Induction of human airway epithelial to mesenchymal transition upon rhinovirus infection

Abstract

Rationale: Airway remodeling collectively refers to a number of structural changes in the airways that are characteristic of asthma. These include epithelial shedding, goblet cell hyperplasia, angiogenesis, increased smooth muscle mass, and thickening of the lamina reticularis. Thickening of the lamina reticularis, is believed to be due to increased deposition of matrix proteins by fibroblasts/myofibroblasts, and is observed even in preschool children. Children who wheeze with human rhinovirus (HRV) during the first three years of life have an increased risk of developing asthma. Children get multiple HRV infections annually, and the primary site of infection for HRV is the airway epithelium. We hypothesized that HRV infection plays a role in airway remodeling by triggering EMT to produce mesenchymal cells that contribute to thickening of the lamina reticularis by depositing matrix proteins. Methods: The BEAS-2B human bronchial epithelial cell line was grown on 6-well plates in bronchial epithelial growth medium (BEGM). Beginning 24 h prior to stimulation, cells were transferred to BEGM from which hydrocortisone was removed. Cells were exposed to medium (control), HRV-16 alone, transforming growth factor-beta 1 (TGF-β1) alone, or HRV + TGF-β1 for 120 h, with medium and TGF-β1 replacement at 48 and 96 h. Cell lysates were collected and analyzed by western blot or ELISA for protein expression of epithelial and mesenchymal markers. HRV-16 rendered replication-deficient by 5 min exposure to ultra-violet (UV) light was also used. Results: Treatment with HRV-16 alone and, even more so, in combination with TGF-β1 caused reduced protein expression of the epithelial marker E-cadherin. Interestingly, HRV was more effective than TGF-β1 alone in stimulating loss of E-cadherin. By contrast, TGF-β1 alone and the combination of HRV + TGF-β1 were more effective in inducing the expression of the mesenchymal markers, fibronectin and vimentin, The ability of HRV to synergize with TGF-β1 was not abolished when HRV-16 was UV-treated. Time-course studies suggest that loss of E-cadherin is evident by 24-48 h. Conclusion: HRV-16 alone, and in combination with TGF-β1, causes EMT-like changes in BEAS-2B cells. Changes in response to treatment with UV-treated HRV-16 suggest that viral replication is not essential and that receptor binding may be sufficient to induce some of the changes observed. Our data suggest that HRV plays a major role in triggering a loss of epithelial phenotype, while TGF-β1 may predominate in inducing mesenchymal markers. The combination of HRV + TGF-β1 was the most effective stimulus for all changes measured.