Alveolus as Incubator: Functional Genomic Dissection of the Host Response to SARS-CoV-2 Infection.

肺泡作为孵化器：宿主对 SARS-CoV-2 感染反应的功能基因组解剖。

• 批准号: 10245986
• 负责人: ANTHONY W ORVEDAHL
• 金额: $ 141.05万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10245986
• 关键词: 2019-nCoV; ACE2; Adenovirus Vector; Adenoviruses; Alveolar; Alveolar Cell; Alveolar Macrophages; Alveolus; Area; Autophagocytosis; Award; Biological Process; COVID-19 cytokine storm; CRISPR library; Cells; Complex; Coronavirus; Cytosol; Degradation Pathway; Development; Disease; Dissection; Epithelial Cells; Event; Genes; Granulocyte-Macrophage Colony-Stimulating Factor; Human; Immune response; Immunity; In Vitro; Incubators; Infection; Inflammation; Inflammatory Response; Injury; Intravenous; Libraries; Lung; Lysosomes; Membrane; Methods; Modeling; Mus; Myeloid Cells; Nonstructural Protein; Pathway interactions; Phenotype; Point Mutation; Population; Precipitating Factors; Role; Signal Transduction; Site; Structure of respiratory epithelium; Syndrome; System; TNF gene; Testing; Tissues; Transgenic Organisms; United States National Institutes of Health; Viral; Viral Pathogenesis; cell type; cytokine; cytokine release syndrome; functional genomics; in vivo; innovation; mouse model; neonate; pathogen; prevent; receptor; recombinant virus; reconstitution; respiratory; response; restraint; screening; stem cells; tool; vector

PROJECT SUMMARY/ABSTRACT The host inflammatory response is a double-edged sword that must vigorously defend against pathogens, but also requires restraint to prevent unintended injury to the host. The Cytokine Storm Syndrome (CSS) represents a state of unbridled inflammation that can be triggered by infections, including Severe Acute Respiratory Syndrome-associated Coronavirus 2 (SARS-CoV-2). While evidence for dysregulated cytokine responses exists for the SARS-CoV-2-associated CSS (S-CSS), the precise cell types and viral factors that precipitate this response remain incompletely understood. Autophagy is a cytosol-to-lysosome degradative pathway that has important functions in host immunity. We have recently shown that autophagy genes in myeloid cells, preliminarily alveolar macrophages (aMΦ), confer protection in a murine model of CSS induced by intravenous TNF. We hypothesize that host autophagy may also have a pleiotropic role in limiting the S-CSS. We are motivated in this hypothesis since coronaviruses (CoVs) manipulate host autophagy-associated membranes for their own replication via the nonstructural protein 6 (nsp6). This proposal for the NIH Director's New Innovator Award will test the role of host autophagy and a viral antagonist in the triggering of S-CSS using both established and innovative methods. The project will utilize a model for SARS-CoV-2 infection in which the human ACE2 receptor (encoded by hAce2) is delivered to mouse lungs via adenovirus (AdV) vector. Additionally, we will determine the role of aMΦ-specific host pathways by utilizing mice deficient for GM-CSF signaling and devoid of aMΦ (Csf2rb-/-), that are durably restored with aMΦ by a single intranasal instillation of progenitor cells in neonates. The role of SARS-CoV-2 nsp6 in viral pathogenesis will be determined with recombinant viruses deleted for this factor or with naturally occurring point mutations hypothesized to facilitate infection. Moreover, we will develop an AdV-hAce2 vector system expressing sgRNAs to edit genes directly in susceptible respiratory cells in Cas9-transgenic recipient mice. We will generate pooled AdV sgRNA libraries via this method for in vivo screening approaches that may identify host pathways important for regulating infection not otherwise recapitulated by in vitro approaches. Further, we will reconstitute Csf2rb-/- mice with aMΦ cell progenitors containing pooled CRISPR libraries to identify host genes important for not only the aMΦ response to SARS- CoV-2 but also for fundamental aspects of aMΦ niche development. These studies have the potential to identify new areas for the development of host- and viral-directed therapies (e.g., the autophagy pathway and nsp6, respectively). The robust and versatile in vivo platforms established for functional genomic studies of a tissue site critical for the proximal response to SARS-CoV-2 have broader implications for the study of complex cell populations in diverse biological processes.

项目摘要/摘要 宿主炎症反应是一把双刃剑，必须为病原体强烈防御， 但也需要克制以防止对宿主的意外伤害。细胞因子风暴综合征（CSS） 代表一种无限制的注射状态，可以由感染触发，包括严重的急性 呼吸综合征相关的冠状病毒2（SARS-COV-2）。而细胞因子失调的证据 对SARS-COV-2相关的CSS（S-CS）的反应存在，即精确的细胞类型和病毒因素 沉淀这种反应仍然不完全理解。自噬是一种胞质到溶质体降解的 在宿主免疫中具有重要功能的途径。我们最近表明髓样中的自噬基因 细胞，初步的肺泡巨噬细胞（AMφ），在CSS的鼠模型中提供保护 静脉内TNF。我们假设宿主自噬可能在限制S-CSS中也可能具有多效性。 由于冠状病毒（COVS）操纵宿主自噬相关，因此我们在这一假设中有动力 通过非结构蛋白6（NSP6）复制自己的复制膜。 NIH导演的这一建议 新的创新者奖将测试宿主自噬和病毒拮抗剂在S-CSS触发中的作用 建立和创新的方法。该项目将利用模型来进行SARS-COV-2感染 人ACE2受体（由HACE2编码）通过腺病毒（ADV）载体传递到小鼠肺。此外， 我们将通过利用缺乏GM-CSF信号传导的小鼠和 没有AMφ（CSF2RB - / - ），它们通过单次鼻内滴注祖细胞持续恢复 在新生儿。 SARS-COV-2 NSP6在病毒发病机理中的作用将由重组病毒确定 为此因素删除或假设自然发生的点突变以促进感染。而且， 我们将开发一个表达SGRNA的Adv-Hace2矢量系统，直接在易感呼吸道中编辑基因 Cas9-转基因受体小鼠中的细胞。我们将通过此方法生成汇总的Adv sgrna库来进行体内 筛选方法可能识别可能对调节感染很重要的宿主途径 通过体外方法概括。此外，我们将用Amφ细胞祖细胞重建CSF2RB - / - 小鼠 包含合并的CRISPR文库，以识别宿主基因不仅对AMφ对SARS-的反应很重要 COV-2，也用于AmφLikeDevelopment的基本方面。这些研究有可能识别 开发宿主和病毒定向疗法的新领域（例如，自噬途径和NSP6， 分别）。为组织的功能基因组研究建立的强大和多功能的体内平台 对SARS-COV-2近端反应至关重要的位点对复杂细胞的研究具有更广泛的影响 潜水员生物学过程中的种群。