SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 Are Expressed in the Microvasculature and Ducts of Human Pancreas but Are Not Enriched in β Cells

Isolated reports of new-onset diabetes in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2 is directly cytotoxic to pancreatic islet β cells. This would require binding and entry of SARS-CoV-2 into β cells via co-expression of its canonical cell entry factors, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2); however, their expression in human pancreas has not been clearly defined. We analyzed six transcriptional datasets of primary human islet cells and found that ACE2 and TMPRSS2 were not co-expressed in single β cells. In pancreatic sections, ACE2 and TMPRSS2 protein was not detected in β cells from donors with and without diabetes. Instead, ACE2 protein was expressed in islet and exocrine tissue microvasculature and in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. These findings reduce the likelihood that SARS-CoV-2 directly infects β cells in vivo through ACE2 and TMPRSS2.


In Brief
Coate et al. examined expression of canonical SARS-CoV-2 entry proteins ACE2 and TMPRSS2 in the human pancreas and report ACE2 expression in the microvasculature, including islet pericytes, whereas both ACE2 and TMPRSS2 are expressed in some ducts. Conversely, neither protein is detected in b cells, arguing against direct b cell viral infection in vivo.
In vitro studies have shown that SARS-CoV-2 entry into human host cells requires binding to the cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as proteolytic cleavage of the viral spike (S) protein by transmembrane serine protease 2 (TMPRSS2) (Hoffmann et al., 2020;Lan et al., 2020;Shang et al., 2020;Wiersinga et al., 2020). In 2010, Yang et al. (2010) examined autopsy samples from a single deceased patient infected by SARS-CoV-1, which uses similar machinery for binding and cellular entry, and reported expression of ACE2 in pancreatic islet cells. Though the identity of these islet cells was not assessed, the authors suggested that binding of ACE2 by SARS-CoV-1 damages islets and causes acute diabetes, A B C Figure 1. ACE2 and TMPRSS2 Expression in Isolated Human Islet Cells and Juvenile Pancreas (A) Relative expression of ACE2 and TMPRSS2 compared with select a (white bars) and b (green bars) cell-type-enriched genes in sorted human islet a and b cells from previously published bulk RNA-seq datasets, reported as transcript per million mapped reads (TPM; n = 7; Blodgett et al., 2015) or reads per kilobase of transcript per million mapped reads (RPKM; n = 8; Arda et al., 2016). Mean expression values are presented as log 2 (TPM+1) or log 2 (RPKM+1) to account for negative values. Dotted line highlights ACE2 and TMPRSS2 expression. which could be reversed after viral recovery (Yang et al., 2010). Further, there have been occasional reports of other viral infections eliciting a diabetogenic effect (reviewed in Filippi and von Herrath, 2008). More recently, Yang and colleagues reported that b-like cells derived from human pluripotent stem cells (hPSCs) as well as b cells of primary human islets express ACE2, raising the possibility of direct infection and cytotoxicity of b cells by SARS-CoV-2 . Importantly, neither of these prior studies (Yang et al., 2010 characterized the expression and localization of TMPRSS2, an obligate co-factor for SARS-CoV-2 cellular entry. Thus, a more detailed analysis of both ACE2 and TMPRSS2 expression and localization in human pancreatic tissue from normal donors and those with diabetes is urgently needed. The purpose of this study was to test the hypothesis that native pancreatic islet b cells possess the cellular machinery that could render them direct targets of SARS-CoV-2. Importantly, we found that ACE2 and TMPRSS2 proteins are not detectable in human islet endocrine cells from normal donors or those with diabetes, making a direct diabetogenic effect of SARS-CoV-2 via ACE2 and TMPRSS2 unlikely.

RESULTS AND DISCUSSION
ACE2 and TMPRSS2 mRNA Expression Is Minimal in Human a or b Cells We first evaluated mRNA expression of ACE2 and TMPRSS2 from two existing bulk RNA sequencing (RNA-seq) datasets Blodgett et al., 2015), where human islet a and b cells were enriched by fluorescence-activated cell sorting, and compared their expression to that of key islet-enriched genes, some of which are normally expressed at relatively low levels in islet cells (e.g., transcription factors). Median expression level of ACE2 and TMPRSS2 mRNA was much less than transcripts of such key islet-enriched genes in human a and b cells (84% and 92% lower than a and b cell-enriched transcripts, respectively) ( Figure 1A; n = 7-8 adult donors per study). In addition, analysis of four single-cell (sc) RNA-seq datasets of human pancreatic cells Camunas-Soler et al., 2020;Kaestner et al., 2019;Segerstolpe et al., 2016) revealed that, in aggregate, less than 1.5% of b cells expressed ACE2 or TMPRSS2, and each transcript was minimally expressed or undetectable in all other endocrine cell subsets (Figures 1B and S1A; Table S1). We note that this included analysis of the robust Human Pancreas Analysis Program (HPAP) dataset that contained more than 25,000 cells from 11 normal donors (Kaestner et al., 2019), the findings of which were confirmed in the analyses of three previously reported, but smaller, datasets (Figures 1B and S1A). Furthermore, given that ACE2 and TMPRSS2 coexpression is required for canonical SARS-CoV-2 host cell entry (Hoffmann et al., 2020), we also evaluated this occurrence but found that no b cells co-expressed ACE2 and TMPRSS2 in any of these four datasets (Table S1).
As for non-endocrine cells, the HPAP dataset (Kaestner et al., 2019) revealed that a small subset (<5%) of endothelial and stellate cells (which include pericytes) expressed moderate to high levels of ACE2, whereas only 1%-3% of either population expressed ACE2 in the datasets by Baron et al. (2016) and Segerstolpe et al. (2016) (Figure 1B). This difference most likely stemmed from the number of cells analyzed; the HPAP dataset contains 2.5and 20-fold more total cells than that of Baron et al. (2016) or Segerstolpe et al. (2016), respectively. In addition, pooled analysis of these datasets Camunas-Soler et al., 2020;Kaestner et al., 2019) showed that less than 1% of acinar or ductal cells expressed ACE2, whereas 35% of both cell types expressed TMPRSS2. In the dataset by Segerstolpe et al. (2016), ACE2 was expressed in 20% of acinar and ductal cells, whereas TMPRSS2 was expressed in greater than 75% of both populations ( Figure 1B). Evaluation of ACE2 and TMPRSS2 co-expression in non-endocrine cells revealed that on average, less than 1% of acinar, ductal, endothelial, and stellate cells co-expressed both transcripts in these datasets Camunas-Soler et al., 2020;Kaestner et al., 2019). In the Segerstolpe et al. (2016) dataset, 5% of acinar cells and 15% of ductal cells co-expressed ACE2 and TMPRSS2, but this difference could represent unintended cell selection bias as this study analyzed the fewest number of cells.
Altogether, these gene expression findings reduce the likelihood that SARS-CoV-2 can bind and enter human b cells via the canonical pathway involving ACE2 and TMPRSS2. However, they do not exclude the possibility of viral entry via non-canonical pathways involving other suggested effector proteases of SARS-CoV-2, such as Cathepsin L (CTSL), ADAM metallopeptidase domain 17 (ADAM17), FURIN, and TMPRSS4 (Breidenbach et al., 2020;Schreiber et al., 2020;Seyedpour et al., 2020;Zang et al., 2020). We evaluated the expression of these transcripts across all four scRNAseq datasets and found that with the exception of TMPRSS4, each is ubiquitously expressed to varying degrees in all cell types (Figures 1B and S1A; Table S1). In aggregate, however, less than 1.3% of b cells co-expressed ACE2 with CTSL, ADAM17, FURIN, or TMPRSS4 (Table S1). An outstanding question is whether expression of these transcripts in even 1% of b cells is sufficient to confer permissiveness to SARS-CoV-2 infection in vivo. Furthermore, heparan sulfate in the extracellular glycocalyx (Clausen et al., 2020) and motile cilia (present on islet endocrine cells)  are mechanisms proposed to assist ACE2-mediated viral entry. Future studies addressing these possibilities in b cells are urgently needed. Notwithstanding, CTSL, ADAM17, FURIN, and TMPRSS4 were more highly expressed in (B) Dot plots of ACE2, TMPRSS2, CTSL, ADAM17, FURIN, TMPRSS4, and DPP4 expression compared with cell-type-enriched genes from three single-cell (sc) RNA-seq datasets Kaestner et al., 2019;Segerstolpe et al., 2016). Dot size indicates percentage of cells in a given population expressing the gene; dot color represents scaled average expression. Dotted lines highlight expression of putative SARS-CoV-2 entry machinery. Percentages of b cells expressing and co-expressing these genes are available in Table S1. (C) Representative images of an isolated islet from an adult human donor (top panels; n = 6 total images analyzed) and a pancreatic section from a juvenile human donor (bottom panels; n = 10 total images analyzed) stained for ACE2 (red; antibody ab15348), insulin (INS; green), glucagon (GCG; white), and DAPI (blue). Dotted yellow line denotes islet area. Human pancreatic donor information is available in Table S2 (donors I1-I3 and J1-J5). See also Figures S1, S2, and S4. Scale bar, 50 mm.

Figure 2. ACE2 and TMPRSS2 Protein in Human Islets and Exocrine Tissue of Adult Pancreas
(A-F 00 ) Immunostaining of SARS-CoV-2 cell entry markers ACE2 (red; antibody AF933; A, C, C, 0 D, F, and F 0 ) or TMPRSS2 (green; B, C, C 00 , E, F, and F 00 ) in islet b cells (insulin, blue; C and C 0 ) and islet a cells (glucagon, blue; F and F 0 ) in native pancreatic sections from adult normal donors without diabetes (n = 14 total images analyzed).
(G-L 00 ) Immunostaining of SARS-CoV-2 cell entry markers ACE2 (red; antibody AF933; G, I, I 0 , J, L, and L 0 ) or TMPRSS2 (green; H, I, I 00 , K, L, and L 00 ) in islet b cells (insulin, blue; I and I 0 ) and islet a cells (glucagon, blue; L and L 0 ) in native pancreatic sections from adult donors with type 2 diabetes (n = 8 total images analyzed).
(legend continued on next page) ll Short Article exocrine, endothelial, and stellate cells, as was ACE2 (Figure 1B). These gene expression patterns raise the possibility that infection of certain pancreatic cell types by SARS-CoV-2 indirectly impacts b cell function.
Obesity is a key metabolic risk factor associated with mortality with COVID-19. A recent report found a positive correlation between BMI and TMPRSS2, but not ACE2, expression (Taneera et al., 2020). We evaluated the expression of ACE2, TMPRSS2, and ADAM17 in b cells from 11 donors according to their BMI in our largest scRNA-seq dataset (Kaestner et al., 2019). We did not observe changes in the expression of ACE2 or TMPRSS2 expression with higher BMI but observed a trend toward higher ADAM17 expression (Figure S1B). However, this dataset included only one patient with a BMI in the obese range. Studies with a larger number of donor islets are needed to address the role of obesity and other pancreas-resident SARS-CoV-2 effector molecules on b cell function in the context of COVID-19.

ACE2 and TMPRSS2 Proteins Are Not Detected in Adult or Juvenile Human Islet a or b Cells
Recently, Yang et al. (2020) reported that ACE2 protein was present in a and b cells from primary human islets and in b-like cells derived from hPSCs. To investigate ACE2 expression, we immunostained cryosections of intact isolated human islets using the same ACE2 antibody as used by Yang et al. (2020) (antibody validation studies in Figure S2; mouse [ Figure S2A] and human [Figures S2J,S2O,and S2T] kidney tissues were utilized as positive controls for ACE2 immunostaining). In accordance with the transcriptomic data reported above, ACE2 was not detected in insulin-positive b cells or glucagon-positive a cells in our samples; instead, ACE2 signal was prominent in surrounding cells such as those of the microvasculature ( Figure 1C, top panels).
Since hPSC-derived b-like cells differentiated in vitro are considered ''juvenile-like'' and not functionally mature b cells (Nair et al., 2019), one possibility is that ACE2 is expressed in less differentiated cells, or in the cultured, immortalized b cells (EndoCbH1) studied by Fignani et al. (2020). We investigated such a possibility by staining for ACE2 in pancreatic sections from juvenile human donors (age range 5 days to 5 years) using the same ACE2 antibody as used by Yang et al. (2020). However, ACE2 was not detected in these juvenile b or a cells ( Figure 1C, bottom panels), making this explanation less likely.
To further characterize ACE2 and TMPRSS2 protein expression in the native pancreas, we next analyzed human pancreatic tissue sections from normal donors (ND, n = 14; age range 18-59 years) and those with type 2 diabetes (T2D, n = 12; age range 42-66 years) or type 1 diabetes (T1D, n = 11; age range 13-63 years) for these proteins on the same sections. ACE2 protein did not co-localize with markers of a or b cells but was detected within the distinct islet areas indicative of microvascular structures in all ND individuals (Figures 2A-2F 00 and S3A-S3D) as well as those with T2D (Figures 2G-2L 00 and S3I-S3L) or T1D (Figures 2M-2R 00 and S3O-S3R). We noted similar findings with three additional ACE2-directed antibodies, including the same antibodies and dilutions reported by Yang et al. (2020) and Fignani et al., (2020), and one antibody validated by the Human Protein Atlas (HPA) (Hikmet et al., 2020) (Figure S2). Three of these antibodies detect epitopes corresponding to the extracellular domain of ACE2 (AF933, MAB933, and HPA000288), while one detects an epitope in the C-terminal domain (ab15348). Staining patterns were similar across all antibodies ( Figure S2). Furthermore, ACE2 antibody specificity (ab15348) was confirmed via peptide competition with the commercially available immunizing peptide ( Figures S2F-S2I).
TMPRSS2 protein was not detected within islets in ND ( We did not detect differences in the signal intensity or spatial distribution of ACE2 or TMPRSS2 between ND and T2D tissue (Figures 2 and S3), but labeling for both proteins appeared to be reduced in T1D pancreatic sections (Figures 2M-2R 00 and S3O-S3V). Altogether, these observations indicate that the cell surface proteins required for canonical SARS-CoV-2 host cell entry were not detected on islet endocrine cells from normal or diabetic donors.
Recent in silico analyses by Vankadari and Wilce (2020) and Li et al. (2020b) suggested that human dipeptidyl peptidase 4 (DPP4) may interact with SARS-CoV-2 and facilitate its entry into host cells. Therefore, we examined the distribution of DPP4 in human pancreas and found that it localized to a cells, but not b cells, of ND, T2D, and T1D islets ( Figures S4A-S4I). This finding is consistent with our analysis of four scRNA-seq datasets showing DPP4-enriched a cells ( Figures 1B and S1A). Given that we did not detect TMPRSS2 within islets, these data suggest that DPP4 is an unlikely mediator of b cell entry by SARS-CoV-2 (Drucker, 2020).
The discrepancy between our findings and those of recent studies identifying ACE2 and TMPRSS2 in human b cells (Fignani et al., 2020;Yang et al., 2010Yang et al., , 2020 is likely explained by important differences in experimental approaches and contexts. Yang et al. (2010) showed ACE2 staining in a single donor islet but did not identify its cellular identity with endocrine markers. In addition, the ACE2 antibody used in their study was not reported, which precluded replication of their finding. Furthermore, Yang et al. (2020) examined ACE2 expression in isolated dispersed human islet cells, whereas our study examined ACE2 in isolated intact islets and in human islets in native pancreas in situ. It is possible that dispersion of pancreatic cells alters the expression   (2020) reported ACE2 protein in some islet endocrine cells from seven non-diabetic donors; however, ACE2 was primarily seen in subcellular compartments rather than the expected cell surface location. Intriguingly, Fignani et al., (2020) observed differential immunostaining patterns in human pancreas and islets upon testing three ACE2 antibodies that recognize different epitopes of the full-length ACE2 protein, leading them to conclude that a recently discovered short isoform of ACE2 (Blume et al., 2020;Ng et al., 2020;Onabajo et al., 2020) is expressed in human b cells. However, at least two of the four ACE2 antibodies (ab15348 and AF933) we used in our study are predicted to recognize both the short and long isoforms of ACE2, yet we did not detect either form of the ACE2 protein in human islet b cells with either of these reagents. One of these antibodies (ab15348) was also used by Fignani et al., (2020). Furthermore, the short isoform of ACE2 appears to be unstable  and, importantly, lacks the residues required for SARS-CoV-2 spike glycoprotein binding (Blume et al., 2020;Ng et al., 2020;Onabajo et al., 2020), making it an unlikely receptor for SARS-CoV-2. Sources of variability in immunostaining techniques can stem from differences in tissue fixation procedures, antigen retrieval methods, antibodies used, and their dilutions. Notwithstanding, the remarkable concordance of our findings with those from a recent independent effort in native human pancreas (Kusmartseva et al., 2020) suggests that differences in experimental conditions may explain the discordance with other studies.

ACE2 Is Localized to Islet and Exocrine Tissue Capillaries
The non-endocrine cell staining pattern of ACE2 in islets prompted us to examine whether ACE2 was expressed in the microvasculature, as described in other organs (Hamming et al., 2004). Indeed, staining of adult and juvenile human pancreatic tissue sections with CD31, an endothelial cell marker, revealed that ACE2 labeling was localized to the perivascular compartment of islet capillaries ( Figures 3A-3D  To address this observation more directly, we visualized ACE2 expression in relationship to known pericyte markers and found that ACE2 was enriched in pericyte populations expressing PDGFRb (Figures 4A-4D). Indeed, colocalization analysis revealed that 60% of ACE2-positive cells were PDGFRb positive ( Figures 4H and 4I). Since PDGFRb labels pericytes and perivascular fibroblasts (Almaç a et al., 2020), we also stained tissues with a more specific pericyte marker, NG2, and found that 30% of ACE2-positive cells were NG2 positive (Figures 4E-4G 0 , 4H, and 4I). These data agree with our scRNA-seq analysis of the HPAP dataset, which identified a subset of pericytes, marked by PDGFRB, that expressed moderate levels of ACE2 ( Figure 1B). In addition, our findings support recent reports of pericyte-specific vascular expression of ACE2 in brain and heart tissue He et al., 2020). Emerging evidence from both pre-clinical models of SARS-CoV-2 infection (Aid et al., 2020) and patients with COVID-19 indicates that cells of the microvasculature (e.g., endothelial cells and pericytes) are requisite contributors to the initiation and propagation of severe disease (Teuwen et al., 2020). Although we found that TMPRSS2 expression was negligible in endothelial and stellate cells (which includes pericytes) of human islets, CTSL, ADAM17, FURIN, and ACE2 were moderately to highly expressed in these populations ( Figure 1B), raising the intriguing possibility of direct infection of cells in the islet microvasculature by SARS-CoV-2. Such an occurrence could trigger b cell dysfunction and the metabolic sequelae of COVID-19. Future studies addressing this possibility in human islets and pancreatic tissue are urgently needed.
Both ACE2 and TMPRSS2 Proteins Are Present in Pancreatic Ducts but Rarely Are Co-expressed Our work indicated that TMPRSS2 protein was localized to the apical surface of intercalated and larger ducts in ND ( Figures  2B, 2C 00 , 2E, 2F 00 , and S3E-S3H), T2D ( Figures 2H, 2I 00 , 2K, 2L 00 , S3M, and S3N), and T1D pancreatic tissues ( Figures 2N,  2O 00 , 2Q, 2R 00 , and S3S-S3V). To further define ductal expression of TMPRSS2 and determine whether ACE2 was expressed beyond exocrine tissue capillaries, we visualized expression of these two proteins in relationship to cytokeratin-19 protein (KRT19), a ductal cell marker. TMPRSS2, but not ACE2, was expressed by KRT19-positive cells on the apical surface of intercalated and larger ducts throughout the exocrine compartment ( Figures 5A-5D 0 ). Rarely, both ACE2 and TMPRSS2 were found on the apical surface of ductal epithelial cells but were spatially distinct ( Figures 5E-5H 0 ). These findings agree with our scRNA-seq analysis in which TMPRSS2 was more highly expressed in ductal cells than ACE2, but cells positive for both ACE2 and TMPRSS2 were rare. In addition, although TMPRSS2 mRNA was detected in acinar cells by scRNA-seq ( Figure 1B), (E-G 0 ) Representative images of an adult human islet labeled for neuron-glial antigen 2 (NG2, red; E and G), ACE2 (green; antibody ab15348; F and G), and insulin (INS, blue; G) in perivascular cells within a normal donor. DAPI (blue; E, F, and G 0 ). Inset areas in (G 0 ) are marked by yellow boxes in (G) and represent a subset of ACE2-expressing islet pericytes (n = 36 total images analyzed). (H) Colocalization of ACE2 with NG2-or PDGFRb-positive cells.
(I) Quantification of ACE2-positive cells expressing pericyte markers PDGFRb and NG2. Shown are average values obtained for 3 confocal planes per islet for a minimum of 5 islets per each donor of a given age. Human pancreatic donor information is available in Table S2 (donors HP1754, HP2041, and HP2091). See also Figure S5. Scale bars, 20 mm (A-C), 40 mm (D-G), and 10 mm (G 0 ).
(legend on next page) ll Short Article TMPRSS2 protein was undetectable by immunofluorescence analysis (Figure 5), indicating that studies of mRNA and protein may be discordant.
Because rare cells in the exocrine compartment co-express ACE2 and TMPRSS2, it is possible that SARS-CoV-2 could infect those cells, induce pancreatitis, and consequently impact islet function. While recent case reports of individuals with COVID-19 have noted mild elevations of amylase and lipase or frank pancreatitis (Karimzadeh et al., 2020;Meireles et al., 2020;Rabice et al., 2020;Wang et al., 2020a), the vast majority of individuals with COVID-19 have neither elevated pancreatic enzymes nor frank pancreatitis (Ashok et al., 2020;Bonney et al., 2020;Bruno et al., 2020). To investigate this possibility, we analyzed the histology of pancreatic tissue obtained at autopsy from seven individuals with COVID-19, three of whom had diabetes. We did not find signs of pancreatitis, interstitial edema, inflammatory infiltrate, hemorrhage, or necrosis ( Figure 5I). It is important that exocrine inflammation and ACE2/TMPRSS2 expression be analyzed in a larger number of COVID-19 autopsy samples, particularly in those with new-onset diabetes. Such studies will be challenging since the pancreas rapidly undergoes autolysis upon death.
In summary, recent reviews, commentaries, and clinical guidelines (Apicella et al., 2020;Bornstein et al., 2020;Goldman et al., 2020;Gupta et al., 2020;Heaney et al., 2020) have highlighted an attractive hypothesis of direct cytotoxicity by SARS-CoV-2 to b cells. Here, we addressed the fundamental question of whether canonical SARS-CoV-2 cell entry machinery is present in b cells of human pancreatic tissue. By examining pancreatic ACE2 and TMPRSS2 expression in normal donors and in those with diabetes, we found that (1) b cells do not coexpress ACE2 and TMPRSS2 transcripts by scRNA-seq analysis and neither protein is detectable in a or b cells by immunofluorescence; (2) ACE2 is primarily expressed in the islet and exocrine tissue capillaries, including pericytes, as well as a subset of ductal cells; (3) TMPRSS2 is primarily expressed in ductal cells; (4) ACE2 and TMPRSS2 are infrequently co-expressed in pancreatic ducts; (5) ACE2 and TMPRSS2 protein expression and distribution appear similar in ND and T2D pancreata, but studies of additional pancreata are needed to definitively answer this question; and (6) alternative pathways facilitating SARS-CoV-2 viral infection in b cells cannot be excluded. These findings align considerably with independent observations from Kusmartseva et al. (2020) and do not support the hypothesis that SARS-CoV-2 binds and infects islet b cells via a canonical pathway mediated by ACE2 and TMPRSS2, eliciting a diabe-togenic effect. As outlined below in the Limitations of Study section, additional studies are needed to investigate whether direct SARS-CoV-2 infection of b cells occurs or is detrimental to b cell health or function by other mechanisms. However, based on current data, it appears that the interaction of diabetes and SARS-CoV-2 is mediated by systemic inflammation and/or metabolic changes in other organs such as liver, muscle, or adipose tissue.

Limitations of Study
We did not directly measure SARS-CoV-2 binding or entry into human b cells but instead assessed expression of canonical co-receptors, ACE2 and TMPRSS2, in the human pancreas. While studies culturing SARS-CoV-2 and human islets in vitro will be of considerable interest, such experiments may be challenging to interpret as the islet isolation process itself may impact ACE2 or TMPRSS2 expression or susceptibility to viral infection. In addition, infection of islet cells within an intact, isolated islet with a variety of viruses has proven difficult, as is selection of an appropriate ratio of viral particles and islet cells reflective of the in vivo environment. Furthermore, it is not clear if infection of an isolated islet accurately models the physiology of a vascularized, innervated islet within the context of the pancreas.
While the preponderance of research suggests that ACE2 and TMPRSS2 are the primary means for SARS-CoV-2 entry into host cells, knowledge about additional pathways and/or mechanisms of SARS-CoV-2 cell entry and infection is rapidly evolving. We sought, but did not find, expression evidence, suggesting other proposed pathways (namely DPP4, CTSL, ADAM17, FURIN, or TMPRSS4) for b cell entry.
We did not assess the presence of SARS-CoV-2, its proteins, or RNA in the pancreas from individuals with COVID-19. It is possible that SARS-CoV-2 could infect pancreatic exocrine cells, ductal cells, endothelial cells, or microvasculature, leading to pancreatic inflammation that negatively impacts b cell health or function. We did not find evidence of inflammation in the pancreas of seven individuals with COVID-19 (three of whom had diabetes). It is also possible that COVID-19 could induce ectopic expression of SARS-CoV-2 cell entry factors in the endocrine pancreas and render islet cells susceptible to direct infection. Future efforts are needed to collect a large number of pancreata from individuals with COVID-19 with and without diabetes (long-standing and recent onset) to search for presence of SARS-CoV-2 or inflammation (islet or exocrine). Since the pancreas undergoes rapid autolysis after death, care must be taken to collect the pancreas very soon after death for this analysis.

DECLARATION OF INTERESTS
The authors declare no competing interests.