S34 Influenza virus infection induces prolonged transcriptional and functional alterations in lung stromal cells

Introduction and Objectives Influenza A virus (IAV) causes respiratory infections that are a significant cause of morbidity and mortality worldwide. Stromal cells serve an important function in innate immune responses by co ordinating with immune cells to clear pathogens. The immediate responses to infection by fibroblasts and epithelial cells have been well characterised, however few studies have investigated longer term consequences. We hypothesise that molecules upregulated by lung stromal cells early in response to IAV infection persist in order to generate/maintain immune memory. Methods C57BL/6 mice were infected intranasally with IAV (WSN, 300PFU). Mice were sacrificed at day 0, 10 and 40 post infection. RNA-sequencing was employed to examine the transcriptional profiles of sorted lung fibroblasts and epithelial cells. Findings were independently validated using Nanostring and flow cytometry. Upstream regulators were identified using Ingenuity Pathway Analysis (IPA). Biological functions were extrapolated using PANTHER and confirmed by flow cytometry and immunofluorescence. The location of altered stromal and immune cells within the IAV infected lung was determined using RNA scope and immunohistochemistry. Results Differential expression of immune-related genes is prolonged in lung fibroblasts and epithelial cells following IAV infection. Many genes (H2-Ab1, Cxcl9, Nlrc5, Ifi47) are triggered by exposure to interferon-gamma (IFNγ) and remain significantly elevated at day 40 post infection. Stromal cells exhibited enrichment in biological processes (antigen processing/presentation and chemokine signalling) and molecular function (antigen binding). IPA identified conserved upstream regulators (IFNγ and CD40 ligand). RNA-scope detected elevated SpiB levels in airway epithelial cells of inflamed lungs at day 40. SpiB+ airway epithelial cells were adjacent to B220+ immune cell clusters in the IAV infected lung. Increased levels of stromal podoplanin were detected in the IAV infected lung, expression was localised in areas adjacent to immune cell clusters. Conclusions Taken together these data indicate that IAV infection induces transcriptional changes in stromal cells during peak T-cell response, these cells fail to return to their resting state for at least 40 days. The persistence of immune cell clusters within the lung suggests a dynamic relationship between immune cells and infection altered stromal cells providing mutual support for their persistence.