Results

Lung Epithelial Cell Expression of Host Factors Used by SARS-CoV-2 in Non-Human Primates and Humans
To investigate which cells within human and NHP tissues represent likely SARS-CoV-2 targets, we analyzed new and existing scRNA-seq datasets to assess which cell types express ACE2, alone or with TMPRSS2. In a previously unpublished dataset consisting of NHP (Macaca mulatta) lung tissue collected after necropsy of healthy adult animals and analyzed by using Seq-Well v1 (Gierahn et al., 2017), we recovered at least 17 distinct major cell types, including various lymphoid, myeloid, and stromal populations (Figures 1 A–1C; Table S1; STAR Methods). ACE2 and TMPRSS2 were primarily expressed in epithelial cells, with 6.7% of type II pneumocytes expressing ACE2 and 3.8% co-expressing ACE2 and TMPRSS2 (Figures 1B and 1C). Notably, the only double-positive cells observed were classified within the type II pneumocyte population; however, we also identified TMPRSS2 expression within club cells, ciliated epithelial cells, and type I pneumocytes, albeit at diminished abundance and frequency compared with type II pneumocytes (Figure 1C; Table S1).
Figure 1 Expression of ACE2 in Type II Pneumocytes in Healthy Lungs of Non-human Primates
(A) Schematic of protocol for isolation of lung tissue at necropsy from healthy non-human primates (M. mulatta, n = 3), creation of scRNA-seq libraries by using Seq-Well v1, and computational analysis to identify cell types by using unbiased methods. UMAP projection of 3,793 single cells, points colored by cell identity (see STAR Methods).
(B) Uniform manifold approximation and projection (UMAP) as in (A), points colored by detection of ACE2 (coronavirus receptor, top) or TMPRSS2 (coronavirus S protein priming for entry, bottom). Color coding is as follows: black, RNA positive; blue, RNA negative.
(C) Dot plot of 2 defining genes for each cell type (Table S1) (Bonferroni-adjusted p < 0.001) and ACE2 and TMPRSS2. Dot size represents fraction of cells within that type expressing a given gene, and color intensity represents binned count-based expression amount (log(scaled UMI+1)) among expressing cells. ACE2 is enriched in type II pneumocytes (6.7% expressing, Bonferroni-adjusted p = 8.62E−33), as is TMPRSS2 (29.5% expressing, Bonferroni-adjusted p = 8.73E−153). Of all type II pneumocytes, 3.8% co-express ACE2 and TMPRSS2 (Table S9). Red arrow indicates cell type with largest proportion of ACE2+TMPRSS2+ cells.
(D) Genes differentially expressed among ACE2+ and ACE2− type II pneumocytes. (SCDE package, FDR-adjusted p < 0.05 for IFNGR2, NT5DC1, ARL6IP1, and TRIM27; full results can be found in Table S1).
See also Table S1.
Next, we compared ACE2 + with ACE2 − type II pneumocytes to explore broader gene programs that differentiate putative SARS-CoV-2 target cells from cells of a similar phenotype and ontogeny (Figure 1D; Table S1). Among genes significantly upregulated in ACE2 + type II pneumocytes, we observed IFNGR2 (false discovery rate [FDR]-adjusted p = 0.022), a receptor for type II IFNs. Notably, previous work has demonstrated limited anti-viral potency of IFN-γ for SARS-associated coronaviruses, compared with that of type I IFNs, at least in vitro (Sainz et al., 2004, Zheng et al., 2004). Other co-regulated genes of potential interest include TRIM27 (FDR-adjusted p = 0.025), as well as NT5DC1 (FDR-adjusted p = 0.003) and ARL6IP1 (FDR-adjusted p = 0.047), which were upregulated in the A549 adenocarcinoma alveolar basal epithelial cell line after exposure to IFN-α and IFN-γ for 6 h (Sanda et al., 2006). We found IFNAR1 consistently expressed among both ACE2 + type II pneumocytes and ACE2 + TMPRSS2 + co-expressing type II pneumocytes, but its level of upregulation compared with all remaining pneumocytes did not meet statistical significance (FDR-adjusted p = 0.11). This analysis finds ACE2 + cells enriched within a rare fraction of secretory cells in NHPs and that ACE2 expression is co-regulated with genes involved in IFN responses.
To assess whether the findings from NHP lung cells were similarly present in humans, we analyzed a previously unpublished scRNA-seq dataset derived from surgical resections of fibrotic lung tissue collected with Seq-Well S3 (Hughes et al., 2019). Unsupervised analysis identified multiple cell types and subtypes of immune cells (Figures 2 A–2C; STAR Methods), as defined by the genes displayed in Figure 2C (full lists available in Table S2). Here, we found that ACE2 and TMPRSS2 were primarily expressed within type II pneumocytes and ciliated cells, in line with our analysis of the NHP-derived cells (Figures 1 and 2A, 2B). In type II pneumocytes (identified by unique expression of surfactant proteins SFTPC, SFTPB, and SFTPA1), we found 1.4% of cells expressing ACE2 (FDR-adjusted p = 1.35E−21), 34.2% expressing TMPRSS2 (FDR-adjusted p < 1E−300), and 0.8% co-expressing both. In ciliated cells, we found 7% were ACE2 + (FDR-adjusted p = 5E−64), 24.6% were TMPRSS2 + (FDR-adjusted p = 3.8E−30), and 5.3% co-expressed both.
Figure 2 Select Lung Epithelial Cells from Control, HIV-1-Infected, and Mycobacterium-tuberculosis-Infected Human Donors Co-Express ACE2 and TMPRSS2
(A) Schematic of protocol for isolation of human lung tissue from surgical excess, creation of scRNA-seq libraries by using Seq-Well S3, and computational analysis to identify cell types by using unbiased methods. Shown on the right is a UMAP projection of 18,915 cells across 8 donors (n = 3 TB+HIV+; n = 3 TB+; n = 2 non-infected patients). Cells represented by points, colored according to cell type (see STAR Methods).
(B) UMAP projection as in (A), points colored by detection of ACE2 (top) or TMPRSS2 (bottom). Color coding is as follows: black, RNA positive; blue, RNA negative.
(C) Dot plot of 2 defining genes for each cell type (FDR-adjusted p < 0.001), and ACE2 and TMPRSS2; dot size represents fraction of cells within cell type expressing a given gene, and color intensity represents binned count-based expression amount (log(scaled UMI+1)) among expressing cells. All cluster-defining genes are provided in Table S2. Red arrow indicates cell types with largest proportion of ACE2+TMPRSS2+ cells.
(D) Volcano plot identifying significantly upregulated genes in ACE2+TMPRSS2+ pneumocytes compared with all remaining pneumocytes. Red points represent genes with a FDR-adjusted p < 0.05, and log2(fold change) >1.5. Text highlighting specific genes; the full list is available in Table S2.
(E) Expression of ACE2 across human donors by HIV and TB status (p = 0.009 by likelihood-ratio test).
See also Table S2.
As above, to assess for cellular pathways significantly co-expressed within putative target cells for SARS-CoV-2, we computed differentially expressed genes between ACE2 + TMPRSS2 + type II pneumocytes and all other type II pneumocytes (Figures 2C and 2D; Table S2). We found significant enrichment of BATF among ACE2 + TMPRSS2 + cells (FDR-adjusted p = 3.25E−7), which has been demonstrated previously to be upregulated by type I and type II IFNs (Murphy et al., 2013). Of note, we also observed TRIM28 co-expressed with ACE2 and TMPRSS2 among type II pneumocytes in this dataset (FDR-adjusted p = 2.34E−9), which might play a role in potentiating an IFN response in lung epithelial cells (Krischuns et al., 2018). Within this cohort of donors, 3 individuals were human immunodeficiency virus (HIV)+ and diagnosed with active tuberculosis, 3 donors had active tuberculosis and were HIV−, and 2 were negative for both pathogens. Surprisingly, we found that all of the ACE2 + cells across all cell types were derived from HIV+ Mycobacterium tuberculosis (Mtb)+ donors despite approximately equivalent recovery of epithelial cell types from all donors (likelihood-ratio test, p = 0.009) (Figure 2E). Given limited cell and patient numbers combined with potential sampling biases, we caution that this observation requires much broader cohorts to validate a potential role for co-infections; still, we note our observation is suggestive of a role for chronic IFNs in the induction of ACE2, given that HIV infection is associated with persistent upregulation of ISGs, and we observed elevated amounts of IFNAR2, IFI30, and IKBKB (Utay and Douek, 2016) (FDR-adjusted p = 1.1E−6, 8.8E−9, 1.57E−7, respectively; HIV+ versus HIV− epithelial cells).
Next, using a previously unpublished scRNA-seq dataset consisting of granuloma and adjacent, uninvolved lung samples from Mtb-infected NHPs (Macaca fascicularis) collected with Seq-Well S3, we identified subsets of epithelial cells expressing ACE2 and TMPRSS2 (Figure S1 ; Table S3; STAR Methods). The majority of ACE2 + TMPRSS2 + cells were, once again, type II pneumocytes (22%) and type I pneumocytes (9.7%) and were largely enriched within granulomatous regions compared with those in adjacent uninvolved lung (Figures S1B and S1C) (p = 0.006, Fisher Exact Test). ACE2 + TMPRSS2 + type II pneumocytes expressed significantly higher amounts of antimicrobial effectors such as LCN2 compared with remaining type II pneumocytes (Figure S1D). Cells with club cell/secretory, type I pneumocyte, and ciliated cell types also contained some ACE2 + TMPRSS2 + cells, but we did not have sufficient power to detect significantly differentially expressed genes between these cells and other cells within those clusters. Altogether, we identify ACE2 + TMPRSS2 + cells in lower airways of humans and NHPs with consistent cellular phenotypes and evidence supporting a potential role for IFN-associated inflammation in upregulation of ACE2.
Figure S1 NHP Tuberculosis Infected Lung and Granuloma, Related to Figures 1 and 2
(A). UMAP projection of epithelial cells (1,099 cells) colored by annotated cell type, tissue source, and gating as ACE2+TMPRSS2+ cells. ACE2+TMPRSS2+ cells comprise 11% of ciliated cells, 16% of club cells, 10% type I pneumocytes, and 22% type II pneumocytes. Data generated using Seq-Well S3 (Table S3).
(B). Number of cells (left) and % (right) ACE2+TMPRSS2+ cells by tissue source (granuloma versus uninvolved lung) and cell type. Ciliated cells and club cells were omitted from this analysis as we detected too few cells (< 7 total cells) belonging to these clusters in the granulomas. Statistical significance assessed by Fisher Exact Test (Table S3).
(C). Dot plot of top cluster defining genes for each epithelial cell type and ACE2 and TMPRSS2. Dot size represents fraction of cells expressing, and color intensity represents average log(normalized UMI + 1) among all cells in each group scaled between 0 and 1 by gene. ACE2 expression is enriched in club cells (Bimodal test, Bonferroni-corrected p < 0.001), ciliated cells (p < 0.005), and type I pneumocytes (p < 0.001). TMPRSS2 expression is enriched in type I pneumocytes (p < 0.001) and ciliated cells (p < 0.001) (Table S3).
(D). Dot plot of genes differentially expressed between ACE2+TMPRSS2+ epithelial cells versus rest (Bimodal test, Bonferroni-corrected p < 0.01, log fold change > 0.5). (Table S3, c = number of cells, n = number of animals).

Ileal Absorptive Enterocytes Express Host Factors Used by SARS-CoV-2
Next, we examined several other tissues for ACE2-expressing cells on the basis of the location of hallmark symptoms of COVID-19, focusing on the gastrointestinal tract due to reports of clinical symptoms and viral shedding (Xiao et al., 2020). Leveraging a previously unpublished scRNA-seq atlas of NHP (M. mulatta) tissues collected with Seq-Well v1, we observed that the majority of ACE2 + cells reside in the small intestine, principally within the ileum, jejunum, and, to a lesser extent, the liver and colon (Figure 3 A; STAR Methods). Critically, we note that, in this experiment, the dissociation method used on each tissue was optimized to preserve immune cell recovery, and therefore under-sampled stromal and epithelial populations, as well as neurons from the brain. Within the ileum, we identified ACE2 + cells as absorptive enterocytes on the basis of specific expression of ACE2 within cells defined by APOA1, SI, FABP6, and ENPEP, among others, by a likelihood-ratio test (Figures 3B and 3C) (p < 1E−300, 62% of all absorptive enterocytes; see Table S4). All other epithelial subtypes expressed ACE2 to a lesser extent, and variably co-expressed ACE2 with TMPRSS2 (see Table S4 for full statistics).
Figure 3 NHP and Human Ileal Absorptive Enterocytes Co-Express ACE2 and TMPRSS2
(A) Expression ACE2 across diverse tissues in healthy NHPs (n = 3 animals; 52,858 cells).
(B) Schematic of protocol for isolation of NHP ileum (n = 5) at necropsy for scRNA-seq using Seq-Well v1, and computational pipeline to identify cell types by using unbiased methods. Shown on the right is a UMAP projection of 4,515 cells colored by cell type.
(C) Dot plot of 2 defining genes for each cell type, with ACE2 and TMPRSS2. Dot size represents fraction of cells within cell type expressing a given gene, and color intensity represents binned count-based expression amounts (log(scaled UMI+1)) among expressing cells. All cluster defining genes are provided in Table S4. Red arrow indicates cell type with largest proportion of ACE2+TMPRSS2+ cells.
(D) Schematic of protocol for isolation of human ileal cells from endoscopic pinch biopsies in non-inflamed regions (n = 13). Shown on the right is a tSNE plot of 13,689 epithelial cells selected from original dataset generated by 10x 3′ v2 (see Figure S2), colored by cellular subsets.
(E). Dot plot of 2 defining genes for each cell type, with ACE2 and TMPRSS2. Dot size represents fraction of cells within cell type expressing a given gene, and color intensity represents binned count-based expression amounts (log(scaled UMI+1)) among expressing cells. All cluster defining genes are provided in Table S5. Red arrow indicates cell type with largest proportion of ACE2+TMPRSS2+ cells.
(F). Expression of ACE2 (left) and TMPRSS2 (right) among all epithelial subsets from human donors.
See also Figure S2 and Tables S4 and S5.
Persistent viral RNA in rectal swabs has been detected in pediatric infection, even after negative nasopharyngeal tests (Xu et al., 2020). In an additional dataset consisting of endoscopic biopsies from the terminal ileum of a human pediatric cohort (n = 13 donors, ranging in age from 10 to 18 years old), collected with 10X 3′ v2, we confirmed a large abundance of ACE2 + cells with selective expression within absorptive enterocytes (29.7% ACE2 +, FDR-adjusted p = 2.46E−100) (Figures 3D and 3E; Table S5; STAR Methods). Furthermore, we identified a subset (888 cells, ∼6.5% of all epithelial cells) that co-express both genes (Figures S2 A–S2C). We performed differential expression testing and GO-term enrichment using these cells relative to matched non-expressers to highlight putative biological functions enriched within them, such as metabolic processes and catalytic activity, and to identify shared phenotypes of ACE2 + TMPRSS2 + ileal cells across both human and NHP cohorts (Table S5). We speculate that viral targeting of these cells, taken from patients without overt clinical viral infection, might help explain intestinal symptoms. Finally, we compared ileal absorptive enterocytes from healthy NHPs and NHPs infected with simian-human immunodeficiency virus (SHIV) and then treated for 6 months with anti-retroviral therapy (animal and infection characteristics published in Colonna et al., 2018) (STAR Methods). We found significant upregulation of ACE2, STAT1, and IFI6 within the absorptive enterocytes of SHIV-infected animals (which maintain chronically elevated amounts of IFNs and ISGs) compared with those of uninfected controls (FDR-adjusted p < 2E-7) (Figure S2D) (Deeks et al., 2017, Utay and Douek, 2016).
Figure S2 Human and NHP Ileum, Related to Figure 3
(A). Top: tSNE projection of all cells from healthy pediatric human ileum within a previously-unpublished 10x 3′ v2 dataset (115,569 cells). Black: higher expression of ACE2 (left), TMPRSS2 (right). Bottom: Corresponding violin plots of expression values for ACE2 (left) and TMPRSS2 (right). Solid line: epithelial cells.
(B). Co-expression of ACE2 and TMPRSS2 by epithelial cell subset. Number indicates % of ACE2+TMPRSS2+ cells by cell subset.
(C). tSNE projection of 13,689 cells as in Figure 3D, cells colored by co-expression of ACE2 and TMPRSS2 (black).
(D). Expression of ACE2 and canonical interferon-responsive genes among absorptive enterocytes from Healthy (n = 2) and SHIV-infected, anti-retroviral treated animals (n = 3). Bonferroni-adjusted p-values by Wilcoxon test (healthy: 510 cells, SHIV-infected: 636 cells).

Upper Airway Expression of Host Factors Used by SARS-CoV-2
To identify potential viral target cells in nasal and sinus tissue, two regions that are frequently primary sites of exposure for coronaviruses, we analyzed existing scRNA-seq datasets from the human upper airway (inferior turbinate and ethmoid sinus mucosa) across a spectrum of healthy donors and individuals with allergic inflammation due to chronic rhinosinusitis (CRS) collected with Seq-Well v1 (Figure 4 A; STAR Methods) (Ordovas-Montanes et al., 2018). We had previously noted a significantly enriched IFN-dominated gene signature in inferior turbinate secretory epithelial cells from both healthy and CRS donors compared with CRS samples from the ethmoid sinus, which were significantly enriched for interleukin-4 (IL-4)/IL-13 gene signatures (Giovannini-Chami et al., 2012, Ordovas-Montanes et al., 2018). We speculate that these cells, taken from clinically non-virally infected patients, yet constantly exposed to environmental viruses, might provide one of the earliest locations for coronaviruses to infect before spreading to other tissues. We observed significant enrichment of ACE2 expression in apical epithelial cells and, to a lesser extent, ciliated cells compared with all cell types recovered from surgically resected mucosa (1% of apical epithelial cells, FDR-adjusted p = 4.55E−6, n.s. in ciliated cells) (Figure 4B; Table S6).
Figure 4 Healthy and Allergic Inflamed Human Nasal Mucosa Co-Express ACE2 and TMPRSS2 in a Subset of Goblet Secretory Cells
(A) Schematic for sampling of n = 12 ethmoid sinus surgical samples and n = 9 inferior turbinate nasal scrapings to generate scRNA-seq libraries by using Seq-Well v1. See Ordovas-Montanes et al., (2018).
(B) Dot plot of all cell types from ethmoid-sinus-derived cells (n = 6 non-polyp CRS samples, n = 6 polyp CRS samples). Two defining genes for each cell type, in addition to CDHR3 (rhinovirus receptor), ACE2, TMPRSS2, and JAK1. Dot size represents fraction of cells within that type expressing a given gene, and color intensity represents binned count-based expression amounts (log(scaled UMI+1)) among expressing cells (see Table S6 for statistics by subset). Red arrow indicates cell types with largest proportion of ACE2+TMPRSS2+ cells.
(C) Dot plot for 2 defining genes for each cell type identified from granular clustering of epithelial cells (18,325 single cells) derived from both ethmoid sinus and inferior turbinate sampling (healthy inferior turbinate [3,681 cells; n = 3 samples], polyp-bearing patient inferior turbinate [1,370 cells; n = 4 samples], non-polyp ethmoid sinus surgical samples [5,928 cells; n = 6 samples], and polyp surgical and scraping samples directly from polyp in ethmoid sinus [7,346 cells; n = 8 samples]). Red arrow indicates cell type with largest proportion of ACE2+TMPRSS2+ cells.
(D) tSNE of 18,325 single epithelial cells from inferior turbinate and ethmoid sinus (omitting immune cells). Colored by cell types 3,152 basal, 3,089 differentiating, 8,840 secretory, 1,105 ciliated, and 2,139 glandular cells.
(E) tSNE as in (D), identifying epithelial cells co-expressing ACE2 and TMPRSS2 (30 cells, black points).
(F) tSNE as in (D), colored by detailed cell types with higher granularity, as in (C).
(G) Individual differentially expressed genes between ACE2+TMPRSS2+ cells and all other secretory epithelial cells (see Table S6 for full gene list with statistics). Bonferroni-adjusted likelihood-ratio test p < 0.02 for all genes displayed.
(H) Stacked bar plot of each subset of epithelial cells among all epithelial cells by donor (each bar) and sampling location (noted below graph) (unpaired t test p < 0.00035 for Secretory Goblet 7 inferior turbinate versus ethmoid sinus; see Table S6 for raw values).
(I) Violin plot of cell clusters in respiratory epithelial cells (from Figures 4C and 4F) ordered by average expression of IFN-α-induced gene signatures, presented as a gene module score; non-normal distribution by Lilliefors test, Mann-Whitney U-test p = 2.2E−16, 1.21 effect size, IFN-α signature for Secretory Goblet Cluster 7 versus all epithelial cells. Arrow indicates cluster containing majority ACE2+TMPRSS2+ cells.
See also Figure S3 and Table S6.
To better map putative SARS-CoV-2 targets among epithelial subsets, we employed a finer-grained clustering method applied to both ethmoid sinus surgical specimens and scrapings from the inferior turbinate and ethmoid sinus (Figures 4C–4F). Once again, we observed selective expression of ACE2 within a minority of cell types, with 1.3% of all secretory cells expressing ACE2 (Figure 4C) (FDR-adjusted p = 0.00023), specifically sub-clusters 7 and 13, which represent two varieties of secretory epithelial cell (Figures 4C, 4F, and 4G). Cluster 7 secretory cells are marked by S100P, LYPD2, PSCA, CEACAM5, and STEAP4; encompass some MUC5AC goblet cells; and contain the most significantly enriched ACE2 and TMPRSS2 expression (4% express ACE2, FDR-adjusted p = 7.32E−28; 28% express TMPRSS2, FDR-adjusted p = 2.15E−132; Table S6). We next explicitly gated cells by their TMPRSS2 and ACE2 expression, identifying a rare subset that co-expresses both, the majority of which fall within the “Secretory Cluster 7” cell type (Figures 4E and 4F) (30 cells, ∼0.3% of all upper airway secretory cells, 1.6% of goblet “Secretory Cluster 7”). These findings are aligned with concurrent work by the HCA Lung Biological Network on human nasal scRNA-seq data, which identified nasal secretory cells to be enriched for ACE2 and TMPRSS2 expression (Sungnak et al., 2020).
Although we identified co-expression of ACE2 and TMPRSS2 in few airway cells overall, we detected ACE2 and TMPRSS2 single- and double-positive cells in over 20 donors and thus posit that these genes are enriched in secretory cells and are not a product of individual-patient-driven variability (Figure S3 A). Inferior turbinate scrapings collected on Seq-Well S3, which increases the resolution of lower-abundance transcripts compared with Seq-Well v1, revealed consistent and specific expression restricted to goblet secretory cells, but at a greater detection frequency in samples from the same donors (Figure S3B) (ACE2 + from 4.7% v1 to 9.8% S3; ACE2 + TMPRSS2 + from 1.9% v1 to 4% S3) (Hughes et al., 2019). Using the gated ACE2 + TMPRSS2 + cells, we tested for differentially expressed genes compared to the remaining secretory epithelial cells (full results provided in Table S6). Notably, we observed significant upregulation of ADAR, GBP2, OAS1, JAK1, and DUOX2 (FDR adjusted, all p < 0.02) within ACE2 + TMPRSS2 + cells, potentially indicative of IFN signaling (Figure 4G). Almost all “Secretory Cluster 7” cells were from inferior turbinate scrapings of healthy and allergically inflamed individuals, few cells were from the ethmoid sinus tissue of patients with chronic rhinosinusitis without nasal polyps, and no cells were detected in polyp tissue (Figure 4H). Gene Ontology (GO) analysis of enriched genes in double-positive cells include processes related to intracellular cytoskeleton and macromolecular localization and catabolism, potentially involved in viral particle entry, packaging, and exocytosis (Fung and Liu, 2019).
Figure S3 Nasal and Sinus Mucosa, Related to Figures 4 and 5
(A). Expression of ACE2 and TMPRSS2 across donors.
(B). Enhanced capture of ACE2 mRNA with second strand synthesis protocol employed in Seq-Well S3. Dot size represents fraction of cells expressing.
(C). Cultured human primary basal epithelial cells at confluence were treated with increasing doses (0.1 to 10ng/mL) of IFNα2, IFNγ, IL-4, IL-13, IL-17A, and IL-1B for 12 h and bulk RNA-seq analysis was performed (Replicate experiment using Human Donor 1 as in Figure 5)
(D). ACE2 expression by stimulation condition. Wilcoxon test between each cytokine (combined doses) versus rest: IFNα Bonferroni-adjusted p = 4.1E-07; IFNγ Bonferroni-adjusted p = 9.3E-03; all else n.s. ∗∗∗ p < 0.001.
(E). ACE2 expression by IFNα2 dose. Bonferroni-corrected t-test compared to 0 ng/mL condition: ∗∗∗ p < 0.001, ∗ p < 0.05.
(F). ACE2 expression by IFNγ dose. Bonferroni-corrected t-test compared to 0 ng/mL condition: ∗∗∗ p < 0.001, ∗ p < 0.05.
(G). IFITM1 expression by IFNα2 dose. Bonferroni-corrected t-test compared to 0 ng/mL condition: ∗∗∗ p < 0.001.
(H). IFITM1 expression by IFNγ dose. Bonferroni-corrected t-test compared to 0 ng/mL condition: ∗∗∗ p < 0.001.
(I). GBP5 expression among cultured human primary basal epithelial cells. Wilcoxon test: IFNα versus IFNγ Bonferroni-adjusted p = 2.94E-07; IFNγ Bonferroni-adjusted p = 9.3E-03. TP10K: transcripts per 10,000 reads. ∗∗∗ p < 0.001.
(J). GBP5 expression by IFNα2 dose. Bonferroni-corrected t-test compared to 0 ng/mL condition: ∗∗∗ p < 0.001.
(K). GBP5 expression by IFNγ dose. Bonferroni-corrected t-test compared to 0 ng/mL condition: ∗∗∗ p < 0.001.
We next utilized IFN-inducible gene sets of relevance to human airway epithelial cells, which we derived from a prior study by performing differential expression on a published dataset where air-liquid interface cultures from primary human nasal epithelial cells were treated with IFN-αA/D, IFN-β1a, IFN-γ, IL-4, or IL-13 (Giovannini-Chami et al., 2012, Ordovas-Montanes et al., 2018). Using these gene lists, we scored the human nasal epithelial cells analyzed by scRNA-seq described in Figures 4C and 4F and found significant concomitant upregulation of the IFN-α-stimulated gene set within ACE2 + TMPRSS2 + secretory goblet cluster 7 (Figure 4I).

Type I Interferon IFN-α Drives ACE2 Expression in Primary Human Nasal Epithelial Cells
The meta-analysis described above consistently identified an association between ACE2 expression and canonical ISGs or components of the IFN-signaling pathway. This prompted us to investigate whether IFNs might play an active role in regulating ACE2 expression levels in specific target cell subsets, thus potentially allowing for a tissue-protective host response or increased viral binding of SARS-CoV-2 through ACE2. Our initial literature search indicated that IFN-γ and IL-4 downregulate the SARS-CoV receptor ACE2 in Vero E6 cells (African green monkey kidney epithelial cells [de Lang et al., 2006]), appearing to invalidate this hypothesis. Relatedly, in vitro stimulation of A549 cells, a commonly used cell line model for lung epithelia, with IFN-α, IFN-γ, and IFN-α+IFN-γ for 24 h did not identify ACE2 as an ISG (Russell et al., 2018). This is potentially explained by recent work that aimed to understand SARS-CoV-2 receptor usage by performing screening studies within cell line models and found that A549 cells did not express ACE2 and therefore represents a poor model to understand regulation of this gene (Letko et al., 2020). While conducting experiments to directly test the hypothesis that ACE2 is an ISG, we noted in our own gene lists used for scoring from Ordovas-Montanes et al., 2018 and in a supplementary extended table available from Giovannini-Chami et al., 2012 that ACE2 was in upregulated gene lists after exposure to Type I IFN.
We directly tested whether IFN-α induces ACE2 in primary human upper airway epithelial cells in greater detail. We cultured human primary basal (stem and progenitors) epithelial cells to confluence and treated them with increasing doses (0.1–10 ng/mL) of IFN-α2, IFN-γ, IL-4, IL-13, IL-17A, or IL-1β for 12 h and then performed bulk RNA-seq (Figure S3C). Only IFN-α2 and IFN-γ led to upregulation of ACE2 over the time period tested, and compared with all other cytokines, IFN-α2 lead to greater and more significant upregulation over all doses tested (Figure S3D,Wilcoxon test: IFN-α2 FDR-adjusted p = 4.1E−07; IFN-γ p = 9.3E-03,Figures S3E and S3F, all statistical tests compared with 0 ng/mL dose). We confirmed substantial and dose-dependent induction of canonical members of the interferon response after IFN-α2 and IFN-γ (Figures S3G and S3H). Conversely, we found that IFN-γ, relative to IFN-α2, induced potent upregulation of GBP5, a GTPase-like protein thought to act as a viral restriction factor through inhibiting furin-mediated protease activity, which could limit viral processing from infected cells, whereas IFN-α2 more robustly induced IFITM1 (Figure S3G–S3K) (Braun and Sauter, 2019).
To further extend and substantiate these findings, as above, we stimulated primary mouse tracheal basal cells, the commonly used human bronchial cell line BEAS-2B, and upper airway basal cells from two human donors (Figure 5 A-D). We confirmed appropriate induction of an IFN response in each cell type by performing differential expression testing between untreated cells and IFN-treated cells for each condition (Table S7). Within each cell type, stimulation with IFN-α2, IFN-γ, or IFN-β resulted in dose-dependent upregulation of canonical ISGs, including STAT1/Stat1, BST2/Bst2, XAF1/Xaf1, IFI35/Ifi35, MX1/Mx1, and GBP2/Gbp2. Notably, Ace2 expression was not robustly induced in basal cells derived from healthy mouse trachea under any interferon stimulation condition (Figure 5A). The magnitude of ACE2 upregulation was diminished in BEAS-2B cells compared to that in our original findings in primary human upper airway epithelial cells, but reached statistical significance compared with that of the untreated condition after IFN-γ exposure (Figure 5B). In primary basal cells derived from healthy nasal mucosa, we confirmed significant induction of ACE2 after IFN-α2 stimulation and, to a lesser extent, after stimulation with IFN-γ (IFN-α2-stimulated: both Bonferroni-adjusted p < 0.001; IFN-γ-stimulated: both Bonferroni-adjusted p < 0.05) (Figures 5C and 5D). Expression of ACE2 was significantly correlated with expression of STAT1 in all human cell types, with a larger effect size and correlation coefficient in primary human basal cells (Figure 5E-H). These experiments support a relationship between induction of the canonical IFN response, including key transcription factors and transcriptional regulation of the ACE2 locus. Finally, among primary human samples, we confirmed the dose-dependence of ACE2 upregulation after IFN-α2 or IFN-γ treatment and significant induction of ACE2 after IFN-α2 stimulation at concentrations as low as 0.1–0.5 ng/mL (Figure 5I-L).
Figure 5 ACE2 is an Interferon-Stimulated Gene in Primary Human Barrier Tissue Epithelial Cells
(A–D) Basal epithelial cells from distinct sources were cultured to confluence and treated with increasing doses (0.1–10 ng/mL) of IFN-α2, IFN-γ, IL-4, IL-17A, and/or IFN-β for 12 h and bulk RNA-seq analysis was performed. Expression of ACE2 (human) or Ace2 (mouse) by cell type and stimulation condition. (A) Primary mouse basal cells from tracheal epithelium are shown. (B) BEAS-2B human bronchial cell line is shown. (C) Primary human basal cells from nasal scraping, Donor 1, is shown. (D) Primary human basal cells from nasal scraping, Donor 2. Abbreviation is as follows: TP10K, transcripts per 10,000 reads. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, Bonferroni-corrected t test compared with untreated condition.
(E–H) Co-expression of STAT1/Stat1 and ACE2/Ace2 by cell type. (E) Primary mouse basal cells from tracheal epithelium are shown. (F) BEAS-2B human bronchial cell line is shown. (G) Primary human basal cells from nasal scraping, Donor 1, are shown. (H) Primary human basal cells from nasal scraping, Donor 2 are shown. Abbreviation is as follows: TP10K, transcripts per 10,000 reads. Statistical significance assessed by Spearman’s rank correlation.
(I–L) Expression of ACE2 in primary human basal cells from nasal scrapings across a range of concentrations of IFN-γ or IFN-α2. (I) IFN-α2 dose response in Donor 1 (p < 0.001 by one-way ANOVA) is shown. (J) IFN-γ dose response in Donor 1 (p < 0.01 by one-way ANOVA) is shown. (K) IFN-α2 dose response in Donor 2 (p < 0.001 by one-way ANOVA) is shown. (L) IFN-γ dose response in Donor 2 (p < 0.001 by one-way ANOVA). Abbreviation is as follows: TP10K, transcripts per 10,000 reads. ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, Bonferroni-corrected post hoc testing compared with 0 ng/mL condition.
See also Figures S3 and S4 and Table S7.
Next, using a publicly available resource (interferome.org) that hosts genomic and transcriptomic data from cells or tissues treated with IFN, we queried ACE2 expression within human and mouse cells, searching for datasets with a log2-fold-change of >1 or < −1 compared with untreated samples, including all IFN types (Rusinova et al., 2013). We recovered 21 datasets spanning 8 distinct primary tissues or cell lines with non-trivial changes in ACE2 expression after both type I and type II IFN treatment (Figure S4 A). We observed substantial upregulation of ACE2 in primary skin and primary bronchial cells treated with either type I or type II IFN (> 5-fold upregulation compared with that in untreated cells), in strong support of our in vitro data (Figures 5C, 5D, 5G–5L, and S3D–S3F). Immune cell types, such as CD4 T cells and macrophages, were noticeably absent from datasets with a significant change in ACE2 expression after IFN stimulation or were even found to downregulate ACE2 (e.g., primary CD4 T cells + type I IFN) (Figure S4A, and in our analysis of scRNA-seq peripheral blood mononuclear cell data from Butler et al., (2018); data not shown).
Figure S4 Published Studies of Epithelial Cells Following Interferon Treatment Related to Figure 5
(A). Fold change of ACE2 expression among human or mouse datasets following Type I or Type II interferon treatment compared to untreated control. Generated from publicly available microarray data curated at interferome.org. Includes all studies with abs(fold-change) > 1.
(B). Location of transcription factors binding regions spanning −1500 bp to +500 bp from the transcription start site of ACE2 (human, top) or Ace2 (mouse, bottom). Generated from TRANSFAC data using the interferome.org database (Matys et al., 2003, Rusinova et al., 2013).
Given that the majority of cells robustly upregulating ACE2 were epithelial, this observation potentially explains why previous analyses to define canonical ISGs within immune populations did not identify ACE2 as an induced gene. Furthermore, using both Transcription Factor database (TRANSFAC) data hosted by the interferome database, as well as chromatin immunoprecipitation sequencing (ChIP-seq) data (provided by the ENCODE Factorbook repository), we found evidence for STAT1, STAT3, IRF8, and IRF1 binding sites within −1500–500 bp of the transcription start site of ACE2 (all in human studies, Figure S4B) (Gerstein et al., 2012, Matys et al., 2003, Wang et al., 2012, Wang et al., 2013). This finding is supportive of our current hypothesis that ACE2 represents a previously unappreciated ISG in epithelial cells within barrier tissues.
Given minimal upregulation of Ace2 among primary mouse basal cells in vitro, we were curious as to whether Ace2 represented a murine ISG in vivo. We treated two mice intranasally with saline and two mice intranasally with 10,000 units of IFN-α (Guerrero-Plata et al., 2005). After 12 h, we isolated the nasal mucosa, consisting of both respiratory and olfactory epithelium, with underlying lamina propria, and performed scRNA-seq using Seq-Well S3 (Figure S5 A). We collected from both tissue sites because of early reports of anosmia in COVID-19 (Lechien et al., 2020). We recovered 11,358 single cells, including epithelial, stromal, neuronal, and immune cell types, generating the largest single-cell atlas of mouse respiratory and olfactory mucosa to date (Figures 6 A and S5B). We annotated all 36 clusters, focusing our attention on epithelial cell clusters, given that we noted enrichment for Ace2 and Tmprss2 within epithelial cell subsets, consistent with our human and NHP results (Table S8). Specifically, we found Ace2 enriched within olfactory epithelial gland cells, Muc5b + Scgb1c1 + goblet cells, basal epithelial cells, and myofibroblasts/pericytes (Bonferroni-corrected p < 0.01) (Figures 6B and S5B) (Brann et al., 2020, Dear et al., 1991, Montoro et al., 2018, Tepe et al., 2018). Notably, Furin was enriched within olfactory epithelial gland cells (Table S8). Next, we asked whether a 12 h stimulation with IFN-α would upregulate Ace2 in vivo. Focusing on basal epithelial cells, which contain the highest abundance of Ace2 + cells, we found that despite robust upregulation of canonical murine ISGs, Ace2 expression was only slightly elevated after IFN-α treatment (Figures 6C, 6D, S5C, and S5D).
Figure S5 Mouse Nasal Epithelium Following Interferon-α Exposure Related to Figure 6
(A). Schematic: mice were exposed to 10,000 units of IFN-α or saline by intranasal application (n = 2 per group). After 12 h, animals were sacrificed and nasal epithelium was dissected and dissociated for scRNA-seq using Seq-Well S3.
(B). Dot plot of 2 defining genes for each cell type, with Ace2, Tmprss2, and Cdhr3. Dot size represents fraction of cells within cell type expressing, and color intensity binned count-based expression level (log(scaled UMI+1)) among expressing cells. All cluster defining genes are provided in Table S8. Red arrows: cell types with largest proportion of Ace2+ cells. Dendrogram (left) by person correlation over differentially expressed genes with Ward clustering.
(C). UMAP of Basal Epithelial Cells (380 cells) across 4 mice. Black: Saline-treated mouse; red: IFN-α treated.
(D). UMAP of Basal Epithelial Cells as in C, points colored by detection of Ace2. Black: RNA positive, blue: RNA negative (6.6% Ace2+, Bonferroni-adjusted p = 1.1E-10 for Basal Epithelial Cell expression versus all other cells).
(E). Schematic: wildtype (WT) and IFNγ-receptor knockout (IFNγR−/−) mice were infected intranasally with murine gamma-herpesvirus-68 (MHV68). Cells from whole lung were digested for scRNA-seq using Drop-seq (yielding 5,558 Epcam+ cells).
(F). Expression of Ace2 by epithelial cell type, wild type (WT) mice. Statistical significance by Wilcoxon rank sum test with Bonferroni correction.
(G). Expression of Ace2 among type II pneumocytes binned by infection status in WT mice. All pairwise comparisons non-significant (p > 0.05) by Wilcoxon rank sum test.
(H). Percent of Ace2+ cells by infection condition (uninfected, bystander cells in MHV68-infected mouse, MHV68 RNA+ cells) and mouse genotype (WT, IFNγR −/−). Black bars: Ace2+ positive cells; white bars: Ace2- cells.
(I). Schematic of RNA-Seq data from (Matos et al., 2019) of human lung explants (n = 5 donors) exposed to influenza A virus (IAV, H3N2) at 24 h post infection.
(J). Expression of SFTPC (surfactant protein C, a marker of type II pneumocytes) versus ACE2 among mock-infected lung explants. Statistical significance assessed by Pearson’s correlation, r = 0.93, p = 0.021. TPM: transcripts per million.
(K). SFTPC expression among matched donors following mock or IAV infection for 24 h. Statistical significance assessed by ratio paired t test, p = 0.86.
(L). ACE2 expression among matched donors following mock or IAV infection for 24 h. Statistical significance assessed by ratio paired t test, p = 0.0054.
(M). Western blot of fully-differentiated air-liquid interface cultures from bronchial cells derived from 4 human donors with asthma. Cells from each donor were treated with 10 ng/mL IFNγ for 24 h, and compared to a matched untreated condition. ACE2 protein: AF933 (R&D). Fold changes quantified for IFNγ treated versus untreated for each patient donor following normalization to GAPDH.
Figure 6 In Vivo Administration of Interferons in Mice Does Not Induce Ace2, and ACE2 Is Induced in Goblet Secretory Cells during Human Influenza Infection
(A) UMAP of 11,358 single cells from mouse nasal epithelium (n = 4).
(B) UMAP projection as in (A), points colored by detection of Ace2 (SARS-CoV-2 receptor homolog). Color coding is as follows: black, RNA positive; blue, RNA negative.
(C) Percent of Ace2+ cells by treatment condition (n = 4 arrays per condition; n = 2 arrays per mouse). Black bars indicate Ace2+ cells; white bars indicate Ace2− cells. p = 0.4 by Student’s t test.
(D) Heatmap of cell-type-defining genes (Trp63 and Krt17), interferon-induced genes (Irf7, Stat1, Irf9, and Oasl2), and Ace2 among basal epithelial cells, separated by cells derived from saline-treated mice (left) and IFN-α-treated mice (right). Statistical significance by likelihood-ratio test with Bonferroni correction is shown. A full list of differentially expressed genes can be found in Table S8.
(E) Schematic for sampling cells derived from nasal washes of n = 18 human donors with and without current influenza A or B infection for Seq-Well v1 (35,840 single cells). See Cao et al., (2020).
(F and G) ACE2 expression among goblet cells (F) and squamous cells (G) by infection status. Shown are Healthy Donor cells from influenza-negative donors (white); Bystander Cells from influenza A (IAV)- or influenza B (IBV)-infected donors, no intracellular viral RNA detected (black); Flu Viral RNA+ Cells with detectable intracellular influenza A or B viral RNA (red). Statistical significance by Wilcoxon test with Bonferroni correction, n.s. for Bystander versus Flu Viral RNA+.
See also Figure S5 and Tables S6 and S8.
This observation was supported by analysis of scRNA-seq data from 5,558 epithelial cells from the lungs of mice 3–6 days after intranasal infection with murine gamma herpesvirus-68 (MHV68) (Figure S5E). Here, we found significant enrichment of Ace2 + cells within type II pneumocytes, in line with our data from NHP and human lungs (Figures S5F). We did not observe changes in Ace2 expression among viral-transcript-positive cells or “bystander” type II pneumocytes (those without detectable cell-associated viral RNA in MHV68-infected animals), nor did we see significant alterations in Ace2 + cell abundance among MHV68-infected mice lacking IFN-γR (Figure S5G and S5H). These observations were in agreement with our in vitro murine basal cell assay (Figure 5A and 5E).
Finally, we sought to validate our hypothesis that ACE2 is upregulated in human epithelial cells during upper airway viral infections, which are known to induce a robust IFN response (Bailey et al., 2014, Everitt et al., 2012, Iwasaki and Pillai, 2014, Jewell et al., 2010, Russell et al., 2018, Steuerman et al., 2018). We re-analyzed a publicly available dataset of RNA-seq from human lung explants isolated after surgical resections that were infected with influenza A virus ex vivo for 24 h. Here, we found that ACE2 expression was significantly correlated with that of SFTPC, supporting our hypothesis that ACE2 is expressed within type II pneumocytes (Figures 1C, 2C, S5I, and S5J) (Matos et al., 2019). Furthermore, although the abundance of SFTPC was not significantly altered by influenza A virus infection, ACE2 expression was significantly upregulated after viral exposure (p = 0.0054, ratio paired t test) (Figures S5K and S5L). This suggests that influenza A virus infection increases ACE2 expression. Nevertheless, these population-level analyses are not able to definitively resolve specific cell subsets of relevance, nor whether they are directly infected cells or bystanders of infection.
In order to address these questions, we leveraged an ongoing scRNA-seq study of nasal washes from 18 individuals with confirmed influenza A virus or influenza B virus infection or healthy controls collected with Seq-Well v1, which yielded 35,840 cells resolved into 17 distinct cell types (Figure 6E; STAR Methods) (Cao et al., 2020). We investigated the cell types with greatest enrichment for ACE2 and TMPRSS2 in non-infected controls and individuals with influenza A and B. Strikingly, ACE2 was most upregulated in samples from influenza-virus-infected individuals within bystander goblet or squamous cells not directly infected by virus (Figures 6F and 6G). ACE2 + TMPRSS2 + goblet cells during influenza infection exhibited enrichment for canonical ISGs such as the CXCL9/CXCL10/CXCL11 gene cluster; correspondence with ACE2 + TMPRSS2 + goblet cells in healthy and allergic nasal scrapings; and a shared overlap in ISGs including GBP2, ZNFX1, ADAR, and ACE2 (significantly differentially expressed gene lists) (Table S6). Together, our data suggest that ACE2 is an ISG in vitro and in vivo in human primary upper airway epithelial basal cells, but that the murine homolog Ace2 is not in airway epithelial basal cells or pulmonary epithelial cells in vitro or in vivo. Collectively, our findings suggest that careful considerations of animal and cellular models will be needed for assessing therapeutic interventions targeting the IFN system when studying ACE2/Ace2-associated biology.
Finally, because our in vivo and in vitro work indicate that IFN might promote human cellular targets for SARS-CoV-2 infection in the human upper airway by inducing ACE2, we attempted to extend our transcriptomic data on IFN-driven expression of ACE2 to protein-level induction of ACE2. As testing of various commercially available polyclonal antibody preparations found broad evidence for non-specific or inconclusive staining in histological immunofluorescent based readouts (data not shown), we assessed whether IFN-γ-stimulated human bronchial air-liquid interface cultures induced ACE2 within 24 h. Our results show that cells from one patient robustly induced ACE2 (+2.02x), cells from another mildly induced ACE2 (+1.21x) and two patient’s cells showed minor changes (+/−1.12x) (Figure S5M). We provide a note of caution as these cells were derived from asthmatic patients, and the overall changes did not reach significance. Furthermore, we could not determine cell surface localization of ACE2 but do note that these results align with our transcriptomic data.