Fig 1.
FGF9 overexpression from club cells after 3 days of DOX administration does not alter lung histology.
(A) Schematic of the doxycycline chow (DOX)-inducible double-transgenic mouse breeding resulting in single-transgenic control mice (Scgb1a1-rtTA or TRE-Fgf9-IRES-eGfp) or double-transgenic FGF9-OE mice (Scgb1a1-rtTA and TRE-Fgf9-IRES-eGfp). (B-C) Representative images of control and FGF9-OE mouse lung sections after 3 days of DOX administration; (B) stained with DAPI (blue), anti-SCGB1A1 (red), and eGFP (green), scale bars = 50 μm, (C) stained with H&E, scale bars = 500 μm. (D) FGF9-OE and control mice were given DOX for 3 days, lungs were harvested, and single cell suspensions were analyzed for epithelial cells by flow cytometry as described in Materials and Methods. Data are represented as mean ± SEM and analyzed by unpaired student’s t test.
Fig 2.
FGF9-OE mice are more susceptible to respiratory virus infection than control mice.
(A-B) FGF9-OE and control mice were administered DOX beginning 3 days prior to infection (d-3), inoculated intranasally (i.n.) on d0 with 6x104 PFU WSN, and monitored for (A) weight loss (control n = 12, FGF9-OE n = 8) and (B) survival (control n = 31, FGF9-OE n = 28). (C-D) DOX-induced FGF9-OE and control mice were inoculated i.n. on d0 with 5 PFU PR8 (control n = 10, FGF9-OE n = 8) (C) or 1x104 PFU SeV-52 (control n = 23, FGF9-OE n = 7) (D) and were monitored for survival. (E) WT FVB/NJ mice were treated i.n. with PBS (n = 7) or 5 μg rFGF9 (n = 7) on d-3 and again concurrently with WSN infection on d0 and were monitored for survival. (F) Survival curve of WSN-infected FGF9-OE mice treated with DOX 1 day pre-infection (d-1, n = 9) or 1 dpi (d+1, n = 9) compared to historic d-3 DOX administration from panel B. For all experiments, weight loss and survival were monitored until 14 dpi. Data were pooled from 2 or more separate experiments. Data are represented as mean ± SEM and were analyzed by Mantel-Cox test (*, p < 0.05; **, p < 0.01; ****, p < 0.0001).
Fig 3.
FGF9-OE mice sustain higher viral titers and have increased lung inflammation.
(A-C) FGF9-OE and control mice were administered DOX beginning on d-3 and infected on d0 with 6x104 PFU WSN i.n. Lungs were harvested at 1, 3, and 6 dpi. At each time point, n = 6–13 control or FGF9-OE mice were analyzed. (A) Infectious virus was quantified by plaque assay; dotted line represents 50 PFU/ml (limit of detection). (B) H&E-stained slides at 1 dpi (control n = 3, FGF9-OE n = 5) and 6 dpi (control n = 1, FGF9-OE n = 3) were scored for peribronchiolitis, squamous epithelial metaplasia, airway epithelial denudation, and alveolitis as described in the Materials and Methods. (C) Representative images of H&E-stained control and FGF9-OE whole lung sections at 1 and 6 dpi; (bottom) corresponding magnified insets (scale bars = 200 μm). Data are represented as mean ± SEM and were analyzed within each time point by unpaired student’s t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
Fig 4.
Cytokine and chemokine expression is elevated in FGF9-OE lungs during IAV infection.
(A-C) FGF9-OE and control mice were administered DOX beginning on d-3 and infected on d0 with 6x104 PFU WSN i.n. (A) Lungs were harvested prior to infection on d0 (0 dpi) and at 1, 3, and 5 dpi (red arrows) and whole lung homogenates were analyzed by multiplex cytokine and chemokine analysis. (B) Data represented in a table as mean fold change of FGF9-OE samples over control samples for each analyte at 0, 1, 3, and 5 dpi. Data statistics were analyzed within each time point using unpaired student’s t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). No symbol indicates no significant difference in analyte expression between FGF9-OE and control samples; ND, not determined. (C) Fold change of 16 selected analytes significantly increased in FGF9-OE samples at 1 dpi compared to control samples, represented as mean ± SEM at 0 and 1 dpi. Data were analyzed across time points by unpaired student’s t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Dotted lines represent limit of detection. At each time point, n = 5–7 control or FGF9-OE mice were analyzed.
Fig 5.
Increased numbers of innate immune cells infiltrate into FGF9-OE lungs at 1 dpi.
(A-J) FGF9-OE and control mice were administered DOX beginning on d-3 and infected on d0 with 6x104 PFU WSN i.n. Lung single cell suspensions were generated prior to infection (0 dpi) and at 1 and 5 dpi, and the total number of live cells was determined for the indicated immune cell subpopulation as described in Materials and Methods. Gating strategy shown in S3 Fig. Data are represented as mean ± SEM and analyzed within each time point by unpaired student’s t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). At each time point, n = 7–8 control or FGF9-OE mice were analyzed.
Fig 6.
FGF9-OE airway epithelial cells display an elevated IFN response at 1 dpi.
(A-D) FGF9-OE and control mice were administered DOX beginning on d-3 and infected on d0 with 6x104 PFU WSN i.n. (A-C) RNA extracted from sorted airway epithelial cells (CD45− CD326+ CD24+) at 1 dpi was analyzed by bulk RNA sequencing as described in Materials and Methods. (A) Top 10 most significant positively-enriched pathways, normalized enrichment scores, and adjusted p-values from GSEA analysis of Gene Ontology Biological Processes terms comparing control and FGF9-OE airway epithelial cells at 1 dpi. (B) Selected representative genes and log fold change from GSEA analysis in FGF9-OE airway epithelial cells at 1 dpi. (C) RT-qPCR validation of Ifnb1, Isg15, and Rsad2 expression in sorted FGF9-OE or control airway epithelial cells at 0 and 1 dpi. Gene expression was normalized to Gapdh and graphed as fold change over control (0 dpi) using the 2−ΔΔCt method. Data are represented as mean ± SEM and analyzed using unpaired student’s t test (**, p < 0.01; ***, p < 0.001). (D) Representative images of lung sections from control or FGF9-OE mice at 0 and 1 dpi stained with anti-ISG15 polyclonal sera (yellow) and analyzed by immunofluorescence microscopy (blue = DAPI, scale bars = 100 μm).
Fig 7.
IAV tropism is shifted from the airway epithelium to the alveolar epithelium in FGF9-OE lungs at 1 dpi.
FGF9-OE and control mice were administered DOX beginning on d-3 and infected on d0 with 6x104 PFU WSN i.n. (A-C). (A) Representative images of multiplex fluorescent RNA-ISH depicting club cells (Scgb1a1, green), AT2 cells (Sftpc, white), and infected cells (Np, red) (blue = DAPI). Images taken with 5X objective (scale bars = 500 μm) and 20X objective (scale bars = 100μm). (B) The number of infected and uninfected airways was quantified from multiple FGF9-OE and control lung slides at 1 dpi, with the total percentage of infected airways per lung section (left) and total number of infected and uninfected cells (right). Infection was defined as any airway with >1 IAV+ cell. (C) The number of IAV+ cells in the alveolar space was quantified from blinded 5X images from FGF9-OE and control lung slides at 1 dpi. Data are represented as mean ± SEM and analyzed using unpaired student’s t test (**, p < 0.01).