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. 2020 Dec 1;32(6):1041-1051.e6.
doi: 10.1016/j.cmet.2020.11.005. Epub 2020 Nov 13.

Expression of SARS-CoV-2 Entry Factors in the Pancreas of Normal Organ Donors and Individuals with COVID-19

Irina Kusmartseva  1 , Wenting Wu  2 , Farooq Syed  3 , Verena Van Der Heide  4 , Marda Jorgensen  1 , Paul Joseph  1 , Xiaohan Tang  5 , Eduardo Candelario-Jalil  6 , Changjun Yang  6 , Harry Nick  6 , Jack L Harbert  7 , Amanda L Posgai  1 , John David Paulsen  8 , Richard Lloyd  9 , Sirlene Cechin  10 , Alberto Pugliese  10 , Martha Campbell-Thompson  11 , Richard S Vander Heide  7 , Carmella Evans-Molina  12 , Dirk Homann  4 , Mark A Atkinson  13
Affiliations

Expression of SARS-CoV-2 Entry Factors in the Pancreas of Normal Organ Donors and Individuals with COVID-19

Irina Kusmartseva et al. Cell Metab. .

Abstract

Diabetes is associated with increased mortality from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Given literature suggesting a potential association between SARS-CoV-2 infection and diabetes induction, we examined pancreatic expression of angiotensin-converting enzyme 2 (ACE2), the key entry factor for SARS-CoV-2 infection. Specifically, we analyzed five public scRNA-seq pancreas datasets and performed fluorescence in situ hybridization, western blotting, and immunolocalization for ACE2 with extensive reagent validation on normal human pancreatic tissues across the lifespan, as well as those from coronavirus disease 2019 (COVID-19) cases. These in silico and ex vivo analyses demonstrated prominent expression of ACE2 in pancreatic ductal epithelium and microvasculature, but we found rare endocrine cell expression at the mRNA level. Pancreata from individuals with COVID-19 demonstrated multiple thrombotic lesions with SARS-CoV-2 nucleocapsid protein expression that was primarily limited to ducts. These results suggest SARS-CoV-2 infection of pancreatic endocrine cells, via ACE2, is an unlikely central pathogenic feature of COVID-19-related diabetes.

Keywords: ACE2; CD34; COVID-19; SARS-CoV-2; TMPRSS2; insulin; islet; pancreas; type 1 diabetes; type 2 diabetes.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
SARS-CoV-2-Associated Gene Expression in Isolated Human Pancreatic Islets (A) Bar graph showing the percentage of cells with detectable ACE2 in islets from pancreata of donors with (n = 2,705 cells) and without type 2 diabetes (n = 12,185 cells). (B) Violin plot showing the distribution of ACE2 normalized expression in islet cells from pancreata of donors without diabetes. (C) Bar graph showing the percentage of cells with detectable TMPRSS2 in islets isolated from pancreata of donors with (n = 2,705 cells) and without type 2 diabetes (n = 12,185 cells); ∗adjusted p < 0.05, paired Student’s t test for indicated comparisons. (D) Violin plot showing the distribution of TMPRSS2 normalized expression in islets cells from pancreata of donors without diabetes; ∗∗∗adjusted p < 0.001, Wilcoxon rank-sum tests. Bonferroni corrections were used to adjust for multiple comparisons. (E) Dot plot of ACE2 and TMPRSS2 expression compared with cell-type-enriched genes in islets from pancreata of donors without type 2 diabetes (n = 12,185 cells). The size of each dot indicates the percentage of cells in a given population expressing the indicated gene. The dot color represents the scaled average expression. (F) Violin plot showing the relative expression of ACE2 and TMPRSS2 in islet α cells from pancreata of donors without diabetes (n = 3,770 cells) compared with select α cell-enriched genes. (G)Violin plot showing the relative expression of ACE2 and TMPRSS2 in islet β cells from pancreata of donors without diabetes (n = 2,985 cells) compared with select β cell-enriched genes. Related to Tables S1 and S2 and Figure S1.
Figure 2
Figure 2
Single Molecular Fluorescent In Situ Hybridization-Based Validation of SARS-CoV-2-Associated Gene Expression in Human Pancreas (A) Representative images of smFISH for ACE2 and TMPRSS2 mRNA in human pancreatic tissue sections counterstained for insulin. Inset highlights mRNA distribution in pancreatic ducts; scale bars, 20 μm. The lower image is high magnification of the boxed region in the merged panel. (B) Representative smFISH images showing the presence of ACE2 and TMPRSS2 in CD34-positive cells in human pancreatic tissue sections; scale bars, 10 μm. The lower image is high magnification of the boxed region in the merged panel. (C) Representative images of smFISH for ACE2 and TMPRSS2 mRNA in human pancreatic tissue sections counterstained for insulin. Inset highlights distribution in the endocrine pancreas; scale bars, 20 μm. The lower image is high magnification of the boxed region in the merged panel. Related to Figure S2.
Figure 3
Figure 3
ACE2 Protein Is Readily Detected in Normal Human Pancreas with Its Expression Largely Restricted to Ductal and Microvascular Structures (A) ACE2 protein structure illustrating the location of respective antibody directed antigen sites. SP, signal peptide; TM, transmembrane domain; CD, cytoplasmic domain. (B) Immunoblot analysis of four commercially available ACE2 antibodies using total pancreas lysates from three control organ donors (p1–p3) with accompanying Actin labeling. 120 and 37 indicate the molecular weight (in kDa) of ACE2 and Actin, respectively. (C) Representative IHC images of human pancreas tissue sections stained for ACE2 and insulin using four commercially available ACE2 antibodies. Scale bars, 200 μm. d, duct; mv, microvasculature. Related to Table S3 and Figures S3 and S4.
Figure 4
Figure 4
ACE2 Protein Expression Is Evident in Normal Pancreata throughout the Human Lifespan and Correlates with BMI (A) Scheme of the experimental setup illustrating human pancreatic tissue processing, whole-stained slide imaging, and machine learning algorithm application for the generation of ACE2 protein expression data. (B) Quantification of ACE2 protein expression in the pancreas of male (blue) and female (orange) control organ donors with ages ranging from birth to 72 years shows progressive developmental changes. Data were analyzed by one-way ANOVA and Tukey’s post hoc test for multiple comparisons and are presented as a percentage, ∗p < 0.05. (C) Quantification of ACE2 protein expression in the pancreas of male (blue) and female (orange) control organ donors with BMI ranging from normal to obese; r = 0.81, n = 22, p < 0.001. (D) Representative confocal images (from n = 14) of ACE2 protein expression in pancreatic ducts of control donors across the different age groups indicated. Scale bars (left to right), 100, 200, 200, 300, 300, and 300 μm. (E) Representative immunofluorescence images (n = 14) show ACE2 protein expression in pancreatic microvasculature from control donors across different age groups. Scale bars, 100 μm. (F) Representative immunofluorescence images (n = 14) of pancreatic islets showing ACE2 protein expression restricted to the islet’s microvasculature in pancreata from control organ donors across different age groups. Scale bars, 50 μm. Related to Table S3 and Figure S4.
Figure 5
Figure 5
Pathological Changes in Pancreata of Individuals with COVID-19 (A) Pancreas tissue section from COVID-19 Case 1 stained for H&E. Inset highlights fibrotic center with residual acinar cells and islet surrounding ductules. Scale bars, 3 mm; inset, 200 μm. (B) Pancreas tissue section from COVID-19 Case 2 stained for H&E. Inset highlights microthrombus without adjacent hemorrhages. Scale bars, 3 mm; inset, 200 μm. (C) Pancreas tissue section of COVID-19 Case 3 stained for H&E. Inset highlights a large, irregularly shaped pancreatic islet surrounded by fibrotic tissue. Scale bars, 3 mm; inset, 200 μm. (D) Representative pancreas tissue sections from three COVID-19 cases stained for ACE2, insulin (INS), and glucagon (GCG). Scale bars, 200 μm. (E) SARS-CoV-2 NP observed in intralobular ducts (d) near an islet in the pancreas of COVID-19 Case 1. Scale bars, 10 μm. (F) Representative image of multiple ducts showing SARS-CoV-2 NP positivity in the pancreas of COVID-19 Case 1. d; duct. Scale bar, 20 μm. Related to Table S4 and Figures S5C and S5D.
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