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A6211 - Targeting CEBPα/BMP4 Signaling to Restore Epithelial-Mesenchymal Homeostasis in Lung Fibrosis
Author Block: Q. Tan, X. Ma, G. Ligresti, A. Haak, J. Meridew, D. J. Tschumperlin; Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, United States.
RATIONALE: Pulmonary fibrosis is accompanied by epithelial cell dysfunction and aberrant epithelial-fibroblast interactions. Recently, single-cell RNA sequencing identified loss of normal epithelial cell identities and gain of abnormal indeterminate states of differentiation that may contribute to pathological processes in pulmonary fibrosis (Xu Y et al. JCI Insight 2017). The goal of this study was to use lung organoids to define mechanisms of how epithelial cell loss of one key factor, CEBPα, modulates fibroblast activation.
METHODS: We characterized interstitial fibroblasts or epithelial cells (Col1-GFP or EpCAM+) during the development and resolution of bleomycin-induced fibrosis and identified Cebpa and Bmp4 as two fibrosis-repressed candidate regulators of epithelial-mesenchymal crosstalk. To study epithelial-mesenchymal interactions, we co-cultured mouse tracheal and bronchial epithelial cells (mTE) with human fibroblasts (hLF) in 3D matrigel-supported organoids. Species specific primers were validated and used to measure cellular gene expression. We employed bleomycin to injure organoids, RNAi and small molecule inhibitors to test loss of function, and multiple assays including in situ extracellular matrix deposition and gene expression analysis to further characterize the roles of Cebpa and Bmp4 in lung epithelial regulation of fibroblast activation.
RESULTS: 3D co-culture of lung fibroblasts with epithelial cells dramatically repressed pro-fibrotic gene and protein expression compared with 3D culture of fibroblasts alone, demonstrating the potent anti-fibrotic role of epithelial cells in maintaining fibroblast homeostasis. Cebpa knockdown in mouse epithelial cells or BMP-4 signaling inhibition potentiated fibroblast activation in this 3D co-culture model. Injury of mouse epithelial cells by in vitro bleomycin treatment also led to fibroblast activation in vitro and in vivo, accompanied by decreased expression of Spc, Cebpa, and Bmp4. Cebpa knockdown also decreased expression of Bmp4 and Spc in the mouse epithelial cells, placing Cebpa upstream of BMP-4 signaling. Inhibition of the histone methyltransferase G9a, responsible for methylating H3K9, increased the expression of Cebpa and Bmp4 and suppressed fibroblast activation in co-culture, suggesting that epigenetic repression of Cebpa and other anti-fibrotic epithelial genes plays a key role in initiating and perpetuating fibroblast activation.
CONCLUSIONS: Normal epithelial cells restrain fibroblast activation and reduce ECM production in a 3D organoid model. Loss of Cebpa expression in epithelial cells and downstream BMP-4 signaling to fibroblasts reduces these anti-fibrotic effects. These data suggest that epithelial cells can act to maintain fibroblast quiescence, and that strategies to restore lung epithelial-mesenchymal interactions to a homeostatic state may attenuate fibroblast activation and pulmonary fibrosis.