Myofibroblast proliferation and heterogeneity are supported by macrophages during skin repair

INTRODUCTION Fibroblasts produce extracellular matrix (ECM) molecules that regulate tissue strength and resilience. Imbalanced ECM maintenance leads to tissue dysfunction. Although multiple populations of fibroblasts support uninjured skin function, the extent of fibroblast diversity and the presence of functionally distinct subsets in adult fibrotic skin are poorly defined. The inability to find a single molecular marker that identifies all activated “myofibroblasts” during wound healing suggests the existence of multiple subsets of ECM-producing myofibroblasts. Furthermore, little is known about the cellular and molecular mechanisms that promote the expansion of individual myofibroblast subsets and support cellular heterogeneity. Although macrophages influence myofibroblast numbers and ECM deposition after injury, subpopulations of wound bed macrophages have only recently been defined, and specific interactions with fibroblasts have not been explored. RATIONALE Variation in healing and scarring rates in multiple tissues suggests that fibroblast diversity exists. To develop therapies targeting fibrotic responses, functionally distinct subsets of myofibroblasts and the mechanisms that support individual populations must be uncovered. We used a comprehensive, hierarchical fluorescence-activated cell sorting strategy to define myofibroblast subsets in skin wound beds from adult mice. This strategy revealed distinct subsets of wound bed myofibroblasts, including an abundant population that contains the cell surface marker profile of adipocyte precursor cells (APs). We examined myofibroblast subsets in different cutaneous fibrotic contexts and explored mechanisms that selectively promote the expansion of APs. RESULTS Genetic lineage tracing and flow cytometry revealed distinct subsets of wound bed myofibroblasts that express smooth muscle actin and collagen. The most abundant populations were CD26-expressing APs and a subset with high cell surface levels of CD29 (CD29 High ). Although transcriptomic analysis revealed that each myofibroblast subset has a distinct gene expression profile, functional analyses suggest that myofibroblast subsets make both overlapping and distinct contributions to repair. APs were significantly reduced and CD29 High cells were more abundant in wound beds from aged mice and skin from mice that underwent bleomycin-induced fibrosis, suggesting that the fibrotic environment influences myofibroblast composition. Injury and repair-related changes in AP transcription implicated macrophage signaling in the modulation of AP gene expression. Genetic ablation and cell transplantations of different myeloid cells revealed that macrophages expressing macrophage galactose N -acetylgalactosamine–specific lectin 2 ( Mgl2 /CD301b) directly stimulate proliferation in a subset of APs and not in other myofibroblast subsets. By combining in vitro cytokine stimulation with in vivo signaling pathway inhibition, we identified multiple CD301b + macrophage–secreted factors (platelet-derived growth factor C and insulin-like growth factor 1) that selectively stimulate AP proliferation, thus supporting the heterogeneity of wound bed myofibroblasts. CONCLUSION We identified multiple populations of skin myofibroblasts and observed that the composition of myofibroblasts is dependent upon the fibrotic environment. Distinct interactions allow CD301b + macrophage–derived signaling to selectively activate the proliferation of APs and not other myofibroblasts. These results have potential for the development of therapies that target multiple cellular populations or signaling pathways under conditions associated with excessive or deficient ECM deposition, such as wound healing and fibrosis.