Airway Basal Stem Cells in COVID-19 Exhibit a Proinflammatory Signature and Impaired Mucocililary Differentiation.

Airway basal stem cells (BSCs) play a critical role in epithelial regeneration. Whether coronavirus disease (COVID-19) affects BSC function is unknown. Here, we derived BSC lines from patients with COVID-19 using tracheal aspirates (TAs) to circumvent the biosafety concerns of live-cell derivation. We show that BSCs derived from the TAs of control patients are bona fide bronchial BSCs. TA BSCs from patients with COVID-19 tested negative for severe acute respiratory syndrome coronavirus 2 RNA; however, these so-termed COVID-19-exposed BSCs in vitro resemble a predominant BSC subpopulation uniquely present in patients with COVID-19, manifested by a proinflammatory gene signature and STAT3 hyperactivation. Furthermore, the sustained STAT3 hyperactivation drives goblet cell differentiation of COVID-19-exposed BSCs in an air-liquid interface. Last, these phenotypes of COVID-19-exposed BSCs can be induced in control BSCs by cytokine cocktail pretreatment. Taken together, acute inflammation in COVID-19 exerts a long-term impact on mucociliary differentiation of BSCs.

Rhinovirus infection induces secretion of endothelin-1 from airway epithelial cells in both in vitro and in vivo models.

BACKGROUND: Rhinovirus (RV) infection of airway epithelial cells triggers asthma exacerbations, during which airway smooth muscle (ASM) excessively contracts. Due to ASM contraction, airway epithelial cells become mechanically compressed. We previously reported that compressed human bronchial epithelial (HBE) cells are a source of endothelin-1 (ET-1) that causes ASM contraction. Here, we hypothesized that epithelial sensing of RV by TLR3 and epithelial compression induce ET-1 secretion through a TGF-ß receptor (TGFßR)-dependent mechanism. METHODS: To test this, we used primary HBE cells well-differentiated in air-liquid interface culture and two mouse models (ovalbumin and house dust mite) of allergic airway disease (AAD). HBE cells were infected with RV-A16, treated with a TLR3 agonist (poly(I:C)), or exposed to compression. Thereafter, EDN1 (ET-1 protein-encoding gene) mRNA expression and secreted ET-1 protein were measured. We examined the role of TGFßR in ET-1 secretion using either a pharmacologic inhibitor of TGFßR or recombinant TGF-ß1 protein. In the AAD mouse models, allergen-sensitized and allergen-challenged mice were subsequently infected with RV. We then measured ET-1 in bronchoalveolar lavage fluid (BALF) and airway hyperresponsiveness (AHR) following methacholine challenge. RESULTS: Our data reveal that RV infection induced EDN1 expression and ET-1 secretion in HBE cells, potentially mediated by TLR3. TGFßR activation was partially required for ET-1 secretion, which was induced by RV, poly(I:C), or compression. TGFßR activation alone was sufficient to increase ET-1 secretion. In AAD mouse models, RV induced ET-1 secretion in BALF, which positively correlated with AHR. CONCLUSIONS: Our data provide evidence that RV infection increased epithelial-cell ET-1 secretion through a TGFßR-dependent mechanism, which contributes to bronchoconstriction during RV-induced asthma exacerbations.

Mechanical Compression of Human Airway Epithelial Cells Induces Release of Extracellular Vesicles Containing Tenascin C.

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-ß 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-ß 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.

Genomic signatures of the unjamming transition in compressed human bronchial epithelial cells.

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.

In well-differentiated primary human bronchial epithelial cells, TGF-ß1 and TGF-ß2 induce expression of furin.

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 factor proteins necessary for the cellular entry of SARS-CoV-2. These proteins include the viral receptor, angiotensin-converting enzyme 2 (ACE2), and the proteases, transmembrane serine protease 2 (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. We assessed the suitability of primary human bronchial epithelial (HBE) cells maintained in an 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 reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blot, and immunofluorescence staining. We also explored the effect of the fibrotic cytokine TGF-ß on the expression of these proteins in well-differentiated HBE cells. 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 ALI are a reliable in vitro system for investigating cellular mechanisms of SARS-CoV-2 infection. We further identified that the profibrotic mediators, TGF-ß1 and TGF-ß2, increase the expression of furin, which is a protease required for the cellular entry of SARS-CoV-2.