Extracellular mitochondria in Inclusion Body Myositis

总结 包涵体肌炎（IBM）是一种无法治愈的炎症性肌病，其发病机制尚不清楚。 线粒体异常在IBM中常见，可能导致肌肉无力。一 一个突出的特点是在一些肌纤维中缺乏线粒体，与局部 炎症然而，目前尚不清楚为什么线粒体不存在，以及它们如何有助于 炎症我们最近发现，线粒体可以从细胞中挤出，有助于炎症 通过cGAS/STING途径。我们的中心假设是IBM患者的线粒体 肌肉细胞挤压导致细胞外线粒体水平升高，促进炎症 和器官损伤为了研究这种生物学，我们有两个具体的目标。为了第一个目标，我们将 确定IBM患者是否有细胞外线粒体循环，以及他们的临床 意义我们将评估各种各样的线粒体成分，包括线粒体（mt）DNA， 氧化的（8-OHdG）DNA、N-甲酰甲硫氨酸肽（fMET）以及线粒体蛋白MT-ND6， 通过qPCR和ELISA分别在具有良好特征的IBM患者（n = 50）、疾病对照（n = 50）、 健康人（n = 50）。线粒体标记物将与疾病活动的标记物相关联 和严重性使用纵向队列（n = 40，10年随访），我们的目的是确定是否 线粒体标记物可以预测疾病进展。为了增加对细胞外 在IBM中，线粒体可能促进炎症和损伤，中性粒细胞将与线粒体抑制剂一起孵育。 已知的mtDNA（cGAS，TLR9）和fMET（FPR1）受体，然后加入含有 线粒体成分结果测量将包括ROS产生和脱粒。第二 目的是调查IBM患者是否有抗线粒体抗体。我们已经开发出一种新颖 基于流式细胞术的测定，以定量抗线粒体抗体（AMA）与细胞膜的结合， 线粒体对线粒体的反应性也将通过蛋白质印迹来评估。的身份 使用凝胶内消化和随后的质谱法来确定线粒体自身抗原。 最后，我们将研究AMA是否可以调理线粒体，增强其炎症反应， 特性.在这项拟议的研究完成后，我们的预期成果将推进 通过提供线粒体挤压的证据来了解线粒体参与IBM， 新的AMA（迄今尚未研究的方面）。我们也希望能展示出 治疗靶点，以调节肌肉损伤后的IBM中神经介导的炎症。我们 我希望这项工作能产生积极的影响，因为它将为评估疾病提供新的生物标志物 进展，以及确定有针对性的途径，以限制炎症和组织损伤的IBM。

Summary Inclusion body myositis (IBM) is an untreatable inflammatory myopathy of unclear pathogenesis. Mitochondrial abnormalities are frequently seen in IBM, likely contributing to the muscle weakness. One prominent feature is the absence of mitochondria in some of the muscle fibers, associated with local inflammation. However, it is not known why the mitochondria are absent, and how they may contribute to inflammation. We recently found that mitochondria can be extruded from cells, contributing to inflammation through the cGAS/STING pathway. Our central hypothesis is that IBM patients have mitochondrial extrusion from muscle cells resulting in elevated levels of extracellular mitochondria promoting inflammation and organ damage. To investigate this biology, we have two specific aims. For the first aim, we will determine whether IBM patients have extracellular mitochondria in circulation, as well as their clinical significance. We will assess a large variety of mitochondrial components, including mitochondrial (mt) DNA, oxidized (8-OHdG) DNA, N-formyl-methionine peptides (fMET), as well as mitochondrial protein MT-ND6, by qPCR and ELISA respectively in well-characterized patients with IBM (n=50), disease controls (n=50), and healthy individuals (n=50). Mitochondrial markers will be associated with markers of disease activity and severity. Using a longitudinal cohort (n=40, 10 years follow-up), we aim to determine whether mitochondrial markers can predict disease progression. To add mechanistic insight into how extracellular mitochondria may promote inflammation and damage in IBM, neutrophils will be incubated with inhibitors of known receptors of mtDNA (cGAS, TLR9) and fMET (FPR1) prior to addition of IBM sera containing mitochondrial components. Outcome measures will include ROS production and degranulation. The second aim will investigate whether IBM patients have anti-mitochondrial antibodies. We have developed a novel flow cytometry-based assay to quantify binding of anti-mitochondrial antibodies (AMAs) to the membrane of mitochondria. Reactivity towards mitochondria will also be assessed by Western blot. The identity of the mitochondrial autoantigen(s) will be defined using in-gel digestion and subsequent mass spectrometry. Finally, we will investigate whether AMAs may opsonize mitochondria enhancing their inflammatory properties. At the completion of this proposed research, our expected outcomes are to have advanced the understanding of mitochondrial involvement in IBM by providing evidence for mitochondrial extrusion and novel AMAs (aspects which have not been studied so far). We also expect to have demonstrated potential therapeutic targets to regulate mitochondrial-mediated inflammation in IBM ensuing muscle damage. We expect this work to have a positive impact because it will offer novel biomarkers for assessment of disease progression, as well as identify targetable pathways to limit inflammation and tissue damage in IBM.