IFNγ causes mitochondrial dysfunction and oxidative stress in myositis.

Idiopathic inflammatory myopathies (IIMs) are severe autoimmune diseases with poorly understood pathogenesis and unmet medical needs. Here, we examine the role of interferon γ (IFNγ) using NOD female mice deficient in the inducible T cell co-stimulator (Icos), which have previously been shown to develop spontaneous IFNγ-driven myositis mimicking human disease. Using muscle proteomic and spatial transcriptomic analyses we reveal profound myofiber metabolic dysregulation in these mice. In addition, we report muscle mitochondrial abnormalities and oxidative stress in diseased mice. Supporting a pathogenic role for oxidative stress, treatment with a reactive oxygen species (ROS) buffer compound alleviated myositis, preserved muscle mitochondrial ultrastructure and respiration, and reduced inflammation. Mitochondrial anomalies and oxidative stress were diminished following anti-IFNγ treatment. Further transcriptomic analysis in IIMs patients and human myoblast in vitro studies supported the link between IFNγ and mitochondrial dysfunction observed in mice. These results suggest that mitochondrial dysfunction, ROS and inflammation are interconnected in a self-maintenance loop, opening perspectives for mitochondria therapy and/or ROS targeting drugs in myositis.

Icos gene disruption in non-obese diabetic mice elicits myositis associated with anti-troponin T3 autoantibodies.

AIMS: Idiopathic inflammatory myopathies (IIM) are autoimmune inflammatory disorders leading to skeletal muscle weakness and disability. The pathophysiology of IIM is poorly understood due to the scarcity of animal disease models. Genetic deletion of Icos or Icosl (inducible T cell co-stimulator/ligand) in non-obese diabetic (NOD) mice leads to muscle disease. Our aim was to characterise Icos-/- NOD myopathy and to search for novel autoantibodies (aAbs) in this model. METHODS: Diabetes, weight, myopathy incidence/clinical score and grip strength were assessed over time. Locomotor activity was analysed with the Catwalk XT gait analysis system. Muscle histology was evaluated in haematoxylin/eosin and Sirius red-stained sections, and immune infiltrates were characterised by immunofluorescence and flow cytometry. 2D gel electrophoresis of muscle protein extracts and mass spectrometry were used to identify novel aAbs. NOD mice were immunised with troponin T3 (TNNT3) in incomplete Freund's adjuvant (IFA) and R848. An addressable laser bead immunoassay (ALBIA) was developed to measure aAb IgG serum levels. RESULTS: Icos-/- NOD mice did not exhibit diabetes but developed spontaneous progressive myositis with decreased muscle strength and altered locomotor activity. Muscle from these mice exhibited myofibre necrosis, myophagocytosis, central nuclei, fibrosis and perimysial and endomysial cell infiltrates with macrophages and T cells. We identified anti-TNNT3 aAbs in diseased mice. Immunisation of NOD mice with murine TNNT3 protein led to myositis development, supporting its pathophysiological role. CONCLUSIONS: These data show that Icos-/- NOD mice represent a spontaneous model of myositis and the discovery of anti-TNNT3 aAb suggests a new autoantigen in this model.

Disease patterns and specific trajectories of anti-MDA5-related disease: a multicentre retrospective study of 70 adult patients.

Introduction: This study aimed to provide an updated analysis of the different prognostic trajectories of patients with anti-melanoma differentiation-associated gene 5 (MDA5) antibodies. Methods: Among a cohort of 70 patients, baseline characteristics and phenotypes, treatments and outcomes were analyzed. A Cox proportional hazards model was used to identify factors associated with poor outcomes, i.e., death or progressive disease at the last follow-up. Results: Among the 70 patients, 45 were women, and 54 were Caucasian. A dermatologic involvement was observed in 58 (83%) patients, including 40 with MDA5 vasculopathy-related skin lesions. Muscular involvement was observed in 39 (56%) patients. Interstitial lung disease (ILD) was observed at baseline in 52 (74%) patients, including 23 (44%) who developed rapidly progressive (RP) ILD. Seven (10%) patients showed thromboembolic complications within the first weeks of diagnosis, and eight (11%) other patients developed a malignancy (4 before the diagnosis of anti-MDA5 disease). Poor outcomes were observed in 28 (40%) patients, including 13 (19%) deaths. Among the 23 patients with RP-ILD, 19 (79%) showed poor outcomes, including 12 (63%) who died. In multivariate analyses, RP-ILD (hazard ratio (HR), 95% CI: 8.24 [3.21-22], p<0.0001), the occurrence of thromboembolic events (HR: 5.22 [1.61-14.77], p=0.008) and the presence of any malignancy (HR: 19.73 [6.67-60], p<0.0001) were the three factors independently associated with poor outcomes. Discussion: This new independent cohort confirms the presence of different clinical phenotypes of anti-MDA5 diseases at baseline and the poor prognosis associated with RP-ILD. Thromboembolic events and malignancies were also identified as prognostic factors.

In vivo pathogenicity of IgG from patients with anti-SRP or anti-HMGCR autoantibodies in immune-mediated necrotising myopathy.

OBJECTIVES: In autoimmunity, autoantibodies (aAb) may be simple biomarkers of disease or true pathogenic effectors. A form of idiopathic inflammatory myopathy associated with anti-signal recognition particle (SRP) or anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) aAb has been individualised and is referred to as immune-mediated necrotising myopathy (IMNM). The level of aAb correlates with IMNM activity and disease may respond to immunosuppression, suggesting that they are pathogenic. We aimed to evaluate the pathogenicity of IgG from patients with anti-SRP or anti-HMGCR aAb in vivo by developing the first mouse model of IMNM. METHODS: IgG from patients suffering from anti-SRP or anti-HMGCR associated IMNM were passively transferred to wild-type, Rag2-/- or complement C3-/- mice. Muscle deficiency was evaluated by muscle strength on electrostimulation and grip test. Histological analyses were performed after haematoxylin/eosin staining or by immunofluorescence or immunohistochemistry analysis. Antibody levels were quantified by addressable laser bead assay (ALBIA). RESULTS: Passive transfer of IgG from patients suffering from IMNM to C57BL/6 or Rag2-/- mice provoked muscle deficiency. Pathogenicity of aAb was reduced in C3-/- mice while increased by supplementation with human complement. Breakage of tolerance by active immunisation with SRP or HMGCR provoked disease. CONCLUSION: This study demonstrates that patient-derived anti-SRP+ and anti-HMGCR+ IgG are pathogenic towards muscle in vivo through a complement-mediated mechanism, definitively establishing the autoimmune character of IMNM. These data support the use of plasma exchanges and argue for evaluating complement-targeting therapies in IMNM.

The Spontaneous Autoimmune Neuromyopathy in ICOSL-/- NOD Mice Is CD4+ T-Cell and Interferon-γ Dependent.

Abrogation of ICOS/ICOS ligand (ICOSL) costimulation prevents the onset of diabetes in the non-obese diabetic (NOD) mouse but, remarkably, yields to the development of a spontaneous autoimmune neuromyopathy. At the pathological level, ICOSL-/- NOD mice show stronger protection from insulitis than their ICOS-/- counterparts. Also, the ICOSL-/- NOD model carries a limited C57BL/6 region containing the Icosl nul mutation, but, in contrast to ICOS-/- NOD mice, no gene variant previously reported as associated to NOD diabetes. Therefore, we aimed at providing a detailed characterization of the ICOSL-/- NOD model. The phenotype observed in ICOSL-/- NOD mice is globally similar to that observed in ICOS-/- and ICOS-/-ICOSL-/- double-knockout NOD mice, manifested by a progressive locomotor disability first affecting the front paws as observed by catwalk analysis and a decrease in grip test performance. The pathology remains limited to peripheral nerve and striated muscle. The muscle disease is characterized by myofiber necrosis/regeneration and an inflammatory infiltrate composed of CD4+ T-cells, CD8+ T-cells, and myeloid cells, resembling human myositis. Autoimmune neuromyopathy can be transferred to NOD.scid recipients by CD4+ but not by CD8+ T-cells isolated from 40-week-old female ICOSL-/- NOD mice. The predominant role of CD4+ T-cells is further demonstrated by the observation that neuromyopathy does not develop in CIITA-/-ICOSL-/- NOD in contrast to ß2microglobulin-/-ICOSL-/- NOD mice. Also, the cytokine profile of CD4+ T-cells infiltrating muscle and nerve of ICOSL-/- NOD mice is biased toward a Th1 pattern. Finally, adoptive transfer experiments show that diabetes development requires expression of ICOSL, in contrast to neuromyopathy. Altogether, the deviation of autoimmunity from the pancreas to skeletal muscles in the absence of ICOS/ICOSL signaling in NOD mice is strictly dependent on CD4+ T-cells, leads to myofiber necrosis and regeneration. It provides the first mouse model of spontaneous autoimmune myopathy akin to human myositis.

Induction of Hematopoietic Microchimerism by Gene-Modified BMT Elicits Antigen-Specific B and T Cell Unresponsiveness toward Gene Therapy Products.

BACKGROUND: Gene therapy is a promising treatment option for hemophilia and other protein deficiencies. However, immune responses against the transgene product represent an obstacle to safe and effective gene therapy, urging for the implementation of tolerization strategies. Induction of a hematopoietic chimerism via bone marrow transplantation (BMT) is a potent means for inducing immunological tolerance in solid organ transplantation. OBJECTIVES: We reasoned, here, that the same viral vector could be used, first, to transduce BM cells for inducing chimerism-associated transgene-specific immune tolerance and, second, for correcting protein deficiencies by vector-mediated systemic production of the deficient coagulation factor. METHODS: Evaluation of strategies to induce B and T cell tolerance was performed using ex vivo gene transfer with lentiviral (LV) vectors encoding coagulation factor IX (FIX) or the SIINFEKL epitope of ovalbumin. Following induction of microchimerism via BMT, animals were challenged with in vivo gene transfer with LV vectors. RESULTS: The experimental approach prevented humoral immune response against FIX, resulting in persistence of therapeutic levels of circulating FIX, after LV-mediated gene transfer in vivo. In an ovalbumin model, we also demonstrated that this approach effectively tolerized the CD8+ T cell compartment in an antigen-specific manner. CONCLUSION: These results provide the proof-of-concept that inducing a microchimerism by gene-modified BMT is a powerful tool to provide transgene-specific B and T cell tolerance in a gene therapy setting.

Impact of recommendations on crushing medications in geriatrics: from prescription to administration.

The practice of crushing drugs is very common in geriatric units. In 2009 a first study, performed in all geriatric units of a university hospital, showed that numerous errors were made during prescription, preparation and administration. The aim of this second prospective study was to assess the impact of regional and national recommendations in the same geriatric units. A survey of 719 patients (85.3 ± 6.7 years) was performed in 2013. For each patient who received crushed drugs, we recorded the reason the drugs were crushed, pharmacological classes, galenic presentations and the technique used for preparation and administration. Results were compared to the previous study. The number of patients receiving drugs after crushing was significantly lower than in the previous study (22.9% vs. 32.3%, P < 0.001). The number of crushed drugs was lower too (594 per 165 patients vs. 966 per 224 patients (P < 0.01). The main indication for crushing drugs remained swallowing disorders. The dosage form prevented crushing in 24.9% of drugs (vs. 42.0% in 2009, P < 0.001), but the drugs generally remained crushed all together. A mortar was used less often (38.6% vs. 92.6%, P < 0.001), with preference for individual-specific cups (56.1%). Mortars were more often cleaned between each patient (56.0% vs. 11.6%). The vehicle was more often neutral (water 88.5% vs. 5.7%, P < 0.001). This second study shows that regional and national recommendations have led to an overall improvement of practices for crushing drugs. Technical improvements are still possible, in association with appropriate pharmacological studies.