Research Article

Myofibroblast proliferation and heterogeneity are supported by macrophages during skin repair

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Science  23 Nov 2018:
Vol. 362, Issue 6417, eaar2971
DOI: 10.1126/science.aar2971

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Myofibroblast diversity with injury and aging

Fibroblasts deposit extracellular matrix (ECM) molecules to regulate tissue strength and function. However, if too much ECM is deposited, fibrosis and scarring results. Shook et al. examined cells during mouse skin wound healing, fibrosis, and aging (see the Perspective by Willenborg and Eming). They identified distinct subpopulations of myofibroblasts, including cells identified as adipocyte precursors (APs). In cellular ablation mouse models, CD301b-expressing macrophages selectively activated proliferation of APs, but not other myofibroblasts. Myofibroblast composition and gene expression changed during aging. Thus, macrophage-fibroblast interactions are important during tissue repair and aging, which may have therapeutic implications for chronic wounds and fibrotic disease.

Science, this issue p. eaar2971; see also p. 891

Structured Abstract

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 (CD29High). 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 CD29High 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.

Regulation of myofibroblast diversity in skin wounds.

After injury, multiple subsets of fibroblasts become activated myofibroblasts that contribute to tissue repair and scar formation. Wound bed macrophages expressing CD301b selectively activate proliferation in APs and not other myofibroblasts. With age, impaired healing is associated with a reduction in CD301b+ macrophages (Mϕ) and APs. These findings identify distinct cellular and molecular interactions that support myofibroblast heterogeneity.

Abstract

During tissue repair, myofibroblasts produce extracellular matrix (ECM) molecules for tissue resilience and strength. Altered ECM deposition can lead to tissue dysfunction and disease. Identification of distinct myofibroblast subsets is necessary to develop treatments for these disorders. We analyzed profibrotic cells during mouse skin wound healing, fibrosis, and aging and identified distinct subpopulations of myofibroblasts, including adipocyte precursors (APs). Multiple mouse models and transplantation assays demonstrate that proliferation of APs but not other myofibroblasts is activated by CD301b-expressing macrophages through insulin-like growth factor 1 and platelet-derived growth factor C. With age, wound bed APs and differential gene expression between myofibroblast subsets are reduced. Our findings identify multiple fibrotic cell populations and suggest that the environment dictates functional myofibroblast heterogeneity, which is driven by fibroblast-immune interactions after wounding.

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