Myeloid mineralocorticoid receptors contribute to skeletal muscle repair in muscular dystrophy and acute muscle injury.

Suppressing mineralocorticoid receptor (MR) activity with MR antagonists is therapeutic for chronic skeletal muscle pathology in Duchenne muscular dystrophy (DMD) mouse models. Although mechanisms underlying clinical MR antagonist efficacy for DMD cardiomyopathy and other cardiac diseases are defined, mechanisms in skeletal muscles are not fully elucidated. Myofiber MR knockout improves skeletal muscle force and a subset of dystrophic pathology. However, MR signaling in myeloid cells is known to be a major contributor to cardiac efficacy. To define contributions of myeloid MR in skeletal muscle function and disease, we performed parallel assessments of muscle pathology, cytokine levels, and myeloid cell populations resulting from myeloid MR genetic knockout in muscular dystrophy and acute muscle injury. Myeloid MR knockout led to lower levels of C-C motif chemokine receptor 2 (CCR2)-expressing macrophages, resulting in sustained myofiber damage after acute injury of normal muscle. In acute injury, myeloid MR knockout also led to increased local muscle levels of the enzyme that produces the endogenous MR agonist aldosterone, further supporting important contributions of MR signaling in normal muscle repair. In muscular dystrophy, myeloid MR knockout altered cytokine levels differentially between quadriceps and diaphragm muscles, which contain different myeloid populations. Myeloid MR knockout led to higher levels of fibrosis in dystrophic diaphragm. These results support important contributions of myeloid MR signaling to skeletal muscle repair in acute and chronic injuries and highlight the useful information gained from cell-specific genetic knockouts to delineate mechanisms of pharmacological efficacy.

Modulating local S1P receptor signaling as a regenerative immunotherapy after volumetric muscle loss injury.

Regeneration of skeletal muscle after volumetric injury is thought to be impaired by a dysregulated immune microenvironment that hinders endogenous repair mechanisms. Such defects result in fatty infiltration, tissue scarring, chronic inflammation, and debilitating functional deficits. Here, we evaluated the key cellular processes driving dysregulation in the injury niche through localized modulation of sphingosine-1-phosphate (S1P) receptor signaling. We employ dimensionality reduction and pseudotime analysis on single cell cytometry data to reveal heterogeneous immune cell subsets infiltrating preclinical muscle defects due to S1P receptor inhibition. We show that global knockout of S1P receptor 3 (S1PR3) is marked by an increase of muscle stem cells within injured tissue, a reduction in classically activated relative to alternatively activated macrophages, and increased bridging of regenerating myofibers across the defect. We found that local S1PR3 antagonism via nanofiber delivery of VPC01091 replicated key features of pseudotime immune cell recruitment dynamics and enhanced regeneration characteristic of global S1PR3 knockout. Our results indicate that local S1P receptor modulation may provide an effective immunotherapy for promoting a proreparative environment leading to improved regeneration following muscle injury.

Inflammatory milieu of muscle biopsies in juvenile dermatomyositis.

Juvenile dermatomyositis (JDM) is an inflammatory myopathy which causes severe morbidity and high mortality if untreated. In this study, we aimed to define the T-helper cell profile in the muscle biopsies of JDM patients. Muscle biopsies of twenty-six patients (50% female) were included in the study. Immunohistochemical expression of CD3, CD20, CD138, CD68, IL-17, Foxp3, IFN-É£, IFN-alpha and IL-4 was studied and muscle biopsies were scored using the JDM muscle biopsy scoring tool. Inflammatory cells were in small clusters in perimysium and perivascular area or scattered throughout the endomysium in most biopsies; however in 2 biopsies, lymphoid follicle-like big clusters were observed, and in one, there was a very dense and diffuse inflammatory infiltration nearly destroying all the muscle architecture. Seventy-three per cent of the biopsies had T cells, 88% had B cells, 57% had plasma cells, and all had macrophages. As for T-helper cell subtypes, 80% of the biopsies were Th1 positive, 92% Th17 positive and 30% Treg positive. No IL-4 positive inflammatory cell was detected, and only 2 biopsies showed IFN-alpha positivity. The mean JDM biopsy score was 17.6, meaning moderate to severe muscular involvement. Visual analogue score of the pathologist was strongly correlated with histopathological features. B cells, macrophages, plasma cells and T cells constitute the inflammatory milieu of the JDM muscle biopsies. As for T cells, JDM is a disease mainly related with Th1 and Th17 T-helper cell subtypes and to some extend Treg. Th2 cells are not involved in the pathogenesis.

TLR9-Activating CpG-B ODN but Not TLR7 Agonists Triggers Antibody Formation to Factor IX in Muscle Gene Transfer.

Innate immune signals that promote B cell responses in gene transfer are generally ill-defined. In this study, we evaluate the effect of activating endosomal Toll-like receptors 7, 8, and 9 (TLR7, TLR7/8, and TLR9) on antibody formation during muscle-directed gene therapy with adeno-associated virus (AAV) vectors. We examined whether activation of endosomal TLRs, by adenine analog CL264 (TLR7 agonist), imidazolquinolone compound R848 (TLR7/8 agonist), or class B CpG oligodeoxynucleotides ODN1826 (TLR9 agonist), could augment antibody formation upon intramuscular administration of AAV1 expressing human clotting factor IX (AAV1-hFIX) in mice. The TLR9 agonist robustly enhanced antibody formation by the 1st week, thus initially eliminating systemic hFIX expression. By contrast, the TLR7 and TLR7/8 agonists did not markedly promote antibody formation, or significantly reduce circulating hFIX. We concurrently investigated the effects of these TLR agonists during muscle gene transfer on mature B cells and dendritic cells (DCs) in the draining lymph nodes including conventional DCs (CD11b+ or CD8α+ cDCs), monocyte-derived dendritic cells (moDCs), and plasmacytoid dendritic cells (pDCs). Only TLR9 stimulation caused a striking increase in the frequency of moDCs within 24 h. The TLR7/8 and TLR9 agonists activated pDCs, both subsets of cDCs, and mature B cells, whereas the TLR7 agonist had only mild effects on these cells. Thus, these TLR ligands have distinct effects on DCs and mature B cells, yet only the TLR9 agonist enhanced the humoral immune response against AAV-expressed hFIX. These new findings indicate a unique ability of certain TLR9 agonists to stimulate B cell responses in muscle gene transfer through enrichment of moDCs.

No Beneficial Effects of Resveratrol on the Metabolic Syndrome: A Randomized Placebo-Controlled Clinical Trial.

Context: Low-grade inflammation is associated with obesity and the metabolic syndrome (MetS). Preclinical evidence suggests that resveratrol (RSV) has beneficial metabolic and anti-inflammatory effects that could have therapeutic implications. Objective: To investigate effects of long-term RSV treatment on inflammation and MetS. Setting and Design: A randomized, placebo-controlled, double-blind, parallel group clinical trial conducted at Aarhus University Hospital. Participants: Middle-aged community-dwelling men (N = 74) with MetS, 66 of whom completed all visits (mean ± standard error of the mean): age, 49.5 ± 0.796 years; body mass index, 33.8 ± 0.44 kg/m2; waist circumference, 115 ± 1.14 cm. Intervention: Daily oral supplementation with 1000 mg RSV (RSVhigh), 150 mg RSV, or placebo for 16 weeks. Main outcome measures: Plasma levels of high-sensitivity C-reactive protein (hs-CRP), circulating lipids, and inflammatory markers in circulation and adipose/muscle tissue biopsy specimens; glucose metabolism; and body composition including visceral fat and ectopic fat deposition. Results: RSV treatment did not lower circulating levels of hs-CRP, interleukin 6, or soluble urokinase plasminogen activator receptor in plasma, and inflammatory gene expression in adipose and muscle tissues also remained unchanged. RSV treatment had no effect on blood pressure, body composition, and lipid deposition in the liver or striated muscle. RSV treatment had no beneficial effect on glucose or lipid metabolism. RSVhigh treatment significantly increased total cholesterol (P < 0.002), low-density lipoprotein (LDL) cholesterol (P < 0.006), and fructosamine (P < 0.013) levels compared with placebo. Conclusion: RSV treatment did not improve inflammatory status, glucose homeostasis, blood pressure, or hepatic lipid content in middle-aged men with MetS. On the contrary, RSVhigh significantly increased total cholesterol, LDL cholesterol, and fructosamine levels compared with placebo.

Effects of 15 weeks of resistance exercise on pro-inflammatory cytokine levels in the vastus lateralis muscle of patients with fibromyalgia.

BACKGROUND: This study aimed at investigating the effect of a resistance exercise intervention on the interstitial muscle levels of pro-inflammatory cytokines in fibromyalgia (FMS) and healthy controls (CON). METHODS: Twenty-four female patients with FMS (54 ± 8 years) and 27 female CON (54 ± 9 years) were subjected to intramuscular microdialysis of the most painful vastus lateralis muscle before and after 15 weeks of progressive resistance exercise twice per week. Baseline dialysates were sampled in the resting muscle 140 min after insertion of the microdialysis catheter. The participants then performed repetitive dynamic contractions (knee extension) for 20 min, followed by 60 min rest. Pain intensity was assessed with a 0-100 mm visual analogue scale (VAS), and fatigue was assessed with Borg's RPE throughout microdialysis. Dialysates were sampled every 20 min and analyzed with Luminex for interleukin (IL)-1ß, tumor necrosis factor (TNF) alpha, IL-6, and IL-8. RESULTS: At both sessions and for both groups the dynamic contractions increased pain (P < 0.012) and fatigue (P < 0.001). The levels of TNF were lower in the FMS group than the CON group at both sessions (P < 0.05), but none of the other cytokines differed between the groups. IL-6 and IL-8 increased after the dynamic contractions in both groups (P < 0.010), while TNF increased only in CON (P < 0.05) and IL-1ß did not change. Overall pain intensity was reduced after the 15 weeks of resistance exercise in FMS (P < 0.05), but there was no changes in fatigue or cytokine levels. CONCLUSION: Progressive resistance exercise for 15 weeks did not affect the interstitial levels of IL-1ß, TNF, IL-6, and IL-8 in the vastus lateralis muscle of FMS patients or CON. TRIAL REGISTRATION: Clinicaltrials.gov NCT01226784 , registered 21 October 2010.

Muscle inflammation susceptibility: a prognostic index of recovery potential after hip arthroplasty?

While elective total hip arthroplasty (THA) for end-stage osteoarthritis (OA) improves pain, mobility function, and quality of life in most cases, a large proportion of patients suffer persistent muscle atrophy, pain, and mobility impairment. Extensive skeletal muscle damage is unavoidable in these surgical procedures, and it stands to reason that poor recovery and long-term mobility impairment among some individuals after THA is linked to failed muscle regeneration and regrowth following surgery and that local muscle inflammation susceptibility (MuIS) is a major contributing factor. Here we present results of two integrated studies. In study 1, we compared muscle inflammation and protein metabolism signaling in elective THA (n=15) vs. hip fracture/trauma (HFX; n=11) vs. nonsurgical controls (CON; n=19). In study 2, we compared two subgroups of THA patients dichotomized into MuIS⁺ (n=7) or MuIS⁻ (n=7) based on muscle expression of TNF-like weak inducer of apoptosis (TWEAK) receptor (Fn14). As expected, HFX demonstrated overt systemic and local muscle inflammation and hypermetabolism. By contrast, no systemic inflammation was detected in elective THA patients; however, local muscle inflammation in the perioperative limb was profound in MuIS⁺ and was accompanied by suppressed muscle protein synthesis compared with MuIS⁻. Muscle from the contralateral limb of MuIS⁺ was unaffected, providing evidence of a true inflammation susceptibility localized to the muscle surrounding the hip with end-stage OA. We suggest MuIS status assessed at the time of surgery may be a useful prognostic index for muscle recovery potential and could therefore provide the basis for a personalized approach to postsurgery rehabilitation.

Association between skeletal muscle inflammatory markers and walking pattern in people with knee osteoarthritis.

OBJECTIVE: Patients with knee osteoarthritis (OA) are characterized by increased muscle inflammation and altered gait. We investigated the association between proinflammatory mediators in the vastus lateralis and physical function and gait in patients with knee OA. METHODS: Nineteen patients with knee OA underwent gait analysis, assessment of self-reported pain and physical function (Western Ontario and McMaster Universities Osteoarthritis Index [WOMAC]), and a muscle biopsy that was taken during their knee replacement surgery. Muscle was analyzed for cellular protein inflammatory mediators, interleukin-6, monocyte chemotactic protein 1 (MCP-1), p65 NF-κB, signal transducer and activator of transcription 3 (STAT-3), and JNK-1. Sagittal plane knee function, including early stance knee range of motion (ROM) and knee sagittal plane impulse, was measured using a motion analysis system. Pearson's correlation was used to assess relationships between selected variables. RESULTS: Significant positive correlations were found between MCP-1 and self-perceived stiffness, physical function, and the total WOMAC score (P < 0.05). MCP-1 was also negatively correlated with early stance knee ROM (r = -0.52, P = 0.023). Reduced velocity was associated with elevated levels of p65 NF-κB and STAT-3 (P < 0.05). Knee sagittal plane impulse was negatively correlated with JNK-1 (P = 0.02), indicating reduction in knee impulse with an increased level of JNK-1. CONCLUSION: Increased levels of several proinflammatory mediators were correlated with altered knee function during walking as well as greater physical disability and slower gait velocity. Identification of the cellular and molecular mechanisms associated with muscle inflammation is important to better understand the underlying mechanism responsible for altered gait and function in patients with knee OA.

Inflammatory cells and apoptosis in respiratory and limb muscles of patients with COPD.

Discrepancies exist regarding the involvement of cellular inflammation and apoptosis in the muscle dysfunction of chronic obstructive pulmonary disease (COPD) patients with preserved body composition. We explored whether levels of inflammatory cells and apoptosis were increased in both respiratory and limb muscles of COPD patients without nutritional abnormalities. In the vastus lateralis, external intercostals, and diaphragms of severe and moderate COPD patients with normal body composition, and in healthy subjects, intramuscular leukocytes and macrophage levels were determined (immunohistochemistry). Muscle structure was also evaluated. In the diaphragm and vastus lateralis of severe and moderate COPD patients and controls, apoptotic nuclei were explored using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, electron microscopy, and caspase-3 expression. In COPD patients compared with controls, diaphragm and intercostal levels of inflammatory cells were extremely low and not significantly different. However, in the vastus lateralis of the severe patients, inflammatory cell counts, although also very low, were significantly greater. In those patients, TUNEL-positive nuclei levels were also significantly greater in diaphragms and vastus lateralis. A significant inverse relationship was found between quadriceps TUNEL-positive nuclei levels and muscle force. Ultrastructural apoptotic nuclei revealed no differences in respiratory or limb muscles between COPD patients and controls. Muscle caspase-3 expression did not differ between patients and controls. In severe COPD patients with preserved body composition, while increased apoptotic nuclei seems to be a contributor to their muscle dysfunction, cellular inflammation does not. The increased numbers of TUNEL-positive nuclei in their muscles suggest that they may also be exposed to a continuous repair/remodeling process.

Increased inflammatory cytokine expression in the vastus lateralis of patients with knee osteoarthritis.

OBJECTIVE: Increased inflammation and pain are inseparable parts of knee osteoarthritis (OA) that may lead to disuse of the affected limb. The aim of this study was to examine the effects of knee OA on inflammation- and atrophy-related genes and proteins in the vastus lateralis muscle of patients with knee OA. METHODS: Nineteen patients with knee OA and 14 asymptomatic control subjects matched for age and body mass index underwent strength measurements and a muscle biopsy. Muscle was analyzed for the total cellular protein of inflammatory kinases (p65 NF-κB, JNK1/2, STAT-3, and suppressor of cytokine signaling 3 [SOCS-3]) and inflammatory intracellular molecules (interleukin-6 [IL-6], IL-8, monocyte chemoattractant protein 1 [MCP-1], tumor necrosis factor α [TNFα], IL-1ß, and atrogin-1). RESULTS: Knee OA resulted in greater levels of IL-6 protein (34%; P = 0.002). The levels of inflammatory kinases, including STAT-3 (187%; P = 0.002), p65 NF-κB (156%; P = 0.002), and JNK1 (179%; P = 0.027), were also elevated. Furthermore, elevated expression of gene transcripts encoding MCP-1 (28%; P = 0.023), TNFα (85%; P < 0.001), and SOCS-3 (38%; P = 0.055) was observed in patients with knee OA compared with control subjects. Patients with knee OA had reduced muscle strength compared with control subjects (mean ± SEM 84.7 ± 8.7 versus 143.1 ± 20.8 Nm; P = 0.005). Negative correlations were observed between muscle strength and MCP-1 protein abundance (r = -0.37 [P = 0.042]) and the gene expression of TNFα and atrogin-1 messenger RNA (r = -0.46 [P = 0.012] and r = -0.36 [P = 0.040], respectively). CONCLUSION: Gene expression and the protein abundance of numerous muscle markers of inflammation and atrophy were elevated in patients with knee OA, and the increase in muscle inflammation was associated with a reduction in muscle strength. Given the role inflammation markers may play in muscle strength and atrophy, further studies are needed to investigate the effect of exercise intervention on skeletal muscle inflammation.

Acute effects of dietary fat on inflammatory markers and gene expression in first-degree relatives of type 2 diabetes patients.

BACKGROUND: Subjects with type 2 diabetes (T2D) and their relatives (REL) carry an increased risk of cardiovascular disease (CVD). Low-grade inflammation, an independent risk factor for CVD, is modifiable by diet. Subjects with T2D show elevated postprandial inflammatory responses to fat-rich meals, while information on postprandial inflammation in REL is sparse. AIM: To clarify whether medium-chain saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) have differential acute effects on low-grade inflammation in REL compared to controls (CON). METHODS: In randomized order, 17 REL and 17 CON ingested two fat-rich meals, with 72 energy percent from MUFA and 79 energy percent from mainly medium-chain SFA, respectively. Plasma high sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), adiponectin, and leptin were measured at baseline, 15 min, 60 min, and 240 min postprandially. Muscle and adipose tissue biopsies were taken at baseline and 210 min after the test meal, and expression of selected genes was analyzed. RESULTS: Plasma IL-6 increased (p < 0.001) without difference between REL and CON and between the meals, whereas plasma adiponectin and plasma hs-CRP were unchanged during the 240 min observation period. Plasma leptin decreased slightly in response to medium-chain SFA in both groups, and to MUFA in REL. Several genes were differentially regulated in muscle and adipose tissue of REL and CON. CONCLUSIONS: MUFA and medium-chain SFA elicit similar postprandial circulating inflammatory responses in REL and CON. Medium-chain SFA seems more proinflammatory than MUFA, judged by the gene expression in muscle and adipose tissue of REL and CON.

Protease supplementation improves muscle function after eccentric exercise.

UNLABELLED: Protease supplementation has been purported to reduce the damaging effects of eccentric exercise and accelerate recovery of muscle function, possibly by regulating inflammation. PURPOSE: To determine the effectiveness of protease supplementation in attenuating eccentric exercise-induced skeletal muscle damage and inflammation. METHODS: After standard physical and hemodynamic assessment and fasting venous blood samples, subjects performed isokinetic extension/flexion of the quadriceps group on a Biodex isokinetic dynamometer at 60°·s(-1), followed by VO2max testing. Subjects were randomly assigned to consume 5.83 g daily of either a cellulose placebo (N = 15; 22.27 ± 3.33 yr, 71.17 ± 2.91 inches, 179.4 ± 24.05 lb, 50.55 ± 5.66 mL·kg(-1)·min(-1)) or a proteolytic supplement containing fungal proteases, bromelain, and papain (N = 14; 22.85 ± 5.9 yr, 70.0 ± 2.67 inches, 173.11 ± 29.94 lb, 49.69 ± 6.15 mL·kg(-1)·min(-1)) for a period of 21 d. After the supplementation period, subjects donated blood samples before performing a 45-min downhill (-17.5%) treadmill protocol at 60% of VO2max. An additional four blood draws and three muscle function tests were performed during the next 48 h. Blood was analyzed using standard hematology and clinical chemistry, enzyme-linked immunosorbent assay, and bead array. Blood data were analyzed using multivariate analysis of variance (MANOVA) with repeated measures, whereas Biodex data were analyzed using a MANOVA on %Δ values. RESULTS: Significant group differences (T1-T3, P = 0.033; T1-T4, P = 0.043) and another strong trend (T1-3 h, P = 0.055) were observed for flexion (peak torque %Δ at 60°·s(-1)) indicating higher force production in the protease group. Significant group × time interactions (P < 0.05) were observed, including elevations in circulating eosinophils and basophils in the protease group coinciding with lower levels of serum cyclooxygenase 2, interleukin 6, and interleukin 12 in this group. CONCLUSIONS: Protease supplementation seems to attenuate muscle strength losses after eccentric exercise by regulating leukocyte activity and inflammation.

[Effect of Myostatin gene vaccine on immunized mice].

AIM: To characterize the immunological function of Myostatin gene vaccine and observe the effect of Myostatin gene vaccine on the immunized animal. METHODS: The mice were immunized with Myostatin gene vaccine. We characterized the antibody titer in the immunized mice serum by the ELISA. The anti-serum was detected by auto-biochemistry analysis software. Meanwhile, the effect anti-serum was detected by auto-biochemistry analysis software. Meanwhile, the effect of Myostatin gene vaccine on skeletal-muscle of the immunized mice was analyzed by HE stain. The cross section area of muscle fiber in immunized mice was analyzed by the Scion Image 4.02 software. RESULTS: Myostatin gene vaccine could induce the anti-serum against Myostatin in immunized mice. Compared with that in the control group, the mean weight in the pVAC-TT-Ms immunized mice group increased with 9.8%. The quadriceps muscle, gastrocnemius muscle and pectoralis magor in immunized mice increased with 24.1%, 10.9% and 20.3%, respectively. CONCLUSION: Myostatin gene vaccine induced the specific antibody against Myostatin, and made the muscle strong.

Potential of transfected muscle cells to contribute to DNA vaccine immunogenicity.

The mechanism(s) by which DNA vaccines trigger the activation of Ag-specific T cells is incompletely understood. A series of in vivo and in vitro experiments indicates plasmid transfection stimulates muscle cells to up-regulate expression of MHC class I and costimulatory molecules and to produce multiple cytokines and chemokines. Transfected muscle cells gain the ability to directly present Ag to CD8 T cells through an IFN-regulatory factor 3-dependent process. These findings suggest that transfected muscle cells at the site of DNA vaccination may contribute to the magnitude and/or duration of the immune response initiated by professional APCs.