缺氧对维甲酸诱导基因-I介导的抗病毒免疫应答的调控及机制研究

The innate immune system provides a first line of defense against invading viruses by releasing multiple inflammatory cytokines, such as interferon-beta, interleukin-6, interleukin-1 beta and tumor necrosis factor-alpha, which directly combat the infectious agent and recruit additional immune responses. However, during pathogenic influenza virus infection, robust cytokine production (cytokine storm) directly contributes to morbidity and mortality. It’s well known that RIG-I is a critical RNA virus sensor that serves to initiate antiviral innate immunity. However, it remains unclear if RIG-I play a role in influenza A viruses-triggered cytokine storm. It’s often observed that cytokine storm occurs with hypoxemia and localizes areas of prominent hypoxia in flu cases. But it is unknown if hypoxia is involved in regulating the production of cytokine storm. Based on our preliminary experiment results: hypoxia significantly up-regulated the anti-H1N1 virus response of macrophages via down-regulating PD-1 expression level, we raise hypothesis: The injury of respiratory epithelial cells due to influenza A viral invasion and replications, thus contributing to hypoxemia and localised areas of prominent hypoxia. Consequently, down-regulation of PD-1 resulted in the decreased SHP-2 phosphorylation, and the subsequent up-regulation of RIG-I expression level, which induces a dysregulated overly exuberant immune response, termed a "cytokine storm". To clarify the role of hypoxia in RIG-I-mediated antiviral response and the formation of cytokine storm is necessary for developing novel methods to combat morbidity and mortality caused by influenza virus infection.

固有免疫系统对病毒过度应答，引起炎症因子过量的“细胞因子风暴”致多器官衰竭是重症流感患者死亡的病理机制之一。RIG-I在病毒免疫中的作用和机制已被深入研究，但是否参与细胞因子风暴尚不清楚；重症流感常并发低氧血症和细胞因子风暴，但它们之间是否存在关联及调控机制尚未可知。我们前期利用小鼠流感病毒感染模型发现，缺氧可显著促进巨噬细胞对H1N1的应答；该作用可能通过调控巨噬细胞表面抑制性受体PD-1及其下游信号而实现。拟在此基础上，通过基因敲除小鼠和体内外实验，验证我们的假说：缺氧抑制病毒感染的巨噬细胞PD-1上调和SHP-2活化，阻断RIG-I信号的负反馈机制（Hypoxia-HIFs-PD-1-SHP-2-RIG-I轴），造成干扰素和炎症因子的过量产生。研究结果有助于阐明缺氧对RIG-I介导的抗病毒应答及细胞因子风暴形成的作用和分子机制，提出新的观点，并可望为临床疾病治疗提供理论依据。

缺氧诱导因子-1α是感受缺氧应激最重要的转录调控因子。缺氧诱导因子-1α与病毒感染的之间有着重要的关联，但是具体关系如何目前还没有定论。PD-1作为重要的免疫检查点蛋白不仅在T淋巴细胞中有表达，其在固有免疫细胞——巨噬细胞中也有一定表达，且与巨噬细胞的功能表型有密切联系。巨噬细胞是最先抵御病毒感染的关键免疫细胞，本研究想要探讨缺氧导致的缺氧诱导因子-1α积累是否能够调节巨噬细胞的抗病毒感染能力以及PD-1在巨噬细胞抗病毒过程中是否也发挥了一定作用。本实验研究了缺氧介导的巨噬细胞抗病毒免疫应答以及PD-1在抗病毒感染中的功能，发现缺氧处理可以进一步降低巨噬细胞表面PD-1的表达，缺氧处理的细胞抗病毒感染能力明显增强。与此一致的是，当敲除PD-1后，巨噬细胞的抗病毒应答也增强，可以分泌更多的炎症因子。而在HIF-1α敲除的巨噬细胞中因无法产生HIF-1α蛋白，其不能表现出野生型细胞缺氧处理后表现出的增强的抗病毒能力。本研究发现了在缺氧环境中，巨噬细胞表面PD-1表达降低，同时HIF-1α的增多可增强巨噬细胞的抗病毒能力，由此推测缺氧可能通过HIF-1α来降低PD-1在巨噬细胞上的表达，从而导致巨噬细胞分泌更多的炎症因子来抵抗病毒的感染。低表达或者不表达PD-1的巨噬细胞表现出一种向M1经典激活的巨噬细胞极化的“活性”状态，细胞内组蛋白乙酰化水平增高，从而更能快速且有效地应对病原体感染。本研究为抗病毒治疗可能提供了一个新的视角。

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