Background/Purpose: Rheumatoid Arthritis (RA) is an autoimmune inflammatory disease causing erosive and disabling joint damage. We recently found that mitochondria are extruded upon formation of neutrophil extracellular traps (NETs). The extruded mitochondria can be a source of danger-associated molecular patterns (DAMPs) that include highly unmethylated CpG motif content, oxidized mitochondrial DNA (mtDNA) and N-formylmethionyl (N-fMet) peptides. In RA, elevated levels of cell-free mtDNA have been reported in both synovial fluid and plasma. However, the clinical utility of mtDNA in RA is yet to be explored.  Nothing has been reported on levels of N-fMet peptides in RA patients. The aim of the current study was to quantitate circulating levels of mitochondrial components in RA patients, and investigate their utility as novel non-invasive biomarkers in monitoring disease activity and severity in RA.

Methods: DNA isolated from plasma samples of healthy individuals (HC, n=20) and RA patients (n=100) was analyzed for mtDNA (Cytochrome C Oxidase Subunit II) and nuclear (nu) DNA (Ribosomal Protein Lateral Stalk Subunit P0) content using qPCR. NETs were analyzed by an in-house MPO-DNA ELISA. Levels of circulating human N-fMet and DNA damage (8-OHdG) were analyzed by ELISA.

Results: Seropositive RA patients had elevated levels of mtDNA compared to seronegative patients (p=0.03). However, no difference was observed for nuDNA (p=0.11). Further, RA patients had extensive DNA oxidation (p=0.0009). Oxidation, mainly seen in mtDNA, is known to increase the inflammatory capacity of DNA. Of note, cfDNA from RA patients had higher inflammatory potential upon in vitro incubation with PBMCs, as compared to cfDNA from HC (p< 0.0001). The inflammatory capacity of DNA correlated with mtDNA levels (r=0.36, p=0.0004), but not with nuDNA levels (r=0.06, p=0.57), suggesting circulating mtDNA as the pro-inflammatory component of cfDNA. Consistent with our hypothesis that NETs could be a potential source of mtDNA, we found a correlation between levels of cf-mtDNA and NETs (r=0.025, p=0.03). RA patients also had significantly elevated levels of circulating N-fMet as compared to controls (p< 0.0001). N-fMet levels correlated with markers of inflammation, including CRP (r=0.56, p< 0.0001), ESR (r=0.32, p=0.01), and calprotectin, (r=0.42, p< 0.0001). Both circulating mtDNA levels and N-fMet levels were elevated in patients with active disease (p=0.001 and p=0.007, respectively). Further, a distinct group of RA patients (n=20) with predominant levels of mtDNA, showed significant correlation between mtDNA levels and CDAI (r=0.56, p=0.03). Circulating N-fMet levels also correlated with joint involvement (N-fMet vs. swollen joints, r= 0.23, p=0.03; N-fMet vs. tender joints, r= 0.22, p=0.04).

Conclusion: Mitochondrial DAMPs are elevated in RA, indicating abnormal mitochondrial extrusion and/or clearance occurring in RA. Levels of mitochondrial DAMPs are related to inflammation and disease activity, suggesting an instrumental role in the RA pathogenesis. In all, pathways involved in mitochondrial extrusion could be considered as novel therapeutic targets as well as biomarkers in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/circulating-mitochondrial-danger-associated-molecular-patterns-as-novel-biomarkers-of-disease-activity-and-inflammation-in-rheumatoid-arthritis/