胶质细胞在猪血凝性脑脊髓炎病毒感染CNS中的免疫应答机制

Gila are key players of the immune response in the central nervous system (CNS) and, being the resident innate immune cells, they are responsible for the early control of infections. Porcine hemagglutinating encephalomyelitis virus (PHEV) mainly invades the CNS of host, and the brain injury with nonsuppurative encephalomyelitis caused by PHEV is an important reason for acute death of infected host. Although the glia's immune resoponses in the CNS during PHEV infection remains poorly understood, we speculate that it should be the host's first line of defense against invading PHEV. Based on this hypothesis, PHEV infection of the BALB/c mice, the primary cultured glia (microglia and astrocytes) and live brain tissue slices from 2-3 day old mice were used as in vivo, in vitro and ex vitro model system, respectively, to investigate innate immune responses during PHEV's encephalitis, including the activation of glia (microglia and astrocytes) and type I interferon signaling and increased expression of inflammatory mediators in this study. The activation of glia will be evidenced by characteristic changes in cellular morphology and expression of astrocyte and microglia-specific proteins.Expression of inflammatory mediators known to be released by activated glia and genes involved in IFN signaling will be detected using the genetic chip, real-time PCR, ELISA, western boltting assays, etc. The prospective results will be helpful to understand deeply the roles of innate immune responses in the CNS during PHEV infection, and the relationship between the neuroinflammation and brain damage caused by PHEV infection.In addition, PHEV infection of the CNS will provide a model for understanding the basic pathogenic mechanisms of virus-induced CNS injury.

在中枢神经系统（CNS）损伤诱发的自身先天性免疫应答中，小胶质细胞和星形胶质细胞发挥重要作用。猪血凝性脑脊髓炎病毒（PHEV）具有典型的嗜神经性，主要侵害CNS导致感染动物死亡。在PHEV感染过程中，这些胶质细胞参与的神经免疫与抗PHEV感染和病毒致脑损伤之间的关系目前尚不清楚。本项目拟以小胶质细胞和星形胶质细胞为研究对象，开展PHEV感染小鼠脑组织中胶质细胞的激活状态及其分泌细胞因子、趋化因子及Ⅰ型干扰素信号的免疫应答研究；应用体外培养的原代胶质细胞，进行重要炎症介质和干扰素信号通路相关基因的检测，结合体内试验结果，分析其在神经免疫和神经病理损伤中的作用；进而以体外建立的小鼠脑活组织培养模型进行PHEV感染，明确炎性细胞浸润对该病毒所致神经炎症的影响程度。预期结果不仅可以系统地阐明胶质细胞在PHEV感染CNS中的免疫应答机制，而且为深入解析PHEV的致病机制奠定基础。

猪血凝性脑脊髓炎病毒（PHEV）为嗜神经性β冠状病毒，主要侵害哺乳仔猪中枢神经系统（CNS）导致其死亡，临床症状以呕吐、衰竭和神经症状为主要特征。PHEV感染过程中，CNS先天性免疫应答与病毒致脑损伤之间存在怎样的关系尚未见报道。本项目利用PHEV感染小鼠、原代神经胶质细胞、脑活组织模型，系统研究PHEV诱导胶质细胞活化、炎症因子分泌、炎性细胞浸润对神经炎症的影响以及神经胶质细胞在神经免疫和神经病理损伤中的作用。利用基因芯片检测PHEV感染诱导差异表达基因，结果显示上调基因主要与免疫过程相关，下调基因主要介导神经元功能障碍及损伤过程。小胶质细胞和星形胶质细胞在PHEV感染3d后开始活化，血脑屏障（BBB）在感染4d后通透性明显增大，CNS发生外周巨噬细胞浸润。对脑组织中细胞因子检测表明，在PHEV感染后3-4d，炎症细胞因子（IL-1α、IL-1β、IL-15、IL-17A、TNF-α）大量表达；感染后3-5d，多效应细胞因子（IL-6、IL-12p40、IL-10）、Th1型细胞因子均显著上调，而Th2型细胞因子未见显著变化。对IFN-I信号检测表明，PHEV感染小鼠3-5d，IFN-I相关PRRs（TLR3、TLR7、RIG-I、MDA5）、IFN-I信号分子（IFNAR、STAT1、STAT2、IRF7）、IFN-I信号下游ISGs及抗病毒蛋白（OAS1、Mx1、ZBP1）均呈不同程度上调。差异基因数据显示，PHEV感染小鼠中后期（3-5d）分泌大量Ⅰ型干扰素，表明PHEV为一种干扰素敏感性病毒。但在本研究中，PHEV感染早期（前24h），RAW264.7细胞中IFN-β处于被抑制状态，对其分子机制研究揭示PHEV通过抑制RIG-I信号通路中IRF3激活从而逃避IFN-β免疫应答。对离体脑活组织及原代神经胶质细胞研究表明，PHEV感染可引起小胶质细胞和星形胶质细胞活化，且小胶质细胞为分泌炎症细胞因子的主要靶细胞；抑制小胶质细胞活化促进PHEV前期复制但降低致死病毒载量，表明PHEV刺激小胶质细胞产生的炎性介质对负载PHEV的神经元具有一定保护力。上述研究阐明胶质细胞在抗PHEV感染的CNS先天免疫应答中的重要作用及分子机制，为深入解析PHEV致病机制奠定基础。