Macrophage plasticity and metabolic control in muscle repair and disease

Abstract

Inflammation is a tightly regulated process essential for skeletal muscle repair, and its dysregulation contributes to chronic disease and impaired regeneration. Following injury, muscle repair involves a coordinated immune response initiated by neutrophil infiltration, followed by macrophage recruitment and diversification. Rather than existing as discrete subsets, macrophages span a continuum of functional states that evolve over time in response to local environmental cues, enabling transitions from clearing debris and pro-inflammatory signaling to supporting resolution of inflammation, and remodeling and regeneration of the tissue. This functional plasticity is closely linked to intracellular metabolic programs. In this review, we examine how metabolic pathways, particularly the balance between glycolysis and oxidative phosphorylation, govern macrophage behavior through epigenetic mechanisms, thereby coupling cellular metabolism to inflammatory and regenerative gene expression. We further explore how these interconnected pathways are disrupted in chronic inflammatory muscle diseases, including muscular dystrophies. Recent transcriptomic studies highlight pathogenic macrophage populations with altered metabolic and epigenetic profiles that contribute to fibrosis and impaired regeneration. By integrating findings from both acute injury and chronic disease contexts, we provide a framework to explore macrophage function through a metabolic and epigenetic lens and discuss emerging strategies aimed at restoring macrophage plasticity and promoting the resolution of inflammation in muscle disease.

Introduction

Inflammation is a unique protective response in multicellular organisms that operates at a tissue, cellular, and molecular level to defend against injury, infection, and cellular damage (1, 2). While often associated with pain or illness, inflammation is essential for tissue healing and recovery, only becoming detrimental when it is uncontrolled, and becomes excessive or persistent, as is the case in a range of chronic health conditions (3–5). While inflammatory response is minimally detected in resting skeletal muscles, inflammation plays a critical and highly regulated role during recovery from strenuous activity and injury (6–8). A variety of immune cells, including leucocytes (e.g. neutrophils and macrophages) and lymphocytes work in concert to receive and transmit signals upon injury that regulate their temporal dynamics and regeneration of the injured muscle (9, 10). This acute inflammatory response helps remove damaged tissue and activate tissue regenerative processes to recover the lost muscle fibers (10–12). When properly controlled, inflammation allows muscle adaptation and improved function, while chronic inflammation, as seen in the context of disease, can interfere with recovery and contribute to muscle weakness and loss over time (10).

In the context of muscle repair, macrophages are widely studied for their role in clearing debris and supporting regeneration. Following their recruitment and activation, macrophages transition through a gradient of phenotypes ranging from pro-inflammatory to pro-regenerative states, which is guided by their niche, metabolism and epigenetic alterations (13–16). The binary pro-inflammatory (M1) and pro-regenerative (M2) macrophage profiles have commonly been used to describe the major polarization of macrophages throughout the waves of inflammation after injury (14). This M1 and M2 nomenclature fails to account for the complexity and diversity of macrophage functional profiles following acute injury (17). Thus, to better characterize this diversity, we propose the term Acute Inflammatory Macrophages (AIMs) to describe the range of transitory states of macrophages during the initial, pro-inflammatory phase of inflammation. Additionally, as the function of macrophages shifts to support tissue repair, we introduce use of the term Acute Regenerative Macrophages (ARMs) to describe these latter transitory phenotypes. Distinct from these acute (transitory) functional profiles, macrophages also appear in a range of chronically inflamed states due to systemic (e.g. metabolic imbalance, aging, etc.) or tissue-specific (e.g. myopathy, muscular dystrophy, etc.) factors. While several of their features overlap with AIMs, including the expression of certain transcripts, their stable persistence and accumulation within chronically-injured tissues, uniquely distinguish them from AIMs, and thus we suggest use of the label Terminal Inflammatory Macrophages (TIMs) to describe this pathogenic fate.

In this review, we will discuss the continuum of macrophage function in healthy acute muscle repair and describe the metabolic-epigenetic axis which helps regulate macrophage polarization and properties (Figure 1). Further, we will discuss the metabolic-epigenetic profiles of macrophages involved in chronic inflammation, revealing therapeutic avenues to support resolution of chronic inflammation and prevent muscle loss.

Figure 1.

Macrophage polarization is impacted by metabolic and epigenetic factors. Following injury, the muscle niche is altered and can impact the metabolism and therefore downstream epigenetic activity in macrophages. These changes alter gene expression and overall macrophage function, contributing to the interplay of several biological processes that must occur in a balanced and temporal manner to allow for proper inflammatory response, resolution and return to homeostasis.

Temporal regulation of inflammatory responses during acute injury and repair of skeletal muscle

A healthy muscle niche is comprised of a complex network of muscle and stromal cells including satellite cells (SCs), endothelial cells (ECs), fibroadipogenic progenitor cells (FAPs), and a variety of immune cells, all of which communicate with one another to maintain homeostasis in health or in response to acute injury (18, 19). Given the mechanical and load-bearing nature of muscle, it is unavoidable that there will be acute bouts of injury even in the healthiest context. Acute injury causes a temporary disruption to the homeostatic muscle niche. The way the niche responds to this disruption, particularly the temporal dynamics of inflammatory response and resolution of injury, governs the efficiency of tissue remodeling and regeneration following acute injury, and any deviation in stromal cell responses can delay repair and the return to homeostasis (10, 12, 20).

To understand the multifaceted role of inflammation within the context of acute muscle repair, it is important to recognize the temporal nature of inflammatory signaling through immune cell accumulation and clearance (Figure 2A). The first immune cells to enter the damaged muscle are neutrophils, which peak over 12-24 hours after injury (10, 12, 18, 20, 22) (Figure 2A). Recruitment of neutrophils from the vasculature after an injury is driven by the sudden and localized release of damage-associated molecular patterns (DAMPs) from the injured myofibers. Tissue-resident macrophages recognize these DAMPs and release inflammatory chemoattractants, including CXCL1/2 and 8, which help recruit neutrophils to the site of injury where they initiate the process of clearing debris (23–25). Additionally, they help reconstruct the damaged vascular network through activation of VEGF signaling to allow local access to required nutrients and an avenue for additional circulating myeloid and lymphoid cells to reach the damaged space (26). These activated neutrophils release factors like sIL-6Rα, which combined with local IL-6, lead to secretion of CCL2, promoting monocyte recruitment and differentiation into macrophages within the tissue for the next stage of the inflammatory process (25, 27–29) (Figure 2A). Although neutrophils are known to clear out relatively quickly after their initial recruitment, in some cases of injury or in disease, neutrophils may linger in areas of damage and continue to secrete pro-inflammatory signals, which can impede the temporal dynamics of inflammatory and repair processes mediated by macrophages and other stromal cell types thereafter (25, 30, 31) (Figure 2B).

Figure 2.

Cellular choreography following acute injury in skeletal muscle. Muscle injury triggers various cells to accumulate transiently for proper inflammatory and regenerative interactions for the eventual repair of the injured muscle. (A) In healthy muscle neutrophil accumulation peaks 12-24 hours following injury and they begin recruiting acute inflammatory macrophages (AIMs) to the site of injury. By 2-3 days, accumulation of AIMs peaks, clearing damaged myofibers and AIMs begin to adopt a more regenerative phenotype. By 5-7 days post injury, acute regenerative macrophages (ARMs) are abundant where they continue to promote the expansion of FAPs and the final stages of myofiber regeneration through SC differentiation and fusion by late timepoints post injury. (B) In dystrophic conditions, after injury response is mounted, AIMs fail to resolve toward an ARM like phenotype and terminal inflammatory macrophages (TIMs) persist at the site of injury, leading to chronic inflammation and fibrotic and adipogenic accumulation rather than regeneration of muscle. This schematic has been adapted from Hogarth et al. (21).

In healthy response to acute injury, after neutrophil activation, circulating monocytes and macrophages infiltrate the damaged area rich with proinflammatory cytokines, like IFNγ and TNF-α (25). These activated monocytes then polarize to take on an acute inflammatory macrophage (AIM) profile, characterized by high Ly6C expression and heightened secretion of pro-inflammatory cytokines (32, 33). After the initial wave of recruitment of Ly6C positive/high monocytes and AIM presence, the overall macrophage profile within the injured niche transitions towards a Ly6C negative/low state (33), where these acute regenerative macrophages (ARMs) support ECM remodeling and myofiber regeneration (16, 34).

The abundance of inflammatory macrophages (AIMs) peak within 3 days post injury and their phagocytic activities help clear additional debris as neutrophils depart the damaged site (10, 16, 35, 36) (Figure 2A). AIMs also release chemokines and cytokines which support subsequent pro-inflammatory processes including phagocytosis, while also activating myogenic processes via SC interactions (10, 11, 37–39). Several factors are involved in facilitating the switch of pro-inflammatory AIMs toward pro-regenerative ARMs, including molecular effectors like MKP1, AMPK, METRNL and IGF-1, in addition to the act of phagocytosing muscle fiber debris, which in itself stimulates AIM polarization towards a pro-regenerative phenotype (14, 40–43). These dynamics indicate the highly plastic nature of the macrophages in various states between AIMs and ARMs.

ARMs, which peak in abundance within the acutely injured muscle niche 5-7 days post injury, support remodeling of the local environment through stromal cell interactions and transient ECM deposition to facilitate myogenesis and the process of regeneration (10, 12, 21, 35, 44, 45) (Figure 2A). These macrophages differ from AIMs in their receptor expression, metabolism, as well as cytokine and chemokine production. Timely polarization of AIM- and ARM-like profiles is essential to the acute inflammatory response, as expression of a variety of anti-inflammatory cytokines like Il4, Il10 and Tgfb by ARMs not only reduces pro-inflammatory signaling but also actively halts recruitment of neutrophils and additionally promotes neutrophil apoptosis (35, 46). This is disrupted during chronic muscle inflammation caused by repeated injury and failed resolution of inflammation, resulting in accumulation and persistence of terminal inflammatory macrophages (TIMs). These cells share some features with AIMs and ARMs, but their inability to resolve contributes to improper muscle repair and long-term inflammation, driving the pathology of chronic inflammatory diseases like muscular dystrophies (47–50) (Figure 2B).

Along with circulating monocytes, which are recruited to the site of damage, prompted to mature into macrophages, and then activated towards inflammatory states, the tissue resident macrophages also participate in regulation of inflammation (51, 52). Resident macrophages are established in embryogenesis and remain in skeletal muscle through adulthood by self-renewal (53–55). Additional resident macrophages can also arise through replenishment via circulating blood monocytes (53, 55). Skeletal muscle resident macrophages help maintain homeostasis by controlling inflammation after small injuries, but their role after major acute injury, when many other macrophages enter the tissue, is not fully understood (51, 56, 57). However, the self-renewing resident macrophages function in the clearance of apoptotic cells in cases of acute injury, and also impact muscle fiber composition in instances of chronic mild injury (54). Additionally, resident macrophages can be activated towards alternative polarization profiles, which indicates that AIMs, ARMs and TIMs do not necessarily arise solely from the circulating monocytes, as demonstrated following inhibition of monocyte infiltration into damaged dystrophic muscle (35, 53, 58). Additional immune cells, including regulatory T lymphocytes and eosinophils also support muscle repair, but the monocytes and macrophages represent the majority of immune cells present in an injured muscle (18, 35, 59).

Macrophage and stromal cell interactions in damaged skeletal muscle repair

In addition to direct functional impacts on inflammatory processes in the injured muscle, over the course of repair, macrophages interact with various other stromal cell types critical for the repair of acutely injured muscle (Figure 2). Of the various non-immune, muscle-resident cells that macrophages interact with, critical cell types include SCs, ECs, and FAPs. Macrophages support the proliferation and/or differentiation of these cells within the damaged niche (10, 16, 45, 60, 61). These interactions are critical to the proper regeneration of muscle as well as remodeling of the ECM and vascular network and are regulated by distinct signaling pathways (Figure 3).

Figure 3.

Stromal cell interactions during acute muscle repair. Macrophages and other cells within the injured muscle niche interact and create a complex regulatory network to allow for the proper balance of pro and anti-inflammatory signaling. The schematic highlights some of the pathways involved in signaling interactions between damaged myofibers, neutrophils, AIMs, ARMs, ECs, FAPs, and SCs following an acute injury in the muscle.

Macrophage interactions with SCs allow for balanced expansion and differentiation of SCs for the regeneration of damaged muscle fibers. AIMs promote SC proliferation, and inhibit their differentiation through the release of cytokines like IL-6 and TNF-α, while secretion of anti-inflammatory cytokines released by ARMs, including IL-4 and IL-13, stimulate myoblast differentiation and fusion (16, 62–67) (Figure 3). In healthy steady state, SCs also recruit ECs via secretion of VEGFA, which in turn aid in the maintenance of SC quiescence (68). (Figure 3).

FAPs are muscle-resident mesenchymal stem cells that proliferate upon injury, where they can differentiate into fibroblasts or adipocytes (69–72). Their fate is tightly regulated by interactions with factors secreted in the muscle environment from cells including macrophages and SCs (71, 73, 74). Early after injury, FAPs rapidly proliferate and adopt a pro-regenerative phenotype enriched in follistatin (Fst), and secrete chemokines that influence immune cell fate and support myogenic cells (75) (Figure 3). As regeneration progresses, FAPs differentiate into fibroblasts to restore the ECM of the repairing muscle, while the absence of FAPs impairs myogenesis (76). Like macrophages, the change in FAP accumulation and activity during repair is tightly regulated. Inflammation supports FAP abundance and differentiation, such that early on following injury, eosinophils promote FAP proliferation via IL-4/IL-13 signaling, while later AIMs promote apoptotic FAP clearance through production of TNF-α (61, 73) (Figure 3). Early in the inflammatory phase, AIMs secrete matrix remodeling enzymes (e.g. MMPs) to locally degrade the ECM, while ARMs support FAP differentiation through TGFβ1 signaling (61) (Figure 3). This blocks TNF–induced FAP apoptosis and supports FAPs to differentiate and secrete the matrix around the newly forming myofibers (61). In chronically inflamed tissues, macrophage-FAP crosstalk causes accumulation and fibrotic differentiation of FAPs via TGFβ and IL-4 signaling (61, 75, 77). Depletion of ARMs or inhibition of TGFβ1 relieves myogenic suppression of FAP-derived Fst (75). Additionally macrophages and injured myofibers release Annexin A2 (AnxA2) which can promote differentiation of FAPs into adipocytes (78, 79) (Figure 3).

Along with being regulated by macrophages, FAPs regulate various cells in the muscle niche. For instance, FAPs increase their expression of Il10 following muscle injury which is a cytokine involved in initiating an ARM-like polarization of macrophages (16, 80, 81). Additionally, FAPs influence healthy SC expansion via WISP1, as well as their differentiation through Fst signaling (82, 83), while suppressing myogenic differentiation in chronically inflamed tissues (84). This highlights the tightly coordinated interplay between immune and myogenic responses, which is disrupted in diseased muscles (21, 84, 85) (Figure 3). In healthy muscles, blocking monocyte recruitment to injury sites contributes to impaired FAP clearance and dysregulated matrix deposition (e.g., collagen overproduction) and fibrosis, while restoring regulation of this in diseased muscles improves the efficiency of myogenesis (58, 61, 84–86).

Macrophage interactions with ECs impact vascularization after injury – culturing ECs with macrophages enhances formation of blood vessels through secreted factors (35, 87, 88). (Figure 3). Priming ECs with AIMs supports angiogenesis (87, 89), but increased ARM recruitment has been associated with improved vascular remodeling and an anti-angiogenic role (87, 90, 91). These findings support macrophage involvement in angiogenic signaling, but this depends on complex local and temporal interactions. Macrophage recruitment and polarization supports EC function and phenotype (35, 92). This includes impact on ECs such as prevention of endothelial to mesenchymal transition (EndMT) causing accumulation of ECs that support fibrosis (92). In particular, macrophage expression of Tnfα and Spp1 contributes to EndMT through TGFβ signaling, which is also activated in muscular dystrophies due to impaired AIM to ARM transition. This impacts EC crosstalk and causes excessive EndMT, fibrotic accumulation, and ineffective muscle regeneration (61, 92–94) (Figure 3). In addition to their interaction with macrophages, ECs also contribute to signaling with other stromal cells further illustrating the critical interplay of various cell-cell interactions within the muscle niche. For example, ECs support SC expansion and differentiation through secretion of growth factors like IGF-1, HGF and PDGF (95–97) (Figure 3).

Along with their role in signaling to other stromal cell populations in the muscle niche, macrophages interact with and are influenced by myofibers themselves. Necrotic myofibers release signals including HMGB1 which recruit phagocytic cells to induce clearance of damaged myofiber elements (98, 99) (Figure 3). AIMs recognize these signals and clear debris, which subsequently induces macrophages to transition from AIM to ARM-like states by decreasing TNF-α secretion and increasing TGFβ secretion, promoting progression towards resolution of inflammation and muscle regeneration (16).

Influence of the metabolic-epigenetic axis on macrophage polarization during muscle repair

Given the variety of roles macrophages play in the process of healing, their polarization needs to be carefully regulated both in terms of magnitude and timing to ensure the efficient regeneration of muscle after injury (35). Unlike the traditional binary approach to describe macrophage polarization defined based on in vitro studies (100), our use of AIMs and ARMs includes the spectrum of phenotypes that lie between the two exaggerated M1 or M2 states. In physiological settings, macrophages manifest a variety of intermediate states where often the canonical markers of both M1 and M2 macrophages can be co-expressed in injured tissue (101). Further, following acute injury in vivo, macrophage profiles that do not align with either binary state, indicating broader macrophage plasticity along their path to polarization from pro-inflammatory to pro-regenerative states (33, 34, 101, 102). This diversity is also noted in several dysregulated microenvironments associated with lipid, disease, tumor, and scar, where macrophages show unique transcriptional profiles related to the specialized state of the tissue where they exist (103–106). Additionally, given the acute nature of the AIM and ARM states, there is a temporal element to their polarizations which contributes to the intermediate phenotypes as they transition along the progression from inflammatory to regenerative.

One of the molecular basis underlying diversity of macrophage polarization across the inflammatory and regenerative spectrum is metabolism, which impacts macrophage profile through various cellular metabolites, whose levels alter downstream epigenetic changes (15, 20, 107) (Figures 1, 4). As metabolic flux can change continually and respond to environmental and internal cues, it allows for the plasticity of states along the gradient of AIMs and ARMs (Figure 4). During the early (inflammatory) state of the injured tissue, macrophages rely on glycolysis for energy, leading to increased production of reactive oxygen species (ROS) (108–110). Downstream impacts of this metabolic feature include IL-6 and IL-1B secretion via the ROS-PKM2-STAT3 axis (111, 112). Additionally, in AIMs the TCA cycle is disrupted due to downregulation of the gene encoding enzyme isocitrate dehydrogenase (IDH), leading to an accumulation of itaconate which inhibits succinate dehydrogenase (SDH) further driving accumulation of succinate (113, 114). The accumulation of these metabolites prevents degradation of HIF1α, a pro-inflammatory associated transcription factor that drives transcription of pro-inflammatory cytokines including Il1β, Il6, and Tnfα (115–117). Increased succinate accumulation has also been found to inhibit lysine demethylases (KDMs) via alpha-ketoglutarate (αKG) antagonization (118). One such regulator, KDM5, demethylates H3K4me3, which is a primary epigenetic marker for active gene transcription (119–121). Therefore, limiting the activity of KDM5, helps maintain H3K4me3 at the promoters of pro-inflammatory cytokines like Il6 and Tnfα to allow their continued expression (122). The Akt-mTOR-HIF1α axis also plays a role in macrophage activation, acting as a master regulator of glucose metabolism. mTOR signaling, specifically in the context of the mTORC1 protein complex, increases HIF1α activation which in turn increases glycolytic activity and the expression of its downstream pro-inflammatory cytokines (123–127) (Figure 4).

Figure 4.

The metabolic epigenetic axis of macrophage found in acutely injured muscles. The AIMs, exhibit increased utilization of glycolysis, mTOR signaling, as well as an accumulation of succinate and citrate increasing stability of HIFα and inhibiting activity of lysine demethylases (KDMs), which supports the transcription of pro-inflammatory factors like Tnfα, Il6 and Il1b. ARMs utilize OXPHOS and the TCA cycle, increasing accumulation of αKG leading to activation of KDMs increasing expression of Retnla and Mrc1. This also increases NAD+, activating SIRTs and lowering expression of pro-inflammatory factors like Tnfα, Il6 and Il1b. Additionally, mTOR signaling in this case can prompt expression of pro-regenerative markers through S6K activation. Increased AMPK promotes Sirt activity as well, further inhibiting the expression of transcription factors Hifα and Nfkb as well as their downstream pro-inflammatory genes.

As the reparative process progresses, macrophages transition into ARMs, where they rely on oxidative phosphorylation (OXPHOS) as the primary source of energy (108, 109, 128). Additionally, while not essential for their pro-regenerative polarization, these macrophages increase fatty acid oxidation utilization (129–131). Unlike AIMs, the TCA cycle remains intact in ARMs, and its function, along with glutaminolysis, leads to accumulation of αKG in these macrophages (132–134). αKG activates KDMs, such as KDM6, which removes the repressive methylation mark H3K27me3 leading to increased expression of pro-regenerative genes like Retnla, Mrc1, and Il6, including in macrophages (134–138). Another cofactor produced during OXPHOS that links metabolism and epigenetics is NAD+, which is more abundant in ARMs than AIMs (139). NAD+ is a cofactor for Sirtuins (Sirt) – a protein modifying enzyme family with various epigenetic modifying functions (140, 141). Sirt1 alters gene expression at the epigenetic level by histone modifications which impact chromatin accessibility, and at the post-translational level through its protein deacetylation activity. Sirt1 activity is largely dependent on NAD+ levels with higher activity promoting a pro-regenerative macrophage phenotype (142). Another metabolic signal that supports the transition from AIMs to ARMs is AMPKα1 (14, 143). Increased Sirt1 activity by NAD+ helps repress inflammatory transcription factors HIF1α and NFKB1 (143). This in turn promotes pro-regenerative polarization of macrophages by AMPKα signaling, reducing chronic inflammatory response and supporting SC differentiation in muscle (142–146). This protein kinase acts as an energy sensor, activating (and preserving) OXPHOS to promote ATP synthesis (147, 148). As a result, AMPK activates Sirt1 through accumulation of NAD+, but also by increasing Sirt1 expression directly, contributing to the pro-regenerative fate of the ARMs (149–151) (Figure 4). Although mTOR signaling supports AIM associated transcriptional pathways, mTOR can also affect pro-regenerative polarization through the Akt-mTOR-S6Kinase axis in the presence of Substance-P or increased αKG (152, 153). However, mTOR signaling impacts a variety of downstream targets, allowing its context-dependent actions that promote AIM or ARM states (124).

Dysregulated macrophage polarization and lack of inflammation resolution in muscle disease

During muscle repair, macrophages transition between a spectrum of AIM to ARM states through metabolic variation and local inflammatory cues. A break in the connection between metabolism and gene regulation can shift the niche into pathogenic states and prevent the normal progression of the tissue reparative process. Such dysregulations are associated with a variety of chronic inflammatory conditions including atherosclerosis, diabetes, rheumatoid arthritis, Alzheimer’s disease, and muscular dystrophies (154–158).

Muscular dystrophy is characterized by a propensity for excessive muscle damage, often accompanied by diminished repair capacity and progressive degeneration, leading to persistent, unresolved inflammation with sustained pro-inflammatory cytokine and chemokine signaling (40, 155–160). These deficits prevent the tissue from regenerating efficiently and are associated with prolonged presence of neutrophils and macrophages, which can further damage healthy muscle tissue, limit subsequent regeneration, and exacerbate fibrotic and adipogenic replacement of muscle (Figure 2B) (6, 22, 23, 31, 161, 162).

Recent single cell, single nuclei, and spatial transcriptomic and proteomic studies have identified disease-specific macrophages across muscular dystrophies and myopathies missing from the healthy muscles (18, 47–49, 92, 162, 163). Characterization of the transcriptional profile of these cells from models of varying disease severity, ranging from models of LAMA2 congenital muscular dystrophy to Duchenne muscular dystrophy, have identified similar macrophage population enriched in disease conditions (47, 49, 50). Here, instead of inflammation being resolved in a timely manner, as is the case in acute injury scenarios (Figure 5), these disease-associated (pathogenic) macrophages persist and progressively accumulate within the tissue with disease progression, leading to chronically inflamed tissue. In this chronic inflammatory state, inflammatory signaling by TIMs prevents the tissue from returning to its homeostatic state (Figure 5). These pathogenic macrophages are detected not only in chronically inflamed muscles, but also in chronically inflamed and pro-fibrotic brain, liver, lungs, and adipose tissues, and are associated with tumors or enriched during aging (103, 169–177). This highlights the broad relevance of TIMs across tissues and disease conditions, as well as the need to better understand their origin and fate. These cells have been shown to arise from both monocytes recruited to damaged tissue, as well as from tissue resident macrophages, suggesting that their origin is associated with signals stemming from within the tissue micro-environment, rather than an innate feature of macrophages or their progenitors (58).

Figure 5.

Temporal and molecular features of macrophages in injured muscles. (A) Following an acute muscle injury, infiltrating monocytes adopt an acute inflammatory macrophage (AIM) phenotype, which peaks around 3 days post injury. Then this phenotypic signature fades, as AIMs adopt an acute regenerative macrophage (ARM) phenotype, through intermediate functional phenotypes. The ARMs become the dominant macrophage around 5 days post injury and their phenotypic signature peaks before the resolution of inflammation. (B) In chronically inflamed tissue, AIMs fail to resolve and give rise to TIMs which not only persist for an extended period in the tissue but also impair muscle regeneration. (C) Aside from their temporal features, these macrophages also exhibit molecular, metabolic and epigenetic features summarized in the table. Gene expression markers of AIMs include Tnfa, Il6, Il1b, Spp1, Lgals3, Trem2, Gpnmb, and Fabp5 (16, 111, 115, 162, 164–167). Markers of ARMs include Mrc1, Arg1, Retnla, Cd163, Tgfb, and Lgals3 (16, 134, 136, 152, 166, 168). Markers of TIMs include Spp1, Lgals3, Trem2, Gpnmb, and Fabp5 (47–50).

Importantly, TIMs are characterized by heightened expression of Spp1, Lgals3, Trem2, Fabp5 and Gpnmb regardless of condition (47–50). Many of these markers upregulated in TIMs are known to contribute to fibrotic or adipogenic signaling through their impact on neighboring cells within the niche. Macrophages expressing Spp1 interact with muscle fibroblasts and drive fibrosis through heightened secretion of osteopontin, fibronectin, and semaphorins (178–180). This is the case in multiple disease conditions throughout various tissues where macrophages with high Spp1 expression contribute to fibrotic deposition in lung, liver, heart, endometrium, kidney, skin and dystrophic muscle (20, 148, 156–158). Expression of Trem2 has also been associated with fibrosis especially in the context of pulmonary diseases, although in other tissues the connection to fibrosis is not clear, and depending on the context, can confer both protective or pathogenic attributes (181–185). In addition, some of the markers associated with TIMs, for instance Lgals3, Spp1, and Trem2, are also known to play a role in promoting adipogenesis and fat accumulation in disease (186–188).

Interestingly however, many of the genes associated with TIMs are transiently expressed by AIMs or ARMs during the acute muscle repair process (Figure 5). For example, macrophages present in a muscle 3 days post injury (AIMs), transiently express Spp1 and Trem2, similar to macrophages in acutely injured spinal cord, but these macrophages transiently expressing Spp1 and Trem2 do not cause fibroadipogenic tissue degeneration (162, 164, 181, 189). Similarly, Gpnmb and Spp1 expressing macrophages are involved in lung repair processes, and it is suggested that their propensity to induce fibrosis is a vestige of a temporal mechanism of their appearance and clearance, not their intrinsic feature (165). Lgals3, another TIM marker, is increased in AIMs and ARMs, indicating that acute Lgals3 expression is not inherently pathogenic (166, 168). Other in vitro studies show Gpnmb and Fapb5 increase upon pro-inflammatory stimulation with LPS or IFNγ, with Gpnmb associated with a negative feedback loop for pro-inflammatory signaling, while Fabp5 deletion promotes transition towards a pro-regenerative phenotype (167, 190). The transient expression of these TIM-enriched genes thus seems to play a role in in a properly regulated acute inflammatory response. This further supports the notion that environmental factors in diseased tissue underlie this inability of TIMs to exit a pro-inflammatory state in disease conditions.

One potential mechanism by which the diseased tissue niche may push macrophages toward the TIM state is through dysregulation of the metabolic–epigenetic axis driven by the aberrant metabolic signature of chronically inflamed tissues (47, 142, 191). Such metabolic disturbances may interfere with epigenetic programs required for gene expression associated with inflammation resolution. For example, Trem2-expressing macrophages in diseased tissues exhibit altered lipid metabolism and related changes that influence metabolite accumulation, thereby reshaping the macrophage epigenetic landscape (191). TIMs also show increased expression of Fabp5, which limits fatty acid accumulation and restricts TCA cycle activity and OXPHOS, which may ultimately restrict their timely transition to ARMs (192). Although the expression of these signature genes is not limited to TIMs, their combined and prolonged expression likely promotes extended macrophage accumulation and pathogenic interactions within the stroma of the diseased tissue niche. The interplay between environmental cues and the metabolic–epigenetic profile of TIMs remains an active area of investigation, and defining the full complement of factors that prevent inflammation resolution is critical to clarify how metabolism contributes to TIMs in disease and whether these cells can be therapeutically redirected toward a more AIM-like phenotype for efficient resolution.

Therapeutic approaches to target dysregulated inflammatory responses in muscle disease

Therapeutic endeavors for chronic inflammatory myopathies, as well as other inflammatory diseases, have primarily focused on the suppression of inflammation. For instance, the use of anti-inflammatory glucocorticoids (GC) is the standard of care for pediatric DMD patients as it improves patients’ longevity and quality of life by limiting inflammation and associated pathologies (193–195). Despite the positive impact of GC therapy, their long term use is associated with a variety of serious adverse effects, and can even increase muscle wasting and weakness over time (196, 197). In fact, broad ablation of inflammatory processes is detrimental to myogenic capacity in dystrophic muscle, where spontaneous damage continues to occur frequently with disease course (198). Although GCs effectively inhibit macrophage polarization toward AIMs, in diseases dominated by tissue inflammation, the use of GCs can also suppress the expression of ARM markers (199) further limiting repair processes. As acute inflammation is critical for efficient muscle regeneration and a return to homeostasis after injury, broad therapeutic inhibition may not be as beneficial as promoting resolution of chronic inflammation in disease. This is supported by the recognition that TIMs are transcriptionally similar to AIMs, but temporally dysregulated as they fail to clear following tissue injury (Figure 5).

A therapeutic approach to target TIMs and stem the impact of chronic inflammation is termed “Resolution Pharmacology”, which leverages existing resolution pathways to clear inflammation and promote repair (200). Such pro-resolution pathways include Resolvins and the Anxa1-FPR2 signaling axis which is involved in regulation of the initial inflammatory signaling of neutrophils and also the timely clearance of both neutrophils and pro-inflammatory macrophages after injury (200–203). AnxA1 signaling has been implicated not just in the appearance of ARMs but plays a role throughout the entire inflammatory process. For example, in the absence of Anxa1, there is reduced inflammatory signals like IL-1β and TNF-α upon LPS stimulation, indicating the necessity for Anxa1 in both the AIM and ARM states (204). Pro-resolving therapies, including small molecule and mimetic peptide agonists of FPR2, have been shown to have beneficial effects following myocardial infarction and on disease pathogenesis in dystrophic cardiac and skeletal muscle, where it effectively cleared inflammation and promoted enhanced repair (143, 205, 206). Additionally, use of the pro-resolving mediator Resolvin D1 showed regenerative improvements following healthy skeletal muscle injury (207). Thus, targeting resolution of inflammation, rather than blunting inflammation altogether, may be the key to balancing the benefits of acute inflammation while preventing the pathogenic effects of chronic TIM persistence.

Metabolic and epigenetic impacts of therapeutic interventions for muscle disease

Treatment with GCs impacts the metabolic-epigenetic axis of macrophages in vitro, such that dexamethasone represses glycolytic usage in macrophages polarized towards AIMs, but also represses mitochondrial respiration of macrophages polarized towards ARMs (208). GCs mitigate inflammatory response of macrophages by promoting TCA cycle flux, thus preventing accumulation of succinate which is a known stabilizer of pro-inflammatory transcription factor HIF1α (209–211). Additionally, GCs have been shown to decrease glycolytic activity of pro-inflammatory macrophages in culture (210). These findings show that treatment with GC can shift the metabolic profile of macrophages away from a pro-inflammatory phenotype.

Pro-resolving therapies also impact the metabolic-epigenetic axis of macrophages. One such therapy that targets the AnxA1-FPR2 axis, promotes macrophage transition from AIMs to ARMs in part by with altering their metabolism via AMPK signaling (143). As previously described, AMPK is known to increase OXPHOS activity and promote Sirt activity. ANXA1-FPR2 binding activates AMPK, connecting this pro-resolution therapy to mechanisms of metabolic and epigenetic regulation of macrophage phenotype (143). Unlike pro-resolving therapies that modulates AMPK activity, GC therapies do not appear to regulate AMPK activity itself (212).

Given the ability of the ANXA1-FPR2 axis to alter the metabolic-epigenetic axis of macrophages through AMPK signaling and Sirt1 activity, an additional approach to promote resolution of inflammation is activating Sirt1 either directly through small molecules or by increasing NAD+ availability to trigger the transition towards ARMs (213–215). This approach has been attempted in a variety of inflammatory diseases and has shown promise in disease models and in pre-clinical trials in dampening the inflammatory response in patients (216–218). Additional testing is needed to confirm the efficacy and specificity of the SIRT1-targeting drugs and develop more potent and specific options to combat longer term inflammation without negative effects on the rest of the system (219).

Discussion

Macrophages in skeletal muscle are dynamic regulators of repair whose behavior emerges from integrated metabolic and epigenetic programs rather than fixed categories. By connecting acute injury responses with chronic disease pathology, we highlight macrophage plasticity as a central requirement for successful regeneration and identify loss of this plasticity as a key feature of persistent inflammation and fibrosis in disease. A major implication of this mode of macrophage action is the need to move beyond marker-based classification toward a continuum-based model in which functional states are linked to metabolic and other molecular transitions. This includes the shift between glycolysis and oxidative phosphorylation being the spectrum that guides epigenetic regulation of inflammatory and regenerative gene programs. This understanding supports greater use of multi-omics and spatial technologies to track macrophage states over time and across tissue niches, with particular focus on how environmental cues drive divergence into resolving or pathogenic trajectories. This is especially relevant in chronic diseases such as muscular dystrophies where these trajectories are stalled or disrupted. Such a framework supports efforts to define disease-associated macrophage populations that arise from disrupted metabolic and epigenetic control and contribute to failed regeneration. Understanding how these states are maintained may guide strategies to interrupt maladaptive signaling loops that sustain inflammation and fibrosis by shifting therapeutic direction from broad immunosuppression toward interventions that restore macrophage plasticity and promote inflammation resolution. Targeting metabolic and epigenetic pathways is therefore positioned as a promising avenue for reestablishing tissue regenerative capacity in muscle and related chronic inflammatory diseases.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. National Institutes of Health (R01NS136349), The Department of Defense Congressionally Directed Medical Research Programs (HT94252510342, HT94252510341), The Jain Foundation, The Muscular Dystrophy Association.

Author contributions

ST-G: Conceptualization, Visualization, Writing – original draft, Writing – review & editing. JN: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing. JJ: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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