Role of CD4 T cells in fatal alphavisus encephalomyelitis

描述(申请人提供)：辛德比斯病毒(SINV)是甲型病毒的原型，可引起小鼠脑脊髓炎，为研究虫媒病毒性脑脊髓炎的发病机制提供了一种模型。我们之前对SINV的研究表明，生存取决于宿主和病毒因素，包括年龄和遗传背景、病毒毒力和宿主免疫反应。神经适应性SINV(NSV)对成年C57BL/6(B6)小鼠是一种致死性脑脊髓炎模型。感染NSV的小鼠会变得虚弱，在7-10天内进展为瘫痪和死亡。病毒在感染后5-6天开始从大脑和脊髓清除，这与神经系统疾病的发病时间一致。虽然抗病毒免疫反应对于病毒清除是必不可少的，但它也可能导致致命的疾病。我们已经证明，感染NSV后，T细胞缺陷小鼠和炎症反应受到药物抑制的小鼠的存活率得到改善。因此，病毒特异性免疫细胞进入中枢神经系统(CNS)导致神经元损伤，但致死性脑脊髓炎期间导致神经元死亡的机制和免疫因素尚不清楚。在初步研究中，我们已经确定，重要的调节细胞因子白介素10(IL-10)的缺乏会加速NSV诱导的致死性麻痹疾病的发生。加速疾病与早期产生IL-17的CD4T细胞(Th17细胞)的中枢神经系统增加和病毒清除延迟有关。确定Th17细胞在新城疫病毒诱导的免疫病理学中的作用并确定它们影响预后的机制(S)对于制定干预措施和确定严重疾病易感性的宿主决定因素非常重要。这项建议的具体目的是：(1)确定在致死性病毒性脑脊髓炎期间进入中枢神经系统的Th17细胞是否具有或进化为“致病”表型。用多色流式细胞术检测IL-10-/-和野生型(WT)B6小鼠Th17细胞转录因子、细胞因子和表面受体的表达。Th17细胞的中枢神经系统环境将使用qRT-PCR和免疫分析进行评估。病毒诱导的CNS Th17细胞的转录组将被确定。(2)确定Th17细胞是否与病毒诱导的免疫病理疾病有关。Th17细胞和结果将在缺乏Th1和Th17分化所需因素的小鼠、具有遗传抗性的BALB/c小鼠、感染SINV无毒株的小鼠以及通过抗病毒抗体从致死性脑炎中拯救出来的小鼠中进行研究。(3)探讨CD4T细胞介导的神经元损伤机制及候选治疗方法。效应器将使用遗传缺陷的小鼠进行鉴定，中和 抗体、免疫组织化学和中枢神经系统切片培养，然后确定预防免疫病理中枢神经系统损伤的治疗方法。(4)确定病毒清除延迟的机制。检测中枢神经系统中干扰素-γ和E2抗体的产生，以及IL-17和其他Th17效应分子对干扰素-γ和抗E2抗体抗病毒作用的影响。

DESCRIPTION (provided by applicant): Sindbis virus (SINV), the prototype alphavirus, causes encephalomyelitis in mice and provides a model for studying the pathogenesis of arboviral encephalomyelitis. Our previous studies with SINV have shown that survival is dependent on host and viral factors that include age and genetic background, virulence of the virus and the host immune response. Neuroadapted SINV (NSV) is virulent for adult C57BL/6 (B6) mice and is a model for virus-induced fatal encephalomyelitis in mature animals. NSV-infected mice develop weakness that progresses to paralysis and death within 7-10 days. Virus clearance from the brain and spinal cord begins 5-6 days after infection and is coincident with the onset of neurological disease. Although the antiviral immune response is essential for virus clearance, it can also contribute to fatal disease. We have shown that survival after NSV infection is improved in T cell-deficient mice and in mice with pharmacologic inhibition of the inflammatory response. Therefore, virus-specific immune cells entering the central nervous system (CNS) contribute to neuronal damage, but the mechanisms and immune factors that cause neuronal death during fatal encephalomyelitis are not known. In preliminary studies, we have determined that deficiency of the important regulatory cytokine interleukin (IL)-10 accelerates the onset of fatal NSV-induced paralytic disease. Accelerated disease is associated with an early increase in the CNS of CD4+ T cells producing IL-17 (Th17 cells) and a delay in virus clearance. Determination of the role of Th17 cells in NSV-induced immunopathology and identification of the mechanism(s) by which they influence outcome is important for developing interventions and for identifying host determinants of susceptibility to severe disease. The specific aims of the proposal are: (1) Determine whether Th17 cells entering the CNS during fatal viral encephalomyelitis have or evolve a "pathogenic" phenotype. Th17 cells in IL-10-/- and wild type (WT) B6 mice will be characterized for expression of transcription factors, cytokines and surface receptors using multicolor flow cytometry. The CNS environment for Th17 cells will be assessed using qRT-PCR and immunoassays. The transcriptome of virus- induced CNS Th17 cells will be determined. (2) Determine whether Th17 cells are responsible for virus- induced immunopathologic disease. Th17 cells and outcome will be studied in mice deficient in factors required for Th1 and Th17 differentiation, genetically resistant BALB/c mice, mice infected with an avirulent strain of SINV and mice rescued from fatal encephalitis by antiviral antibody. (3) Determine the mechanism of CD4+ T cell-mediated neuronal damage and test candidate therapeutics. Effectors will be identified using genetically deficient mice, neutralizing antibodies, immunohistochemistry and CNS slice cultures followed by identification of therapies that prevent immunopathologic CNS damage. (4) Determine the mechanism for delayed virus clearance. Production of IFN-γ and E2 antibody in the CNS and the effect of IL-17 and other Th17 effector molecules on the antiviral effects of IFN-γ and anti-E2 antibody will be measured.