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: 10597608
• 负责人: Caleb Stokes
• 金额: $ 19.39万
• 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/10597608
• 关键词: Affect; Americas; Area; Attenuated; Award; Bioinformatics; Biological Models; Brain; Career Mobility; Cell Differentiation process; Cells; Central Nervous System; Cerebrum; Clustered Regularly Interspaced Short Palindromic Repeats; Communicable Diseases; Defect; Detection; Development; Development Plans; Disease Outbreaks; ES Cell Line; Encephalitis; Faculty; Family member; Flavivirus; Flow Cytometry; 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; Neuroglia; Neuronal Differentiation; Neurons; Neurophysiology - biologic function; Neurosciences; Organoids; Pathogen detection; Pattern recognition receptor; Predisposition; Pregnancy; Production; Proteins; Protocols documentation; Receptor Signaling; Resistance; Resources; Role; Signal Induction; Signal Transduction; System; Techniques; Testing; Therapeutic Intervention; Time; Toll-like receptors; Training; Tretinoin; Up-Regulation; Viral; Viral Encephalitis; Virus; Virus Diseases; West Nile virus; Work; ZIKV infection; Zika Virus; career development; cell type; congenital zika syndrome; differential expression; embryonic stem cell; experimental study; fetal; gene induction; gene network; in utero; innate immune mechanisms; insight; mosquito-borne; nerve stem cell; nervous system development; neural; neurogenesis; novel strategies; overexpression; progenitor; receptor; receptor expression; receptor function; response; self-renewal; single-cell RNA sequencing; 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.

项目总结/摘要 2016年在美洲爆发的寨卡病毒（ZIKV）证明了蚊子传播的病毒感染对人类生活的影响是多么迅速和巨大。与相关的黄病毒家族成员（如西尼罗河病毒）一样，ZIKV可以侵入并感染中枢神经系统（CNS），但在引起子宫内感染方面是独特的，这导致发育异常，包括小头畸形。ZIKV似乎偏好感染神经祖细胞（NPC），并且在子宫内感染后在胎儿CNS中持续数月，这表明病毒的免疫控制无效。我们假设神经祖细胞的先天免疫受损是ZIKV感染易感性增加的基础，并导致小头畸形。 先天免疫系统包括模式识别受体（PRR），其检测病原体并通过包括干扰素（IFN）的效应分子发出信号，干扰素驱动数百种抗病毒干扰素刺激基因（ISG）的表达。使用诱导的神经祖细胞（iNPC）作为胎儿CNS发育的模型，我们已经确定了与成熟神经元和神经胶质细胞相比在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驱动的两种ISG（IFIT 1和IFITM 1）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，确定PRRs在神经分化过程中的表达和功能的发育变化;（2）我们将在散装iNPC和脑类器官模型中使用CRISPR敲除或RIG-I过表达来测试是否存在RIG-I不足。（3）我们将对ZIKV感染的神经祖细胞、神经元和神经胶质进行单细胞RNA-seq以鉴定差异表达的基因和基因网络，揭示赋予对ZIKV的易感性或抗性的先天免疫组分;以及（4）使用IFIT 1和IFITM 1的CRISPR敲除或过表达，我们将测试这些蛋白质在限制祖细胞和脑类器官中的ZIKV感染中的作用。这些实验将确定影响ZIKV易感性或抗性的关键先天免疫蛋白，确定治疗靶点以在ZIKV感染期间保护胎儿大脑。 博士斯托克斯的发展计划建立在神经科学的背景上，包括免疫学、神经干细胞和生物信息学的课程和实践培训。该提案建立了一个指导委员会，包括免疫学，神经科学和传染病教师提供指导和职业发展。K 08奖将使斯托克斯博士能够最大限度地利用华盛顿大学广泛的科学资源，实现科学独立，推进他的职业目标，开发治疗干预措施，保护神经功能免受病毒性脑炎损伤。