Background/Purpose: Though mainly found intracellularly, we recently observed mitochondrial extrusion upon cell death, contributing to inflammation and organ damage in lupus-prone mice. Of note, mitochondria are immunogenic, promoting development of anti-mitochondrial antibodies (AMAs). Mitochondrial remnants, including DNA, are observed in the circulation of many systemic diseases, including lupus, and thought to partake in the disease pathogenesis. However, the role of mitochondrial extrusion in children with juvenile dermatomyositis (JDM) has not been addressed. The aim of this study was to investigate markers of mitochondrial extrusion, including mtDNA and AMAs, in JDM children to determine their clinical utility.

Methods: AMAs, as well as cell-free mtDNA levels were analyzed in healthy children (HC, n=22), pediatric lupus (n=10), polymyositis (n=7), JDM patients (n=61), and RNP+ myositis (12), by a state-of-the-art flow cytometry technique as well as an in-house qPCR assay. Mitochondrial markers were associated with disease activity score (DAS), calcinosis, as well as autoantibody profiles. Muscle biopsies were analyzed using electron microscopy.

Results: Electron microscopy imaging demonstrated profound mitochondrial abnormalities in JDM muscle, including intramitochondrial calcification associated with degenerate muscle fibers and mitochondrial extrusion. As compared to healthy controls, JDM patients had increased levels of cell-free mtDNA (p=0.02) but not genomic DNA (p=0.09) in peripheral blood, especially in children with calcinosis (p=0.002). As determined by Western blot, JDM patients had autoantibodies reacting towards mitochondrial antigens of 60 kDa, similar to what was seen in jSLE. By flow cytometry, 40% of JDM patients were found to be positive for AMAs (p< 0.001). Consistent with the immunogenic nature of mitochondria, AMA levels correlated with presence of antigen, e.g. mtDNA, in peripheral blood (r=0.28, p< 0.05), as well as with immune complex (IC) levels (r=0.56, p< 0.0001) and complement C4 consumption (r=-0.59, p=0.01). Upon activation with mitochondrial ICs, neutrophils induced IL-8 production (p< 0.01) as well as underwent formation of neutrophil extracellular traps (NETs, p< 0.05), clearly demonstrating the inflammatory potential of mitochondrial ICs. AMA positivity was associated with calcinosis (OR=6.1, p=0.006). Of importance, AMA became elevated prior to the clinical diagnosis of calcinosis (OR=11.1, p< 0.05), with a sensitivity and specificity of 80% and 73.5%, respectively, to identify calcinosis-prone individuals within the group of children with JDM, suggesting a prognostic potential of AMA in JDM calcinosis development. Conclusion: JDM patients have obvious mitochondrial abnormalities in tissue and periphery. Our novel findings of mitochondrial antibodies in JDM, preceding clinical diagnosis of calcinosis, support i) mitochondrial extrusion as a potential therapeutic targetable pathway, and ii) the use of AMAs as prognostic markers, allowing for early, preventive treatment, reducing development of disabling calcinosis in those children.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mitochondrial-contribution-to-juvenile-dermatomyositis-pathogenesis/