Monocyte/Macrophage Heterogeneity during Skin Wound Healing in Mice

Monocytes (Mos)/macrophages (M phi s) orchestrate biological processes critical for efficient skin wound healing. However, current understanding of skin wound Mo/M phi heterogeneity is limited by traditional experimental approaches such as flow cytometry and immunohistochemistry. Therefore, we sought to more fully explore Mo/M phi heterogeneity and associated state transitions during the course of excisional skin wound healing in mice using single-cell RNA sequencing. The live CD45(+)CD11b(+)Ly6G(-) cells were isolated from skin wounds of C57BL/6 mice on days 3, 6, and 10 postinjury and captured using the 10x Genomics Chromium platform. A total of 2813 high-quality cells were embedded into a uniform manifold approximation and projection space, and eight clusters of distinctive cell populations were identified. Cluster dissimilarity and differentially expressed gene analysis categorized those clusters into three groups: early-stage/proinflammatory, late-stage/prohealing, and Ag-presenting phenotypes. Signature gene and Gene Ontology analysis of each cluster provided clues about the different functions of the Mo/M phi subsets, including inflammation, chemotaxis, biosynthesis, angiogenesis, proliferation, and cell death. Quantitative PCR assays validated characteristics of early- versus late-stage Mos/M phi s inferred from our single-cell RNA sequencing dataset. Additionally, cell trajectory analysis by pseudotime and RNA velocity and adoptive transfer experiments indicated state transitions between early- and late-state Mos/M phi s as healing progressed. Finally, we show that the chemokine Ccl7, which was a signature gene for early-stage Mos/ M phi s, preferentially induced the accumulation of proinflammatory Ly6C(+)F4/80(lo/-) Mos/M phi s in mouse skin wounds. In summary, our data demonstrate the complexity of Mo/M phi phenotypes, their dynamic behavior, and diverse functions during normal skin wound healing.