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Airway epithelial responses to models of SARS-CoV-2 Guo, Tony Ju Feng

Abstract

RATIONALE: The airway epithelium, which serves as the physical and immune barrier of the airways, may be injured by pathogens such as SARS-CoV-2, requiring repair to restore function. It is unclear whether and how SARS-CoV-2 can impact airway epithelial functions, such as wound repair. This project will validate models of both the airway epithelium and SARS-CoV-2, explore their interaction, and assess the impact of spike S1 subunit treatment and full-length spike transfection on airway epithelial function. We hypothesize that spike S1 subunit and spike transfection of airway epithelial cells impair repair following injury. METHODS: 16HBE14o- (16HBE) airway epithelial cells in monolayer culture were evaluated for ACE2, TMPRSS2, and furin expression, proteins crucial for SARS-CoV-2 entry. Dexamethasone treatment and airway differentiation were introduced to assess their effects on viral entry factor expression in airway epithelial cultures. 16HBE cultures were treated with recombinant spike S1 subunit or transfected with full-length spike-expressing plasmid. Ki67 and p21ᶜͥᵖ¹ (p21), markers for proliferation and cellular senescence, respectively, were assessed. Culture proliferation and secreted interleukin-6 (IL-6) and -8 were assayed. Mechanical injury was induced in treated or transfected cultures to monitor wound closure. RESULTS: 16HBE cultures expressed ACE2, TMPRSS2, and furin, indicating susceptibility to SARS-CoV-2. Dexamethasone treatment of 16HBE monolayer cultures and differentiation of primary epithelial culture altered viral entry factor expression. Spike S1 treatment of 16HBE reduced Ki67 expression, increased p21 expression, and decreased culture proliferation. Secreted IL-6 but not IL-8 increased with treatment. Spike transfection reduced Ki67 expression, coinciding with peak spike expression at 48 hours post-transfection. Transfection inhibited culture proliferation and increased IL-6 and IL-8 secretion. Wound closure was inhibited by spike treatment or transfection, with significant reductions compared to control (76.6 ± 1.5 % vs. 99.5 ± 3.9 % and 33.4 ± 8.8 % vs. 93.7 ± 3.1 %, respectively). CONCLUSION: We show that SARS-CoV-2 spike S1 treatment and full-length spike transfection can alter measures of proliferation, cellular senescence, and inflammation and impairs wound closure of 16HBE14o- airway epithelial cells. These results highlight how components of SARS-CoV-2 can impair functions of the airway epithelium in a viral-replication independent manner.

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Attribution-NonCommercial-NoDerivatives 4.0 International