COVID-19: Elucidating the Role of the NasalEpithelium in SARS-CoV-2 Infection, Transmission, and Prevention

摘要 冠状病毒病原体SARS-CoV-2 疾病2019(新冠肺炎)导致了一场大流行，死亡率约为3.5%，范围广泛 发病率结果受先前存在的疾病的负面影响。考虑到预先存在的 退伍军人并发疾病，了解SARS-CoV-2入侵机制势在必行 并在鼻子的屏障防御细胞内复制，这是病毒进入的主要门户。此外， 目前的数据表明，鼻腔携带病毒具有潜在的病毒持久性和 在严重呼吸道症状出现之前和期间传播(即脱落) 症状。该项目利用一个独特的生物库，从1000多个鼻细胞库中收集冷冻保存的鼻细胞库。 超过15年的个人了解围绕SARS-CoV-2与人类相互作用的关键问题 鼻腔上皮细胞。 矛盾的是，虽然SARS-CoV-2可以在鼻拭子中检测到，但在痰中检测到它之前， 与新冠肺炎有关的鼻科症状稀少(5%伴有鼻塞)，但 30%-70%的患者出现可逆性嗅觉障碍。这尤其成问题，因为高达25%的感染者 个人仍然没有症状，但可以通过空气中的飞沫继续传播SARS-CoV-2。这部作品 旨在阐明控制哪些上皮细胞系感染病毒和 在受感染的或邻近的上皮细胞内产生的免疫反应的类型。通过这种方式，我们将 阐明为什么某些人从不出现症状，而另一些人则进展为严重的问题 呼吸衰竭和死亡。 我们将重点关注SARS-CoV-2受体血管紧张素转换酶2(ACE2)，这是必不可少的 并且足以让病毒进入细胞。我们的初步数据来自于对原代细胞的单细胞RNA分析 人类鼻窦组织表明，ACE2在离散的鼻上皮团中表达。ACE2- 人鼻上皮细胞和原代纤毛气液界面的特异性免疫染色 培养证实了sc-RNAseq数据。此外，我们的数据显示，接种原代ALI培养物 SARS-CoV-2的感染导致大约1%-25%的细胞被感染，这表明这是一个选择性的过程。 这些数据表明，我们唯一准备检验的假设是，表达ACE2的细胞构成 独特的病毒复制储存库，可能会启动一种独特的炎性细胞因子反应 来自未感染的上皮细胞。利用我们在鼻腔上皮细胞生物学方面的专家团队，病毒 发病机制、炎性细胞因子生物学和遗传学我们将确定以下内容：a)哪些类型的 上皮细胞是病毒感染的，B)感染的细胞与未感染的细胞的局部炎性级联反应是什么，以及 C)对上皮天然防御通路的药理学操作是否会显著改变SARS-CoV-2 人鼻上皮细胞感染、复制和释放的能力。这项工作很有可能成功完成 对制定新的战略以抗击COVID19疾病的发展产生重大影响 普通人群，特别是美国退伍军人。

ABSTRACT Severe acute respiratory syndrome coronavirus SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19) has led to a pandemic with a mortality of approximately 3.5% and a wide range of morbidity outcomes negatively impacted by pre-existing conditions. Given the prevalence of pre-existing comorbid conditions in Veterans, it is imperative to understand the mechanisms of how SARS-CoV-2 invades and replicates within the barrier defense cells of the nose, which is the primary portal for viral entry. Furthermore, current data suggests that the nasal carriage functions as a potential reservoir for viral persistence and transmission (i.e., shedding) at times that are both prior to and during the manifestation of severe respiratory symptoms. This project utilizes a unique biobank of cryopreserved nasal cells collected from over 1000 individuals over 15 years to understand the critical issues surrounding SARS-CoV-2 interaction with the human nasal epithelia. Paradoxically, while SARS-CoV-2 can be detected in nasal swabs prior to its detection in sputum, there is a paucity of rhinologic symptoms (<5% with nasal congestion) associated with COVID-19, with the exception of reversible anosmia in 30-70% of patients. This is particularly problematic because up to 25% of infected individuals remain asymptomatic, but can continue to spread SARS-CoV-2 through airborne droplets. This work seeks to elucidate both the mechanisms controlling which epithelial cell lineages become infected with virus and the type of immune response generated within infected or neighboring epithelia. Through this approach, we will shed light on the issue of why certain individuals never develop symptoms while others progress to severe respiratory failure and death. We will focus on the SARS-CoV-2 receptor Angiotensin Converting Enzyme 2 (ACE2), which is essential and sufficient for the virus to enter cells. Our preliminary data generated from single cell RNA analysis of primary human sinonasal tissue demonstrates that ACE2 is expressed in discrete clusters of nasal epithelia. ACE2- specific immunostaining of human nasal epithelial cells ex vivo and primary ciliated air liquid interface (ALI) cultures corroborates the sc-RNAseq data. Furthermore, our data show that inoculation of primary ALI cultures with SARS-CoV-2 results in approximately 1%-25% of cells becoming infected, suggesting a selective process. These data indicate that we are uniquely poised to test the hypothesis that ACE2 expressing cells constitute a unique reservoir of viral replication and are likely to mount an inflammatory cytokine response that is distinct from non-infected epithelia. Using our established team of experts in nasal epithelial cell biology, viral pathogenesis, inflammatory cytokine biology and genetics we will determine the following: A) which types of epithelia are virally infected, B) what are the local inflammatory cascades in infected vs. non-infected cells, and C) will pharmacologic manipulation of the epithelial innate defense pathways significantly alter SARS-CoV-2 ability to infect, replicate and be released from human nasal epithelia. Successful completion of this work is likely to have a major impact on development of novel strategies to combat COVID19 disease progression within the general population and especially in the U.S. Veteran population.