CHEMOKINE-MEDIATED NEUROPROTECTION AND REPAIR DURING WNV ENCEPHALITIS

描述（由申请人提供）：该竞争性更新的长期实验目的是评价趋化因子及其受体在WNV脑炎小鼠模型中促进病毒清除、免疫病理学和修复的功能作用。鼠WNV感染是公认的黄病毒脑炎模型，其中用低接种量的强毒株皮下感染导致免疫功能低下或失调的个体中的神经侵袭性疾病。因此，鼠实验研究已经确定了决定人类WNV脑炎的神经发病机制和结果的病毒和宿主因素，包括决定浸润免疫细胞进入和命运的几种趋化因子受体。在已发表的研究中，我们已经确定趋化因子CXCL 12和CXCL 10分别以区域特异性方式控制白细胞进入血脑屏障（BBB）处的中枢神经系统（CNS）实质和CNS实质内。在初步研究中，我们还观察到，西尼罗河病毒感染导致神经发生区内BrDU掺入增加，表明成年神经发生受到病毒感染的影响。此外，我们已经确定TLR 3信号传导影响神经前体细胞转录因子Pax 6和巢蛋白在几个脑区的表达，提供了一个连接病毒感染和先天免疫分子的激活已知调节趋化因子的表达和神经发生。目前的建议旨在了解血脑屏障运输保护性与致病性白细胞和病毒进入的免疫学基础。由于趋化因子上调也招募神经前体细胞进行修复，我们将另外评估趋化因子如何影响WNV损伤的CNS内的神经发生。我们将解决这些问题在三个目标：1）确定促炎途径负责迁移的保护性与致病性白细胞通过血脑屏障在西尼罗河病毒脑炎。这一目标将确定白细胞进入血脑屏障和特定中枢神经系统区域内运输的分子线索。此外，我们将确定CXCR 4在T细胞亚群进入BBB中的作用; 2）确定参与WNV进入的分子机制和在BBB中的作用。该目标将使用体外和体内方法来确定WNV对CNS内皮和病毒侵袭的影响;和3）检查WNV脑炎期间趋化因子受体介导的神经保护和修复策略。这一目标将确定西尼罗河病毒感染如何影响成年中枢神经系统中神经细胞的产生以及控制这一过程的趋化因子。总之，这些研究将扩展我们目前对趋化因子及其受体如何控制CNS炎症并最终在WNV脑炎期间修复的理解。 公共卫生相关性：病毒性脑炎是一种新兴的全球健康威胁，缺乏有效的治疗策略。特别地，节肢动物传播的黄病毒、西尼罗河病毒（WNV）、日本脑炎病毒（JEV）、蜱传脑炎病毒（TBEV）、墨累谷脑炎病毒（MVEV）和圣路易斯脑炎病毒（SLEV）引起全世界脑炎的爆发，总的年病例率超过50，000（www.who.int）。关于黄病毒脑炎的神经发病机制的重要问题之一是，个体发展神经侵袭性疾病是否是因为外周的病毒控制受损和/或因为血脑屏障（BBB）生物学的根本差异，以及病毒介导的内源性修复机制的作用是否改变功能结果。此外，尽管这些嗜细胞性神经病毒引起的中枢神经系统感染（CNS）需要T淋巴细胞的实质穿透以清除病毒，但也有数据表明CNS内这些免疫细胞的存在诱导免疫病理学损伤。因此，了解控制病毒和白细胞亚群进入BBB的机制，对于开发可以预防高危患者神经侵袭并在神经侵袭发生后促进CNS内适当的抗病毒免疫应答的治疗方法至关重要。本提案旨在了解BBB运输保护性与致病性白细胞和病毒进入的免疫学基础，并进一步评估WNV受损CNS内的成人神经发生。这些研究将扩展我们目前对控制西尼罗河病毒脑炎期间CNS炎症和修复的分子机制的理解。此外，从这些实验中获得的数据可以鉴定用于治疗性治疗患有黄病毒脑炎的人的潜在靶标。

DESCRIPTION (provided by applicant): The long term experimental objective of this competitive renewal is to evaluate the functional roles of chemokines and their receptors in contributing to viral clearance, immunopathology and repair in a murine model of WNV encephalitis. Murine WNV infection is well-accepted flavivirus encephalitis model whereby subcutaneous infection with low inoculum of virulent strains leads to neuroinvasive disease in individuals with depressed or dysregulated immune function. Thus, murine experimental studies have identified viral and host factors that determine the neuropathogenesis and outcome of WNV encephalitis in humans, including several chemokine receptors that determine the entry and fate of infiltrating immune cells. In published studies we have determined that the chemokines CXCL12 and CXCL10 control leukocyte access to the central nervous system (CNS) parenchyma at the blood-brain barrier (BBB) and within the CNS parenchyma in a region-specific manner, respectively. In preliminary studies we have also observed that WNV infection led to increased BrDU incorporation within neurogenic zones, suggesting that adult neurogenesis is affected by viral infection. In addition, we have determined that TLR3 signaling affects the expression of neural precursor cell transcription factors Pax6 and nestin in several brain regions, providing a link between viral infection and activation of innate immune molecules known to regulate both chemokine expression and neurogenesis. The present proposal seeks to understand the immunological basis of BBB trafficking for protective versus pathogenic leukocytes and for virus entry. Because chemokine up-regulation also recruits neural precursor cells for repair, we will additionally evaluate how chemokines influence neurogenesis within the WNV-damaged CNS. We will address these questions in three aims: 1) Identify the proinflammatory pathways responsible for the migration of protective versus pathogenic leukocytes across the BBB during WNV encephalitis. This aim will determine molecular cues for leukocyte entry at the BBB and trafficking within specific CNS regions. Additionally, we will determine the role of CXCR4 in T cell subset entry at the BBB; 2) Identify molecular mechanisms involved in WNV entry and effects at the BBB. This aim will use in vitro and in vivo approaches to determine WNV effects on CNS endothelium and viral invasion; and 3) Examine chemokine receptor-mediated strategies for neuroprotection and repair during WNV encephalitis. This aim will determine how WNV infection affects the generation of neural cells in the adult CNS and the chemokines that control this process. Together, these studies will extend our current understanding of how chemokines and their receptors control CNS inflammation and ultimately repair during WNV encephalitis. PUBLIC HEALTH RELEVANCE: Viral encephalitis is an emerging global health threat without effective therapeutic strategies. In particular, the arthropod-borne flaviviruses, West Nile (WNV), Japanese encephalitis (JEV), Tick-borne encephalitis (TBEV, Murray Valley encephalitis (MVEV) and St. Louis encephalitis (SLEV) viruses, cause outbreaks of encephalitis worldwide, with a total annual case rate of over 50,000 (www.who.int). Among the important questions regarding the neuropathogenesis of flavivirus encephalitis is whether individuals that develop neuroinvasive disease do so because of impaired virologic control in the periphery and/or because of fundamental differences in blood-brain barrier (BBB) biology and whether viral-mediated effects on endogenous repair mechanisms alter functional outcomes. In addition, although central nervous system infections (CNS) with these cytopathic neurotropic viruses require the parenchymal penetration of T lymphocytes for virus clearance, there are also data suggesting that the presence of these immune cells within the CNS induces immunopathologic damage. Thus, understanding the mechanisms that govern the entry of virus and leukocyte subsets at the BBB is essential for developing treatments that can prevent neuroinvasion in patients at risk and promote appropriate antiviral immune responses within the CNS after neuroinvasion has occurred. The present proposal seeks to understand the immunological basis of BBB trafficking for protective versus pathogenic leukocytes and for virus entry and to additionally evaluate adult neurogenesis within the WNV-damaged CNS. These studies will extend our current understanding of the molecular mechanisms that control CNS inflammation and repair during WNV encephalitis. Further, the data obtained from these experiments may identify potential targets for therapeutic treatment of humans with flavivirus encephalitides.