Extracellular mitochondria in Inclusion Body Myositis

摘要 包涵体肌炎(IBM)是一种发病机制不明的不可治疗的炎症性肌病。 线粒体异常在IBM中很常见，可能是肌肉无力的原因之一。一 突出的特征是在一些肌肉纤维中没有线粒体，与局部 发炎。然而，目前还不知道线粒体为什么没有，以及它们可能如何对 发炎。我们最近发现，线粒体可以从细胞中挤出，从而导致炎症 通过cGAS/STING途径。我们的中心假设是IBM患者有线粒体 从肌肉细胞中排出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，我们有两个明确的目标。对于第一个目标，我们将 确定IBM患者是否有循环中的细胞外线粒体，以及他们的临床 意义。我们将评估大量的线粒体成分，包括线粒体(MT)DNA， 氧化(8-OHdG)DNA，N-甲酰-蛋氨酸肽(FMET)，以及线粒体蛋白MT-ND6， 分别对特征良好的IBM患者(n=50)、疾病对照组(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 和严肃性。使用纵向队列(n=40，10年随访)，我们的目标是确定 线粒体标志物可以预测疾病的进展。增加对细胞外的机械洞察力 线粒体可能促进IBM的炎症和损伤，中性粒细胞将与抑制剂孵育 在加入IBM血清之前已知的mtDNA受体(cGAS，TLR9)和fMET(FPR1)，其中包含 线粒体成分。结果措施将包括ROS的产生和脱颗粒。第二 AIM将调查IBM患者是否有抗线粒体抗体。我们开发了一部小说 基于流式细胞术的抗线粒体抗体(AMA)与细胞膜结合的定量分析 线粒体。对线粒体的反应性也将通过蛋白质印迹进行评估。这些人的身份 线粒体自身抗原(S)将使用凝胶内消化和随后的质谱分析来确定。 最后，我们将研究AMAs是否可能调理线粒体，增强其炎症反应。 属性。在这项拟议的研究完成后，我们的预期结果将使 通过提供线粒体挤出和线粒体参与的证据来理解IBM的线粒体参与 新AMA(到目前为止还没有研究的方面)。我们还希望有展示出的潜力 调节IBM继发性肌肉损伤中线粒体介导的炎症的治疗目标。我们 预计这项工作将产生积极影响，因为它将为疾病评估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在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.