A clear view of encephalitis: a single cell approach to determine the basis of flaviviral pathogenesis in the central nervous system

脑黄病毒感染每年影响全球数千人，导致急性脑脊髓炎，并给卫生保健系统带来重大负担。目前，还没有针对这些危及生命的疾病的病毒特异性治疗方法。中枢神经系统（CNS）包含数十种具有不同性质和功能的细胞类型。由于缺乏将通量、深度和分辨率结合起来的适当工具，病毒与这种复杂环境之间的相互作用在很大程度上仍然是一个谜。更复杂的是，中枢神经系统还通过物理神经投射和外周免疫信号与外周广泛连接。我们的初步研究表明，重要的脑炎病毒西尼罗病毒（WNV）在小鼠直接颅内接种后在中枢神经系统内的趋向性与外周感染后扩散到中枢神经系统后的趋向性不同。我们假设中枢神经系统的偏向性在很大程度上是由常驻的神经和胶质先天免疫谱决定的，这些免疫谱很容易被外周感染过程中产生的免疫信号所改变。为了解决这一假设，我们将利用西尼罗河病毒在体内小鼠模型和体外人类诱导多能干细胞（hPSC）模型中研究细胞类型之间的免疫相互作用。组织清除技术和整个安装成像将用于可视化病毒抗原在整个大脑和脊髓使用薄片显微镜，创建一个完整的时间分辨的三维感染图。单细胞的反应将使用尖端的核RNA测序和基于显微镜的空间转录组学的组合进行检查。hpsc衍生的神经元和胶质细胞的共培养将使用高通量显微镜和测序来鉴定人类细胞类型的抗性因素和反应。将使用阻断抗体和crispr介导的敲除机制研究hit。最后，通过使用系统和细胞类型特异性敲除动物或细胞因子中和抗体，我们将研究I型干扰素在调节中枢神经系统向性和疾病中的作用。该项目将以无与伦比的分辨率提供关于黄病毒脑炎的新数据，通过在单细胞水平上定义细胞趋向性和中枢神经系统炎症反应，并评估外周信号的变化如何影响大脑感染，帮助弥合当前的信息空白，提高基础知识。已确定的限制中枢神经系统感染的外周因素可能是免疫调节治疗的靶点，从而促进可能改善其他形式病毒性脑炎治疗的研究。最后，由此产生的实验管道将广泛适用于中枢神经系统应激和炎症的研究，与莱姆病等其他疾病或创伤性脑损伤等慢性衰弱性疾病相关。

Encephalitic flavivirus infections affect thousands of people globally every year causing acute encephalomyelitis and placing significant burden on healthcare systems. Currently, no virus-specific treatments are available for these life-threatening conditions. The central nervous system (CNS) encompasses dozens of cell types with diverse properties and functions. Limited by a lack of adequate tools that combine throughput, depth and resolution, the interactions between viruses and this complex environment remain largely a mystery. To complicate matters, the CNS is also extensively connected to the periphery by both physical neural projections and peripheral immune signaling. Our preliminary studies demonstrate that tropism of the important encephalitic virus West Nile (WNV) within the CNS after direct intracranial inoculation of mice differs from that seen after spread to the CNS following peripheral infection. We hypothesize that CNS tropism is largely determined by resident neural and glial innate immune profiles, which can be readily modified by immune signals generated during peripheral infection. To address this hypothesis, we will utilize WNV to study immune interactions between cell types in an in vivo mouse model and in vitro using human induced pluripotent stem cell (hPSC) models. Tissue clearing techniques and whole mount imaging will be used to visualize viral antigens across the entire brain and spinal cord using light sheet microscopy, creating a complete time-resolved 3-dimensional map of infection. Responses of single cells will be examined using a combination of cutting-edge nuclear RNA sequencing and microscopy-based spatial transcriptomics. Co-cultures of hPSC-derived neurons and glia will be interrogated using high-throughput microscopy and sequencing to identify resistance factors and responses in human cell types. Hits will be mechanistically studied using blocking antibodies and CRISPR-mediated knockouts. Lastly, by using systemic and cell-type specific knock out animals or cytokine neutralizing antibodies, we will investigate the role of type I interferon in modulating CNS tropism and disease. This project will provide new data on flaviviral encephalitis at unparalleled resolution to help bridge current information gaps and improve fundamental knowledge by defining cellular tropism and CNS inflammatory responses at the single cell level and evaluating how changes in peripheral signaling influence infection of the brain. Identified peripheral factors restricting CNS infection are possible targets for immunomodulatory therapy, thus promoting research that may improve treatment for other forms of viral encephalitis. Finally, the resulting experimental pipeline will be broadly applicable to the study of CNS stress and inflammation, with relevance to other diseases like Lyme neuroborreliosis or chronic debilitating conditions like traumatic brain injury.