Clec9A介导的坏死细胞清除在COPD获得性免疫中的作用机制研究

As a result of a variety of stimuli in chronic obstructive pulmonary disease (COPD), such as cigarette smoke, induce the accumulation of necrotic cells in lung tissue, which expose or release a variety of DAMPs such as HMGB-1, F-actin, etc., resulting in adaptive immune response. Our previous work demonstrated that apoptotic and necrotic cells were increased in lung tissue of mice exposed by cigarette smoke, leading to DAMPs such as HMGB-1 release. Moreover, cigarette smoke exposure leads to interstitial and peritracheal lymphoid neogenesis (lymphoid follicle formation) in lung. Clec9A as a dendritic cell-restricted receptor specific for F-actin that is exposed by necrotic cells, DC phagocytes necrotic cells and present necrosis-related antigens to CD8+ T cells. In this study, we speculated that pulmonary migratory CD103+ DC and CD8α+ DC in neonatal lymph nodes of lung, which expressed Clec9A, bind F-actin exposed by necrotic cells, present necrotic cell-associated antigens to CD8+ T cells and activate cytotoxic T lymphocyte (CTL), which is the main mechanism of CD8+ T cell is adaptive immunity activation of COPD. In this study, we will utilize specimens of COPD patients and cigarette smoke exposed model mice to investigate the cellular and molecular mechanism of Clec9A-mediated phagocytosis of necrotic cells and CD8+ T cell accumulation in COPD.

COPD病人受到多种环境因素刺激导致肺组织中坏死细胞累积，暴露或释放多种DAMPs如HMGB-1、F-actin等，启动COPD病人获得性免疫应答反应。我们前期研究发现香烟烟雾刺激导致肺组织中凋亡及坏死细胞增多，导致HMGB-1的释放；且肺实质及支气管旁新生淋巴组织显著性增多。树突状细胞表面Clec9A结合坏死细胞表面暴露的F-actin，内吞坏死细胞并将坏死相关抗原提呈给CD8+ T细胞。本课题假设肺组织中迁移性CD103+ DC及新生淋巴组织中CD8α+ DC表达Clec9A结合肺组织坏死细胞暴露的F-actin，吞噬坏死细胞并将相关的抗原提呈给CD8+ T细胞，是COPD病人CD8+ T细胞免疫应答的机制。本项目将利用临床样本及香烟烟雾暴露小鼠模型，揭示COPD病人Clec9A介导坏死细胞清除引起CD8+ T细胞增加的细胞及分子机制。

CD8+ T淋巴细胞是介导COPD发病的关键机制之一。未被机体及时清除的凋亡或坏死细胞暴露或释放多种DAMPs如F-actin等，与PRRs结合后启动COPD患者获得性免疫应答。我们前期研究发现香烟烟雾刺激导致肺组织凋亡及坏死细胞增多，释放HMGB-1分子。cDC1s上表达的DAMPs受体Clec9A能特异性识别坏死细胞暴露的F-actin，交叉提呈至CD8+ T淋巴细胞并激活CTL。然而，Clec9A能否介导COPD患者CD8+ T细胞活化尚不清楚。本课题首先检测COPD患者肺组织内死亡细胞及DAMPs的释放情况，并分析COPD患者CD8+ T淋巴细胞的活化情况，明确F-actin是启动CD8+ T淋巴细胞应答的激活分子；在此基础上进一步研究COPD患者肺组织内主要抗原交叉提呈细胞Clec9A+ DC的表达含量；然后探索COPD患者肺组织内Clec9A+ DC与CTL的相关性；最后利用COPD小鼠模型明确Clec9A+ DC和CTL的相互作用。研究结果证实COPD患者肺组织内大量浸润的Clec9A+ DC参与交叉提呈坏死细胞抗原，导致CD8+ T淋巴细胞过度活化及CTL产生；CTL释放的IFN-γ和Granzyme-B则进一步攻击肺组织细胞，形成DAMPs释放和CD8+ T淋巴细胞持续激活的恶性循环。该课题研究结果首次揭示COPD患者肺组织中Clec9A介导的CD8+ T淋巴细胞活化，为COPD获得性免疫启动提供新的靶点，同时从全新的角度诠释了COPD获得性免疫应答持续激活的机制，为阐明COPD持续性气道炎症反应提供重要理论依据，有利于设计针对性的措施来干预COPD的疾病进展。