自噬促进宿主抵抗力和耐受力对脓毒症的保护效应及其机制研究

The prevention and treatment of microbial sepsis continue to be a major clinical challenge, particularly in intensive care units. Upon bacterial infection, resistance and tolerance are the two distinct defense strategies that the host can employ to protect itself against microbial threats. Our previous work has shown that LC3b-deficient mice were more susceptible to CLP-induced polymicrobial sepsis; the induction of autophagy in vivo affords strong protection against CLP-induced polymicrobial sepsis, which is closely associated with accelerated bacterial clearance and attenuated tissue and organ damage. We further revealed that induction of autophagy in macrophages by C2250 strongly augmented their bacterial phagocytosis and intracellular bacterial killing；and induction of autophagy in endothelial cells by C2250 leads to increased tolerance to LPS-induced cell apoptosis and damage in endothelial monolayer integrity. However, the precise molecular mechanism involved is still unclear. In this proposed research project, we will use clinical samples and in vivo animal model to investigate the protection of autophagy against microbial sepsis. We will also use the Atg16L1fl/fl-LyzCre and Atg16L1fl/fl-Tie2Cre mice to clarify the autophagy inducer-afforded protection against sepsis-associated lethality is predominantly dependent on autophagy-augmented host resistance and/or tolerance. Finally, we will use the in vitro experiments to verify the underlying mechanism responsible for autophagy-augmented resistance and tolerance of the host to microbial infection. By identifying the significance of autophagy confers protection against microbial sepsis by promoting both resistance and tolerance of the host to bacterial infection, this study will help us to clarify a new immuno-therapeutic strategies for prevention and treatment of microbial sepsis.

严重感染所致脓毒症发病机制及其防治策略是亟待解决的重大难题。研究发现，宿主可通过抵抗力和耐受力两种策略抵抗病原菌感染。我们前期研究结果显示：自噬缺失小鼠对病原菌感染和脓毒症更加易感，自噬的体内诱导能够提高CLP脓毒症动物存活率，并且促进了对细菌的清除以及减轻组织和器官的损伤。进一步体外实验显示，诱导自噬后，巨噬细胞的吞噬作用和杀菌活性显著增强，内皮细胞对细胞损伤的耐受力提高。提示自噬可通过促进宿主对细菌感染的抵抗力和耐受力两种机制发挥保护作用，但确切机制尚待研究。本项目拟利用临床标本及动物模型进一步明确自噬对脓毒症感染的作用，并通过条件性基因敲除小鼠，证明自噬对脓毒症小鼠的保护作用有赖于自噬所促进的宿主抵抗力和耐受力，同时进一步探讨自噬增强宿主抗细菌感染抵抗力和耐受力的确切分子机制。自噬通过促进宿主抵抗力和耐受力对脓毒症的保护作用机制的阐明，将为脓毒症的免疫防治提供新的策略。

脓毒症、脓毒症休克及其后遗症是重症监护病房的主要死亡原因，治疗方案有限。脓毒症时，细菌内毒素刺激机体后，促使炎性细胞特别是中性粒细胞和巨噬细胞在器官组织中募集和活化，产生释放大量的炎症介质如细胞因子、趋化因子、氧自由基及蛋白酶等，互成反馈，不断扩大炎性反应，形成瀑布样级联反应，并共同作用于肺内皮细胞和肠上皮细胞等，增加了细胞的通透性，导致细胞损伤。抵抗力和耐受力是保护宿主免受微生物感染的两种进化保守而又独特的防御策略。自噬是对抗微生物挑战的一种天然免疫防御机制。在脓毒症中，自噬调节对多器官损伤具有保护作用。这在一定程度上是通过防止细胞凋亡、维持促炎细胞因子和抗炎细胞因子的产生之间的平衡以及保护线粒体功能来实现的。本研究发现：在脓毒症发生前6小时给予陶罗立定Taurolidine，通过促进宿主的抵抗力和耐受力，对脓毒症提供了强有力的保护，其特点是加快细菌清除、改善器官损伤、降低血管和肠道通透性。同时发现，在脓毒症发生后6小时给药的Taurolidine也可通过增强宿主对微生物感染的疾病耐受力，从而减轻脓毒症导致的致命损害。Taurolidine可保护野生型小鼠，但对自噬缺陷小鼠缺乏保护作用，证明Taurolidine在体内提供的这种保护作用依赖于完整的自噬途径。在体外实验中，Taurolidine启动了固有吞噬细胞、血管内皮细胞和消化道上皮细胞的自噬诱导，从而增强了吞噬细胞的杀菌活性，增强了血管内皮和消化道上皮细胞对内毒素诱导的损伤的耐受力。这些结果表明，Taurolidine可以通过促进宿主的抵抗力和耐受力来提供对脓毒症的保护，而这一作用依赖于完整的自噬通路。利用自噬激活剂作为脓毒症治疗的可行性是通过成功地最小化器官损伤，从而减少脓毒症导致的死亡率。这种临床转化思路给脓毒症的治疗提供了新的潜力。