Macrophage Regulation of Mesenchymal Subpopulations During Healing

Abstract

Healing is a response to tissue injury. It is a complex and highly coordinated process involving multiple cell types. Among these, macrophages and mesenchymal cells play critical roles in orchestrating repair. In this dissertation, I specifically focus on two models of tissue repair: wound healing and fracture healing. Fibrosis commonly occurs during adult skin wound healing and is characterized by excessive extracellular matrix (ECM) deposition that leads to scarring. Fracture healing is a process that relies on mesenchymal cell–driven regeneration of bone. Although macrophages are known to influence both fibrotic responses in skin and regenerative processes in bone, the specific mesenchymal subpopulations they regulate—and the mechanisms by which this regulation occurs—remain poorly defined.To elucidate these mechanisms, we performed single-cell RNA sequencing (scRNA-seq) on mice with conditional macrophage depletion during early phase of healing to identify mesenchymal subpopulations responsive to macrophages. Functional relevance was tested through targeted ablation of specific mesenchymal populations. Rescue experiments was performed via cell reintroduction and administration of macrophage-derived factors. In wound healing, early-phase macrophage depletion led to reduced fibrosis and a significant decrease in a Fcer1g-expressing mesenchymal subpopulation. This cell population were highly proliferative and localized to the wound bed. Targeted ablation of Fcer1g-expressing mesenchymal cells reduced cutaneous scarring without impairing wound closure. Moreover, less-fibrotic wounds from EDA fibronectin-deficient mice contained significantly fewer Fcer1g-expressing mesenchymal cells, suggesting that Fcer1g-expressing mesenchymal cells drives fibrosis in this context. Our findings reveal a macrophage-induced adult mesenchymal subpopulation responsible for fibrosis during adult wound healing. In fracture healing, early-phase macrophage depletion resulted in reduced callus formation and a loss of a Col2a1-expressing mesenchymal subpopulation. Ablation of these cells delayed fracture repair, whereas their reintroduction restored bone regeneration. Cell–cell interaction analysis identified thrombospondin-1 (TSP1)–CD47 as the top ligand–receptor pair mediating communication between macrophages and mesenchymal cells. Targeted inhibition of this interaction using TAX2 impaired both chondrogenesis and osteogenesis. Consistently, CD47null mice exhibited a reduction in Col2a1-expressing cells at 7 days post-fracture. Furthermore, local delivery of TSP1 induced Col2a1-expressing mesenchymal cells and rescued the impaired healing phenotype in macrophage-depleted mice. These findings reveal a novel mechanism by which early-phase macrophages promote fracture healing through TSP1-mediated activation of Col2a1-expressing mesenchymal cells.