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RNA sequencing of sorted conducting airway epithelial cells from FGF9-overexpressing and control mouse lungs at 1 day post-infection with IAV


Hiller, Bradley (2022), RNA sequencing of sorted conducting airway epithelial cells from FGF9-overexpressing and control mouse lungs at 1 day post-infection with IAV, Dryad, Dataset,


Influenza A virus (IAV) preferentially infects conducting airway and alveolar epithelial cells in the lung. The outcome of these infections is impacted by the host response, including the production of various cytokines, chemokines, and growth factors. Fibroblast growth factor-9 (FGF9) is required for lung development, can display antiviral activity in vitro, and is upregulated in asymptomatic patients during early IAV infection. We therefore hypothesized that FGF9 would protect the lungs from respiratory virus infection and evaluated IAV pathogenesis in mice that overexpress FGF9 in club cells in the conducting airway epithelium (FGF9-OE mice). However, we found that FGF9-OE mice were highly susceptible to IAV and Sendai virus infection compared to control mice. FGF9-OE mice displayed elevated and persistent viral loads, increased expression of cytokines and chemokines, and increased numbers of infiltrating immune cells as early as 1 day post-infection (dpi). Gene expression analysis showed an elevated type I interferon (IFN) signature in the conducting airway epithelium and analysis of IAV tropism uncovered a dramatic shift in infection from the conducting airway epithelium to the alveolar epithelium in FGF9-OE lungs. These results demonstrate that FGF9 signaling primes the conducting airway epithelium to rapidly induce a localized, protective IFN and proinflammatory cytokine response during viral infection. Although this response protects the airway epithelial cells from IAV infection, it allows for early and enhanced infection of the alveolar epithelium, ultimately leading to increased morbidity and mortality. Our study illuminates a novel role for FGF9 in regulating respiratory virus infection and pathogenesis.


Mice were sacrificed and perfused with PBS by intracardiac injection. Lungs were harvested and single cell suspensions were generated by incubation for 1 h at 37°C in 5 ml digestion buffer with manual shaking every 10-15 min. Whole lungs were combined from 2 mice for each single sample. Digestion buffer for epithelial cell analysis consisted of RPMI (Sigma), DNase I (10 mg/ml, Sigma), 15 mM HEPES buffer (Corning), and 10% fetal bovine serum (FBS) (BioWest). Digested tissues were passed through a 70-µm cell strainer and washed once with 40 ml PBS containing 5% FBS and treated with red blood cell lysing buffer (Sigma). The number of viable cells was quantified by trypan blue staining. Single cell suspensions were incubated with anti-mouse CD16/CD32 (Clone 93; BioLegend) for 15 min at 4°C and then surface stained in PBS containing 5% FBS for 1 h at 4°C. All antibodies were diluted 1:200 and are from BioLegend: anti-CD45 Alex Fluor 700 (30-F11), anti-CD326 (EpCAM) PE (phycoerythrin) (G8.8), anti-CD24 APC (allophycocyanin) (M1/69).

After staining, cells were washed in PBS containing 5% FBS and CD45− EpCAM+ CD24+ cells were sorted using a Sony Cell Sorter SH800S and Cell Sorter Software V2.1.5 (Sony Biotechnology, Sony Biotechnology). RNA was extracted using TRIzol Reagent and an RNeasy Mini Kit (Qiagen). The TRIzol Reagent manufacturer’s protocol was followed up until removal of the RNA-containing aqueous phase, to which an equal volume of 100% RNase-free ethanol was added. This solution was added to the RNeasy column, after which the RNeasy Mini Kit instructions were followed. 3 FGF9-OE and 2 control RNA samples were submitted to the Genome Technology Access Center at the McDonnell Genome Institute at Washington University School of Medicine for the following analyses. Total RNA integrity was determined using Agilent Bioanalyzer. Library preparation was performed with 10 ng of total RNA with a Bioanalyzer RIN score greater than 8.0. ds-cDNA and was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per manufacturer's protocol. cDNA was fragmented, blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases. High quality gene reads were mapped to the Mus musculus genome.