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Obesity is associated with low-grade chronic inflammation and impaired glucose metabolism, both of which are detrimental to wound healing. C-C motif chemokine receptor 2 (CCR2) plays an important role in cell recruitment during healing, and our recent studies revealed the significance of CCR2-CCL2 signaling in promoting the proliferation of pro-inflammatory monocytes/macrophages in wounds. Therefore, we sought to determine whether diet-induced obesity increases monocyte/macrophage proliferation and their accumulation in skin wounds. We first confirmed that wound closure was delayed in obese CCR2 RFP/+ mice fed with a high-fat diet (HFD) compared to mice fed with a normal diet (ND). Using in vivo imaging and flow cytometry analysis, we found that HFD mice had significantly increased accumulation of CCR2+ monocytes/macrophages, particularly pro-inflammatory CCR2+Ly6C+ cells in wounds compared to their ND counterparts. Importantly, HFD mice exhibited an increased proliferation of wound CCR2+Ly6C+ compared to ND mice. Together, our data suggest that obesity leads to an increased proliferation and accumulation of pro-inflammatory CCR2+Ly6C+ monocytes/macrophages in skin wounds, which may contribute to delayed healing.

Monocytes (Mo) and macrophages (Mφ) play important roles in the function of tissues, organs, and systems of all animals during homeostasis, infection, injury, and disease. For decades, conventional wisdom has dictated that Mo and Mφ are end-stage cells that do not proliferate and that Mφ accumulation in tissues is the result of infiltration of Mo from the blood and subsequent differentiation to Mφ. However, reports from the early 1900s to the present describe evidence of Mo and Mφ proliferation in different tissues and contexts. The purpose of this review is to summarize both historical and current evidence for the contribution of Mφ proliferation to their accumulation in different tissues during homeostasis, infection, injury, and disease. Mφ proliferate in different organs and tissues, including skin, peritoneum, lung, heart, aorta, kidney, liver, pancreas, brain, spinal cord, eye, adipose tissue, and uterus, and in different species including mouse, rat, rabbit, and human. Mφ can proliferate at different stages of differentiation with infiltrating Mo-like cells proliferating in certain inflammatory contexts (e.g. skin wounding, kidney injury, bladder and liver infection) and mature resident Mφ proliferating in other inflammatory contexts (e.g. nematode infection, acetaminophen liver injury) and during homeostasis. The pathways involved in stimulating Mφ proliferation also may be context dependent, with different cytokines and transcription factors implicated in different studies. Although Mφ are known to proliferate in health, injury, and disease, much remains to be learned about the regulation of Mφ proliferation in different contexts and its impact on the homeostasis, injury, and repair of different organs and tissues., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Monocytes and macrophages (Mo/MΦ) rapidly accumulate in skin wounds after injury, then disappear as healing progresses. However, the mechanisms underlying their ultimate fate in wounds remain to be elucidated. Here, we show that apoptosis of Mo/MΦ parallels their reduction as wound healing progresses in non-diabetic mice. scRNAseq analysis confirmed enriched apoptosis GO pathways on day 6 post-injury in wound Mo/MΦ from non-diabetic mice. In contrast, there was significantly less Mo/MΦ apoptosis in wounds from diabetic mice, particularly in the pro-inflammatory Ly6C+ population, which may contribute to persistent Mo/MΦ accumulation and chronic inflammation. scRNAseq analysis implicated TNF, MAPK, Jak-STAT, and FoxO signaling pathways in promoting wound Mo/MΦ apoptosis in non-diabetic mice while cell proliferation related pathways appeared to be activated in diabetic mice. These novel findings indicate that reduced apoptosis is a contributor to persistent Mo/MΦ accumulation in diabetic wounds. These findings also highlight pathways that may regulate Mo/MΦ apoptosis during wound healing, which could be targeted to help resolve inflammation and improve healing., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Diabetic wounds are characterized by persistent accumulation of proinflammatory monocytes (Mo)/macrophages (MΦ) and impaired healing. However, the mechanisms underlying the persistent accumulation of Mo/MΦ remain poorly understood. In this study, we report that Ly6C + F4/80 lo/- Mo/MΦ proliferate at higher rates in wounds of diabetic mice compared with nondiabetic mice, leading to greater accumulation of these cells. Unbiased single cell RNA sequencing analysis of combined nondiabetic and diabetic wound Mo/MΦ revealed a cluster, populated primarily by cells from diabetic wounds, for which genes associated with the cell cycle were enriched. In a screen of potential regulators, CCL2 levels were increased in wounds of diabetic mice, and subsequent experiments showed that local CCL2 treatment increased Ly6C + F4/80 lo/- Mo/MΦ proliferation. Importantly, adoptive transfer of mixtures of CCR2 -/- and CCR2 +/+ Ly6C hi Mo indicated that CCL2/CCR2 signaling is required for their proliferation in the wound environment. Together, these data demonstrate a novel role for the CCL2/CCR2 signaling pathway in promoting skin Mo/MΦ proliferation, contributing to persistent accumulation of Mo/MΦ and impaired healing in diabetic mice., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Monocytes and macrophages (Mo/MΦ) play critical roles in all phases of skin wound healing. The majority of these cells are thought to be recruited from blood Mo; however, the role local proliferation of Mo/MΦ in the wound has not been defined. Therefore, we tested the hypothesis that local proliferation of Mo and/or MΦ contributes to their accumulation during wound healing. Male C57Bl/6 mice (N = 4-9/group) were subjected to excisional skin wounding. Proliferating Mo/MΦ (F4/80+Ki67+) were observed in wound cryosections, peaking on day 5 post-wounding. Cell cycle analysis on cells isolated from skin tissue revealed that wounding increased both the number and percentage of inflammatory Ly6C+F4/80lo/- Mo/MΦ in the S/G2/M phases, peaking on day 6 post-wounding. In contrast, more mature Ly6C-F4/80+ cells were found predominantly in the G0 phase with less than 1% cells in S/G2/M phase following injury. In peripheral blood, Mo were very rarely found in the S/G2/M phase, suggesting that the wound environment triggered the Ly6C+F4/80lo/- Mo proliferative response. Furthermore, injury induced several potential regulators of proliferation in wounds, including IL-1β and IL-6, and wound Mo/MΦ expressed surface receptors for these cytokines. However, wound Mo/MΦ proliferation was not altered in IL-1R1 knockout (KO) or IL-6 KO mice. In summary, our findings indicate that proliferation contributes to Mo/MΦ accumulation in wounds and, contrary to findings in other pathophysiologic conditions, Ly6C+/F4/80lo/- Mo/MΦ proliferate during skin wound healing whereas mature Ly6C-F4/80+ MΦ do not., (©2019 Society for Leukocyte Biology.)

The aim of this study was to determine whether skin wounding induces monocyte (Mo) expansion in bone marrow and whether IL-1R1 signaling regulates this process. Our data show that skin wounding increases myeloid lineage-committed multipotent progenitors (MPP3 subset) and Mo in bone marrow, but this expansion is not impaired in Il1r1 -/- mice. We also demonstrate that M-CSF-induced differentiation of myeloid progenitors into Mo is not impaired by the loss of IL-1R1 ex vivo, indicating that IL-R1 deficiency does not abrogate myeloid progenitor differentiation potential. In addition, we observed modestly delayed wound closure in Il1r1 -/- mice associated with higher frequency of Ly6C lo Mo in the circulation at baseline and in wounds early after injury. Thus, in contrast to other models of inflammation that involve IL-1R1-dependent monopoiesis, our results demonstrate that skin wounding induces Mo progenitor and Mo expansion independently of IL-1R1 signaling., (Copyright © 2019 by The American Association of Immunologists, Inc.)

The hypothesis of this study was that cells of the monocyte/macrophage lineage (Mo/Mp) exhibit an impaired transition from pro-inflammatory to pro-healing phenotypes in wounds of diabetic mice, which contributes to deficient healing. Mo/Mp isolated from excisional wounds in non-diabetic db/+ mice exhibited a pro-inflammatory phenotype on day 5 post-injury, with high level expression of the pro-inflammatory molecules interleukin-1β, matrix metalloprotease-9 and inducible nitric oxide synthase. Wound Mo/Mp exhibited a less inflammatory phenotype on day 10 post-injury, with decreased expression of the pro-inflammatory molecules and increased expression of the alternative activation markers CD206 and CD36. In contrast, in db/db mice, the pro-inflammatory phenotype persisted through day 10 post-injury and was associated with reduced expression of insulin-like growth factor-1, transforming growth factor-β1 and vascular endothelial growth factor. Reduced levels of these growth factors in wounds of db/db mice may have contributed to impaired wound closure, reduced granulation tissue formation, angiogenesis and collagen deposition. The persistent pro-inflammatory wound Mo/Mp phenotype in db/db mice may have resulted from elevated levels of pro-inflammatory interleukin-1β and interferon-γ and reduced levels of anti-inflammatory interleukin-10 in the wound environment. Our findings are consistent with the hypothesis that dysregulation of Mo/Mp phenotypes contributes to impaired healing of diabetic wounds., (Copyright © 2011 Elsevier Ltd. All rights reserved.)