A Macrophage Cation Channel in Prevention and Recovery from Inflammatory Injury

描述（由申请人提供）： 现有的自身免疫性和炎症性疾病的治疗方法对慢性损伤的消退和恢复影响甚微。在理解巨噬细胞如何调节细胞表型以介导损伤或修复方面也存在根本差距。长期目标是开发预防和逆转慢性炎性损伤的治疗方法。 使用细胞，基因组，蛋白质组学和体内方法的组合，一种新的剪接变体的人钠通道基因，SCN 5A，已被确定为抗炎巨噬细胞信号传导的中央调节器。这种剪接变体编码一种新的内体阳离子通道，其将细胞内钙流与下游信号传导和基因转录偶联。由于这种剪接变体在小鼠中不表达，因此开发了转基因敲入模型C57BL6cfms-hSCN 5A小鼠以研究其在巨噬细胞中的体内功能。从这些小鼠转移骨髓衍生的巨噬细胞介导多发性硬化（MS）的实验性自身免疫性脑脊髓炎（EAE）模型的临床恢复。本研究的目的是表征巨噬细胞中的SCN 5A信号网络，并确定它如何调节慢性炎症性疾病EAE模型的临床恢复。 核心假设是巨噬细胞中SCN 5A的持续表达启动并维持了增强炎症损伤恢复的细胞表型。这项研究的基本原理是，了解这种机制将导致创新的方法来预防和治疗慢性炎症性疾病。该假设将在两个特定的目标进行测试：1。确定SCN 5A变体阳离子通道如何调节巨噬细胞表型。2.分析EAE期间SCN 5A+巨噬细胞如何介导恢复。 基于初步数据，工作模型是内体通道活性介导决定和维持细胞表型的短期生化信号传导和长期基因表达模式。在目标1中，将检查细胞溶质钙、环AMP和激活转录因子2（ATF 2）通路的通道依赖性调节。实验方法将包括现有的生物化学，成像和分子技术在小鼠和人的原代巨噬细胞的组合。在目的2中，EAE模型将用于评估体内该途径并确定SCN 5A+巨噬细胞如何介导从炎性损伤中恢复。将分析体内基因和蛋白质表达，并与SCN 5A调节巨噬细胞囊泡分泌和细胞间通讯的体外评估相关。 这项工作是创新的，因为它代表了一种调节巨噬细胞功能和治疗慢性炎症性疾病的新方法。这是重要的，因为促进MS和其他慢性炎性疾病的恢复代表了未满足的治疗需求。

DESCRIPTION (provided by applicant): Existing therapies for autoimmune and inflammatory diseases have minimal effects on resolution and recovery from chronic injury. There is also a fundamental gap in understanding how macrophages regulate cellular phenotype to mediate either injury or repair. The long-term goal is to develop treatments that prevent and reverse chronic inflammatory injury. Using a combination of cellular, genomic, proteomic and in vivo approaches, a novel splice variant of the human sodium channel gene, SCN5A, has been identified as a central regulator of anti-inflammatory macrophage signaling. This splice variant encodes a novel, endosomal cation channel that couples intracellular calcium flux to downstream signaling and gene transcription. Because this splice variant is not expressed in mice, a transgenic knock-in model, the C57BL6cfms-hSCN5A mouse, was developed to study its in vivo function in macrophages. Transfer of bone marrow derived macrophages from these mice mediates clinical recovery in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). The objective of this study is to characterize the SCN5A signaling network in macrophages and determine how it regulates clinical recovery in the EAE model of chronic inflammatory disease. The central hypothesis is that persistent expression of SCN5A in macrophages initiates and maintains a cellular phenotype that enhances recovery from inflammatory injury. The rationale for this study is that understanding this mechanism will lead to innovative approaches to prevent and treat chronic inflammatory disease. The hypothesis will be tested in two specific aims: 1. Determine how the SCN5A variant cation channel regulates macrophage phenotype. 2. Analyze how SCN5A+ macrophages mediate recovery during EAE. Based on preliminary data, the working model is that endosomal channel activity mediates short-term biochemical signaling and long-term gene expression patterns that determine and maintain cellular phenotype. In Aim 1, channel-dependent regulation of cytosolic calcium, cyclic AMP, and activating transcription factor 2 (ATF2) pathways will be examined. The experimental approaches will include a combination of existing biochemical, imaging and molecular techniques in mouse and human primary macrophages. In Aim 2, the EAE model will be utilized to assess this pathway in vivo and determine how SCN5A+ macrophages mediate recovery from inflammatory injury. In vivo gene and protein expression will be analyzed and correlated with in vitro assessment of SCN5A regulation of macrophage vesicular secretion and intercellular communication. The proposed work is innovative because it represents a new approach to regulate macrophage function and treat chronic inflammatory disease. It is significant because promotion of recovery in MS and other chronic inflammatory diseases represents an unmet therapeutic need.