Key Points

Mos/Mϕs exhibit complex and dynamic heterogeneity during skin wound healing.

Mos/Mϕs transit from proinflammatory to healing-associated transcriptional states.

An early increase of Ccl7 preferentially induces accumulation of proinflammatory Mos/Mϕs.

Abstract

Monocytes (Mos)/macrophages (Mϕs) orchestrate biological processes critical for efficient skin wound healing. However, current understanding of skin wound Mo/Mϕ heterogeneity is limited by traditional experimental approaches such as flow cytometry and immunohistochemistry. Therefore, we sought to more fully explore Mo/Mϕ 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/Mϕ subsets, including inflammation, chemotaxis, biosynthesis, angiogenesis, proliferation, and cell death. Quantitative PCR assays validated characteristics of early- versus late-stage Mos/Mϕs 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/Mϕs as healing progressed. Finally, we show that the chemokine Ccl7, which was a signature gene for early-stage Mos/Mϕs, preferentially induced the accumulation of proinflammatory Ly6C+F4/80lo/− Mos/Mϕs in mouse skin wounds. In summary, our data demonstrate the complexity of Mo/Mϕ phenotypes, their dynamic behavior, and diverse functions during normal skin wound healing.

Footnotes

This work was supported by National Institute of General Medical Sciences Grant R35GM136228 (to T.J.K.). M.M.-C. was supported in part by National Center for Advancing Translational Sciences Grant UL1TR002003.

The online version of this article contains supplemental material.

J.P. designed the study, conducted the experiments, performed data and statistical analysis, and wrote the manuscript; M.M.-C. helped design the study, performed data and statistical analysis, and helped write the manuscript; and T.J.K. helped design the study, write the manuscript, and provided all materials.

Abbreviations used in this article:

ANOSIManalysis of similaritiesBMbone marrowcDC1conventional type 1 DCcDC2conventional type 2 DCDCdendritic cellDEdifferentially expressedDPBSDulbecco’s PBSGEMgel beads in emulsionGOGene OntologyMϕmacrophageMomonocyteMoDCMo-derived DCmregDCmature DC enriched in immunoregulatory moleculesNAbneutralizing AbPAGApartition-based graph abstractionqPCRquantitative PCRRNA-seqRNA sequencingscRNA-seqsingle-cell RNA-seqUMAPuniform manifold approximation and projectionReceived May 18, 2022.Accepted September 7, 2022.Copyright © 2022 by The American Association of Immunologists, Inc.