**3. The role of macrophages in normal wound healing**

Wound healing is a very complex process encompassing 4 specific phases: hemostasis, inflammation, proliferation and remodeling. A successful wound closure requires the process to be well orchestrated by multiple cell types including keratinocytes, fibroblasts, endothelial cells, mesenchymal cells and inflammatory cells (macrophages, lymphocytes, neutrophils, NK cells, etc.) in a dynamic interactive way [10]. These 4 phases occur in a coordinated, linear and partially overlapping manner, in which one earlier phase is required for the completion of later phases [11]. The hemostasis phase is initiated immediately after injury, which involves vasoconstriction, platelet aggregation and activation of the clotting cascade resulting in clot formation and degranulation of platelets to convert soluble fibrinogen to insoluble fibrin and to release factors like P-selectin to recruit neutrophils to initiate the inflammatory phase [27, 28]. Next, the recruited neutrophils (peaking at 2 days after injury) release ROS, antimicrobial peptides and neutrophil extracellular traps in addition to chemokines to attract monocytes/macrophages [27]. Meanwhile,

tissue-resident macrophages and other antigen-presenting cells like dendritic cells are activated and release factors [11]. The monocytes/macrophages from either the tissue or the circulation thus become the cell type dominating the inflammatory phase and are the central regulators of this inflammatory phase [29]. Here, the macrophages interact with many cell types, activate a number of signaling pathways, and release many soluble mediators, such as growth factors, chemokines, cytokines and metabolites that signal to other cell types. Thus, the macrophages orchestrate the complex tasks and biological activities to enhance wound healing [30]. At this phase of wound healing, which typically lasts 3 days, macrophages are mainly pro-inflammatory, or M1-like, and produce cytokines such as IL-1β, IL-6, IL-12 and TNFα [10]. However, as early as day 1 after initiation of wound healing, anti-inflammatory or M2-like macrophages start to appear and their numbers increase little by little until they become dominant by about 4 days after wounding when the proliferative phase starts [14]. During the proliferative phase, M2-like macrophages (peaking at 7 days after wound) produce and secrete factors to activate fibroblasts and keratinocytes to proliferate, differentiate and migrate to the wound and deposit collagen and other ECM proteins, though keratinocytes start to proliferate immediately after wound due to loss of contact inhibition [13]. Moreover, M2-like macrophages also promote angiogenesis through interactions with vascular endothelial cells [13]. A requirement for macrophages in the activation and functions of fibroblasts at this phase was proven by a study applying macrophage-depletion [31]. Interestingly, a very recent report showed a myeloid origin of about 10% of wound fibroblasts in a mouse skin wounding model, further supporting the important role and plasticity of macrophages during wound healing [32]. The final remodeling phase is the longest phase of wound healing, during which type I collagen gradually replaces type III collagen to increase tensile strength [33].

Macrophages are key regulators for the overall wound repair process [34]. The wound monocytes/macrophages have different origins. Before injury occurs, skin monocytes/macrophages consist of the tissue-resident macrophages as predominantly Langerhans cells in the epidermal layer, and also as dermal macrophages in the dermis [35]. Wounds without macrophages have less cell proliferation, inadequate differentiation, compromised migration, delayed re-epithelialization, impaired angiogenesis and reduced collagen deposition [27]. Moreover, reduced secretion of vascular endothelial growth factor (VEGF)-A and transforming growth factor (TGF)β1 renders the wounds less conducive to angiogenesis and cell proliferation, which are critical for a proper completion of wound repair [36]. The plasticity of macrophages allows them to be first polarized to a pro-inflammatory M1-like phenotype, and then transition or repolarize to an anti-inflammatory M2-like phenotype [29]. Indeed, M1 macrophages are primarily responsible for destruction of pathogens and production and release of inflammatory cytokines in the wound. Meanwhile, M2 macrophages are associated with the late repair and regeneration phases of wound healing, and they are critical for proper angiogenesis, regeneration and remodeling of ECM, cell growth and replacement, production of anti-inflammatory and trophic cytokines and resolution of the inflammation [37]. Actually, as stated above, macrophages are not all polarized uniformly throughout wound healing [23]. At any given time point they may be in a wide spectrum of phenotypes, and only the predominant phenotype presented as the overall macrophage phenotype at any given time in the repair process (**Figure 1**) [10, 23, 29, 38].

Dermal wound healing is a very complex process encompassing 4 specific phases: hemostasis, inflammation, proliferation and remodeling. These 4 phases occur in a coordinated, linear and partially overlapping manner, in which one earlier phase is

*Macrophages as a Target for Treating Diabetic Foot Ulcers DOI: http://dx.doi.org/10.5772/intechopen.106613*

**Figure 1.** *Role of macrophages in wound healing.*

required for the completion of later phases. The hemostasis phase is initiated immediately after injury, and involves vasoconstriction, platelet aggregation and activation of the clotting cascade, resulting in clot formation and degranulation of platelets to convert soluble fibrinogen into insoluble fibrin and to release factors like P-selectin to recruit neutrophils to initiate the inflammatory phase. Next, the recruited neutrophils (peaking at 2 days after injury) release chemokines to attract monocytes/ macrophages. Meanwhile, tissue-resident macrophages and other antigen-presenting cells like dendritic cells are activated and release factors. The monocytes/macrophages from either the local tissue or the circulation thus dominate this phase of wound healing, which typically lasts 3 days. At this time, macrophages are mainly proinflammatory or M1-like, and produce cytokines such as IL-1β, IL-6, IL-12 and TNFα. However, as early as day 1 after the initiation of wound healing, anti-inflammatory or M2-like macrophages start to appear and their numbers increase little by little until they become dominant by about 4 days after wounding, the point when the proliferative phase starts. In the proliferative phase, M2-like macrophages (peaking at 7 days after wounding) produce and secrete factors to activate fibroblasts and keratinocytes to proliferate, differentiate and migrate to the wound and deposit of collagen and other ECM proteins. Moreover, M2-like macrophages also regulate angiogenesis through interactions with vascular endothelial cells. The final remodeling phase is the longest phase of wound healing, during which type III collagen is gradually replaced with type I collagen to increase tensile strength.
