key: cord-0878178-hdgew8i1 authors: Wu, Chien-Ting; Lidsky, Peter V.; Xiao, Yinghong; Lee, Ivan T.; Cheng, Ran; Nakayama, Tsuguhisa; Jiang, Sizun; Demeter, Janos; Bevacqua, Romina J.; Chang, Charles A.; Whitener, Robert L.; Stalder, Anna K.; Zhu, Bokai; Chen, Han; Goltsev, Yury; Tzankov, Alexandar; Nayak, Jayakar V.; Nolan, Garry P.; Matter, Matthias S.; Andino, Raul; Jackson, Peter K. title: SARS-CoV-2 infects human pancreatic β-cells and elicits β-cell impairment date: 2021-05-18 journal: Cell Metab DOI: 10.1016/j.cmet.2021.05.013 sha: eeebb6464cc851fd10bce8b4fee84a47c7c18856 doc_id: 878178 cord_uid: hdgew8i1 Emerging evidence points towards an intricate relationship between the pandemic of coronavirus disease 2019 (COVID-19) and diabetes. While pre-existing diabetes is associated with severe COVID-19, it is unclear if 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, 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 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. 137 Next, we determined whether SARS-CoV-2 tropism for β-cells is also observed in patients with 138 COVID-19. We obtained pancreatic autopsy samples from 9 patients who died from severe 139 COVID-19-related complications. The characteristics of these patients are summarized in Table 3 . Histological analysis revealed lipomatosis, fibrosis, or autolysis in some of the samples, whereas acute 141 or chronic pancreatitis was not observed in any patient (Table 1) , tending to exclude that broad 142 pancreatic damage is a universal feature. The pancreas of 7 out of 9 of these patients had 143 SARS-CoV-2 viral positivity as measured by RT-PCR. We observed SARS-CoV-2 NP staining 144 selective to insulin-positive β-cells in 4 of 7 patients, while the remaining 3 pancreatic samples and 145 healthy control samples were negative for NP staining ( Figure 3A ). The specificity of the anti-NP 146 antibody was validated through peptide blocking assays (Figures S2D). The 3 negative samples 147 (staining not shown) from patients with COVID-19 had extensive autolysis/atrophy (Table 3) , which 148 may explain the lack of NP signal due to rapid proteolysis of tissue by digestive enzymes. As an 149 orthogonal confirmation of our observations of viral presence in β-cells, we performed in situ β-cells of patients with COVID-19 compared with non-COVID-19 donors as a potential explanation for why β-cells are more susceptible to viral infection. ACE2 expression remained low in individuals with non-COVID-19 donors ( Figures S3C and S3D) . These results support a potential role of NRP1 in β-cell 163 susceptibility of viral infection. However, it is also possible that this is due to the increased NRP1 164 expression in the organ caused by SARS-CoV-2 infection, which in turn causes the cells to be more 165 susceptible to infection. Further research is needed to establish the generality and mechanism by 166 which SARS-CoV-2 may require either (1) preexisting or (2) virally induced upregulation of NRP1 167 levels. To determine whether SARS-CoV-2 infection affected pancreatic islet function, we quantified the 170 insulin content and glucose-stimulated insulin secretion (GSIS), a functional assay for β cell insulin 171 release, in infected islets. We observed a dramatic decrease in insulin content and GSIS in this effect is partially reversed upon treatment with the NRP1 antagonist EG00229 ( Figure S3E ). In protein-treated human islets was calculated by comparing them to vehicle control, and KSEA was 207 performed using a kinase-substrate database created using PhosphoSitePlus (Hornbeck et al., 2015) 208 and NetworKin (Linding et al., 2008) . Quantification of the activity of 67 kinases ( Figure S4B , Table S1 , and Of note, while we found SARS-CoV-2 localization within β-cells from 4 autopsied patients, 250 SARS-CoV-2 NP was not detected in pancreatic islets from 3 autopsied patient samples from a 251 separate report (Kusmartseva et al., 2020) . This discrepancy is likely due to the issue that pancreatic 252 tissues are highly prone to autolysis, resulting in the rapid proteolysis of proteins due to the abundance 253 of digestive enzymes. Indeed, we also did not detect SARS-CoV-2 NP signal in 3 out of 7 pancreas 254 tissues with extensive pathologist-verified autolysis/atrophy, further suggesting the importance of rapid Table 1 ). **p < 0.001, One-way ANOVA with Tukey's post-test. Each dot represents one donor. Scale bars: 5 μm (A) and (C and D) Error bars represent mean ± s.d. (500~1000 cells were quantified from healthy isolated 399 human islets from Donor 1-5, see Table 2 ). Table 2 ). *p < 0.05, two-tailed Student's t-test. Each dot represents one donor. Scale bars: 5 μm (A, B, and E), 2 μm (insets). See also Figure S3 and Table 3 . (A) Insulin content is decreased in SARS-CoV-2 infected islets compared to mock-treated islets. (B) GSIS is decreased in SARS-CoV-2 infected islets compared to mock-treated islets. (A and B) Error bars represent mean ± s.d. (Data was collected from 7 healthy isolated human islets, Donor 2-8, see Table 2 ). *p < 0.05, two-tailed Student's t-test. Table 2 ). *p < 0.05, **p < 0.01, two-tailed Student's t-test. Scale bars: 5 μm (C and E). See also Figure S3 -5, Table 2, and Table S1 -3. Fluorescence-immunolabeled images were acquired using a Zeiss AxioImager Z1 microscope or a GSIS was performed 6 days post infection. Batches of 25 islets were used for in vitro secretion assays. Islets were incubated at a glucose concentration of 2.8 mM for 1 hour as an initial equilibration period. Subsequently, islets were incubated at 2.8 mM glucose concentration for 1 h. Supernatant was taken 557 and stored for insulin quantification. Islets were incubated at 16.7 mM glucose concentration for 558 another 1 h. Supernatant was taken and stored for insulin quantification. Islets were then lysed in an 559 acid-ethanol solution (1.5% HCL in 75% ethanol) to extract the total cellular insulin or glucagon content. Secreted human insulin in the supernatants and islet lysates were quantified using a human insulin 561 ELISA kit (Mercodia). Secreted insulin levels were divided by total insulin content and presented as a 562 percentage of total insulin content and normalized to values obtained at 2.8 mM glucose. All secretion 563 assays were carried out in RPMI 1640 (Gibco) and the above-mentioned glucose concentrations. vortexed, 100 µl water, vortexed, and centrifuged at 13,000g for 2 minutes. The top aqueous layer was 577 removed, 200 µl of methanol was added, vortexed, and centrifuged at 13,000g for 3 minutes. Methanol 578 was removed and dried proteins were resuspended using 200mM HEPES pH 8.5. Proteins were 579 digested using Trypsin/Lys-C overnight at 37ºC 250 RPM. Sample was acidified using TFA and 580 cleaned using stage tips. Stage tips were created using 5 layers of C18 filters packed into a P200 tip. exploratory nature of the work and the limited availability of tissue specimens. No pancreatic samples U test was used when the data were not normally distributed by When the normal 638 distribution and equal variance were confirmed, Student's t test were used. Kruskal-Wallis 639 test and post-hoc Dunn's multiple comparison test were used for comparisons of more than 640 two groups. Analyses were performed with GraphPad Prism 6.0 (GraphPad Software Supplemental Table 1. Phosphoproteomic Data, Related to Figure 4 and S4 Contains raw count of filtered phosphoproteomic data and fold change analysis of phosphosites for 644 human islet cells treated with purified Spike protein (PhosphoSpike) or SARS-CoV-2 (PhosphoSars) Unfiltered phosphoproteomic raw count data is also included (UnfilteredPhospho) Kinase activity as predicted by KSEA of various kinases in human islet cells treated with purified Spike 648 protein (KSEA.Spike) or SARS-CoV-2 (KSEA.Sars). Kinase activity is reported as Enrichment Score 649 (ES) as calculated by weighted Kolmogorov-Smirnov statistics Gene ontology enrichments for upregulated/downregulated kinases upon treatment by Spike protein or 652 SARS-CoV-2 Age-Dependent Pancreatic Gene Regulation Reveals Mechanisms Governing Human beta 657 Cell Function Autoantibody-negative insulin-dependent diabetes mellitus 715 after SARS-CoV-2 infection: a case report 2014: mutations, PTMs and recalibrations Molecular and genetic regulation of pig pancreatic islet cell 720 development Expression of SARS-CoV-2 Entry Factors in the 723 Pancreas of Normal Organ Donors and Individuals with COVID-19 SARS-CoV-2 productively infects 726 human gut enterocytes ACE2 localizes to the respiratory cilia and is not increased by ACE 729 inhibitors or ARBs SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial derived cells and cardiomyocytes NetworKIN: a resource for exploring cellular phosphorylation networks A brain serine/threonine protein 738 kinase activated by Cdc42 and Rac1 Online Parallel Accumulation-Serial Fragmentation (PASEF) 741 with a Novel Trapped Ion Mobility Mass Spectrometer Postmortem examination of COVID-19 patients reveals diffuse 744 alveolar damage with severe capillary congestion and variegated findings in lungs and other organs 745 suggesting vascular dysfunction SARS-CoV-2 infects and replicates in cells of the human 748 endocrine and exocrine pancreas New onset diabetes with diabetic ketoacidosis 750 in a child with multisystem inflammatory syndrome due to COVID-19 Loss of intra-islet heparan sulfate is a highly 763 sensitive marker of type 1 diabetes progression in humans Hyperglycemia without diabetes and new-onset diabetes are both 765 associated with poorer outcomes in COVID-19 Intestinal Inflammation Modulates the Expression of ACE2 and 768 TMPRSS2 and Potentially Overlaps With the Pathogenesis of SARS-CoV-2-related Disease Gene set enrichment analysis: a 772 knowledge-based approach for interpreting genome-wide expression profiles Transferrin receptor is another receptor for SARS-CoV-2 entry Requirement of JNK for stress-induced activation of the cytochrome 778 c-mediated death pathway New-Onset Type 1 Diabetes in Children During COVID-19: Multicenter 781 Regional Findings in the U.K Mitogen-activated protein kinases in apoptosis regulation Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a 786 single-centered, retrospective, observational study clusterProfiler: an R package for comparing 788 biological themes among gene clusters