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

脑炎的清晰认识：用单细胞方法确定中枢神经系统黄病毒发病机制的基础

• 批准号: 10446620
• 负责人: Charles M Rice
• 金额: $ 68.19万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-21 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10446620
• 关键词: 3-Dimensional; Acute; Address; Adverse reactions; Affect; Antibodies; Area; Astrocytes; Attenuated; Autoantibodies; Blocking Antibodies; Brain; Brain region; Cell Culture Techniques; Cell Nucleus; Cells; Cellular Tropism; Central Nervous System Infections; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Complex; Data; Data Set; Disease; Encephalitis; Encephalomyelitis; Endocrine; Environment; Event; Exhibits; Flavivirus; Flavivirus Infections; Fluorescent in Situ Hybridization; Frequencies; Gene Expression; Healthcare Systems; Human; IFNAR1 gene; Image; Immune; Immune response; Immune signaling; Immunity; Immunologic Factors; In Vitro; Individual; Induced pluripotent stem cell derived neurons; Infection; Inflammation; Inflammatory Response; Innate Immune Response; Integration Host Factors; Interferon Type I; Interferons; Kinetics; Knock-out; Knockout Mice; Knowledge; Life; Light; Literature; Lyme Neuroborreliosis; Maps; Mediating; Microglia; Microscopy; Modeling; Mus; Neuraxis; Neuroglia; Neurons; Nuclear RNA; Outcome; Pathogenesis; Pathogenicity; Pattern; Peripheral; Persons; Play; Population; Process; Property; Research; Research Personnel; Resistance; Resolution; Role; Route; Shapes; Signal Transduction; Spinal Cord; Stress; System; Techniques; Testing; Time; Tissues; Traumatic Brain Injury; Tropism; Viral; Viral Antigens; Viral Encephalitis; Viral Pathogenesis; Virus; Virus Diseases; Virus Replication; West Nile Encephalitis; West Nile virus; Yellow Fever Vaccine; autocrine; base; brain tissue; cell type; cytokine; human model; immunomodulatory therapies; immunoreaction; improved; in vivo; induced pluripotent stem cell; knockout animal; molecular imaging; mouse model; neural model; neuroinflammation; neutralizing antibody; novel therapeutic intervention; paracrine; permissiveness; prevent; relating to nervous system; resistance factors; response; severe COVID-19; single molecule; stem cell model; tissue tropism; tool; transcriptome sequencing; transcriptomics; tumor-immune system interactions; virology

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.

脑炎黄病毒感染每年影响全球数千人，导致急性脑脊髓炎，并给医疗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)包括数十种具有不同性质和功能的细胞类型。由于缺乏将吞吐量、深度和分辨率结合在一起的适当工具，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广泛连接到外周。我们的初步研究表明，重要的脑炎病毒西尼罗河病毒(WNV)在小鼠脑内直接接种后在中枢神经系统内的趋向性与在外周感染后传播到中枢神经系统后的不同。我们假设中枢神经系统的趋向性在很大程度上是由常驻的神经和神经胶质天然免疫特性决定的，而这种天然免疫特性很容易被外周感染期间产生的免疫信号所改变。为了解决这一假设，我们将利用WNV在体内小鼠模型和使用人类诱导多能干细胞(HPSC)模型的体外研究细胞类型之间的免疫相互作用。组织清除技术和整体成像将用于使用光片显微镜显示整个大脑和脊髓中的病毒抗原，创建完整的时间分辨三维感染地图。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学相结合的方法进行检测。将使用高通量显微镜和测序来询问hPSC来源的神经元和神经胶质细胞的共培养，以确定人类细胞类型中的阻力因素和反应。将使用封闭抗体和CRISPR介导的基因敲除对HITS进行机械研究。最后，通过使用系统和细胞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趋向性和疾病中的作用。该项目将以无与伦比的分辨率提供有关黄病毒脑炎的新数据，通过在单细胞水平上定义细胞嗜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差距并提高基础知识。已确定的限制中枢神经系统感染的外围因素可能是免疫调节治疗的目标，从而促进可能改进其他形式的病毒性脑炎治疗的研究。最后，由此产生的实验流水线将广泛适用于中枢神经系统应激和炎症的研究，并与莱姆氏脑疏松症等其他疾病或创伤性脑损伤等慢性衰弱疾病相关。