Expression of interleukin-18 in muscle tissue of patients with polymyositis or dermatomyositis and effects of conventional immunosuppressive treatment.

Objectives: To investigate the expression of IL-18 in symptomatic and asymptomatic muscle tissues of patients with PM and DM and the effects of conventional immunosuppressive treatment on such expression. Methods: Two cohorts of patients were included in this study. The first cohort consisted of 10 new-onset myositis patients. IL-18 expression was compared between symptomatic and asymptomatic muscle biopsies that were taken prior to treatment. The second cohort consisted of another 10 patients with repeated muscle biopsies before and after 8 months with conventional immunosuppressive treatment. Using immunohistochemistry, IL-18 expression in muscle tissues was compared before and after treatment. Biopsies from seven healthy individuals were included as controls. Results: IL-18 expression was predominantly localized to inflammatory cells and capillaries in patients and mostly to capillaries in healthy controls. Total IL-18 expression in muscle tissues from the new-onset patients, at both symptomatic and asymptomatic sites, was significantly higher compared with healthy controls (P = 0.007 and P = 0.002) with no statistical difference in appearances between symptomatic and asymptomatic sites. The number of IL-18 positive capillaries was not different among symptomatic, asymptomatic and healthy muscles. Total IL-18 expression appeared lower in biopsies from patients receiving and improving with immunosuppressive treatment, particularly the number of IL-18 positive inflammatory cells but not the number of IL-18 positive capillaries, which was consistent with significantly decreased expression of CD68+ macrophages (P = 0.04). Conclusion: IL-18 is highly expressed in muscle tissue in the context of inflammatory myopathies and based on its plausible effector functions could provide a novel therapeutic target in future.

Effect of endurance exercise on microRNAs in myositis skeletal muscle-A randomized controlled study.

OBJECTIVE: To identify changes in skeletal muscle microRNA expression after endurance exercise and associate the identified microRNAs with mRNA and protein expression to disease-specific pathways in polymyositis (PM) and dermatomyositis (DM) patients. METHODS: Following a parallel clinical trial design, patients with probable PM or DM, exercising less than once a week, and on stable medication for at least one month were randomized into two groups at Karolinska University Hospital: a 12-week endurance exercise group (n = 12) or a non-exercised control group (n = 11). Using an Affymetrix microarray, microRNA expression was determined in paired muscle biopsies taken before and after the exercise intervention from 3 patients in each group. Ingenuity pathway analysis with a microRNA target filter was used to identify microRNA transcript targets. These targets were investigated at the mRNA (microarray) and protein (mass spectrometry) levels in patients. RESULTS: Endurance exercise altered 39 microRNAs. The microRNAs with increased expression were predicted to target transcripts involved in inflammatory processes, metabolism, and muscle atrophy. Further, these target transcripts had an associated decrease in mRNA expression in exercised patients. In particular, a decrease in the NF-κB regulator IKBKB was associated with an increase in its target microRNA (miR-196b). At the protein level, there was an increase in mitochondrial proteins (AK3, HIBADH), which were associated with a decrease in microRNAs that were predicted to regulate their expression. CONCLUSION: Improvement in disease phenotype after exercise is associated with increasing microRNAs that target and downregulate immune processes at the transcript level, as well as decreasing microRNAs that target and upregulate mitochondrial content at the protein level. Therefore, microRNAs may improve disease by decreasing immune responses and increasing mitochondrial biogenesis. TRIAL REGISTRATION: ClinicalTrials.gov NCT01184625.

Effects on muscle tissue remodeling and lipid metabolism in muscle tissue from adult patients with polymyositis or dermatomyositis treated with immunosuppressive agents.

BACKGROUND: Polymyositis (PM) and dermatomyositis (DM) are autoimmune muscle diseases, conventionally treated with high doses of glucocorticoids in combination with immunosuppressive drugs. Treatment is often dissatisfying, with persisting muscle impairment. We aimed to investigate molecular mechanisms that might contribute to the persisting muscle impairment despite immunosuppressive treatment in adult patients with PM or DM using gene expression profiling of repeated muscle biopsies. METHODS: Paired skeletal muscle biopsies from six newly diagnosed adult patients with DM or PM taken before and after conventional immunosuppressive treatment were examined by gene expression microarray analysis. Selected genes that displayed changes in expression were analyzed by Western blot. Muscle biopsy sections were evaluated for inflammation, T lymphocytes (CD3), macrophages (CD68), major histocompatibility complex (MHC) class I expression and fiber type composition. RESULTS: After treatment, genes related to immune response and inflammation, including inflammasome pathways and interferon, were downregulated. This was confirmed at the protein level for AIM-2 and caspase-1 in the inflammasome pathway. Changes in genes involved in muscle tissue remodeling suggested a negative effect on muscle regeneration and growth. Gene markers for fast type II fibers were upregulated and fiber composition was switched towards type II fibers in response to treatment. The expression of genes involved in lipid metabolism was altered, suggesting a potential lipotoxic effect on muscles of the immunosuppressive treatment. CONCLUSION: The anti-inflammatory effect of immunosuppressive treatment was combined with negative effects on genes involved in muscle tissue remodeling and lipid metabolism, suggesting a negative effect on recovery of muscle performance which may contribute to persisting muscle impairment in adult patients with DM and PM.

Effects of conventional immunosuppressive treatment on CD244+ (CD28null) and FOXP3+ T cells in the inflamed muscle of patients with polymyositis and dermatomyositis.

BACKGROUND: T-cell infiltrates may persist in muscle tissue of polymyositis (PM) and dermatomyositis (DM) patients despite aggressive immunosuppressive treatment. Here, we investigated to what extent persistent T cells in affected muscle were FOXP3+, a marker for regulatory T cells (Tregs), or CD244+, a marker for CD28null T cells, and whether their presence correlated to clinical outcome. The sensitivity of CD28null T cells towards glucocorticoid and Treg-mediated immunosuppression was also investigated. METHODS: Muscle biopsies from 16 newly diagnosed or untreated patients with PM/DM were investigated by immunohistochemistry for expression of CD3, FOXP3 and CD244 before and after treatment with glucocorticoids and immunosuppressive agents. For clinical evaluation, serum levels of creatine kinase, muscle performance (FI and MMT8), disease activity (MITAX) and disability (HAQ) were measured. In vitro suppressive effects of glucocorticoids and Tregs on T-cell activation were measured by CD69 upregulation. RESULTS: Before treatment, CD244+ cells were present at higher proportions compared to FOXP3+ cells in the inflamed muscle. Following treatment, FOXP3+ cell numbers decreased while CD244+ cells persisted. Patients with impaired muscle function (<75 % FI) post-treatment had higher levels of CD244+ cells in the follow-up biopsy compared to those with FI >75 %. MITAX and HAQ correlated with the number of CD244+ cells post-treatment. CD4+CD28null T cells displayed lower sensitivity towards both glucocorticoid and Treg-mediated immunosuppression in vitro compared to their CD28+ counterparts. CONCLUSIONS: Poor outcome in patients with myositis following immunosuppressive therapy was linked to persistence of CD244+ (CD28null) T cells in muscle tissue, suggesting their resistance against immunosuppression. A relative loss of regulatory T cells could also contribute to poor clinical outcome given their recently ascribed role in muscle tissue regeneration.

Endurance Exercise Improves Molecular Pathways of Aerobic Metabolism in Patients With Myositis.

OBJECTIVE: Endurance exercise demonstrates beneficial effects in polymyositis/dermatomyositis (PM/DM); however, the molecular effects of exercise on skeletal muscle are incompletely understood. We undertook this controlled pilot study to investigate the effects of a 12-week endurance exercise training program on the molecular profile of skeletal muscle in patients with established PM/DM compared to a nonexercised control group of patients with established PM/DM. METHODS: Fifteen patients (7 in the exercise group and 8 in the control group) with paired baseline and 12-week follow-up muscle biopsy samples were included. Messenger RNA expression profiling, mass spectrometry-based quantitative proteomics, and immunohistochemical analyses were performed on muscle biopsy samples to determine molecular adaptations associated with changes in clinical measurements induced by endurance exercise. RESULTS: Compared to the control group, the exercise group improved in minutes of cycling time (P < 0.01) and Vo2 max (P < 0.05). The exercise group also had reduced disease activity (P < 0.05) and reduced lactate levels at exhaustion (P < 0.05). Genes related to capillary growth, mitochondrial biogenesis, protein synthesis, cytoskeletal remodeling, and muscle hypertrophy were up-regulated in the exercise group, while genes related to inflammation/immune response and endoplasmic reticulum stress were down-regulated. Mitochondrial pathways including the oxidative phosphorylation metabolic pathway were most affected by the endurance exercise, as demonstrated by proteomics analysis. The exercise group also showed a higher number of capillaries per mm(2) in follow-up biopsy samples (P < 0.05). CONCLUSION: Our data indicate that endurance exercise in patients with established PM and DM may activate an aerobic phenotype and promote muscle growth and simultaneously suppress the inflammatory response in these patients' muscles, as supported by a combination of data on gene expression, proteomics, and capillary density in repeated muscle biopsies.

Resistive home exercise in patients with recent-onset polymyositis and dermatomyositis -- a randomized controlled single-blinded study with a 2-year followup.

OBJECTIVE: To evaluate the outcome of resistive home exercise and its possible longterm influence on health, disability, and disease activity in patients with active polymyositis (PM) or dermatomyositis (DM). METHODS: Nineteen patients with recent-onset PM/DM were included after introduction of high-dose prednisolone. They were assessed by independent assessors as to perceived health, muscle performance, aerobic capacity, and serum creatine phosphokinase (CPK) at baseline and after 24 weeks, including repeated muscle biopsies at 24 weeks (single-blinded randomized controlled study), and in an open-label followup at 52, 78, and 104 weeks. Patients were randomized to 12 weeks, 5 days/week resistive home exercise with telephone support and encouragement for another 12 weeks of twice-a-week home or gym exercise (EG, n = 10) or to 24 weeks, 5 days/week range of motion exercise (CG, n = 9). Patients in the CG group without inflammatory infiltrates in muscle biopsies at 24 weeks were invited to the 12-week resistive home exercises. RESULTS: At baseline, the EG had poorer perceived health, but otherwise the groups were comparable. At 24 weeks, both groups improved in muscle performance and aerobic capacity (p < 0.001 to < 0.05) with no signs of increased inflammation assessed by CPK levels or muscle biopsies. Both groups improved in muscle performance and aerobic capacity up to 52 weeks (p < 0.05) lasting to 104 weeks in the EG (p < 0.05) and presented minor improvements in perceived health. CONCLUSION: Our study supports the safety of resistive exercise in patients with active PM/DM but did not reveal any between-group differences in exercise effects. An individually adapted physical therapist-supervised home exercise program might be recommended in early active PM/DM, with regular evaluation of muscle performance and health.

Improved exercise performance and increased aerobic capacity after endurance training of patients with stable polymyositis and dermatomyositis.

INTRODUCTION: This randomized, controlled study on patients with polymyositis or dermatomyositis was based on three hypotheses: patients display impaired endurance due to reduced aerobic capacity and muscle weakness, endurance training improves their exercise performance by increasing the aerobic capacity, and endurance training has general beneficial effects on their health status. METHODS: In the first part of this study, we compared 23 patients with polymyositis or dermatomyositis with 12 age- and gender-matched healthy controls. A subgroup of patients were randomized to perform a 12-week endurance training program (exercise group, n = 9) or to a non-exercising control group (n = 6). We measured maximal oxygen uptake (VO2 max) and the associated power output during a progressive cycling test. Endurance was assessed as the cycling time to exhaustion at 65% of VO2 max. Lactate levels in the vastus lateralis muscle were measured with microdialysis. Mitochondrial function was assessed by measuring citrate synthase (CS) and ß-hydroxyacyl-CoA dehydrogenase (ß-HAD) activities in muscle biopsies. Clinical improvement was assessed according to the International Myositis Assessment and Clinical Studies Group (IMACS) improvement criteria. All assessors were blinded to the type of intervention (that is, training or control). RESULTS: Exercise performance and aerobic capacity were lower in patients than in healthy controls, whereas lactate levels at exhaustion were similar. Patients in the exercise group increased their cycling time, aerobic capacity and CS and ß-HAD activities, whereas lactate levels at exhaustion decreased. Six of nine patients in the exercise group met the IMACS improvement criteria. Patients in the control group did not show any consistent changes during the 12-week study. CONCLUSIONS: Polymyositis and dermatomyositis patients have impaired endurance, which could be improved by 12 weeks of endurance training. The clinical improvement corresponds to increases in aerobic capacity and muscle mitochondrial enzyme activities. The results emphasize the importance of endurance exercise in addition to immunosuppressive treatment of patients with polymyositis or dermatomyositis. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01184625.

Activated LTB4 pathway in muscle tissue of patients with polymyositis or dermatomyositis.

OBJECTIVE: To investigate the involvement of the leukotriene B4 (LTB4) pathway in polymyositis (PM) and dermatomyositis (DM) and the effect of immunosuppressive treatment on the LTB4 pathway. METHODS: 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP) and LTB4 receptor-1 (BLT1) expression was analysed by immunohistochemistry in muscle tissue from patients with PM/DM before and after immunosuppressive treatment and from healthy individuals. In vivo LTB4 in thigh muscle was measured by microdialysis at rest and after acute exercise in another cohort of patients and healthy controls. RESULTS: The number of 5-LO-positive cells and BLT1-positive capillaries was higher in patients with PM/DM than in healthy individuals. The number of FLAP-expressing cells divided the patients into two groups (high/low expression). Treatment reduced the number of FLAP-positive cells in the group with initial high levels, however the expression remained high compared with healthy individuals. The number of BLT1-positive cells was also reduced while staining for 5-LO was unchanged. An inverse correlation was observed between the number of 5-LO or FLAP-positive cells in muscle tissue and muscle performance. LTB4 could be detected in dialysate of muscle tissue in vivo in both patients and healthy controls and was significantly increased after exercise in patients. CONCLUSION: The LTB4 pathway is upregulated in muscle tissue from patients with PM/DM and this upregulation correlated negatively to muscle performance, suggesting a role for LTB4 in myositis muscle weakness. The immunosuppressive treatment was insufficient on the LTB4 pathway and, for patients with high expression of FLAP, FLAP inhibitors may be considered as possible therapy.

Effects of immunosuppressive treatment on interleukin-15 and interleukin-15 receptor α expression in muscle tissue of patients with polymyositis or dermatomyositis.

OBJECTIVES: To investigate the expression of interleukin (IL)-15 and IL-15 receptor α (IL-15Rα) in muscle tissue from patients with polymyositis or dermatomyositis before and after conventional immunosuppressive (IS) treatment. METHODS: Muscle biopsies from 17 patients before and after conventional IS treatment and seven healthy individuals were investigated by immunohistochemistry using antibodies against IL-15 and IL-15Rα. Quantification was performed by computerised image analysis. Cellular localisation of IL-15 was determined by double immunofluorescence. Clinical outcome was measured by the functional index and serum creatine kinase. Human myotubes were cultured and IL-15 staining was performed by immunocytochemistry. RESULTS: IL-15 was observed in mononuclear inflammatory cells of muscle tissue while IL-15Rα was localised to mononuclear inflammatory cells, capillaries and large vessels. Double staining showed localisation of IL-15 to CD163+ macrophages. A significantly larger number of IL-15 and IL-15Rα-positive cells were seen in muscle tissue of patients compared with healthy individuals. Baseline IL-15 expression correlated negatively with improvement in muscle function. After conventional IS treatment, a significantly lower number of IL-15 and IL-15Rα-positive cells was found. However, compared with controls, eight of 17 patients still had more IL-15-positive cells and less muscle function improvement was shown in this group of patients, both in short-term and long-term observations. Human differentiated myotubes were negative for IL-15 staining. CONCLUSIONS: IL-15 and its receptor are expressed in the muscle tissue of patients with myositis and IL-15 expression is correlated with improvement in muscle function. IL-15 may play a role in the pathogenesis of myositis and could be a biological treatment target, at least in a subgroup of patients with polymyositis or dermatomyositis.

Effect of physical training on the proportion of slow-twitch type I muscle fibers, a novel nonimmune-mediated mechanism for muscle impairment in polymyositis or dermatomyositis.

OBJECTIVE: To compare muscle fiber type composition and muscle fiber area in patients with chronic polymyositis or dermatomyositis and healthy controls, and to determine whether physical training for 12 weeks could alter these muscle characteristics. METHODS: Muscle fiber type composition and muscle fiber area were investigated by biochemical and immunohistochemistry techniques in repeated muscle biopsy samples obtained from 9 patients with chronic myositis before and after a 12-week exercise program and in healthy controls. Muscle performance was evaluated by the Functional Index (FI) in myositis and by the Short Form 36 (SF-36) quality of life instrument. RESULTS: Before exercise, the proportion of type I fibers was lower (mean +/- SD 32% +/- 10%) and the proportion of type IIC fibers was higher (3% +/- 3%) in patients compared with healthy controls. After exercise, percentage of type I fiber increased to 42% +/- 13% (P < 0.05), and type IIC decreased to 1% +/- 1%. An exercise-induced 20% increase of the mean fiber area was also observed. The functional capacity measured by the FI in myositis and the physical functioning subscale of the SF-36 increased significantly. Improved physical functioning was positively correlated with the proportion of type I fibers (r = 0.88, P < 0.01) and type II muscle fiber area (r = 0.70, P < 0.05). CONCLUSION: Low muscle endurance in chronic polymyositis or dermatomyositis may be related to a low proportion of oxidative, slow-twitch type I fibers. Change in fiber type composition and increased muscle fiber area may contribute to improved muscle endurance and decreased muscle fatigue after a moderate physical training program.