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.