Transcription factor FOXF1 identifies compartmentally distinct mesenchymal cells with a role in lung allograft fibrogenesis
Russell R. Braeuer, Natalie M. Walker, Keizo Misumi, Serina Mazzoni-Putman, Yoshiro Aoki, Ruohan Liao, Ragini Vittal, Gabriel G. Kleer, David S. Wheeler, Jonathan Z. Sexton, Carol F. Farver, Joshua D. Welch, Vibha N. Lama
Russell R. Braeuer, Natalie M. Walker, Keizo Misumi, Serina Mazzoni-Putman, Yoshiro Aoki, Ruohan Liao, Ragini Vittal, Gabriel G. Kleer, David S. Wheeler, Jonathan Z. Sexton, Carol F. Farver, Joshua D. Welch, Vibha N. Lama
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Concise Communication Cell biology Pulmonology

Transcription factor FOXF1 identifies compartmentally distinct mesenchymal cells with a role in lung allograft fibrogenesis

Abstract

In this study, we demonstrate that forkhead box F1 (FOXF1), a mesenchymal transcriptional factor essential for lung development, was retained in a topographically distinct mesenchymal stromal cell population along the bronchovascular space in an adult lung and identify this distinct subset of collagen-expressing cells as key players in lung allograft remodeling and fibrosis. Using Foxf1-tdTomato BAC (Foxf1-tdTomato) and Foxf1-tdTomato Col1a1-GFP mice, we show that Lin–Foxf1+ cells encompassed the stem cell antigen 1+CD34+ (Sca1+CD34+) subset of collagen 1–expressing mesenchymal cells (MCs) with a capacity to generate CFU and lung epithelial organoids. Histologically, FOXF1-expressing MCs formed a 3D network along the conducting airways; FOXF1 was noted to be conspicuously absent in MCs in the alveolar compartment. Bulk and single-cell RNA-Seq confirmed distinct transcriptional signatures of Foxf1+ and Foxf1– MCs, with Foxf1-expressing cells delineated by their high expression of the transcription factor glioma-associated oncogene 1 (Gli1) and low expression of integrin α8 (Itga), versus other collagen-expressing MCs. FOXF1+Gli1+ MCs showed proximity to Sonic hedgehog–expressing (Shh-expressing) bronchial epithelium, and mesenchymal expression of Foxf1 and Gli1 was found to be dependent on paracrine Shh signaling in epithelial organoids. Using a murine lung transplant model, we show dysregulation of epithelial-mesenchymal SHH/GLI1/FOXF1 crosstalk and expansion of this specific peribronchial MC population in chronically rejecting fibrotic lung allografts.

Authors

Russell R. Braeuer, Natalie M. Walker, Keizo Misumi, Serina Mazzoni-Putman, Yoshiro Aoki, Ruohan Liao, Ragini Vittal, Gabriel G. Kleer, David S. Wheeler, Jonathan Z. Sexton, Carol F. Farver, Joshua D. Welch, Vibha N. Lama

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Figure 4

FOXF1 and Gli1 expression and expansion of Gli1+ MCs in an orthotopic mouse lung transplant model.

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FOXF1 and Gli1 expression and expansion of Gli1+ MCs in an orthotopic mo...
(A) Generation of 3D epithelial organoids in coculture of Foxf1-tdTomato+Col1GFP+ MCs and BASCs from Rosa26mTmG mice. Scale bars: 500 μm. (B) Flow cytometry–sorted cell populations from organoids were analyzed by real-time PCR. (C) Foxf1+_MCs were cocultured with BASCs, with or without the Shh signaling inhibitor LDE225 (0.75 μM), and compared with Foxf1_MCs cultured in Matrigel alone. (D) Mesenchymal (Lin–PDGFRα+) and epithelial (Lin–EpCAM+) cells sorted from isografts and allografts in the orthotopic mouse lung transplant model were analyzed for the indicated genes by real-time PCR. D28, day 28. (E) Schema for the generation of Gli1CreERT2/WT Rosa26mTmG donor mice and lung transplant experiments. Tam, tamoxifen. (F) Representative immunofluorescence images of lineage-traced Gli1GFP+ MCs in 2 control and 3 transplanted lungs. Scale bars: 100 μm (zoom, cropped: 50 μm). Data indicate the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed unpaired t test (B and D) and 1-way ANOVA with Bonferroni’s test (C).