High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa

The study investigates whether the oral mucosa serves as a potential route for 2019-nCoV (SARS-CoV-2) infection by examining the expression and cellular distribution of its entry receptor, ACE2, in oral tissues. Contextually, COVID-19 emerged in late 2019 with significant respiratory and systemic symptoms. Prior work established ACE2 as the cellular receptor for SARS-CoV-2, similar to SARS-CoV and HCoV-NL63, underscoring the importance of mapping ACE2 expression to infer tissue susceptibility. The purpose of this study is to determine if ACE2 is expressed in oral mucosa and to identify the specific oral sites and cell types with higher ACE2 expression, thereby assessing the potential infection risk via the oral cavity and informing dental clinical practice and public health precautions.

Previous research using single-cell RNA-seq reported high ACE2 expression in multiple tissues, including type II alveolar cells of the lung, esophageal upper and stratified epithelial cells, absorptive enterocytes from the ileum and colon, cholangiocytes, myocardial cells, kidney proximal tubule cells, and bladder epithelial cells. These findings suggest that tissues with higher ACE2-expressing cells are potentially more susceptible to SARS-CoV-2 infection. The literature also indicated that SARS-CoV-2 uses ACE2 for cellular entry and does not utilize other coronavirus receptors such as aminopeptidase N or dipeptidyl peptidase IV, reinforcing the centrality of ACE2 in host susceptibility mapping.

The study combined analyses of public bulk RNA-seq datasets with in-house single-cell RNA-seq (scRNA-seq) data. Bulk RNA-seq: Expression profiles from The Cancer Genome Atlas (TCGA) included 695 para-carcinoma normal tissues across 13 organ types, with a focus on oral cavity samples. ACE2 expression means across organs were compiled (e.g., oral cavity mean 6.23). Site-specific analysis of ACE2 within oral tissues used TCGA annotations: 13 adjacent normal tissues from oral tongue, 2 from base of tongue, 3 from floor of mouth, and 14 categorized as oral cavity without a specific site. The FANTOM5 CAGE dataset (normal tissues across 14 organs) served to validate ACE2 expression patterns observed in TCGA. Single-cell RNA-seq: Four oral tissues were profiled in-house. After preprocessing, 22,969 cells were retained and clustered using UMAP into seven major cell types identified by canonical markers: epithelial cells (e.g., SFN, KRT6A, KRT10), fibroblasts (e.g., FAP, PDPN, COL1A2, COL3A1, COL6A1, TGFB1), immune cells (including T and B cells; e.g., CD1G, CST3, CFB, CD68, FCGR2A), and mesenchymal cells (e.g., COL1A1, MMP2, THBS2). ACE2 expression was mapped across oral sites (tongue, buccal, gingiva) and cell types to quantify ACE2-positive cell proportions and their distribution. Visualization included violin plots, scatter plots, heatmaps, and UMAPs to present ACE2 expression by organ, site, and cell type.

- Bulk RNA-seq showed ACE2 expression across multiple organs; oral cavity tissues expressed ACE2 (TCGA oral cavity mean expression 6.23). Patterns were consistent with normal-tissue data from the FANTOM5 CAGE dataset. - Within oral tissues (TCGA), ACE2 mean expression tended to be higher in oral tongue (13 tissues) compared with other oral sites combined (base of tongue, floor of mouth, and unspecified oral cavity; 19 tissues), though the difference did not reach statistical significance (p = 0.062), likely due to limited sample sizes. - scRNA-seq of four oral tissues (22,969 cells) confirmed ACE2 expression in oral mucosa. Overall, 0.52% of cells were ACE2-positive; 95.86% of ACE2-positive cells were located in oral tongue, indicating higher ACE2 expression in tongue compared to buccal and gingival tissues. - ACE2-positive cells were identified among epithelial cells (1.19% ACE2-positive within epithelial cells), as well as T cells, B cells, and fibroblasts (each <0.5%). Epithelial cells accounted for 93.38% of all ACE2-positive cells, demonstrating strong enrichment of ACE2 expression in tongue epithelial cells.

The findings directly address the research question by demonstrating that ACE2, the entry receptor for SARS-CoV-2, is expressed in the oral mucosa and is particularly enriched in epithelial cells of the tongue. This cellular and site-specific distribution suggests that the oral cavity—especially the tongue epithelium—may be a susceptible site for viral entry and early infection. The concordance between bulk RNA-seq (TCGA and FANTOM5) and in-house scRNA-seq strengthens the evidence that oral epithelial cells contribute substantially to ACE2 expression. These results have practical implications for infection control in dental and oral healthcare settings and highlight the importance of protective strategies aimed at reducing exposure of oral mucosal surfaces to the virus.

This study shows that ACE2 is expressed in the oral mucosa and is highly enriched in epithelial cells, with the tongue exhibiting higher ACE2 levels than buccal and gingival tissues. These findings imply that the oral cavity, particularly the tongue epithelium, may represent a high-risk route for SARS-CoV-2 infection and should be a focus of preventive measures in clinical dental practice and daily life. Future research could expand sample sizes for site-specific comparisons, evaluate protein-level ACE2 expression, and investigate functional susceptibility and viral entry dynamics in oral epithelial subtypes.

The oral site comparison in TCGA was limited by small sample sizes for certain sites, and the observed higher ACE2 expression in oral tongue did not achieve statistical significance (p = 0.062). The study primarily relies on transcriptomic measurements (bulk and single-cell RNA-seq) without functional assays, and details of donor variability for the in-house scRNA-seq were not provided, which may limit generalizability.