Hyperbaric Oxygen Exposure Increases Spheroid Formation and Decreases SARS-CoV-2 Viral Receptor Expression Via NRF2 Upregulation as Well as Inhibits Mucociliary Differentiation and Increases Expression of Stem/Progenitor Cell Markers Via HIF2A Upregulation in the Air-Liquid Interface-Cultured Normal Human Bronchial Epithelial Cells

Rationale: It has been reported that hyperbaric oxygen (HBO) therapy is an effective treatment for various types of tissue injury and diseases, including central airway stenosis, and that HBO may promote migration and proliferation of various types of adult tissue-specific stem/progenitor cells. Notably, in 2020 more than 30 COVID-19 patients in China have been reported to be successfully discharged from the hospital after 38 HBO treatments. Therefore, it is of interest to study the effects of HBO exposure on the expression of both the stem/progenitor cell markers and SARS-CoV-2 viral receptors in human bronchial epithelial cells (HBECs). Methods: The HBECs were obtained from the Lonza Biotechnology Company and were derived from the tracheo-bronchial junction surrounding the airway bifurcation of the same race and age-matched healthy subjects (i.e., NHBE cells) and COPD patients (i.e., DHBE cells). The HBECs at passage 3 were grown on the Millicell® PTFE cell culture inserts and submerged in the CELLnTEC® Prime Airway Proliferation medium for 3 days followed by replacing with the CELLnTEC® Prime Airway Differentiation medium for 1 day and subsequently cultured in the air-liquid interface (ALI) with the upper layer medium aspirated. The ALI cultures of HBECs were then exposed daily to 100% O2 at 2.5 ATA for 40 min for 28 days in total. Results: After 28-day HBO exposure, the numbers of spheroids formed per cm2 on the cell culture inserts were 257.60±58.80 in NHBE cells, which were more than 19 folds of the numbers in DHBE cells (13.26±5.31) and more than 10 folds of the numbers in the NHBE and DHBE cells without HBO exposure (22.99±3.54 and 22.10±8.71, respectively). Interestingly, the HBO-induced spheroids expressed stem/progenitor cell markers OCT4, SOX2 and TP63 but did not express the ciliated cell marker FOXJ1 or the goblet cell marker MUC5AC. The mRNA levels of NRF2, HIF1A and HIF2A were all increased in the NHBE cells whereas decreased in the DHBE cells after HBO exposure, and were reversely associated with ACE2, TMPRSS2, CC10, and FOXJ1 expression in the ALI-cultured NHBE and DHBE cells. In the NHBE cells, transfection with NRF2 siRNA inhibited spheroid formation and increased ACE2 and TMPRSS2 expression, while transfection with HIF2A siRNA increased CC10, FOXJ1 and MUC5AC expression whereas decreased OCT4, SOX2 and TP63 expression. Conclusions: Our study shows for the first time that HBO differentially regulates bronchial spheroid formation, mucociliary differentiation and viral receptor gene expression via distinct NRF2 and HIF2A regulatory modules.