Proliferation of Ly6C+ monocytes/macrophages contributes to their accumulation in mouse skin wounds

Abstract

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.

Macrophage Dysregulation and Impaired Skin Wound Healing in Diabetes

“…After infiltrating wounds, novel recent findings demonstrate that inflammatory Mo/Mϕ (Ly6C hi F4/80 –/lo ) proliferate rapidly peaking on day 6 post-wounding. In contrast, the majority of mature wound Mϕ (Ly6C – F4/80 + ) remain at resting G0 phase indicating that proliferation of infiltrating inflammatory Mo followed by their differentiation into mature Mϕ results in wound Mϕ expansion ( Pang et al, 2019 ). In addition, several studies have demonstrated that bone marrow-derived Mo contribute to skin wound Mϕ and that similar to other tissue injuries such as myocardial infarction and hindlimb ischemia, skin wounding also promotes bone marrow monopoiesis in mice ( Ishida et al, 2008 ; Sager et al, 2015 ; Fang et al, 2018 ; Barman et al, 2019a ).…”

Local proliferation of monocytes

“…Countering this view, Pang et al. show that Ly6C‐expressing myeloid cells recruited to the site of an excisional skin wound undergo extensive proliferation 6 . These findings complement two recent studies that found urinary tract 7 and hepatic 8 monocytes to proliferate during bacterial and helminth infection, respectively, suggesting bone marrow‐derived monocytes are able to proliferate to a significant degree within certain inflamed tissues.…”

Skin Wound Healing: Normal Macrophage Function and Macrophage Dysfunction in Diabetic Wounds

“…Factors in the local wound environment stimulate these bone marrow-derived monocytes to differentiate into macrophages and the precise combination of these factors is what appears to dictate macrophage phenotype, although the original phenotype of the bone marrow monocytes may also dictate the macrophage phenotype. In addition, some immune cells can proliferate in the wound [ 39 ]. It is the inflammatory monocytes/macrophages derived from the circulating monocytes, but not the mature wound macrophages, that are able to proliferate in the wound and, at the mid-stages of healing, these cells constitute around 25% of macrophage population.…”