Defense Mechanisms Against CMV Brain Infection

描述（申请人提供）：巨细胞病毒是艾滋病患者重要的机会性病原体，也是先天性脑部疾病最常见的感染原因。我们最近建立了一种小鼠巨细胞病毒（MCMV）脑炎模型，其中免疫正常和免疫缺陷小鼠通过脑室内（icv）途径感染。与人类的情况类似，免疫能力强的小鼠对CMV脑炎不敏感，而具有严重免疫缺陷的小鼠则会发生与晚期艾滋病患者相似的无法控制的脑感染。小鼠脾细胞过继移植对免疫缺陷动物有保护作用，这种保护作用与脑内几种淋巴细胞化学引诱剂水平升高有关。在本研究中，需要验证的中心假设是，神经胶质细胞产生的CXCR3配体招募淋巴细胞，通过产生抗病毒细胞因子来控制MCMV在脑内的扩散。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单独消耗脾淋巴细胞亚群（即T淋巴细胞[CD4+和CD8+]， NK细胞和b细胞），并确定随后的icv感染对病毒传播的影响。然后，我们将研究如何通过给药CXCR3配体的中和抗体来破坏趋化因子网络，影响病毒表达和淋巴细胞运输。此外，我们将研究CXCR3配体在将CXCR3敲除小鼠的脾细胞过继转移后，在淋巴细胞运输到感染大脑中的作用。淋巴细胞对胶质细胞趋化因子产生的影响将通过确定过继性转移是否放大CXCR3配体水平和确定哪种胶质细胞类型在MCMV感染时产生这些配体来研究。将白细胞介素-10敲除小鼠的淋巴细胞进行转移，以确定该细胞因子是否用于抑制胶质细胞趋化因子的产生，以应对感染。抗病毒细胞因子在抑制病毒在大脑中的复制中的作用将通过检查肿瘤坏死因子(TNF)- α和干扰素(IFN)- γ的差异来解决，这些差异是在有和没有过继转移脾细胞的感染小鼠的大脑中。最后，将tnf - α和ifn - γ敲除小鼠的脾淋巴细胞亚群过继转移用于确定这些细胞因子是否改变病毒在脑内的复制和传播。在此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的运输以及趋化因子诱导的细胞因子网络的产生等方面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.