Comprehensive characterization of myeloid cells during wound healing in healthy and healing‐impaired diabetic mice

Abstract

Wound healing involves the concerted action of various lymphoid and in particular myeloid cell populations. To characterize and quantitate different types of myeloid cells and to obtain information on their kinetics during wound healing, we performed multiparametric flow cytometry analysis. In healthy mice, neutrophil numbers increased early after injury and returned to near basal levels after completion of healing. Macrophages, monocyte‐derived dendritic cells (DCs), and eosinophils were abundant throughout the healing phase, in particular in early wounds, and Langerhans cells increased after wounding and remained elevated after epithelial closure. Major differences in healing‐impaired diabetic mice were a much higher percentage of immune cells in late wounds, mainly as a result of neutrophil, macrophage, and monocyte persistence; reduced numbers and percentages of macrophages and monocyte‐derived DCs in early wounds; and of Langerhans cells, conventional DCs, and eosinophils throughout the healing process. Finally, unbiased cluster analysis (PhenoGraph) identified a large number of different clusters of myeloid cells in skin wounds. These results provide insight into myeloid cell diversity and dynamics during wound repair and highlight the abnormal inflammatory response associated with impaired healing.

Macrophages in Healing Wounds: Paradoxes and Paradigms

“…Easily accessible, and thus easy to study, skin provides an excellent prototype in which to study the complete wound healing response from start to finish. Macrophages in wounds come from two primary sources—resident cells and infiltrating cells derived from the circulation [ 16 ]. The exact time point of maximal macrophage content varies depending upon wound size and depth but most generally occurs just prior to full epithelial closure [ 17 ].…”

Dermal Drivers of Injury-Induced Inflammation: Contribution of Adipocytes and Fibroblasts

“…These studies produced prospective targets for improved healing outcomes, including administration of mesenchymal stem cells to dampen inflammation and promote ECM production [ 156 ]. Interestingly, new lines of investigation have uncovered a need for robust, efficient recruitment of leukocytes to support proper repair [ 33 , 34 , 157 ], making factors that impact early inflammation a critical area of research. In particular, delayed or sustained neutrophil or macrophage function can have detrimental effects on multiple facets of downstream wound resolution and healing [ 158 , 159 ].…”

“…Although early healing time points can be challenging to obtain from humans, diabetic mouse studies have detected epigenetic- [ 160 ] and chemokine-mediated [ 157 ] delays in macrophage recruitment and activation at early time points after injury. Thorough analysis of wound bed myeloid cells revealed a marked delay in peak macrophage numbers of diabetic mice as well as various changes in transitioning immune cells [ 33 , 34 ]. In the elderly population, lower basal hematopoiesis [ 161 ] may compound decreased macrophage responsiveness and inflammatory polarization [ 162 , 163 ].…”

“…In addition to a delay in the initial macrophage response, a second influx of inflammatory macrophages impairs healing in high-fat diet-induced diabetic mice [ 166 ]. Both diabetes and aging are characterized by systemic inflammation [ 167 , 168 ], likely contributing to persistence of inflammatory neutrophils and macrophages at later time points after injury [ 28 , 33 ]. Pro-inflammatory skewing of diabetic macrophages [ 169 , 170 ] begins in the bone marrow [ 171 ], and macrophages from aged mice have a diminished capacity to respond to external polarization signals [ 162 ], reducing their ability to transition during repair.…”

“…These hard-to-treat wounds pose a significant medical challenge; as their prevalence has steadily increased over time and only modest therapeutic advancements have come from animal studies [ 30 , 31 ]. While tremendous efforts have uncovered defects in cellular composition and function during the proliferative phase of repair, animal models have recently revealed that reduced activation of early inflammatory responses is associated with delayed healing [ 32 , 33 , 34 ]. Due to their role in ECM production, dermal mesenchymal cells have been studied in the context of ECM formation and maturation; however, emerging evidence has revealed that adipocytes and fibroblasts can also promote inflammation.…”