Multiple Fibroblast Subtypes Contribute to Matrix Deposition in Pulmonary Fibrosis.

Progressive pulmonary fibrosis results from a dysfunctional tissue repair response and is characterized by fibroblast proliferation, activation, invasion, and extracellular matrix accumulation. Lung fibroblast heterogeneity is well recognized. With single cell RNA-sequencing (scRNA-seq), fibroblast subtypes have been reported by recent studies. However, the roles of fibroblast subtypes in effector functions in lung fibrosis are not well understood. In this study, we incorporated the recently published scRNA-seq datasets on murine lung samples of fibrosis models and human lung samples of fibrotic diseases and analyzed fibroblast gene signatures. We identified and confirmed the novel fibroblast subtypes we reported recently across all samples of both mouse models and human lung fibrotic diseases including idiopathic pulmonary fibrosis, systemic sclerosis associated interstitial lung disease, and COVID-19. Furthermore, we identified specific cell surface proteins for each fibroblast subtype through differential gene expression analysis which enabled us to isolate primary cells representing distinct fibroblast subtypes by flow cytometry sorting. We compared matrix production including fibronectin, collagen, and hyaluronan after pro-fibrotic factor stimulation and assessed invasive capacity of each fibroblast subtype. Our results suggested that in addition to myofibroblasts, lipofibroblasts and Ebf1+ fibroblasts are two important fibroblast subtypes that contribute to matrix deposition and also have enhanced invasive, proliferative, and contraction phenotypes. The histological locations of fibroblast subtypes are identified in healthy and fibrotic lungs by these cell surface proteins. This study provides new insights to inform approaches to targeting lung fibroblast subtypes to promote the development of therapeutics for lung fibrosis.