Changes in coagulation parameters and plasma cytokine concentration under the formation of neutrophil extracellular traps in vitro: 616-092.4

Abstract

Summary. Background. Intravascular neutrophil extracellular traps (NETs) releasing represents one of the mechanisms of immunothrombosis — thrombi formation associated with immune reactions. NETs act as integrating link between immunity and hemostasis, thus acquiring particular importance in the sites of malignancy. Objectives: to reveal whether the suicide NETosis can modify clotting parameters and the plasma level of essential immunoregulatory factors in vitro. Patients/Methods. The plasma concentration of interleukins (IL) — IL-1β, IL-2, IL-4, IL-6, IL-10, free active transforming growth factor (TGF-β1), tumor necrosis factor- alpha (TNF-α), interferon-γ (IFN-γ), chemokines CXCL8, CCL2, CXCL10 and I, II, III, IX, XI, XIII coagulation factors were measured before and after of experimental modeling of suicide NETosis in vitro. Coagulation process was assessed by thrombodynamics method. Cell culture of neutrophils and plasma were obtained from whole venous blood of 30 patients with newly diagnosed stage II–III colon cancer without metastases. The comparison group consisted of 40 healthy, age and gender matched volunteers. Plasma level of cytokines and clotting factors were measured by flow cytometry using multiplex assay panels. Results. Increasing of clotting rate by 5.5% and prolongation of lag-time were detected in both groups. There were no variations of coagulation factors level. The significant increase of plasma level of CXCL8, IL2 and free active TGF-β1 were detected in both groups in samples with netotic cells, when compared with their baseline concentration and with samples, contained resting cells. Conclusions. Suicide NETosis is accompanied by acceleration of initial stage of fibrin clot formation through external way and by strong increase in CXCL8 concen- tration and less increase in IL-2 and TGF-β1 that can be considered as one of the ways how NETs contribute to hypercoagulation state and chronic inflammation development and also implement their tumorgenicity.

References

1. Brinkmann V., Reichard U., Goosmann C. et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303(5663):1532–5. DOI: 10.1126/science.1092385. 2. Thiam H.R., Wong S.L., Wagner D.D., Waterman C.M. Cellular mechanisms of NETosis. Annu Rev Cell Dev Biol. 2020;36:91–218. DOI: 10.1146/annurev-cellbio-020520–111016. 3. Goldmann O., Medina E. The expanding world of extracellular traps: not only neutrophils but much more. Front Immunol. 2012;3:420. DOI: 10.3389/fimmu.2012.00420. 4. Dabrowska D., Jabłońska E., Garley M. et al. New aspects of the biology of neutrophil extracellular traps. Scand J Immunol. 2016;84(6):317–22. DOI: 10.1111/sji.12494. 5. Garley M., Jabłońska E. Heterogeneity among neutrophils. Arch Immun Ther Exp (Warsz). 2017;66(1):21–30. DOI: 10.1007/s00005–017–0476–4. 6. Potapnev M. P., Gushchina L. M., Moroz L. A. Human neutrophils subpopulations and functions heterogeneity in norm and pathology. Immunologiya. 2019;40(5):84–96. (In Russ.). DOI: 10.24411/0206–4952–2019–15009. 7. Demers M., Wong S.L., Martinod K. et al. Priming of neutrophils toward NETosis promotes tumor growth. Oncoimmunology. 2016;5: e1134073. DOI: 10.1080/2162402X.2015.1134073. 8. Shaul M.E., Fridlender Z.G. Cancer-related circulating and tumorassociated neutrophils — subtypes, sources and function. FEBS J. 2018;285(23):4316–42. DOI: 10.1111/febs.14524. 9. Demers M., Wagner D.D. Neutrophil extracellular traps: A new link to cancer-associated thrombosis and potential implication for tumor progression. Oncoimmunology. 2013;2(2): e22946. DOI: 10.4161/onci.22946. 10. Sanz-Moreno V., Balkwill F.R. Mets and NETs: the awakening force. Immunity. 2018;49(5):798–800. DOI: 10.1016/j.immuni.2018.11.009. 11. Fuchs T.A., Brill A., Wagner D.D. Neutrophil extracellular trap (NET) impact on deep vein thrombosis. Atheroskler Thromb Vasc Biol. 2012;3(8):1777–83. DOI: 10.1161/ATVBAHA.111.242859. 12. Gould T.J., Vu T.T., Swystun L.L. et al. Neutrophil extracellular traps promote thrombin generation through platelet-dependent and platelet-independent mechanisms. Atheroskler Thromb Vasc Biol. 2014;34(9):1977–84. DOI: 10.1161/ATVBAHA.114.304114. 13. Roitman E.V. Pathophysiological peculiarities of cancer-associated thrombosis specifying opportunities of therapy with direct oral anticoagulants. Tromboz, gemostaz i reologiya. 2019;(2):22–30. (In Russ.). DOI: 10.25555/THR.2019.2.0876. 14. de Los Reyes-García A., Aroca A., Arroyo A.B. Neutrophil extracellular trap components increase the expression of coagulation factors. Biomed Rep. 2019;10(3):195–201. DOI: 10.3892/br.2019.1187. 15. Bogdanov I.G., Kuznik B.I., Smolyakov Yu.N. et al. Thrombodynamic properties of fibrin clots in patients with IHD coronary bypass grafting. Zabajkal’skij medicinskij vestnik. 2021;(2):18–28. (In Russ.). DOI: 10.52485/19986173_2021_2_18. 16. Abbas A.K., Trotta E., Simeonov D.R. et al. Revisiting IL-2: biology and therapeutic prospects. Sci Immunol. 2018;3(25):eaat1482. DOI: 10.1126/sciimmunol.aat1482. 17. Bendickova K., Fric J. Roles of IL-2 in bridging adaptive and innate immunity, and as a tool for cellular immunotherapy. J Leukoc Biol. 2020;108(1):427–37. DOI: 10.1002/JLB.5MIR0420–055R. 18. Bu H.Q., Shen F., Cui J. The inhibitory effect of oridonin on colon cancer was mediated by deactivation of TGF-β1/Smads-PAI-1 signaling pathway in vitro and vivo. Onco Targets Ther. 2019;12:7467– 76. DOI: 10.2147/OTT.S220401. 19. Martin C.J., Datta A., Littlefield C. et al. Selective inhibition of TGFβ1 activation overcomes primary resistance to checkpoint blockade therapy by altering tumor immune landscape. Sci Trans Med. 2020;12(536):eaay8456. DOI: 10.1126/scitranslmed.aay8456. 20. De Streel G., Lucas S. Targeting immunosuppression by TGF-β1 for cancer immunotherapy. Biochem Pharmacol. 2021;192:114697. DOI: 10.1016/j.bcp.2021.114697. 21. Yang L., Liu L., Zhang R., Hong J. et al. IL-8 mediates a positive loop connecting increased neutrophil extracellular traps (NETs) and colorectal cancer liver metastasis. J Cancer. 2020;11(15):4384– 96. DOI: 10.7150/jca.44215. 22. Cristinziano L., Modestino L., Antonelli A. et al. Neutrophil extracellular traps in cancer. Semin Cancer Biol. 2022;79:91–104. DOI: 10.1016/j.semcancer.2021.07.011. 23. Pausch T.M., Aue E., Wirsik N.M. et al. Metastasis-associated fibroblasts promote angiogenesis in metastasized pancreatic cancer via the CXCL8 and the CCL2 axes. Sci Rep. 2020;10(1):5420. DOI: 10.1038/s41598–020–62416-x. 24. Li J., Liu Q., Huang X., Cai Y. et al. Transcriptional profiling reveals the regulatory role of CXCL8 in promoting colorectal cancer. Front Genet. 2020;10:1360. DOI: 10.3389/fgene.2019.01360. 25. Xiao Y.-C., Yang Z.-B., Cheng X.-S. et al. CXCL8 overexpressed in colorectal cancer, enhances the resistance of colorectal cancer cells to anoikis. Cancer Lett. 2015;361(1):22–32. DOI: 10.1016/j. canlet.2015.02.021.