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 严重急性呼吸综合征冠状病毒 SARS-CoV-2，冠状病毒的病原体 2019 年疾病 (COVID-19) 已导致一场大流行，死亡率约为 3.5%，并且范围广泛 发病结果受到原有疾病的负面影响。鉴于先前存在的普遍性 退伍军人的共病，必须了解 SARS-CoV-2 的入侵机制 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， 目前的数据表明，鼻腔运输是病毒持续存在的潜在储存库， 在出现严重呼吸道疾病之前和期间有时会传播（即脱落） 症状。该项目利用了一个独特的冷冻保存鼻细胞生物库，该生物库收集自 1000 多个鼻腔细胞。 15 年以上的个人了解 SARS-CoV-2 与人类相互作用的关键问题 鼻上皮。 矛盾的是，虽然 SARS-CoV-2 可以在鼻拭子中检测到，然后再在痰液中检测到，但 与 COVID-19 相关的鼻科症状很少（<5% 伴有鼻塞），但以下情况除外 30-70% 的患者患有可逆性嗅觉丧失。这尤其成问题，因为高达 25% 的感染者 个体仍然无症状，但可以继续通过空气飞沫传播 SARS-CoV-2。这部作品 试图阐明控制哪些上皮细胞谱系被病毒感染的机制以及 受感染或邻近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却发展为严重症状的问题 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)，它是必不可少的 并足以让病毒进入细胞。我们的初步数据来自原代细胞的单细胞 RNA 分析 人类鼻腔组织表明 ACE2 在离散的鼻上皮细胞簇中表达。 ACE2- 离体人鼻上皮细胞和初级纤毛气液界面 (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，原代 ALI 培养物的接种 SARS-CoV-2 导致大约 1%-25% 的细胞被感染，这表明这是一个选择性过程。 这些数据表明我们完全准备好检验以下假设：表达 ACE2 的细胞构成了 独特的病毒复制库，并可能引发独特的炎症细胞因子反应 来自未感染的上皮细胞。利用我们在鼻上皮细胞生物学、病毒方面的成熟专家团队 通过发病机制、炎症细胞因子生物学和遗传学，我们将确定以下内容：A) 哪些类型 上皮细胞被病毒感染，B) 感染细胞与未感染细胞的局部炎症级联反应是什么，以及 C) 对上皮先天防御途径的药理学操作会显着改变 SARS-CoV-2 感染、复制和从人鼻上皮细胞释放的能力。这项工作很可能成功完成 对制定抗击新冠肺炎疾病进展的新策略产生重大影响 一般人群，尤其是美国退伍军人人群。