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.

项目概要/摘要 宿主炎症反应是一把双刃剑，必须大力防御病原体， 但也需要克制以防止对宿主造成意外伤害。 代表一种可由感染引发的肆无忌惮的炎症状态，包括严重急性感染 呼吸综合征相关冠状病毒 2 (SARS-CoV-2) 的证据表明细胞因子失调。 SARS-CoV-2 相关 CSS (S-CSS) 存在反应，确切的细胞类型和病毒因子 沉淀这种反应仍不完全清楚，自噬是一种细胞质到溶酶体的降解。 我们最近发现骨髓中的自噬基因具有重要的功能。 细胞，最初是肺泡巨噬细胞（aMΦ），在CSS诱导的小鼠模型中提供保护 我们推测宿主自噬可能在限制 S-CSS 方面也具有多效性作用。 我们之所以提出这一假设，是因为冠状病毒（CoV）操纵宿主自噬相关的 膜通过非结构蛋白 6 (nsp6) 进行自身复制 这是 NIH 主任的提案。 新创新奖将测试宿主自噬和病毒拮抗剂在触发 S-CSS 中的作用 该项目将利用一种 SARS-CoV-2 感染模型，其中 人 ACE2 受体（由 hAce2 编码）通过腺病毒 (AdV) 载体递送至小鼠肺部。 我们将利用缺乏 GM-CSF 信号传导的小鼠来确定 aMΦ 特异性宿主途径的作用， 缺乏 aMΦ (Csf2rb-/-)，通过单次鼻内滴注祖细胞即可持久恢复 aMΦ SARS-CoV-2 nsp6 在病毒发病机制中的作用将通过重组病毒来确定。 删除该因素或自然发生的点突变以促进感染。 我们将开发表达 sgRNA 的 AdV-hAce2 载体系统，以直接编辑易感呼吸道中的基因 我们将通过这种方法在体内生成混合的 AdV sgRNA 文库。 可以识别对调节感染很重要的宿主途径的筛选方法 此外，我们将用 aMΦ 细胞祖细胞重建 Csf2rb-/- 小鼠。 包含汇集的 CRISPR 文库，用于识别宿主基因，这些基因不仅对 aMΦ 对 SARS 的反应很重要 CoV-2 也适用于 aMΦ 生态位发展的基本方面，这些研究有可能确定。 开发宿主和病毒定向疗法的新领域（例如自噬途径和 nsp6， 为组织功能基因组研究而建立的强大且多功能的体内平台。 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 不同生物过程中的种群。