells (MDCs), defined here a mixed population of dendritic cells and macrophages differentiated from monocytes during the autoimmune-driven neuroinflammation, are required for the pathogenesis of EAE. The overall goal of this proposal is to identify signaling pathways and their downstream effector mediators that regulate the interactions of CD4+ T cells and MDCs within CNS that promote the pathogenesis of EAE. Signal transducers and activators of transcription 5A and 5B (STAT5A and STAT5B) play a critical role in mediating cellular responses following stimulation of cytokines, interferons, growth factors. The activated STAT5 proteins can form dimers and tetramers. The biological functions of STAT5 tetramers are not fully understood. Using a Stat5a-Stat5b N-domain double knock-in (DKI) mouse strain, in which STAT5 tetramers cannot be formed but STAT5 dimers are unaffected, we found that STAT5 tetramers promote the pathogenesis of EAE. The mild EAE phenotype in DKI mice correlates with reduced interactions of CD4+ T cells and monocytes/MDCs in the spinal cord meninges during EAE. We further demonstrated that STAT5 tetramer activation by cytokine GM-CSF promotes monocyte differentiation into dendritic cells and the expression of chemokine CCL17. Correspondingly, the expression of CCL17 in the spinal cords is significantly reduced in DKI mice during EAE. Importantly, mice receiving STAT5 tetramer-deficient Th17 cells treated with CCL17 developed more severe EAE with earlier onset of the disease compared with mice receiving cells without CCL17 treatment. Our central hypothesis is that GM-CSF-mediated STAT5 tetramerization is critical for facilitating the interactions of pathogenic Th17 cells and monocytes/MDCs in the spinal cord meninges via a CCL17-dependent mechanism, and these cell interactions are required to promote the pathogenesis of EAE. We will test our hypothesis in three specific aims. In Aim 1, we will utilize different EAE induction strategies to determine the pathogenic role of STAT5 tetramers in CD4+ T cells and monocytes/MDCs during EAE. In Aim 2, we will test our hypothesis that STAT5 tetramer signaling promotes monocyte differentiation and Th17 cell-MDC interactions at the spinal cord meninges during EAE using intravital imaging technique. In Aim 3, we will investigate the mechanism by which CCL17 promotes the pathogenesis of EAE. Specifically, we will test our hypothesis that CCL17 increases the affinity of integrin LFA-1 on Th17 cells, thereby facilitates Th17 cell-MDC interactions. Targeting the pathways and mediators that control the interactions between CD4+ T cells and MDCs within CNS may become a novel strategy for the treatments of MS.