Abstract Rationale Healthy children are more likely to require hospitalization for influenza virus infection than healthy adults, suggesting age-related differences in the host immune response may influence illness severity. The respiratory epithelium is the primary site of influenza infection, driving the initial immune response. In a mouse model of influenza A virus (IAV) pneumonia, juvenile mice have higher mortality than adult mice despite similar rates of viral clearance as measured by plaque assay. However, since both infectious virions and viral RNA can activate the immune response, we aimed to investigate whether viral transcripts were comparable in alveolar epithelial cells from juvenile and adult mice, and to test whether age-related differences in the early alveolar epithelial response to IAV were independent of viral RNA. Methods Adult mice (8-12-week-old) and juvenile mice (4-week-old) were anesthetized and infected intratracheally with 30 PFU IAV. Lungs were harvested 2 days post infection (dpi) and Alveolar Type 2 (AT2) cells were isolated using flow cytometry. Total RNA was isolated and enriched for mRNA. Bulk RNA sequencing was performed and sequences aligned with mouse and IAV genomes for quantification. Since there was high correlation between the expression of all IAV genes, host gene expression was normalized to the influenza hemagglutinin (HA) gene. Analysis was performed using Deseq2 package in R. Results Following IAV infection at 2 dpi, more IAV transcripts were detected in juvenile AT2 cells than adult AT2 cells. When normalized to HA transcripts. Juvenile AT2 cells had increased expression of genes enriched for Type I and Type II interferon signaling pathways, Signal Transducer And Activator of Transcription 1 (STAT1) signaling, and Inhibitor Of Nuclear Factor Kappa B Kinase Subunit Beta (Ikbkb) signaling (all adjusted P values 0.05) compared to adult AT2 cells. Multiple interferon stimulated genes in these pathways were 2-3 log 2-fold change higher in juvenile AT2 cells compared to adult AT2 cells (including CXCL10, CXCL11, ZBP1, SOCS1, OAS3, ISG15) (all adjusted P values 0.05), even after normalization to the amount of IAV transcripts present. Conclusions In mice infected with IAV, juvenile AT2 cells have a more robust early interferon response than adult AT2 cells, even when controlling for the amount of viral RNA present. This supports intrinsic age-related differences in the epithelial response to IAV which may contribute to influenza susceptibility. Further studies are needed to understand temporal differences in this response and how modulation may affect disease severity to inform potential therapeutic targets. This abstract is funded by: 5R01HL168672-03
Setar et al. (Fri,) studied this question.