Neutrophils mediate the early innate immune response through extracellular traps comprising intracellular protein and DNA. These traps play a pivotal role in both immunity against invading pathogens and the development of immunopathological reactions through the production of reactive oxygen species (ROS). Proteins serve as the main target for ROS, resulting in the formation of protein adducts. Herein, we report that the superoxide anion radical (O2˙-) plays a vital role in neutrophil function through sequential events involving 5-lipoxygenase (5-LOX) and NADPH oxidase (NOX). More specifically, differences in NOX homologs expression were observed post-stimulation with PMA and LPS. Differentiation conditions and O2˙- generation were confirmed using flow cytometry. Immunoblotting analysis confirmed the time-dependent expression of NOX underlying its requirement and 5-LOX-mediated lipid peroxidation events in neutrophil function. Protein-malondialdehyde (MDA) adducts formed were detected using immunoblotting, and quercetin was evaluated for its ability to scavenge free radicals through electron paramagnetic resonance (EPR) spin-trapping spectroscopy and results were confirmed with blotting analysis. Free radical-mediated protein oxidation events influence neutrophil function and protein adducts formed serve as markers of neutrophil activation upon infection and inflammation. The study warrants further corroboration and the study of specific proteins involved in neutrophil activation and their role in inflammation.

Department of Biophysics Faculty of Science Palacký University Šlechtitelů 27 783 71 Olomouc Czech Republic 420 585225737 420 585634752

Department of Immunology Faculty of Medicine and Dentistry Palacký University Hněvotínská 3 775 15 Olomouc Czech Republic

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de Buhr N. von Köckritz-Blickwede M. How Neutrophil Extracellular Traps Become Visible. J. Immunol. Res. 2016;2016:4604713. doi: 10.1155/2016/4604713. doi: 10.1155/2016/4604713. PubMed DOI PMC

Kolaczkowska E. Kubes P. Neutrophil recruitment and function in health and inflammation. Nat. Rev. Immunol. 2013;13:159–175. doi: 10.1038/nri3399. doi: 10.1038/nri3399. PubMed DOI

Neeli I. Radic M. Opposition between PKC isoforms regulates histone deimination and neutrophil extracellular chromatin release. Front. Immunol. 2013;4:38. doi: 10.3389/fimmu.2013.00038. PubMed DOI PMC

Schauer C. Janko C. Munoz L. E. Zhao Y. Kienhöfer D. Frey B. Lell M. Manger B. Rech J. Naschberger E. et al., Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines. Nat. Med. 2014;20:511–517. doi: 10.1038/nm.3547. doi: 10.1038/nm.3547. PubMed DOI

Clarke J. NETs directly injure cartilage in RA. Nat. Rev. Rheumatol. 2020;16:410. doi: 10.1038/s41584-020-0459-4. doi: 10.1038/s41584-020-0459-4. PubMed DOI

Dwyer M. Shan Q. D'Ortona S. Maurer R. Mitchell R. Olesen H. Thiel S. Huebner J. Gadjeva M. Cystic Fibrosis Sputum DNA Has NETosis Characteristics and Neutrophil Extracellular Trap Release Is Regulated by Macrophage Migration-Inhibitory Factor. J. Innate Immun. 2014;6:765–779. doi: 10.1159/000363242. doi: 10.1159/000363242. PubMed DOI PMC

Björnsdottir H. Welin A. Michaëlsson E. Osla V. Berg S. Christenson K. Sundqvist M. Dahlgren C. Karlsson A. Bylund J. Neutrophil NET formation is regulated from the inside by myeloperoxidase-processed reactive oxygen species. Free Radicals Biol. Med. 2015;89:1024–1035. doi: 10.1016/j.freeradbiomed.2015.10.398. doi: 10.1016/j.freeradbiomed.2015.10.398. PubMed DOI

Vorobjeva N. V. Chernyak B. V. NETosis: Molecular Mechanisms, Role in Physiology and Pathology. Biochemistry. 2020;85:1178–1190. doi: 10.1134/S0006297920100065. PubMed DOI PMC

Masuda S. Nakazawa D. Shida H. Miyoshi A. Kusunoki Y. Tomaru U. Ishizu A. NETosis markers: Quest for specific, objective, and quantitative markers. Clin. Chim. Acta. 2016;459:89–93. doi: 10.1016/j.cca.2016.05.029. doi: 10.1016/j.cca.2016.05.029. PubMed DOI

Petroniene J. Morkvenaite-Vilkonciene I. Miksiunas R. Bironaite D. Ramanaviciene A. Rucinskas K. Ramanavicius A. Scanning electrochemical microscopy for the investigation of redox potential of human myocardium-derived mesenchymal stem cells grown at 2D and 3D conditions. Electrochim. Acta. 2020;360:136956. doi: 10.1016/j.electacta.2020.136956. doi: 10.1016/j.electacta.2020.136956. 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. doi: 10.1126/science.1092385. PubMed DOI

Stoiber W. Obermayer A. Steinbacher P. Krautgartner W.-D. The Role of Reactive Oxygen Species (ROS) in the Formation of Extracellular Traps (ETs) in Humans. Biomolecules. 2015;5:702–723. doi: 10.3390/biom5020702. doi: 10.3390/biom5020702. PubMed DOI PMC

Ravindran M. Khan M. A. Palaniyar N. Neutrophil Extracellular Trap Formation: Physiology, Pathology, and Pharmacology. Biomolecules. 2019;9:365. doi: 10.3390/biom9080365. doi: 10.3390/biom9080365. PubMed DOI PMC

Wilkie-Grantham R. P. Magon N. J. Harwood D. T. Kettle A. J. Vissers M. C. Winterbourn C. C. Hampton M. B. Myeloperoxidase-dependent Lipid Peroxidation Promotes the Oxidative Modification of Cytosolic Proteins in Phagocytic Neutrophils. J. Biol. Chem. 2015;290:9896–9905. doi: 10.1074/jbc.M114.61342. doi: 10.1074/jbc.M114.613422. PubMed DOI PMC

Rådmark O. Samuelsson B. 5-Lipoxygenase: mechanisms of regulation. J. Lipid Res. 2009;50:S40–S45. doi: 10.1194/jlr.R800062-JLR200. doi: 10.1194/jlr.R800062-JLR200. PubMed DOI PMC

Blanter M. Gouwy M. Struyf S. Studying Neutrophil Function in vitro: Cell Models and Environmental Factors. J. Inflammation Res. 2021;14:141–162. doi: 10.2147/JIR.S284941. doi: 10.2147/JIR.S284941. PubMed DOI PMC

Gibellini L. Pinti M. Nasi M. De Biasi S. Roat E. Bertoncelli L. Cossarizza A. Interfering with ROS Metabolism in Cancer Cells: The Potential Role of Quercetin. Cancers. 2010;2:1288–1311. doi: 10.3390/cancers2021288. doi: 10.3390/cancers2021288. PubMed DOI PMC

Hartung N. M. Fischer J. Ostermann A. I. Willenberg I. Rund K. M. Schebb N. H. Garscha U. Impact of food polyphenols on oxylipin biosynthesis in human neutrophils. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids. 2019;1864:1536–1544. doi: 10.1016/j.bbalip.2019.05.002. doi: 10.1016/j.bbalip.2019.05.002. PubMed DOI

Hougee S. Sanders A. Faber J. Graus Y. M. F. Van Den Berg W. B. Garssen J. Smit H. F. Hoijer M. A. Decreased pro-inflammatory cytokine production by LPS-stimulated PBMC upon in vitro incubation with the flavonoids apigenin, luteolin or chrysin, due to selective elimination of monocytes/macrophages. Biochem. Pharmacol. 2005;69:241–248. doi: 10.1016/j.bcp.2004.10.002. doi: 10.1016/j.bcp.2004.10.002. PubMed DOI

Comalada M. Camuesco D. Sierra S. Ballester I. Xaus J. Gálvez J. Zarzuelo A. In vivo quercitrin anti-inflammatory effect involves release of quercetin, which inhibits inflammation through down-regulation of the NF-κB pathway. Eur. J. Immunol. 2005;35:584–592. doi: 10.1002/eji.200425778. doi: 10.1002/eji.200425778. PubMed DOI

Bhaskar S. Shalini V. Helen A. Quercetin regulates oxidized LDL induced inflammatory changes in human PBMCs by modulating the TLR-NF-κB signaling pathway. Immunobiology. 2011;216:367–373. doi: 10.1016/j.imbio.2010.07.011. doi: 10.1016/j.imbio.2010.07.011. PubMed DOI

Awasthi D. Nagarkoti S. Kumar A. Dubey M. Singh A. K. Pathak P. Chandra T. Barthwal M. K. Dikshit M. Oxidized LDL induced extracellular trap formation in human neutrophils via TLR-PKC-IRAK-MAPK and NADPH-oxidase activation. Free Radicals Biol. Med. 2016;93:190–203. doi: 10.1016/j.freeradbiomed.2016.01.004. doi: 10.1016/j.freeradbiomed.2016.01.004. PubMed DOI

Guo Y. Gao F. Wang Q. Wang K. Pan S. Pan Z. Xu S. Li L. Zhao D. Differentiation of HL-60 cells in serum-free hematopoietic cell media enhances the production of neutrophil extracellular traps. Exp. Ther. Med. 2021;21:353. doi: 10.3892/etm.2021.9784. doi: 10.3892/etm.2021.9784. PubMed DOI PMC

Abubaker A. A. Vara D. Eggleston I. Canobbio I. Pula G. A novel flow cytometry assay using dihydroethidium as redox-sensitive probe reveals NADPH oxidase-dependent generation of superoxide anion in human platelets exposed to amyloid peptide β. Platelets. 2019;30(2):181–189. doi: 10.1080/09537104.2017.1392497. doi: 10.1080/09537104.2017.1392497. PubMed DOI

Sul O.-J. Ra S. W. Quercetin Prevents LPS-Induced Oxidative Stress and Inflammation by Modulating NOX2/ROS/NF-kB in Lung Epithelial Cells. Molecules. 2021;26:6949. doi: 10.3390/molecules26226949. doi: 10.3390/molecules26226949. PubMed DOI PMC

Gotham J. P. Li R. Tipple T. E. Lancaster J. R. Liu T. Li Q. Quantitation of spin probe-detectable oxidants in cells using electron paramagnetic resonance spectroscopy: To probe or to trap? Free Radicals Biol. Med. 2020;154:84–94. doi: 10.1016/j.freeradbiomed.2020.04.020. doi: 10.1016/j.freeradbiomed.2020.04.020. PubMed DOI PMC

Manda-Handzlik A. Bystrzycka W. Wachowska M. Sieczkowska S. Stelmaszczyk-Emmel A. Demkow U. Ciepiela O. The influence of agents differentiating HL-60 cells toward granulocyte-like cells on their ability to release neutrophil extracellular traps. Immunol. Cell Biol. 2018;96:413–425. doi: 10.1111/imcb.12015. doi: 10.1111/imcb.12015. PubMed DOI

Prasad A. Manoharan R. R. Sedlářová M. Pospíšil P. Free Radical-Mediated Protein Radical Formation in Differentiating Monocytes. Int. J. Mater. Sci. 2021;22:9963. doi: 10.3390/ijms22189963. PubMed DOI PMC

Rincón E. Rocha-Gregg B. L. Collins S. R. A map of gene expression in neutrophil-like cell lines. BMC Genomics. 2018;19:573. doi: 10.1186/s12864-018-4957-6. doi: 10.1186/s12864-018-4957-6. PubMed DOI PMC

Gupta D. Shah H. P. Malu K. Berliner N. Gaines P. Differentiation and Characterization of Myeloid Cells. Curr. Protoc. Immunol. 2014;104 doi: 10.1002/0471142735.im22f05s104. PubMed DOI PMC

Chen F. Haigh S. Barman S. Fulton D. J. R. From form to function: the role of Nox4 in the cardiovascular system. Front. Physiol. 2012;3:412. doi: 10.3389/fphys.2012.00412. PubMed DOI PMC

Yuan K. Zhu Q. Lu Q. Jiang H. Zhu M. Li X. Huang G. Xu A. Quercetin alleviates rheumatoid arthritis by inhibiting neutrophil inflammatory activities. J. Nutr. Biochem. 2020;84:108454. doi: 10.1016/j.jnutbio.2020.108454. doi: 10.1016/j.jnutbio.2020.108454. PubMed DOI

Papayannopoulos V. Neutrophil extracellular traps in immunity and disease. Nat. Rev. Immunol. 2018;18:134–147. doi: 10.1038/nri.2017.105. doi: 10.1038/nri.2017.105. PubMed DOI

Di Martino O. Welch J. S. Retinoic Acid Receptors in Acute Myeloid Leukemia Therapy. Cancers. 2019;11:1915. doi: 10.3390/cancers11121915. doi: 10.3390/cancers11121915. PubMed DOI PMC

Manda-Handzlik A. Bystrzycka W. Cieloch A. Glodkowska-Mrowka E. Jankowska-Steifer E. Heropolitanska-Pliszka E. Skrobot A. Muchowicz A. Ciepiela O. Wachowska M. et al., Nitric oxide and peroxynitrite trigger and enhance release of neutrophil extracellular traps. Cell. Mol. Life Sci. 2020;77:3059–3075. doi: 10.1007/s00018-019-03331-x. doi: 10.1007/s00018-019-03331-x. PubMed DOI PMC

Dömer D. Walther T. Möller S. Behnen M. Laskay T. Neutrophil Extracellular Traps Activate Proinflammatory Functions of Human Neutrophils. Front. Immunol. 2021;12:636954. doi: 10.3389/fimmu.2021.636954. doi: 10.3389/fimmu.2021.636954. PubMed DOI PMC

Lorne E. Zmijewski J. W. Zhao X. Liu G. Tsuruta Y. Park Y.-J. Dupont H. Abraham E. Role of extracellular superoxide in neutrophil activation: interactions between xanthine oxidase and TLR4 induce proinflammatory cytokine production. Am. J. Physiol.: Cell Physiol. 2008;294:C985–C993. doi: 10.1152/ajpcell.00454.2007. doi: 10.1152/ajpcell.00454.2007. PubMed DOI

Sasaki Y. Does oxidative stress participate in the development of hepatocellular carcinoma? J. Gastroenterol. 2007;41:1135–1148. doi: 10.1007/s00535-006-1982-z. doi: 10.1007/s00535-006-1982-z. PubMed DOI

Li G. Ye C. Zhu Y. Zhang T. Gu J. Pan J. Wang F. Wu F. Huang K. Xu K. et al., Oxidative Injury in Ischemic Stroke: A Focus on NADPH Oxidase 4. Oxid. Med. Cell. Longevity. 2022:1–12. doi: 10.1155/2022/1148874. PubMed DOI PMC

Manoharan R. R. Sedlářová M. Pospíšil P. Prasad A. Detection and characterization of free oxygen radicals induced protein adduct formation in differentiating macrophages. Biochim. Biophys. Acta, Gen. Subj. 2023;1867:130324. doi: 10.1016/j.bbagen.2023.130324. doi: 10.1016/j.bbagen.2023.130324. PubMed DOI

Hamam P. Post-Translational Modifications in NETosis and NETs-Mediated Diseases. Biomolecules. 2019;9:369. doi: 10.3390/biom9080369. doi: 10.3390/biom9080369. PubMed DOI PMC

Hamam H. J. Palaniyar N. Histone Deacetylase Inhibitors Dose-Dependently Switch Neutrophil Death from NETosis to Apoptosis. Biomolecules. 2019;9:184. doi: 10.3390/biom9050184. doi: 10.3390/biom9050184. PubMed DOI PMC

Wroński A. Gęgotek A. Skrzydlewska E. Protein adducts with lipid peroxidation products in patients with psoriasis. Redox Biol. 2023;63:102729. doi: 10.1016/j.redox.2023.102729. doi: 10.1016/j.redox.2023.102729. PubMed DOI PMC

Kim S. Y. Kim T.-B. Moon K. Kim T. J. Shin D. Cho Y. S. Moon H.-B. Lee K.-Y. Regulation of pro-inflammatory responses by lipoxygenases via intracellular reactive oxygen species in vitro and in vivo. Exp. Mol. Med. 2008;40:461. doi: 10.3858/emm.2008.40.4.461. doi: 10.3858/emm.2008.40.4.461. PubMed DOI PMC

Awasthi D. Nagarkoti S. Sadaf S. Chandra T. Kumar S. Dikshit M. Glycolysis dependent lactate formation in neutrophils: A metabolic link between NOX-dependent and independent NETosis. Biochim. Biophys. Acta, Mol. Basis Dis. 2019;1865:165542. doi: 10.1016/j.bbadis.2019.165542. doi: 10.1016/j.bbadis.2019.165542. PubMed DOI

Prasad A. Rossi C. Manoharan R. R. Sedlářová M. Cangeloni L. Rathi D. Tamasi G. Pospíšil P. Consumi M. Bioactive Compounds and Their Impact on Protein Modification in Human Cells. Int. J. Mol. Sci. 2022;23:7424. doi: 10.3390/ijms23137424. doi: 10.3390/ijms23137424. PubMed DOI PMC