ORMDL3 Regulation of Dendritic Cells in Asthma

项目摘要/摘要：哮喘是儿童最常见的慢性疾病， 给美国儿童带来沉重的临床和经济负担，使美国医疗保健系统损失约501亿美元 每年1美元。儿童哮喘的发病机制是复杂的，继发于两种遗传易感性。 以及环境/感染性暴露。多个强大的遗传关联研究(Gwas)和顺式- 表达数量性状基因座研究(cis-eQTL)已将ORMDL3基因的过度表达与 儿童发作性哮喘。不幸的是，该基因在哮喘发病机制中的作用尚不清楚。多重 研究表明，ORMDL蛋白调节鞘磷脂代谢的关键方面，但潜在的 ORMDL3风险变异体调节失调的免疫机制目前尚不清楚。这一差距在我们的基础上 知识阻碍了针对这一重要的新哮喘途径的新疗法的合理靶向。我有过 最近进行的初步研究表明，ORMDL3在树突状细胞(DC)中过表达 降低基底细胞神经酰胺水平，增强促炎细胞因子分泌。我假设 ORMDL3在这些细胞中的过度表达可能参与了该基因在哮喘发病机制中的作用。这个 我提出的研究项目的主要科学目标是：1)建立细胞和分子 树突状细胞中ORMDL3的过度表达与肺的促炎反应有关的机制 建立ORMDL3风险变异体对神经鞘脂脂调节的人DC免疫反应的影响。至 为了实现这些目标，我将获得生物统计学、脂肪组学分析和高级 免疫学的长期目标是建立一个实验室，重点研究鞘脂在 肺部发炎。 在随附的建议书中，我概述了一套综合的多学科研究，以评估 ORMDL3在树突状细胞在小鼠和人类系统哮喘发病机制中的作用。在《目标1》中，我将 检测ORDML3过表达对DC免疫功能的影响。我们将利用一个 转基因ORMDL3过表达小鼠模型的建立是为了探讨OMDL3的功能效应 ORMDL3对DC抗原处理、T细胞活化和DC迁移的影响。在目标2中，我们将确定 通过检测ORMDL3基因对细胞脂和蛋白表达的影响来调节树突状细胞的过度表达 DC成熟过程中的基因表达。我们将结合使用猎枪和靶向脂质组学。 通过RNA测序来确定由ORMDL3改变的关键基因和脂质代谢产物途径，导致 到增强DC介导的炎症。在目标3中，我们将确定ORMDL3风险等位基因是否调节 ORMDL3基因分型受试者中人单核细胞来源的树突状细胞中的鞘磷脂水平。把这些研究放在一起 将直接解决我们关于鞘脂和DC功能的知识差距，并预计将 提供对儿童哮喘易感机制的见解。

Project Summary/Abstract: Asthma is the most common chronic illness of childhood and imposes a profound clinical and financial burden on American children, costing the US health care system ~$50.1 billion dollars annually. The pathogenesis of pediatric asthma is complex, secondary to both genetic predisposition and environmental/infectious exposures. Multiple well-powered genetic association studies (GWAS) and cis- expression quantitative trait loci studies (cis-eQTL) have linked over-expression of the gene ORMDL3 to pediatric-onset asthma. Unfortunately, the role of this gene in asthma pathogenesis remains unclear. Multiple studies have shown ORMDL proteins regulate critical aspects of sphingolipid metabolism, but the underlying immune mechanisms dysregulated by ORMDL3 risk variants are currently undefined. This gap in our basic knowledge impedes the rational targeting of novel therapies to this important new asthma pathway. I have recently performed preliminary studies that have shown ORMDL3 over-expression in dendritic cells (DCs) reduced basal cellular ceramide levels and enhances pro-inflammatory cytokine secretion. I hypothesize that ORMDL3 over-expression in these cells may contribute to the gene's role in asthma pathogenesis. The primary scientific goals of my proposed research project are 1) to establish the cellular and molecular mechanisms linking ORMDL3 over-expression in DCs to pro-inflammatory responses in the lung and 2) to establish the effects of ORMDL3 risk variants on sphingolipid-regulated immune responses in human DCs. To achieve these goals, I will acquire additional training in biostatistics, lipidomic analysis and advanced immunology with the long-term goal of establishing a laboratory focused on the role of sphingolipids in pulmonary inflammation. In the attached proposal, I have outlined an integrated set of multidisciplinary studies to evaluate the role of ORMDL3 in DCs during asthma pathogenesis in both mouse and human systems. In Aim 1, I will determine the in vitro and in vivo effects of ORDML3 over-expression on DC immune function. We will utilize a transgenic ORMDL3 over-expressing mouse model I have developed to probe the functional effects of ORMDL3 on DC antigen processing, T cell activation and DC migration. In Aim 2, we will identify the regulatory effects of ORMDL3 over-expression in DCs by examining the gene's effects on cellular lipids and gene expression during DC maturation. We will utilize both shotgun and targeted lipidomics in combination with RNA sequencing to identify key gene and lipid metabolite pathways that are altered by ORMDL3, leading to enhanced DC-mediated inflammation. In Aim 3, we will determine whether ORMDL3 risk alleles regulate sphingolipid levels in human monocyte-derived DC in ORMDL3 genotyped subjects. Together these studies will directly address the gap in our knowledge regarding sphingolipids and DC function and are predicted to offer insights into the susceptibility mechanisms of pediatric asthma.