Macrophages Protect against Muscle Atrophy and Promote Muscle Recovery in Vivo and in Vitro : A Mechanism Partly Dependent on the Insulin-Like Growth Factor-1 Signaling Molecule

Hindlimb unloading and reloading are characterized by a major loss of muscle force and are associated with classic leukocyte infiltration during recovery from muscle atrophy. Macrophages act as a cellular cornerstone by playing both pro- and anti-inflammatory roles during muscle recovery from atrophy. In the present study, we investigated the role of macrophages in muscle atrophy and regrowth using in vivo and in vitro models. Mice depleted in monocytes/macrophages and submitted to a hindlimb unloading and reloading protocol experienced a significant delay in muscle force recovery compared with matched placebo mice at 7 and 14 days after reloading. Furthermore, an in vitro myotube/macrophage coculture showed that anti-inflammatory macrophages, which contain apoptotic neutrophils and express low levels of cyclooxygenase-2, completely prevented the loss of protein content and the myotube atrophy observed after 2 days in low serum medium. The presence of macrophages also protected against the decrease in myosin heavy chain content in myotubes exposed to low serum medium for 1 day. Interestingly, the addition of an anti-IGF-1 antibody to the coculture significantly decreased the ability of macrophages to protect against myotube atrophy and myosin heavy chain loss after 2 days in low serum medium. These results clearly indicate that macrophages and, more precisely, the release of IGF-1 by macrophages, play an important role in recovery from muscle atrophy.Monocytes are large mononuclear cells that circulate in the blood and differentiate into macrophages in invaded tissues in response to various stimuli.¹ Macrophages have a strong phagocytic capacity and can orchestrate the inflammatory process via the release of a wide variety of cytokines and chemokines such as interleukin (IL)-1, tumor necrosis factor-α, and macrophage inflammatory protein-2.^2,3 Numerous studies have also demonstrated that macrophages play a direct role in tissue recovery through the release of the anti-inflammatory molecules and anabolic growth factors IL-10, basic fibroblast growth factor, and insulin-like growth factor-1 (IGF-1).^4,5,6The regulation of the multiple and occasionally opposing functions of macrophages is very complex and poorly understood.^7,8,9 To add to this complexity, the diversity of experimental models can also lead to different conclusions regarding the roles of each macrophage phenotype in muscle recovery from atrophy or damage. For example, in a model of eccentric contractions, in which injured muscle is basically devoid of neutrophils, macrophage invasion contributes to secondary damage to the skeletal muscle.¹⁰ On the other hand, the phagocytosis of apoptotic neutrophils and necrotic cells by macrophages induces a change in macrophage phenotype from pro- to anti-inflammatory, which has a strong modulatory effect on cytokine profiles and is essential for dampening environmental inflammatory signals.^11,12 Regarding the different subsets of macrophages, coculture experiments have shown that pro-inflammatory macrophages (Ly-6C^hi) stimulate myogenic cell proliferation, whereas anti-inflammatory macrophages (Ly-6C^lo) exert a strong differentiating influence on myogenic cells.^12,13 Nonetheless, in a model in which rodent hindlimbs are deprived of mechanical loading for 10 days followed by reloading, the high concentration of leukocytes in reloaded muscles regardless of the absence of significant muscle damage raises intriguing questions about the detrimental and beneficial roles of leukocytes in muscle dysfunction and recovery from atrophy.^14,15,16 It is thus tempting to speculate that the roles of macrophages may vary as a function of the type of insult.In the present study, mice depleted in macrophages were submitted to hindlimb unloading and reloading to evaluate the roles of macrophages in muscle atrophy and regrowth. Our results showed that macrophages neither prevent the loss in muscle force nor promote recovery during the early inflammatory phase (1 and 3 days after reloading). However, they play a key role in muscle growth and recovery at later times (7 and 14 days after reloading). In addition, an in vitro coculture model in which atrophied myotubes were combined with macrophages expressing an anti-inflammatory phenotype showed that the presence of macrophages protects myotubes from atrophy and that this protective effect is partly mediated by the release of IGF-1.