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

Abstract

Background As circulating DNA (cirDNA) is 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 studied (1) the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; (2) the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; and (3) the in vitro NET degradation in serum; for this, we exploited the synergistic analytical information provided by specifically quantifying DNA by qPCR, and used shallow WGS and capillary electrophoresis to perform fragment size analysis. 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 24 h 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 h 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 8 h 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 describes the mechanisms by which NET and cirDNA are linked. In doing so, we demonstrate that NET are a major source of mono-N cirDNA independent of apoptosis and establish a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions. We also demonstrate a link between immune response and cirDNA.

Long-read sequencing reveals aberrant fragmentation patterns and origins of circulating DNA in cancer

“…These fragments could be distinguished by a shorter length distribution (peaking at 900-4300bp or 5-25 nucleosomes) and had end motif patterns consistent with typical DNASE1L3 fragmentation. The length distribution observed in these cancers is broadly consistent with that of Neutrophil Extracellular Traps (NETs) exposed to circulating nucleases [36], suggesting they might derive from NETs fragmented in circulation. NETs have been associated with myeloid hyperproliferation [37] and linked to accumulation of total circulating DNA in cancer [38,39].…”

Association of vascular netosis with COVID-19 severity in asymptomatic and symptomatic patients

“…We previously validated the use of a set of markers, consisting of two protein markers (NE and MPO) and cir-nDNA concentrations, to indirectly quantify NETs formation in metastatic colorectal cancer 25 , 26 and in COVID-19 26 , 27 patients. First, we demonstrated that the quantitative analysis of cir-nDNA empowers the assessment of NETs formation when combined with the quantification of the granular enzymes (NE and MPO) that are essential in NETs production and in the digestion of trapped microbes.…”

“…First, we demonstrated that the quantitative analysis of cir-nDNA empowers the assessment of NETs formation when combined with the quantification of the granular enzymes (NE and MPO) that are essential in NETs production and in the digestion of trapped microbes. 25 , 26 Second, we have recently demonstrated that the degradation of NETs’ chromatin fibers are degraded by DNases in blood and could be autocatalytic, leading to DNA fragmentation, thus producing mainly mono-nucleosomes as well as a very small proportion of di-nucleosomes. 26 Several studies showed that plasma-extracted DNA is mainly associated with mono-nucleosomes and to a lesser extent di-nucleosomes (>90% in healthy individuals).…”

Association of the immediate perioperative dynamics of circulating DNA levels and neutrophil extracellular traps formation in cancer patients

“…cir-mtDNA may cause an inflammatory response by acting as damage-associated molecular patterns, in combination with other circulating NETs by-products (histones, granule proteins like NE or MPO, and structural proteins). Although HI have lower amounts of cir-mtDNA than cir-nDNA, cir-mtDNA could be a marker for inflammatory diseases [ 41 , 42 ].…”