Role of Cytosolic DNA-multiprotein Interactome in Allergic Airway Inflammation

项目概要/摘要 过敏性鼻炎和哮喘是以过敏性气道炎症为特征的高度流行的疾病。的 气道上皮是抵御过敏原的第一道防线。我们报告说，过敏性提取物会刺激 这些细胞中的氧化 DNA 损伤和先天免疫反应。在这里我们展示了胞质双 (ds)DNA 链与过敏性鼻炎患者鼻粘膜中的多种蛋白质形成复合物， 过敏小鼠的肺部，但不敏感的人和小鼠的肺部。我们将这种细胞质 DNA 命名为： 相互作用组“过敏体”。过敏体中的 DNA 相互作用蛋白是： 干扰素调节因子 3 (IRF3)、DNA 糖基化酶 Nei-Like 2 (Neil2) 和环 GMP-AMP 合酶 (cGAS)。值得注意的是，这些因素 与干扰素基因刺激物 (STING)、TANK 结合激酶 1 (TBK1) 和促过敏相关 变态反应体中的细胞因子 IL-33。过敏体在过敏性炎症中的形成和作用是空白 了解我们在本提案中讨论的内容。该提案的中心假设是“过敏的人类 小鼠的气道粘膜和肺部的细胞质中会产生过敏体，从而刺激过敏 气道炎症”。在目标 1 中，我们将检验以下假设：患有过敏性鼻炎的人类受试者，但不患有过敏性鼻炎 健康对照受试者，在鼻粘膜细胞中开发出具有隔离 IL-33 的过敏体。细胞质和 将从豚草过敏者和健康者的鼻孔活检中制备交联核提取物 科目。鼻活检的胞质提取物将使用抗 IRF3 进行免疫沉淀 (IP) 抗体，并通过 Chip-seq 分析相关 DNA 序列，以及 过敏体。在第二个目标中，我们将检验 Neil2 在 Allergosome 中的隔离假设 降低其保护基因组中启动子位点免于结合 NFκB 并刺激过敏性气道的能力 炎症。胞质和核提取物将按目标 1 中的方法制备。胞质提取物将 使用抗 Neil2 抗体进行 IP，并按照目标 1 进行分析。核提取物将进行 使用抗 Neil2 和抗 NFκB 抗体对 IP d DNA 进行 ChIP-Seq 以确定其启动子占据情况， 和凝胶位移分析来检测 NFκB 结合。重组 WT 和催化失活 Neil2 将交付 通过工程肽载体进入致敏的野生型小鼠的气道，过敏性炎症将发生 重新评估。在目标 3 中，我们将检验以下假设：STING 和 cGAS 稳定过敏体并 刺激过敏性气道炎症。 WT 中变应体的形成和 CDE 诱导的过敏性炎症 将小鼠与 StingKO 小鼠、肺上皮特异性诱导型 cGasKO 小鼠和 WT 小鼠进行比较 用药物 cGAS 抑制剂治疗。这些研究将首次明确 过敏性气道炎症中的过敏体。我们将提供针对目标组件的原理证明 过敏体可减少过敏性炎症。在未来的研究中，这些策略可能会发展成治疗方法 减轻过敏性鼻炎和哮喘的过敏性炎症。

PROJECT SUMMARY/ABSTRACT Allergic rhinitis and asthma are highly prevalent diseases characterized by allergic airway inflammation. The airway epithelium is the first line of defense against allergens. We reported that allergenic extracts stimulate oxidative DNA damage and innate immune responses in these cells. Here we show that cytosolic double strand (ds)DNA forms a complex with several proteins in the nasal mucosa of humans with allergic rhinitis and lungs of allergic mice, but not in non-sensitized humans and mice. We named this cytoplasmic DNA- interactome “Allergosome”. The DNA-interacting proteins in the Allergosome are: interferon regulatory factor 3 (IRF3), the DNA glycosylase Nei-Like 2 (Neil2), and Cyclic GMP-AMP synthase (cGAS). Notably, these factors associate with Stimulator of Interferon Genes (STING), TANK-binding kinase-1 (TBK1), and the pro-allergic cytokine IL-33 in the Allergosome. The formation and role of the Allergosome in allergic inflammation are gaps in knowledge that we address in this proposal. The central hypothesis of this proposal is that “Allergic humans and mice develop an Allergosome in the cytoplasm of the airway mucosa and lungs that stimulates allergic airway inflammation”. In Aim 1 we will test the hypothesis that human subjects with allergic rhinitis, but not healthy control subjects, develop an Allergosome with sequestered IL-33 in nasal mucosal cells. Cytosolic and crosslinked nuclear extracts will be prepared from the nasal punch biopsies of ragweed-allergic and healthy subjects. The cytosolic extract of the nasal biopsies will be subjected to immunoprecipitation (IP) with anti-IRF3 antibody, and analyzed for associated DNA sequences by Chip-seq, and for associated proteins in the Allergosome. In the second Aim, we will test the hypothesis that sequestration of Neil2 in the Allergosome reduces its ability to protect promoter sites in the genome from binding NFκB and stimulating allergic airway inflammation. Cytosolic and nuclear extracts will be prepared as in Aim 1. The cytosolic extracts will be subjected to IP with anti-Neil2 antibody, and analyzed as in Aim 1. The nuclear extracts will be subjected to ChIP-Seq of the IP’d DNA with anti-Neil2 and anti-NFκB antibodies to determine their promoter occupancy, and gel shift analysis to detect NFκB binding. Recombinant WT and catalytically inactive Neil2 will be delivered to the airways of sensitized WT mice by engineered peptide carriers, and allergic inflammation will be reassessed. In Aim 3, we will test the hypothesis that STING and cGAS stabilize the Allergosome and stimulate allergic airway inflammation. Allergosome formation and CDE-induced allergic inflammation in WT mice will be compared to that of StingKO mice, lung epithelium-specific inducible cGasKO mice, and WT mice treated with pharmacologic cGAS inhibitors. These studies will define, for the first time, the central role of the Allergosome in allergic airway inflammation. We will provide proof of principle that targeting components of the Allergosome reduces allergic inflammation. In future studies, these strategies may develop into therapies that mitigate allergic inflammation in allergic rhinitis and asthma.