Experimental and natural SARS-CoV-2 infection of the human pancreas

人类胰腺的实验性和自然 SARS-CoV-2 感染

• 批准号: 10319841
• 负责人: DIRK HOMANN
• 金额: $ 45.33万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-17 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319841
• 关键词: 2019-nCoV; ACE2; Address; Animal Experiments; Area; Autopsy; Beta Cell; Biological; Biomedical Research; Blood Glucose; COVID-19; COVID-19 mortality; COVID-19 patient; Cell physiology; Cells; Clinical; Cohort Studies; Cytometry; Data; Detection; Diabetes Mellitus; Endocrine; Epidemiology; Experimental Designs; Flow Cytometry; Foundations; Future; Generations; Glucose; Glucose tolerance test; Goals; Histologic; Histology; Human; Immune; Immunodeficient Mouse; Immunologics; Impairment; In Situ; In Vitro; Infection; Insulin; Insulin-Dependent Diabetes Mellitus; Intervention; Investigation; Islets of Langerhans; Islets of Langerhans Transplantation; Journals; Letters; Logistics; Mediating; Medicine; Messenger RNA; Metabolic; Modeling; Mus; Nature; New England; Non-Insulin-Dependent Diabetes Mellitus; Pancreas; Pathologic; Pathway interactions; Pattern; Physiological; Plant Roots; Plasma; Population; Predisposition; Proteins; Proteome; Protocols documentation; Publications; Publishing; Reporting; Research; Research Personnel; Resolution; Risk Assessment; Role; SARS-CoV-2 infection; Sampling; Series; Severities; Slice; Stains; Therapeutic Intervention; Time; Tissues; Tropism; Viral; Viral Load result; Viral Proteins; Virus Diseases; Work; base; blood glucose regulation; case control; cell type; comorbidity; demographics; diabetogenic; epidemiologic data; experimental study; high dimensionality; human model; humanized mouse; in vitro Model; in vivo; influenza infection; insight; insulin secretion; islet; mRNA Expression; mouse model; non-diabetic; patient registry; prophylactic; receptor; response; transcriptome; virology

ABSTRACT The possibility that infection with SARS-CoV-2 may trigger diabetes emerged as a concern in mid 2020. We have addressed this issue by developing a research roadmap that identified complementary areas of investigation according to several broad topics in the realm of relevant basic biomedical research. Based on our recent publication about the expression of SARS-CoV-2 entry factors in the non-diabetic human pancreas, we have now developed a Research Strategy that will assess the principal capacity of pancreatic SARS-CoV-2 infection and its primary consequences in a series of in vitro, in vivo and ex vivo studies distributed across three Specific Aims: In Aim 1 we will define expression patterns of ACE2 and other viral entry factors in dispersed human pancreatic cell populations; will subject islets to in vitro SARS-CoV-2 infection and quantify infection patterns as well as alterations of single-cell proteomes and transcriptomes by flow cytometry, high-dimensional mass cytometry, and scRNAseq; will determine functional islet responses under conditions of viral infection by dynamic glucose-stimulated insulin secretion; will conduct mechanistic studies with targeted ACE2 blockade and by generation of ACE2-deficient beta-like cells; and will explore SARS-CoV-2 infection in the context of living pancreas slices that provide a more “physiological” experimental platform for in vitro studies. In Aim 2, we will use the robust “minimal mass” model of human islet transplantation into immunodeficient mice to delineate metabolic perturbations (blood glucose values, plasma insulin, intraperitoneal glucose tolerance test) accrued in the wake of an in vivo SARS-CoV-2 challenge. To address the considerable experimental and logistical challenges in these studies, we have developed a tiered approach that progressively refines experimental designs (including use of a recently generated, sequenced and characterized mouse-adapted [ma] SARS-CoV-2 strain) with the goal to imbue our models with escalating biological relevance. In Aim 3, we will draw on our access to COVID-19 and control autopsy cases to reveal the ex vivo viral burden of natural SARS-CoV-2 infection in the pancreas. These studies, which build on recently published pilot data as well as extensive preparatory studies to adjust our staining protocols to the demands of at times suboptimal tissue quality, will define SARS-CoV-2 protein and mRNA abundance, cellular association and distribution patterns across the COVID-19 pancreas. We will further leverage an established immunohistochemical multiplexing strategy to define the identity, quantity and distribution of major immune cell subsets throughout COVID-19 and control pancreata. Thus, the proposed work will generate an inclusive perspective on the cardinal viral and immunological components of the potentially altered pancreatic histology in COVID-19. Collectively, the proposed work addresses, and is expected to resolve at least in part, key aspects of the hypothesis that SARS-CoV-2 infection may precipitate diabetes onset. Together with emerging epidemiological data, it may therefore provide an important foundation for future risk assessment and the prioritization of prophylactic and/or therapeutic intervention strategies.

摘要 2020年年中，感染SARS-CoV-2可能引发糖尿病的可能性成为一个令人担忧的问题。我们有 通过制定确定互补调查领域的研究路线图解决了这一问题 涉及相关基础生物医学研究领域的几个广泛主题。基于我们最近出版的关于 SARS-CoV-2进入因子在非糖尿病人胰腺中的表达 将评估胰腺SARS-CoV-2感染的主要能力及其在 一系列体外、体内和体外研究分布在三个具体目标上： 在目标1中，我们将确定ACE2和其他病毒进入因子在分散的人胰腺细胞中的表达模式 将使胰岛在体外受到SARS-CoV-2感染，并量化感染模式以及 单细胞蛋白质组和转录体的流式细胞术、高维质量细胞术和scRNAseq； 通过动态葡萄糖刺激的胰岛素分泌来确定病毒感染条件下的胰岛功能反应； 将通过靶向阻断血管紧张素转换酶2和产生缺乏血管紧张素转换酶2的类β细胞进行机制研究；以及 将在探索SARS-CoV-2感染的背景下，为活体胰腺切片提供更多的“生理学” 体外研究的实验平台。在目标2中，我们将使用人类胰岛的健壮“最小质量”模型。 移植到免疫缺陷小鼠以描绘代谢紊乱(血糖值，血浆胰岛素， 腹膜糖耐量试验)在体内SARS-CoV-2攻击后累积。要解决这个问题 在这些研究中面临着相当大的实验和后勤挑战，我们开发了一种分层方法， 逐步改进实验设计(包括使用最近生成、排序和表征的 小鼠适应的SARS-CoV-2毒株)，目的是使我们的模型具有不断升级的生物学相关性。在……里面 目标3，我们将利用我们的新冠肺炎和对照尸检病例来揭示天然肺炎病毒的体外病毒负担 胰腺的SARS-CoV-2感染。这些研究建立在最近公布的试点数据以及广泛的 调整我们的染色方案以适应有时不理想的组织质量要求的预备研究，将确定 新冠肺炎地区SARS-CoV-2蛋白和信使核糖核酸丰度、细胞关联及分布格局 胰腺。我们将进一步利用已建立的免疫组织化学多路传输策略来定义身份， 主要免疫细胞亚群在新冠肺炎和对照胰腺的数量和分布。因此，拟议的 工作将产生关于潜在的病毒和免疫学主要组成部分的包容性观点 新冠肺炎胰腺组织学改变。 总的来说，拟议的工作涉及并有望至少部分解决这一假说的关键方面 SARS-CoV-2感染可能诱发糖尿病的发病。结合新出现的流行病学数据，它可能会 因此，为今后的风险评估和确定预防性和/或 治疗性干预策略。