SARS-CoV-2 infects human pancreatic β cells and elicits β cell impairment

Abstract

Emerging evidence points toward an intricate relationship between the pandemic of coronavirus disease 2019 (COVID-19) and diabetes. While preexisting diabetes is associated with severe COVID-19, it is unclear whether COVID-19 severity is a cause or consequence of diabetes. To mechanistically link COVID-19 to diabetes, we tested whether insulin-producing pancreatic β cells can be infected by SARS-CoV-2 and cause β cell depletion. We found that the SARS-CoV-2 receptor, ACE2, and related entry factors (TMPRSS2, NRP1, and TRFC) are expressed in β cells, with selectively high expression of NRP1. We discovered that SARS-CoV-2 infects human pancreatic β cells in patients who succumbed to COVID-19 and selectively infects human islet β cells in vitro. We demonstrated that SARS-CoV-2 infection attenuates pancreatic insulin levels and secretion and induces β cell apoptosis, each rescued by NRP1 inhibition. Phosphoproteomic pathway analysis of infected islets indicates apoptotic β cell signaling, similar to that observed in type 1 diabetes (T1D). In summary, our study shows SARS-CoV-2 can directly induce β cell killing.

Keywords: ACE2; COVID-19; SARS-CoV-2; SARS-CoV-2 spike protein; apoptosis; insulin; neuropilin 1; pancreatic beta cell; phosphoproteomics; type 1 diabetes.

Graphical abstract

Figure 1

SARS-CoV-2-associated receptors are expressed in pancreatic β cells (A) Representative double immunofluorescence staining of ACE2, TMPRSS2, NRP1, and TFRC with the β cell marker, insulin (INS), and α cell marker, glucagon (GLU), in the normal human pancreas, donor 1. See Table 1. (B) Quantification of ACE2, TMPRSS2, NRP1, and TFRC in β cells (INS +) and α cells (GLU +) from a normal pancreas. No statistically significant changes in ACE2 and TMPRSS2 expression were detected between β and α cells. NRP1 and TFRC expression was statistically significantly higher in β cells compared with α cells. Rabbit anti-NRP1 (Abcam, ab81321, 1:200) and mouse anti-TFRC (Thermo Fisher, # 13-6800, 1:200) were used for the experiments shown here. Error bars represent mean ± SD (~10–15 islets from the pancreas of 5 non-COVID-19 donors; see Table 1). ^∗∗p < 0.001, one-way ANOVA with Tukey’s post-test. Each dot represents one donor. Scale bars, 5 μm (A) and 2 μm (insets). See also Figures S1 and S2 and Table 1.

Figure 2

SARS-CoV-2 preferentially infects β cells of human pancreatic islets ex vivo (A–D) Mock-treated or SARS-CoV-2-infected human pancreatic islets were stained after 2 or 6 dpi. (A) Representative double immunofluorescence staining of SARS-CoV-2 nucleocapsid protein (NP) in combination with β cell marker, insulin (INS); ɑ cell marker, glucagon (GLU); δ cell marker, somatostatin (SST); and endothelial cell marker (CD31). (B) Representative double immunofluorescence staining of SARS-CoV-2 spike protein (SP) in combination with a similar combination of markers as (A). The nuclei were stained using DAPI (blue) as a counterstain. (C) Quantified percentages of SARS-CoV-2 NP and SP within α, β, δ, and endothelial cells of pancreatic islets. Around 40% to 60% NP and SP staining, respectively, are present within β cells. (D) Quantified percentages of SARS-CoV-2 NP- and SP-positive α, β, δ, and endothelial cells. (C and D) Error bars represent mean ± SD (~500–1,000 cells were quantified from healthy isolated human islets from donors 1–5; see Table 2). (E) Representative double immunofluorescence staining of SARS-CoV-2 NP in combination with insulin after pre-treating islets with dimethyl sulfoxide (DMSO) or 100 μM EG00229 for 1 h before infection with SARS-CoV-2. Islets were fixed at 2 dpi and stained for SARS-CoV-2 NP and β cell marker, insulin (INS). Quantification of the percentages of β cells containing NP-positive β cells (right). Error bars represent mean ± SD (~500–1,000 cells were quantified from healthy isolated human islets from donors 10–13; see Table 2). ^∗p < 0.05, two-tailed Student’s t test. Each dot represents one donor. Scale bars, 5 μm (A, B, and E) and 2 μm (insets). See also Table 2.

Figure 3

SARS-CoV-2 infects pancreatic β cells of patients with COVID-19 (A) Representative double immunofluorescence staining of pancreatic islets from patients with COVID-19 and healthy controls using antibodies against SARS-CoV-2 NP and INS. (B) Representative multiplexed images of in situ hybridization against the SARS-CoV-2 spike mRNA, in combination with immunofluorescence staining of insulin (INS). SARS-CoV-2 spike mRNA expression (red dots) was detected within pancreatic β cells. The nuclei were stained using DAPI (blue) as a counterstain. Scale bars, 5 μm (A and B) and 2 μm (insets). See also Figure S3 and Table 3.

Figure 4

SARS-CoV-2 infection interferes with insulin content/secretion and induces β cell apoptosis (A–F) Pancreatic islet functionality was analyzed by insulin content, glucose-stimulated insulin secretion (GSIS), and TUNEL staining ex vivo. (A) Insulin content is decreased in SARS-CoV-2-infected islets compared with mock-treated islets. (B) GSIS is decreased in SARS-CoV-2-infected islets compared with mock-treated islets. (A and B) Error bars represent mean ± SD (data were collected from 7 healthy isolated human islets, donors 2–8; see Table 2). ^∗p < 0.05, two-tailed Student’s t test. (C) Representative staining of β cell apoptosis by in situ TUNEL and DAPI staining in β cells (INS) of mock- or SARS-CoV-2-treated human islets. DNase-treated sections were used as a positive control in the TUNEL assay. (D and F) Quantification of the percentages of islets containing TUNEL-positive β cells. Error bars represent mean ± SD (~500–1,000 cells were quantified from each of 3–5 separate healthy isolated human islets, donors 1–5 [D] and 7–9 [F]; see Table 2). (E) Representative staining of β cell apoptosis by in situ TUNEL and DAPI staining in β cells (INS) of mock-treated versus SARS-CoV-2-SP-treated human islets. ^∗p < 0.05, ^∗∗p < 0.01, two-tailed Student’s t test. Scale bars, 5 μm (C and E). See also Figures S3–S5 and Tables 2, S1, S2, and S3.