DMD is the most common form of dystrophinopathy, a severe, X-linked recessive genetic disorder characterized by progressive muscle degeneration, chronic inflammation, and fibrosis. DMD is the most prevalent type of muscular dystrophy and results from mutations in the dystrophin gene, which is an essential structural component of muscle fibers [7].

Dystrophin plays a critical role in maintaining the structural integrity of the muscle cell membrane by linking the actin cytoskeleton to the dystrophin-associated glycoprotein complex (DGC), a multiprotein complex crucial for muscle function and membrane stability during contraction [8, 9]. In the absence of functional dystrophin, muscle cells cannot withstand the mechanical stress caused by repeated contractions, leading to cellular damage and leakage of intracellular components. This triggers degeneration and regeneration, where damaged muscle fibers attempt to repair, but ultimately fail, resulting in progressive muscle weakness [10]. The weakness primarily affects proximal muscles, including those of the hips, thighs, and shoulders, and is often noticeable in early childhood as delayed motor milestones, such as difficulty in standing, walking, and running [8].

As the disease advances, muscle weakness worsens, leading to loss of ambulation by early adolescence. Most patients are unable to walk between the age of 12 and 14 [11]. DMD is often accompanied by severe complications, including respiratory failure and cardiomyopathy, which are the primary causes of morbidity and mortality in affected individuals [8, 12]. As muscle fibers continue to deteriorate, they are progressively replaced by fibrosis and adipose tissue, further impairing muscle function and mobility [13]. This replacement of healthy muscle tissue exacerbates the disability and significantly reduces the patient’s quality of life, ultimately leading to premature death, typically due to cardiac and respiratory failure in the late teens or early twenties [13].

The mdx mouse strain serves as a widely used animal model for human DMD. Identified in the early 1980s within a colony of C57Bl/10ScSn mice, the primary mdx mouse model (C57Bl/10ScSn-Dmd^mdx) carries a spontaneous nonsense point mutation (C>T) within exon 23 in the dystrophin (Dmd) gene, resulting in a premature stop codon [14, 15]. Consequently, mdx mice exhibit a deficiency of functional dystrophin in their muscles. Similar to human patients, these mice display muscle fiber necrosis and degeneration, elevated levels of serum creatine kinase, and a gradual onset of muscle weakness [14]. The mdx mice closely mirrors the pathology observed in affected humans, making it an invaluable tool for studying disease mechanisms and potential therapeutic interventions [16].

Dysferlinopathy refers to a group of muscle disorders caused by mutations in the DYSF gene, which encodes the dysferlin protein. These disorders include limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM) [17]. Dysferlin is 237 kDa membrane protein that plays a crucial role in repairing muscle cell membranes, stabilizing calcium signaling, and supporting the development of the T-tubule system essential for muscle function [18–20]. The absence or dysfunction of dysferlin disrupts these processes, leading to progressive muscle degeneration, impaired calcium signaling, and chronic inflammation [18, 21, 22].

LGMD2B primarily affects the proximal muscles of the shoulders, hips, and limbs, causing weakness that typically manifests in adolescence or early adulthood. In contrast, MM often begins with weakness in the distal muscles, particularly in the calves [17]. While LGMD2B progresses more slowly than DMD, it can still result in severe disability and loss of ambulation in later stages. Unlike DMD, which predominantly affects boys, dysferlinopathy affect both men and women equally [18, 23].

In dysferlin-deficient muscles, the inability to repair sarcolemma damage leads to repeated phases of muscle breakdown and regeneration, exacerbated by inflammatory responses. These mechanisms further contribute to muscle atrophy and functional impairment over time [24]. Although no definitive cure exists, ongoing research into gene therapy, stem cell treatments, and other therapeutic approaches shows promise for managing symptoms and improving quality of life for patients with dysferlinopathy. Dysferlinopathy has been extensively studied using the A/J mouse strain and its backcross variants [25]. The deficiency of dysferlin in A/J mice arises. This condition in A/J mice arises from a homozygous retrotransposon insertion in the dysferlin (Dysf) gene [26]. Muscular dystrophy in these mice typically manifests later in life, around four to five months of age, and is progressive. The muscle pathology is characterized by repeated myofiber degeneration and regeneration, with centrally positioned nuclei [25]. Notably, the proximal muscles in A/J mice are more severely affected by this condition compared to the distal muscles.

In DMD, macrophages infiltrate the damaged muscle tissue in response to continuous muscle degeneration and regeneration. The persistent muscle damage leads to the recruitment of various immune cells, with macrophages being one of the most significant contributors. Initially, pro-inflammatory M1 macrophages dominate the damaged area, releasing a cascade of cytokines, including TNF-α, IL-1β, and IL-6. These cytokines not only amplify the inflammatory response but also directly contribute to the degradation of muscle fibers by inducing oxidative stress and promoting apoptosis of myocytes [40].

M1 macrophages are particularly specialized for free-radical production, driven by the expression of inducible nitric oxide synthase (iNOS). iNOS is induced by TNF-α through the activation of the NF-κB signaling pathway, resulting in the production of nitric oxide (NO) and citrulline [41]. The high levels of NO generated by iNOS disrupt the redox environment within the injured tissue, react with other free radicals, and amplify their reactivity and toxicity. This contributes to the lysis of muscle cell membranes, which has been demonstrated in vitro using mdx muscle-derived macrophages, where iNOS inhibition prevented muscle cell damage [41]. Similarly, mdx mice, a murine model of human DMD, with an iNOS-null mutation exhibited reduced muscle membrane damage during the acute phase of pathology [41]. However, the partial reduction of damage highlights the involvement of iNOS-independent mechanisms, emphasizing the multifactorial role of M1 macrophages in driving muscle pathology [42].

The regulation of M1 macrophage activation in DMD involves multiple interconnected signaling pathways. The activation of M1 macrophages in DMD is regulated through multiple signaling pathways triggered by DAMPs and pro-inflammatory cytokines. DAMPs, such as high-mobility HMGB1 and extracellular ATP released from damaged muscle fibers, bind to PRRs like Toll-like receptor 4 (TLR4) on macrophages [43–45]. This interaction activates downstream signaling cascades, including the MyD88-dependent pathway, which leads to the activation of NF-κB and the production of pro-inflammatory cytokines such as TNF-α and IL-1β [46]. Additionally, ATP binding to purinergic receptors (e.g., P2X7) enhances NLRP3 inflammasome assembly, promoting the secretion of IL-1β and IL-18, further amplifying the inflammatory response [47, 48]. Furthermore, cytokines such as IFN-γ, predominantly secreted by infiltrating T cells, reinforce the M1 phenotype by enhancing STAT1 signaling in macrophages and also suppressing M2 macrophage activation [49–52]. Together, these pathways sustain the pro-inflammatory state of M1 macrophages in DMD, perpetuating oxidative stress and muscle damage.

Additionally, macrophage-secreted chemokines such as CCL2 play a pivotal role in enhancing the recruitment of more immune cells, perpetuating the inflammatory environment and sustaining the dominance of M1 macrophages during early stages of the response [53]. This inflammatory state leads to further damage by recruiting neutrophils and other cytotoxic immune cells into the dystrophic tissue.

In parallel, M2 macrophages, which are involved in anti-inflammatory and reparative processes, are recruited as a counter-regulatory mechanism. These macrophages produce IL-10 and TGF-β to attenuate inflammation and support muscle regeneration [54]. However, in DMD, the dynamic balance between M1 and M2 macrophages is disrupted, resulting in a chronic inflammatory state. This disruption is exacerbated by chronic deterioration and attempted muscle renewal, which prevent the resolution of inflammation, leading to fibrosis and impaired tissue repair. Consequently, prolonged M1 macrophage activity amplifies muscle damage and perpetuates the pathological cycle [54].

This imbalance is exacerbated by the repeated cycles of degeneration and regeneration, which prevent effective resolution of inflammation and hinder proper tissue repair. Consequently, the prolonged presence of M1 macrophages and their cytotoxic activity further aggravates muscle damage, perpetuating the pathological cycle of inflammation and fibrosis.

In dysferlinopathy, defective membrane repair caused by mutations in the dysferlin gene results in chronic muscle damage, creating a sustained trigger for macrophage recruitment and activation [31]. Pro-inflammatory M1 macrophages are predominant during the early stages of inflammation, releasing cytokines such as TNF-α and IL-1β, which amplify the inflammatory response and exacerbate muscle fiber damage [55, 56]. The persistent activation of M1 macrophages leads to the production of reactive oxygen species (ROS), further contributing to oxidative stress and destabilizing the muscle environment [57].

Macrophage activity in dysferlinopathy is significantly influenced by dysregulated signaling pathways, particularly those involving TLRs. MyD88, an adapter protein critical for TLR signaling, plays a central role in the recruitment of macrophages to dysferlin-deficient muscles. When muscles are exposed to DAMPs such as ssRNA, MyD88-dependent signaling through TLR7 and TLR8 is upregulated, driving macrophage activation and invasion. Although MyD88 deletion reduces inflammatory cell recruitment in the presence of DAMPs, its ablation has minimal effect in the absence of external stimuli, suggesting that additional mechanisms contribute to macrophage-driven inflammation in dysferlinopathy [57].

Thrombospondin-1 (TSP1), a TLR4 ligand, further modulates macrophage activity in dysferlin-deficient muscles. TSP1 is expressed at elevated levels in dysferlin-deficient myotubes and activates TLR4/NF-κB signaling in macrophages, enhancing their inflammatory responses. Importantly, this increase in TSP1 expression is directly linked to dysferlin deficiency rather than being secondary to muscle degeneration. Moreover, TSP1 also acts as a chemoattractant for macrophages, and its neutralization significantly reduces monocyte/macrophage recruitment to dysferlin-deficient muscle cultures, underlining its specific role in macrophage-driven inflammation [57–59].

Ultimately, the persistent activation of macrophages and their dysregulated signaling pathways contribute to the chronic inflammatory environment in dysferlinopathy. This prolonged inflammation not only exacerbates muscle degeneration but also impairs effective tissue repair. Thus, macrophage-driven inflammation emerges as a critical driver of disease progression, reinforcing the need for therapeutic interventions targeting these pathways [57–59].

Fibrosis is a pathological hallmark of dystrophinopathy and dysferlinopathy, resulting from the complex interaction between immune responses and tissue repair mechanisms, with macrophages playing a pivotal role. These immune cells regulate the local microenvironment, influencing both inflammation and fibrosis through dynamic functional phenotypes.

In DMD, macrophages undergo dynamic polarization, transitioning between the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype. Early in the disease process, M2 macrophages promote tissue repair by aiding in the removal of debris and stimulating muscle regeneration. However, chronic activation of M2 macrophages over time results in excessive secretion TGF-β, a key cytokine for fibroblast activation [60, 61]. TGF-β signaling enhances the synthesis and deposition of ECM components, such as collagen, which progressively replaces functional muscle tissue with fibrotic scar tissue. This pathological remodeling disrupts muscle elasticity and impedes effective force transmission, worsening muscle weakness and loss of function. As the disease progresses, the continuous cycle of muscle injury and ineffective regeneration fosters the accumulation of fibroadipose tissue, further impairing muscle performance and mobility [62], reinforcing the cycle of fibrosis and muscle degeneration in DMD.

Unlike typical muscle injury, where macrophage polarization follows a clear timeline towards tissue repair and regeneration, DMD features an ongoing cycle of muscle damage with insufficient repair. This results in a sustained inflammatory response, where macrophages often fail to transition fully into the regenerative M2 phenotype. Instead, there is a persistent presence of both M1 and M2-like macrophages, with a shift towards a pro-fibrotic M2 phenotype in the later stages of the disease. This shift is marked by the upregulation of arginase-1 (Arg1) and other fibrosis-associated molecules, which promote collagen deposition in the muscle, exacerbating fibrosis and impairing the ability of muscle satellite cells to regenerate myofibers [41, 63].

In dysferlinopathy, a similar inflammatory and fibrotic response is observed, though the underlying mechanisms differ due to the dysferlin deficiency. Dysferlin is essential for proper membrane repair in muscle fibers, and its absence leads to chronic muscle damage and inflammation [64]. Macrophages in dysferlinopathy also exhibit polarized phenotypes, with an initial M2 response attempting repair and regeneration. However, the sustained muscle damage and ineffective repair in dysferlinopathy, similar to DMD, promote the shift of macrophages toward a more fibrotic M2 phenotype. This results in chronic secretion of TGF-β and subsequent collagen deposition, contributing to fibrosis. While the exact contribution of dysferlin to macrophage activation is still under investigation, the fibrotic response in dysferlinopathy involves both macrophages and fibroblasts in a similar manner to DMD.

The immune response in both dystrophinopathy and dysferlinopathy, the profibrotic actions of macrophages are further amplified by secondary damage driven by the immune response. Fibrinogen, a soluble acute phase protein released during stress, plays a critical role in this process. It not only contributes to blood clotting following vascular injury but also extravasates into sites of inflammation, where it can interact with macrophages through receptors such as Mac-1. This interaction induces the production of pro-inflammatory cytokines such as IL-1β, which in turn stimulates TGF-β production. TGF-β then promotes collagen synthesis by fibroblasts, leading to the excessive deposition of ECM proteins, particularly type I and III collagen, which drive fibrosis in the dystrophic muscle [65].

Recent studies in the mdx mouse model of DMD have shown that macrophages play an integral role in sustaining inflammation and promoting fibrosis through their diverse phenotypes. During the early stages of the disease, macrophages contribute to the removal of damaged tissue, but chronic inflammation triggers a shift towards a more fibrotic environment. This ongoing fibrotic remodeling creates a pro-fibrotic niche that impedes muscle regeneration by satellite cells, contributing to the progressive loss of muscle function [4, 41].

In both dystrophinopathy and dysferlinopathy, macrophages interact dynamically with other immune cells, creating a complex inflammatory microenvironment that influences disease progression (Figure 2).

T cells, particularly the CD4⁺ and CD8⁺ subsets, play an essential role in regulating macrophage activity within inflammatory environments, especially during muscle pathology. Th1 cytokines, such as IFN-γ, which activates the CIITA transcription factor and leads to MHC-II⁺ upregulation and promote the activation of M1 macrophages, which are pro-inflammatory and can exacerbate muscle damage by increasing the release of inflammatory mediators and cytotoxic molecules [66]. Conversely, Th2 cytokines, such as IL-4 and IL-13, drive a shift toward the M2 macrophage phenotype, characterized by anti-inflammatory properties and the promotion of tissue repair [67, 68]. While M2 macrophages are beneficial in acute tissue injury by aiding repair, under chronic inflammatory conditions, they can contribute to fibrosis by promoting ECM deposition and scar formation. This dynamic interplay between T cells and macrophages underscores the complex immune modulation that influences disease progression in MDs.

Neutrophils, recruited early during muscle damage, release pro-inflammatory mediators such as IL-8 and ROS, which enhance macrophage activation and recruitment, amplifying the inflammatory response. In vitro studies reveal that neutrophils induce muscle cell death via superoxide-dependent mechanisms, while macrophages rely on NO-dependent pathways. When neutrophils and macrophages co-exist with muscle cells in proportions seen in injured muscle, cytotoxicity shifts to a NO-dependent, superoxide-independent mechanism, with their combined presence significantly increasing muscle cell death compared to either cell type alone [69].

In addition to T cells and neutrophils, other immune cell interactions with macrophages play a critical role in the pathogenesis of dystrophinopathy and dysferlinopathy. B cells, for example, contribute to immune modulation not only through antibody production but also via cytokine secretion. IL-10 produced by B cells can promote the polarization of macrophages toward an M2 phenotype, which may be beneficial in acute injury but could exacerbate fibrosis under chronic conditions [70].

Finally, non-immune cells such as fibroblasts and satellite cells also interact with macrophages, influencing muscle regeneration and repair. Macrophages regulate satellite cell activation, which is essential for muscle regeneration, and interact with fibroblasts to mediate ECM remodeling. These interactions highlight the importance of considering both immune and non-immune components of the inflammatory microenvironment when exploring therapeutic targets for MDs [38].

Together, these interactions not only sustain chronic inflammation but also create a dysregulated immune environment that impairs muscle regeneration, highlighting the need for therapies targeting the crosstalk between macrophages and other immune cells in monogenic MDs.