Abstract
BackgroundChronic increases in the levels of the inflammatory cytokine interleukin-6 (IL-6) in serum and skeletal muscle are thought to contribute to the progression of muscular dystrophy. Dystrophin/utrophin double-knockout (dKO) mice develop a more severe and progressive muscular dystrophy than the mdx mice, the most common murine model of Duchenne muscular dystrophy (DMD). In particular, dKO mice have smaller body sizes and muscle diameters, and develop progressive kyphosis and fibrosis in skeletal and cardiac muscles. As mdx mice and DMD patients, we found that IL-6 levels in the skeletal muscle were significantly increased in dKO mice. Thus, in this study, we aimed to analyze the effects of IL-6 receptor (IL-6R) blockade on the muscle pathology of dKO mice.MethodsMale dKO mice were administered an initial injection (200 mg/kg intraperitoneally (i.p.)) of either the anti-IL-6R antibody MR16-1 or an isotype-matched control rat IgG at the age of 14 days, and were then given weekly injections (25 mg/kg i.p.) until 90 days of age.ResultsTreatment of dKO mice with the MR16-1 antibody successfully inhibited the IL-6 pathway in the skeletal muscle and resulted in a significant reduction in the expression levels of phosphorylated signal transducer and activator of transcription 3 in the skeletal muscle. Pathologically, a significant increase in the area of embryonic myosin heavy chain-positive myofibers and muscle diameter, and reduced fibrosis in the quadriceps muscle were observed. These results demonstrated the therapeutic effects of IL-6R blockade on promoting muscle regeneration. Consistently, serum creatine kinase levels were decreased. Despite these improvements observed in the limb muscles, degeneration of the diaphragm and cardiac muscles was not ameliorated by the treatment of mice with the MR16-1 antibody.ConclusionAs no adverse effects of treatment with the MR16-1 antibody were observed, our results indicate that the anti-IL-6R antibody is a potential therapy for muscular dystrophy particularly for promoting skeletal muscle regeneration.
Highlights
Chronic increases in the levels of the inflammatory cytokine interleukin-6 (IL-6) in serum and skeletal muscle are thought to contribute to the progression of muscular dystrophy
While the number of Pax7 +/Ki67+ satellite cells was not different, Pax7+/Ki67− satellite cells were significantly increased in MR16-1-treated dKO mice (P < 0.05). c A representative image of Embryonic myosin heavy chain (eMyHC)-positive regenerating muscle fibers in dKO mice treated with rat IgG or MR16-1. d The percentage of eMyHC-positive area per total area. e The number of eMyHC-positive regenerating muscle fibers per field (× 200 magnification). f The percentage of average eMyHC-positive muscle fiber size. g Gene expression levels of myh3, normalized by Lbr, was expressed as fold increase of wild-type mice (n = 3–4 per group). h The percentage of number of myonuclei per fiber. *P < 0.05, **P < 0.01 macrophages, and we found approximately 1.6% of the total area were F4/80-positive macrophages
Phosphorylated STAT3 and total STAT3 levels in the skeletal and cardiac muscles from dKO mice treated with rat IgG and MR16-1 were compared with samples from wild-type mice (Additional file 1: Figure S2)
Summary
Chronic increases in the levels of the inflammatory cytokine interleukin-6 (IL-6) in serum and skeletal muscle are thought to contribute to the progression of muscular dystrophy. Dystrophin/utrophin double-knockout (dKO) mice develop a more severe and progressive muscular dystrophy than the mdx mice, the most common murine model of Duchenne muscular dystrophy (DMD). As mdx mice and DMD patients, we found that IL-6 levels in the skeletal muscle were significantly increased in dKO mice. The skeletal muscle in DMD patients is eventually replaced with non-functional tissues [4, 5], and preventing the accumulation of connective and adipose tissues is an important factor for delaying disease progression. Several inflammatory factors are increased in the DMD skeletal muscle, including interleukin-6 (IL-6), TNF-alpha, and NF-kappaB [6]. IL-6 plays multiple biological roles in different signaling pathways through the IL-6 receptor (IL-6R) and activates downstream intracellular signaling cascades including the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway
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