Functional profiling and visualization of stromal cell dynamics in the influenza virus infected lung

Introduction: Influenza A virus (IAV) respiratory infections are a major cause of morbidity and mortality. Stromal cells co-ordinate with immune cells to clear IAV. While the immediate responses of stromal cells to IAV are well characterised, few studies have investigated the long-term consequences of infection. Methods: To address this, we performed RNA-seq on FACS sorted lung epithelial cells and fibroblasts from naive animals and at early (day 10) and late time points (day 40) following intranasal IAV infection. Biological functions were investigated by flow cytometry. The location of altered stromal and immune cells in the lung was determined using RNAscope and immunohistochemistry. Results: Analysis of differentially expressed genes (DEGs) demonstrated an enrichment in cell cycle and extracellular matrix genes at day 10. More strikingly, immune related genes were enriched in the DEGs at day 10 and 40; many, including MHCII and CXCL9/10, are regulated by the inflammatory cytokine interferon-γ. SpiB, a transcription factor that regulates genes involved in antigen processing/presentation, was found in epithelial cells in infected mice using RNAscope, but only in airways in close proximity to B220+ immune clusters. Furthermore, immunohistochemistry demonstrated that expression of the immunomodulatory molecule, podoplanin, was limited to cluster‑adjacent fibroblasts. Conclusions: Our functional and geographical analysis of the post-IAV lung indicate a prolonged dynamic relationship between immune cells and infection altered stromal cells. These data have important implications for understanding the altered dynamic communications between lung immune and stromal cells during and following subsequent lung infections.