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...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-11-01
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| Series: | Genome Medicine |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13073-022-01125-8 |
| _version_ | 1828123412632961024 |
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| author | Ekaterina Pisareva Lucia Mihalovičová Brice Pastor Andrei Kudriavtsev Alexia Mirandola Thibault Mazard Stephanie Badiou Ulrich Maus Lena Ostermann Julia Weinmann-Menke Elmo W. I. Neuberger Perikles Simon Alain R. Thierry |
| author_facet | Ekaterina Pisareva Lucia Mihalovičová Brice Pastor Andrei Kudriavtsev Alexia Mirandola Thibault Mazard Stephanie Badiou Ulrich Maus Lena Ostermann Julia Weinmann-Menke Elmo W. I. Neuberger Perikles Simon Alain R. Thierry |
| author_sort | Ekaterina Pisareva |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T14:50:38Z |
| format | Article |
| id | doaj.art-5a60bc2545e44dbbb874dd10acdbe5c7 |
| institution | Directory Open Access Journal |
| issn | 1756-994X |
| language | English |
| last_indexed | 2024-04-11T14:50:38Z |
| publishDate | 2022-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Genome Medicine |
| spelling | doaj.art-5a60bc2545e44dbbb874dd10acdbe5c72022-12-22T04:17:27ZengBMCGenome Medicine1756-994X2022-11-0114112410.1186/s13073-022-01125-8Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNAEkaterina Pisareva0Lucia Mihalovičová1Brice Pastor2Andrei Kudriavtsev3Alexia Mirandola4Thibault Mazard5Stephanie Badiou6Ulrich Maus7Lena Ostermann8Julia Weinmann-Menke9Elmo W. I. Neuberger10Perikles Simon11Alain R. Thierry12IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut Régional du Cancer de MontpellierIRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut Régional du Cancer de MontpellierIRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut Régional du Cancer de MontpellierIRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut Régional du Cancer de MontpellierIRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut Régional du Cancer de MontpellierIRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut Régional du Cancer de MontpellierLaboratoire de Biochimie Et Hormonologie, PhyMedExp, Université de Montpellier, INSERM, CNRS, CHU de MontpellierDivision of Experimental Pneumology, Hannover Medical School, and German Center for Lung Research, Partner Site BREATH (Biomedical Research in Endstage and Obstructive Lung Disease)Division of Experimental Pneumology, Hannover Medical School, and German Center for Lung Research, Partner Site BREATH (Biomedical Research in Endstage and Obstructive Lung Disease)Department of Rheumatology and Nephrology, University Medical Center MainzDepartment of Sports Medicine, University of MainzDepartment of Sports Medicine, University of MainzIRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de Montpellier, Institut Régional du Cancer de MontpellierAbstract 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.https://doi.org/10.1186/s13073-022-01125-8Circulating DNAElastaseMyeloperoxidaseNeutrophilNETNucleosome |
| spellingShingle | Ekaterina Pisareva Lucia Mihalovičová Brice Pastor Andrei Kudriavtsev Alexia Mirandola Thibault Mazard Stephanie Badiou Ulrich Maus Lena Ostermann Julia Weinmann-Menke Elmo W. I. Neuberger Perikles Simon Alain R. Thierry Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA Genome Medicine Circulating DNA Elastase Myeloperoxidase Neutrophil NET Nucleosome |
| title | Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA |
| title_full | Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA |
| title_fullStr | Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA |
| title_full_unstemmed | Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA |
| title_short | Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA |
| title_sort | neutrophil extracellular traps have auto catabolic activity and produce mononucleosome associated circulating dna |
| topic | Circulating DNA Elastase Myeloperoxidase Neutrophil NET Nucleosome |
| url | https://doi.org/10.1186/s13073-022-01125-8 |
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