Role of CD4 T cells in fatal alphavisus encephalomyelitis

描述（申请人提供）：辛德比斯病毒（SINV），原型甲病毒，引起小鼠脑脊髓炎，并为研究虫媒病毒性脑脊髓炎的发病机制提供了模型。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 SINV (NSV) 对成年 C57BL/6 (B6) 小鼠具有毒性，是成熟动物中病毒诱导的致命性脑脊髓炎的模型。感染 NSV 的小鼠会出现虚弱，并在 7-10 天内发展至瘫痪和死亡。病毒从感染后 5-6 天开始从大脑和脊髓中清除，并且与神经系统疾病的发作同时发生。尽管抗病毒免疫反应对于清除病毒至关重要，但它也可能导致致命疾病。我们已经证明，T 细胞缺陷小鼠和通过药物抑制炎症反应的小鼠，NSV 感染后的存活率得到改善。因此，进入中枢神经系统（CNS）的病毒特异性免疫细胞会导致神经元损伤，但在致命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，我们已确定重要的调节细胞因子白细胞介素 (IL)-10 的缺乏会加速致命性 NSV 诱发的麻痹性疾病的发作。疾病加速与 CNS 中产生 IL-17（Th17 细胞）的 CD4 T 细胞早期增加和病毒清除延迟有关。确定 Th17 细胞在 NSV 诱导的免疫病理学中的作用并确定它们影响结果的机制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。该提案的具体目标是：（1）确定致命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出“致病”表型。使用多色流式细胞术对 IL-10-/- 和野生型 (WT) B6 小鼠中的 Th17 细胞的转录因子、细胞因子和表面受体的表达进行表征。将使用 qRT-PCR 和免疫测定来评估 Th17 细胞的 CNS 环境。将确定病毒诱导的 CNS Th17 细胞的转录组。 (2)确定Th17细胞是否与病毒诱导的免疫病理学疾病有关。将在缺乏 Th1 和 Th17 分化所需因子的小鼠、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命性脑炎中拯救的小鼠中研究 Th17 细胞和结果。 (3)确定CD4 T细胞介导的神经元损伤的机制并测试候选疗法。将使用遗传缺陷小鼠来鉴定效应器，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性中枢神经系统损伤的疗法。 (4)确定延迟病毒清除的机制。将测量CNS中IFN-γ和E2抗体的产生以及IL-17和其他Th17效应分子对IFN-γ和抗E2抗体的抗病毒作用的影响。

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.