Double-stranded RNA sensing in airway basal cells impairs subsequent epithelial barrier formation via TLR3/TRIF

Abstract Background Double-stranded viral RNA disrupts the airway epithelial barrier; however, its specific impact on basal progenitor cells remains unknown. In this study, we stimulated undifferentiated normal human bronchial epithelial cells and VA10 cells, which function as basal cells, with the Toll-like receptor 3 (TLR3) ligand polyinosinic: polycytidylic acid (poly I: C). The cells were subsequently cultured at an air-liquid interface (ALI) to evaluate the airway epithelial barrier integrity and perform global gene expression analysis. Results Poly I: C stimulation significantly decreased transepithelial electrical resistance and reduced the expression of barrier-related genes. This barrier dysfunction was attenuated by silencing TLR3 or TIR-domain-containing adapter-inducing interferon-β (TRIF). Transcriptomic analysis identified distinct gene-expression patterns at the two time points examined. At day 10, replicated microarray analysis showed increased expression of inflammation- and remodeling-associated genes. At day 3, an exploratory pooled analysis, with individual genes validated by RT-qPCR in separate, non-pooled wells, showed induction of interferon-stimulated genes together with reduced expression of genes required for epithelial differentiation and barrier formation. Conclusions In airway basal cells, double-stranded RNA impaired subsequent epithelial barrier formation, and this impairment required TLR3 and TRIF during the exposure window. The accompanying transcriptional changes differed between the two time points examined: an antiviral program at day 3, coinciding with reduced expression of differentiation- and barrier-associated genes, and increased expression of inflammation- and remodeling-associated genes at day 10. As a hypothesis to be tested directly, we propose that dsRNA sensing during this early window redirects basal cell differentiation. These findings suggest a mechanism by which dsRNA sensing in airway basal cells may contribute to persistent barrier vulnerability. Validation using live respiratory viruses, and in primary cells from multiple donors, is required before this mechanism can be extended to viral infections.