Notch信号通路介导白细胞介素-22分泌在乙型肝炎病毒感染炎症应答中的作用

Notch signaling promotes production of endogenous stimulators for aryl hydrocarbon receptor (AHR), which further augment interleukin-22 (IL-22) secretion. Notch-AHR-IL-22 axis fine-tunes immune system and controls inflammatory responses. Studies indicated that RBP-J-deficient mice, which completely block the Notch signaling, were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4+ T cells. However, there are no reports about the Notch signaling, which drives IL-22 secretion, regulates liver inflammatory responses in HBV infection. Our previous studies demonstrated that IL-22 plays a proinflammatory role in the immune response to HBV infection, and Notch signaling takes part in the mantenance of liver homeostasis. However, it is still not fully understood that whether Notch signaling could drive IL-22 secretion in HBV-specific CD4+ T cells and whether liver inflammation caused by HBV infection could be ameliorated by blockade of Notch signaling pathway. Thus, in the present study, we will examine the hypothesis that Notch-AHR-IL-22 axis plays an important role in the inflammatory responses to HBV. To evaluate the hypothsis, we will carry out the experiments to determine the effect of overexpression of intracellular domain of Notch on the secretion of IL-22 in HBV-specific CD4+ T cells by using peripheral blood mononuclear cells from HBV-infected patients, and to clarify the inflammatory responses to HBV infection in RBP-J-deficient mice and wild type C57BL/6 mice. These experiments are important, as they may identify Notch-AHR-IL-22 axis as a new pathway that regulates HBV related liver inflammation. A complete understanding of these processes may also lead to improved therapies for the liver damage caused by HBV infection, which would have the potential to prevent liver cirrhosis and hepatocellular carcinoma.

Notch信号通路可通过刺激内源性芳香烃受体（AHR）激动剂，促进CD4+T细胞分泌IL-22，Notch-AHR-IL-22轴具有精细调控炎症应答的作用。然而目前尚未见Notch信号介导IL-22分泌如何影响HBV感染肝脏炎症应答的研究报道。我们前期研究结果表明，IL-22在HBV感染应答中发挥促炎因子的作用，Notch信号也参与调节肝脏内环境稳态的维持，但Notch信号是否参与介导HBV特异性CD4+T细胞分泌IL-22，阻断Notch通路是否能减轻HBV感染所致的肝脏炎症损伤尚不清楚。因此，本研究拟选择HBV感染者和RBP-J基因敲除小鼠为研究对象，分析增强Notch信号对HBV特异性IL-22分泌的影响和RBP-J基因敲除小鼠对HBV感染的炎症应答效应，以期揭示Notch-AHR-IL-22轴在HBV感染炎症应答中的作用，为全面阐明HBV感染诱导肝脏炎症应答的机制提供实验依据。

HBV感染介导肝脏炎症损伤的机制尚未完全阐明。我们前期研究结果表明，IL-22在HBV感染应答中发挥促炎因子的作用，Notch信号通路可促进CD4+T细胞分泌IL-22，Notch-IL-22轴具有精细调控炎症应答的作用，但未见Notch信号介导IL-22分泌如何影响HBV感染肝脏炎症应答的研究报道。本研究通过检测细胞因子、趋化因子水平和免疫细胞亚群变化评估慢性HBV感染者的免疫状态，检测HBV感染者外周血CD4+T细胞中Notch信号通路相关分子的表达水平，分析抑制Notch信号通路对CD4+T细胞分泌IL-22的影响。利用HBV高压尾静脉注射小鼠模型，观察HBV感染对Notch信号通路的影响，并检测在体内抑制Notch信号通路对IL-22分泌和炎症细胞向肝脏浸润的影响，评估抑制Notch信号通路对HBV感染炎症应答的影响。研究发现，调节性T细胞的比例在慢乙肝患者中较健康志愿者显著升高，血清中CXCL9、CXCL10、CXCL11和IL-10的水平在慢乙肝患者中升高，且与ALT水平呈显著正相关，而G-CSF、MCP-3和IFN-γ的水平在慢乙肝患者中降低，这提示HBV感染者中存在免疫紊乱，抑制性免疫细胞的水平增加，导致机体发生免疫耐受，病毒感染慢性化，而炎症相关的细胞因子和趋化因子的水平亦增加，介导肝脏炎症损伤。而在CD4+T细胞中，Notch1和Notch2 mRNA的表达水平在HBV感染者中显著升高，抑制Notch信号通路不影响Notch1和Notch2的表达，而Hes1和Hes5的表达显著降低，同时，在非特异性刺激条件下，CD4+T细胞分泌IL-22的水平明显降低，而抑制Notch信号通路对HBV特异性CD4+T细胞分泌IL-22的水平无显著影响。在高压尾静脉注射小鼠模型中，HBV感染可上调Notch分子、Notch分子配体和Notch信号通路下游分子的表达水平，抑制Notch信号通路对血清中HBsAg和HBeAg的分泌无影响，但可显著降低脾细胞中Th17和Th22细胞的比例，同时抑制非特异性炎症细胞向肝脏的浸润。本研究从体内和体外两方面研究了Notch-IL-22轴对HBV介导炎症应答的调节作用，初步揭示了Notch信号通路介导IL-22的分泌在HBV感染炎症应答中发挥的作用，为HBV感染后肝脏炎症反应的机制提供新的思路和实验依据。