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

摘要 感染SARS-CoV-2可能引发糖尿病的可能性在2020年年中成为一个关注点。我们有 通过制定研究路线图来解决这个问题，该路线图根据 在相关的基础生物医学研究领域的几个广泛的主题。根据我们最近发表的关于 SARS-CoV-2进入因子在非糖尿病人胰腺中的表达，我们现在已经开发了一种研究 评估胰腺SARS-CoV-2感染的主要能力及其主要后果的策略， 一系列体外、体内和离体研究，分布于三个特定目的： 在目的1中，我们将确定ACE 2和其他病毒进入因子在分散的人胰腺细胞中的表达模式， 将使胰岛经受体外SARS-CoV-2感染，并量化感染模式以及 单细胞蛋白质组和转录组的流式细胞术，高维质谱仪，和scRNAseq;将 通过动态葡萄糖刺激的胰岛素分泌确定病毒感染条件下的功能性胰岛反应; 将通过靶向ACE 2阻断和生成ACE 2缺陷的β样细胞进行机制研究;以及 将探索SARS-CoV-2感染的情况下，活胰腺切片，提供了一个更“生理”的 用于体外研究的实验平台。在目标2中，我们将使用人体胰岛的鲁棒“最小质量”模型 移植到免疫缺陷小鼠中以描述代谢扰动（血糖值，血浆胰岛素， 腹膜内葡萄糖耐量试验）在体内SARS-CoV-2攻击后产生。解决 在这些研究中存在相当大的实验和后勤挑战，我们开发了一种分层方法， 逐步完善实验设计（包括使用最近生成的，测序和表征的 小鼠适应的SARS-CoV-2菌株），目标是使我们的模型具有不断升级的生物相关性。在 目标3，我们将利用我们对COVID-19的访问和控制尸检病例，以揭示天然药物的体外病毒负荷。 胰腺中的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 准备研究，以调整我们的染色协议，有时次优的组织质量的要求，将定义 SARS-CoV-2蛋白和mRNA丰度、细胞关联和COVID-19中的分布模式 胰腺我们将进一步利用已建立的免疫组化多重策略来确定身份， 整个COVID-19和对照胰腺中主要免疫细胞亚群的数量和分布。因此，拟议的 这项工作将产生一个包容性的观点，对潜在的主要病毒和免疫成分， 胰腺组织学改变 总的来说，拟议的工作地址，并预计将解决至少部分，假设的关键方面 SARS-CoV-2感染可能促使糖尿病发病。再加上新出现的流行病学数据， 因此，为今后的风险评估和预防和/或 治疗干预策略。

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.