Lung structural cell dynamics are altered by influenza virus infection experience leading to rapid immune protection following viral re-challenge

Lung structural cells, including epithelial cells and fibroblasts, form barriers against pathogens and trigger immune responses following infections such as influenza A virus. This response leads to the recruitment of innate and adaptive immune cells required for viral clearance. Some of these recruited cells remain within the lung following infection and contribute to enhanced viral control following subsequent infections. There is growing evidence that structural cells can also display long-term changes following infection or insults. Here we investigate long-term changes to mouse lung epithelial cells, fibroblasts, and endothelial cells following influenza virus infection and find that all three cell types maintain an imprint of the infection, particularly in genes associated with communication with T cells. Lung epithelial cells from IAV-infected mice display functional changes by more rapidly controlling influenza virus than cells from naïve animals. This rapid anti-viral response and increased expression of molecules required to communicate with T cells demonstrates sustained and enhanced functions following infection. These data suggest lung structural cells could be effective targets for vaccines to boost durable protective immunity. Overall design: Lungs were taken from C57BL/6 mice that were either naïve (control) or had been infected with influenza A virus 10 or 40 days previously. Lungs were digested with Collagenase P, Dispase and DNase and CD45+ cells removed using magnetic beads. Cells were stained with antibodies to Epithelial cells (EpCAM1), Fibroblasts (CD140a) or blood endothelial cells (CD31) and live cells of each types sorted ex vivo by FACS. For each sample, cells were combined from 6-8 mice. Cells were pelleted, resuspended in a small volume and centrifuged through a QiaShredder. RNA was prepared using a mini or micro RNAeasy kit from Qiagen. RNA was purified from total RNA (100ng) using poly-T oligo-attached magnetic beads (Life Technologies, CA, USA). Sequencing libraries were generated using NEB Next Ultra Directional RNA Library Prep Kit for Illumina (NEB) in accordance with the manufacturer's recommendations. Products were purified and quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system. After the clustering of the index-coded samples, each library preparation was sequenced on an Illumina NextSeq™ 500 platform with 2x75bp paired end sequencing. An average sequencing depth of 20M reads per sample was achieved.