Author:
Pacheco Esmeralda Vásquez,Marega Manuela,Lingampally Arun,Fassy Julien,Truchi Marin,Goth Kerstin,Trygub Lisa,Bartkuhn Marek,Alexopoulos Ioannis,Dong Ying,Lebrigand Kevin,Gunther Andreas,Chen Chengshui,Chao Cho-Ming,Alam Denise Al,Agha Elie El,Mari Bernard,Bellusci Saverio,Rivetti Stefano
Abstract
AbstractBackgroundMyofibroblasts (MYFs) are generally considered the principal culprits in excessive extracellular matrix deposition and scar formation in the pathogenesis of lung fibrosis. Lipofibroblasts (LIFs), on the other hand, are defined by their lipid-storing capacity and are predominantly found in the alveolar regions of the lung. They have been proposed to play a protective role in lung fibrosis. We previously reported that a LIF to MYF reversible differentiation switch occurred during fibrosis formation and resolution. In this study, we tested whether WI-38 cells, a human embryonic lung fibroblast cell line, could be used to study fibroblast differentiation towards the LIF or MYF phenotype and whether this could be relevant for idiopathic pulmonary fibrosis (IPF).Methodsusing WI-38 cells, MYF differentiation was triggered using TGF-β1 treatment and LIF differentiation using Metformin treatment. We analyzed the LIF to MYF and MYF to LIF differentiation by pre-treating the WI-38 cells with TGF-β1 or Metformin first, followed by treatment with Metformin and TGF-β1, respectively. We used IF, qPCR and bulk RNA-Seq to analyze the phenotypic and transcriptomic changes in the cells. We correlated our in vitro transcriptome data from WI-38 cells (obtained via bulk RNA sequencing) with the transcriptomic signature of LIFs and MYFs derived from the IPF cell atlas as well as with our own single-cell transcriptomic data from IFP patients-derived lung fibroblasts (LF-IPF) culturedin vitro. We also carried out alveolosphere assays to evaluate the ability of the proposed LIF and MYF cells to support the growth of alveolar epithelial type 2 cells.ResultsWI-38 and LF-IPF display similar phenotypical and gene expression responses to TGF-β1 and Metformin treatment. Bulk RNA-Seq analysis of WI-38 and LF-IPF treated with TGF-β1, or Metformin indicate similar transcriptomic changes. We also show the partial conservation of the LIF and MYF signature extracted from the Habermann et al. scRNA-seq dataset in WI-38 cells treated with Metformin or TGF-β1, respectively. Alveolosphere assays indicate that LIFs enhance organoid growth, while MYFs inhibit organoid growth. Finally, we provide evidence supporting the LIF to MYF reversible switch using WI-38 cells.ConclusionsWI-38 cells represent a versatile and reliable model to study the intricate dynamics of fibroblast differentiation towards the MYF or LIF phenotype associated with lung fibrosis formation and resolution, providing valuable insights to drive future research.Graphical abstractin vitroapproach using WI-38 cells as a versatile and reliable model to study the MYF or LIF phenotype associated with lung fibrosis formation and resolution observedin vivo. WI-38 are providing valuable insights to drive future research on lung fibrosis.
Publisher
Cold Spring Harbor Laboratory
Reference51 articles.
1. Mechanisms of Pulmonary Fibrosis
2. Todd NW , Luzina IG , Atamas SP . Molecular and cellular mechanisms of pulmonary fibrosis. Fibrogenesis Tissue Repair. 2012; 5: 1.
3. Habiel DM , Hogaboam CM . Heterogeneity of Fibroblasts and Myofibroblasts in Pulmonary Fibrosis. Vol. 5, Current Pathobiology Reports. Springer; 2017.
4. Rock JR , Barkauskas CE , Cronce MJ , et al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc Natl Acad Sci U S A. 2011; 108.
5. Desmoulibre A , Geinoz A , Gabbiani F , Gabbiani G . Transforming Growth Factor-ill Induces u-Smooth Muscle Actin Expression in Granulation Tissue Myofibroblasts and in Quiescent and Growing Cultured Fibroblasts.