Influenza virus-induced type I interferons disrupt alveolar epithelial repair and tight junction integrity in the developing lung
Type I interferons (IFN-I) are key innate immune mediators which control viral infection in adults. Recently, using a neonatal murine model of influenza A infection (IAV), we demonstrated that IAV-infected murine neonates lacking a functional IFN-I receptor (IFNAR-/-) have significantly improved survival and reduced lung pathology relative to wild-type (WT) neonates. However, the opposite is observed in adults, with IAV-infected IFNAR-/- adults exhibiting enhanced morbidity relative to WT adults, indicating an age-specific IFN-I toxicity in neonates. Therefore, we hypothesized that IAV-induced IFN-I signaling in primary neonatal type II alveolar epithelial cells (TIIECs), the main cell type of IAV infection and initiator of host response in the lung, contributed to age-specific viral pathogenesis.  To investigate the role of IFN-I signaling in TIIECs, we performed RNA-Seq on purified TIIECs in a multifactorial design, comparing 1. IFNAR-/- and WT genotypes, 2. neonatal and adult ages, 3. IAV-infected and uninfected, 4. over a three-day time course.Analysis of purified TIIECs revealed age, not infection status, as the primary driver of transcriptional differences in TIIECs. Subsequent pathway analysis demonstrated IAV-infected IFNAR-/- neonates significantly upregulate cell proliferation, tissue repair and tight junction genes at 2-days post-infection (dpi), compared to WT neonates. Next, to determine if these growth and repair differences persisted later in infection, targeted analysis of repair gene expression and immunofluorescent quantification of pulmonary sealing tight junction molecules ZO-1 and occludin was performed at 6-dpi. Relative to WT neonates, IFNAR-/- neonates had significantly higher whole lung occludin staining and repair gene expression. Together, our data demonstrates IFN-I signaling is extremely pathogenic in the developing lung because by disrupting alveolar repair and pulmonary barrier integrity. Overall design: To investigate the role of IFN-I signaling within TIIECs during influenza infection, 3-day-old wild-type (WT) and IFNAR-/- neonates, and 8-week-old WT and IFNAR-/- adults were infected intranasally with PR8-IAV. Primary TIIECs were isolated from the lungs of infected mice at 1-day (24h), 2-days (48h), and 3-days (72h) -post infection. TIIECs were harvetsed from timepoint-matched uninfected WT and IFNAR-/- neonates (4-, 5-, and 6-days old), and uninfected 8-week-old WT and IFNAR adults to serve as controls. RNA was extracted from Type II alveolar epithelial cell samples and gene expression analysis was performed using RNA-seq. Comparative gene expression analysis was determined based on differences in genotype (WT vs IFNAR-/-), age (neonate vs adult), treatment (flu-infection vs uninfected), and timepoint (1-, 2-, 3-days post infection).