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.