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Corticosteroids are highly prescribed and effective anti-inflammatory drugs but the burden of side effects with chronic use significantly detracts from patient quality of life, particularly in children. Developing safer steroids amenable to long-term use is an important goal for treatment of chronic inflammatory diseases such as Duchenne muscular dystrophy (DMD). We have developed vamorolone (VBP15), a first-in-class dissociative glucocorticoid receptor (GR) ligand that shows the anti-inflammatory efficacy of corticosteroids without key steroid side effects in animal models. miRNAs are increasingly recognized as key regulators of inflammatory responses. To define effects of prednisolone and vamorolone on the muscle miRNAome, we performed a preclinical discovery study in the mdx mouse model of DMD. miRNAs associated with inflammation were highly elevated in mdx muscle. Both vamorolone and prednisolone returned these toward wild-type levels (miR-142-5p, miR-142-3p, miR-146a, miR-301a, miR-324-3p, miR-455-5p, miR-455-3p, miR-497, miR-652). Effects of vamorolone were largely limited to reduction of proinflammatory miRNAs. In contrast, prednisolone activated a separate group of miRNAs associated with steroid side effects and a noncoding RNA cluster homologous to human chromosome 14q32. Effects were validated for inflammatory miRNAs in a second, independent preclinical study. For the anti-inflammatory miRNA signature, bioinformatic analyses showed all of these miRNAs are directly regulated by, or in turn activate, the inflammatory transcription factor NF-κB. Moving forward miR-146a and miR-142 are of particular interest as biomarkers or novel drug targets. These data validate NF-κB signaling as a target of dissociative GR-ligand efficacy in vivo and provide new insight into miRNA signaling in chronic inflammation.

New Findings: What is the central question of this study? What is the functional relevance of OPN isoform expression in muscle pathology? What is the main finding and its importance? The full-length human OPN-a isoform is the most pro-inflammatory isoform in the muscle microenvironment, acting on macrophages and myoblasts in an RGD-integrin-dependent manner. OPN-a upregulates expression of tenascin-C (TNC), a known Toll-like receptor 4 (TLR4) agonist. Blocking TLR4 signalling inhibits the pro-inflammatory effects of OPN-a, suggesting that a potential mechanism of OPN action is by promoting TNC-TLR4 signalling. Although osteopontin (OPN) is an important mediator of muscle remodelling in health and disease, functional differences in human spliced OPN variants in the muscle microenvironment have not been characterized. We thus sought to define the pro-inflammatory activities of human OPN isoforms (OPN-a, OPN-b and OPN-c) on cells present in regenerating muscle. OPN transcripts were quantified in normal and dystrophic human and dog muscle. Human macrophages and myoblasts were stimulated with recombinant human OPN protein isoforms, and cytokine mRNA and protein induction was assayed. OPN isoforms were greatly increased in dystrophic human (OPN-a > OPN-b > OPN-c) and dog muscle (OPN-a = OPN-c). In healthy human muscle, mechanical loading also upregulated OPN-a expression (eightfold; P < 0.01), but did not significantly upregulate OPN-c expression (twofold; P > 0.05). In vitro, OPN-a displayed the most pronounced pro-inflammatory activity among isoforms, acting on both macrophages and myoblasts. In vitro and in vivo data revealed that OPN-a upregulated tenascin-C (TNC), a known Toll-like receptor 4 (TLR4) agonist. Inhibition of TLR4 signalling attenuated OPN-mediated macrophage cytokine production. In summary, OPN-a is the most abundant and functionally active human spliced isoform in the skeletal muscle microenvironment. Here, OPN-a promotes pro-inflammatory signalling in both macrophages and myoblasts, possibly through induction of TNC-TLR4 signalling. Together, our findings suggest that specific targeting of OPN-a and/or TNC signalling in the damaged muscle microenvironment may be of therapeutic relevance., (© 2016 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The dysferlin-deficient A/J mouse strain represents a homologous model for limb-girdle muscular dystrophy 2B. We evaluated the disease phenotype in 10 month old A/J mice compared to two dysferlin-sufficient, C57BL/6 and A/JOlaHsd, mouse lines to determine which functional end-points are sufficiently sensitive to define the disease phenotype for use in preclinical studies in the A/J strain. A/J mice had significantly lower open field behavioral activity (horizontal activity, total distance, movement time and vertical activity) when compared to C57BL/6 and A/JoIaHsd mice. Both A/J and A/JOIaHsd mice showed decreases in latency to fall with rotarod compared to C57BL/6. No changes were detected in grip strength, force measurements or motor coordination between these three groups. Furthermore, we have found that A/J muscle shows significantly increased levels of the pro-inflammatory cytokine TNF-α when compared to C57BL/6 mice, indicating an activation of NF-κB signaling as part of the inflammatory response in dysferlin-deficient muscle. Therefore, we assessed the effect of celastrol (a potent NF-κB inhibitor) on the disease phenotype in female A/J mice. Celastrol treatment for four months significantly reduced the inflammation in A/J muscle; however, it had no beneficial effect in improving muscle function, as assessed by grip strength, open field activity, and in vitro force contraction. In fact, celastrol treated mice showed a decrease in body mass, hindlimb grip strength and maximal EDL force. These findings suggest that inhibition of inflammation alone may not be sufficient to improve the muscle disease phenotype in dysferlin-deficient mice and may require combination therapies that target membrane stability to achieve a functional improvement in skeletal muscle., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A deficiency of the dysferlin protein results in limb girdle muscular dystrophy type 2B and Miyoshi myopathy, with resulting plasma membrane abnormalities in myofibers. Many patients show muscle inflammation, but the molecular mechanisms that initiate and perpetuate this inflammation are not well understood. We previously showed abnormal activation of macrophages and hypothesized that activation of the inflammasome pathway may play a role in disease progression. To test this, we studied the inflammasome molecular platform in dysferlin-deficient human and mouse muscle. Consistent with our model, components of the NACHT, LRR and PYD-containing proteins (NALP)-3 inflammasome pathway were specifically up-regulated and activated in dysferlin-deficient but not in dystrophin-deficient and normal muscle. We demonstrate for the first time that normal primary skeletal muscle cells are capable of secreting IL-1beta in response to combined treatment with lipopolysaccharide and the P2X7 receptor agonist, benzylated ATP, suggesting that not only immune cells but also muscle cells can actively participate in inflammasome formation. In addition, we show that dysferlin-deficient primary muscle cells express toll-like receptors (TLRs; TLR-2 and TLR-4) and can efficiently produce IL-1beta in response to lipopolysaccharide and benzylated ATP. These data indicate that skeletal muscle is an active contributor of IL-1beta and strategies that interfere with this pathway may be therapeutically useful for patients with limb girdle muscular dystrophy type 2B.

Mutations in the dysferlin gene cause limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. Dysferlin-deficient cells show abnormalities in vesicular traffic and membrane repair although onset of symptoms is not commonly seen until the late teenage years and is often associated with subacute onset and marked muscle inflammation. To identify molecular networks specific to dysferlin-deficient muscle that might explain disease pathogenesis, muscle mRNA profiles from 10 mutation-positive LGMD2B/MM patients were compared with a disease control [LGMD2I; (n = 9)], and normal muscle samples (n = 11). Query of inflammatory pathways suggested LGMD2B-specific increases in co-stimulatory signaling between dendritic cells and T cells (CD86, CD28, and CTLA4), associated with localized expression of both versican and tenascin. LGMD2B muscle also showed an increase in vesicular trafficking pathway proteins not normally observed in muscle (synaptotagmin-like protein Slp2a/SYTL2 and the small GTPase Rab27A). We propose that Rab27A/Slp2a expression in LGMD2B muscle provides a compensatory vesicular trafficking pathway that is able to repair membrane damage in the absence of dysferlin. However, this same pathway may release endocytotic vesicle contents, resulting in an inflammatory microenvironment. As dysferlin deficiency has been shown to enhance phagocytosis by macrophages, together with our findings of abnormal myofiber endocytosis pathways and dendritic-T cell activation markers, these results suggest a model of immune and inflammatory network over-stimulation that may explain the subacute inflammatory presentation.

Dysferlin deficiency causes limb-girdle muscular dystrophy type 2B (LGMD2B; proximal weakness) and Miyoshi myopathy (distal weakness). Muscle inflammation is often present in dysferlin deficiency, and patients are frequently misdiagnosed as having polymyositis. Because monocytes normally express dysferlin, we hypothesized that monocyte/macrophage dysfunction in dysferlin-deficient patients might contribute to disease onset and progression. We therefore examined phagocytic activity, in the presence and absence of cytokines, in freshly isolated peripheral blood monocytes from LGMD2B patients and in the SJL dysferlin-deficient mouse model. Dysferlin-deficient monocytes showed increased phagocytic activity compared with control cells. siRNA-mediated inhibition of dysferlin expression in the J774 macrophage cell line resulted in significantly enhanced phagocytosis, both at baseline and in response to tumor necrosis factor-alpha. Immunohistochemical analysis revealed positive staining for several mononuclear cell activation markers in LGMD2B human muscle and SJL mouse muscle. SJL muscle showed strong up-regulation of endocytic proteins CIMPR, clathrin, and adaptin-alpha, and LGMD2B muscle exhibited decreased expression of decay accelerating factor, which was not dysferlin-specific. We further showed that expression levels of small Rho family GTPases RhoA, Rac1, and Cdc 42 were increased in dysferlin-deficient murine immune cells compared with control cells. Therefore, we hypothesize that mild myofiber damage in dysferlin-deficient muscle stimulates an inflammatory cascade that may initiate, exacerbate, and possibly perpetuate the underlying myofiber-specific dystrophic process.

Objective: To identify stage-specific induction of molecular pathology pathways in Duchenne muscular dystrophy (DMD)., Methods: We performed mRNA profiling using muscles from fetopsies, infants (aged 8 to 10 months), and symptomatic patients (aged 5 to 12 years) with DMD, and age- and sex-matched controls. We performed immunohistochemistry to determine changes at the protein level and protein localization., Results: Activated tissue dendritic cells, expression of toll-like receptor 7, and strong induction of nuclear factor-kappaB pathways occurred soon after birth in DMD muscle. Two muscle wasting pathways, atrogin-1 and myostatin, were not induced at any stage of the disease. Normal muscle showed accumulation of glycolytic and oxidative metabolism capacity with increased age, but this accumulation failed in DMD. The transforming growth factor (TGF)-beta pathway was strongly induced in symptomatic patients, with expression of TGFbeta type II receptor and apoptosis signal-regulating kinase 1 proteins on subsets of mature DMD myofibers., Conclusions: Our data show stage-specific remodeling of human dystrophin-deficient muscle, with inflammatory pathways predominating in the presymptomatic stages and acute activation of TGFbeta and failure of metabolic pathways later in the disease.

Geographic tongue or benign migratory glossitis is usually an asymptomatic inflammatory disorder of the tongue mucosa of unknown etiology. It is characterized by circinate, erythematous, ulcer-like lesions of the dorsum and lateral border of the tongue due to loss of filiform papillae of the tongue epithelium. The condition usually is discovered on routine clinical examination. The lesion may recur at different sites on the tongue, creating a migratory appearance, and in many cases, will resolve completely. We report an unusual case of symptomatic geographic tongue in a 3-year-old child. [ABSTRACT FROM AUTHOR]

Abstract Background IL-6 is a pleiotropic cytokine that modulates inflammatory responses and plays critical roles in muscle maintenance and remodeling. In the mouse model (mdx) of Duchenne Muscular Dystrophy, IL-6 and muscle inflammation are elevated, which is believed to contribute to the chronic inflammation and failure of muscle regeneration in DMD. The purpose of the current study was to examine the effect of blocking IL-6 signaling on the muscle phenotype including muscle weakness and pathology in the mdx mouse. Methods A monoclonal antibody against the IL-6 receptor (IL-6r mAb) that blocks local and systemic IL-6 signaling was administered to mdx and BL-10 mice for 5 weeks and muscle function, histology, and inflammation were examined. Results IL-6r mAb treatment increased mdx muscle inflammation including total inflammation score and ICAM-1 positive lumens in muscles. There was no significant improvement in muscle strength nor muscle pathology due to IL-6r mAb treatment in mdx mice. Conclusions These results showed that instead of reducing inflammation, IL-6 signaling blockade for 5 weeks caused an increase in muscle inflammation, with no significant change in indices related to muscle regeneration and muscle function. The results suggest a potential anti-inflammatory instead of the original hypothesized pro-inflammatory role of IL-6 signaling in the mdx mice.