Unraveling Alveolar Fibroblast and Activated Myofibroblast Heterogeneity and Differentiation Trajectories During Lung Fibrosis Development and Resolution in Young and Old Mice.

Idiopathic pulmonary fibrosis (IPF) is an age-associated disease characterized by the irreversible accumulation of excessive extracellular matrix components by activated myofibroblasts (aMYFs). Following bleomycin administration in young mice, fibrosis formation associated with efficient resolution takes place limiting the clinical relevance of this model for IPF. In this study, we used aged mice in combination with bleomycin administration to trigger enhanced fibrosis formation and delayed resolution as a more relevant model for IPF. Alveolosphere assays were carried out to compare the alveolar resident mesenchymal niche activity for AT2 stem cells in young versus old mice. Lineage tracing of the Acta2+ aMYFs in old mice exposed to bleomycin followed by scRNAseq of the lineage-traced cells isolated during fibrosis formation and resolution was performed to delineate the heterogeneity of aMYFs during fibrosis formation and their fate during resolution. Integration of previously published similar scRNAseq results using young mice was carried out. Our results show that alveolar resident mesenchymal cells from old mice display decreased supporting activity for AT2 stem cells. Our findings suggest that the cellular and molecular mechanisms underlying the aMYFs formation and differentiation towards the Lipofibroblast phenotype are mostly conserved between young and old mice. In addition to persistent fibrotic signaling in aMYF from old mice during resolution, we also identified differences linked to interleukin signaling in old versus young alveolar fibroblast populations before and during bleomycin injury. Importantly, our work confirms the relevance of a subcluster of aMYFs in old mice that is potentially relevant for future management of IPF.