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.

PROJECT NARRATIVE While convincing epidemiological evidence supports the notion that type 1 and type 2 diabetes constitute co-morbidities that can promote an aggravated COVID-19 course, the notion that SARS-CoV-2 infection may precipitate diabetes onset, prominently advanced in June 2020, remains controversial. Here, building on our recent work on the expression of SARS-CoV-2 entry factors in the human pancreas, we propose to elucidate the susceptibility of islet endocrine and other pancreatic cell types in models of in vitro and in vivo SARS-CoV-2 infection, and to conduct expanded histopathological interrogations of pancreatic autopsy samples from COVID-19 and control cases. Together, these investigations are expected to provide essential insights into the role of SARS-CoV-2 infection as a potential trigger for.