Role of non-immune mechanisms of muscle damage in idiopathic inflammatory myopathies

Idiopathic inflammatory myopathies (IIMs) comprise a group of autoimmune diseases that are characterized by symmetrical skeletal muscle weakness and muscle inflammation with no known cause. Like other autoimmune diseases, IIMs are treated with either glucocorticoids or immunosuppressive drugs. However, many patients with an IIM are frequently resistant to immunosuppressive treatments, and there is compelling evidence to indicate that not only adaptive immune but also several non-immune mechanisms play a role in the pathogenesis of these disorders. Here, we focus on some of the evidence related to pathologic mechanisms, such as the innate immune response, endoplasmic reticulum stress, non-immune consequences of MHC class I overexpression, metabolic disturbances, and hypoxia. These mechanisms may explain how IIM-related pathologic processes can continue even in the face of immunosuppressive therapies. These data indicate that therapeutic strategies in IIMs should be directed at both immune and non-immune mechanisms of muscle damage

Idiopathic inflammatory myopathies: Pathogenic mechanisms of muscle weakness

Idiopathic inflammatory myopathies (IIMs) are a heterogenous group of complex muscle diseases of unknown etiology. These diseases are characterized by progressive muscle weakness and damage, together with involvement of other organ systems. It is generally believed that the autoimmune response (autoreactive lymphocytes and autoantibodies) to skeletal muscle-derived antigens is responsible for the muscle fiber damage and muscle weakness in this group of disorders. Therefore, most of the current therapeutic strategies are directed at either suppressing or modifying immune cell activity. Recent studies have indicated that the underlying mechanisms that mediate muscle damage and dysfunction are multiple and complex. Emerging evidence indicates that not only autoimmune responses but also innate immune and non-immune metabolic pathways contribute to disease pathogenesis. However, the relative contributions of each of these mechanisms to disease pathogenesis are currently unknown. Here we discuss some of these complex pathways, their inter-relationships and their relation to muscle damage in myositis. Understanding the relative contributions of each of these pathways to disease pathogenesis would help us to identify suitable drug targets to alleviate muscle damage and also improve muscle weakness and quality of life for patients suffering from these debilitating muscle diseases

An amphipathic trans-acting phosphorothioate RNA element delivers an uncharged phosphorodiamidate morpholino sequence in mdx mouse myotube

An efficient method for the delivery of uncharged polyA-tailed phosphorodiamidate morpholino sequences (PMO) in mammalian cells consists of employing a synthetic 8-mer amphipathic trans-acting poly-2′-O-methyluridylic thiophosphate triester element (2′-OMeUtaPS) as a transfection reagent. Unlike the dTtaPS DNA-based element, this RNA element is potent at delivering polyA-tailed PMO sequences to HeLa pLuc 705 cells or to myotube muscle cells. However, much like dTtaPS, the 2′-OMeUtaPS-mediated internalization of PMO sequences occurs through an energy-dependent mechanism; macropinocytosis appears to be the predominant endocytic pathway used for cellular uptake. The transfected PMO sequences induce alternate splicing of either the pre-mRNA encoding luciferase in HeLa pLuc 705 cells or the excision of exon 23 from the pre-mRNA encoding dystrophin in myotube muscle cells of the mdx mouse model of muscular dystrophy with an efficiency comparable to that of commercial cationic lipid reagents but without detrimental cytotoxicity

Daily supplementation of D-ribose shows no therapeutic benefits in the MHC-I transgenic mouse model of inflammatory myositis

Background Current treatments for idiopathic inflammatory myopathies (collectively called myositis) focus on the suppression of an autoimmune inflammatory response within the skeletal muscle. However, it has been observed that there is a poor correlation between the successful suppression of muscle inflammation and an improvement in muscle function. Some evidence in the literature suggests that metabolic abnormalities in the skeletal muscle underlie the weakness that continues despite successful immunosuppression. We have previously shown that decreased expression of a purine nucleotide cycle enzyme, adenosine monophosphate deaminase (AMPD1), leads to muscle weakness in a mouse model of myositis and may provide a mechanistic basis for muscle weakness. One of the downstream metabolites of this pathway, D-ribose, has been reported to alleviate symptoms of myalgia in patients with a congenital loss of AMPD1. Therefore, we hypothesized that supplementing exogenous D-ribose would improve muscle function in the mouse model of myositis. We treated normal and myositis mice with daily doses of D-ribose (4 mg/kg) over a 6-week time period and assessed its effects using a battery of behavioral, functional, histological and molecular measures. Results Treatment with D-ribose was found to have no statistically significant effects on body weight, grip strength, open field behavioral activity, maximal and specific forces of EDL, soleus muscles, or histological features. Histological and gene expression analysis indicated that muscle tissues remained inflamed despite treatment. Gene expression analysis also suggested that low levels of the ribokinase enzyme in the skeletal muscle might prevent skeletal muscle tissue from effectively utilizing D-ribose. Conclusions Treatment with daily oral doses of D-ribose showed no significant effect on either disease progression or muscle function in the mouse model of myositis

Idiopathic inflammatory myopathies: pathogenic mechanisms of muscle weakness

Idiopathic inflammatory myopathies (IIMs) are a heterogenous group of complex muscle diseases of unknown etiology. These diseases are characterized by progressive muscle weakness and damage, together with involvement of other organ systems. It is generally believed that the autoimmune response (autoreactive lymphocytes and autoantibodies) to skeletal muscle-derived antigens is responsible for the muscle fiber damage and muscle weakness in this group of disorders. Therefore, most of the current therapeutic strategies are directed at either suppressing or modifying immune cell activity. Recent studies have indicated that the underlying mechanisms that mediate muscle damage and dysfunction are multiple and complex. Emerging evidence indicates that not only autoimmune responses but also innate immune and non-immune metabolic pathways contribute to disease pathogenesis. However, the relative contributions of each of these mechanisms to disease pathogenesis are currently unknown. Here we discuss some of these complex pathways, their inter-relationships and their relation to muscle damage in myositis. Understanding the relative contributions of each of these pathways to disease pathogenesis would help us to identify suitable drug targets to alleviate muscle damage and also improve muscle weakness and quality of life for patients suffering from these debilitating muscle diseases

Sources of variability and effect of experimental approach on expression profiling data interpretation

BACKGROUND: We provide a systematic study of the sources of variability in expression profiling data using 56 RNAs isolated from human muscle biopsies (34 Affymetrix MuscleChip arrays), and 36 murine cell culture and tissue RNAs (42 Affymetrix U74Av2 arrays). RESULTS: We studied muscle biopsies from 28 human subjects as well as murine myogenic cell cultures, muscle, and spleens. Human MuscleChip arrays (4,601 probe sets) and murine U74Av2 Affymetrix microarrays were used for expression profiling. RNAs were profiled both singly, and as mixed groups. Variables studied included tissue heterogeneity, cRNA probe production, patient diagnosis, and GeneChip hybridizations. We found that the greatest source of variability was often different regions of the same patient muscle biopsy, reflecting variation in cell type content even in a relatively homogeneous tissue such as muscle. Inter-patient variation was also very high (SNP noise). Experimental variation (RNA, cDNA, cRNA, or GeneChip) was minor. Pre-profile mixing of patient cRNA samples effectively normalized both intra- and inter-patient sources of variation, while retaining a high degree of specificity of the individual profiles (86% of statistically significant differences detected by absolute analysis; and 85% by a 4-pairwise comparison survival method). CONCLUSIONS: Using unsupervised cluster analysis and correlation coefficients of 92 RNA samples on 76 oligonucleotide microarrays, we found that experimental error was not a significant source of unwanted variability in expression profiling experiments. Major sources of variability were from use of small tissue biopsies, particularly in humans where there is substantial inter-patient variability (SNP noise)

Muscle myeloid type I interferon gene expression may predict therapeutic responses to rituximab in myositis patients.

Objective. To identify muscle gene expression patterns that predict rituximab responses and assess the effects of rituximab on muscle gene expression in PM and DM. Methods. In an attempt to understand the molecular mechanism of response and non-response to rituximab therapy, we performed Affymetrix gene expression array analyses on muscle biopsy specimens taken before and after rituximab therapy from eight PM and two DM patients in the Rituximab in Myositis study. We also analysed selected muscle-infiltrating cell phenotypes in these biopsies by immunohistochemical staining. Partek and Ingenuity pathway analyses assessed the gene pathways and networks. Results. Myeloid type I IFN signature genes were expressed at higher levels at baseline in the skeletal muscle of rituximab responders than in non-responders, whereas classic non-myeloid IFN signature genes were expressed at higher levels in non-responders at baseline. Also, rituximab responders have a greater reduction of the myeloid and non-myeloid type I IFN signatures than non-responders. The decrease in the type I IFN signature following administration of rituximab may be associated with the decreases in muscle-infiltrating CD19 + B cells and CD68 + macrophages in responders. Conclusion. Our findings suggest that high levels of myeloid type I IFN gene expression in skeletal muscle predict responses to rituximab in PM/DM and that rituximab responders also have a greater decrease in the expression of these genes. These data add further evidence to recent studies defining the type I IFN signature as both a predictor of therapeutic responses and a biomarker of myositis disease activity

Improving Reproducibility of Phenotypic Assessments in the DyW Mouse Model of Laminin-α2 Related Congenital Muscular Dystrophy.

Laminin-α2 related Congenital Muscular Dystrophy (LAMA2-CMD) is a progressive muscle disease caused by partial or complete deficiency of laminin-211, a skeletal muscle extracellular matrix protein. In the last decade, basic science research has queried underlying disease mechanisms in existing LAMA2-CMD murine models and identified possible clinical targets and pharmacological interventions. Experimental rigor in preclinical studies is critical to efficiently and accurately quantify both negative and positive results, degree of efficiency of potential therapeutics and determine whether to move a compound forward for additional preclinical testing. In this review, we compare published available data measured to assess three common parameters in the widely used mouse model DyW, that mimics LAMA2-CMD, we quantify variability and analyse its possible sources. Finally, on the basis of this analysis, we suggest standard set of assessments and the use of available standardized protocols, to reduce variability of outcomes in the future and to improve the value of preclinical research

Enhanced autoantigen expression in regenerating muscle cells in idiopathic inflammatory myopathy

Unique autoantibody specificities are strongly associated with distinct clinical phenotypes, making autoantibodies useful for diagnosis and prognosis. To investigate the mechanisms underlying this striking association, we examined autoantigen expression in normal muscle and in muscle from patients with autoimmune myositis. Although myositis autoantigens are expressed at very low levels in control muscle, they are found at high levels in myositis muscle. Furthermore, increased autoantigen expression correlates with differentiation state, such that myositis autoantigen expression is increased in cells that have features of regenerating muscle cells. Consistent with this, we found that cultured myoblasts express high levels of autoantigens, which are strikingly down-regulated as cells differentiate into myotubes in vitro. These data strongly implicate regenerating muscle cells rather than mature myotubes as the source of ongoing antigen supply in autoimmune myositis. Myositis autoantigen expression is also markedly increased in several cancers known to be associated with autoimmune myositis, but not in their related normal tissues, demonstrating that tumor cells and undifferentiated myoblasts are antigenically similar. We propose that in cancer-associated myositis, an autoimmune response directed against cancer cross-reacts with regenerating muscle cells, enabling a feed-forward loop of tissue damage and antigen selection. Regulating pathways of antigen expression may provide unrecognized therapeutic opportunities in autoimmune diseases

A population-based study of children suggests blunted morning cortisol rhythms are associated with alterations of the systemic inflammatory state

Background: In children, digital media, lifestyle, and the COVID pandemic have impacted sunlight exposure, exercise, and diet patterns - cues that entrain the circadian clock. We hypothesized that low morning cortisol reflects a weak circadian clock, impacting the pro-inflammatory state. The primary objective was to test relationships between diurnal cortisol fluctuations and the inflammatory state in children as a means of providing indirect support for this hypothesis. Methods: The Cardiovascular Health Intervention Program (CHIP) was a population-based cross-sectional and longitudinal study of circadian health in public elementary school children in Southern Maine, USA (recruitment period 2012–2017). Participants were 689 students in 4th grade (baseline; age=9.2 ± 0.4 years), and 647 students in 5th grade (age=10.5 ± 0.5 years). Nine salivary cortisol measures per child (2 awakening and 1 prior to bed for 3 sequential days) (n = 1336 child phenotype days; n = 7987 cortisol assays), 10 cytokines measured in morning and evening saliva samples (n = 202 child phenotype days), and lipids were measured. Clinical outcomes were blood pressure, weight and height (body mass index [BMI]; BMI = kg/m2), among others. Findings: Upon-waking cortisol levels were 0.28 ± 0.13 µg/dL, 30-minute post-waking 0.33 ± 0.15 µg/dL, and evening 0.08 ± 0.10 µg/dL. Salivary cytokine levels (n = 202) showed interleukins (IL) IL-1β and IL-8 were highest in early morning (upon awakening; AM), and IL-6 and tumor necrosis factor (TNF) TNF-α highest before bed (PM) (IL-1β AM \u3e PM [−4.02 fold; p \u3c 0.001]; IL-8 AM \u3e PM [−1.36 fold; p \u3c 0.001]; IL-6 AM \u3c PM [+1.49 fold; p \u3c 0.001]; TNF-α AM \u3c PM [+1.73 fold; p = 0.03]. Regression modeling showed high morning cortisol was associated with high morning IL-1β (p = 3.82 ×10−6), but low evening IL-1β (p = 6.27 ×10−4). Regression modeling of BMI z-score as the response variable showed the expected significant relationships to high density lipoprotein (HDL) (negative; p \u3c 0.001), mean arterial pressure (positive; p \u3c 0.001), and morning cortisol (negative; p = 0.01) but only weak relationships to either evening cortisol (p = 0.1) or cytokine (positive; p = 0.02; from the model with smallest Rsquared) levels. Interpretation: We provide preliminary data on diurnal fluctuations of inflammatory cytokines in saliva in a population-based cohort of children. Correlation of morning and evening cortisol levels with inflammatory cytokines in the same saliva samples showed that high morning cortisol was associated with high morning IL-1β and low evening IL-1β. Future studies may test the hypothesis that strong diurnal cycling of IL-1β may serve as a homeostatic mechanism keeping the immune system in check, and that low morning cortisol (possible circadian misalignment) may lead to less stringent control of inflammatory networks