Monocyte/macrophage heterogeneity during skin wound healing in mice

Abstract

Abstract Cells of the monocyte/macrophage (Mo/Ma) lineage orchestrate diverse biological processes required for efficient skin wound healing. Current understanding of Mo/Ma heterogeneity during wound healing has been established by traditional experimental approaches such as flow cytometry, qPCR, and IHC, which indicates a phenotype switch from pro-inflammatory to pro-healing cells as wound healing progresses. However, these phenotypes are limited by the relatively small number of markers assessed. Therefore, our study aimed to generate an unbiased atlas of Mo/Ma phenotypes over the course of skin wound healing by scRNAseq. Cells were dissociated from excisional skin wounds of normal healing (WT) mice on day 3, 6, and 10 post-injury and live CD11b+Ly6G− cells were isolated by FACS sorting. Using 10× Genomics Chromium system, we pooled data from a total of 2,813 high-quality cells and performed clustering using Seurat which resulted in 8 subpopulations of cells. Cluster dissimilarity and differential expression analysis categorized those clusters into three groups: early stage/pro-inflammatory, antigen-presenting, and late-stage/pro-healing phenotypes that changed over the course of healing. Signature genes and gene ontology analysis of each cluster were used to further characterize the cell populations. Moreover, Ly6C+ vs Ly6C− Mo/Ma were FACS sorted and qPCR was used to validate select marker genes from our scRNAseq clusters. Finally, cell trajectory analysis by pseudotime and RNA velocity indicated the transitions between different Mo/Ma clusters over the course of healing. In summary, our data demonstrated the complexity of Mo/Ma phenotypes and their diverse functions during skin wound healing in mice. Funded by NIH/NIGMS: R35GM136228