Fig 2: TMPRSS2 is expressed at higher levels in goblet cells. (A) The airway epithelia (described in Fig. 1) were stained for cilia (anti-acetyl-alpha tubulin, cyan), TMPRSS2 (anti-TMPRSS2, green), and F-actin (rhodamine phalloidin, red); the nuclei were stained with DAPI (blue). Bar =15 µm. (B) The airway epithelia were stained for the identification of MUC5AC+ goblet cells (anti-MUC5AC, cyan), TMPRSS2 (anti-TMPRSS2, green), F-actin (rhodamine phalloidin, red), and nuclei (DAPI, blue). Bar =15 µm. (C) The airway epithelia were stained to identify MUC5B+ goblet cells (anti-MUC5B, cyan), TMPRSS2 (anti-TMPRSS2, green), F-actin (rhodamine phalloidin, red), and nuclei (DAPI, blue). Bar = 15 µm.

Fig 3: The airway epithelium model recapitulates the bronchial characteristics of COPD. (A) Airway epithelium derived from differentiated NHBE cells obtained from a healthy adult or COPD patient were fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The nuclei are stained dark purple, and the cytoplasmic components are pink. Bar = 50 µm. (B) The sectioned epithelia were stained with alcian blue. Mucosubstances are stained blue, whereas the cytoplasmic components are pale pink, and the nuclei are pink/red. Bar = 50 µm. (C) The apical sites of the airway epithelia were fixed, permeabilized, and stained for the goblet cell marker MUC5AC (anti-MUC5AC, green), the ciliated cell marker acetyl-alpha tubulin (anti-acetyl-alpha tubulin, cyan), and F-actin (rhodamine phalloidin, red); the nuclei were also stained with DAPI (blue). Bar = 10 µm. (D) The sectioned epithelia were stained for the ciliated cell marker acetyl-alpha tubulin (anti-acetyl-alpha tubulin, cyan), the goblet cell marker MUC5AC (anti-MUC5AC, green), and F-actin (rhodamine phalloidin, red); the nuclei were also stained with DAPI (blue). Bar = 20 µm. (E) The apical sites of the airway epithelia were fixed and stained for two goblet cell markers, MUC5AC and MUC5B (anti-MUC5AC [green] and anti-MUC5B [cyan], respectively) and F-actin (rhodamine phalloidin, red); the nuclei were also stained with DAPI (blue). Bar = 10 µm. (F) The sectioned epithelia were stained for the basal cell marker P63 (anti-P63, green), the goblet cell marker MUC5B (anti-MUC5B, cyan), and F-actin (rhodamine phalloidin, red); the nuclei were also stained with DAPI (blue). Bar = 20 µm. (G) The MUC5B+ goblet cells were counted from tiled images of the airway epithelium stained for MUC5B (anti-MUC5B, cyan) (described above) obtained with a Leica DMi8 epifluorescence microscope. A total of approximately 3,000 cells were counted using ImageJ software from each epithelium to determine the ratio. The error bar represents the SEMs. The statistical significance was determined by unpaired two-tailed t tests. The results from one independent experiment are shown. (H) The MUC5AC+ goblet cells were counted using ImageJ software from random fields in confocal images of the airway epithelium stained for MUC5AC (anti-MUCAC, cyan) (described above). A total of approximately 2,000 cells were counted from each epithelium to determine the ratio. The error bar represents the SEMs. The statistical significance was determined by unpaired two-tailed t tests. The results from two independent experiments are shown. (I) The sectioned epithelia were stained for MUC5B (anti-MUC5B, cyan); the nuclei were stained with DAPI (blue). Bar = 20 µm. (J) Transepithelial electrical measurements (TEERs) of the airway epithelia were obtained. The data were obtained by combining three independent Transwell reads, and each Transwell read was an average of three independent reads. The error bars represent the SEMs. (K) The ciliary beat frequency (CBF) was measured on the airway epithelia. The data were obtained by combining three independent Transwell reads, and each Transwell read was an average of six random point reads. The error bars represent the SEMs. The statistical significance was determined by unpaired two-tailed t tests. The results from one independent experiment are shown.

Fig 4: SARS-CoV-2 induces syncytia and cell sloughing. (A) The airway epithelia (mock or infected; described in Fig. 4) were stained for SARS-CoV-2 N (anti-N, green) and F-actin (rhodamine phalloidin, red); the nuclei were also stained with DAPI (blue). The arrows indicate syncytia. Bar = 5 µm. (B) At 4 dpi, the sectioned epithelia were stained for the goblet cell marker MUC5AC (anti-MUC5AC, cyan) and SARS-CoV-2 N (anti-N, green), and the nuclei were also stained with DAPI (blue). The arrows indicate syncytia. Bar = 20 µm. (C) At 4 dpi, the number of syncytia (at least two nuclei in the fused infected cells) were quantified in the infected airway epithelium (healthy or COPD) (at least two independent donors). The distribution of nuclei in the syncytium is indicated by different colors in the same bar. The data were obtained from two independent experiments. No statistical analysis was done. (D) At 4 dpi, the airway epithelium was stained for SARS-CoV-2 N (anti-N, green) and F-actin (rhodamine phalloidin, red), and the nuclei were stained with DAPI (blue). The arrows indicate cell sloughing. Bar = 5 µm. (E) At 4 dpi, the sectioned epithelium was stained for cilia (anti-acetyl-alpha tubulin, cyan) and SARS-CoV-2 N (anti-N, green), and the nuclei were stained with DAPI (blue). The arrows indicate cell sloughing. Bar = 20 µm.

Fig 5: HDAC4 deficiency attenuates mucus production in IL-13-treated hNECs. (A) RT-qPCR and western blot analysis of MUC5AC expression. (B) MUC5AC expression was further tested by IF assay. HDAC4, histone deacetylase 4; IL-13, interleukin-13; MUC5AC, Mucin-5AC; RT-qPCR, quantitative reverse-transcription polymerase chain reaction