Mechanisms of cytokine-mediated viral clearance from neurons

细胞因子介导的神经元病毒清除机制

• 批准号: 7483902
• 负责人: LAUREN Alene O'DONNELL
• 金额: $ 4.68万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7483902
• 关键词: Address; Adult; Age; Brain; CD46 Antigen; Cell Line; Cells; Defense Mechanisms; Dependence; Development; Disease; Fibroblasts; Heterogeneity; Hippocampus (Brain); Image; Immune response; Immune system; In Vitro; Interferon Type II; Interferons; Knock-out; Laboratories; Measles virus; Measures; Mediating; Modeling; Mus; Neuraxis; Neuroglia; Neurons; Neuropathogenesis; Phosphorylation; Play; Property; Recruitment Activity; Reverse Transcriptase Polymerase Chain Reaction; Role; STAT1 gene; Signal Pathway; Signal Transduction; Subacute Sclerosing Panencephalitis; T-Lymphocyte; Transcription Coactivator; Transgenic Organisms; Viral; Viral Physiology; Virus Diseases; Virus Replication; Western Blotting; cell type; cytokine; in vivo; mouse model; neonate; neuropathology; receptor; response

DESCRIPTION (provided by applicant): Measles virus (MV) infection in the central nervous system (CMS) is associated with neuropathological disorders, such as subacute sclerosing panencephalitis (SSPE), in which the immune system fails to control MV replication in the brain. Our laboratory uses a transgenic mouse model of CNS-restricted MV infection via targeted neuronal expression of one of the measles virus receptors (CD46) to study interactions between MV-infected neurons and the immune response. In this model, adult mice successfully clear MV from neurons in the brain by mounting an immune response that requires interferon-gamma (IFN?) and involves T-cell recruitment to the CNS, whereas MV-infected neonates recruit similar levels of T-cells to the brain, but are unable to control MV replication in neurons, develop extensive neuropathology, and rapidly succumb to MV infection. While IFN? is necessary for MV clearance from neurons in adult brains in vivo, IFN? triggers a distinct signaling cascade in primary cultured neurons in comparison to the more extensively studied fibroblasts and cell lines. A paradox emerges from these preliminary observations: how does IFN? mediate viral clearance from neurons, when the canonical signaling components (as measured by phosphorylation/activation of the transcriptional activator STAT1) are restricted? We hypothesize that limited STAT1 activation by IFN? in neurons contributes to viral clearance, but that other signaling pathways contribute to anti-viral activity in neurons. To address this hypothesis, Aim 1 dissects the mechanisms IFN?-mediated MV clearance in explanted primary hippocampal neurons in vitro. Through western blot, confocal imaging, and quantitative RT-PCR, the intrinsic ability of primary neurons to mediate viral clearance will be addressed independently of the complicating contribution of glial cells found in mixed CNS cultures. Aim 2 will extend the observations made in primary cultured neurons by establishing the role of IFN? signal transduction in the complicated microenvironment of the brain in vivo. These studies will be aided by genetically modified mice (e.g. IFN? knockouts, STAT1 knockouts) and will allow us to address the age-dependence of neuropathogenesis. By determining the role of IFN? signaling in MV clearance in neurons, these studies will clarify how IFN? triggers anti-viral defense mechanisms in a unique, non-renewable cell type, but also aim to provide a mechanism for the cell-specific heterogeneity of responses to IFN?. In addition, these studies will also address how intrinsic developmental neuronal properties play a role in dictating whether or not viral clearance occurs in CNS neurons.

描述（由申请人提供）：中枢神经系统（CMS）中的麻疹病毒（MV）感染与神经病理性疾病有关，例如亚急性硬化性全脑炎（SSPE），其中免疫系统无法控制大脑中的 MV 复制。我们的实验室通过麻疹病毒受体之一（CD46）的靶向神经元表达，使用 CNS 限制性 MV 感染的转基因小鼠模型来研究 MV 感染的神经元与免疫反应之间的相互作用。在该模型中，成年小鼠通过产生需要干扰素γ（IFN？）的免疫反应并涉及将T细胞募集到中枢神经系统，成功地从大脑神经元中清除MV，而感染MV的新生儿将相似水平的T细胞募集到大脑，但无法控制神经元中的MV复制，出现广泛的神经病理学，并迅速死于MV感染。而干扰素？对于成人大脑体内神经元的 MV 清除是必需的，IFN？与更广泛研究的成纤维细胞和细胞系相比，在原代培养的神经元中触发独特的信号级联。这些初步观察结果出现了一个悖论：干扰素是如何发挥作用的？当经典信号成分（通过转录激活剂 STAT1 的磷酸化/激活来测量）受到限制时，介导病毒从神经元中清除？我们假设 IFN 限制了 STAT1 的激活？神经元中的信号通路有助于病毒清除，但其他信号通路有助于神经元中的抗病毒活性。为了解决这一假设，目标 1 剖析了体外移植的原代海马神经元中 IFNα 介导的 MV 清除机制。通过蛋白质印迹、共聚焦成像和定量 RT-PCR，将独立于混合 CNS 培养物中发现的神经胶质细胞的复杂贡献来解决初级神经元介导病毒清除的内在能力。目标 2 将通过确定 IFN? 的作用来扩展在原代培养神经元中进行的观察。体内大脑复杂微环境中的信号转导。这些研究将得到基因改造小鼠（例如 IFN? 敲除、STAT1 敲除）的帮助，并使我们能够解决神经发病机制的年龄依赖性问题。通过确定IFN的作用？神经元MV清除中的信号传导，这些研究将阐明IFN如何发挥作用？在独特的、不可再生的细胞类型中触发抗病毒防御机制，但也旨在为细胞特异性的 IFN? 反应异质性提供机制。此外，这些研究还将探讨内在的发育神经元特性如何在决定中枢神经系统神经元中是否发生病毒清除方面发挥作用。