Background: The COVID-19 pandemic is an ongoing threat to public health. Since the identification of COVID-19, the disease caused by SARS-CoV-2, no drugs have been developed to specifically target SARS-CoV-2. To develop effective and safe treatment options, a better understanding of cellular mechanisms underlying SARS-CoV-2 infection is required. To fill this knowledge gap, researchers require reliable experimental systems that express the host proteins necessary for the cellular entry of SARS-CoV-2. These proteins include the viral receptor, ACE2 and the proteases TMPRSS2 and furin. A number of studies have reported cell-type specific expression of the genes encoding these molecules. However, less is known about the protein expression of these molecules. Methods: We assessed the suitability of primary human bronchial epithelial (HBE) cells maintained in air-liquid interface (ALI) as an experimental system for studying SARS-CoV-2 infection in vitro. During cellular differentiation, we measured the expression of ACE2, TMPRSS2, and furin over progressive ALI days by RT-qPCR, western blot and immunofluorescence staining. We also explored the effect of the fibrotic cytokine TGF-b on the expression of these proteins in well-differentiated HBE cells. Results/Discussion: Like ACE2, TMPRSS2 and furin proteins are localized in differentiated ciliated cells as confirmed by immunofluorescence staining. These data suggest that well-differentiated HBE cells maintained in air-liquid interface is a reliable in vitro system for investigating cellular mechanisms of SARS-CoV-2 infection. We further identified that profibrotic mediators, TGF-β1 and TGF-β2, increase the expression of furin, which is a protease required for the cellular entry of SARS-CoV-2.

Epithelial tissue can transition from a jammed, solid-like, quiescent phase to an unjammed, fluid-like, migratory phase, but the underlying molecular events of the unjamming transition (UJT) remain largely unexplored. Using primary human bronchial epithelial cells (HBECs) and one well-defined trigger of the UJT, compression mimicking the mechanical effects of bronchoconstriction, here, we combine RNA sequencing data with protein-protein interaction networks to provide the first genome-wide analysis of the UJT. Our results show that compression induces an early transcriptional activation of the membrane and actomyosin network and a delayed activation of the extracellular matrix (ECM) and cell-matrix networks. This response is associated with a signaling cascade that promotes actin polymerization and cellular motility through the coordinated interplay of downstream pathways including ERK, JNK, integrin signaling, and energy metabolism. Moreover, in nonasthmatic versus asthmatic HBECs, common genomic patterns associated with ECM remodeling suggest a molecular connection between airway remodeling, bronchoconstriction, and the UJT.

Aberrant remodeling of the asthmatic airway is not well understood but is thought to be attributable in part to mechanical compression of airway epithelial cells. Here, we examine compression-induced expression and secretion of the extracellular matrix protein tenascin C (TNC) from well-differentiated primary human bronchial epithelial (HBE) cells grown in an air–liquid interface culture. We measured TNC mRNA expression using RT-qPCR and secreted TNC protein using Western blotting and ELISA. To determine intracellular signaling pathways, we used specific inhibitors for either ERK or TGF-β receptor, and to assess the release of extracellular vesicles (EVs) we used a commercially available kit and Western blotting. At baseline, secreted TNC protein was significantly higher in asthmatic compared to non-asthmatic cells. In response to mechanical compression, both TNC mRNA expression and secreted TNC protein was significantly increased in both non-asthmatic and asthmatic cells. TNC production depended on both the ERK and TGF-β receptor pathways. Moreover, mechanically compressed HBE cells released EVs that contain TNC. These data reveal a novel mechanism by which mechanical compression, as is caused by bronchospasm, is sufficient to induce the production of ECM protein in the airway and potentially contribute to airway remodeling.

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