Patients with mutations in the DMD gene have Duchenne muscular dystrophy (DMD), which is a progressive muscle degenerative disease that leads to loss of skeletal muscle cardiomyopathy and eventual death (Bushby et al., 2010). In DMD, defective dystrophin protein causes muscle membrane fragility, leading to contraction-induced injury caused by high forces on the cell membrane and leading to cycles of muscle fiber degeneration and regeneration (Vetrone et al., 2009; Heydemann et al., 2009). Inflammatory cells invade in response to the repeated waves of damage and repair and release cytokines, which promote pathological fibrosis (Borthwick et al., 2013). Fibrosis leads to loss of mobility and function and has the potential to interfere with gene and stem cell therapies. Inflammatory cells also secrete promyogenic factors that enable proper myogenic differentiation and are absolutely necessary for successful muscle repair (Cantini et al., 2002; Dumont and Frenette, 2010; Saclier et al., 2013). Thus, modulating the inflammatory cell infiltrate in dystrophic muscle has the potential to target both fibrosis and regeneration and represents a therapeutic target for DMD. Such treatment may attenuate disease and improve the efficacy of other therapies if used in combination. Macrophages are the predominant immune cell type found in the inflammatory infiltrate of human DMD and mdx muscles (McDouall et al., 1990; Wehling et al., 2001). Macrophages that infiltrate dystrophic muscles are a heterogeneous mix of classically (M1) and alternatively (M2) activated types (Villalta e t al., 2009; Mann et al., 2011). M1 macrophages are classified as proinflammatory because they express known proinflammatory mediators (tumor necrosis factor, interleukin [IL]-1β, and IL-6)

In the degenerative disease Duchenne muscular dystrophy, inflammatory cells enter muscles in response to repetitive muscle damage. Immune factors are required for muscle regeneration, but chronic inflammation creates a profibrotic milieu that exacerbates disease progression. Osteopontin (OPN) is an immunomodulator highly expressed in dystrophic muscles. Ablation of OPN correlates with reduced fibrosis and improved muscle strength as well as reduced natural killer T (NKT) cell counts. Here, we demonstrate that the improved dystrophic phenotype observed with OPN ablation does not result from reductions in NKT cells. OPN ablation skews macrophage polarization toward a pro-regenerative phenotype by reducing M1 and M2a and increasing M2c subsets. These changes are associated with increased expression of pro-regenerative factors insulin-like growth factor 1, leukemia inhibitory factor, and urokinase-type plasminogen activator. Furthermore, altered macrophage polarization correlated with increases in muscle weight and muscle fiber diameter, resulting in long-term improvements in muscle strength and function in mdx mice. These findings suggest that OPN ablation promotes muscle repair via macrophage secretion of pro-myogenic growth factors.