Architectural structure and regulation of TOLLIP in IPF

项目摘要 特发性肺纤维化(IPF)每年导致5万名患者死亡。发病机制涉及异常修复 不同原因的肺损伤后的反应，由涉及Toll样受体(TLRs)的先天免疫所介导。 Toll相互作用蛋白(TOLLIP)对TLRs有负抑制作用。单核苷酸多态(SNPs) TOLLIP与易感性和IPF的生存相关。调控TOLLIP活性的遗传变异 而它们对肺间质纤维化肺损伤的先天免疫反应的影响目前尚不清楚。 在我们的初步数据中，我们通过下一代测序(NGS)对192名IPF患者的TOLLIP进行了测序 多个功能SNPs在体外能够调节TOLLIP的表达，简单重复 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免疫反应，从而 影响IPF易感性和存活率。我们的总体目标是了解TOLLIP在 IPF易感性和病程通过测定其基因座结构多样性和 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 对1000例IPF病例和1000名匹配的健康对照进行了分析。我们将验证并 使用剩余的队列重复我们的发现，并进行回归分析以确定易感性- 预测生存的相关SNPs。不常见的变体将按预测函数和 分析了易感性和存活率。SNPs还将被评估为与mRNAs肺表达相关。 和其他肺分子标志物，并将优先进行进一步研究。我们将确定哪些SNPs 用荧光素酶分析调节mRNA的表达水平。在我们的初步数据中，我们发现了几个 在管理区域(即3‘非编码区、5’非编码区或外显子)内使用电子方法的候选SNP，确认于 细胞培养模型。这暗示了多种潜在的监管模式。与TLR配体的刺激也将 确定涉及的潜在TLR。最后，我们将确定TOLLIP基因型是否与Tollip相关 IPF患者前瞻性队列中血液单核细胞和B细胞中的蛋白质水平。我们还将 从大鼠肺巨噬细胞中确定TOLLIP基因型与Tollip蛋白水平是否相关 对非IPF供体肺进行队列，并将这些结果与IPF外植体肺的样本进行比较。最后，我们将 确定TOLLIP变异体刺激单核细胞和肺对TLR信号的影响 具有TLR配体的巨噬细胞。TLR术后Tollip蛋白水平的TOLLIP变异依赖性差异 在血液和疾病器官中的刺激将解决TOLLIP变体功能的作用 对IPF中无节制纤维化反应的影响。 拟议的工作将确定与IPF相关的TOLLIP功能变体并描述其机制 通过它们调节IPF中关键的先天TLR反应。对TOLLIP遗传变异的认识 可在开发IPF治疗方法方面提供直接的临床评估，既有害处，也有好处。

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.