At this time point, 51 6% of nuclei were BrdU-positive in WT muscle, whereas only 23 8% of nuclei were BrdU-positive in dysferlin-deficient muscle (Fig.?3c, d). the co-cultures and transcriptome analysis implicated two soluble factors in mediating the effects: IL-1 and IL-4, which during acute injury are secreted from M1 and M2a macrophages, respectively. To test the roles of these two factors in dysferlin-deficient muscle, myoblasts were treated with IL-4, which improved muscle differentiation, or IL-1, which inhibited it. Importantly, blockade of IL-1 signaling significantly improved differentiation of dysferlin-deficient cells. Conclusions We propose that the inhibitory effects of M1 macrophages on myogenesis are mediated by IL-1 signals and suppression of the M1-mediated immune response may improve muscle regeneration in dysferlin deficiency. Our studies identify a potential therapeutic approach to promote muscle regeneration in dystrophic muscle. mouse model of Duchenne muscular dystrophy [9]. Macrophages have been classified into two major categories: the classically activated (M1) and the alternatively activated (M2a). M1 macrophages, identified by expression and secretion of TNF, Cox-2, IL-1, IL-12, and iNOS, respond to TLR ligands such as lipopolysaccharide (LPS) and demonstrate phagocytic and bacteriocidal activity [10]. In contrast, M2a macrophages, identified by expression of mannose receptor (Mrc1), resistin-like (Retnla, Fizz1), and chitinase 3-like 3 (Chi3l3, Ym1), are activated by IL-4 or IL-13 signals that arise during Th2 immune responses and participate in wound-healing processes (reviewed in [11, 10]). Immune cells are sequentially recruited to sites of acute injury, with a wave of neutrophils followed by M1-polarized macrophages that phagocytose necrotic material. M2a macrophages are also recruited at the time of M1 macrophage infiltration [12] and remain at the site of injury as M1 macrophages transdifferentiate to M2a macrophages in situ [13, 14], thereby resolving the injury and promoting myoblast differentiation. Studies of the macrophage populations in mice determined that L-ANAP M1 macrophages predominate during early phases to be replaced L-ANAP by M2a macrophages during the later regenerative/fibrotic stages of the disease [15]. Additionally, regulatory macrophages, M2b, are a third category MRX47 of macrophages and are characterized by IL-10 secretion and anti-inflammatory activity. The effects of the two types of differentially polarized macrophages on muscle cells were previously studied in the context of wild-type muscle [16]. Direct co-culture of myoblasts with M2 macrophages, or conditioned medium from them, increased myogenin-positive cells and myotubes, whereas co-culture or conditioned medium from M1 macrophages had no effect [16], whereas other studies have suggested that M1 macrophages promote myoblast proliferation and M2a promote myotube fusion [14]. These studies implicate macrophages as playing a major role in the process of muscle regeneration. However, the role of macrophages in chronic myopathies remains to be elucidated. To L-ANAP examine myoblast-macrophage interactions, we used an co-culture system of macrophages and immortalized myoblasts (H-2K cells), focusing on the molecular effects of macrophage-released soluble factors on myoblasts. We hypothesized that macrophage-secreted factors can influence differentiation of dysferlin-deficient muscle and proceeded to identify such factors and examine their effects on myoblasts. To approach these questions, we (1) examined the effects of co-culture with M1 and M2a macrophages on the differentiation of wild-type (WT) mouse myoblasts, (2) compared the effects of macrophages in dysferlin-deficient dystrophic myoblasts, and (3) identified IL-1 as the M1-derived factor inhibiting muscle differentiation in dysferlin deficiency. Importantly, we show for the first time that use of a blocking antibody to inhibit IL-1 improves muscle differentiation in dysferlin-deficient myotubes. Our studies delineate the effects of the pro-inflammatory environment on muscle regeneration in dysferlinopathy and raise the possibility of modulating this environment to promote muscle regeneration. Methods Animals All animal protocols were reviewed and approved by the local Institutional Animal Care and Use Committee of the Childrens National Medical Center, Washington, DC. Four- to seven-month-old Bla/J mice (B6.A-Dysfprmd/GeneJ) (stock # 012767) and BALB/c (stock# 000651) were obtained from The Jackson Laboratory (Bar Harbor, ME). The Bla/J model of dysferlin deficiency, produced by crossing the A/J naturally occurring dysferlin-mutated mice onto the C57Bl6/J.