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

ACE2 and TMPRSS2 Gene Expression Is Low in Human Pancreatic Endocrine Cells

Diabetes, obesity, and advanced age increase the risk of COVID-19 mortality (Zhou et al., 2020). Autopsy studies of individuals infected with SARS-CoV-2 demonstrate systemic viral dissemination with persistence in multiple organs, including the lungs and kidneys (Hanley et al., 2020; Liu et al., 2020; Menter et al., 2020; Wichmann et al., 2020), but there was an apparent limitation of pronounced inflammatory alterations to the lung and reticulo-endothelial system (Dorward et al., 2020). Recent studies (Barron et al., 2020; Fignani et al., 2020; Goldman et al., 2020; Holman et al., 2020; Li et al., 2020; Marchand et al., 2020; Unsworth et al., 2020; Wang et al., 2020) spurred interest in ACE2 expression in the pancreas, particularly the endocrine compartment, to address a potential relationship between diabetes and COVID-19, including the potential for either direct β cell infection or β cell damage via indirect mechanisms. To date, studies of ACE2 expression in the pancreas have been limited and contradictory, and analysis of autopsy specimens from COVID-19 cases have not been published, likely due to challenges associated with tissue procurement and post-mortem autolysis.

SARS-CoV-2 entry into cells via the ACE2 receptor requires S protein priming by the mucosal serine proteases (Lee et al., 2020b; Zang et al., 2020). We thus investigated expression patterns of ACE2 and several proteases linked with SARS-CoV-2 processing by conducting an integrated analysis of scRNA-seq data from five public datasets including 22 non-diabetic and 8 T2D individuals (Baron et al., 2016; Grün et al., 2016; Lawlor et al., 2017; Muraro et al., 2016; Segerstolpe et al., 2016). This analysis revealed a low frequency of ACE2-expressing cells and low ACE2 expression levels in the majority of islet cell subsets (Figures 1A and 1B). In islets from donors without diabetes, ACE2 was expressed in <2% of endocrine, endothelial, and select innate immune cells. ACE2 was detectable in 4.11% of acinar cells and 5.54% of ductal cells in non-diabetic donors as compared to 8.07% of acinar and 8.13% of ductal cells in donors with T2D (Figures 1A and 1B; Table S1). Expression levels of ACE2 were not different between non-diabetic donors and those with T2D in any of the islet cell subtypes (Figure 1A).

We next investigated the expression patterns of the serine protease TMPRSS2, which is thought to be the main serine protease required for SARS-CoV-2 infectivity. TMPRSS2 was detectable in 53.73% of acinar and 50.55% of ductal cells in donors without diabetes, and 71.43% of acinar and 58.74% of ductal cells in donors with T2D (Figure 1C; Table S1). Apart from α cells, which demonstrated 16.55% positivity in non-diabetic donors and 32.07% positivity in donors with T2D, TMPRSS2 expression was low in the majority of other endocrine cell subsets (Figure 1C). TMPRSS2 was detectable in 5.46% of β cells, and neither ACE2 nor TMPRSS2 expression differed significantly in β cells from non-diabetic versus T2D donors (Figures 1A, 1C, S1A, and S1B). TMPRSS2 showed higher relative expression levels in ductal and acinar cells compared to β cells (adjusted p = 1.31 × 10⁻²⁹¹ and p < 1 × 10⁻³⁰⁰, respectively), and was detectable in an elevated proportion of these cells in non-diabetic donors (p < 0.05) (Figures 1C and 1D). The frequency and expression of ACE2 and TMPRSS2 relative to a subset of cellular identity marker genes across each of the analyzed cell populations highlight, in general, the low expression of ACE2 and TMPRSS2 across pancreatic cells (Figure 1E). We also analyzed ACE2 and TMPRSS2 levels relative to an expanded list of highly expressed genes in α and β cells, including GCG, TTR, SCG2, INS, IAPP, and PDX1 (Figures 1F and 1G).

Because the expression of ACE2 in combination with an associated protease appears to be required for cellular entry (Lee et al., 2020b; Zang et al., 2020), we calculated the percentage of each cell type that expressed both ACE2 and TMPRSS2. Notably, only 0.24% and 0.10% of α cells and β cells were positive for ACE2 and TMPRSS2 in islets from donors without diabetes, while no α or β cells from donors with T2D expressed both (Table S2). Fewer than 4% of acinar and ductal cells in donors without diabetes were positive for ACE2 and TMPRSS2, while co-expression was observed in 8.07% and 6.10% of acinar and ductal cells, respectively, in donors with T2D (Table S2).

We also investigated expression patterns for other SARS-CoV-2-associated proteases, including TMPRSS4, CTSL, ADAM17, and TMPRSS11D (Table S1; Figures S1C–S1H). In addition, we quantitated the percentage of cells that co-expressed ACE2 and each of these proteases (Table S2). TMPRSS4 was expressed in a similar proportion of α and β cells; however, relative expression levels tended to be low in the endocrine pancreas (Figures S1C and S1D). CTSL and ADAM17 were detected at higher levels in α and β cells (Figures S1E and S1F). The relative expression of TMPRSS11D was low in most cell types examined (Figure S1G). Within the β cells, only CTSL showed higher expression in donors with T2D compared to donors without diabetes (adjusted p = 8.94 × 10⁻³²; Figure S1H). Notably, less than 1% of β cells expressed ACE2 in combination with any of these identified proteases (Table S2).

To visualize ACE2 and TMPRSS2 mRNA expression patterns in situ, we used smFISH on non-diabetic, SARS-CoV-2-negative, “normal” human pancreata from seven donors across a wide age-span (Table S3) with duodenum, ileum, and kidney used as positive controls (Figures S2A and S2B). ACE2 mRNA was observed in pancreatic ducts, acinar tissue, and CD34⁺ endothelial cells (Figures 2A and 2B), but was observed to a much lesser extent in the islet endocrine cells (Figure 2C). TMPRSS2 mRNA showed a similar pattern but was expressed at higher frequency (Figure 2). We observed limited expression of TMPRSS2 and/or ACE2 in insulin-positive β cells (Figure 2C, inset), which was consistent with quantification of ACE2 and TMPRSS2 expression and co-expression from scRNA-seq data (Table S2). smFISH analysis of other SARS-CoV-2-associated proteases revealed TMPRSS4 mRNA in the exocrine pancreas, with rare TMPRSS4 expression within the islet, while CTSL, ADAM17, and TMPRSS11D expression was observed in both the endocrine and exocrine pancreas (Figures S2C–S2F).

Extensive Reagent Validation Confirms High Level of ACE2 Protein Expression in the Human Pancreas

Information regarding ACE2 protein expression in pancreatic tissue sections remains limited and unfortunately contradictory. A 2010 study on SARS-CoV and its relationship with diabetes interrogated ACE2 expression in a single donor (43 years of age), with an unspecified antibody, and reported weak ACE2 staining in exocrine tissues but pronounced expression in pancreatic islets (Yang et al., 2010). Fignani et al. (2020) recently described heterogenous ACE2 expression across donors, pancreatic lobes, and islets, and identified three main ACE2-expressing cell types in formalin fixed, paraffin embedded (FFPE) pancreatic sections from seven donors (aged 22–59 years) probed with three ACE2 antibodies (MAB933, ab15348, and ab108252): endothelial cells and pericytes, ductal cells, and in an analysis of 128 islets, β cells that often presented with a granular staining pattern, partially overlapping with insulin. In contrast, an analysis of tissue microarrays containing pancreatic FFPE sections from 10 donors (aged 30–79 years) utilizing two ACE2 antibodies (MAB933 and HPA000288) reported ACE2 expression restricted to endothelial cells and pericytes and interlobular ducts while ACE2 was not detectable in islets, acinar glandular cells, intercalated ducts, or intralobular ducts (Hikmet et al., 2020). This study, together with a publicly available preprint employing six ACE2 antibodies (Lee et al., 2020a), is noteworthy for the broad range of major human tissues and organs investigated as well as the delineation of mostly shared but, also, some distinctive ACE2 antibody staining properties. In light of these diverging observations, it is imperative to utilize multiple ACE2 antibodies on larger donor cohorts to gather a comprehensive description of ACE2 expression in the pancreas.

We selected four widely referenced, commercially available antibodies recognizing specific epitopes and both the short and long isoforms of the ACE2 protein (Figure 3 A) (Blume et al., 2020; Ng et al., 2020; Onabajo et al., 2020) to evaluate by immunoblot (Figures 3B, S3A, and S3B) using protein extracts from three non-diabetic, SARS-CoV-2-negative, “normal” pancreas donors (Table S3). ACE2 is an 805 amino acid protein (UniProt Q9BYF1) with a theoretical molecular mass of 94.2 kDa but an actual mass of ∼120 kDa due to glycosylation at N terminus sites (Tipnis et al., 2000). Accordingly, a pronounced ∼120 kDa band was readily visualized by AF933 and ab108252 while ab15348 and especially MAB933 revealed a much weaker band (Figures 3B and S3A). Nevertheless, in our efforts, all four antibodies demonstrated robust and essentially commensurate staining in human duodenum and kidney FFPE sections (Figure S3C), consistent with two recent reports (Hikmet et al., 2020; Lee et al., 2020a).

We next visualized ACE2 via chromogen-based IHC in FFPE pancreata from non-diabetic, SARS-CoV-2-negative, “normal” donors using all four antibodies and consistently observed positive staining in the microvasculature and ductal epithelium (Figure 3C). To further validate specificity, we utilized a peptide-blocking assay wherein ab108252 was pre-incubated with an ACE2 peptide prior to IHC staining (Figure S3D). Taking western blot and IHC validation into account, ab108252 produced a clearly detectable 120 kDa band (Figures 3B and S3A) and crisp in situ staining (Figures 3C and S3C) that was completely blocked by pre-incubation with the ACE2 peptide (Figure S3D). This is a monoclonal antibody, which offers a high degree of specificity and consistency between lots. Hence, we elected to use ab108252 in subsequent IHC and IF assays.

Human Pancreatic Expression of ACE2 Protein Is Primarily Detected in the Duct Epithelium and Microvasculature Across the Lifespan and Positively Associated with Obesity

To quantitatively evaluate pancreatic ACE2 protein expression, an FFPE tissue cross-section from each of 36 SARS-CoV-2-negative donors without diabetes (aged 0–72 years; Table S3) was stained for insulin and ACE2 (ab108252) and scanned to produce a whole-slide image. Co-localization of ACE2 protein with insulin was not observed (Figures 3C and S4A). The tissue area staining positive for ACE2 was analyzed using the HALO area quantification algorithm (Figure 4 A). Body mass index (BMI)-matched donors were binned into six age groups: neonate (0–0.25 years, n = 6), infant and toddler (0.25–2 years, n = 6), child (2–11 years, n = 6), adolescent (11–15 years, n = 6), young adult (20–33 years, n = 6), and senior adult (51–72 years, n = 6). Collectively, these data demonstrated the percentage of tissue staining positive for ACE2 increased steadily from birth throughout childhood, peaking in adolescence and maintained through early adulthood, followed by a decline in persons over 50 years of age (Figure 4B). The ACE2-positive area did not appear different for male versus female subjects (Figure 4B), but with n = 6 per age group, statistical analysis was not performed and will be the subject of future efforts.

Obesity is associated with an increased risk of COVID-19-associated morbidity and mortality (Hussain et al., 2020; Nakeshbandi et al., 2020; Tartof et al., 2020). Taneera et al. (2020) observed no relationship between ACE2 and BMI in isolated pancreatic islets analyzed by microarray. To analyze this in situ, we examined ACE2 protein expression in the aforementioned six young adult donors together with pancreata from 16 additional donors (20–33 years of age, SARS-CoV-2 negative, no diabetes, for a total n = 22) with BMI ranging from normal (19.5 kg/m²) to obese (35.6 kg/m²) (Table S3). Notably, we observed a positive correlation between ACE2-positive area and BMI (Figure 4C).

To more precisely visualize pancreatic ACE2 localization, we performed IF staining for ACE2 (again using ab108252) in conjunction with insulin and glucagon (Figures 4D–4F and S4B) or with CD34 (Figure S4C). Across all ages, we observed ACE2 expression in the pancreatic ductal epithelium (Figure 4D), but not major blood vessels (Figure S4B), based on their morphology and geographic positioning. ACE2 was highly expressed in the microvasculature within acinar and islet regions, with no evidence of α cells or β cells expressing ACE2 protein (Figures 4E, 4F, S4A, and S4C). Our data corroborate findings by two groups (Coate et al., 2020; Hikmet et al., 2020), yet contrast with two others (Fignani et al., 2020; Yang et al., 2010). Moreover, our IHC analyses do not support the notion that islet endocrine cells may preferentially express a recently described short ACE2 isoform (Fignani et al., 2020). Beyond this, we note that this isoform lacks the SARS-CoV-2-binding domain (Blume et al., 2020; Ng et al., 2020; Onabajo et al., 2020) and, thus, would not render endocrine cells susceptible to infection. These disparate results may be influenced by technical (e.g., tissue processing, antigen retrieval, reagent), material (e.g., isolated islets versus tissue sections), or donor differences. However, in aggregate, our analysis of 52 donors across a wide age and BMI range provides a comprehensive perspective on pancreatic ACE2 protein distribution that demonstrates limited to no ACE2 expression by pancreatic endocrine cell types, including β cells (Figures 3 and 4), corroborating scRNA-seq and smFISH gene expression data (Figures 1 and 2).

SARS-CoV-2 NP Is Localized to Pancreatic Ductal Epithelium from COVID-19 Cases With and Without T2D

Following autopsy, we reviewed pancreatic pathology by H&E-stained sections in three individuals with fatal COVID-19 (aged 45–72 years; Figures 5A–5C), two of whom had a previous diagnosis of T2D (Table S4). Case 1 was an individual without history of diabetes. The major findings included severe fatty replacement of acinar cell mass and moderate arteriosclerosis (Figure 5A). Lobules contained fibrotic centers with residual acinar cells and islets surrounding ductules (Figure 5A, insert). Islets were observed primarily within fibrotic regions. Case 2 was an individual with T2D whose pancreas had moderate fatty replacement and limited centrolobular fibrosis (Figure 5B). Dystrophic calcification of adipocytes was rare. We observed numerous islets and one microthrombus without adjacent hemorrhage (Figure 5B, insert). The pancreas of another individual with T2D (Case 3) showed mild to moderate arteriosclerosis with acinar regions containing mild centrolobular fibrosis (Figure 5C). Moderate numbers of islets were present of varying sizes (Figure 5C, insert). None of these individuals showed islet amyloidosis or acute polymorphonuclear cell infiltrates. These histopathological findings were compatible with the normal range of expected lesions within the exocrine compartment in pancreata from aged persons and those with T2D.

IHC showed islets containing insulin-positive (INS+) β cells with mostly spherical profiles, small to medium sizes, and varying proportions of β to α cells in all three cases (Figure 5D). We observed numerous single and clustered INS+ and glucagon-positive (GCG+) cells in ductules of fibrotic foci. The endothelium showed moderate ACE2 staining intensity within both endocrine and exocrine compartments. The ductal epithelium showed low to moderate ACE2 staining intensity throughout the cytoplasm (Figure S5A), similar to that observed in non-diabetic pancreas donors (Figure 3C).

IHC for SARS-CoV-2 NP was also conducted to investigate the cellular distribution of the virus. A lung sample from a person with COVID-19 pneumonia was used to optimize staining conditions. Immunopositive alveolar epithelial cells and macrophages were observed with numerous viral inclusions (Figure S5B). In COVID-19 Case 1, SARS-CoV-2 NP was present in some intralobular and interlobular ductal epithelial cells shown near an islet and widely scattered throughout the exocrine regions (Figures 5E, 5F, S5C, and S5D). Pancreata from Cases 2 and 3, who had T2D, showed little to no immunopositivity for SARS-CoV-2 NP.

We believe these data provide an important foundation for considerations of pancreatic SARS-CoV-2 infection as a potential trigger for diabetes. Indeed, the notion of ACE2 expression on pancreatic β cells as a potential mechanism linking the two conditions may, in our view, promote a quasi-dogmatic perspective (Koch et al., 2020) that is grounded in limited experimental evidence (Yang et al., 2010). However, the histopathology data presented herein do not support a simple model of direct and widespread islet endocrine, including β cell infection via the ACE2 receptor. Rather, preferential ACE2 gene and protein expression in microvascular and ductal structures suggests these cells constitute a more likely target for viral infection, a notion that is in principle supported by our observation of very limited SARS-CoV-2 NP expression in ductal epithelium, but not islet endocrine cells, of pancreata from individuals with COVID-19. While scarce ACE2 protein expression and limited SARS-CoV-2 propagation, as most recently documented for the lung (Hönzke et al., 2020), do not preclude extensive tissue damage mediated by innate immune responses in particular, the absence of polymorphonuclear infiltrations in the three COVID-19 pancreata interrogated here indicates a very limited degree of inflammation consistent with that reported for many tissues other than the lung and reticulo-endothelial system (Dorward et al., 2020). More detailed investigations of pancreatic tissue from fatal COVID-19 cases, to date a very limited resource, will be necessary to answer outstanding questions about the potential pathological involvement of the pancreas in COVID-19.

Limitations of Study

Our delineation, localization, and quantification of pancreatic ACE2 is largely based on in silico and ex vivo in situ analyses of gene (public scRNA-seq data, smFISH) and protein (IHC, IF) expression in pancreata of a broad cohort of organ donors without diabetes and a more limited number of organs collected from COVID-19 autopsy subjects. As such, our pancreatic tissue analyses can only provide approximate predictions regarding ACE2 protein expression in isolated islets and islet cells or their susceptibility to in vitro SARS-CoV-2 infection. Although the aggregate scRNA-seq data demonstrate relatively little ACE2 mRNA in endocrine cells, Yang et al., in a wide-ranging report on the development of a stem cell-based platform for SARS-CoV-2 infection studies, recently demonstrated both ACE2 protein expression by endocrine cells in isolated human islets and their productive infection with SARS-CoV-2 (Yang et al., 2020). Without question and in addition to stem cell-derived endocrine cells and islet organoids, isolated human islets constitute an important model system for research discovery. Further work will be needed to precisely quantify the fractions and expression levels of ACE2 by islet endocrine cells in these islet preparations, to evaluate possible modulation of ACE2 by the prolonged and varied islet handling requirements (e.g., isolation, culture under different conditions, dispersion), to define the exact role of ACE2 in mediating SARS-CoV-2 infection in vitro and in vivo using extended islet xenotransplantation approaches, and to interrogate the precise relation of scarce ACE2 expression and immunopathology as most recently demonstrated for the lung in a manuscript available for public review (Hönzke et al., 2020). Beyond this, contrasting epidemiological reports from the United Kingdom and Germany (Tittel et al., 2020; Unsworth et al., 2020) not only underscore the requirement for data on diabetes incidence and SARS-CoV-2 infection rates in defined populations over time, but in addition raise the need for studying pancreatic tissues from donor cohorts of different demographics, ethnicities, and geographic populations. Indeed, it is possible that COVID-19 may cause an upregulation of ACE2 in endocrine or other cell types, but as of yet, gene and protein expression levels cannot be reliably quantified from autopsy tissues due to pancreatic autolysis and the relative scarcity of high-quality pancreas tissues obtained from SARS-CoV-2-infected individuals. Only in the larger context of expanded investigations into pancreatic histopathology of individuals with COVID-19, with carefully controlled in vitro infection studies, judicious use of animal models to directly test the ability of SARS-CoV-2 to infect the endocrine pancreas, and crucially, a balanced assessment of emerging and future epidemiological studies (DiMeglio et al., 2020) can we provide a complete framework for an informed assessment of diabetes risk and potential prevention in the wake of SARS-CoV-2 infection.

Key Resources Table

Resource Availability

Experimental Model and Subject Details

Method Details

Quantification and Statistical Analysis

For scRNA-seq analysis, n represents the number of cells as indicated in the figure legends. Analyses were performed in R as described in Method Details above. Differences in the average gene expression levels between pancreatic cell subsets or within each cell subset from non-diabetic donors versus donors with T2D were compared using Wilcoxon rank sum tests, requiring a minimum 1.19-fold change between the two groups and expression in at least 10% of cells from either group. Bonferroni corrections were used to adjust for multiple comparisons. Paired Student’s t tests were used to compare proportions of cells with detectable gene expression. p values < 0.05 were considered significant. Violin plot limits show maxima and minima, and the dots represent individual data points. smFISH data were not quantitatively evaluated. For the quantification of ACE2 protein expression throughout the human lifespan and for the analysis of correlation between ACE2 protein expression and BMI, digitized images of ACE2 and insulin co-stained slides were analyzed using the HALO quantitative image analysis platform V3.0.311.262 (Indica Labs, Corrales, NM) (Figure 4A). The annotation pen tool was used to outline the tissue section to determine total tissue area (mm²). The Area quantification algorithm v2.1.3 based on red, blue, green (RBG) spectra was employed to detect ACE2 positive tissue area stained for 3,3′-Diaminobenzidine (DAB, brown). The algorithm detected DAB IHC positivity and calculated percentage of ACE2 positive area per total tissue area. For the ACE2 expression as factor of age study, BMI-matched donors (n = 36) were binned into six age groups representing major stages of human life: neonatal (0-0.25 years), infant and toddler (0.25-2 years), childhood (2-11 years), puberty (11-15 years), young adult (20-33 years), and senior adult (51-72 years). Data were analyzed in GraphPad Prism v8.3 (GraphPad Software, San Diego, CA) by one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons with significance defined as p < 0.05 and graphed with the median percent ACE2 area shown for each group in a box and whisker plot. To assess for an association between ACE2 expression and BMI, we reviewed the 34 nPOD donors aged 20-33 years without diabetes with samples available and selected those without significant underlying pancreatic pathology. This resulted in evaluation of 22 adult organ donors with BMI ranging from 19.5-35.6 kg/m²; data were analyzed in R using Spearman’s correlation test. All image analysis data were collected in blinded fashion. The remaining IF and IHC data were not quantitatively evaluated.