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

摘要 严重急性呼吸综合征冠状病毒SARS-CoV-2，冠状病毒的病原体 2019冠状病毒病（COVID-19）导致了一场大流行，死亡率约为3.5%， 发病率结果受到既存疾病的负面影响。鉴于普遍存在的 退伍军人的共病状况，必须了解SARS-CoV-2如何入侵的机制 并在鼻的屏障防御细胞内复制，鼻是病毒进入的主要门户。此外，委员会认为， 目前的数据表明，鼻携带作为病毒持久性的潜在储存库， 传输（即，在严重呼吸道疾病表现之前和期间， 症状该项目利用了一个独特的生物银行的冷冻保存的鼻细胞收集超过1000 15年以上的个人，以了解围绕SARS-CoV-2与人类相互作用的关键问题 鼻上皮 奇怪的是，虽然SARS-CoV-2可以在痰液中检测到之前在鼻拭子中检测到， 与COVID-19相关的鼻粘膜症状很少（<5%伴有鼻塞），但 30-70%的患者出现可逆性嗅觉丧失。这是一个特别的问题，因为高达25%的感染者 这些人仍然没有症状，但可以继续通过空气飞沫传播SARS-CoV-2。这项工作 试图阐明控制上皮细胞谱系被病毒感染的机制， 在受感染的或邻近的上皮细胞内产生的免疫反应类型。通过这种方法，我们将 阐明了为什么某些人从未出现症状，而另一些人则进展到严重的问题。 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（ACE 2），这是必不可少的 足以让病毒进入细胞我们的初步数据来自原发性肝癌的单细胞RNA分析。 人鼻窦组织证实ACE 2在鼻上皮细胞的离散簇中表达。ACE 2- 体外人鼻上皮细胞和原代纤毛气液界面（ALI）的特异性免疫染色 培养物证实了sc-RNAseq数据。此外，我们的数据表明，接种原代ALI培养物 SARS-CoV-2导致大约1%-25%的细胞被感染，这表明了一个选择性过程。 这些数据表明，我们是唯一准备测试的假设，表达ACE 2的细胞构成了一个新的细胞。 病毒复制的独特储存库，并且可能引起与病毒复制不同的炎症细胞因子反应。 从未感染的上皮细胞。利用我们在鼻上皮细胞生物学方面的专家团队， 发病机制、炎症细胞因子生物学和遗传学，我们将确定以下内容： 上皮细胞被病毒感染，B）感染细胞与未感染细胞中的局部炎性级联反应是什么，以及 C）药物对上皮细胞天然防御途径的操作是否会显著改变SARS-CoV-2 感染、复制和从人鼻上皮细胞释放的能力。成功完成这项工作是可能的 对开发新的策略以对抗COVID 19疾病进展产生重大影响， 尤其是在美国的退伍军人群体中。

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.