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

项目概要/摘要 宿主的炎症反应是一把双刃剑，必须积极防御病原体， 而且还需要约束以防止对宿主的意外伤害。细胞因子风暴综合征（CSS） 代表一种可由感染引发的无节制炎症状态，包括严重急性 呼吸综合征相关冠状病毒2（SARS-CoV-2）。虽然有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， 这种反应仍然不完全理解。自噬是一种从细胞质到溶酶体的降解过程， 在宿主免疫中具有重要功能的途径。我们最近的研究表明，骨髓细胞中的自噬基因 细胞，最初是肺泡巨噬细胞（aMΦ），在由 静脉注射TNF。我们推测宿主自噬在限制S-CSS中也可能具有多效性作用。 我们在这个假设的动机，因为冠状病毒（冠状病毒）操纵宿主自噬相关的 膜通过非结构蛋白6（NSP 6）进行自身复制。这份针对NIH主任的提案 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， 既有创新的方法，也有创新的方法。该项目将利用SARS-CoV-2感染模型， 通过腺病毒（AdV）载体将人ACE 2受体（由hAce 2编码）递送至小鼠肺。此外，本发明还 我们将利用缺乏GM-CSF信号传导的小鼠来确定aMΦ特异性宿主通路的作用， 缺乏aMΦ（Csf 2 rb-/-），通过单次鼻内滴注祖细胞可持久恢复aMΦ 在新生儿中。SARS-CoV-2 nsp 6在病毒发病机制中的作用将用重组病毒确定 缺失该因子或具有天然存在的点突变，假设其促进感染。此外，委员会认为， 我们将开发一个表达sgRNA的AdV-hAce 2载体系统，在易感的呼吸道疾病中直接编辑基因， Cas9转基因受体小鼠中的细胞。我们将通过这种方法产生合并的AdV sgRNA文库，用于体内研究。 筛选方法，可以确定宿主途径的重要性，调节感染，而不是其他 通过体外方法概括。此外，我们将用aMΦ细胞祖细胞重建Csf 2 rb-/-小鼠， 包含合并的CRISPR文库，以鉴定不仅对aMΦ对SARS的反应重要的宿主基因， CoV-2，而且还用于aMΦ生态位发展的基本方面。这些研究有可能发现 开发宿主和病毒导向疗法的新领域（例如，自噬途径和NSP 6， 分别）。为组织的功能基因组研究建立的稳健和通用的体内平台 对SARS-CoV-2的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意义。 在不同的生物过程中。

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.