Defense Mechanisms Against CMV Brain Infection

描述(申请人提供)：巨细胞病毒是艾滋病患者的一种重要的机会性病原体，也是导致先天性脑部疾病的最常见的感染原因。我们最近建立了一种小鼠巨细胞病毒(MCMV)脑炎模型，其中免疫活性和免疫缺陷的小鼠通过脑室内(ICV)途径感染。与人类的情况类似，免疫功能正常的小鼠不容易感染巨细胞病毒脑炎，而免疫缺陷严重的小鼠会出现类似于晚期艾滋病患者的失控脑感染。过继转移来自巨细胞病毒感染的小鼠的脾细胞可以保护免疫缺陷的动物，这种保护与几种淋巴细胞趋化剂的脑水平升高有关。在这项提议中，需要检验的中心假设是，胶质细胞产生的CXCR3配体招募淋巴细胞，通过产生抗病毒细胞因子来控制MCMV在脑内的传播。为了验证这一假设，我们将在过继转移到免疫缺陷小鼠之前分别耗尽脾淋巴细胞亚群(即T淋巴细胞[CD4+和CD8+]、NK细胞和B细胞)，并确定在随后ICV感染后对病毒传播的影响。然后，我们将研究通过注射针对每个CXCR3配体的中和抗体来破坏趋化因子网络如何影响病毒的表达和淋巴细胞的运输。此外，我们将研究CXCR3配体在过继转移CXCR3基因敲除小鼠的脾细胞后，将淋巴细胞运输到受感染的脑中的作用。淋巴细胞对胶质细胞产生趋化因子的影响将通过确定过继转移是否增强CXCR3配体水平以及确定哪些胶质细胞类型在MCMV感染时产生这些配体来研究。将进行白介素10基因敲除小鼠的淋巴细胞转移，以确定这种细胞因子是否用于抑制感染反应中胶质细胞趋化因子的产生。抗病毒细胞因子在抑制脑内病毒复制方面的作用将通过检测有和没有过继转移脾细胞的感染小鼠大脑中肿瘤坏死因子(TNF)-α和干扰素(干扰素)-γ水平的差异来解决。最后，过继转移来自肿瘤坏死因子-α和干扰素-γ基因敲除小鼠的脾淋巴细胞亚群将被用来确定这些细胞因子是否改变了病毒的复制和在大脑中的传播。在这项拨款申请中提出的体内MCMV脑感染模型为我们提供了研究病毒性脑炎期间发生的神经发病机制和大脑防御的能力。在这一竞争性更新应用中提出的研究将为激活的神经胶质细胞产生CXCR3配体在控制病毒复制、趋化因子介导的淋巴细胞进入大脑以及产生趋化因子诱导的细胞因子网络中的作用提供新的见解。

DESCRIPTION (provided by applicant): Cytomegalovirus is a significant opportunistic pathogen in AIDS patients as well as the most common infectious cause of congenital brain disorders. We have recently developed a model of murine cytomegalovirus (MCMV) encephalitis in which immunocompetent and immunodeficient mice are infected through the intracerebroventricular (icv) route. Analogous t o t he situation in humans, immunocompetent m ice a re not susceptible t o M CMV encephalitis, w whereas mice with a severe immune deficit develop uncontrolled brain infection similar to that seen in patients with advanced AIDS. Adoptive transfer of splenocytes from MCMV-primed mice protects immunodeficient animals and this protection is associated with elevated brain levels of several lymphocyte chemoattractants. In this proposal, the central hypothesis to be tested is that glial cell-produced CXCR3 ligands recruit lymphocytes that control the intracerebral spread of MCMV via the production of antiviral cytokines. To test this hypothesis, we will individually deplete splenic lymphocyte subpopulations (i.e., T lymphocytes [CD4+ and CD8+], NK cells, and B-cells) prior to adoptive transfer into immunodeficient mice and determine the effect on viral spread following subsequent icv infection. We will then examine how disruption of chemokine networks by administration of neutralizing antibodies to each of the CXCR3 ligands affects viral expression and lymphocyte trafficking. Additionally, we will examine the role of CXCR3 ligands in trafficking of lymphocytes into infected brains following adoptive transfer of splenocytes from CXCR3 knockout mice. The effect of lymphocytes on chemokine production by glial cells will be investigated by determining if adoptive transfer amplifies CXCR3 ligand levels and identifying which glial cell types produce these ligands in response to MCMV infection. Transfer of lymphocytes from interleukin-10 knockout mice will be performed to determine if this cytokine is used to dampen glial cell chemokine production in response to infection. The role of antiviral cytokines in inhibiting viral replication in the brain will be addressed by examining differences in tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma, levels in the brains of infected mice with and without adoptively transferred splenocytes. Finally, adoptive transfer of splenic lymphocyte subpopulations, from TNF-alpha and IFN-gamma knockout mice will be used to determine if these cytokines alter viral replication and spread within the brain. The in vivo MCMV brain-infection model presented in this grant application provides us with the ability to investigate neuropathogenesis and defense of the brain occurring during viral encephalitis. The studies proposed in this competitive renewal application will provide new insights into the role of CXCR3 ligand production by activated glial cells in the control of viral replication, chemokine-mediated trafficking of lymphocytes into the brain, and the generation of chemokine-induced cytokine networks.