胞外杀菌网调控COPD气道炎症及粘液高分泌的机制研究

Now there is a growing awareness that COPD is a disease with immunologic disorder，and progressive airway inflammation and mucus hypersecretion are closely correlated with the immune inflammatory response. However, the underling mechanism is not fully understood. Persistent airway inflammation is the most essential characteristic of chronic obstructive pulmonary disease (COPD). Under certain pathological circumstances, nuclear DNA or mtDNA based extracellular traps (ETs) are formed excessively, with various granular proteins, and have been proved to modulate native immune response, aggravating tissue damage. Our preliminary studies indicate that, in COPD patients, extracellular mtDNA in serum and expression of β-defensin in granular proteins in lung tissue increase significantly. We also found that in vitro blockage of specific mtDNA receptors inhibits the cigarette-smoke-extract (CSE) induced cell signaling activating and inflammatory mediators releasing from human airway epithelial cells and neutrophils. Therefore, We assume that extracellular traps (ETs) may play essential roles in modulating airway inflammation and mucus hypersecretion in COPD. Our goal in this proposal is to elucidate roles of nuclear DNA or mtDNA based extracellular traps (ETs) in COPD airway immune inflammatory response,using scanning/confocal electron microscoping imaging, proteomics, mtDNA receptor knockout mice in vivo and ex vivo. The study may provide valuable clues for the treatment of COPD. from human airway epithelial cells and neutrophils. Therefore, We assume that extracellular traps (ETs) may play essential roles in modulating airway inflammation and mucus hypersecretion in COPD. Our goal in this proposal is to elucidate roles of nuclear DNA or mtDNA based extracellular traps (ETs) in COPD airway immune inflammatory response,using scanning/confocal electron microscoping imaging, proteomics, mtDNA receptor knockout mice in vivo and ex vivo. The study may provide valuable clues for the treatment of COPD.

目前认为COPD患者机体存在免疫紊乱，慢性气道炎症及粘液高分泌可能与持续的免疫-炎症反应密切相关，但机制尚未明确。最新研究发现病理状态下，免疫细胞会释放出过多的胞外杀菌网，一种以细胞核DNA或线粒体mtDNA为骨架、多种抗生物肽为蛋白组分的网状结构，能介导免疫炎症反应，进一步造成组织损伤。我们前期发现COPD患者循环mtDNA水平显著升高，气道上皮中颗粒蛋白β防御素表达也明显上调；体外阻断mtDNA与其受体的结合能抑制香烟提取物诱导的气道上皮细胞、中性粒细胞信号通路激活及炎性介质的释放。因此胞外杀菌网可能是调控COPD气道免疫-炎症反应的重要机制。本项目拟通过构建体内外气道炎症及粘液高分泌模型，并结合临床样本，利用扫描\激光共聚焦电子显微镜成像、蛋白组学、基因敲除等关键技术，阐明胞外杀菌网对COPD气道炎症-免疫反应的调控机制，探索其在粘液高分泌中的作用，以期为临床治疗COPD提供新思路。

本课题提出的科学假说是：慢阻肺患者吸烟引发氧化应激环境，诱导肺组织中大量浸润的中性粒细胞释放过量的胞外杀菌网NETs（neutrophil extracellular traps，NETs），其主要成分DNA作为损伤相关分子（damage-associated molecular patterns，DAMPs）可通过绑定气道上皮细胞中DNA感应蛋白TRL9激活共同的下游Type I IFN炎症通路，参与并维持慢阻肺的气道炎症-免疫反应；抑制NETs的释放或阻断感应DNA的自免疫通路可能是降低慢阻肺炎症-免疫水平的新途径。围绕上述假设，我们从细胞、动物和慢阻肺临床样本等层面展开研究，实验结果表明：香烟烟雾提取物（cigarette smoke extract，CSE）诱导人中性粒细胞释放NETs，其释放依赖线粒体ROS表达；NETs主要成分是源于染色体和线粒体的自体氧化损伤DNA；NETs直接刺激人气道上皮细胞活力/增殖能力增高、Type I IFN相关炎症因子的基因表达；在细胞水平或动物层面沉默或敲除TLR9，可有效抑制NETs诱导的气道上皮细胞功能的变化，并改善熏烟诱导的慢阻肺小鼠模型肺部Type I IFN炎症响应、整体免疫-炎症水平，从而阻止肺气肿和气道阻塞症状的发生；对慢阻肺小鼠模型给予NETs抑制剂或降解剂，可一定程度抑制NETs的表达及其所致Type I IFN炎症响应和整体气道炎症水平，改善慢阻肺小鼠肺气肿程度；临床样本检测发现，慢阻肺患者BALF和外周血中NETs主要成分mtDNA和MPO表达增高，相应的Type I IFN炎症响应及相关促炎因子表达也较健康对照有所升高。上述研究结果提示：香烟烟雾诱导的NETs的氧化损伤DNA成分可通过TLR9介导Type I IFN炎症响应，是吸烟所致慢阻肺中启动和维持慢性气道炎症的重要环节，抑制NETs的释放或阻止其被识别有望作为改善慢阻肺慢性气道炎症的治疗新靶标。