Effect of antioxidants on the production of MCP-1 chemokine by EA.hy926 cells in response to IL-6

Elevated level of circulatory interleukin 6 (IL-6) is a biomarker for cytokine storm of various etiologies including COVID-19 and contributes to poor prognosis. Vascular endothelial cells are one of the main targets of pathological action of IL-6. IL-6 activates trans-signaling pathway via the formation of the IL-6/sIL-6Ra/gp130 receptor complex and subsequent activation of the JAK/STAT3 signaling pathway, and in some cases PI3K/AKT and MEK/ERK kinases. Previously, by our group and other researchers, it was shown that reactive oxygen species (ROS) including mitochondrial ROS (mito-ROS) contribute to the induction of IL-6 expression in the endothelium, mainly due to increased activation of the transcription factor NF-kB. We have also shown that the mitochondria-targeted antioxidant SkQ1 (plastoquinolyl-10(6’-decyltriphenyl) phosphonium) prevented tumor necrosis factor (TNF)-induced cytokine storm and death in mice. In the aortas of these animals, SkQ1 also prevented the increase in the expression of NF-kB-dependent genes, including the cytokine IL-6 and the chemokine MCP-1. In the current work, we have tested the hypothesis of mito-ROS involvement in the IL-6-signaling-mediated pro-inflammatory gene expression in endothelial cells. SkQ1 suppressed the expression and secretion of the MCP-1 chemokine, induced by IL-6 in combination with sIL-6-Ra, but not the expression of ICAM1 adhesion molecules in EA.hy926 human endothelial cells. Using specific inhibitors, we have shown that in EA.hy926 cells, IL-6-induced expression of MCP-1 and ICAM-1 depends on the signaling protein and transcription activator STAT3 and, in some cases, on JNK, PI3K, and MEK1/2 kinases and is independent of p38 kinase. In this model, IL-6 induced rapid STAT3 activation while ERK1/2 activation was less pronounced, and there was no IL-6 effect on Akt and JNK activation. SkQ1 partially suppressed STAT3 and ERK1/2 activation. Thus, we have shown that SkQ1 suppresses not only NF-kB-dependent expression of IL-6 and other proinflammatory genes, but also IL-6-induced activation of JAK/STAT3 and STAT3-dependent expression of MCP-1, which probably contributes to the overall therapeutic effect of SkQ1.

1. Uciechowski P., Dempke W.C.M. Interleukin-6: A masterplayer in the cytokine network // Oncology. 2020. Vol. 98. N 3. P. 131–137.

2. Hou T., Tieu B.C., Ray S., Recinos III A., Cui R., Tilton R.G., Brasier A.R. Roles of IL-6-gp130 signaling in vascular inflammation // Curr. Cardiol. Rev. 2008. Vol. 4. N 3. P. 179–192.

3. Kang S., Kishimoto T. Interplay between interleukin-6 signaling and the vascular endothelium in cytokine storms // Exp. Mol. Med. 2021. Vol. 53. N 7. P. 1116–1123.

4. Fajgenbaum D.C., June C.H. Cytokine storm // N. Engl. J. Med. 2020. Vol. 383. N 23. P. 2255–2273.

5. Liu Y., Chen D., Hou J., Li H., Cao D., Guo M., Ling Y., Gao M., Zhou Y., Wan Y., Zhu Z. An intercorrelated cytokine network identified at the center of cytokine storm predicted COVID-19 prognosis // Cytokine. 2021. Vol. 138: 155365.

6. Chen G., Wu D.I., Guo W., et al. Clinical and immunological features of severe and moderate coronavirus disease 2019 // J. Clin. Invest. 2020. Vol. 130. N 5. P. 2620–2629.

7. Herold T., Jurinovic V., Arnreich C., Lipworth B.J., Hellmuth J.C., von Bergwelt-Baildon M., Klein M., Weinberger T. Elevated levels of IL-6 and CRP predict the need for mechanical ventilation in COVID-19 // J. Allergy Clin. Immunol. 2020. Vol. 146. N 1. P. 128-136.e4.

8. Ruan Q., Yang K., Wang W., Jiang L., Song J. Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China // Intensive Care Med. 2020. Vol. 46. N 5. P. 846–848.

9. Ascierto P.A., Fu B., Wei H. IL-6 modulation for COVID-19: the right patients at the right time? // J. Immunother. Cancer. 2021. Vol. 9. N 4: e002285.

10. Alsaffar H.., Martino N., Garrett J.P., Adam A.P. Interleukin-6 promotes a sustained loss of endothelial barrier function via Janus kinase-mediated STAT3 phosphorylation and de novo protein synthesis // Am. J. Physiol. Cell Physiol. 2018. Vol. 314. N 5. P. C589–C602.

11. Desai T.R., Leeper N.J., Hynes K.L., Gewertz B.L. Interleukin-6 causes endothelial barrier dysfunction via the protein kinase C pathway // J. Surg. Res. 2002. Vol. 104. N 2. P. 118–123.

12. Watson C., Whittaker S., Smith N., Vora A.J., Dumonde D.C., Brown K.A. IL-6 acts on endothelial cells to preferentially increase their adherence for lymphocytes // Clin. Exp. Immunol. 1996. Vol. 105. N 1. P. 112–119.

13. Romano M., Sironi M., Toniatti C., Polentarutti N., Fruscella P., Ghezzi P., Faggioni R., Luini W., Van Hinsbergh V., Sozzani S., Bussolino F., Poli V., Ciliberto G., Mantovani A. Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment // Immunity. 1997. Vol. 6. N 3. P. 315–325.

14. Wung B.S., Ni C.W., Wang D.L. ICAM-1 induction by TNFα and IL-6 is mediated by distinct pathways via Rac in endothelial cells // J. Biomed. Sci. 2005. Vol. 12. N 1. P. 91–101.

15. Wung B.S., Hsu M.C., Wu C.C., Hsieh C.W. Resveratrol suppresses IL-6-induced ICAM-1 gene expression in endothelial cells: Effects on the inhibition of STAT3 phosphorylation // Life Sci. 2005. Vol. 78. N 4. P. 389–397.

16. McLoughlin R.M., Jenkins B.J., Grail D., Williams A.S., Fielding C.A., Parker C.R., Ernst M., Topley N., Jones S.A. IL-6 trans-signaling via STAT3 directs T cell infiltration in acute inflammation // Proc. Natl. Acad. Sci. U.S.A. 2005. Vol. 102. N 27. P. 9589–9594.

17. Kang S., Tanaka T., Inoue H., Ono C., Hashimoto S., Kioi Y., Matsumoto H., Matsuura H., Matsubara T., Shimizu K., Ogura H. IL-6 trans-signaling induces plasminogen activator inhibitor-1 from vascular endothelial cells in cytokine release syndrome // Proc. Natl. Acad. Sci. U.S.A. 2020. Vol. 117. N 36. P. 22351–22356.

18. Xu S., Ilyas I., Little P.J., Li H., Kamato D., Zheng X., Luo S., Li Z., Liu P., Han J., Harding I.C. Endothelial dysfunction in atherosclerotic cardiovascular diseases and beyond: From mechanism to pharmacotherapies // Pharmacol. Rev. 2021. Vol. 73. N 3. P. 924–967.

19. Aird W.C. Review article The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome // Blood. 2003. Vol. 101. N 10. P. 3765–3777.

20. Prasad M., Leon M., Lerman L.O., Lerman A. Viral endothelial dysfunction: A unifying mechanism for COVID-19 // Mayo Clin. Proc. 2021. Vol. 96. N 12. P. 3099–3108.

21. Jin Y., Ji W., Yang H., Chen S., Zhang W., Duan G. Endothelial activation and dysfunction in COVID-19: from basic mechanisms to potential therapeutic approaches // Signal Transduct. Target. Ther. 2020. Vol. 5: 293.

22. Birnhuber A., Fliesser E., Gorkiewicz G., Zacharias M., Seeliger B., David S., Welte T., Schmidt J., Olschewski H., Wygrecka M., Kwapiszewska G. Between inflammation and thrombosis: Endothelial cells in COVID-19 // Eur. Respir. J. 2021. Vol. 58. N 3: 2100377.

23. Otifi H.M., Adiga B.K. Endothelial dysfunction in Covid-19 infection // Am. J. Med. Sci. 2022. Vol. 363. N 4. P. 285–291.

24. Panieri E., Santoro M.M. ROS signaling and redox biology in endothelial cells // Cell. Mol. Life Sci. 2015. Vol. 72. N 17. P. 3281–3303.

25. Aldosari S., Awad M., Harrington E.O., Sellke F.W., Abid M.R. Subcellular reactive oxygen species (ROS) in cardiovascular pathophysiology // Antioxidants. 2018. Vol. 7. N 1: 14.

26. Didion S.P. Cellular and oxidative mechanisms associated with interleukin-6 signaling in the vasculature // Int. J. Mol. Sci. 2017. Vol. 18. N 12: 2563.

27. Volk T., Hensel M., Schuster H., Kox W.J. Secretion of MCP-1 and IL-6 by cytokine stimulated production of reactive oxygen species in endothelial cells // Mol. Cell. Biochem. 2000. Vol. 206. N 1–2. P. 105–112.

28. Ali M.H., Schlidt S.A., Chandel N.S., Hynes K.L., Schumacker P.T., Gewertz B.L. Endothelial permeability and IL-6 production during hypoxia: role of ROS in signal transduction. // Am. J. Physiol. – Lung Cell. Mol. Physiol. 1999. Vol. 277. Pt. 1. P. L1057–L1065.

29. Lee Y.W., Lee W.H., Kim P.H. Oxidative mechanisms of IL-4-induced IL-6 expression in vascular endothelium // Cytokine. 2010. Vol. 49. N 1. P. 73–79.

30. Pearlstein D.P., Ali M.H., Mungai P.T., Hynes K.L., Gewertz B.L., Schumacker P.T. Role of mitochondrial oxidant generation in endothelial cell responses to hypoxia // Arterioscler. Thromb. Vasc. Biol. 2002. Vol. 22. N 4. P. 566–573.

31. Zinovkin R.A., Romaschenko V.P., Galkin I.I., Zakharova V.V., Pletjushkina O.Y., Chernyak B.V., Popova E.N. Role of mitochondrial reactive oxygen species in agerelated inflammatory activation of endothelium // Aging (Albany N.Y.). 2014. Vol. 6. N 8. P. 661–674.

32. Zakharova V.V., Pletjushkina O.Y., Galkin I.I., Zinovkin R.A., Chernyak B.V., Krysko D.V., Bachert C., Krysko O., Skulachev V.P., Popova E.N. Low concentration of uncouplers of oxidative phosphorylation decreases the TNF-induced endothelial permeability and lethality in mice // Biochim. Biophys. Acta – Mol. Basis Dis. 2017. Vol. 1863. N 4. P. 968–977.

33. Wassmann S., Stumpf M., Strehlow K., Schmid A., Schieffer B., Böhm M., Nickenig G. Interleukin-6 induces oxidative stress and endothehal dysfunction by overexpression of the angiotensin II type 1 receptor // Circ. Res. 2004. Vol. 94. N 4. P. 534–541.

34. Chopra A., Willmore W.G., Biggar K.K. Protein quantification and visualization via ultraviolet-dependent labeling with 2,2,2-trichloroethanol // Sci. Rep. 2019. Vol. 9: 13923.

35. Li R., Ren T., Zeng J. Mitochondrial coenzyme Q protects sepsis-induced acute lung injury by activating PI3K/Akt/GSK-3 β /mTOR pathway in rats // Biomed Res. Int. 2019. Vol. 2019: 5240898.

36. Zakharova V.V., Pletjushkina O.Y., Zinovkin R.A., Popova E.N., Chernyak B.V. Mitochondria-targeted antioxidants and uncouplers of oxidative phosphorylation in treatment of the systemic inflammatory response syndrome (SIRS) // J. Cell. Physiol. 2017. Vol. 232. N 5. P. 904–912.

37. Rademann P., Weidinger A., Drechsler S., et al. Mitochondria-targeted antioxidants SkQ1 and MitoTEMPO failed to exert a long-term beneficial effect in murine polymicrobial sepsis // Oxid. Med. Cell. Longev. 2017. Vol. 2017: 6412682.

38. Ding J., Song D., Ye X., Liu S.F. A pivotal role of endothelial-specific NF-κB signaling in the pathogenesis of septic shock and septic vascular dysfunction // J. Immunol. 2009. Vol. 183. N 6. P. 4031–4038.

39. Demyanenko I.A., Popova E.N., Zakharova V.V., Ilyinskaya O.P., Vasilieva T.V., Romashchenko V.P., Fedorov A.V., Manskikh V.N., Skulachev M.V., Zinovkin R.A., Pletjushkina O.Y., Skulachev V.P., Chernyak B.V. Mitochondria-targeted antioxidant SkQ1 improves impaired dermal wound healing in old mice // Aging (Albany N.Y.). 2015. Vol. 7. N 7. P. 475–485.

40. Galkin I.I., Pletjushkina O.Y., Zinovkin R.A., Zakharova V.V., Chernyak B.V., Popova E.N. Mitochondriatargeted antioxidant SkQR1 reduces TNF-induced endothelial permeability in vitro // Biochemistry (Mosc.). 2016. Vol. 81. N 10. P. 1188–1197.

41. Galkin I.I., Pletjushkina O.Y., Zinovkin R.A., Zakharova V.V., Birjukov I.S., Chernyak B.V., Popova E.N. Mitochondria-targeted antioxidants prevent TNFα-induced endothelial cell damage // Biochemistry (Mosc.). 2014. Vol. 79. N 2. P. 124–130.

42. Chelombitko M.A., Averina O.A., Vasilyeva T.V., Pletiushkina O.Y., Popova E.N., Fedorov A.V., Chernyak B.V., Shishkina V.S., Ilinskaya O.P. Mitochondria-targeted antioxidant SkQ1 (10-(6′-plastoquinonyl)decyltriphenylphosphonium bromide) inhibits mast cell degranulation in vivo and in vitro // Biochemistry (Mosc.). 2017. Vol. 82. N 12. P. 1493–1503.

43. Chelombitko M.A., Averina O.A., Vasil’eva T.V., Dvorianinova E.E., Egorov M.V., Pletjushkina O.Y., Popova E.N., Fedorov A.V., Romashchenko V.P., Ilyinskaya O.P. Comparison of the effects of mitochondria-targeted antioxidant 10-(6’-plastoquinonyl)decyltriphenylphosphonium bromide (SkQ1) and a fragment of its molecule dodecyltriphenylphosphonium on carrageenan-induced acute inflammation in mouse model of subcuteneo // Bull. Exp. Biol. Med. 2017. Vol. 162. N 6. P. 730–733.