SARS-CoV-2 Receptor Angiotensin I-Converting Enzyme Type 2 (ACE2) Is Expressed in Human Pancreatic β-Cells and in the Human Pancreas Microvasculature

Abstract

Increasing evidence demonstrated that the expression of Angiotensin I-Converting Enzyme type 2 (ACE2) is a necessary step for SARS-CoV-2 infection permissiveness. In light of the recent data highlighting an association between COVID-19 and diabetes, a detailed analysis aimed at evaluating ACE2 expression pattern distribution in human pancreas is still lacking. Here, we took advantage of INNODIA network EUnPOD biobank collection to thoroughly analyze ACE2, both at mRNA and protein level, in multiple human pancreatic tissues and using several methodologies. Using multiple reagents and antibodies, we showed that ACE2 is expressed in human pancreatic islets, where it is preferentially expressed in subsets of insulin producing β-cells. ACE2 is also highly expressed in pancreas microvasculature pericytes and moderately expressed in rare scattered ductal cells. By using different ACE2 antibodies we showed that a recently described short-ACE2 isoform is also prevalently expressed in human β-cells. Finally, using RT-qPCR, RNA-seq and High-Content imaging screening analysis, we demonstrated that pro-inflammatory cytokines, but not palmitate, increase ACE2 expression in the β-cell line EndoC-βH1 and in primary human pancreatic islets. Taken together, our data indicate a potential link between SARS-CoV-2 and diabetes through putative infection of pancreatic microvasculature and/or ductal cells and/or through direct β-cell virus tropism.

Keywords: COVID-19; SARS-CoV-2; angiotensin I-converting enzyme type 2 (ACE2); beta-cell; diabetes; human pancreatic islets; inflammation.

Figure 1

ACE2 staining pattern in human pancreas. Immunohistochemistry for ACE2 in human pancreatic tissue sections (case #110118) using R&D MAB933 antibody. ACE2 is markedly expressed in microvasculature associated cells (A, B) in some rare ductal cells (C, D) and in a subset of endocrine cells within pancreatic islets (E, F). Scale bars in (A, C, E) 150 µm. Scale bars in (B, D, F) 70 µm. Zoom-in images are reported in (B, D, F).

Figure 2

In microvasculature, ACE2 is putatively expressed in pericytes. (A) Representative image of human pancreatic Formalin-Fixed Paraffin Embedded (FFPE) section stained for ACE2 in case #301118. In panel-a, a representative image of a pancreatic section showing two adjacent lobules (blue and red dotted lines) with different staining for ACE2 in endothelial cells/pericytes. A specific segmentation of the two lobules with high (blue) (zoom-in, panel-b) and low or null expression of ACE2 (red) (zoom-in, panel-c) is shown, suggesting lobularity of ACE2 expression in exocrine endothelial cells/pericytes of human pancreas. Scale bar in panel-a: 100 µm. Scale bar in panels-b and -c: 30 µm. (B) Double immunofluorescence staining of ACE2 (green) and CD31 (red) in FFPE pancreas sections from Body01A of Case #110118 (panels-a to -d) and of Body01B of Case #141117 (panels-e to -i). Digital zoom-in overlay images are shown in panels-d, -h and -i. Scale bar in panels-d and -g: 100 μm.

Figure 3

ACE2 immunohistochemistry staining pattern in human pancreatic islets using MAB933, Ab15348, and Ab108252 antibodies. (A) Aligned sequences and structures of recently described ACE2 isoforms, long-ACE2 (805aa, ~110 kDa) and short-ACE2 (459aa, ~50kDa). Main ACE2 protein domains are reported with different colors. (B) R&D MAB933, (C) Abcam Ab15348 and (D) Ab108252 antibody predicted target sequence within the two ACE2 isoforms, alongside with immunohistochemistry staining distribution in pancreatic islets. Scale bars in (B–D) are 100 μm.

Figure 4

In human pancreatic islets, ACE2 is preferentially expressed in insulin-producing β-cells. Triple immunofluorescence staining and image analysis of FFPE human pancreatic section stained for insulin (red), glucagon (blue) and ACE2 (green). (A) Representative islets of two different cases. Panels-a to -g: representative pancreatic islet of FFPE pancreas block Body01A of case #110118. Panels-h to -m: representative pancreatic islet of FFPE pancreas block Body01B of case #141117. Panels-e to -g: digital zoom in images of the pancreatic islet shown in panel-d. Panels-l and -m: digital zoom in images of the pancreatic islet shown in panel-k. Scale bar in panels-d and -k: 100 µm. (B) Colocalization rate analysis of overlapping ACE2-insulin and ACE2-glucagon in 128 single pancreatic islets of seven different cases. p-value was calculated using Wilcoxon matched-pairs signed rank test. On the right: colocalization rate analysis of ACE2-insulin and ACE2-glucagon in each of the seven cases analyzed. For each case, a total of 7–11 islets/section were analyzed. (C) Analysis of the intensity of ACE2 islet-related signals in the cases analyzed. Values are shown as fluorescence intensity of each islet reported as the sum of gray-scale values for each pixel normalized for the islets area (ROI, μm²).

Figure 5

A short-ACE2 isoform is prevalently expressed in human β-cell line EndoC-βH1. Western Blot and Immunofluorescence analysis of EndoC-βH1 using (A) R&D monoclonal MAB933, (B) Abcam polyclonal Ab15348 and (C) Abcam monoclonal Ab108252 anti-ACE2 antibody. For each antibody adopted, specific target sequence is reported within the aligned ACE2 isoforms. In western blot analysis, molecular weight markers (from 15 kDa to 250 kDa) are reported; red arrows indicate long-ACE2 isoform (expected band of ~110kDa), while blue arrows indicate short-ACE2 isoform (~50kDa). (D) ACE2 (R&D MAB933) and insulin double immunofluorescence analysis in EndoC-βH1 cultured cells. Negative isotype primary antibody control (relative to ACE2 primary antibody) is shown in panel-a. Insulin (red) and ACE2 (green) are reported in panels-b and -c, while overlay is reported in panel-d. Digital zoom-in images are reported from panels-e to -h. Scale bar in panel-h = 15 µm.

Figure 6

qRT-Real Time PCR analysis of ACE2 mRNA. (A–D) ACE2 raw Ct values results in lung tissue (n = 1, in duplicate), in enzymatic-isolated human pancreatic islets samples (n = 4), in LCM-microdissected islets (n = 5) and in EndoC-βH1 (n = 4). (E–H) ACE2 expression values normalized using GAPDH and β2-microglobulin of the samples analysed in A–D. Values are reported as 2^−dCt. Mean ± S.D. values are shown.

Figure 7

ACE2 expression is increased by inflammatory stress. ACE2 mRNA RT-Real-Time PCR analysis in EndoC-βH1 treated or not (CTR) with Palmitate (2.0 mM) (A) or with cytokines (IL-1β + IFNγ + TNFα) (Cyt. Mix) (B) for 24 h. Results are reported as mean ± S.D of 2^−dCT normalized values. p-values were calculated using Wilcoxon matched-pairs signed rank test. (C) Immunofluorescence analysis of insulin (red, panels-a and -g) and ACE2 (green, panels-b and -h) in EndoC-βH1 not-treated (panels-a to -f) or treated (panels-g to -l) with cytokines for 24 h. Digital zoom-in images are reported in panels-d to -f and in panels-j to -l. Scale bar in panel-f: 10 µm. Scale bar in panel-l: 15 µm. (D) Representative images of ACE2 staining (green) and analysis using micro-confocal High-content screening in EndoC-βH1 treated or not with cytokines (IL-1β + IFNγ+TNFα). Panels-a and -b: ACE2 signal (green) and automated cell cytoplasm segmentation and identification. Panels-c and -d: identification of ACE2 granular spots within segmented cytoplasm in panels-a and -b. Each ACE2 granular spot intensity was measured and analyzed. (E) Mean intensity imaging analysis related to data reported in (C) of EndoC-βH1 treated or not with cytokines. Data are reported as individual values alongside with mean ± S.D of RGB gray-intensity measures of 6–11 different experimental points related to two different independent experiments. Individual values alongside with mean ± S.D are reported. P-value was calculated using Mann–Whitney U test. (F) High content screening analysis of Corrected Median Spot intensity of ACE2 signal in EndoC-βH1 treated or not with cytokines. Median intensity values of six different experimental points are reported. P-value was calculated using Mann–Whitney U test (p < 0.05).