Background/Purpose: We recently described a fundamental role for mitochondrial (mt)-mediated inflammation in systemic lupus erythematosus (SLE). Briefly, mtROS promoted formation of neutrophil extracellular traps (NETs), and extrusion of cell-free mitochondria and highly oxidized interferogenic mtDNA propagating disease in lupus-prone mice. However, though our data clearly demonstrate a role of mtDNA in vitro as well as in animal models, the clinical utility of cell-free mtDNA in human SLE was not known. Thus, the aim of the current study was to investigate the clinical utility of cell-free mtDNA as a non-invasive biomarker in SLE patients.

Methods: DNA isolated from plasma samples of healthy individuals (HC, n=20) and SLE patients (n=39) was analyzed for mtDNA (Cytochrome C Oxidase Subunit II) and nuclear (nu) DNA (Ribosomal Protein Lateral Stalk Subunit P0) using qPCR. NETs were analyzed by an in-house MPO-DNA ELISA. Anti-mitochondrial antibodies (anti-cardiolipin antibodies, aCL) were analyzed by ELISA. Patients were stratified based on mtDNA, aCL and NET levels using classification and regression tree (CART) analysis.

Results: SLE patients had significantly elevated levels of mtDNA as compared to HC (p<0.0001, 166576 vs. 36781 copies/ml of plasma). Surprisingly, in contrast to the elevated mtDNA levels, SLE patients had significantly lowered nuDNA compared to HC (p=<0.001, 6799 vs. 15551 copies/ml of plasma), resulting in a skewed mtDNA/nuDNA ratio (p<0.0001, 30.82 vs. 2.602). Consistent with our hypothesis, mtDNA levels were particularly elevated in patients with evidence of NETosis as compared to patients with no NETs (p<0.01, 227404 vs. 124422 copies/ml of plasma). Further, mtDNA levels were associated with markers of inflammation and disease activity: erythrocyte sedimentation rate (r=0.49, p<0.01), hemoglobin (r=-0.59, p<0.01) and complement consumption (p<0.05). In addition to mtDNA, mitochondrial-specific autoantibodies are also indicative of mitochondrial extrusion. Accordingly, we found a significant correlation between mtDNA levels and autoantibodies specific for cardiolipin, an important mitochondrial phospholipid (r=0.38, p<0.05). A subgroup analysis revealed that aCL-positive patients had elevated mtDNA levels compared to aCL-negative patients (p<0.05, 183291 vs. 128096 copies/ml of plasma). In CART analysis, aCL levels, mtDNA copies/mL and NETs were identified as major predictors to stratify patients into non-active (SLEDAI 0-5) and active disease groups (SLEDAI ≥6 to 18) with a sensitivity and specificity of 79% and 71% respectively.

Conclusion:

SLE patients have evidence of markedly elevated levels of mtDNA associated with mitochondrial autoimmunity and disease activity, indicating exaggerated mitochondrial extrusion and impaired mitochondrial clearance partaking in the lupus pathogenesis. Though a limited number of patients, the ability to stratify patients into active and inactive disease suggest that mtDNA may be a promising novel biomarker in SLE. Thus, therapies targeting mitochondrial extrusion are expected to reduce inflammation, as well as long-term morbidities, including cardiovascular disease.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/cell-free-mitochondrial-dna-as-a-novel-biomarker-in-systemic-lupus-erythematosus/