Novel muscular anti-inflammatory mechanisms of adiponectin and their implication in Duchenne Muscular Dystrophy

Adiponectin (ApN), which is decreased in the metabolic syndrome, is a hormone that has antidiabetic properties by acting on various organs. In skeletal muscle, ApN reduces inflammation and oxidative stress caused by various stimuli (LPS, obesogenic diet) or by the absence of dystrophin. In this work, we investigated the anti-inflammatory mechanisms of ApN in skeletal muscle and their implication in Duchenne muscular dystrophy. On one hand, we have characterized microRNAs regulated by this hormone and, on the other hand, studied the role of the NLRP3 inflammasome complex in the development of chronic inflammation that characterizes dystrophic muscles.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

Downregulation of the NLRP3 inflammasome by adiponectin rescues Duchenne muscular dystrophy

BACKGROUND: The hormone adiponectin (ApN) exerts powerful anti-inflammatory effects on skeletal muscle and can reverse devastating myopathies, like Duchenne muscular dystrophy (DMD), where inflammation exacerbates disease progression. The NLRP3 inflammasome plays a key role in the inflammation process, and its aberrant activation leads to several inflammatory or immune diseases. Here we investigated the expression of the NLRP inflammasome in skeletal muscle and its contribution to DMD. RESULTS: We find that NLRP3 is expressed in skeletal muscle and show that ApN downregulates NLRP3 via its anti-inflammatory mediator, miR-711. This repression occurs both in vitro in C2C12 myotubes and in vivo after either local (via muscle electrotransfer) or systemic (by using transgenic mice) ApN supplementation. To explore the role of the NLRP3 inflammasome in a murine model of DMD, we crossed mdx mice with Nlrp3-knockout mice. In mdx mice, all components of the inflammasome were upregulated in muscle, and the complex was overactivated. By contrast, in mdx mice lacking Nlrp3, there was a reduction in caspase-1 activation, inflammation and oxidative stress in dystrophic muscle, and these mice showed higher global muscle force/endurance than regular mdx mice as well as decreased muscle damage. To investigate the relevance of NLPR3 regulation in a human disease context, we characterized NLRP3 expression in primary cultures of myotubes from DMD subjects and found a threefold increase compared to control subjects. This overexpression was attenuated by ApN or miR-711 mimic treatments. CONCLUSIONS: The NLRP3 inflammasome plays a key pathogenic role in DMD and muscle inflammation, thereby opening new therapeutic perspectives for these and other related disorders

Potential Therapeutic Action of Adiponectin in Duchenne Muscular Dystrophy.

Adiponectin (ApN) is a hormone that exhibits anti-inflammatory effects on skeletal muscle exposed to acute and chronic inflammation. We have previously tested the implication of ApN in Duchenne muscular dystrophy (DMD) using mdx mice, a model of DMD, and by generating transgenic mdx mice overexpressing ApN. We showed that ApN can act as a preventive agent and delay disease progression by reducing muscle inflammation/injury and improving force/myogenesis. Herein, we took an opposite approach and crossed mdx mice with ApN knockout mice, to obtain mdx mice with ApN depletion. The aims were to test whether ApN deficiency could worsen the mdx phenotype and whether ApN supplementation can reverse several muscle abnormalities once the disease is settled. mdx-knockout mice exhibited lower muscle force/endurance as well as increased muscle damage when compared to regular mdx mice. Local administration of the ApN gene significantly reduced the expression of several oxidative stress/inflammatory markers and increased the expression of myogenic markers in the skeletal muscle. Finally, the presence of ApN markedly reduced the activity of NF-κB, a key player in muscle inflammation and myogenesis. ApN proves to be a powerful protector of the skeletal muscle capable of reversing the disease progression, thus making it a potential therapeutic agent for DMD

Skeletal muscle secretome in Duchenne muscular dystrophy: a pivotal anti-inflammatory role of adiponectin

BACKGROUND: Persistent inflammation exacerbates the progression of Duchenne muscular dystrophy (DMD). The hormone, adiponectin (ApN), which is decreased in the metabolic syndrome, exhibits anti-inflammatory properties on skeletal muscle and alleviates the dystrophic phenotype of mdx mice. Here, we investigate whether ApN retains its anti-inflammatory action in myotubes obtained from DMD patients. We unravel the underlying mechanisms by studying the secretome and the early events of ApN. METHODS: Primary cultures of myotubes from DMD and control patients were treated or not by ApN after an inflammatory challenge. Myokines secreted in medium were identified by cytokine antibody-arrays and ELISAs. The early events of ApN signaling were assessed by abrogating selected genes. RESULTS: ApN retained its anti-inflammatory properties in both dystrophic and control myotubes. Profiling of secretory products revealed that ApN downregulated the secretion of two pro-inflammatory factors (TNFα and IL-17A), one soluble receptor (sTNFRII), and one chemokine (CCL28) in DMD myotubes, while upregulating IL-6 that exerts some anti-inflammatory effects. These changes were explained by pretranslational mechanisms. Earlier events of the ApN cascade involved AdipoR1, the main receptor for muscle, and the AMPK-SIRT1-PGC-1α axis leading, besides alteration of the myokine profile, to the upregulation of utrophin A (a dystrophin analog). CONCLUSION: ApN retains its beneficial properties in dystrophic muscles by activating the AdipoR1-AMPK-SIRT1-PGC-1α pathway, thereby inducing a shift in the secretion of downstream myokines toward a less inflammatory profile while upregulating utrophin. ApN, the early events of the cascade and downstream myokines may be therapeutic targets for the management of DMD

New targets to alleviate skeletal muscle inflammation: role of microRNAs regulated by adiponectin.

Muscle inflammation worsens metabolic disorders as well as devastating myopathies. The hormone adiponectin (ApN) has emerged has a master regulator of inflammation/immunity in several tissues including the skeletal muscle. In this work, we explore whether microRNAs regulated by ApN may represent novel mechanisms for controlling muscle inflammation. By screening arrays, we found miR-711 as a strong candidate for mediating ApN action. Thus, ApN-knockout mice showed decreased muscular expression of miR-711 together with enhanced inflammation/oxidative stress markers, while mice overexpressing ApN showed increased miR-711 levels. Likewise, electrotransfer of the ApN gene in muscle of ApN-knockout mice upregulated miR-711 while reducing inflammation and oxidative stress. Similar data were obtained in murine C2C12 cells or in human primary myotubes treated with ApN. MiR-711 overexpression downregulated several components of the Toll-like receptor-4 (TLR4) pathway, which led to repression of NF-κB activity and downstream pro-inflammatory cytokines. MiR-711 blockade had opposite effects. Moreover, muscle electrotransfer of pre-miR-711 recapitulated in vivo the anti-inflammatory effects observed in vitro. Thus, miR-711, which is upregulated by ApN represses TLR4 signaling, acting therefore as a major mediator of the anti-inflammatory action of ApN. This novel miRNA and its related target genes may open new therapeutic perspectives for controlling muscle inflammation

New targets to control skeletal muscle inflammation: MicroRNAs regulated by adiponectin

Background and aims: Low-grade pro-inflammatory state contributes to the metabolic syndrome (MS). Adiponectin (ApN), which is reduced in the MS, has emerged as a master regulator of inflammation/immunity. We wished to identify whether microRNAs (miRNAs) may mediate the antiinflammatory action of ApN on skeletal muscle. Materials and methods: miRNA expression profiling was performed in tibialis anterior muscles of ApN-knockout (ApN-KO) mice: one leg was electrotransferred with a plasmid coding for the ApN gene, while the contralateral leg received an empty plasmid and served as control. Mice were next challenged by lipopolysaccharide (LPS) to induce inflammation. Role of specific miRNAs was analyzed by gain-of or loss-of function approaches in C2C12 myotubes and in vivo by muscle electrotransfer. miRNA expression was also studied in human myotubes. Results: Expression of miR-711 was up-regulated by muscle electrotransfer of ApN, which concomitantly reduced inflammation (TNFα, IL-1β) and oxidative stress (peroxiredoxin-3) markers. Likewise, in C2C12 cells, ApN treatment upregulated miR-711 expression. Transfection of miR-711 mimic reproduced the anti-inflammatory effects of ApN, while miR-711 blockade attenuated its protective effects. We found that miR-711 repressed the expression of 4 genes belonging to the Toll-like receptor-4 (TLR4) pathway. This pathway is activated by LPS and ultimately leads to stimulation of NF-kB, a pro-inflammatory transcription factor. As expected, NF-kB activity, measured via a luciferase reporter plasmid, was reduced by the miR mimic and enhanced by miR silencing. This protection against inflammation was recapitulated in ApN-KO mice by in vivo muscle electrotransfer of a plasmid coding for miR-711. Eventually, miR-711 expression was also upregulated in human myotubes after ApN treatment. Conclusion: miR-711, which is up-regulated by ApN, represses TLR4 signaling and NF-kB, acting therefore as a major mediator of the antiinflammatory action of ApN on muscle. This novel miRNA may open new therapeutic perspectives for the MS or other inflamed muscle condition

Potential therapeutic action of adiponectin in Duchenne muscular dystrophy

Adiponectin (ApN) is a hormone tightly linked to the metabolic syndrome where it exerts anti-diabetic actions. It is also known to possess powerful anti-inflammatory effects on skeletal muscle exposed to acute and chronic inflammation. We have previously tested the implication of ApN in Duchenne Muscular Dystrophy (DMD) by generating transgenic mdx mice overexpressing ApN. We showed that ApN can act as preventive agent and delay disease progression. ApN upregulation was capable of reducing muscle inflammation/injury and improving force/myogenesis. Here we took an a contrario approach and crossed mdx mice with ApN knockout mice, in order to obtain mdx with ApN depletion (mdx-KO). The aims were to (1) test whether ApN deficiency could worsen the mdx phenotype and (2) test if ApN supplementation can counteract all abnormalities once the disease has settled. Mdx-KO mice exhibited lower global muscle force/endurance as well as increased muscle damage when compared to regular mdx mice. We next injected and electro-transferred one tibialis anterior with the ApN gene, while the contralateral muscle received and empty plasmid and served as a control. Local administration of the ApN gene significantly reduced the expression of two oxidative stress markers (PRDX3, HNE), two inflammatory markers (TNFα, IL-1β), as well as the number of M1 macrophages. In addition, the expression of an anti-inflammatory cytokine (IL-10) was markedly increased. Local administration of ApN was also able to increase the expression of two major factors of muscle differentiation (Myogenin, Mrf4), one key marker of muscle regeneration (Myh3), plus induce a switch towards a resistant oxidative fiber phenotype. Finally, the presence of ApN markedly reduced NF-κB, a maestro of muscle inflammation and myogenesis. ApN proves to be, yet again, a powerful protector of the skeletal muscle, thus making it a potential therapeutic agent for DMD, and for other diseases where inflammation plays a crucial role. [Award: the Duchenne research fund prize, for the best presentation by a young researcher on the treatment of Duchenne muscular dystrophy

Adiponectin hinders the NLRP3 inflammasome in a murine model of Duchenne Muscular Dystrophy

NLRP3 inflammasome activation cleaves several pro-inflammatory cytokines including pro-IL-1β, resulting in their activation and secretion. Several myopathies result in excessive inflammation that plays a worsening pathogenic role. IL-1β is overexpressed in skeletal muscle of mdx mice, a murine model of Duchenne muscular dystrophy. However, NLRP3 expression/activity has not been yet investigated in skeletal muscle. Adiponectin (ApN) is a hormone mainly secreted by adipose tissue, which exerts anti-inflammatory properties on skeletal muscle. Recently, transgenic mdx mice overexpressing ApN exhibited higher global force/endurance as well as decreased muscle damage/inflammation. We have shown that APN upregulated miR-711 in skeletal muscle, thereby contributing to its anti-inflammatory effects via the inhibition of NF-κB activity. In this study, we investigated the presence of NLRP3 in myofibers and whether its expression is potentially regulated by ApN and miR-711 in vivo and in vitro. One tibialis anterior of ApN-KO mice was electroporated with a plasmid coding for the ApN gene or the miR-711, while the contralateral one received a respective control plasmid. Mice were next challenged by lipopolysaccharide (LPS) to induce inflammation. Muscle electrotransfer of either the ApN gene or miR-711 induced anti-inflammatory effects: downregulation (~50%) of inflammation (TNFα, IL-1β) and oxidative stress (peroxiredoxin-3) stress markers. NLRP3 labeling found as cluster stains in sarcoplasm was also reduced (40%), while being undetectable in NLRP3-KO mice. In C2C12 myotubes, both ApN treatment and transfection of miR-711 mimic reduced gene expression of TNFα, IL-1β and NLRP3 after LPS challenge. In mdx mice, the expression of NLRP3 was 4-fold higher than in WT mice, while mdx mice overexpressing APN presented a 30% reduction. These novel data show that NLRP3 is present within myofibers and may open new therapeutic perspectives to control muscle inflammatio

Abolition of the NLRP3 inflammasome improves the dystrophic phenotype in a murine model of DMD

Assembly of the NLRP3 inflammasome leads to caspase-1 activation and mediates the cleaving and release of several inflammatory cytokines. The NLRP3 inflammasome can amplify inflammatory responses and thus worsen several diseases. Duchenne muscular dystrophy (DMD) is one of the most devastating muscle disease and it is known to harbor a severe inflammation. We have recently shown that NLRP3 was more expressed in skeletal muscle fibers of mdx mice (a murine model of DMD) than in Wild-Type (WT) mice. Adiponectin (ApN) is a hormone known to possess powerful anti-inflammatory effects on skeletal muscle. Interestingly, transgenic mdx mice that overexpress ApN exhibited lower muscle inflammation/damage as well as higher globular muscle force/endurance when compared to regular mdx mice. These beneficial effects of ApN were associated with a reduction in NLRP3 expression in skeletal muscle. In this study, we investigated the effects of the absence of NLRP3 on the dystrophic phenotype by crossing mdx mice with NLRP3-knockout (NLRP3-KO) mice. First, functional in vivo studies (grip test, wire test and treadmill exercise) were performed on 4 groups of mice: WT, NLRP3-KO, mdx and NLRP3-KO-mdx. Compared to WT, mdx mice presented a strong decrease of global force and endurance that was partially restored in NLRP3-KO-mdx mice. In addition, NLRP3-KO-mdx mice also exhibited a significant decrease in muscle damage, oxidative stress and inflammation as well as a reduction in caspase-1 activation, when compared to regular mdx mice. Furthermore, satellite cells obtained from control and DMD subjects were cultured and differentiated into myotubes. We found that NLRP3 basal expression was 3.5-fold higher in DLD myotubes than in control myotubes. This expression was then reduced after ApN treatment. These novel data show that NLRP3 is implicated in DMD where it plays a key pathogenic role, thus opening new therapeutic perspectives to control muscle inflammation and damag