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

COVID-19：阐明鼻上皮在 SARS-CoV-2 感染、传播和预防中的作用

• 批准号: 10350632
• 负责人: Noam A Cohen
• 金额: --
• 依托单位: PHILADELPHIA VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10350632
• 关键词: 2019-nCoV; ACE2; Address; Age; Air; Anosmia; Apical; Asthma; Basal Cell; Biology; Bronchitis; COVID-19; COVID-19 impact; COVID-19 risk; Caring; Cell Lineage; Cells; Cellular biology; Cessation of life; Chronic Obstructive Pulmonary Disease; Collaborations; Congestive; Cryopreservation; Data; Detection; Development; Disease Progression; Double-Stranded RNA; Epithelial; Epithelial Cells; Functional disorder; Future; Gender; General Population; Genetic; Genotype; Goals; Goblet Cells; Growth; Harvest; Health; Healthcare Systems; Human; Immune; Immune response; Immunologic Receptors; Individual; Infection; Inflammatory; Inflammatory Response Pathway; Invaded; Kinetics; Knowledge; Lead; Light; Liquid substance; Malignant neoplasm of lung; Medical; Morbidity - disease rate; Mucins; Mucous Membrane; Mucous body substance; Nasal Epithelium; Natural Immunity; Nitric Oxide; Nose; Nucleocapsid Proteins; Outcome; Pathway interactions; Patients; Pennsylvania; Peptide Hydrolases; Pharmacology; Predisposition; Prevalence; Prevention; Process; Production; Prophylactic treatment; Protocols documentation; Pulmonary Emphysema; RNA analysis; Race; Resources; Respiration Disorders; Respiratory Disease; Respiratory Failure; Respiratory Signs and Symptoms; Respiratory Tract Infections; Rhinitis; Role; SARS coronavirus; SARS-CoV-2 infection; Sampling; Sinusitis; Sputum; Symptoms; TMPRSS2 gene; Testing; Time; Tissues; Type 2 Angiotensin II Receptor; Universities; Veterans; Viral; Viral Genome; Viral Pathogenesis; Viral reservoir; Virus; Virus Diseases; Virus Replication; Virus Shedding; Vulnerable Populations; Work; airway epithelium; antimicrobial peptide; asthma exacerbation; biobank; biosafety level 3 facility; burden of illness; combat; community transmission; comorbidity; cytokine; demographics; discrete time; experimental study; high risk; in vitro Model; innate immune pathways; insight; military veteran; mortality; nasal swab; novel strategies; pandemic disease; particle; post SARS-CoV-2 infection; prophylactic; racial disparity; receptor; screening; single-cell RNA sequencing; success; targeted treatment; transcriptome sequencing; transmission process; ultraviolet irradiation

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.

抽象的 严重的急性呼吸综合征冠状病毒SARS-COV-2，冠状病毒的病毒药物 2019年疾病（COVID-19）导致大流行，死亡率约为3.5％，范围广泛。 先前存在的条件对发病结果负面影响。鉴于先前存在的流行率 在退伍军人中的合并条件，必须了解SARS-COV-2如何入侵的机制 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， 当前的数据表明，鼻托架是病毒持续性的潜在储层和 有时在严重呼吸的表现之前和表现期间的传播（即脱落） 症状。该项目利用了从1000多个收集的冷冻保存的鼻细胞的独特生物库 个人超过15年，了解与人类互动有关的关键问题 鼻上皮。 矛盾的是，虽然在鼻拭子发现鼻拭子中可以检测到SARS-COV-2，但 是与COVID-19相关的鼻症状症状缺乏（<5％），例外 30-70％的患者中可逆的厌食症。这尤其有问题，因为多达25％的感染 个体仍然无症状，但可以继续通过机载液滴传播SARS-COV-2。这项工作 旨在阐明控制上皮细胞谱系感染病毒和 在感染或相邻上皮中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永远不会出现症状，而其他人则陷入严重的问题 呼吸衰竭和死亡。 我们将专注于SARS-COV-2受体血管紧张素转化酶2（ACE2），这是必不可少的 足以使病毒进入细胞。我们的初步数据是由单细胞RNA分析产生的 人鼻窦组织表明，ACE2在鼻上皮的离散簇中表达。 ace2-- 人类鼻上皮细胞的特异性免疫染色和原发性空气液体界面（ALI） 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 随着SARS-COV-2，大约1％-25％的细胞被感染，这表明有选择性过程。 这些数据表明，我们有唯一准备测试ACE2表达细胞的假设 独特的病毒复制储层，可能会安装炎症性细胞因子反应，该反应不同 来自未感染的上皮。使用我们既定的鼻皮细胞生物学专家团队，病毒 发病机理，炎症性细胞因子生物学和遗传学我们将确定以下内容：a）哪种类型 上皮受到病毒感染，b）在感染与未感染的细胞中，什么是局部炎症级联反应，以及 c）上皮先天防御途径的药理学操纵会显着改变SARS-COV-2 能够从人类鼻上皮中感染，复制和释放的能力。成功完成这项工作可能是 要对制定新策略的发展产生重大影响 普通人群，尤其是在美国退伍军人人口中。