NIK及其介导的非经典NF-κB通路活化在慢性HBV感染中的作用和机制研究

The non-canonical NF-kB pathway plays important roles in the infection and immunity, as well as inflammation development, but its role in chronic hepatitis B virus (HBV) infection remains largely unknown. NF-κB-inducing kinase (NIK) is a central player that controls the activation of non-canonical pathways and its expression is regulated by cellular inhibitor of apoptosis proteins (cIAPs). We have found that cIAPs antagonist injection in mouse model with chronic HBV-infection resulted in enhancement of HBV-specific T cell response to clear HBV infection, accompanied with increased NIK and its downstream protein p52 expression in liver. Further, NIK is able to inhibit HBV replication and gene expression in hepatocytes directly. Thus, we speculate that NIK may represent an intrinsic host restriction factor of HBV replication in hepatocytes and its mediated activation of non-classical NF-kB pathway may contribute to immune control in HBV infection. This study intends to use clinical samples to test the correlation between the expression of NIK and its pathway molecules with viral or inflammatory markers in patients with chronic hepatitis B. Simultaneously, the underlying mechanism of antiviral action of NIK, as well as the regulatory effect on HBV-specific immune responses mediated by the non-canonical pathways will be explored in cell and animal models. The study will be helpful to further elucidate the mechanism of chronic hepatitis B immunopathogenesis and provides experimental and theoretical basis for the clinical usage of cIAPs antagonists for HBV therapy.

非经典NF-κB通路广泛参与机体抗感染免疫和炎症发生，但其在乙肝病毒（HBV）感染中的作用尚缺少研究。NF-κB诱导激酶（NIK）是控制非经典通路活化的中心分子，其表达受到细胞凋亡抑制蛋白（cIAPs）的调控，我们前期发现cIAPs拮抗剂注射可以增强慢性HBV感染小鼠模型体内HBV特异性免疫应答清除HBV，肝内的NIK及其下游通路蛋白p52的表达亦增加。进一步研究发现，NIK过表达可以抑制肝细胞HBV复制和基因表达。由此，我们推测NIK可能是肝细胞HBV复制的内源性限制因子，其介导的非经典通路活化可能参与HBV免疫控制。本研究拟采用临床样本，检测慢乙肝患者NIK及其通路分子的表达与病毒和炎症指标的相关性，同时在细胞和动物模型中探讨NIK的抗病毒作用机制及其介导的非经典通路对HBV特异性免疫应答的调控效应，从而进一步阐明慢乙肝免疫致病机制和为cIAPs拮抗剂用于乙肝治疗提供实验和理论依据。

NF-κB诱导激酶(NF-κB-inducing kinase, NIK)是宿主非经典NF-κB通路活化的关键分子，参与DNA和RNA病毒复制的调控，但其在乙型肝炎病毒（HBV）感染中的作用尚无报道。研究结果表明慢乙肝患者PBMC及T细胞中存在cIAP2高表达及NIK低表达。与NIKfl/fl小鼠相比，肝细胞特异性NIK敲除（NIKΔhep）小鼠中建模后血清HBV DNA、HBsAg、HBeAg和肝组织HBcAg表达水平显著高于对照小鼠，说明肝细胞NIK缺失可促进HBV复制和基因表达；T细胞特异性敲除NIK（NIKΔT）小鼠中，NIK缺陷导致HBV复制增加和清除障碍。反之，野生型小鼠肝内过表达NIK后，上述HBV病毒学指标明显低于对照组，表明NIK可以抑制HBV复制和转录。体外细胞模型研究显示，HepG2细胞中NIK过表达以剂量依赖性抑制HBV复制和转录。此外，NIK激酶活性位点KK431/432AA突变后不能发挥抗HBV效应，说明NIK发挥抗HBV效应依赖其激酶活性及其下游通路的激活。通过RNA-seq测序筛选差异表达基因及进行基因通路富集分析，NIK过表达后干扰素应答通路和TNFα/NF-κB信号通路被显著激活。荧光素酶报告基因和Realtime PCR检测证实IFNβ通路活化，干扰素刺激基因如ISG15/MX1/IFIT1等表达显著增加，Western blot检测证实非经典NF-κB通路活化，p100/52的表达显著增加。最后，利用CRISPR-CAS9技术分别构建p100/52敲除细胞系和IRF9敲除细胞系，发现IRF9的敲除NIK抗病毒效应显著减弱，而p100/52的敲除不影响NIK的抗病毒效应。本研究证实体内肝细胞及T细胞介导的干扰素和非经典NF-κB通路激活可以抑制HBV感染，研究成果进一步阐明了慢乙肝免疫致病机制，且为NIK激动剂用于慢乙肝的治疗提供了实验和理论依据。