S1PR3调控吞噬体功能促进细菌清除对脓毒症发生发展的影响及机制研究

Macrophages, as the tissue-residence sentinel cells, play a key role in the innate immune response, whose function changes attract special attention in the early immunotherapy of sepsis. Phagosome is the direct place of pathogens digestion. Oxygen-dependent and oxygen-independent pathways are the mean manners of bacterial clearance by early phagosome and mature phagosome, respectively. Sphingosine-1 phosphate receptor 3 (S1PR3), which is constitutively expressed on macrophages, plays a pivotal role in a variety of pathophysiologic process by regulating oxidative stress response. We applied S1PR3 overall knockout model and pharmacological intervention methods in our pilot study and found that S1PR3 could up-regulate macrophages NOX2 activity and enhance ROS production after Escherichia coli (E. coli) infection on the one hand, on the other hand, S1PR3 could promote phagolysosome formation, which accelerate E. coli clearance together, and improve the organ damage of septic mice. Therefore, we hypothesis that S1PR3 will be involved in the pathogenesis of sepsis via modulating the phagosome functions and promoting bacterial clearance. The present project will, from thesystemic, cellular and molecular levels, analyze the function of S1PR3 in modulating the phagosome functions and promoting bacterial clearance, to clarify the roles of S1PR3 during bacterial sepsis, and sebsquent to explore the signal pathway how S1PR3 regulate phagosome functions. This study will provide new targets in the management of sepsis.

巨噬细胞作为组织居留型前哨细胞是固有免疫的关键抗感染细胞，其功能变化在脓毒症早期免疫治疗中备受关注。吞噬体是巨噬细胞消化病原体的直接场所，氧依赖和非氧依赖途径分别是早期吞噬体和成熟吞噬体清除致病菌的主要方式。S1PR3组成型表达于巨噬细胞，参与调节多种病生过程中的氧化应激反应。我们前期运用S1PR3敲除模式动物及药理学干预等方法发现：大肠杆菌感染后，S1PR3一方面通过增加巨噬细胞NOX2活性及ROS水平，另一方面通过促进吞噬溶酶体形成，加快大肠杆菌清除，改善脓毒症小鼠预后。因此，我们推测：S1PR3通过调控吞噬体功能参与脓毒症发生发展。本项目拟从整体、细胞和分子水平， 明确S1PR3通过调控巨噬细胞吞噬体功能促进细菌清除的作用，及其对脓毒症预后的影响；深入探讨S1PR3调控吞噬体功能的分子机制，为脓毒症免疫防治提供新靶标。

病原微清除及炎症反应控制是决定脓毒症预后的两大关键因素。目前研究表明“Sphingosine-1-phosphate (S1P) - Sphingosine-1-phosphate receptor 2/3 (S1PR2/3)” 通路参与脓毒症炎症反应调控，然而S1P-S1PR2/3通路在炎性体活化及病原体清除中的作用目前仍不明确。我们研究发现：（1）脓毒症模型后，巨噬细胞S1PR3表达水平显著上调。S1PR3-/-小鼠脓毒症模型后全身及局部脏器细菌负荷较WT小鼠显著增加，生存率下降。骨髓过继回输WT来源骨髓细胞给予S1PR3-/-小鼠，可显著改善S1PR3-/-小鼠脓毒症预后，同时给予S1PR3高表达巨噬细胞可进一步改善脓毒症小鼠预后。体外研究发现，S1PR3-/-巨噬细胞较WT巨噬细胞杀菌能力显著减弱。体内外应用S1PR3特异性激动小肽可增强巨噬细胞杀菌能力同时改善脓毒症小鼠预后。机制上，S1PR3通过促进Vps34转移到吞噬体，参与吞噬体细菌清除过程。临床研究发现，脓毒症患者外周血单核细胞S1PR3表达水平较健康对照者显著升高，外周血单核细胞S1PR3表达水平与其杀菌能力及免疫功能正相关。因此，S1PR3信号通路促进细菌清除改善细菌性脓毒症预后，靶向干预S1PR3信号通路在调控固有免疫介导的病原体清除中具有临床转化前景。（2）巨噬细胞S1PR2缺失可以显著改善革兰阴性菌感染脓毒症预后(野生鼠组2/10 [20%] vs. S1pr2-/-组7/10 [70%], P = 0.004)，同时模型后腹腔巨噬细胞pyroposis水平降低（(pyroptosis比例: 野生鼠组35 ± 3% vs. S1pr2-/-组10 ± 3%, P <0.001）。S1pr2-/-巨噬细胞非经典炎性体Caspase-11活化水平降低是改善pyroptosis水平的关键。此外，RhoA抑制剂可以抑制S1PR2-过表达导致的Caspase-11的活化。临床革兰阴性菌感染脓毒症患者，单核细胞Caspase-4表达水平较非脓毒症患者显著上调，并与S1PR2表达水平呈正相关。因此，S1PR2及Caspase-11可能成为脓毒症治疗新靶标。