鼠伤寒沙门菌诱发巨噬细胞胞外诱捕网的分子机制

The formation of extracellular traps (ETs) has been recently recognized as a novel defense mechanism in several types of innate immune cells. However, studies of macrophages extracellular traps (METs) were insufficient , and the formative mechanism of METs is still ambiguous. In our previously work, we have firstly showed Salmonella Typhimurium induced murine macrophages cell line trigger METs formation. In view of some reports of neutrophil extracellular traps (NETs) and mast cell extracellular traps (MCETs), the formation and characteristic of METs in macrophages-infected with Salmonella Typhimurium were investigated, and also the signal pathways and the antimicrobial activity of METs were evaluated via regulation of HIF-1α expression by the use of hypoxia-inducer, inhibitors and RNA interference and blocking PI3K/Akt/ mTOR signaling pathway in vitro and in vivo. This study might illuminate the molecular mechanism of the formation of METs induced by Salmonella Typhimurium and provide new ideals for development of the innate immune bactericidal effect by METs.

细胞外诱捕网是新近发现的第三种先天性免疫细胞杀菌机制。鉴于我们在前期研究中首次发现鼠伤寒沙门菌感染小鼠巨噬细胞可诱导细胞外诱捕网（macrophages extracellar traps, METs）的产生，而巨噬细胞METs的报道缺乏，且形成机制不清。本研究拟结合有关中性粒细胞和肥大细胞ETs产生的报道，以鼠伤寒沙门菌为研究对象，从HIF-1α和METs、PI3K/Akt/mTOR和HIF-1α关系入手，在体内、体外利用低氧诱导剂、抑制剂和RNA干扰技术调控巨噬细胞HIF-1α表达以及特异性阻断PI3K/Akt/mTOR信号通路后观察METs形成及其特性的变化情况，分析鼠伤寒沙门菌诱发METs的信号通路及其介导METs杀菌作用，旨在明确鼠伤寒沙门菌诱发METs形成的特性与分子机制，并为开发巨噬细胞利用ETs发挥先天性免疫杀菌作用提供新的思路。

细胞外诱捕网（Extracellular traps, ETs）是一种除吞噬和自噬之外的固有免疫防御机制。目前关于ETs形成的分子调控机制仍然不清楚。低氧诱导因子-1α（Hypoxia-inducible factor 1, HIF-1α）是重要的固有免疫调节因子，且HIF-1α的表达和活性受PI3K/Akt/mTOR信号通路调控。本项目以鼠伤寒沙门菌和小鼠巨噬细胞互作为研究模型，从HIF-1α和巨噬细胞胞外诱捕网（Macrophages extracellar traps, METs）、PI3K/Akt/mTOR和HIF-1α关系入手，阐明了PI3K/Akt/mTOR信号通路在调控HIF-1α参与METs形成中的作用，通过本项目的研究，主要取得了以下研究进展：.（1）证实了鼠伤寒沙门菌可诱导巨噬细胞释放METs，巨噬细胞产生METs时伴随ROS形成、LDH释放和细胞凋亡。.（2）初步探究了鼠伤寒沙门菌SL1344株鞭毛蛋白FliC和5'-nucleotidase在鼠伤寒沙门菌激发巨噬细胞释放METs产生中的作用。.（3）低氧应激提高巨噬细胞HIF-1α的表达可增强鼠伤寒沙门菌诱发巨噬细胞释放METs及其介导的杀菌能力。.（4）低氧应激可增加鼠伤寒沙门菌刺激巨噬细胞PI3K/Akt/mTOR信号通路相关分子HIF-1α、PI3K、p-PI3K、Akt、p-Akt、mTOR、p-mTOR、4EBP1、p-4EBP1、p70s6k和p-p70s6k的表达水平。.（5）PI3K/Akt/mTOR信号通路对鼠伤寒沙门菌诱导巨噬细胞METs的形成及其介导的杀菌起到正向作用。.（6）初步建立了鼠伤寒沙门菌感染小鼠模型，分析了PI3K/Akt/mTOR信号通路对小鼠杀伤鼠伤寒沙门菌的影响。.本项目基本完成了原计划内容，明确了低氧应激对鼠伤寒沙门菌诱发巨噬细胞释放METs及其介导杀菌作用的影响，探究了PI3K/Akt/mTOR信号通路在鼠伤寒沙门菌诱导巨噬细胞METs中的作用。