Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA

Background Because circulating DNA (cirDNA) are mainly detected as mononucleosome-associated circulating DNA (mono-N cirDNA) in blood apoptosis has until now been considered as the main source of cirDNA. The mechanism of cirDNA release into the circulation, however, is still not fully understood. This work addresses that knowledge gap, working from the postulate that neutrophil extracellular traps (NET) may be a source of cirDNA, and by investigating whether NET may directly produce mono-N cirDNA Methods We used the synergistic analytical information provided by specifically quantifying DNA by qPCR, and analyzing fragment size analysis by shallow WGS, and capillary electrophoresis to unequivocally study the following: the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; in vitro NET degradation in serum. We also performed an in vivo study in knockout mice, and an in vitro study of gHMW DNA degradation, to elucidate the role of NE and MPO in effecting DNA degradation and fragmentation. We then compared the NET associated markers and fragmentation size profiles of cirDNA in plasma obtained from patients with inflammatory diseases found to be associated with NET formation and high levels of cirDNA (COVID-19, N= 28; systemic lupus erythematosus, N= 10; metastatic colorectal cancer, N= 10; and from healthy individuals, N= 114). Results Our studies reveal that: gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24H incubation in serum corresponded to mono-N DNA); “ex vivo” NET formation, as demonstrated by a concurrent 5-, 5- and 35-fold increase of NE, MPO, and cell-free DNA (cfDNA) concentration in PMA-activated neutrophil culture supernatant, leads to the release of high molecular weight DNA that degrades down to mono-N in serum; NET mainly in the form of gHMW DNA generate mono-N cirDNA (2% and 41% of the remaining DNA after 2 hours in serum corresponded to 1-10 kbp fragments and mono-N, respectively) independent of any cellular process when degraded in serum; NE and MPO may contribute synergistically to NET autocatabolism, resulting in a 25-fold decrease in total DNA concentration and a DNA fragment size profile similar to that observed from cirDNA following 8h incubation with both NE and MPO; the cirDNA size profile of NE KO mice significantly differed from that of the WT, suggesting NE involvement in DNA degradation; and a significant increase in the levels of NE, MPO and cirDNA was detected in plasma samples from lupus, COVID-19 and mCRC, showing a high correlation with these inflammatory diseases, while no correlation of NE and MPO with cirDNA was found in HI. Conclusions Our work thus describes the mechanisms by which NET and cirDNA are linked, by demonstrating that NET are a major source of mono-N cirDNA independent of apoptosis, and thus establishing a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions, as well as demonstrating a link between immune response and cirDNA. ### Competing Interest Statement The authors have declared no competing interest. * NET : neutrophil extracellular traps; gHMW DNA : genomic high molecular weight DNA; NE : neutrophil elastatse; MPO : myeloperoxidase; mono-N : mononucleosome; di-N : dinucleosome; tri-n : trinucleosome; cirDNA : circulating DNA; cir-nDNA : circulating DNA of nuclear origin; cir-mtDNA : circulating DNA of mitochondrial origin; LPS : lipopolysaccharides; PMA : Phorbol 12-myristate 13- acetate; qPCR : quantitative polymerase chain reaction; sWGS : shallow whole genome sequencing; SLE : systemic lupus erythematosus; mCRC : metastatic colorectal cancer; HI : healthy individuals.