β-catenin dependent transcription mediates upregulation of <i>Fgfr2b</i> expression, which is inhibited in PR/8-infected, but not in non-infected lung epithelial cells.

<p>(A) EpCam⁺ lung epithelial cells derived from <i>Rosa26</i>^{<i>ERTCre/ERTCre</i>}<i>;Ctnnb1</i>^{<i>flox/flox</i>} mice were grown to confluency and treated with tamoxifen or DMSO control prior to infection with PR/8 (MOI = 0.1; 24h). mRNA expression of β-catenin (<i>Ctnnb1</i>) (left) or of <i>Fgfr2b</i> (right) was quantified and normalized to values of DMSO-treated control. (B) Wt distal lung epithelial cells in confluent culture were PR/8-infected (MOI 0.1) and treated with an activator (LiCl) or inhibitor (XAV939) of β-catenin signaling. Expression of the viral M segment was quantified at 16 h pi and normalized to LiCl-treated cultures (left). The right plot shows representative photomicrographs of these cultures stained for IV nucleoprotein (NP) after 6 h of PR/8 infection. (C) Wt mice were infected with PR/8 for 7d and infected (IV hemagglutinin⁺, HA⁺) vs. non-infected (HA-) EpCam⁺ cells were flow-sorted. Expression of the β-catenin-dependent transcripts <i>Axin2</i>, <i>Fgfr2b</i>, and <i>Ccnd1</i> was quantified in HA- cells and normalized to values from HA⁺ cells. All bar graphs represent means ± SD of n = 3–4 independent experiments; * <i>p</i><0.05; **<i>p</i><0.01; Tam, tamoxifen.</p

Therapeutic treatment with recombinant Fgf10 improves influenza virus-induced lung injury and improves re-epithelialization and barrier repair.

<p>Wt mice were infected with PR/8 and treated with a single dose of either 5μg recombinant Fgf10 (rFgf10) or diluent (PBS^-/-) at d6 pi. (A) The proliferative response of EpCam⁺ epithelial cell subsets was determined by flow cytometry at d7 pi. (B) Lung sections were stained with hematoxylin-eosin at d10 and d21 pi. Arrows depict non-epithelialized alveolar tissue; arrowheads depict areas of ongoing re-epithelialization. (C) Immunofluorescence staining of lung sections for E-cadherin (green), Ki67 (red), and Dapi (blue) at d21 pi. The top row shows lung tissue from mock-infected, untreated mice at d21. (D) Quantification of total lung epithelial cells (EpCam⁺) in lung homogenates at d14 pi. (E) Lung sections were stained for krt5 (green) and Dapi (blue) at d21 pi. (F) Lung barrier function was analysed by quantification of alveolar leakage of FITC-labeled albumin at d14 pi. Values are given in arbitrary units (AU) and represent ratios of FITC fluorescence in BALF and serum. (G) Survival of n = 8 mice per treatment group was analysed until d21 pi. Bar graphs represent means ± SD of n = 5–6 independent experiments; * <i>p</i><0.05; **<i>p</i><0.01. Photomicrographs are representative for n = 3–4 independent experiments; bars = 200 μm. (H) Summary: IV with high pathogenicity infect a substantial fraction of EpiSPC, resulting in inhibition of β-catenin-dependent Fgfr2b upregulation and impaired epithelial repair mediated by rMC-expressed Fgf10. Therapeutic application of excess Fgf10 antagonizes IV-induced regeneration failure by engagement of non-infected, Fgfr2b^high EpiSPC.</p

Influenza virus infected EpiSPC are impaired in their regenerative response due to restricted Fgfr2b expression.

<p>(A) Wt mice were infected with 500pfu of the indicated influenza virus strains, or mock-infected, and lung sections were stained with hematoxylin-eosin at d21 pi (arrows indicate areas of non-epithelialized tissue). (B) Infected and non-infected epithelial cell subsets of PR/8 infected wt mice were quantified by flow cytometry for their proliferative response. (C) Quantification of Fgfr2b expression in infected (NP⁺) and non-infected (NP^-) EpiSPC by flow cytometry at d4 pi. (D) Flow-sorted EpiSPC were <i>ex vivo</i> infected with the indicated MOI of PR/8 and seeded in matrix for 3D cultures. At d6 of culture, the number of formed organoids was quantified. (E) Infected (hemagglutinin⁺; HA⁺) or non-infected (hemagglutinin^neg; HA^-) EpiSPC or control HA^- SAEC were flow-sorted from the lungs of PR/8-infected tdtomato⁺ mice at d4 pi for intrapulmonary transplantation into 7d PR/8-infected wt mice. Lung sections were obtained at d7 and d14 after transplantation. Representative micrographs show overlays of brightfield and red staining of tdtomato⁺ transplanted cells. Overlay of tdtomato⁺ transplanted cells (red) and the type I AEC cell marker T1α (green) is shown in the right panels (arrows indicate co-expression of T1α and tdtomato); bars = 100μm. Bar graphs represent means ± SD of n = 3–4 independent experiments; * <i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; HA, hemagglutinin; Tx, transplantaion.</p

EpiSPC are resistant to apoptosis and show a high proliferative response after PR/8 infection which is mediated by Fgf10/Fgfr2b signaling.

<p>(A) Proliferation rates of the given epithelial cell subsets was analysed in PR/8 infected wt mice by FACS quantification of Ki67⁺ cells at the indicated time points pi. (B) Apoptosis of each EpCam⁺ subset was quantified by FACS (Annexin V⁺ proportions) at d7 post PR/8 infection and of non-infected wt mice. (C) Expression of Fgfr2b on EpiSPC at the given time points post PR/8 or mock infection was quantified by FACS and is given as MFI (median fluorescence intensity) of Fgfr2b ab minus MFI of matched isotype control. The proliferative response of the EpCam⁺ cell subsets was quantified by FACS at d7 pi in <i>Rosa26</i>^{<i>rtTA/+</i>}<i>;tet(O)sFgfr2b/+</i> (D) <i>Rosa26</i>^{<i>rtTA/+</i>}<i>;tet(O)Fgf10/+</i> mice (E) and <i>Fgf7</i>^<i>-/-</i> mice (F) compared to non-dox-induced or wt littermates. Bar graphs represent means ± SD of n = 4–6 independent experiments; * <i>p</i><0.05; **<i>p</i><0.01; +dox, doxycycline food; -dox, normal diet.</p