Macrophage Phenotypes in Normal and Diabetic Wound Healing and Therapeutic Interventions

Abstract

Macrophage differentiation and polarization are essential players in the success of the wound-healing process. Acute simple wounds progress from inflammation to proliferation/regeneration and, finally, to remodeling. In injured skin, macrophages either reside in the epithelium or are recruited from monocytes. Their main role is supported by their plasticity, which allows them to adopt different phenotypic states, such as the M1-inflammatory state, in which they produce TNF and NO, and the M2-reparative state, in which they resolve inflammation and exhibit a reparative function. Reparative macrophages are an essential source of growth factors such as TGF-β and VEGF and are not found in nonhealing wounds. This review discusses the differences between macrophage phenotypes in vitro and in vivo, how macrophages originate, and how they cross-communicate with other cellular components in a wound. This review also highlights the dysregulation of macrophages that occurs in nonhealing versus overhealing wounds and fibrosis. Then, the therapeutic manipulation of macrophages is presented as an attractive strategy for promoting healing through the secretion of growth factors for angiogenesis, keratinocyte migration, and collagen production. Finally, Hoxa3 overexpression is discussed as an example of the therapeutic repolarization of macrophages to the normal maturation state and phenotype with better healing outcomes.

Keywords: differentiation; inflammation; macrophage polarization; tissue regeneration; tissue remodeling.

Figure 1

Summary of the coagulation and inflammation phase of healing. The repair process begins with the formation of fibrin-based clots and the inflammatory response. Neutrophils were recruited in response to products released from platelet degranulation releasing IL-8 and CXCL8 and also by chemotactic signals produced from DAMP, MAMP, HSP, and HMGB and by complement opsonization. Monocytes migrate later in this phase from the circulation into the tissue due to LPS produced by invading bacteria, which are bound to TLR on the surface of monocytes. This pathway forms one example of the PAMP-stimulated migration of monocytes. Products released from damaged tissue such as ATP, extracellular RNA, or DNA influence monocyte migration. PAMP- or DAMP-mediated monocyte recruitment can stimulate interleukins or chemokines such as CCL20 and CCL2, which bind to their receptors—CCR6 and CCR2, respectively—on monocytes. Wound resident macrophages also play a role in this phase as they are activated by DAMP, including ATP and HMGB. ATP, adenosine triphosphate; DAMP, damage-associated molecular pattern; HMGB, high-mobility group box protein; LPS, lipopolysaccharides; PMN, polymorphonuclear neutrophil; PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptors; HSP, heat shock protein; MAMP, microbes-associated molecular patterns.

Figure 2

Summary of the late inflammation and proliferation phase of healing. Keratinocytes migrate to the wound and proliferate around the outermost area of the wound to close the wound surface by re-epithelialization. Underneath the keratinocyte layer, granulation tissue is formed and replaces the damaged dermis in a process that depends on growth factors such as FGF and TGF-β. Macrophages are differentiated from monocytes in response to TGF-β and VEGF growth factors released from keratinocytes and fibroblasts. In addition, during this phase, resident macrophages (purple-colored in the dermis) are directly involved in the elimination of inflammatory cells from the wound by engulfing apoptotic PMNs (efferocytosis). This process contributes to the switch of the macrophage’s phenotype from M1 to M2. The damaged blood vessels are replaced by neovascularization through angiogenesis, in which new blood vessels sprout from healthy vessels regulated via alterations in the oxygen gradient through HIF. Alternatively, it can form via vasculogenesis, in which endothelial cells are combined to create a new branch of the blood vessel in a process that requires EPC migration from the BM. VEGF produced from reparative macrophages or fibroblasts stimulates both processes of neovascularization. TGF-β, transforming growth factor beta; VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor; HIF, hypoxia-inducible factor; CBP/p300, core-binding factor/protein 300; HRE, hypoxia response element.

Figure 3

Summary of the tissue-regeneration phase of healing. In this phase, re-epithelialization and the formation of collagen-based ECM occur. Keratinocytes and fibroblasts can migrate to the wound in response to releasing H₂O₂, exposed Ca2+, and serum from damaged tissue. Fibroblasts are provided to the injured tissue either from the healthy dermis, BM-derived fibrocytes, or multipotent precursor cells. Both fibrocytes and multipotent cells can differentiate into fibroblasts before they are recruited to the wound. Reparative macrophages (green-colored in the wound) play an essential role in this phase by producing several growth factors, including FGF, TGF-β, IGF, and VEGF. TGF-β promotes the differentiation of fibroblasts into myofibroblasts, a type of contractile cell that can re-approximate the wound edges. Additionally, it can negatively regulate the re-epithelialization process. FGF and HGF are associated with keratinocyte migration to the wound edge to perform re-epithelialization. BM, bone marrow; FGF, fibroblast growth factor; IGF, insulin-like growth factor; PMN, polymorphonuclear neutrophil; TGF-β, transforming growth factor beta; VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor; HGF, hepatocytes growth factor.

Figure 4

Summary of the process that occurs during the tissue-remodeling phase of healing. The release of MMP from fibroblasts, macrophages, and myofibroblasts helps to remodel the ECM by converting type III collagen into type I collagen. Fibrin-based ECM made during the first phase of healing is replaced by collagen-based ECM. The very few cells that remain in the wound are removed by apoptosis and neovascularization ceases. ECM, extracellular matrix; MMP, matrix metalloproteinase.