Reciprocal regulation of fibroblast-macrophage equilibrium governs skin integrity

Fibroblast-macrophage crosstalk is well-established in vitro and fibroblast-derived colony-stimulating factor 1 (CSF1) supports macrophages in select tissues. However, whether macrophages regulate fibroblasts in vivo remains unknown. Leveraging genetic mouse models, single-cell transcriptomics, CITE-seq, flow cytometry, and imaging, we show that fibroblast depletion or loss of fibroblast-derived growth factors impacts skin macrophage populations in the dermis and hypodermis. Conditional deletion of Csf1 in Dpt+ fibroblasts progressively decreases CD64+ and CD11c+ macrophages, impairing skin wound healing. Reduced macrophage abundance disrupts fibroblast cell cycle regulation, metabolism, immune signaling, and increases fibroblast abundance, affirming a reciprocal relationship. In human systemic sclerosis (scleroderma), elevated fibroblast-derived CSF1 and increased macrophage abundance correlate with disease severity, implicating CSF1-CSF1R axis in pathology. These findings provide in vivo evidence of macrophage regulation of fibroblasts, revealing a bidirectional interplay that advances understanding of tissue homeostasis and immune regulation in skin.Fibroblasts and macrophages were isolated by FACS from skin tissue of 5x control (iDPT;R26.YFP/DTR treated with PBS) and 5x DT (iDPT;R26.YFP/DTR treated with Diphtheria toxin) treated mice for single-cell profiling (total n=10). Samples were indexed using TotalSeq-B hashtag antibodies (B0101-B0110) and the TotalSeq-A antibody cocktail for CITE-Seq. For CSF1 deletion steady-state and wound healing studies, total skin tissue cells were isolated from 5x wild type (iDPT;R26.YFP.CSF1 wt/wt) and 5x floxed mice (iDPT;R26.YFP.CSF1 fl/fl) for single-cell profiling (total n=10) without cell enrichment. Timepoints included day 4 (control and wounded skin) and day 7 post wounding. Samples were indexed using TotalSeq-B hashtag antibodies (B0101-B0110) and the TotalSeq-A antibody cocktail for CITE-Seq.