Innate Immunity of Zika Virus Infection In Human Neural Progenitors

人类神经祖细胞对寨卡病毒感染的先天免疫

• 批准号: 10372056
• 负责人: Caleb Stokes
• 金额: $ 19.5万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-16 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10372056
• 关键词: Affect; Americas; Area; Attenuated; Award; Bioinformatics; Biological Models; Brain; Cell Differentiation process; Cells; Cerebrum; Clustered Regularly Interspaced Short Palindromic Repeats; Communicable Diseases; Defect; Detection; Development; Development Plans; Disease Outbreaks; ES Cell Line; Embryo; Encephalitis; Faculty; Family member; Flavivirus; Gene Expression; Genes; Goals; Human; IFITM1 gene; Immune; Immune signaling; Immune system; Immunology; Impairment; In Vitro; Infant; Infection; Injury; Innate Immune Response; Innate Immune System; Interferons; Invaded; Knock-out; Life; Link; Mediating; Mentors; Microcephaly; Modeling; Natural Immunity; Neuraxis; Neuroglia; Neuronal Differentiation; Neurons; Neurophysiology - biologic function; Neurosciences; Organoids; Pattern recognition receptor; Predisposition; Pregnancy; Production; Proteins; Protocols documentation; Receptor Signaling; Resistance; Resources; Role; Signal Transduction; System; Techniques; Testing; Therapeutic Intervention; Time; Toll-like receptors; Training; Tretinoin; Up-Regulation; Viral Encephalitis; Virus; Virus Diseases; West Nile virus; Work; ZIKV infection; Zika Virus; career; career development; cell type; congenital zika syndrome; differential expression; experimental study; fetal; gene induction; gene network; in utero; innate immune mechanisms; insight; mosquito-borne; nerve stem cell; nervous system development; neurogenesis; novel strategies; overexpression; pathogen; progenitor; receptor; receptor expression; receptor function; relating to nervous system; response; self-renewal; single-cell RNA sequencing; stem; therapeutic target; therapy design; viral RNA; viral detection

PROJECT SUMMARY / ABSTRACT The 2016 outbreak of Zika virus (ZIKV) in the Americas demonstrated how quickly and dramatically a mosquito-borne viral infection can affect human life. Like related flavivirus family members such as West Nile virus, ZIKV can invade and infect the central nervous system (CNS), but is unique in causing in utero infection which leads to developmental abnormalities including microcephaly. ZIKV seems to have a predilection for infecting neural progenitor cells (NPCs) and persists for months in the fetal CNS after in utero infection, which suggests an ineffective immune control of the virus. We hypothesize that impaired innate immunity in neural progenitor cells underlies increased susceptibility to infection by ZIKV and contributes to microcephaly. The innate immune system includes pattern recognition receptors (PRRs) that detect pathogens and signal through effector molecules including interferon (IFN), which drives the expression of hundreds of antiviral interferon-stimulated genes (ISGs). Using induced neural progenitor cells (iNPCs) as a model for fetal CNS development, we have identified key innate immune signals that are attenuated in neural progenitors compared to mature neurons and glia: the expression levels of retinoic acid-inducible gene I (RIG-I, a PRR that detects viral RNA); and the IFN-driven upregulation of two ISGs (IFIT1 and IFITM1). We now propose to extend these findings as follows: (1) we will define the developmental changes in expression and function of PRRs during neural differentiation using the iNPC system and an established embryonic stem cell line as a control; (2) we will use CRISPR knockout or overexpression of RIG-I in bulk iNPCs and in a cerebral organoid model to test whether insufficient RIG-I signaling underlies iNPC susceptibility to ZIKV infection and microcephaly; (3) we will perform single cell RNA-seq on ZIKV-infected neural progenitors, neurons and glia to identify differentially expressed genes and gene networks, revealing innate immune components that confer susceptibility or resistance to ZIKV; and (4) using CRISPR knockout or overexpression of IFIT1 and IFITM1 we will test the role of these proteins in limiting ZIKV infection in progenitors and cerebral organoids. These experiments will define key innate immune proteins that influence susceptibility or resistance to ZIKV, identifying therapeutic targets to protect the fetal brain during ZIKV infection. Dr. Stokes’ development plan builds on a background in neurosciences with coursework and hands-on training in immunology, neural stem cells, and bioinformatics. The proposal establishes a mentoring committee including faculty in immunology, neurosciences, and infectious disease to provide guidance and career development. A K08 award will allow Dr. Stokes to make maximal use of UW’s extensive scientific resources to achieve scientific independence, advancing his career goal to develop therapeutic interventions that protect neural function from injury during viral encephalitis.

项目概要/摘要 2016 年美洲爆发的寨卡病毒 (ZIKV) 证明了蚊媒病毒感染对人类生活的影响有多快、有多严重。与西尼罗河病毒等相关黄病毒家族成员一样，寨卡病毒可以侵入并感染中枢神经系统（CNS），但其独特之处在于引起子宫内感染，从而导致包括小头畸形在内的发育异常。 ZIKV 似乎倾向于感染神经祖细胞 (NPC)，并在子宫内感染后在胎儿中枢神经系统中持续数月，这表明对该病毒的免疫控制无效。我们假设神经祖细胞先天免疫受损导致对 ZIKV 感染的易感性增加，并导致小头畸形。 先天免疫系统包括模式识别受体（PRR），可检测病原体并通过效应分子（包括干扰素（IFN））发出信号，干扰素驱动数百种抗病毒干扰素刺激基因（ISG）的表达。使用诱导神经祖细胞（iNPC）作为胎儿中枢神经系统发育的模型，我们确定了与成熟神经元和神经胶质细胞相比，神经祖细胞中减弱的关键先天免疫信号：视黄酸诱导基因 I（RIG-I，一种检测病毒 RNA 的 PRR）的表达水平；以及 IFN 驱动的两个 ISG（IFIT1 和 IFITM1）的上调。我们现在建议将这些发现扩展如下：（1）我们将使用 iNPC 系统和已建立的胚胎干细胞系作为对照，定义神经分化过程中 PRR 表达和功能的发育变化； (2) 我们将在大量 iNPC 和脑类器官模型中使用 CRISPR 敲除或过表达 RIG-I，以测试 RIG-I 信号传导不足是否是 iNPC 对 ZIKV 感染和小头畸形的易感性的基础； (3) 我们将对感染 ZIKV 的神经祖细胞、神经元和神经胶质细胞进行单细胞 RNA-seq，以识别差异表达的基因和基因网络，揭示赋予 ZIKV 易感性或抗性的先天免疫成分； (4) 使用 CRISPR 敲除或过度表达 IFIT1 和 IFITM1，我们将测试这些蛋白质在限制祖细胞和脑类器官中 ZIKV 感染中的作用。这些实验将定义影响 ZIKV 易感性或耐药性的关键先天免疫蛋白，确定在 ZIKV 感染期间保护胎儿大脑的治疗靶点。 斯托克斯博士的发展计划建立在神经科学背景之上，包括免疫学、神经干细胞和生物信息学方面的课程和实践培训。该提案建立了一个由免疫学、神经科学和传染病领域的教师组成的指导委员会，以提供指导和职业发展。 K08 奖将使 Stokes 博士能够最大限度地利用华盛顿大学广泛的科学资源来实现科学独立，从而推进他的职业目标，即开发保护神经功能免受病毒性脑炎期间损伤的治疗干预措施。