Systematic analysis of cell-type differences in the epithelial secretome reveals insights into the pathogenesis of respiratory syncytial virus-induced lower respiratory tract infections

Lower respiratory tract infections from respiratory syncytial virus (RSV) are due, in part, to secreted signals from lower airway cells that modify the immune response and trigger airway remodeling. To understand this process, we applied an unbiased quantitative proteomics analysis of the RSV-induced epithelial secretory response in cells representative of the trachea versus small airway bronchiolar cells. A workflow was established using telomerase-immortalized human epithelial cells that revealed highly reproducible cell type-specific differences in secreted proteins and nanoparticles (exosomes). Approximately one third of secretome proteins are exosomal; the remainder are from lysosomal and vacuolar compartments. We applied this workflow to three independently derived primary human cultures from trachea versus bronchioles. A total of 577 differentially expressed proteins from control supernatants and 966 differentially expressed proteins from RSV-infected cell supernatants were identified at a 1% false discovery rate. Fifteen proteins unique to RSV-infected primary human cultures from trachea were regulated by epithelialspecific ets homologous factor. A total of 106 proteins unique to RSV-infected human small airway epithelial cells was regulated by the transcription factor NF-kB. In this latter group, we validated the differential expression of CCL20/macrophage-inducible protein 3a, thymic stromal lymphopoietin, and CCL3-like 1 because of their roles in Th2 polarization. CCL20/macrophageinducible protein 3a was the most active mucin-inducing factor in the RSV-infected human small airway epithelial cell secretome and was differentially expressed in smaller airways in a mouse model of RSV infection. These studies provide insights into the complexity of innate responses and regional differences in the epithelial secretome participating in RSV lower respiratory tract infection-induced airway remodeling.