Role of Macrophages in Wound Healing

Abstract

Monocytes/macrophages are key components of the body's innate ability to restore tissue function after injury. In most tissues, both embryo-derived tissue-resident macrophages and recruited blood monocyte–derived macrophages contribute to the injury response. The developmental origin of injury-associated macrophages has a major impact on the outcome of the healing process. Macrophages are abundant at all stages of repair and coordinate the progression through the different phases of healing. They are highly plastic cells that continuously adapt to their environment and acquire phase-specific activation phenotypes. Advanced omics methodologies have revealed a vast heterogeneity of macrophage activation phenotypes and metabolic status at injury sites in different organs. In this review, we highlight the role of the developmental origin, the link between the wound phase-specific activation state and metabolic reprogramming as well as the fate of macrophages during the resolution of the wounding response.

MECHANISTIC PRINCIPLES OF THE WOUND HEALING PROCESS

Tissue damage can be caused by diverse acute or chronic insults, including mechanical injury, autoimmune reactions, and infections with pathogens. Yet, the mechanisms of the damage-induced repair response depend not only on the nature of the injury, but also vary in different organisms and range from regeneration of the affected tissue toward replacement of the original tissue by connective tissue and scar formation (Poss 2010). In humans, as in most other mammals, organ regeneration is limited and declines after birth; soft tissue and organ repair occurs primarily via the deposition of an extracellular matrix (ECM) in the injured site resulting in fibrosis and scar formation (Ding et al. 2021).

The skin serves as a practical and scientifically relevant model to study mechanistic principles of the tissue repair response. In the skin, as in many other organs, wound healing is characterized by a highly dynamic and complex biological process engaging various nonimmune (stromal and parenchymal cells) and immune cell types, both resident or recruited to the site of tissue damage (Martin 1997; Gurtner et al. 2008). Cellular interplay results in the production of soluble mediators and ECM molecules, orchestrating complex cell–cell and cell–matrix interactions that are essential for a rapid restoration of tissue integrity and homeostasis (Eming et al. 2017).

Mice are a highly valuable preclinical model for investigating regenerative responses because of established genetic manipulation techniques and relatively low maintenance costs. However, when evaluating translation of preclinical findings in mice, different physiological aspects of wound closure in mice and men must be taken into account. Skin wounds close more rapidly in rodents compared with humans. The difference in the temporal course is primarily attributable to the different anatomical skin structure in mice and man. In rodents, the skin is mobile, and its contraction facilitates rapid wound closure in addition to reepithelialization and the deposition of granulation tissue. In humans, in addition to reepithelialization, wound closure occurs primarily through granulation tissue formation and ECM deposition, processes that require more time. Therefore, for example, a defective formation of granulation tissue in mice affects the timing of wound closure less than in humans in which the impact is more dramatic. In the following, wound healing mechanisms are described that were mainly established in the mouse model and that particularly emphasize the role of macrophages in the sequential stages of the healing process.

The course of the injury response consists of temporally and spatially overlapping phases. For simplicity, a classification is made between hemostasis, inflammation, proliferation, ECM deposition, and remodeling (Fig. 1). The primary goal of the healing process is to achieve rapid wound closure and restoration of the initial tissue architecture and function. The biology and duration of each phase of the wounding response depend on multiple factors, such as wound size, age, gender, anatomical location, bacterial burden, and individual underlying health conditions (e.g., diabetes mellitus, vascular disease), which can significantly impact the rate of tissue repair and lead to delayed healing or ultimately nonhealing (chronic) wounds (Eming et al. 2014). Hemostasis and inflammation occur during the early stage, proliferation, deposition of ECM during the mid-stage, and ECM remodeling and scar formation during the late stage after restoration of the tissue barrier is completed.

Figure 1.

Schematic overview of different stages of wound healing. Shown are the sequential and overlapping phases of the injury response. Macrophages play an important role in all stages; their function changes in the early stages (hemostasis, inflammation) compared with the latter ones (proliferation, remodeling) because of their high functional plasticity and communication with different cell subsets. During hemostasis and inflammation, macrophages together with polymorphonuclear neutrophils (PMNs) ensure bacterial clearance engaging Toll-like receptors (TLRs) to recognize and internalize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). During the transition of the inflammatory phase into the proliferative phase, macrophages together with other wound cells release multiple growth factors (in orange), stimulating the proliferation of stromal and parenchymal cells and the production of extracellular matrix (ECM) components to ensure the restoration of the original tissue. Growth factors: epidermal growth factor (EGF), transforming growth factor β (TGF-β), fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and placental growth factor (PLGF).

MACROPHAGES ARE KEY AND VERSATILE PLAYERS OF THE REPAIR RESPONSE

The essential role of innate immunity in repairing damaged tissues was recognized more than a century ago by Élie Metchnikoff when he discovered macrophages as the professional cells performing phagocytosis (Metchnikoff 1905). His discovery provided the basis for the investigations of monocyte/macrophage function in different injury scenarios in multiple model organisms and diverse tissues (Leibovich and Ross 1975; Martin et al. 2003; Stramer et al. 2005; Lucas et al. 2010; Godwin et al. 2013; Gurevich et al. 2018). Yet, a seminal study by Martin et al. (2003) in neonatal transcription factor PU.1 (PU.1)-null mice, which lack macrophages, revealed that at this early stage of life macrophages are not essential for timely healing of small wounds and that PU.1-null mice compared with wild-type siblings heal with minimal scarring. In a microarray approach, the same group compared gene expression in wounds of PU.1-null and wild-type mice and identified differentially expressed genes, which might become interesting therapeutic targets attenuating scar formation (Cooper et al. 2005). However, almost a decade later, other groups showed that in adult mice, using cell-type-specific and time-controlled depletion of myeloid cells, monocytes/macrophages contribute to the repair response with specific functions in each phase (Goren et al. 2009; Mirza et al. 2009; Lucas et al. 2010). Indeed, it is now well established that macrophages in the early stage contribute to critical aspects of hemostasis, acute inflammation, and clearance of bacteria/cell debris, whereas in the mid-stage, they promote tissue vascularization, granulation tissue formation, and wound epithelization (Mirza et al. 2009; Lucas et al. 2010). In the late stage, macrophages control scar formation (Mirza et al. 2009; Lucas et al. 2010; Knipper et al. 2015; Pakshir et al. 2019). Macrophages can perform these different functions owing to their highly dynamic activation diversity.

MACROPHAGE HETEROGENEITY DETERMINES THE QUALITY OF THE REPAIR RESPONSE

A major discovery of the past decade was that macrophages are highly heterogeneous immune cells. There is strong evidence that this heterogeneity controls and determines the quality of the healing process. The heterogeneity is based in particular on (1) different developmental origins of monocytes/macrophages, and (2) different activation phenotypes. Both aspects will be outlined in the following.

ORIGIN AND DEVELOPMENT OF MONOCYTES/MACROPHAGES

Different Origin and Development of Monocytes and Macrophages

One important finding in macrophage biology is that the developmental origin of a macrophage determines its function in tissue (Fig. 2). Both tissue-resident macrophages and freshly recruited monocytes/macrophages from the blood contribute to the healing process. Their origin has been untangled over the past decades.

Figure 2.

Origin of macrophages during homeostasis and inflammation. Under homeostatic conditions, organs contain tissue-resident macrophages that originate from both the yolk sac/liver during embryogenesis and from bone marrow–derived circulating monocytes postnatally. Upon injury, the recruitment of blood monocytes will quickly outweigh the resident populations and will constitute the main source of proinflammatory macrophages at the wound site. Reprogramming of inflammatory toward repair macrophages is required for resolution and healing.

For a long time, a dogma in immunology was that macrophages are exclusively derived from blood monocytes that develop from precursor cells in the bone marrow (van Furth et al. 1972). This view was challenged in past decades, as studies revealed that erythromyeloid progenitors (EMPs) develop in the yolk sac before the appearance of hematopoietic stem cells (HSCs) and that the EMPs represent precursor cells of tissue-resident macrophages in adult tissues (Schulz et al. 2012). Importantly, yolk sac–derived tissue-resident macrophages were shown to proliferate and renew in adult mice independently of the bone marrow (Schulz et al. 2012; Hashimoto et al. 2013). This finding led to the concept that in adult mice at least two macrophage lineages with different developmental histories coexist: monocyte-derived macrophages and yolk sac–derived macrophages (Cox et al. 2021). Later, it was shown that these different lineages impact the quality of the injury response and repair.

Fate-mapping studies of cells in the developing mouse embryo identified EMPs from embryonic day 8.5 (E8.5) in the yolk sac, which are characterized by expression of the macrophage colony-stimulating factor 1 receptor (Csf1r) (Gomez Perdiguero et al. 2015). For the development of EMPs, the transcription factor myeloblastosis oncogene (Myb) is dispensable, in contrast to HSCs, whose development requires Myb (Sarrazin and Sieweke 2004; Schulz et al. 2012). EMPs migrate to the nascent fetal liver before E10.5, where they expand and initiate a first wave of hematopoiesis. They give rise to F4/80^bright macrophages, monocytes, granulocytes, and red blood cells (Gomez Perdiguero et al. 2015). When organogenesis starts from E9.5, EMP-derived macrophage precursors colonize tissues in the entire embryo and acquire a core macrophage transcriptional program (Mass et al. 2016). These macrophage precursors up-regulate the chemokine receptor CX₃CR1 (CX₃C chemokine receptor 1), which is critical for the colonization within the embryo (Mass et al. 2016). To perform tissue-specific functions, macrophages require organ-specific differentiation, which is achieved by the expression of tissue-specific transcriptional regulators (e.g., inhibitor of DNA binding 3 [Id3] for the development of Kupffer cells in the liver or runt-related transcription factor 3 [Runx3] for the development of Langerhans cells in the epidermis) (Mass et al. 2016). Tissue-specific transcriptional programs in macrophages are associated with specific phagocytic activities (e.g., erythrocyte clearance by red pulp macrophages in the spleen) and specific growth factor supply (e.g., production of nerve growth factor by microglia to promote neural outgrowth and survival in the brain) (Kohyama et al. 2009; Pollard 2009; Cox et al. 2021). An important hallmark of yolk sac–derived tissue-resident macrophages is that they are locally maintained under homeostatic conditions attributable to their self-renewal ability. However, recently, it was shown that, in adults, HSC-derived blood monocytes also contribute to the replenishment of tissue-resident macrophages at different proportions that vary with the tissues considered (Cox et al. 2021).

The transition from yolk sac– to HSC-derived hematopoiesis occurs for monocytes at E14.5 (Gomez Perdiguero et al. 2015). HSCs emerge in large arteries, migrate first to the fetal liver, and then shift at E17.5 to the bone marrow (Cox et al. 2021). Here, they persist, self-renew, and give rise to all blood cell types including myeloid cells such as granulocytes, monocytes, and dendritic cell subsets (Rieger and Schroeder 2012; Cox et al. 2021). Monocytes develop in the bone marrow through different stages of progenitor cells, including a common myeloid progenitor and a granulocyte–macrophage progenitor (Rieger and Schroeder 2012). The release of monocytes into the circulation depends on the expression of the chemokine receptor CCR2 (C-C chemokine receptor type 2) (Serbina and Pamer 2006). Within the circulation, two main monocyte subsets have been identified, which can be differentiated by their surface markers and their functions (Geissmann et al. 2003; Sunderkötter et al. 2004; Auffray et al. 2009; Narasimhan et al. 2019). The major (classical) monocyte subset is defined by the expression of high levels of lymphocyte antigen 6C (Ly6C), CCR2, the adhesion molecule L-selectin (CD62L), and low levels of CX₃CR1 (Auffray et al. 2009). This subset has been termed “inflammatory” because it is rapidly recruited into inflamed tissues (Geissmann et al. 2003; Sunderkötter et al. 2004). Importantly, injuries in various organs such as liver, lung, heart, and skin lead to the recruitment of these Ly6C⁺CCR2⁺CD62L⁺ monocytes from the circulation into the tissue (Nahrendorf et al. 2007; Karlmark et al. 2009; Willenborg et al. 2012; Epelman et al. 2014; Misharin et al. 2017). The second (nonclassical) subset has been termed “resident” because it can be found in both resting and inflamed tissues (Geissmann et al. 2003). It is characterized by high expression levels of CX₃CR1, lymphocyte function-associated antigen 1 (LFA-1), and the lack of expression of Ly6C, CCR2, and CD62L (Geissmann et al. 2003). Intravital imaging of resident monocytes in CX₃CR1-green fluorescent protein (GFP) reporter mice revealed that these cells are crawling inside of blood vessels on the resting endothelium in a CX₃CR1- and LFA-1-dependent manner (Auffray et al. 2007). Nonclassical “patrolling” monocytes exert critical housekeeping functions and maintain vascular homeostasis by clearing dying endothelial cells (Carlin et al. 2013). However, nonclassical monocytes can also rapidly invade a site of injury or infection, differentiate into macrophages, initiate an early inflammatory response, and promote the resolution of inflammation (Auffray et al. 2007; Narasimhan et al. 2019).

Origin of Macrophages Determines Their Function in Wound Healing

In various injury models, it has been shown that the developmental origin of macrophages correlates with their function in tissue. Generally, the influx of blood CCR2⁺Ly6C⁺ inflammatory monocytes is highly associated with increased inflammation and later tissue fibrosis. For example, studies in Ccr2^−/− mice revealed that deficiency in CCR2 signaling results in diminished liver fibrosis after carbon tetrachloride (CCl₄)-induced injury and protection from pulmonary fibrosis after treatment with bleomycin (Moore et al. 2001; Karlmark et al. 2009). Furthermore, in experimental models of myocardial infarction, inflammation was reduced/resolved faster and ventricular remodeling was attenuated when Ccr2 was deleted or when Ccr2 in apolipoprotein E^−/− mice (modeling myocardial ischemia caused by hypercholesterolemia and atherosclerosis) was silenced by nanoparticle-encapsulated small interfering RNA (siRNA) (Kaikita et al. 2004; Majmudar et al. 2013). In skin, diphtheria toxin-mediated selective and transient depletion of monocytes/macrophages in the early granulation tissue, which is dominated by CCR2⁺Ly6C^high monocytes/macrophages, led to a significant reduction in granulation tissue and scar formation (Lucas et al. 2010; Willenborg et al. 2012). CCR2⁺Ly6C^high monocytes rapidly invade skin wounds and give rise to proinflammatory and proangiogenic macrophages, characterized by high expression of vascular endothelial growth factor A (Vegfa). Blocking entry of CCR2⁺Ly6C^high monocytes in skin wounds by using global or myeloid cell–specific CCR2-deficient mice resulted in severely impaired formation of vascularized granulation tissue and delayed healing (Willenborg et al. 2012).

Compared with blood-recruited inflammatory monocytes, tissue-resident macrophages appear to be less inflammatory and less fibrotic while promoting repair and recovery. Distinct functions of macrophages with different origins have been carefully dissected in experimental models of cardiac injury. The neonatal heart contains CCR2⁻ embryo-derived tissue-resident macrophages, which after injury produce only minimal inflammation and promote cardiac repair by inducing angiogenesis and myofibroblast proliferation (Lavine et al. 2014; Bajpai et al. 2019). In the adult heart, embryo-derived tissue-resident macrophages are replaced with CCR2⁺ inflammatory monocyte-derived macrophages, leading to inflammation and fibrosis (Lavine et al. 2014). When monocyte influx was blocked by treating mice with a CCR2 inhibitor, the embryonic macrophage was preserved, resulting in less inflammation and an accelerated repair response (Lavine et al. 2014). Moreover, in lung, it was shown that tissue-resident alveolar macrophages are dispensable for the development of lung fibrosis (Misharin et al. 2017).

MACROPHAGE ACTIVATION PHENOTYPES ORCHESTRATE THE HEALING PROCESS

Concepts of Macrophage Activation

To categorize and understand functional macrophage plasticity, different activation concepts have been described. In recent years, macrophage activation patterns were primarily based on the two activation phenotypes, type 1 (M1) or type 2 activation (M2) (Mosser and Edwards 2008). Thereby, the M1 activation phenotype resembles macrophages obtained through lipopolysaccharide (LPS) and interferon γ (IFN-γ) stimulation in vitro, whereas M2 macrophages (also named alternatively activated macrophages) are obtained using exposure to interleukin (IL)-4 and IL-13. This concept has been helpful and practical to classify activation states of the macrophage and to link these activation states to specific repair functions (Fig. 3). However, based on further analysis of macrophages using advanced omics methodologies, it is now clear that the M1/M2 concept is too simplistic, and in tissues macrophage function comprises a broad spectrum of activation phenotypes (Xue et al. 2014; Chávez-Galán et al. 2015; MacParland et al. 2018; Aran et al. 2019; Dick et al. 2019; Askenase et al. 2021; Willenborg et al. 2021). The current consensus emphasizes to precisely describe the state/condition of macrophage activation rather than using a simplified model (Murray et al. 2014). Thus, a broadly accepted, general macrophage activation concept and how this might apply to the wound healing process is still pending.

Figure 3.

Early- and late-stage reprogramming of macrophages. Macrophages are highly versatile cells being able to ensure both proinflammatory and pro-resolution functions in the tissues depending on the environmental stimuli. Proinflammatory macrophages are characterized as highly glycolytic cells, thus producing lactate that will lower the extracellular pH and contribute to acidosis. They are characterized by an increased production of mitochondrial reactive oxygen species (mtROS), stabilizing transcription factor hypoxia inducible factor 1α (HIF1α) that enables the production of angiogenic factors, such as vascular endothelial growth factor A (VEGF-A), and proinflammatory cytokines, such as interleukin 1β (IL-1β), and enhances the use of glucose metabolism. Moreover, proinflammatory macrophages are professional phagocytes ensuring both the removal of apoptotic polymorphonuclear cells (PMNs) and other cell debris and the killing of pathogenic bacteria using Toll-like receptors (TLRs) and nicotinamide adenine dinucleotide phosphate + hydrogen (NADPH) oxidase 2 (NOX2). The recognition of pathogen-associated molecular patterns (PAMPs) will lead to nuclear factor κ light-chain enhancer of activated B-cell (NF-κB) stabilization and the transcription of major proinflammatory cytokines (tumor necrosis factor α [TNF-α], IL-1β, IL-6, interferon γ [IFN-γ], C-C motif chemokine ligand 2 [CCL2]) and mediators (nitric oxide [NO] by inducible NO synthase [iNOS]), sustaining the inflammatory reaction. Pro-resolution macrophages (cluster of differentiation 163⁺ [CD163⁺], CD206⁺, IL-4Rα, arginase 1 high [Arg1^high]) are characterized by an enhanced mitochondrial metabolism, relying mostly on fatty acid (FA) oxidation coupled to oxidative phosphorylation (OXPHOS). They ensure the restoration of tissue integrity by producing anti-inflammatory cytokines (IL-10, TGF-β) tempering the proinflammatory response. Moreover, they produce mediators that stimulate extracellular matrix (ECM) production in fibroblasts and ECM stabilization.

Early Wound Phase Activation: Controlling the Immediate Danger

Upon tissue injury, blood components are spilled into the wound triggering the clotting cascade and the formation of the hemostatic plug. Platelets are the first cellular components to contribute to hemostasis and attract myeloid cells (neutrophils and monocytes). Monocyte recruitment is achieved by the activation of the complement cascade and the secretion of wound stabilizing agents, such as C-C-type chemokines (e.g., C-C motif chemokine ligand 3 [CCL3], CCL5, CCL7, CCL8, CCL13), growth factors (e.g., platelet-derived growth factor [PDGF], transforming growth factor β [TGF-β]), inflammatory cytokines (e.g., IL-1β, tumor necrosis factor α [TNF-α]), and fibronectin (DiPietro et al. 1998; Frank et al. 2000; Wetzler et al. 2000; Werner and Grose 2003; Barrientos et al. 2008; Rees et al. 2015; Cognasse et al. 2019).

To initiate monocyte transendothelial migration from the peripheral circulation, endothelial cells up-regulate adhesion-related receptors, such as selectins (P- and E-) and immunoglobulins (intercellular adhesion molecule 1 and 2 [ICAM-1, -2], vascular cell adhesion molecule 1 [VCAM-1], melanoma cell adhesion molecule [MCAM], and junctional adhesion molecule A and B [JAM-A, -B]). These receptors will then interact with ligands expressed by monocytes (L-selectin, macrophage-1 antigen [Mac-1], very late antigen 4 [VLA-4]) leading to their vascular rolling followed by firm adhesion onto the endothelium, allowing the migration to occur (Shi and Pamer 2011; Nourshargh and Alon 2014; Gerhardt and Ley 2015; Teh et al. 2019). Dysregulation of adhesion molecule expression has been described in recurrent infections (Shuster et al. 1992), models of peritonitis (Wilson et al. 1993), and in impaired wound healing (Subramaniam et al. 1997; DiPietro et al. 1998; Nagaoka et al. 2000; Wetzler et al. 2000; Mori et al. 2004). Because patrolling, nonclassical Ly6C^lo monocytes interact closely with the endothelium, they are considered faster migrators than classical Ly6C^high monocytes (1–2 vs. 24–48 h). However, Rodero and colleagues showed in a mouse skin wound model that Ly6C^high monocytes can directly enter the injured tissue in the presence of microhemorrhages, explaining their presence in inflamed tissues within hours (Rodero et al. 2014).

Once in the wound, monocytes encounter microbial products, such as LPS recognized by Toll-like receptors (TLRs), and proinflammatory cytokines produced by local cells, triggering their differentiation into early wound-phase “M1-like” proinflammatory macrophages. The major role of M1-like proinflammatory macrophages (cluster of differentiation 11b⁺ [CD11b⁺] F4/80⁺Ly6C^high) is to orchestrate in parallel host defense and initiation of the repair process. To overcome the risk of bacterial infection without further tissue damage, the inflammatory response needs to be finely tuned and limited. Both an excessive and a deficient inflammatory response can have devastating effects in wound healing. The impact of a perturbed injury-induced immune response has been characterized in several acute and chronic disease models (e.g., acute skin wound healing, chronic venous leg ulcers) (Lucas et al. 2010; Sindrilaru et al. 2011) and is considered to be the predominant pathology underlying impaired healing conditions in patients (Loots et al. 1998; Barrientos et al. 2008; Eming et al. 2014).

The accumulation of M1-like macrophages in skin wounds starts shortly after injury and peaks at mid-stage during the phase of granulation tissue formation. At this stage, proinflammatory macrophages represent 40%–50% of all wound cells (Daley et al. 2010; Willenborg et al. 2012). They release a multitude of proinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-12, CCL2) and use antimicrobial functions to clear the wound of pathogens and cell detritus. Macrophages are professional phagocytes that, owing to their high number of cell surface receptors, recognize and internalize a variety of different components, both foreign and endogenous. For example, bacterial internalization occurs through a TLR receptor-based mechanism (Blander and Medzhitov 2004; Doyle et al. 2004) and apoptotic cell removal occurs via a phosphatidylserine-mediated mechanism (Fadok et al. 1998). Phagocytosis of apoptotic cells is important for the initiation of the mid-stage of wound healing because it represses the production of proinflammatory cytokines and stimulates the release of cell growth and proliferation factors by macrophages (Voll et al. 1997; Poon et al. 2014; Bosurgi et al. 2017; Marwick et al. 2018). Moreover, late-stage M1-like macrophages are also known to release mediators, such as nitric oxide (NO), and growth factors, such as PDGF-stimulating fibroblast and keratinocyte proliferation and contributing to granulation tissue formation (Witte and Barbul 2002; Schultz and Wysocki 2009).

Mid-Wound Phase Activation: Formation of Granulation Tissue

When the immediate danger has passed, the number of polymorphonuclear cells (PMNs) declines and macrophages initiate their adaption of a proinflammatory M1-like toward a reparative M2-like activation phenotype. In this stage of repair, macrophages coordinate the formation of vascularized granulation tissue and induce fibroplasia by the secretion of multiple growth factors. The granulation tissue provides oxygen and nutrient supply supporting the expansion of the newly forming tissue; therefore, rapid and sufficient vascularization is essential. Wound healing studies in VEGF-β-galactosidase (lacZ) reporter mice revealed that macrophages represent the major fraction of Vegf-expressing cells in the granulation tissue in the early and mid-stages of healing (Willenborg et al. 2012). Importantly, VEGF-A from classical CCR2⁺ blood monocyte–derived wound macrophages is required for timely vascular sprouting (Willenborg et al. 2012). In addition, nonclassical monocytes were shown to home to perivascular niches and give rise to M2-like wound macrophages, supporting vascular remodeling after injury (Olingy et al. 2017). Furthermore, mid-stage wound macrophages are critical sources of TGF-β and PDGF, which have important roles, for example, in fibroplasia, ECM synthesis, and myofibroblast differentiation (Barrientos et al. 2008; Pakshir et al. 2019). Cell-type-specific depletion of myeloid cells in the mid-phase of healing abrogates these processes, resulting in impaired formation of granulation tissue, reduced tissue vascularization, impaired differentiation of α-smooth muscle actin (aSMA)-expressing myofibroblasts, and delayed epithelialization (Goren et al. 2009; Mirza et al. 2009; Lucas et al. 2010). Similar results were observed when a subpopulation of wound macrophages, which express the lectin CD301b (encoded by macrophage galactose N-acetyl-galactosamine-specific lectin 2 [Mgl2]) and expands in the mid-stage of healing, was specifically ablated by diphtheria toxin (Shook et al. 2016).

Late Wound Phase Activation: Stabilizing the New Wound Tissue

During the late wound phase, in most organs, the granulation tissue is converted into a scar. Hallmarks of this process are the decline in cell number, the increase in ECM deposition and ECM stabilization, and conversion of macrophages into an M2-like reparative phenotype. The transition of M1 toward M2 activation over healing progression has been shown to be critical in physiological tissue repair. Defects of this transition have been associated with delayed healing in mice and humans (Mirza et al. 2009; Sindrilaru et al. 2011; Knipper et al. 2015; Schönborn et al. 2020). However, which signals are specifically required for macrophage transition from proinflammatory clearance of infection or tissue injury toward a pro-resolution phenotype that rebuilds tissue architecture and restores homeostasis are still elusive.

Detection and phagocytosis of apoptotic cells have been identified as critical mechanisms for converting macrophages into a pro-resolution phenotype, as shown in lung repair after helminth infections as well as in skin wound healing (Chen et al. 2015; Bosurgi et al. 2017). Induction of M2 gene expression is also critically regulated by epigenetic mechanisms. Lactate-derived lactylation of histone lysine residues in proinflammatory macrophages was shown to initiate the expression of homeostatic genes associated with M2-like macrophages (Zhang et al. 2019). In addition, IL-4 activates the protein kinase B (Akt)-mammalian target of rapamycin complex 1 (mTORC1) axis, which regulates acetyl coenzyme A production, leading to increased histone acetylation and induction of a distinct subset of M2 genes (Covarrubias et al. 2016). In vivo, Katherine Gallagher's group found that the methyltransferase Setdb2 (suppressor of variegation 3-9, enhancer of zeste, and trithorax [SET] domain, bifurcated 2) is induced in wound macrophages by IFN-β. Setdb2 trimethylates histone 3 at NF-κB (nuclear factor κ light-chain enhancer of activated B cells) binding sites and thereby dampens the inflammatory response (Kimball et al. 2019). Notably, the investigators showed that IFN-β-induced Setdb2 expression is significantly lower in macrophages isolated from diabetic mice compared with controls, so that the macrophages remained in a proinflammatory state, eventually resulting in impaired repair (Kimball et al. 2019; Knipper et al. 2019).

In addition, over the past decade in multiple tissues, type 2 immunity has emerged as a key regulator of resolution, repair, and fibrosis (Gause et al. 2013; Knipper et al. 2015). In a model of skin injury, mice with myeloid cell–restricted deletion of the IL-4Rα chain (required for IL-4 and IL-13 signaling) show perturbed M2-like macrophage activation, associated with hemorrhages in the mid-stage of healing, perturbations in collagen fibril synthesis and cross-linking, resulting finally in attenuated mechanical stabilization of the newly formed ECM (Knipper et al. 2015). Furthermore, ECM components in the wound bed have emerged as critical regulators of macrophage activation, for example, overexpression of collagen XII in skin wounds leads to impaired transition of M1-like toward M2-like macrophages, resulting in prolonged inflammation and delayed healing (Schönborn et al. 2020). Together, these findings highlight the close interaction between macrophages and fibroblasts not only for quantitative ECM synthesis, but also for matrix maturation and stabilization.

MACROPHAGE METABOLISM DETERMINES ACTIVATION IN WOUND HEALING

Over the past decade, it has been shown that macrophage activation states are tightly linked with specific metabolic profiles (O'Neill and Pearce 2016). However, how cellular metabolism in macrophages might impact the wound healing process is poorly understood (Eming et al. 2021). A recent study provided novel insights into how the dynamic of metabolic reprogramming in wound macrophages orchestrates the healing process in skin (Willenborg et al. 2021). Similar to neutrophils, early-phase wound macrophages are highly glycolytic (Eming et al. 2021; Willenborg et al. 2021). A key regulator of the glycolytic pathway in early-phase wound macrophages is hypoxia-inducible factor 1α (HIF1α), which can be activated in injured tissues by both hypoxia and inflammatory signals such as LPS (Crowther et al. 2001; Lin and Simon 2016). Increased glucose metabolism in macrophages results in large quantities of lactic acid, a metabolite that drives vascular sprouting and contributes to tissue acidosis, a hallmark of inflammation (Trabold et al. 2003; Hunt et al. 2007; Porporato et al. 2012). Glycolytic metabolism in macrophages is associated with a “broken” tricarboxylic acid cycle (TCA cycle), which further promotes inflammation through the accumulation of TCA cycle intermediates: citrate, succinate, and itaconate (O'Neill et al. 2016). Citrate supports lipid synthesis necessary for the production of inflammatory mediators (Infantino et al. 2013), succinate stabilizes HIF1α, inducing IL-1β expression and enhancing glycolysis (Tannahill et al. 2013), and itaconate possesses a direct antimicrobial activity (McFadden and Purohit 1977) and supports succinate accumulation (Lampropoulou et al. 2016; Németh et al. 2016). Moreover, the increased glucose uptake and metabolism enables the activation and use of major glucose-dependent antibacterial functions, such as phagocytosis and reactive oxygen species (ROS) production (Viola et al. 2019). Furthermore, mitochondria have emerged recently as a central regulator of HIF1α-mediated VEGF-A expression in wound macrophages (Willenborg et al. 2021). A subpopulation of glycolytic early-phase wound macrophages repurposes mitochondria from adenosine triphosphate (ATP) production toward mitochondrial ROS production, which stabilizes HIF1α, thereby inducing a proangiogenic program (Willenborg et al. 2021).

The decline in inflammation toward the late phase of healing is tightly linked with profound changes in macrophage metabolism. Late-phase wound macrophages down-regulate genes of the glycolytic pathway and up-regulate genes involved in lipolysis and fatty acid oxidation (Willenborg et al. 2021). The TCA cycle is intact and generates high amounts of NADH (nicotinamide adenine dinucleotide + hydrogen), which is oxidized at the electron transport chain in the inner mitochondrial membrane, resulting in increased oxygen consumption and ATP production (oxidative phosphorylation, OXPHOS) (Eming et al. 2021; Willenborg et al. 2021). Induction of oxidative metabolism in late-phase wound macrophages, accompanied by mitochondrial biogenesis, is regulated by type 2 cytokines (Willenborg et al. 2021). From a functional perspective, increased oxidative metabolism helps wound macrophages in the late phase to dampen the production of inflammatory cytokines and to produce anti-inflammatory IL-10 and uridine diphosphate N-acetylglucosamine (UDP-GlcNAC, required for the glycosylation of type-2 cytokine-induced scavenger receptors such as CD206) (Jha et al. 2015; Knipper et al. 2015; Malandrino et al. 2015; Willenborg et al. 2021). Further, increased OXPHOS might increase the life span of macrophages in the late wound tissue, as shown for type 2–activated macrophages in vitro and memory T cells (Vats et al. 2006; Michalek et al. 2011).

FATE OF WOUND MACROPHAGES

The number of wound macrophages gradually decreases over the time course of the injury response. This decline is critical to enter the resolution phase and to avoid prolonged tissue damage. Yet, mechanisms of macrophage removal from injured tissues have been little analyzed and remain a topic of debate. Emerging evidence suggests that not all early-/mid-stage macrophages share the same fate and that different mechanisms might exist with currently unknown dynamics and functional relevance for the outcome of the healing process.

Evidence for the lymphatic removal of macrophages was shown by Bellingan et al. (1996) by the isolation of viable macrophages from the lymph nodes during wound healing. The mechanism was later explained by a direct interaction between macrophages and the mesothelium overlying the lymphatic vessels via integrin β1 (Bellingan et al. 2002). Although controversy still exists regarding the nature of these isolated cells (Hashimoto et al. 2011), another study showed a delayed migration of peritoneal macrophages into the lymphatics following LPS stimulation in an integrin Mac-1-deficient mouse model (Cao et al. 2005) confirming that the lymphatics do in part contribute to the removal of inflammatory cells to restore tissue homeostasis.

In addition, similarly to neutrophils, a portion of the proinflammatory macrophages are likely to die in situ. Cell death of injury-associated macrophages has been suspected by the presence of macrophage intracellular components in wound extracellular fluid (Albina et al. 1990). Although the exact death trigger and the mode of cell death in wound macrophages remains elusive, it has been suggested that M1-like macrophages undergo apoptosis owing to a toxic accumulation of endogenous NO coupled with death-ligand-induced cell death (Janssen et al. 2011). Thus, although resistant to cell death in the early stage of activation, macrophage susceptibility to cell death is likely to change over the time course of healing. Interestingly, recent evidence suggests that wound macrophages can give rise to other differentiated cell types including vascular cells and fibroblasts (Meng et al. 2016; Sinha et al. 2018; Haider et al. 2019; Li et al. 2021). Yet, the functional impact of this transition on the time course and the quality of the healing process needs to be further understood and solidified.

CLINICAL TRANSLATION AND PERSPECTIVES

Poor wound healing in the clinic is often associated with immune dysfunction leading to prolonged inflammation and tissue damage with a broad spectrum of pathological outcomes (Eming et al. 2014). An ulcerative healing defect, for example, in the skin (e.g., chronic skin ulcer associated with vascular disease, diabetes mellitus, or aging) and the excessive formation of ECM with perturbed architecture leading to organ fibrosis (e.g., hypertrophic scarring of the skin and keloid formation) represent extremes on this spectrum. Therapeutic interventions in diseases with poor wound healing are still insufficient and of high unmet medical need. Perturbed macrophage activation and function have been associated with the impaired wound healing scenarios described above (Sindrilaru et al. 2011; Zhu et al. 2016; Ud-Din et al. 2019; Theocharidis et al. 2022), and the normalization of their function is a promising therapeutic approach. Yet, although shown in preclinical model systems, their causative relationship in wound healing pathologies in the clinical setting has yet to be shown. A current obstacle in monocyte/macrophage-specific drug development is the lack of approaches to specifically target monocytes/macrophages in diseased tissues. It is likely that with the identification of monocyte/macrophage-specific druggable targets, the current lack of efficient therapies for impaired healing conditions will greatly improve.

In addition, exploration of the mechanistic effects behind current effective therapeutic approaches (e.g., local application of β-glucan, systemic blocking of type 2 cytokines), which are believed to mediate their effects through modulation of macrophage activation, will advance the field of regenerative therapies (Allanore et al. 2020; Yamaguchi et al. 2021). Furthermore, repurposing the rapidly growing number of potent novel immunomodulators, not yet licensed for the treatment of disease conditions with poor wound healing outcome, should be an interesting and rewarding approach in therapy development in tissue regeneration. For example, injury-associated macrophages are both a target and a rich source for TNF-α during the healing response (Willenborg et al. 2012, 2021). Interestingly, a recent preclinical study showed that global inhibition of TNF-α in the wound environment promotes the activation of tissue reparative macrophages (Dichtl et al. 2022). Thus, the targeted modulation of central cytokine pathways opens new avenues in the treatment of dysregulated repair responses.