Evidence for a lipofibroblast-to-Cthrc1pos myofibroblast reversible switch during the development and resolution of lung fibrosis

ABSTRACT: Question addressed in the study: Genetic lineage tracing of alpha smooth muscle actin-positive (ACTA2+) cells in the context of bleomycin-induced pulmonary fibrosis in mice has shown that activated myofibroblasts (aMYFs) differentiate into lipofibroblasts (LIFs) during fibrosis resolution. However, the heterogeneity of the different mesenchymal cell subclusters during fibrosis formation and their contribution to the aMYFs as well as the fate of aMYFs during fibrosis resolution are still unclear. Materials and methods: Two-month-old Tg(Acta2-CreERT2); tdTomatoflox mice were used to label Acta2pos cells before (Tam-Bleo condition) or after (Bleo-Tam condition) bleomycin administration. Using scRNA-seq, the origin and fate of Acta2pos cells converging on the aMYF/Cthrc1pos myofibroblast lineage, were analyzed at the peak of fibrosis formation (day 14) as well as during resolution (day 60). Mining of human IPF scRNA-seq data was also carried out. The function of resident mesenchymal cells derived from saline or bleomycin-injured lungs during fibrosis formation or resolution in supporting AT2 progenitor cell renewal was assessed using alveolar organoid assays. Results: By employing the dimensionality reduction and UMAP visualization MYF/Cthrc1pos cells were identified and characterized in three scRNA-seq conditions: Saline, Tam-Bleo and Bleo-Tam. Acta2pos cells isolated from saline-treated mice revealed the presence of Acta2pos cells in the expected airway and vascular smooth muscle cells, but also in alveolar fibroblasts, peribronchial and adventitial fibroblasts as well as in a restricted number of Cthrc1pos cells indicating a primed pro-fibrotic status of the non-injured lung. In Tam-Bleo lungs, minimal contribution of these cells to the Cthrc1pos pool was observed. By contrast, labeling of Acta2pos cells following bleo administration (Bleo-Tam) led to massive labeling of Cthrc1pos cells, which following fine clustering were grouped into 4 subclusters named F1-F4. Interestingly, cells in cluster F1 were identified as Cthrc1low and displayed a strong LIF signature (LIFhigh) while cells in cluster F2 were LIFlow and Cthrc1high. Examination of F1 and F2 subclusters during fibrosis resolution suggested that F2 transition back to an F1 status and eventually differentiate into Cthrc1low LIFhigh alveolar fibroblasts. Analysis of the alveolar fibroblast cluster revealed the presence of three subclusters (A, B and C). Cluster C appeared only in Bleo-Tam and displayed a fibrotic signature (Sfrp1, Eln, Ltbp2, Spp1high). These cells, which originate from Acta2neg alveolar fibroblasts are suggested to differentiate into the Cthrc1low LIFhigh F1 subcluster. Data mining of human scRNA-seq from fibrotic lungs supported the conservation of the heterogeneity of the CTHRC1pos population. Alveolosphere assays indicated that resident mesenchymal cells did not reacquire their stem cell niche function at day 60, during fibrosis resolution. Answer to the question: Our results support a LIF-to-aMYF reversible switch during fibrosis formation and resolution.

ORGANISM(S): Mus musculus

PROVIDER: GSE223664 | GEO | 2024/12/31

REPOSITORIES: GEO