Abstract 18: Circulating Extracellular DNA Mediates Vaso-Occlusions in Sickle Cell Disease

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Rationale: It was recently reported that circulating extracellular DNA (cirDNA) is increased in sickle cell disease (SCD). CirDNA apears to be associated to intravascular hemolysis and cell-free heme in plasma. But nothing is known of the physiopathological impact of cirDNA in SCD. Here, we aimed to define the role of cirDNA and hypothesized that it may favor red blood cell (RBC) aggregation, vaso-occlusive crises (VOC) and ischemic events. Material and Methods: A blood collection from SCD patients and matched healthy volunteers was built. RBCs and neutrophils were separated from plasma by centrifugation. We cultured human neutrophils and endothelial cells in vitro. We quantified extracellular DNA with a fluorescent intercalant probe. We also used our model of renal vaso-occlusions induced by infusion of heme-loaded RBC microparticles in SAD transgenic mice. Results: In SCD patients at steady state, cirDNA was increased by 300% over health, and a further 100% during VOC. Next, we treated cultured neutrophils and endothelial cells with purified heme. Heme induced neutrophil extracellular traps (NETs) and endothelial apoptosis, with release of genomic DNA in supernatants. We purified neutrophil DNA, added it to whole blood and studied RBC aggregation by laser-assisted optical red blood cell aggregometry. DNA induced RBC aggregation in SCD blood, but not healthy blood. Conversely, DNase-1 added to SCD blood collected during VOC reduced RBC aggregation back to steady state levels. Next, we triggered renal vaso-occlusions in SAD transgenic mice, and cirDNA levels were increased by 200% within 30 minutes. Moreover, one intravenous bolus of DNase-1, administered after inducing the occlusions, accelerated renal reperfusion back to normal levels in under 20 minutes. In contrast, spontaneous reperfusion with vehicle injection took over 90 minutes. Conclusion: Our data reveal a novel mechanism of vascular occlusion in SCD: CirDNA can bind RBCs in blood, promote aggregation and small vessel occlusions. CirDNA and NETs may thus bridge intravascular hemolysis, inflammation and ischemic tissue injury in SCD. Interestingly, DNase digestion of plasma DNA and NETs may prove an unexpected but powerful novel pharmaceutical approach to treat vaso-occlusive events.