2.1 Historical perspective on macrophage plasticity

Macrophage plasticity emerged in the 1960s when Mackaness et al.^26-31 reported two distinct macrophage activation states responding to cytokines. Type 1 macrophages, now known as M1, exhibited enhanced microbicidal activity against intracellular pathogens like Mycobacterium tuberculosis. Type 2 macrophages, now called M2, dampened inflammation and promoted extracellular matrix remodeling. In the 1980 and 1990s, the phenotypic and functional differences between M1 and M2 macrophages came into sharper focus. Stein et al.³² found that M1 macrophages produced high levels of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), IL-1, IL-6, and IL-12. This enabled them to stimulate T-cell responses and potently unleash oxidative attacks against pathogens. Conversely, M2 macrophages secreted anti-inflammatory cytokines like IL-10 and expressed high arginase-1 (ARG-1) levels, allowing them to suppress immune responses and promote tissue repair.³² Based on arginine metabolism, the M1/M2 classification system was consolidated by Mills et al.¹⁵ in 2000, drawing parallels with T helper 1 (Th1) and Th2 lymphocyte polarization. While this dichotomy provided a helpful framework, it oversimplified the complex spectrum of macrophage activation states observed in vivo (Table 1).

2.2 The spectrum of macrophage activation states: beyond M1/M2 dichotomy

The advent of single-cell technologies, such as single-cell RNA sequencing (scRNA-seq) and mass cytometry (CyTOF), has revolutionized our understanding of macrophage heterogeneity.^109-111 These high-resolution techniques have allowed researchers to profile the transcriptomes and proteomes of individual macrophages, uncovering a continuum of activation states that extend beyond the M1/M2 dichotomy.¹¹² A seminal study by Xue et al.⁴¹ used scRNA-seq to analyze human macrophages stimulated with 28 different activation conditions. They identified 49 distinct macrophage subsets, each with a unique transcriptional signature, highlighting the incredible diversity of macrophage responses to environmental cues. Similarly, a CyTOF study by Roussel et al.⁴⁴ demonstrated that human macrophages exhibit a spectrum of activation states in response to various stimuli, with some cells coexpressing both M1 and M2 markers. Recent studies have also revealed novel macrophage subsets with unique functions. For example, Angel et al.¹¹³ identified a population of antigen-presenting macrophages in human lymph nodes that express high levels of MHC class II and costimulatory molecules, suggesting a role in adaptive immunity. Another study by Zilionis et al.⁴⁵ discovered a subset of mouse lung macrophages expressing M1 and M2 markers that play a critical role in maintaining lung homeostasis. These findings underscore the limitations of the M1/M2 classification system and emphasize the need for a more nuanced understanding of macrophage plasticity. The spectrum of activation states revealed by single-cell technologies highlights the remarkable adaptability of macrophages to diverse environmental cues and their multifaceted roles in health and disease.^{8, 114, 115}

2.3 Tissue-specific imprinting of macrophage identity and function

One of the most significant advances in macrophage biology over the past decade has been recognizing the profound influence of tissue microenvironments on macrophage development, phenotype, and function. Macrophages are present in virtually all tissues, performing specialized functions tailored to the unique demands of their local niche.¹¹⁶ Recent studies have shown that tissue-specific factors, such as cytokines, metabolites, and cell–cell interactions, can imprint distinct transcriptional and epigenetic signatures on resident macrophages, giving rise to specialized subsets with unique functions.^{117, 118} For example, Lavin et al.⁴⁰ demonstrated that macrophages from different tissues, such as the lung, liver, and spleen, possess distinct enhancer landscapes shaped by tissue-specific transcription factors. The gut microbiome has also emerged as a critical regulator of intestinal macrophage function.^{119, 120} Studies have shown that microbial metabolites, such as short-chain fatty acids and taurine, can modulate the phenotype and activity of intestinal macrophages, promoting homeostasis and protecting against enteric infections.^121-123 These findings highlight the importance of studying macrophages in their native tissue context and underscore the limitations of extrapolating conclusions from in vitro studies to in vivo settings. The tissue-specific imprinting of macrophage identity and function has important implications for our understanding of immune regulation and disease pathogenesis, as dysregulation of these processes may contribute to developing inflammatory and metabolic disorders.^{116, 124, 125}

2.4 Ontogenetic diversity of tissue-resident macrophages

Another significant paradigm shift in macrophage biology has been the discovery of the ontogenetic diversity of tissue-resident macrophages (TrMΦ).^{126, 127} Contrary to the traditional view that all tissue macrophages are derived from circulating monocytes, recent fate mapping studies have revealed that many TrMΦ are established during embryonic development and maintain themselves through local proliferation, independent of adult monocyte input.^{128, 129} For example, microglia, the resident macrophages of the central nervous system, have been shown to originate from yolk sac-derived progenitors that seed the brain early in embryonic development.¹³⁰ Similarly, Kupffer cells, the resident macrophages of the liver, are derived from a combination of yolk sac and fetal liver progenitors.¹³¹ The ontogenetic origin of TrMΦ has important implications for their function and response to environmental challenges. Embryonically derived macrophages have been shown to possess unique transcriptional and epigenetic profiles compared with their monocyte-derived counterparts, which may confer distinct functional properties.^{132, 133} The discovery of the ontogenetic diversity of TrMΦ has also prompted a reevaluation of the contribution of monocyte-derived macrophages to tissue homeostasis and inflammation. While monocyte-derived macrophages play a crucial role in the response to injury and infection, their contribution to the maintenance of TrMΦ populations appears context dependent. It may vary across different organs and disease states.^{134, 135}

2.5 Trained immunity: long-term reprogramming of macrophages

In addition to short-term plasticity, macrophages can undergo long-term functional reprogramming in response to microbial stimuli, known as trained innate immunity.^{136, 137} This process involves epigenetic and metabolic changes that enhance the responsiveness of macrophages to subsequent challenges, providing a form of innate immune memory.^{138, 139} A landmark study by Quintin et al.³⁹ demonstrated that exposure to the fungal cell wall component β-glucan induces epigenetic modifications in human monocytes, leading to increased production of proinflammatory cytokines upon restimulation. This trained immunity is mediated by changes in histone methylation, acetylation, and a metabolic shift toward glycolysis.^{140, 141} Subsequent studies have shown that other microbial stimuli, such as the bacillus Calmette-Guérin (BCG) vaccine and the bacterial component muramyl dipeptide, can also induce trained immunity in macrophages.^{139, 142-144} Arts et al.⁴³ identified a critical role for ATF7 in mediating the epigenetic reprogramming of macrophages during β-glucan-induced training. Another study by Cheng et al.⁴² demonstrated that the metabolic enzyme glutamine synthetase is essential for the induction of trained immunity by β-glucan, highlighting the link between metabolism and epigenetic reprogramming. The discovery of trained immunity has important implications for developing novel immunotherapies. For example, Moorlag et al.¹⁴⁵ showed that BCG vaccination induces trained immunity in human monocytes, enhancing their ability to eliminate the respiratory syncytial virus. Harnessing trained immunity could be a promising strategy for boosting host defense against infectious diseases.

In summary, the study of macrophage plasticity has come a long way since the initial discovery of these versatile immune cells by Élie Metchnikoff in 1882. The traditional M1/M2 classification system provided a valuable framework for understanding macrophage polarization, but recent advances have revealed a spectrum of activation states that extend beyond this dichotomy. Single-cell technologies have uncovered macrophages’ remarkable heterogeneity and ability to adapt to diverse tissue microenvironments. The functional significance of macrophage plasticity is evident in their roles in maintaining tissue homeostasis, orchestrating immune responses, and contributing to disease pathogenesis. Tissue-specific imprinting and trained immunity further highlight the adaptability of macrophages to their local environment and their capacity for long-term functional reprogramming.

3.1 Toll-like receptors: sentinels of macrophage polarization

TLRs are a family of pattern recognition receptors (PRRs) that play a pivotal role in the innate immune response by recognizing conserved molecular patterns associated with pathogens (PAMPs) and endogenous danger signals (DAMPs).^{146, 147} TLR signaling is a crucial driver of macrophage polarization, particularly in M1 activation.^148-150 Engagement of TLRs by their respective ligands triggers the recruitment of adaptor proteins, such as myeloid differentiation primary response 88 (MyD88) and TIR-domain-containing adapter-inducing IFN-β (TRIF), which initiate downstream signaling cascades.^151-154 These cascades lead to the activation of transcription factors, including nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and IFN regulatory factors (IRFs), which drive the expression of proinflammatory genes and shape the M1 macrophage phenotype.^155-160 TLR4, the receptor for bacterial LPS, is a potent inducer of M1 polarization.^{159, 161} Upon LPS recognition, TLR4 activates both MyD88-dependent and TRIF-dependent pathways, producing proinflammatory cytokines and type I IFNs, respectively.^{162, 163} The MyD88-dependent pathway involves the activation of NF-κB and mitogen-activated protein kinases (MAPKs), such as p38, JNK, and ERK, which promote the expression of proinflammatory genes.^164-166 The TRIF-dependent pathway, on the other hand, activates IRF3 and IRF7, leading to the production of type I IFNs and the induction of IFN-stimulated genes.^167-169 Other TLRs, such as TLR2 (which recognizes bacterial lipoproteins) and TLR3 (which detects viral double-stranded RNA), also contribute to macrophage polarization. TLR2 signaling predominantly activates NF-κB and MAPKs, driving M1 polarization, while TLR3 activation leads to the production of type I IFNs and proinflammatory cytokines via the TRIF-dependent pathway.^170-172 Recent studies have revealed that TLR signaling can also modulate M2 polarization. For instance, activation of TLR2 and TLR4 has been shown to enhance the expression of M2 markers, such as ARG-1 and Ym1, in the presence of IL-4.^{150, 173} This suggests that TLR signaling can fine-tune macrophage polarization depending on the microenvironmental context and the presence of other polarizing stimuli. Targeting TLR signaling pathways has emerged as a promising therapeutic strategy for modulating macrophage polarization in various disease settings. For example, inhibition of TLR4 signaling has been shown to attenuate M1 polarization and promote M2-like phenotypes in models of inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease.^174-176 Conversely, activation of TLR3 signaling has been explored to boost antitumor immunity by promoting M1 polarization in tumor-associated macrophages (TAMs).^{177, 178}

3.2 STAT signaling: a key regulator of macrophage polarization

STAT proteins are transcription factors that play critical roles in cytokine signaling and macrophage polarization.¹⁷⁹ Different STAT proteins are activated by specific cytokines and regulate distinct aspects of macrophage function, with STAT1 and STAT6 being particularly important for M1 and M2 polarization, respectively.¹⁸⁰ STAT1 is activated by IFN-γ, a potent inducer of M1 polarization.^{181, 182} Upon IFN-γ binding to its receptor, Janus kinases (JAKs) are activated, leading to the phosphorylation and dimerization of STAT1. Activated STAT1 dimers translocate to the nucleus, where they bind to gamma-activated sequences in the promoters of target genes, driving the expression of proinflammatory and microbicidal factors, such as inducible iNOS and IL-12.^{182, 183} In contrast, STAT6 is activated by the Th2 cytokines IL-4 and IL-13, critical drivers of M2 polarization.^{184, 185} Engagement of IL-4 or IL-13 with their respective receptors leads to the activation of JAKs and the phosphorylation of STAT6. Phosphorylated STAT6 dimers translocate to the nucleus and bind to specific DNA sequences, promoting the expression of M2-associated genes, such as ARG-1, mannose receptor (CD206), and resistin-like molecule-α (FIZZ1).^{185, 186} The balance between STAT1 and STAT6 activation is a critical determinant of macrophage polarization, with the relative abundance of IFN-γ and IL-4/IL-13 in the microenvironment playing a key role.^{187, 188} Interestingly, STAT1 and STAT6 have been shown to antagonize each other's functions, with STAT1 activation suppressing M2 polarization and STAT6 activation inhibiting M1 responses.^{189, 190} This antagonism highlights the complex interplay between signaling pathways in shaping macrophage activation states. Targeting STAT signaling pathways has emerged as a potential therapeutic strategy for modulating macrophage polarization in various disease contexts. For example, inhibition of STAT1 signaling has been explored to attenuate M1 polarization and promote tissue repair in models of inflammatory diseases, such as multiple sclerosis and inflammatory bowel disease.^{189, 191} Conversely, activation of STAT6 signaling has been investigated as a potential approach to promote M2 polarization and resolve inflammation in conditions such as obesity and atherosclerosis.^{192, 193}

3.3 Nuclear receptors: transcriptional regulators of macrophage polarization

Nuclear receptors are a family of ligand-activated transcription factors that regulate macrophage polarization and function.¹⁹⁴ Two nuclear receptors, peroxisome proliferator-activated receptor-γ (PPARγ) and liver X receptors (LXRs), have been particularly implicated in modulating macrophage activation states. PPARγ is a master regulator of M2 polarization, promoting the expression of anti-inflammatory and tissue repair genes.¹⁹⁵ Activation of PPARγ by endogenous ligands, such as polyunsaturated fatty acids and eicosanoids, or synthetic agonists, such as thiazolidinediones, leads to the formation of heterodimers with retinoid X receptors.¹⁹⁶ These heterodimers bind to specific DNA sequences called PPAR response elements in the promoters of target genes, driving the expression of M2-associated factors, such as ARG-1, CD206, and IL-10.¹⁹⁷ PPARγ activation has been shown to antagonize M1 polarization by inhibiting the activity of proinflammatory transcription factors, such as NF-κB and AP-1.¹⁹⁸ This antagonism is mediated through various mechanisms, including direct protein–protein interactions, competition for coactivators, and induction of anti-inflammatory genes. Consequently, PPARγ agonists have been explored as potential therapeutic agents for modulating macrophage polarization in inflammatory diseases, such as atherosclerosis, obesity, and insulin resistance.^198-200 LXRs, including LXRα and LXRβ, are another nuclear receptor class regulating macrophage polarization and function. LXRs are activated by oxysterols and oxidized cholesterol derivatives and play critical roles in lipid metabolism and inflammation. Activation of LXRs has been shown to promote an anti-inflammatory M2-like phenotype in macrophages, characterized by increased expression of genes involved in lipid efflux, such as ATP-binding cassette transporters A1 and G1, and reduced production of proinflammatory cytokines.^{201, 202} LXR agonists have demonstrated anti-inflammatory and immunomodulatory effects in various disease models, including atherosclerosis, Alzheimer's, and autoimmune disorders.^{203, 204} These effects are mediated, in part, by the ability of LXRs to inhibit NF-κB signaling and promote the resolution of inflammation.²⁰⁵ As such, targeting LXR signaling has emerged as a potential therapeutic strategy for modulating macrophage polarization and function in inflammatory diseases.²⁰⁶

3.4 MicroRNAs: posttranscriptional regulators of macrophage polarization

miRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level by binding to complementary sequences in the 3′ untranslated regions of target mRNAs, leading to their degradation or translational repression.^207-209 Evidence suggests that miRNAs play crucial roles in regulating macrophage polarization and function. Several miRNAs have been identified as key regulators of M1 polarization, including miR-155, miR-125b, and miR-146a.^210-212 miR-155 is upregulated in M1 macrophages and promotes the expression of proinflammatory genes by targeting negative regulators of NF-κB signaling, such as suppressor of cytokine signaling 1 (SOCS1) and Src homology 2 domain-containing inositol-5-phosphatase 1.^{213, 214} miR-125b, on the other hand, inhibits M1 polarization by targeting the transcription factor IRF4, which is involved in the induction of proinflammatory cytokines.²¹⁵ miR-146a acts as a negative feedback regulator of M1 responses by targeting key components of the NF-κB signaling pathway, such as IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6.²¹⁶ Similarly, several miRNAs have been implicated in regulating M2 polarization, including miR-21, miR-124, and miR-223.²¹⁷ miR-21 promotes M2 polarization by targeting programmed cell death 4 (PDCD4), a negative regulator of IL-10 production. miR-124 is upregulated in M2 macrophages and promotes the expression of M2-associated genes, such as ARG-1 and FIZZ1, by targeting the transcription factor CCAAT/enhancer-binding protein-α.²¹⁸ miR-223 has been shown to promote M2 polarization by targeting Pknox1, a transcription factor that suppresses the expression of M2-associated genes.^{219, 220} The therapeutic potential of targeting miRNAs to modulate macrophage polarization has been explored in various disease models. For example, inhibition of miR-155 has been shown to attenuate M1 polarization and promote M2-like phenotypes in models of inflammatory diseases, such as rheumatoid arthritis and.^{221, 222} Conversely, overexpression of miR-21 or miR-124 has been investigated to promote M2 polarization and resolve inflammation in conditions such as sepsis and spinal cord injury.²²³

3.5 Metabolic regulation of macrophage polarization

Macrophage polarization is closely linked to metabolic reprogramming, with distinct metabolic profiles associated with M1 and M2 phenotypes.^{5, 224, 225} M1 macrophages rely on glycolysis and the pentose phosphate pathway to meet their energy demands and support their proinflammatory functions.²²⁶ In contrast, M2 macrophages primarily utilize oxidative phosphorylation and fatty acid oxidation for energy production.²²⁷ The mechanistic target of the rapamycin (mTOR) pathway is a central regulator of macrophage metabolism and polarization. mTOR complex 1 (mTORC1) is activated in M1 macrophages and promotes glycolysis through the induction of hypoxia-inducible factor-1α (HIF-1α) and the expression of glycolytic enzymes.^228-231 Inhibition of mTORC1 by rapamycin or genetic deletion of its component Raptor skews macrophages toward an M2 phenotype.²³² Adenosine monophosphate-activated protein kinase (AMPK), a key energy sensor, is crucial in regulating macrophage polarization.²³³ AMPK activation promotes M2 polarization by inhibiting mTORC1 and enhancing oxidative metabolism.^{234, 235} Metformin, an AMPK activator, has been shown to promote M2 polarization and alleviate inflammatory responses in various disease models.^{236, 237} Recent studies have also highlighted the role of lipid metabolism in macrophage polarization. Fatty acid synthesis is upregulated in M1 macrophages, while fatty acid oxidation is associated with M2 polarization.^{238, 239} PPARs, particularly PPARγ and PPARδ, are key regulators of lipid metabolism and have been implicated in promoting M2 polarization.²⁴⁰

3.6 Epigenetic regulation of macrophage polarization

Epigenetic modifications, such as DNA methylation and histone modifications, are crucial in regulating macrophage polarization by modulating the accessibility of polarization-associated genes.^{4, 240} M1 and M2 macrophages exhibit distinct epigenetic signatures contributing to their phenotypic stability and plasticity. Histone deacetylases (HDACs) have emerged as essential regulators of macrophage polarization.^{241, 242} HDAC3 has been shown to promote M1 polarization by deacetylating and activating NF-κB, while its inhibition skews macrophages toward an M2 phenotype.^243-245 In contrast, HDAC4 and HDAC5 have been implicated in promoting M2 polarization through the deacetylation of STAT6.^246-249 DNA methylation also plays a role in macrophage polarization. The DNA methyltransferase DNMT3b is upregulated in M1 macrophages and mediates the silencing of M2-associated genes.^{250, 251} Conversely, the demethylase TET2 promotes M2 polarization by demethylating and activating M2-associated genes.²⁵² Noncoding RNAs, such as miRNAs and long noncoding RNAs (lncRNAs), have also emerged as critical epigenetic regulators of macrophage polarization.²⁵³ For example, miR-21 promotes M1 polarization by targeting the anti-inflammatory cytokine IL-10, while miR-146a promotes M2 polarization by inhibiting NF-κB signaling.^{216, 254} LncRNAs, such as lncRNA-Cox2 and lncRNA-Mirt2, have been shown to regulate macrophage polarization by modulating the expression of polarization-associated genes^{255, 256} (Table 2).

3.7 Polarization of macrophages in some pathological processes

Macrophage polarization is crucial in various physiological and pathological processes, including host defense, tissue homeostasis, inflammatory diseases, and cancer. Understanding the signaling pathways that govern macrophage polarization can provide valuable insights into disease pathogenesis and guide the development of targeted therapies. In inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease, an imbalance between M1 and M2 macrophages contributes to chronic inflammation and tissue damage.^295-298 Targeting the signaling pathways that promote M1 polarization, such as TLR and NF-κB signaling, has shown promise in alleviating inflammatory responses in preclinical models. Conversely, promoting M2 polarization through activating STAT6 or PPARγ has been explored as a strategy to resolve inflammation and promote tissue repair. In cancer, TAMs often exhibit an M2-like phenotype and contribute to tumor progression by promoting angiogenesis, immunosuppression, and metastasis.²⁹⁹ Targeting the signaling pathways that drive M2 polarization in TAMs, such as colony-stimulating factor (CSF)-1/CSF-1R and IL-4/IL-13 signaling, has emerged as a promising therapeutic approach.^{300, 301} Reprogramming TAMs toward an M1-like phenotype through TLR or STING signaling activation has also shown potential in enhancing antitumor immunity.^{302, 303} In tissue regeneration and wound healing, M2 macrophages are crucial in promoting tissue repair and resolving inflammation. Harnessing the signaling pathways that promote M2 polarization, such as IL-4/STAT6 and IL-10/STAT3 signaling, has been explored to enhance tissue regeneration and limit fibrosis.^304-306

In summary, macrophage polarization is a dynamic and finely tuned process orchestrated by a complex network of signaling pathways. The integration of signals from TLRs, cytokines, and metabolic pathways shapes the functional phenotype of macrophages, allowing them to adapt to various microenvironmental cues. Recent advances in understanding the molecular mechanisms governing macrophage polarization have provided valuable insights into the role of these cells in health and disease. However, several challenges and opportunities remain in the field of macrophage polarization. The dichotomous M1/M2 classification, while applicable as a conceptual framework, oversimplifies the spectrum of macrophage activation states. Future studies should focus on delineating the complex heterogeneity of macrophage phenotypes and their functional implications in specific tissue contexts. Moreover, the crosstalk between signaling pathways and the influence of the tissue microenvironment on macrophage polarization warrants further investigation. Integrating multiomics approaches, such as transcriptomics, proteomics, and metabolomics, can provide a comprehensive understanding of the regulatory networks governing macrophage polarization. Translating the knowledge of macrophage polarization signaling into clinical applications remains a major challenge. Developing targeted therapies that modulate specific signaling pathways in macrophages while minimizing off-target effects is crucial. Nanoparticle-based drug delivery systems and engineered exosomes have shown promise in selectively targeting macrophages and modulating their polarization state.

4.1 The inflammatory phase: M1 macrophages as first responders

4.1.3 Debris removal and tissue remodeling

M1 macrophages are crucial in removing cellular debris and initiating tissue remodeling during the inflammatory phase.³³¹ They are essential for the clearance of apoptotic cells, a process known as efferocytosis, which prevents the release of potentially harmful intracellular contents and promotes the resolution of inflammation.^{314, 332} Furthermore, M1 macrophages initiate the remodeling of the extracellular matrix (ECM) by producing proteolytic enzymes, such as MMPs, and cytokines that regulate ECM turnover.³³³ This ECM degradation facilitates the removal of damaged or necrotic tissue and creates space for the subsequent influx of new cells and the deposition of a provisional ECM.^{334, 335} M1 macrophages also contribute to the initiation of angiogenesis, the formation of new blood vessels, by producing proangiogenic factors like VEGF, bFGF, and TNF-α.^{336, 337} These factors stimulate endothelial cell proliferation, migration, and the assembly of functional vascular structures (Figure 1).

4.2 The proliferation and remodeling phase: M2 macrophages facilitate tissue regeneration

4.2.3 Extracellular matrix remodeling and tissue regeneration

The proliferation and remodeling phase is characterized by the deposition and remodeling of the ECM, a complex network of proteins and polysaccharides that provide structural support and signaling cues for cell migration, proliferation, and differentiation.^362-366 M2 macrophages play a crucial role in regulating ECM remodeling and promoting tissue regeneration through the production of ECM components, regulation of ECM-remodeling enzymes, and modulation of fibroblast and stem cell behavior.^367-370

M2 macrophages contribute to the deposition and remodeling of the ECM by producing various ECM components, including: (a) Collagens: M2 macrophages secrete different types of collagens, such as collagen I, III, and IV, which are essential for the formation of the provisional ECM and the subsequent deposition of the mature ECM during tissue repair.³⁷¹ (b) Fibronectin: a glycoprotein that plays a crucial role in cell adhesion, migration, and ECM assembly. M2 macrophages produce fibronectin, which helps create a provisional matrix for cell migration and proliferation during tissue repair.³⁷¹ (c) Tenascin-C: an ECM glycoprotein highly expressed during tissue repair that promotes cell migration, proliferation, and angiogenesis. M2 macrophages secrete tenascin-C, which modulates the activity of various growth factors and cytokines, thereby regulating cellular processes in tissue regeneration.^{372, 373} (d) GAGs: M2 macrophages produce various GAGs, such as hyaluronic acid, heparan sulfate, and chondroitin sulfate, essential ECM components. GAGs interact with growth factors, cytokines, and ECM proteins, modulating their activity and regulating cellular processes in tissue repair and regeneration.^{97, 374} The deposition of a provisional ECM provides a scaffold for the recruitment and organization of various cell types, including endothelial cells, fibroblasts, and stem cells, enabling the formation of new tissue and restoring tissue integrity.^375-377 Furthermore, the ECM components produced by M2 macrophages play a crucial role in modulating the behavior of other cells involved in tissue repair, such as cell migration, proliferation, and differentiation, through interactions with integrin receptors and the modulation of growth factor and cytokine activity.

M2 macrophages modulate the activity of various ECM-remodeling enzymes, such as MMPs and tissue TIMPs, essential for ECM turnover and remodeling.³⁷⁸ During the proliferation and remodeling phase, M2 macrophages produce specific MMPs, such as MMP-2 and MMP-9, which facilitate the breakdown of existing ECM components, creating space for the deposition of new ECM and the migration of cells involved in tissue regeneration.^{379, 380} However, excessive and uncontrolled MMP activity can lead to excessive ECM degradation and impair tissue repair. To maintain a balance between ECM degradation and deposition, M2 macrophages also produce TIMPs, which are endogenous inhibitors of MMPs. TIMPs bind to and inactivate MMPs, regulating their proteolytic activity and preventing excessive ECM breakdown.^{381, 382} The production of specific MMPs and TIMPs by M2 macrophages is tightly controlled and depends on the stage of tissue repair and the microenvironmental cues present. For example, during the early stages of the proliferation and remodeling phase, M2 macrophages may produce higher levels of MMPs to facilitate the initial breakdown of the ECM and create space for new tissue formation.^{383, 384} As the tissue repair process progresses, M2 macrophages may shift toward producing higher levels of TIMPs to stabilize the newly formed ECM and promote tissue maturation.³⁸⁵ In addition to regulating MMPs and TIMPs, M2 macrophages modulate the activity of other ECM-remodeling enzymes, such as lysyl oxidases (LOXs) and transglutaminases. LOXs catalyze the cross-linking of collagen and elastin fibers, increasing the stability and mechanical strength of the ECM. In contrast, transglutaminases catalyze the formation of covalent cross-links between ECM proteins, further contributing to ECM stabilization and maturation^386-388 (Figure 2).

4.2.4 Macrophage plasticity and phenotypic transitions

While the M1 and M2 phenotypes represent the extremes of the macrophage activation spectrum, it is crucial to recognize that macrophages exhibit a remarkable degree of plasticity, capable of adopting a wide range of functional states along a continuum. This plasticity allows macrophages to adapt dynamically to the changing microenvironmental cues encountered during tissue repair and regeneration.^389-391 Recent studies have subdivided M2 macrophages into subgroups, including M2a, M2b, M2c, and M2d, based on their upstream activators and downstream gene expression patterns.^{103, 104, 392} For example, M2a macrophages are activated by IL-4 and IL-13, exhibiting increased expression of IL-10, TGF-β, and chemokines like CCL17, CCL18, and CCL22. In contrast, M2c macrophages are activated by glucocorticoids, IL-10, and TGF-β and exhibit increased transcription of IL-10, TGF-β, CCL16, and CCL18.^393-395 This classification highlights macrophages’ complex nature and ability to modify their gene transcription profiles along a continuous spectrum, especially in pathological situations. It is important to note that the M1 and M2 phenotypes represent simplified extremes of a heterogeneous and dynamic functional continuum rather than distinct and mutually exclusive populations. Furthermore, macrophages can undergo phenotypic transitions in response to changing microenvironmental signals, allowing them to adapt their functional programs to the evolving needs of the tissue repair process. For example, M1 macrophages may transition to an M2-like phenotype during the later stages of tissue repair, facilitating the resolution of inflammation and promoting tissue regeneration^396-398 (Figure 3).

4.3 Macrophages in tissue-specific repair and regeneration

While the general principles of macrophage involvement in tissue repair and regeneration are consistent across various organ systems, some tissue-specific nuances and mechanisms highlight the versatility and adaptability of these cells. Here, we will briefly discuss the roles of macrophages in the repair and regeneration of selected tissues, including skeletal muscle, liver, heart, and skin.

4.4 Macrophage dynamics in chronic wounds

Chronic wounds, characterized by their persistent inflammatory state and impaired healing, pose a significant challenge in clinical settings. Among the various types of chronic wounds, diabetic wounds stand out as a significant concern due to their increasing prevalence and the unique microenvironment that hinders the healing process.⁴⁴³ In recent years, the role of macrophages in the pathogenesis and resolution of diabetic wounds has garnered significant attention.^{62, 444} This section delves into the complex interplay between macrophages and the diabetic wound microenvironment, highlighting the mechanisms that influence macrophage phenotype and function and exploring potential therapeutic strategies targeting these interactions.

Diabetic wounds, particularly diabetic foot ulcers, are a common and severe complication of diabetes mellitus. The global prevalence of diabetic foot ulcers is estimated to be 6.3%, with a lifetime incidence of up to 25% among diabetic patients.^{445, 446} These wounds are characterized by a prolonged inflammatory phase, impaired angiogenesis, and delayed re-epithelialization, leading to a chronic nonhealing state. The unique microenvironment of diabetic wounds, shaped by hyperglycemia, oxidative stress, and the accumulation of advanced glycation end products (AGEs), significantly influences the behavior and function of macrophages, which are critical players in the wound healing process.^{447, 448}

5.1 Targeting the CSF-1/CSF-1R signaling pathway

The macrophage CSF-1 and its receptor (CSF-1R) signaling pathway play a crucial role in the maturation and transformation of TrMΦ, making it an attractive target for therapeutic^498-501 interventions. Stutchfield et al.⁵⁰² demonstrated that the administration of CSF1-Fc, an exogenous form of CSF-1, enhanced the recruitment and conversion of monocytes into protective hepatic macrophages, facilitating liver recovery after acute injury and partial hepatectomy. Furthermore, a systematic review and meta-analysis by Wei et al.⁵⁰³ provided robust evidence supporting the efficacy and safety of CSF in accelerating wound healing. In addition to CSF-related approaches, targeting chemokine receptors on monocytes and macrophages has shown promise in altering their migration patterns and impacting their function in tissue repair.^{169, 504} Promoting the survival of M2 macrophages, which play an essential role in tissue repair and angiogenesis, represents another promising strategy for enhancing tissue healing.

5.2 Modulating macrophage function through signaling pathways and transcription factors

Manipulating macrophage function by targeting specific signaling pathways and transcription factors has emerged as a promising therapeutic strategy for promoting tissue repair and regeneration. The TLR9 signaling pathway has been found to encourage macrophage M2 polarization in various models.⁵⁰⁵ Activation of the TLR9 pathway, such as with the agonist cobitolimod, induces a prohealing phenotype in macrophages, enhancing macrophage-mediated tissue healing in conditions like ulcerative colitis.⁵⁰⁶ Transcription factors, including NF-κB and MAPK, play critical roles in macrophage activation and polarization.^{507, 508} Inhibiting these signaling pathways has shown promising results in promoting M2 polarization and reducing inflammation. Puerarin, a compound from traditional Chinese medicine, has demonstrated the ability to inhibit NF-κB and MAPK pathways, leading to decreased production of inflammatory cytokines and promotion of M2 polarization in macrophages.^{99, 509, 510} Noncoding RNAs, particularly miRNAs, have also emerged as important macrophage polarization and activation regulators. Targeting specific noncoding RNAs provides a means to modulate macrophage function and influence tissue repair processes.^{511, 512} For instance, inhibiting the pro-M1 polarization molecule CRMP2 through small interfering RNA has demonstrated reduced local inflammation and fibrosis following MI.⁵¹³

5.3 Relay transfer and cell transplantation of macrophages

Targeting specific subpopulations of macrophages through relay transfer and cell transplantation has shown great potential for clinical treatment aimed at tissue repair and regeneration.⁵¹⁴ Lopes et al.⁵¹⁵ demonstrated the efficacy of modifying macrophages to express the M2 phenotype in vitro and subsequently transferring these M2 macrophages to a colitis mouse model, reducing inflammation and pathological damage. Similarly, Zheng et al.⁵¹⁶ stimulated macrophages to adopt the M2 phenotype using IL-4 and IL-13. They transplanted these M2 macrophages into a streptozotocin-induced diabetic mouse model, significantly reducing damage in the islets and kidneys. These studies highlight the potential of relay transfer and polarized macrophage cell transplantation to promote tissue repair and regeneration. However, further research is needed to fully understand the mechanisms of macrophage polarization and develop more precise and effective strategies for modulating macrophage function in vivo.⁵¹⁷ This includes identifying specific markers and signaling pathways that regulate macrophage polarization and investigating methods to optimize the survival and functionality of transplanted macrophages.

5.4 Biomaterial-based strategies for precise regulation of macrophage polarization

Recent advancements in biomaterials have enabled precise regulation of macrophage polarization phenotypes, specifically M1/M2, leading to enhanced tissue regeneration and accelerated wound healing.^518-523 Hydrogel-based constructs, known for their biocompatibility, tunable physical properties, and drug-delivery capabilities, have emerged as valuable tools for tissue repair.^524-526 Huang et al.⁵²⁷ developed curcumin-based metal-organic framework hydrogels that effectively downregulated M1 macrophage-related gene expression while upregulating anti-inflammatory gene expression, promoting the polarization of macrophages toward the M2 phenotype and facilitating the regeneration of blood vessels and nerves in chronic wounds. Henn et al.⁵²⁸ investigated xenotransplantation-mediated activation of Trem2+ macrophages, which promoted re-epithelialization and angiogenesis through growth factor secretion and contributed to collagen remodeling by secreting MMPs.³⁷⁸ By designing a soft pullulan-collagen hydrogel, delivery of Trem2+ macrophages obtained after vitamin D3 treatment to the wound bed showed great potential for clinical translation. Biomaterials offer a platform for precise modulation of macrophage function and polarization through tailored design and incorporation of specific cues, such as drug delivery systems or bioactive molecules.^529-532 Further research is needed to optimize the design and functionality of biomaterials to achieve better control over macrophage polarization and ultimately improve clinical outcomes in tissue repair and regeneration (Table 3).

5.5 Therapeutic potential and clinical applications

The therapeutic potential of macrophages in cell-based therapies has been demonstrated in various clinical trials and preclinical studies.^{522, 559, 560} Macrophages have outperformed stem cells in specific target diseases, showcasing their outstanding regenerative capacity.⁵⁶¹ Conditions such as kidney disease, stroke, arterial disease, and cancer have been targeted using macrophage-based therapies. Genetic modification of macrophages, such as the development of chimeric antigen receptor-macrophages (CAR-M), has further expanded the potential of genetically engineered macrophages for cell therapy.^562-564 The use of induced pluripotent stem cell (iPSC)-derived macrophages, macrophages loaded with nanoparticles, ex vivo polarization and adoptive transfer of macrophages, and surface-anchoring engineering of macrophages have also shown promising results in preclinical studies.^565-567 The therapeutic applications of macrophage CSF-1 have been explored in various contexts, including tissue repair after ischemia in the kidney and heart, promotion of angiogenesis, and elimination of amyloid deposits in the brain.^568-570 CSF-1 has been shown to promote a resident-type macrophage phenotype, making it a potential treatment for tissue repair (Table 4).

In summary, macrophages play a crucial role in tissue repair, regeneration, and fibrosis, making them attractive targets for therapeutic interventions. The ability to harness macrophages through various strategies, such as targeting specific signaling pathways, modulating macrophage function, relay transfer and cell transplantation, and biomaterial-based approaches, holds great promise for enhancing tissue repair and regeneration. Future research should further elucidate the mechanisms that instruct macrophages to adopt specific phenotypes and identify novel targets for therapeutic modulation. The development of more precise and effective strategies for modulating macrophage function in vivo and optimizing the survival and functionality of transplanted macrophages will be crucial for translating these findings into clinical applications. Additionally, the potential of genetically engineered macrophages, such as CAR-M, and using iPSC-derived macrophages warrant further exploration. Combining macrophage-based therapies with other therapeutic modalities, such as biomaterials and drug delivery systems, may also provide synergistic effects and improve clinical outcomes.