Expanding Excellence in Developmental Biology in Oklahoma Supplement: 3D Human Lung Tissue Model to Dissect Cellular Responses upon SARS-CoV-2 Infection

摘要 SARS-CoV-2感染是2019冠状病毒病（COVID-19）的病原体，这是一种全球大流行病 导致全球超过600万人死亡。人类气道的粘膜纤毛上皮是主要的 SARS-CoV-2感染部位。在病毒暴露后，上皮细胞启动应答以募集免疫细胞 并协调先天免疫反应。每种细胞类型数量的平衡 (i.e.,纤毛，分泌）是维持健康上皮，和气道上皮损伤和变化的关键 细胞类型丰度（称为重塑）在与SARS-CoV-2相关的病理学中起重要作用 感染然而，我们对免疫细胞相互作用和新出现的SARS-CoV-2变异的理解， 影响病毒诱导的气道上皮损伤和重塑是有限的。我们的项目将测试中央 假设骨髓细胞反应和与上皮细胞的相互作用是炎症的主要驱动因素- 在SARS-CoV-2感染的上气道中介导的上皮细胞重塑。使用新型体外3D模型 人上呼吸道上皮细胞，包括原代分化的气道上皮细胞，肺成纤维细胞，肺成纤维细胞， 微血管内皮细胞和骨髓免疫细胞，结合尖端的scRNA-seq技术， 我们的研究小组将解决以下关于宿主对SARS-CoV-2的免疫反应的未解决的问题。 在目标1中，我们将确定在骨髓增生的初始阶段，上皮细胞和先天性骨髓细胞之间的串扰是如何发生的。 SARS-CoV-2感染影响细胞分化过程，导致病理性重塑和损伤 呼吸道上皮细胞。在目标2中，我们将确定SARS-CoV-2祖先毒株和Delta和 Omicron变异体在初始感染时引起不同的宿主免疫应答和致病性上皮重塑 相同的主机环境。我们的新型3D气道模型的优势包括跟踪动力学的能力 宿主细胞在感染后确定时间的反应，以捕获早期和后期事件， 比较每种SARS-CoV-2变异体对同一组供体人类细胞的影响。为了实现这一 在项目中，我们组建了一个多学科团队，拥有非重叠和协同的专业知识。博士 肺细胞生物学家和病毒学家Matthew Walters（联合项目负责人）将监督SARS-CoV-2感染 研究和表征上皮重塑表型。Susan Kovats博士（联合项目负责人）， 一位在呼吸道病毒引起的肺骨髓反应方面具有专长的免疫学家将监督肺模型 构建并表征组织免疫应答。Willard Freeman博士（共同研究者）， 转录组分析，将监督scRNA-seq分析。收集的数据将构成新的多- PI NIH R 01提案的目标是了解不同的宿主环境（例如，整个生命周期的年龄， 性别和慢性肺病）调节宿主免疫应答和气道上皮重塑 呼吸道病毒如SARS-CoV-2、RSV和流感病毒感染。

Abstract SARS-CoV-2 infection is the causative agent of coronavirus disease 2019 (COVID-19), a global pandemic responsible for >6 million deaths worldwide. The mucociliary epithelium lining the human airways is the primary site of SARS-CoV-2 infection. Following viral exposure, the epithelium initiates responses to recruit immune cells to the site of infection and coordinate the innate immune response. A balance in the number of each cell type (i.e., ciliated, secretory) is critical to maintaining a healthy epithelium, and airway epithelial damage and changes in cell type abundance (termed remodeling) play important roles in the pathology associated with SARS-CoV-2 infection. However, our understanding of how immune cell interactions and emergent SARS-CoV-2 variants impact virus-induced damage and remodeling of the airway epithelium is limited. Our project will test the central hypothesis that myeloid cell responses and interactions with epithelial cells are major drivers of inflammation- mediated epithelial cell remodeling in SARS-CoV-2 infection of the upper airway. Using a novel in vitro 3D model of the human upper airway, composed of primary differentiated airway epithelial cells, lung fibroblasts, pulmonary microvascular endothelial cells and myeloid immune cells, combined with cutting edge scRNA-seq technology, our team will address the following unresolved questions regarding the host immune response to SARS-CoV-2. In Aim 1, we will determine how cross-talk between epithelial and innate myeloid cells in the initial stages of SARS-CoV-2 infection impacts the cell differentiation processes that drive pathological remodeling and damage of the airway epithelium. In Aim 2, we will determine if the SARS-CoV-2 ancestral strain and the Delta and Omicron variants elicit distinct host immune responses and pathogenic epithelial remodeling upon initial infection of the same host environment. The strengths of our novel 3D airway model include the ability to track the kinetics of the host cell response at defined times post-infection to capture early and later events and the ability to compare the impact of each SARS-CoV-2 variant on the same set of donor human cells. To accomplish this project, we have assembled a multi-disciplinary team with non-overlapping and synergistic expertise. Dr. Matthew Walters (Co-Project Lead), a lung cell biologist and virologist, will oversee the SARS-CoV-2 infection studies and characterize the epithelial remodeling phenotypes. Dr. Susan Kovats (Co-Project Lead), an immunologist with expertise in the pulmonary myeloid response to respiratory viruses, will oversee lung model construction and characterize the tissue immune response. Dr. Willard Freeman (Co-Investigator), an expert in transcriptome profiling, will oversee the scRNA-seq analyses. The data collected will form the basis of new multi- PI NIH R01 proposals with the goal to understand how different host environments (e.g., age across the lifespan, gender and chronic lung disease) modulate host immune responses and airway epithelium remodeling upon infection by respiratory viruses such as SARS-CoV-2, RSV and influenza virus.