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.

Biomedical Center Martin Jessenius Faculty of Medicine Comenius University in Bratislava 03601 Martin Slovakia

Comenius University Science Park Univerzita Komenského 84104 Bratislava Slovakia

Department of Molecular Biology Faculty of Natural Sciences Comenius University in Bratislava 84215 Bratislava Slovakia

Department of Pathophysiology Jessenius Faculty of Medicine Comenius University in Bratislava 03601 Martin Slovakia

Department of Physiology 3rd Faculty of Medicine Charles University 10000 Prague Czech Republic

Geneton Ltd 84104 Bratislava Slovakia

Institute of Clinical and Translational Research Biomedical Research Center Slovak Academy of Sciences 84505 Bratislava Slovakia

Institute of Molecular Biomedicine Faculty of Medicine Comenius University in Bratislava 81108 Bratislava Slovakia

Institute of Physiology Faculty of Medicine Comenius University in Bratislava 81372 Bratislava Slovakia

Slovak Centre of Scientific and Technical Information 81104 Bratislava Slovakia

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Ng S.C., Shi H.Y., Hamidi N., Underwood F.E., Tang W., Benchimol E.I., Panaccione R., Ghosh S., Wu J.C.Y., Chan F.K.L., et al. Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: A systematic review of population-based studies. Lancet. 2018;390:2769–2778. doi: 10.1016/S0140-6736(17)32448-0. PubMed DOI

Pieczyńska J., Prescha A., Zabłocka-Słowińska K., Neubauer K., Smereka A., Grajeta H., Biernat J., Paradowski L. Occurrence of dietary risk factors in inflammatory bowel disease: Influence on the nutritional status of patients in clinical remission. Adv. Clin. Exp. Med. 2019;28:587–592. doi: 10.17219/acem/78590. PubMed DOI

Gagliardi A., Totino V., Cacciotti F., Iebba V., Neroni B., Bonfiglio G., Trancassini M., Passariello C., Pantanella F., Schippa S. Rebuilding the gut microbiota ecosystem. Int. J. Environ. Res. Public Health. 2018;15:1679. PubMed PMC

De Meij T.G.J., De Groot E.F.J., Peeters C.F.W., De Boer N.K.H., Kneepkens C.M.F., Eck A., Benninga M.A., Savelkoul P.H.M., Van Bodegraven A.A., Budding A.E. Variability of core microbiota in newly diagnosed treatment-naïve paediatric inflammatory bowel disease patients. PLoS ONE. 2018;13:e0197649. doi: 10.1371/journal.pone.0197649. PubMed DOI PMC

Song C., Yang J., Ye W., Zhang Y., Tang C., Li X., Zhou X., Xie Y. Urban–rural environmental exposure during childhood and subsequent risk of inflammatory bowel disease: A meta-analysis. Expert Rev. Gastroenterol. Hepatol. 2019;13:591–602. doi: 10.1080/17474124.2018.1511425. PubMed DOI

O’Driscoll L. Extracellular nucleic acids and their potential as diagnostic, prognostic and predictive biomarkers. Anticancer Res. 2007;27:1257–1265. PubMed

Zhong X.Y., Burk M.R., Troeger C., Kang A., Holzgreve W., Hahn S. Fluctuation of maternal and fetal free extracellular circulatory DNA in maternal plasma. Obstet. Gynecol. 2000;96:991–996. PubMed

Lui Y.Y.N., Woo K.-S., Wang A.Y.M., Yeung C.-K., Li P.K.T., Chau E., Ruygrok P., Lo Y.M.D. Origin of plasma cell-free DNA after solid organ transplantation. Clin. Chem. 2003;49:495–496. doi: 10.1373/49.3.495. PubMed DOI

Nishimoto S., Fukuda D., Sata M. Emerging roles of Toll-like receptor 9 in cardiometabolic disorders. Inflamm. Regen. 2020;40:1–13. doi: 10.1186/s41232-020-00118-7. PubMed DOI PMC

Krieg A.M. CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol. 2002;20:709–760. doi: 10.1146/annurev.immunol.20.100301.064842. PubMed DOI

Sharma S., Fitzgerald K.A. Innate immune sensing of DNA. PLoS Pathog. 2011;7:e1001310. doi: 10.1371/journal.ppat.1001310. PubMed DOI PMC

Avriel A., Wiessman M.P., Almog Y., Perl Y., Novack V., Galante O., Klein M., Pencina M.J., Douvdevani A. Admission cell free DNA levels predict 28-day mortality in patients with severe sepsis in intensive care. PLoS ONE. 2014;9:e100514. doi: 10.1371/journal.pone.0100514. PubMed DOI PMC

Clementi A., Virzi G.M., Brocca A., Pastori S., de Cal M., Marcante S., Granata A., Ronco C. The Role of Cell-Free Plasma DNA in Critically Ill Patients with Sepsis. Blood Purif. 2016;41:34–40. doi: 10.1159/000440975. PubMed DOI

Schneck E., Samara O., Koch C., Hecker A., Padberg W., Lichtenstern C., Weigand M.A., Uhle F. Plasma DNA and RNA differentially impact coagulation during abdominal sepsis-an explorative study. J. Surg. Res. 2017;210:231–243. doi: 10.1016/j.jss.2016.11.044. PubMed DOI

Ahmed A.I., Soliman R.A., Samir S. Cell Free DNA and Procalcitonin as Early Markers of Complications in ICU Patients with Multiple Trauma and Major Surgery. Clin. Lab. 2016;62:2395–2404. doi: 10.7754/Clin.Lab.2016.160615. PubMed DOI

Nishimoto S., Fukuda D., Higashikuni Y., Tanaka K., Hirata Y., Murata C., Kim-Kaneyama J.R., Sato F., Bando M., Yagi S., et al. Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance. Sci. Adv. 2016;2:e1501332. doi: 10.1126/sciadv.1501332. PubMed DOI PMC

Koike Y., Uchida K., Tanaka K., Ide S., Otake K., Okita Y., Inoue M., Araki T., Mizoguchi A., Kusunoki M. Dynamic pathology for circulating free DNA in a dextran sodium sulfate colitis mouse model. Pediatr. Surg. Int. 2014;30:1199–1206. doi: 10.1007/s00383-014-3607-6. PubMed DOI

Maronek M., Gromova B., Liptak R., Klimova D., Cechova B., Gardlik R. Extracellular DNA is Increased in Dextran Sulphate Sodium-Induced Colitis in Mice. Folia Biol. (Praha) 2018;64:167–172. PubMed

Sipos F., Műzes G., Fűri I., Spisák S., Wichmann B., Germann T.M., Constantinovits M., Krenács T., Tulassay Z., Molnár B. Intravenous administration of a single-dose free-circulating DNA of colitic origin improves severe murine DSS-colitis. Pathol. Oncol. Res. 2014;20:867–877. PubMed

Műzes G., Kiss A.L., Tulassay Z., Sipos F. Cell-free DNA-induced alteration of autophagy response and TLR9-signaling: Their relation to amelioration of DSS-colitis. Comp. Immunol. Microbiol. Infect. Dis. 2017;52:48–57. doi: 10.1016/j.cimid.2017.06.005. PubMed DOI

Constantinovits M., Sipos F., Kiss A.L., Műzes G. Preconditioning with cell-free DNA prevents DSS-colitis by promoting cell protective autophagy. J. Investig. Med. 2020;68:992–1001. doi: 10.1136/jim-2020-001296. PubMed DOI

Brinkmann V., Reichard U., Goosmann C., Fauler B., Uhlemann Y., Weiss D.S., Weinrauch Y., Zychlinsky A. Neutrophil Extracellular Traps Kill Bacteria. Science. 2004;303:1532–1535. doi: 10.1126/science.1092385. PubMed DOI

Vaibhav K., Braun M., Alverson K., Khodadadi H., Kutiyanawalla A., Ward A., Banerjee C., Sparks T., Malik A., Rashid M.H., et al. Neutrophil extracellular traps exacerbate neurological deficits after traumatic brain injury. Sci. Adv. 2020;6:eaax8847. doi: 10.1126/sciadv.aax8847. PubMed DOI PMC

Meijenfeldt F.A., von Jenne C.N. Netting Liver Disease: Neutrophil Extracellular Traps in the Initiation and Exacerbation of Liver Pathology. Semin. Thromb. Hemost. 2020;46:724–734. doi: 10.1055/s-0040-1715474. PubMed DOI

Carmona-Rivera C., Carlucci P.M., Goel R.R., James E., Brooks S.R., Rims C., Hoffmann V., Fox D.A., Buckner J.H., Kaplan M.J. Neutrophil extracellular traps mediate articular cartilage damage and enhance cartilage component immunogenicity in rheumatoid arthritis. JCI Insight. 2020;5:e139388. PubMed PMC

Lin E.Y.H., Lai H.J., Cheng Y.K., Leong K.Q., Cheng L.C., Chou Y.C., Peng Y.C., Hsu Y.H., Chiang H. Sen Neutrophil extracellular traps impair intestinal barrier function during experimental colitis. Biomedicines. 2020;8:275. doi: 10.3390/biomedicines8080275. PubMed DOI PMC

Huang H., Zhang H., Onuma A.E., Tsung A. Advances in Experimental Medicine and Biology. Volume 1263. Springer; Cham, Switzerland: 2020. Neutrophil Elastase and Neutrophil Extracellular Traps in the Tumor Microenvironment; pp. 13–23. PubMed PMC

Yang L., Liu L., Zhang R., Hong J., Wang Y., Wang J., Zuo J., Zhang J., Chen J., Hao H. IL-8 mediates a positive loop connecting increased neutrophil extracellular traps (NETs) and colorectal cancer liver metastasis. J. Cancer. 2020;11:4384–4396. doi: 10.7150/jca.44215. PubMed DOI PMC

Laukova L., Konecna B., Babickova J., Wagnerova A., Meliskova V., Vlkova B., Celec P. Exogenous deoxyribonuclease has a protective effect in a mouse model of sepsis. Biomed. Pharmacother. 2017;93:8–16. PubMed

Vokálová L., Lauková L., Čonka J., Melišková V., Borbélyová V., Bábíčková J., Tóthová L., Hodosy J., Vlková B., Celec P. Deoxyribonuclease partially ameliorates thioacetamide-induced hepatorenal injury. Am. J. Physiol. Liver Physiol. 2017;312:G457–G463. doi: 10.1152/ajpgi.00446.2016. PubMed DOI

Mondal S., Thompson P.R. Protein Arginine Deiminases (PADs): Biochemistry and Chemical Biology of Protein Citrullination. Acc. Chem. Res. 2019;52:818–832. PubMed PMC

Mutua V., Gershwin L.J. A Review of Neutrophil Extracellular Traps (NETs) in Disease: Potential Anti-NETs Therapeutics. Clin. Rev. Allergy Immunol. 2020;1:1–18. doi: 10.1007/s12016-020-08804-7. PubMed DOI PMC

Malik A.N., Czajka A., Cunningham P. Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences. Mitochondrion. 2016;29:59–64. doi: 10.1016/j.mito.2016.05.003. PubMed DOI

Zhang T., Mei Y., Dong W., Wang J., Huang F., Wu J. Evaluation of protein arginine deiminase-4 inhibitor in TNBS- induced colitis in mice. Int. Immunopharmacol. 2020;84:106583. PubMed

Kim J.J., Shajib M.S., Manocha M.M., Khan W.I. Investigating intestinal inflammation in DSS-induced model of IBD. J. Vis. Exp. 2012:1–6. doi: 10.3791/3678. PubMed DOI PMC

Dinallo V., Marafini I., Di Fusco D., Laudisi F., Franzè E., Di Grazia A., Figliuzzi M.M., Caprioli F., Stolfi C., Monteleone I., et al. Neutrophil Extracellular Traps Sustain Inflammatory Signals in Ulcerative Colitis. J. Crohns. Colitis. 2019;13:772–784. doi: 10.1093/ecco-jcc/jjy215. PubMed DOI

Bolger A.M., Lohse M., Usadel B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. doi: 10.1093/bioinformatics/btu170. PubMed DOI PMC

Malíčková K., Duricová D., Bortlík M., Hrušková Z., Svobodová B., Machková N., Komárek V., Fučíková T., Janatková I., Zima T., et al. Impaired deoxyribonuclease I activity in patients with inflammatory bowel diseases. Autoimmune Dis. 2011;2011:945861. doi: 10.4061/2011/945861. PubMed DOI PMC

Babraham A. Bioinformatics. [(accessed on 31 October 2020)]; Available online: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

Yuzhalin A.E. Citrullination in cancer. Cancer Res. 2019;79:1274–1284. doi: 10.1158/0008-5472.CAN-18-2797. PubMed DOI

Xu H., Luo X., Qian J., Pang X., Song J., Qian G., Chen J., Chen S. FastUniq: A Fast De Novo Duplicates Removal Tool for Paired Short Reads. PLoS ONE. 2012;7:e52249. doi: 10.1371/journal.pone.0052249. PubMed DOI PMC

Li P., Wang D., Yao H., Doret P., Hao G., Shen Q., Qiu H., Zhang X., Wang Y., Chen G., et al. Coordination of PAD4 and HDAC2 in the regulation of p53-target gene expression. Oncogene. 2010;29:3153–3162. doi: 10.1038/onc.2010.51. PubMed DOI PMC

Langmead B., Salzberg S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods. 2012;9:357–359. PubMed PMC

Babickova J., Conka J., Janovicova L., Boris M., Konecna B., Gardlik R. Extracellular DNA as a Prognostic and Therapeutic Target in Mouse Colitis under DNase I Treatment. Folia Biol. (Praha) 2018;64:10–15. PubMed

Wood D.E., Lu J., Langmead B. Improved metagenomic analysis with Kraken 2. Genome Biol. 2019;20:257. doi: 10.1186/s13059-019-1891-0. PubMed DOI PMC

Kubiritova Z., Radvanszky J., Gardlik R. Cell-Free Nucleic Acids and their Emerging Role in the Pathogenesis and Clinical Management of Inflammatory Bowel Disease. Int. J. Mol. Sci. 2019;20:3662. PubMed PMC

Chumanevich A.A., Causey C.P., Knuckley B.A., Jones J.E., Poudyal D., Chumanevich A.P., Davis T., Matesic L.E., Thompson P.R., Hofseth L.J. Suppression of colitis in mice by Cl-amidine: A novel peptidylarginine deiminase inhibitor. Am. J. Physiol. Gastrointest. Liver Physiol. 2011;300:G929–G938. doi: 10.1152/ajpgi.00435.2010. PubMed DOI PMC

Dreyton C., Jones J., Knuckley B., Subramanian V., Anderson E., Brown S., Fernandez-Vega V., Eberhart C., Spicer T., Zuhl A., et al. Probe Reports from the NIH Molecular Libraries Program [Internet] National Center for Biotechnology Information; Bethesda, MD, USA: 2013. Optimization and characterization of a pan protein arginine deiminase (PAD) inhibitor. PubMed