Role of Cytosolic DNA-multiprotein Interactome in Allergic Airway Inflammation

项目摘要/摘要 过敏性鼻炎和哮喘是以过敏性呼吸道炎症为特征的高发疾病。这个 呼吸道上皮是对抗过敏原的第一道防线。我们报告了过敏性提取物刺激 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的双倍体 变应性鼻炎患者鼻黏膜中的双链DNA与多种蛋白质形成复合体。 过敏小鼠的肺，但非致敏的人和小鼠不能。我们将这种细胞质DNA命名为- 互动体“Allergoome”。等位基因中与DNA相互作用的蛋白质是：干扰素调节因子3 (IRF3)、DNA糖基酶Nei-like 2(Neil2)和环GMP-AMP合成酶(CGAS)。值得注意的是，这些因素 与干扰素基因刺激物(STING)、坦克结合蛋白-1(TBK1)和促过敏相关 变态小体中的细胞因子IL-33。变态反应小体的形成及其在变态反应性炎症中的作用 在了解我们在这项提案中提到的情况下。这项提议的中心假设是“过敏性人类 小鼠在呼吸道粘膜和肺的细胞质中形成一个过敏小体，刺激过敏 呼吸道发炎“。在目标1中，我们将测试人类过敏性鼻炎受试者的假设，但不是 健康对照受试者，在鼻黏膜细胞中形成具有隔离的IL-33的变应体。胞质和胞质 将从豚草鼻腔穿孔活检中制备交联核提取液--过敏和健康 研究对象。鼻活检的胞浆提取物将与抗IRF3一起进行免疫沉淀(IP)。 抗体，并通过芯片序列分析相关的DNA序列和相关的蛋白质在 Allergoome。在第二个目标中，我们将检验这样的假设，即Neil2在等位基因中的隔离 降低其保护基因组中的启动子位置的能力，使其免受结合NFκB和刺激过敏性呼吸道的影响 发炎。如目标1所示，将制备胞浆和核提取物。胞液提取物将 用抗Neil2抗体进行IP，分析如目标1。核提取液将接受 用抗Neil2和抗NFκB抗体对IPdDNA进行芯片测序以确定其启动子占有率， 凝胶位移分析检测核因子κB结合情况。重组WT和无催化活性的Neil2将被交付 通过工程多肽载体致敏WT小鼠的呼吸道，过敏性炎症 重新评估。在目标3中，我们将检验刺痛和cGAs稳定变态小体和 刺激过敏性呼吸道炎症。WT变态反应体形成与CDE诱导的变态反应性炎症 小鼠将与StingKO小鼠、肺上皮特异性诱导的cGasKO小鼠和WT小鼠进行比较 用药物cGAS抑制剂治疗。这些研究将第一次确定 过敏性呼吸道炎症中的过敏性小体。我们将提供针对目标组件的原则证明 Allergoome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 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.