Cell-Free DNA Promotes Thrombin Autolysis and Generation of Thrombin-Derived C-Terminal Fragments

Saravanan, Rathi; Choong, Yeu Khai; Lim, Chun Hwee; Lim, Li Ming; Petrlová, Jitka; Schmidtchen, Artur

doi:10.3389/fimmu.2021.593020

Cited by 8 publications

(5 citation statements)

References 40 publications

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“…Conversely, other NET constituents may be involved in protecting DNA from nucleases such as the antimicrobial peptide LL-37 [ 72 ]. Alternatively, thrombin may bind to NET, thus conferring mutual protection against nuclease and protease degradation [ 73 ], illustrating the complex interplay between coagulation and NET formation [ 74 ].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2022

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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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2022

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“…Conversely, other NET constituents may be involved in protecting DNA from nucleases such as the antimicrobial peptide LL-37 [70]. Alternatively, thrombin may bind to NET, thus conferring mutual protection against nuclease and protease degradation [71], illustrating the complex interplay between coagulation and NET formation [72].…”

Section: Discussionmentioning

confidence: 99%

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

Pisareva

Mihalovičová

Pastor

et al. 2022

Preprint

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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.

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“…cfDNA can also be used for non-invasive prenatal Down syndrome screening ( Vrachnis et al, 2014 ; Malmir et al, 2021 ) and immune rejection surveillance after organ transplantation ( Snyder et al, 2016 ; Danovitch et al, 2021 ). The third source is the release of auto-activated DNA, cfDNA content is related to trauma, burn, infection, heart failure, stroke, and organ IRI level ( Tian et al, 2019 ; Dinsdale et al, 2020 ; Yokokawa et al, 2020 ; Zuo et al, 2020 ; Saravanan et al, 2021 ). The fourth source is tumor-derived ctDNA, which comprises a very small proportion of cfDNA.…”

Section: Cfdna Sources Distribution Content and Detection Methodsmentioning

confidence: 99%

Effects of cell-free DNA on kidney disease and intervention strategies

Zhang,

Cai,

Zhong

et al. 2024

Front. Pharmacol.

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Kidney disease has become a global public health problem. Patients with end-stage kidney disease must rely on dialysis or undergo renal transplantation, placing heavy burdens on their families and society. Therefore, it is important to develop new therapeutic targets and intervention strategies during early stages of chronic kidney disease. The widespread application of liquid biopsy has led to an increasing number of studies concerning the roles of cell-free DNA (cfDNA) in kidney disease. In this review, we summarize relevant studies concerning the roles of cfDNA in kidney disease and describe various strategies for targeted removal of cfDNA, with the goal of establishing novel therapeutic approaches for kidney disease.

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Cell-Free DNA Promotes Thrombin Autolysis and Generation of Thrombin-Derived C-Terminal Fragments

Cited by 8 publications

References 40 publications

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

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

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

Effects of cell-free DNA on kidney disease and intervention strategies

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