FGF10-FGFR2B Signaling Generates Basal Cells and Drives Alveolar Epithelial Regeneration by Bronchial Epithelial Stem Cells after Lung Injury

Abstract

Idiopathic pulmonary fibrosis is a common form of interstitial lung disease resulting in alveolar remodeling and progressive loss of pulmonary function because of chronic alveolar injury and failure to regenerate the respiratory epithelium. Histologically, fibrotic lesions and honeycomb structures expressing atypical proximal airway epithelial markers replace alveolar structures, the latter normally lined by alveolar type 1 (AT1) and AT2 cells. Bronchial epithelial stem cells (BESCs) can give rise to AT2 and AT1 cells or honeycomb cysts following bleomycin-mediated lung injury. However, little is known about what controls this binary decision or whether this decision can be reversed. Here we report that inactivation of Fgfr2b in BESCs impairs their contribution to both alveolar epithelial regeneration and honeycomb cysts after bleomycin injury. By contrast overexpression of Fgf10 in BESCs enhances fibrosis resolution by favoring the more desirable outcome of alveolar epithelial regeneration over the development of pathologic honeycomb cysts.

Keywords: Fgf signaling; lung fibrosis; lung regeneration; lung stem cells.

Figure 1

Fgf10-Fgfr2b Signaling Is Required for AT2 Stem Cell Maintenance (A) β-Gal (blue) and oil red (LIF marker) staining on frozen sections of 3-month-old Fgf10^LacZ lungs shows that LIFs express Fgf10. (B) β-Gal (blue) and immunostaining for SFTPC (brown) on paraffin sections of 3-month-old Fgf10^LacZ lungs shows LIFs located adjacent to AT2 cells. (C and D) β-Gal and eosin staining on Fgf10^LacZ lungs 21 days after saline (C) and bleomycin treatment (D). Inset in (D) shows coimmunostaining for α-SMA (MYF marker) and β-gal (FGF10), demonstrating that LIFs transdifferentiate into MYFs after bleomycin injury. (E) Single-cell RNA-seq analysis on GFP⁺, EPCAM⁻, CD45⁻, TER119⁻ sorted fibroblasts isolated from Tbx4^CreERT2;mTmG mice show fewer LIFs expressing higher levels of Fgf10 in noninjured lungs compared with lungs 3 weeks after bleomycin injury. (F) Immunostaining for GFP and SFTPC (AT2 cell) on Sftpc^CreERT2;mTmG and Sftpc^CreERT2;Fgfr2b^f/f;mTmG lungs 4 weeks after being placed on tamoxifen chow. (G) Immunostaining for GFP (AT2 cell and descendant) on Sftpc^CreERT2;mTmG and Sftpc^CreERT2;Fgfr2b^f/f;mTmG lungs 6 weeks after bleomycin treatment. Scale bars, 10 μm (A), 100 μm (C), 50 μm, and (F) 500 μm (G).

Figure 2

Distal Bronchial Epithelial Cells Reprogram into Neo-BCs and AT1/2 Cells upon Bleomycin Injury in Response to Fgf10-Fgfr2b Signaling (A) Experimental strategy of (C, J, and K). (B) Western blotting for FGF10 on wild-type (wt) saline-treated lungs and wt lungs 3 and 6 weeks after bleomycin injury. (C) Immunostaining on ctrl, Sox2-Fgfr2b^f/f, and Acta2-Fgf10^f/f lungs for SCGB1A1 (green) and keratin 5 (K5) (red) at 3 weeks after bleomycin injury. (D) Immunostaining on Sox2-mTmG and Sox2-Fgfr2b^f/f-mTmG lungs for SFTPC (white), RAGE (red), and GFP (green) at 6 weeks after bleomycin injury. (E) Experimental strategy of (D and F–H). (F) Immunostaining on Scgb1a1ER-Confetti lung for K5 (red), SCGB1A1 (white), or K14 (red), p63 (white), and G/Y/CFP (green) at 6 weeks after bleomycin injury. White arrowheads show K14, p63, and G/Y/CFP triple-positive cells, indicating the dedifferentiation of distal airway club cells into neo-BCs. (G) Immunostaining on Scgb1a1ER-Fgfr2bf/f-Confetti lung for Scgb1a1ER-Confetti lung for K5 (red), SCGB1A1 (white), and G/Y/CFP (green), showing no basal cells near injury. (H) Immunostaining on Scgb1a1ER-Confetti and Scgb1a1ER-Fgfr2bf/f-Confetti lungs for SFTPC (red), or RAGE (red) and RFP (white) and G/Y/CFP (green) (RFP and G/Y/CFP label the four different confetti clone cells) at 6 weeks after bleomycin injury. (I) Flexivent pulmonary function analysis (static compliance) on ctrl and Scgb1a1ER-Fgfr2b^f/f lungs 6 weeks after bleomycin injury. (J) Relative mRNA levels of K5 and p63 in ctrl, Sox2-Fgfr2b^f/f, and Acta2-Fgf10^f/f lungs 3 and 6 weeks after bleomycin injury. (K) Quantification of BC numbers in ctrl, Sox2-Fgfr2b^f/f, and Acta2-Fgf10^f/f lungs at 3 weeks after bleomycin injury. (L) qPCR of relative mRNA levels of K5 and p63 in ctrl and Scgb1a1ER-Fgfr2b^f/f lungs 6 weeks after bleomycin injury. (M) Flexivent pulmonary function analysis (static compliance) on ctrl and Acta2^CreERT2-Fgfr10^f/f lungs 5 weeks after bleomycin injury. Nuclei were stained with DAPI (blue). ^∗∗p < 0.01, ^∗p < 0.05; n ≥ 14; error bars mean ± SEM. Scale bars, 100 μm (C and H), 200 μm (F, left and G), 20 μm (F, right), 250 μm (D).

Figure 3

Single-Cell RNA-Seq Analysis of Lineage-Traced Sox2^CreERT2;mTmG Cells 6 Weeks after Saline or Bleomycin Treatment t-SNE plots show neuroendocrine cells, ciliated cells, hillock cells, tuft cells, ionocytes (IO), distal and proximal club cells, goblet cells, UPK3A cells, AT2, AT1, and BCs. Heatmaps show differential gene expression between the different subpopulations and/or AT1, AT2, and neo-BCs specifically highlighting cell-type signatures among both injured and uninjured lungs.

Figure 4

Overexpression of Fgf10 in Bronchial Epithelial Cells after Bleomycin Injury Promotes AT2 Cell Differentiation (A and B) Coimmunostaining for RAGE (AT1 cell) and/or SFTPC (AT2 cell) on Sox2^CreERT2;LSL-rtTa;Tet-Fgf10 lungs that received intratracheal bleomycin at 2 months of age and were placed on doxycycline-containing chow 2 weeks after bleomycin injury for 3 weeks and were harvested either at 5 (A) or 6 (B) weeks after injury. (C) Hydroxyproline measurements for collagen content in ctrl and Sox2CreERT2; LSL-rtTa;Tet-Fgf10 mice 6 weeks after bleomycin injury (n ≥ 15). (D) Relative mRNA levels of K5, p63 in ctrl and Sox2^CreERT2;LSL-rtTa;Tet-Fgf10 mice 6 weeks after bleomycin injury (n ≥ 15). (E–J) Coimmunostaining for SFTPC (green) and p63 (red) (E and F) or α-SMA (green) and K5 (red) and (G and H) on honeycomb regions in lungs from IPF patients and mouse lungs 18 days after bleomycin injury. White arrowheads (E and F) show SFTPC and p63 double-positive cells. (I and J) Immunostaining on Sftpc^CreERT2;mTmG lungs for SCGB1A1 (red) or K5 (red) and GFP (green) at 3 weeks after bleomycin injury. Nuclei were stained with DAPI (blue). White arrowheads (H) show α-SMA (green) and K5 (red) double-positive cells. ^∗p < 0.05; n ≥ 14; error bars mean ± SEM. Scale bars, 500 μm (A and B), 100 μm (G and H), and 50 μm (E, F, I, and J).

Figure 5

Neo-BCs Require Fgfr2b Signaling for their Maintenance (A) Experimental strategy of (B). Induction with tamoxifen was performed at days 17, 19, and 21 after bleomycin injury. (B and C) Immunostaining on Krt5-mTmG lungs and Krt5-Fgfr2b^f/f-mTmG lungs to localize keratin 5 (K5) (red), SCGB1A1 (white), and GFP (green) (left) at 4 (B) or 8 weeks (C) after bleomycin injury. White arrowheads (B and C) show K5 (red) positive and SCGB1A1 (white) negative cells. Nuclei were stained with DAPI (blue). Scale bars, 100 μm (B and C).

Figure 6

BCs that Appear through Reprogramming of Scgb1a1⁺ Cells after Bleomycin Injury Require Fgfr2b Signaling for their Maintenance and to Differentiate into Alveolar Epithelial Cells (A) Experimental strategy of (B–F). Tamoxifen induction was performed on days 17, 19, and 21 after bleomycin injury. (B) Immunostaining on Krt5-mTmG lungs and Krt5-Fgfr2b^f/f-mTmG lungs for p63 (red), K5 (white), and GFP (green) or RAGE (red) and GFP (green) (middle) or SFTPC (red) and GFP (green) (right) at 4 and/or 8 weeks after bleomycin injury. White arrowheads show p63 (red), K5 (white), and GFP (green) triple-positive cells. (C) Quantification of the distribution of GFP⁺ cells in (B). (D) Relative mRNA levels of K5 and p63 at 3, 4, 6, 8, and 12 weeks after bleomycin injury showing time course of BC abundance. (E) Relative mRNA levels of K5, p63, and Gfp in control and Krt5-Fgfr2b^f/f lungs at 4 weeks after bleomycin injury. (F) Flexivent pulmonary function analysis measured static compliance on control and Krt5-Fgfr2b^f/f lungs at 4 weeks after bleomycin injury. Nuclei were stained with DAPI (blue). ^∗∗p < 0.01, ^∗p < 0.05; n ≥ 14; error bars mean ± SEM. Scale bars, 50 μm (B).

Figure 7

BCs in Precision Cut IPF Lung Slices Treated with Fgf10 Differentiate into AT2 Cells (A) Flow sorting for alveolar AT2 marker HTII-280 and CK5 on precision-cut lung slices cultured in the absence or presence of FGF10. Graph displayed as the log of the fold change shows an increase in CK5⁻ HTII-280⁺ and CK5⁺ HTII-280⁺ double-positive cells but a decrease in CK5⁺ HTII-280⁻ cells after FGF10 treatment. ^∗∗p < 0.01, ^∗p < 0.05; n = 5; error bars mean ± SEM. (B and C) Model showing how FGF10-FGFR2B maintains and generates basal-like stem cells by activating the stromal niche during homeostasis and epithelial regeneration. (B) During homeostasis, BCs are only found in the trachea, where they depend on FGF10 secreted by the intercartilage stromal tissue. (C) The dedifferentiation of distal airway club cells after major lung epithelial injury critically depends on FGF10-FGFR2B signaling. Note that, in addition to Fgf10-expressing ASMCs, activated myofibroblasts may further provide a strong additional source of FGF10 to allow for BC mobilization to injured regions. At injured sites, neo-BCs give rise to AT cells.