A fibroblast-dependent TGF-β1/sFRP2 noncanonical Wnt signaling axis promotes epithelial metaplasia in idiopathic pulmonary fibrosis
Max L. Cohen, … , Harold A. Chapman, Claude Jourdan Le Saux
Max L. Cohen, … , Harold A. Chapman, Claude Jourdan Le Saux
Published July 9, 2024
Citation Information: J Clin Invest. 2024;134(18):e174598. https://doi.org/10.1172/JCI174598.
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Research Article Pulmonology

A fibroblast-dependent TGF-β1/sFRP2 noncanonical Wnt signaling axis promotes epithelial metaplasia in idiopathic pulmonary fibrosis

Abstract

Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate, we administered the fibroblast-selective TGF-β1 signaling inhibitor epigallocatechin gallate (EGCG) to interstitial lung disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA-Seq on spare tissue. Biopsies from untreated patients showed higher fibroblast TGF-β1 signaling compared with nondisease donor or end-stage ILD tissues. In vivo, EGCG downregulated TGF-β1 signaling and several proinflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted frizzled-related protein 2 (sFRP2), an unrecognized TGF-β1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s) in situ. Using AEC2-fibroblast coculture organoids and precision-cut lung slices (PCLSs) from nondiseased donors, we found TGF-β1 signaling promotes a spread AEC2 KRT17+ basaloid state, whereupon sFRP2 then activates a mature cytokeratin 5+ (Krt5+) basal cell program. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin signaling were required for sFRP2-induced nuclear NFATc3 accumulation and KRT5 expression. These findings highlight stage-specific TGF-β1 signaling in ILD and the therapeutic potential of EGCG in reducing idiopathic pulmonary fibrosis–related (IPF-related) transcriptional changes and identify TGF-β1/noncanonical Wnt pathway crosstalk via sFRP2 as a mechanism for dysfunctional epithelial signaling in IPF/ILD.

Authors

Max L. Cohen, Alexis N. Brumwell, Tsung Che Ho, Kiana Garakani, Genevieve Montas, Darren Leong, Vivianne W. Ding, Jeffrey A. Golden, Binh N. Trinh, David M. Jablons, Michael A. Matthay, Kirk D. Jones, Paul J. Wolters, Ying Wei, Harold A. Chapman, Claude Jourdan Le Saux

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Figure 4

Impact of EGCG on distribution of sFRP2 expression in fibroblast subpopulations, biopsy tissues, and PCLS cultures.

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Impact of EGCG on distribution of sFRP2 expression in fibroblast subpopu...
(A) Ridge plot of sfrp2 and col1a1 gene expression in alveolar and pathologic fibroblast subtypes from untreated and EGCG biopsy samples. (B) Relative expression of sfrp2 mRNA in human fibroblasts treated with TGF-β1 (1 ng/ml) and/or TGF-β inhibitor SB4331542 (5 μM) for 48 hours (n = 5). (C) Feature plots of sfrp2 gene expression (green) in various fibroblast subsets characterized by cthrc1 or ccl2 gene expression (red). Cells expressing both sfrp2 and cthrc1 or ccl2 are indicated in yellow. Additional fibroblast markers are shown in Supplemental Figure 4B. (D and E) RNA in situ hybridization was performed for col1a1 (yellow) and sfrp2 (red) genes in untreated and EGCG biopsies (D). The signal intensity of sfrp2 was quantified for each image (E). Representative images of n = 5 samples per group, 4–6 images per sample. Original magnification, ×100 (top images). Bottom images represent a region of interest as indicated by white rectangle. (F and G) PCLSs from IPF lung donors cultured for 7 days with EGCG (1 μM) and analyzed by Western blot. Additional samples are shown in Supplemental Figure 5C. (G) Graphical representation of the level of expression of selected proteins for all samples (see Supplemental Figure 5C). n = 6. Statistical significance was determined by the Kruskal-Wallis test (B) and 2-tailed t test (E and G).