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

项目摘要/摘要 宿主的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，以防止对东道主的意外伤害。细胞因子风暴综合征(CS) 表示可由感染(包括严重急性呼吸道感染)触发的不受控制的炎症状态 呼吸综合征相关冠状病毒2(SARS-CoV-2)。而失调的细胞因子的证据 存在对SARS-CoV-2相关css(S-css)的反应，确切的细胞类型和病毒因素 这一反应尚不完全清楚。自噬是一种从细胞溶质到溶酶体的降解剂 在宿主免疫中具有重要功能的途径。我们最近证明了髓系细胞中的自噬基因 细胞，初步肺泡巨噬细胞(AMΦ)，提供保护作用的小鼠模型，由 静脉注射肿瘤坏死因子。我们推测，宿主自噬也可能在限制S-css中起到多效性作用。 我们之所以提出这一假设，是因为冠状病毒(CoV)操纵与宿主自噬相关的 通过非结构蛋白6(NSP6)进行自身复制的膜。这项针对美国国立卫生研究院院长的提案 新创新者奖将测试宿主自噬和病毒拮抗剂在触发S-css中的作用 既有既定的方法，也有创新的方法。该项目将利用SARS-CoV-2感染模型，在该模型中 人ACE2受体(由hAce2编码)通过腺病毒(AdV)载体进入小鼠肺内。另外， 我们将通过利用缺乏GM-Φ信号的小鼠和 不含AmΦ(Csf2Rb-/-)，可通过单次鼻腔注入造血祖细胞用AmΦ持久修复 在新生儿身上。SARS-CoV-2 nsp6在病毒致病机制中的作用将通过重组病毒来确定 因该因子而缺失或有自然发生的点突变以促进感染的假说。此外， 我们将开发一种表达sgRNAs的AdV-hAce2载体系统，以直接编辑易感呼吸道的基因 在Cas9转基因受体小鼠中的细胞。我们将通过这种方法为体内构建池的Adv sgRNA文库 可能识别对调节感染重要的宿主途径而不是其他途径的筛选方法 通过体外实验方法进行总结。此外，我们将用AMΦ细胞前体细胞重建Csf2rb-/-小鼠 包含共用的CRISPR文库，用于识别宿主基因，这些基因不仅对AMΦ对SARS的反应至关重要- CoV-2，但也用于AmΦ利基发展的基本方面。这些研究有可能确定 发展宿主和病毒导向疗法的新领域(例如，自噬途径和NSP6， )。为组织功能基因组研究建立的强大而通用的体内平台 SARS-CoV-2近端应答的关键部位对复杂细胞的研究具有更广泛的意义 在不同的生物过程中的种群。

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.