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

基本信息

批准号：
10553697
负责人：
Charles M Rice
金额：
$ 62.82万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
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/10553697
关键词：
3-Dimensional Acute Address Adverse reactions Affect Antibodies Area Astrocytes Attenuated Autoantibodies Blocking Antibodies Brain Brain region Cell Culture Techniques Cells Cellular Tropism Central Nervous System Central Nervous System Diseases 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 Invaded Kinetics Knock-out Knockout Mice Knowledge Life Light Literature Lyme Neuroborreliosis Maps Mediating Microglia Microscopy Modeling Mus 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 Visualization West Nile Encephalitis West Nile viral infection West Nile virus Yellow Fever Vaccine autocrine cell type cytokine human model immunomodulatory therapies immunoreaction improved in vivo induced pluripotent stem cell knockout animal molecular imaging mouse model neural neural model neuroinflammation neutralizing antibody novel therapeutic intervention paracrine permissiveness prevent resistance factors response severe COVID-19 single molecule single nucleus RNA-sequencing stem cell model tissue tropism tool transcriptome sequencing transcriptomics tumor-immune system interactions virology

项目摘要

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.

每年全球成千上万的全球范围内，脑脑膜炎会导致急性脑脊髓炎，并给医疗保健系统带来巨大负担。当前，对于这些威胁生命的疾病，尚无病毒特异性治疗。中枢神经系统（CNS）包括数十种具有不同特性和功能的细胞类型。由于缺乏结合吞吐量，深度和分辨率的足够工具的限制，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。使事情复杂化，中枢神经系统也通过物理神经投影和周围免疫信号传导广泛连接到周围。我们的初步研究表明，在周围感染后传播后传播后传播后传播后，颅内接种直接颅内接种后，中枢神经系统内的重要脑病病毒（WNV）的偏见与中枢神经系统有所不同。我们假设中枢神经系统的向流主要取决于常驻神经和神经胶质的先天免疫特征，可以通过周围感染期间产生的免疫信号轻易修饰。为了解决这一假设，我们将利用WNV研究体内小鼠模型中细胞类型之间的免疫相互作用，并使用人类诱导的多能干细胞（HPSC）模型在体外进行体外。组织清除技术和整个安装成像将用于使用轻度显微镜可视化整个大脑和脊髓的病毒抗原，从而形成完整的时间分辨的3维感染图。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来检查单细胞的反应。 HPSC衍生的神经元和神经胶质的共培养将使用高通量显微镜和测序进行询问，以识别人类细胞类型中的抗性因子和反应。将使用阻断抗体和CRISPR介导的敲除对命中进行机械研究。最后，通过使用全身和细胞类型的特异性敲除动物或中和抗体中和抗体，我们将研究I型干扰素在调节中枢神经系统的疗法和疾病中的作用。该项目将在无与伦比的分辨率下提供有关黄素脑炎的新数据，以帮助弥合当前信息差距并改善基本知识，通过定义单个细胞水平上的细胞向热和CNS炎症反应，并评估周围信号传导的变化如何影响大脑的感染。确定的限制中枢神经系统感染的外围因素是免疫调节治疗的可能靶标，从而促进研究可以改善其他形式的病毒性脑炎的治疗方法。最后，由此产生的实验管道将广泛适用于CNS应力和炎症的研究，与其他疾病有关，例如莱姆神经性疾病或慢性衰弱状况，例如创伤性脑损伤。