DOI: 10.5061/dryad.j3tx95xgv
title: RNA sequencing of sorted conducting airway epithelial cells from FGF9-overexpressing and control mouse lungs at 1 day post-infection with IAV

description: 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.
description: 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.