Immune cell dynamics during central nervous system viral infection

项目总监/首席调查员(最后、第一、中间)：De La Torre，胡安C.1R01 AI075298-01A2 病毒可以在中枢神经系统中诱导多种疾病状态。脑膜炎是一种由包括病毒在内的一长串人类病原体引起的潜在致命疾病，通常与发烧、头痛、颈部僵硬和癫痫等症状有关。目前，除了缓解症状外，病毒性脑膜炎患者几乎无能为力。我们认为，对这一致病过程的实时详细了解可能会促进新干预措施的发展，以缓解症状和预防永久性神经功能障碍和死亡。为了实现这一目标，我们建议研究由淋巴细胞性脉络膜脑膜炎病毒(LCMV)引起的脑膜炎，LCMV是一种非细胞病变的小鼠，也是人类的病原体。小鼠脑内接种LCMV可在6天内导致致死性脑膜炎，几乎完全由细胞毒性淋巴细胞(CTL)介导。重要的是，这种疾病可以通过事先接种疫苗或免疫完全预防。在感染控制失败或有效的过程中，脑膜中细胞相互作用的动力学以前还没有研究过。此外，在这个模型中调节致命性损伤的确切机制还不完全清楚。我们将利用最先进的病毒反向遗传学、荧光标记的免疫细胞和双光子激光扫描显微镜结合不同分子物种的原位染色来跟踪感染LCMV的小鼠大脑皮层和脑膜空间中的局部免疫细胞动态。我们的假设是，在急性巨细胞病毒诱导的脑膜炎过程中，CTL对中枢神经系统星形胶质细胞网络的破坏会导致全身性致死性癫痫发作，而快速反应和通过激活的记忆T细胞在免疫小鼠中使用替代效应机制可以对星形胶质细胞网络造成有限的破坏，维持血脑屏障，并提高存活率。这一假说将通过以下两个特定目标来解决：1)首次实时分析CTL与感染荧光标记LCMV的CNS靶标之间的相互作用；2)在体内评估免疫突触形成和参与CNS CTL靶向和损伤的分子机制。

Program Director/Principal Investigator (Last, First, Middle): De La Torre, Juan C. 1R01 AI075298-01A2 Viruses can induce a variety of disease states in the central nervous system. Meningitis is a potentially fatal disorder induced by a long list of human pathogens, including viruses, and is often associated with symptoms that include fever, headache, stiffness of the neck, and seizures. Presently, very little can be done for patients with viral meningitis other than to relieve symptoms. We propose that a detailed understanding of this pathogenic process in real time may foster the development of novel interventions to alleviate symptoms and prevent permanent neurological dysfunction and fatalities. To accomplish this goal we propose to study the well-described meningitis induced by lymphocytic choriomeningitis virus (LCMV), a noncytopathic mouse as well as human pathogen. Intracerebral inoculation of mice with LCMV results in a fatal meningitis within 6 days that is mediated almost entirely by cytotoxic lymphocytes (CTL). Importantly, this disease can be completely prevented by prior vaccination or immunization. The dynamics of cellular interactions in the meninges during failed or effective control of infection have not been studied previously. Moreover, the precise mechanisms that mediate fatal injury in this model are not entirely understood. We will utilize a combination of state-of-the art viral reverse genetics, fluorescently-tagged immune cells, and two-photon laser scanning microscopy in combination with in situ staining for different molecular species to follow the local immune cell dynamics in the LCMV-infected mouse cerebral cortex and meningeal space. Our hypothesis is that CTL damage to astrocyte networks in the CNS leads to generalized fatal seizure during acute LCMV-induced meningitis, and that rapid responsiveness and the use of alternative effector mechanisms by activated memory T cells in vaccinated mice results in limited damage to astrocyte networks, maintenance of the blood brain barrier, and survival. This hypothesis will be addressed by the two focused following specific aims: 1) Completion of the first real time analyses of interactions between CTL and CNS targets infected by fluorescently-tagged LCMV, and 2) in vivo evaluation of immunological synapse formation and molecular mechanisms involved in CNS CTL targeting and damage.