Muscle, myeloid cells, and complement: a complex interaction

The complement system is a critical arm of the immune system that plays an important role not only in maintaining health but also contributes to disease. Complement activation generates biologically active products C3a and C5a, which are important links between complement and cellular immunity. Recent studies have revealed that C5a/C5aR signaling is critical in both humoral and inflammatory responses, resulting in functional renal insufficiency in immune complex-mediated glomerulonephritis.¹ Consistent with these studies and considering the complexity of complement system interactions based on location and setting, a recent article by Zhang et al.² has shed light on C3a/C3aR signaling as the link that regulates monocyte function and trafficking and facilitates skeletal muscle regeneration. This commentary highlights the important themes originating from the work described, which include the following: (1) complement C3 depletion reduced muscle regeneration, (2) C3a signaling through its G-coupled receptor C3aR facilitated muscle regeneration by regulating monocyte/macrophage infiltration through CCL5, and (3) C5a/C5aR signaling does not affect monocyte/macrophage infiltration in this particular setting.

The complement cascade consists of three pathways that are initiated differently but ultimately form C5b-9, or the membrane attack complex. The pathways consist of approximately 30 proteins, both circulating and membrane bound, with regulators at strategic locations.³ Although complement remained unexplored for a long time after its discovery, different relevant proteins have been identified over time. More recently, complement research has become a dynamic area of study with discoveries of novel functions, and there is an urgency to identify effective complement therapies. Together with its traditionally understood role in immune defense,⁴ studies have revealed that complement plays an important role in differentiation, development, and disease.^{5, 6} The complement system is ubiquitous in the body, and its function is oriented towards the local microenvironment. In addition, too much or too little complement activation can be detrimental. Therefore, understanding its role in different organs and settings is important for enhancing health and in disease therapy.

During development, myogenic precursor cells undergo proliferation and differentiation coordinated by the interplay of different regulatory mechanisms to ultimately form muscle fibers.⁷ This developmental process is repeated during muscle regeneration in adults, especially during injury. Muscle has the remarkable property of regeneration after an injury caused by either normal wear and tear or by trauma and strain. Both the developmental and regenerative processes are orchestrated by different factors in the surrounding microenvironment. However, although the mechanisms regulating development and regeneration resemble one another, the microenvironments differ significantly, with relatively few immune cells in the former condition and excessive immune cells in the latter condition.⁸ Both proteomic and transcriptomic analyses have supported a key role for the core complement protein C3 in myogenesis.⁹ A “yin and yang” exists in the complement system, which is beneficial during development and defense and detrimental during inflammation and disease; therefore, activation of the system must be critically regulated to achieve a beneficial outcome. In the muscle setting, complement participates in muscle destruction in disorders such as myocarditis, while the same complement system participates in regeneration during muscle tear or injury.¹⁰

Dysferlin is a protein involved in the smooth function and membrane repair of muscle. Mutations of the dysferlin gene result in muscle wasting disorders.¹¹ Gene expression profiling in muscles from both mice and humans has revealed increased expression of classical and alternative complement components in the context of dysferlin deficiency, which can be normalized by transgenic expression of dysferlin in the muscle. Dysferlinopathy is associated with a prominent inflammatory response, with dysferlin-deficient monocytes displaying increased phagocytic activity.¹² Deletion of C3 alleviated the muscle pathology, while deletion of C5 had no effect, indicating that the upstream early components rather than the terminal component are critical in complement-related muscle disorders. In contrast, C5a/C5aR1 signaling is upregulated in hSOD1 transgenic rodent models of ALS,¹³ and deletion of C5aR1 resulted in a significant reduction in infiltrating macrophages in the muscle with reduced neuromuscular denervation and improved muscle strength.¹⁴ Interestingly, complement did not participate in mdx-associated muscle disease,¹⁵ indicating that it is disease-specific.

The extensive work of Zhang et al.² addresses the roles of C3 and C5 and delineates the potential effects of C3a and C3b. C3a is generated during complement activation, and manipulation of C3a and subsequent signaling does not affect C3b, C5a, and C5b-9 or their roles in defense. C3a regulates monocyte trafficking and function, and small-molecule C3a and C3a receptor (R) antagonists are available; therefore, C3a is a viable therapeutic target.

Earlier studies from this group revealed that complement participated in muscle regeneration. The studies concluded that C1q reduced regeneration in aging muscle through Wnt signaling, independent of complement activation.¹⁶ The role of complement in muscle well-being was further substantiated by microarray data showing enhanced C3aR1 and C1qa expression in cardiotoxin-mediated skeletal muscle injury and regeneration. In the present study, using cobra venom factor to deplete complement at defined time points, complement gene-knockout models, and reconstitution of knockout models with wild-type serum, complement activation was demonstrated to occur early after muscle injury. The myofiber cross-sectional area (mCSA) is known to correlate with muscle strength.¹⁷ Based on the mCSA, the absence of C3 reduced muscle regeneration after injury. However, C3 depletion 24 h after injury did not cause this effect, indicating that C3 was critical in the early phase after injury. To delineate the effects of the classical and alternative pathways, C4-knockout models and factor B (Cfb)-knockout models were used, respectively. The mCSA was similar in the wild-type mice and C4-knockout mice but was reduced in Cfb-knockout mice, indicating that the classical complement pathway had no effect on muscle regeneration, while the alternative pathway had a significant effect.

In addition, the group’s results demonstrated that the alternative pathway was essential for the macrophage infiltration required for the restorative process. During muscle injury, the “first responders” are neutrophils, which promote revascularization in the muscle cells, followed by eosinophils¹⁸ and macrophages,¹⁹ which stimulate regeneration. Eosinophils remove cellular debris and activate progenitor cells, while macrophages promote membrane repair and muscle growth. This study focused on the role of complement in macrophage infiltration in this setting. The results show that C3a/C3aR signaling but not C5a/C5aR signaling was critical for macrophage infiltration, while the apoptosis and proliferation of these cells remained unaffected. The mCSA was reduced in C3aR−/− mice and remained unchanged in C5aR−/− and wild-type mice. These results suggest that C3a/C3aR signaling is essential for macrophage infiltration into muscle and regeneration of muscle. On further investigation, consistent with in vivo signaling, agonist stimulation of C3a/C3aR in monocytes in vitro resulted in increased transcriptional and translational expression of CCL5. Reconstitution experiments proved that C3a/C3aR signaling is critical for muscle regeneration and that the signaling occurred through CCL5. This study connecting C3a/C3aR, macrophage infiltration, and muscle regeneration suggests modulation of C3a/C3aR signaling as an effective therapeutic target in different myopathies.

Macrophages are classified into different myeloid populations, including CD206−/F4/80+ inflammatory M1 macrophages rich in inducible nitric oxide synthase and arginase and CD206+/F4/80+ regenerative M2 macrophages rich in interleukin-4 (IL-4) and IL-10.²⁰ The specific macrophage population affected in these studies was not defined. However, the complex interplay between the different myeloid populations and the responses based on the phagocytosed material, whether apoptotic or necrotic, highlights the importance of understanding the macrophage population associated with injury and the time from injury to define effective therapeutic strategies since the therapy required for one population of myeloid cells may not be appropriate for another population of myeloid cells. In addition, the roles of other signaling pathways involved in myogenesis, muscle growth, homeostasis, and repair must be assessed. Therefore, although the complement system is involved in both inflammation and regeneration, its activity depends on the specific microenvironment. Accordingly, therapeutic complement inhibition must be applied cautiously considering that reducing inflammation could also reduce necessary regeneration.