Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated.

Alcohol-related liver disease (ALD) is a leading cause of chronic liver conditions globally. Chronic alcohol consumption induces liver damage through various mechanisms, including neutrophil extracellular trap (NET) formation. Extracellular DNA (ecDNA), released from damaged hepatocytes and NETotic neutrophils, has emerged as a potential biomarker and contributor to liver disease pathology. Enzyme DNases could be an effective therapy for the denaturation of immunogenic ecDNA. This study investigated the circulating ecDNA and NET markers in ALD and therapeutic effect of DNase I in a murine model of ALD. Female C57BL/6J mice were fed a control diet (n = 13) or Lieber–DeCarli ethanol diet for 10 days followed by a binge ethanol dose to mimic acute-on-chronic alcoholic liver injury. From day 5, mice fed ethanol were randomized into an ethanol diet group (n = 17) and ethanol + DNase group (n = 5), which received additional DNase I treatment every 12 h. Liver damage markers were analyzed. Circulating ecDNA and NETosis were measured by fluorometry and cytometry, respectively. DNase I activity was analyzed with single radial enzyme dispersion assay. The ethanol-fed mice exhibited increased mortality, neutrophil infiltration and structural damage in the liver. Total circulating ecDNA levels and NET markers did not differ between groups. DNase activity was higher in ethanol-fed mice compared to controls and additional daily administration of DNase prevented liver injury. These findings suggest that alcohol-induced liver injury modestly influences systemic NETosis and ecDNA levels. However, increased DNase activity can prevent disease progression and enhanced systemic degradation of ecDNA using DNase I.

Deoxyribonucleases (DNases) cleave extracellular DNA (ecDNA) and are under intense research as interventions for diseases associated with high ecDNA, such as acute live injury. DNase I treatment decreases morbidity and mortality in this animal model. Endogenous DNase activity has high interindividual variability. In this study, we tested the hypothesis that high endogenous DNase activity is beneficial in an animal model of acute liver failure. DNase activity was measured in the plasma of adult male mice taken before i.p. injection of thioacetamide to induce acute liver failure. The survival of mice was monitored for 48 h. Mice were retrospectively divided into two groups based on the median DNase activity assessed using the gel-based single-radial enzyme diffusion assay. In acute liver failure, mice with a higher baseline DNase activity had lower mortality after 48 h (by 25%). Different protection of ecDNA against nucleases by vesicles or DNA-binding proteins could play a role and should be further evaluated. Similarly, the role of endogenous DNase activity should be analyzed in other disease models associated with high ecDNA.