肺巨噬细胞与内皮微粒的交互作用参与脓毒症肺损伤的发生：Mer-KLF13 机制

Previous work by us demonstrated that endothelial microparticles (EMPs) generated from the cytoskeletal alteration participated in the pathophysiology of sepsis-induced lung injury. Moreover, EMPs can even regulate the function of lung macrophages. The established mechanism of which is that the phagocytosis of EMPs by lung macrophages is facilitated through the Mer receptor, and the microRNAs derived from EMPs regulates an important regulatory protein, Krüppel-like factor (KLF)-13, secreted by lung macrophages. These events subsequently modulate lung macrophages and the downstream inflammatory responses in the lung. Thus, phagocytosis of EMPs by lung macrophages, and its regulation on the phenotypes of lung macrophages are critical in seeking the therapeutic strategies in alleviating lung injury. In the proposed study, Mer receptor knock out (KO) and KLF13 KO mice will be used to investigate the mechanism of the “EMPs-Mer-KLF13” pathway on the interaction between EMPs and lung macrophages. In vitro experiments, plasmid transfection, protein binding assay, protein kinase assay, and RNA silencing will be used to explore how the phagocytosis of EMPs is enhanced by lung macrophages in septic shock patients. We will also investigate the regulatory mechanisms of EMPs on lung macrophages, and research the role of KLF13 and Mer in this process. In summary, the proposed study in this grant application will provide us with a much more clear and comprehensive picture on how the interaction between EMPs and lung macrophages could lead to critical physiopathology in the lung; and will also provide us with new insights for the treatment of sepsis-induced lung injury.

预实验发现：由内皮细胞骨架改变而产生的内皮微粒参与脓毒症肺损伤的发生，并且，内皮微粒对肺巨噬细胞表型或有调节作用，推测其机制为：巨噬细胞通过膜受体Mer吞噬内皮微粒，被吞噬的内皮微粒通过其包含的microRNAs作用于巨噬细胞KLF13转录因子，参与调节巨噬细胞的表型及其下游炎症反应。因此，促进巨噬细胞对内皮微粒的吞噬、调节巨噬细胞的表型转化是减轻肺损伤的有效手段。本研究拟以Mer-/-、KLF13-/-鼠分析内皮微粒和巨噬细胞的相互作用及“内皮微粒-Mer-KLF13”通路在其中的机制，并在体外实验中以质粒转染、蛋白质体外结合实验、体外激酶实验及RNA静默等方法，探讨促进巨噬细胞吞噬内皮微粒的途径，分析内皮微粒调节巨噬细胞的机制，研究Mer、KLF13在其中的具体作用，完善内皮微粒和巨噬细胞的相互作用在脓毒症肺损伤中的机制，为肺损伤的治疗提供新的思路。

小鼠盲肠结扎穿孔术(CLP)后24h 小鼠肺组织qPCR结果显示，IL-1β、TNF-α等炎症因子显著上调，CLP小鼠24h后肺组织中Mertk表达显著下调；CLP小鼠24h肺组织NLRP3炎性小体活化；LPS+ATP处理后小鼠骨髓来源巨噬细胞Mertk表达显著下调；LPS+ATP处理后小鼠骨髓来源巨噬细胞NLRP3炎性小体活化；Mertk激动剂抑制MH-S肺泡巨噬细胞NLRP3炎性小体活化；Mertk受体抑制剂UNC2250抑制 MH-S肺泡巨噬细胞增殖，促进MH-S细胞凋亡。