Gestational and Postnatal Age Define Airway Epithelial Maturation and RSV Responses During the First Year of Life: Results of an ex-vivo/in-vitro Airway Epithelial Cell Study

Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants globally.  Newborns, especially those born prematurely, are at increased risk of severe RSV-related illness, yet the influence of age on early-life airway epithelium innate immune responses remains relatively understudied.  We established ex-vivo/in-vitro well-differentiated paediatric nasal epithelial cell (WD-PNEC) cultures derived from preterm and term infants at birth and again at one-year-old.  Infection characteristics and innate immune responses were determined following in vitro RSV infection.  RSV growth kinetics and cytopathology were similar irrespective of gestation or age.  Secretion of interferon lambda-1 (IFN-λ1), CXCL10, CCL5, and CXCL8 were comparable between RSV-infected preterm- and term-newborn WD-PNECs.  Importantly, following RSV infection, secretion of RSV-induced IFN-λ1 (p<0.05) and CCL5 (p<0.01) and expression of the IFN-stimulated gene, IFI6 (p<0.05) were significantly higher in one-year- compared to newborn-derived WD-PNECs. Interestingly, secretion of the cell signalling cytokine pleiotrophin increased significantly with longer gestation (p<0.01) and older age (p<0.001). Differential gene expression analysis demonstrated 156 differentially expressed genes (DEGs) with age and revealed transcriptomic changes linked to gestation in newborns that persisted at 1-year. These findings indicate two major conclusions.  Firstly, we report transcriptomic differences in airway epithelial cell (AEC) development between term and preterm infants across the first year of life.  Secondly, we demonstrate that RSV infection responses of AECs become more robust with age in early-life.  Further investigation of identified DEGs and differentially expressed chemokines/cytokines is warranted to clarify their role in increased susceptibility of very young infants to severe RSV disease, as well as the significance of these factors in the emergence of wheezing phenotypes and long-term respiratory outcomes.