1458 Dissection of the molecular and cellular heterogeneity of dermal fibroblasts in skin fibrosis

Skin fibrosis, a common scarring condition that currently has no curative treatments, is caused by persistent activation of fibroblasts after tissue injury or inflammation that results in excessive connective tissue deposition, leading to severe disability, organ malfunction and increased morbidity. This study aims to uncover the early and advanced changes in different fibroblast subsets and molecular pathways that are integral to pathological fibroblast activation using a bleomycin-induced skin fibrosis mouse model that recapitulates well the human disease. We have shown that 2 weeks (early) and 4 weeks (advanced) of subcutaneous bleomycin injections result in an increasing severity of skin fibrosis, characterized by increased dermal thickness, collagen deposition, inflammation and loss of dermal fibroblasts and adipocytes. We performed single cell RNA-sequencing and successfully isolated 14944 control, 16344 early and 6860 advanced fibrotic cells. We identified the changes in fibroblast subclusters and gene signatures that are altered across the time course. Using CellChat, we are dissecting the specific signaling crosstalk between different fibroblast subpopulations and other skin cells, promoting a profibrotic phenotype and locking fibroblasts in an activated state. To understand the impact that changes in extracellular matrix biomechanics elicit on profibrotic fibroblast fate, we are correlating our histological and transcriptomic analysis with nanoindentation atomic force microscopy imaging throughout the fibrotic time course. This novel multidisciplinary approach allows us to compare the cellular and molecular changes in early and advanced fibrotic lesions, identifying the key fibroblast subpopulations, molecular regulators and connective tissue alterations promoting fibrotic tissue repair. Our findings will support the development of new fibroblast-targeted fibrosis therapies in skin and potentially other organs.