Architectural structure and regulation of TOLLIP in IPF

项目摘要 特发性肺纤维化（IPF）每年导致50，000名患者死亡。发病机制涉及异常修复 在各种原因的肺损伤后，由涉及Toll样受体（TLR）的先天免疫介导的免疫应答。 TLR受到Toll相互作用蛋白（TOLLIP）的负抑制。单核苷酸多态性 与IPF的易感性和生存率相关的TOLLIP。调节TOLLIP活性的遗传变异 目前尚不清楚它们对IPF中肺损伤的先天免疫应答的影响。 在我们的初步数据中，我们通过下一代测序（NGS）对192例IPF患者的TOLLIP进行了测序 和鉴定;能够在体外调节TOLLIP表达的多个功能性SNP， 与易感性高度相关的区域，以及与生存相关的几个SNP。 我们假设TOLLIP内的遗传变异调节先天性免疫应答， 影响IPF易感性和生存率。我们的总体目标是了解TOLLIP在 通过确定IPF基因座的结构多样性及其 通过遗传变异进行调节。我们的方法是对TOLLIP基因进行测序，并进行病例对照。 在1,000例IPF病例沿着以及1,000例匹配的健康对照的亚组中进行分析。我们将验证和 用剩下的队列重复我们的发现，并进行回归分析，以确定易感性- 预测生存率的SNPs。不常见的变体将按预测函数进行聚合， 分析易感性和存活率。还将评估SNP与mRNA肺表达的相关性 和其他肺部分子标志物，并将优先用于进一步研究。我们将确定哪些SNP 使用荧光素酶测定调节mRNA表达水平。在我们的初步数据中，我们已经确定了几个 使用计算机模拟方法确定调控区（即3 'UTR、5' UTR或外显子）内的候选SNP， 细胞培养模型这表明有多种潜在的监管模式。用TLR配体刺激也将 识别潜在的TLR。最后，我们将确定TOLLIP基因型是否与Tollip IPF患者前瞻性队列中血液单核细胞和B细胞的蛋白水平。我们还将 确定TOLLIP基因型是否与人肺巨噬细胞中的Tollip蛋白水平相关， 将这些结果与来自IPF外植体肺的样品进行比较。最后，我们将 通过刺激血液单核细胞和肺来确定TOLLIP变体对TLR信号传导的影响 巨噬细胞与TLR配体。TLR后Tollip蛋白水平的TOLLIP变体依赖性差异 刺激，在血液和器官的疾病，将解决的作用，TOLLIP变体的功能 对IPF中不受控制的纤维化反应的影响。 拟议的工作将确定与IPF相关的功能性TOLLIP变体，并描述其机制 它们通过该机制调节IPF中关键的先天TLR应答。了解TOLLIP的遗传变异 在开发IPF治疗方法时，可提供直接的临床评估，包括危害和获益。

Project Summary Idiopathic pulmonary fibrosis (IPF) kills 50,000 patients annually. Pathogenesis involves an aberrant repair response after various causes of lung injury, mediated by innate immunity involving toll-like receptors (TLRs). TLRs undergo negative inhibition by toll interacting protein (TOLLIP). Single nucleotide polymorphisms (SNPs) in TOLLIP associated with susceptibility, and survival in IPF. The genetic variants regulating TOLLIP activity and their effect on the innate immune response to lung injury in IPF is currently unknown. In our preliminary data, we sequenced TOLLIP by next generation sequencing (NGS) in 192 patients with IPF and identified; multiple functional SNPs capable of regulating TOLLIP expression in vitro, a simple repeating region highly associated with susceptibility, as well as several SNPs that correlate with survival. We hypothesize that genetic variants within TOLLIP modulate the innate immune response, thereby influencing IPF susceptibility and survival. Our overall goal is to understand the role of TOLLIP in the susceptibility and disease course of IPF through determining the architectural diversity of its locus and its regulation by genetic variation. Our approach is to sequence the TOLLIP gene, and perform a case-control analysis in a subset of 1,000 IPF cases along with 1,000 matched healthy controls. We will validate and replicate our findings using the remaining cohort and perform regression analyses to determine susceptibility- associated SNPs that predict survival. Uncommon variants will be aggregated by predicted function and analyzed for susceptibility and survival. SNPs will also be assessed for association with mRNA lung expression and other lung molecular markers and will be prioritized for further study. We will determine which SNPs regulate mRNA expression levels using luciferase assays. In our preliminary data, we have identified several candidate SNPs within regulatory regions (i.e. 3'UTR, 5'UTR, or exonic) using in silico methods, confirmed in cell culture models. This suggests multiple potential modes of regulation. Stimulation with TLR ligands will also identify potential TLRs involved. Lastly, we will determine whether TOLLIP genotypes correlate with Tollip protein levels, in blood monocytes and B cells in a prospective cohort of patients with IPF. We will also determine if TOLLIP genotypes correlate with Tollip protein levels in human lung macrophages from a large cohort of non-IPF donor lungs and compare these results to samples from IPF explant lungs. Lastly, we will determine the effect of TOLLIP variants on TLR signaling by stimulating blood monocytes and lung macrophages with TLR ligands. TOLLIP variant-dependent differences in Tollip protein levels after TLR stimulation, in both the blood and the organ of disease, will address the role of TOLLIP variant functional effects on the uncontrolled fibrotic response in IPF. The proposed work will identify functional TOLLIP variants associated with IPF and delineate the mechanisms by which they regulate critical innate TLR responses in IPF. Understanding the genetic variation of TOLLIP may provide direct clinical assessment, for both harm and benefit, in developing treatments for IPF.