Characterization of combined effects of reactive oxygen metabolites, complement system, and antimicrobial peptides In Vitro

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Abstract

Phagocytes activation results in the production of reactive oxygen metabolites (ROM) exerting antimicrobial and host-damaging activity. Although the main pool of papers shows their potentiating action on a key humoral nexus of innate immunity, complement system, the data are controversial. Combined action of ROM with antimicrobial peptides of phagocytes also remains poorly characterized. We have investigated the influence of oxidative burst products on complement activation in different in vitro models. Hydrogen peroxide, including that in medium with Fe-EDTA did not affect parameters of complement activity in human blood serum. HOCl in millimolar concentrations stimulated production of C3a and C5a anaphylatoxins in 80% serum, the effect was inhibited by EDTA. We have identified bivalent ions-independent C5 cleavage in the presence of 16 mM HOCl. At the same time, HOCl served as an inhibitor of the alternative complement pathway in the model of surface-associated activation on rabbit erythrocytes in 5% serum. It inhibited production of C3a (IC50 ~ 4 mM) and C5a as well as serum hemolytic activity (IC50 ~ 0.2 mM); the inhibition of C5a generation was less pronounced in the presence of 4–16 mM HOCl. Decrease in anaphylatoxins generation was also observed in the system with zymosan in 5% serum. Under similar conditions but without activating surfaces, moderate HOCl concentrations enhanced C3a and C5a accumulation; EDTA inhibited this effect completely (C3a) or partially (C5a). Finally, in 70% serum, 16 mM HOCl enhanced the anaphylatoxins accumulation but in the presence of zymosan it inhibited this process almost completely. We hypothesize that HOCl can attack the thioester bond in C3 protein to form C3(HOCl) adduct which is capable of fluid-phase convertases formation; however, the attack of C3b can prevent its covalent fixation on membranes and blocks the complement amplification loop. Besides, we have demonstrated the additive character of the combined action of HOCl with antimicrobial peptides (LL-37 cathelicidin and α-defensins) towards Listeria monocytogenes and Escherichia coli. The data obtained precise the picture of the interaction between bactericidal factors of phagocytes and complement as key participants of the immune defense and host damage.

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About the authors

I. A. Krenev

Institute of Experimental Medicine

Author for correspondence.
Email: il.krenevv13@yandex.ru
Russian Federation, St. Petersburg

E. V. Egorova

Institute of Experimental Medicine; St. Petersburg State University

Email: il.krenevv13@yandex.ru
Russian Federation, St. Petersburg; St. Petersburg

N. P. Gorbunov

Institute of Experimental Medicine; St. Petersburg Pasteur Research Institute of Epidemiology and Microbiology

Email: il.krenevv13@yandex.ru
Russian Federation, St. Petersburg; St. Petersburg

V. A. Kostevich

Institute of Experimental Medicine

Email: il.krenevv13@yandex.ru
Russian Federation, St. Petersburg

A. V. Sokolov

Institute of Experimental Medicine; St. Petersburg State University

Email: il.krenevv13@yandex.ru
Russian Federation, St. Petersburg; St. Petersburg

A. S. Komlev

Institute of Experimental Medicine

Email: il.krenevv13@yandex.ru
Russian Federation, St. Petersburg

Y. A. Zabrodskaya

Peter the Great St. Petersburg Polytechnic University; A. A. Smorodintsev Research Institute of Influenza

Email: il.krenevv13@yandex.ru
Russian Federation, St. Petersburg; St. Petersburg

O. V. Shamova

Institute of Experimental Medicine; St. Petersburg State University

Email: il.krenevv13@yandex.ru
Russian Federation, St. Petersburg; St. Petersburg

M. N. Berlov

Institute of Experimental Medicine; St. Petersburg State University

Email: il.krenevv13@yandex.ru
Russian Federation, St. Petersburg; St. Petersburg

References

  1. Uribe-Querol E., Rosales C. // Front. Immunol. 2020. V. 11. P. 1066. https://doi.org/10.3389/fimmu.2020.01066
  2. Burn G.L., Foti A., Marsman G., Patel D.F., Zychlinsky A. // Immunity. 2021. V. 54. P. 1377–1391. https://doi.org/10.1016/j.immuni.2021.06.006
  3. Katsuyama M. // J. Pharmacol. Sci. 2010. V. 114. P. 134–146. https://doi.org/10.1254/jphs.10r01cr
  4. Zheng M., Liu Y., Zhang G., Yang Z., Xu W., Chen Q. // Antioxidants (Basel). 2023. V. 12. P. 1675. https://doi.org/10.3390/antiox12091675
  5. Andrés C.M.C., Pérez de la Lastra J.M., Juan C.A., Plou F.J., Pérez-Lebeña E. // Stresses. 2022. V. 2. P. 256–274. https://doi.org/10.3390/stresses2030019
  6. Aratani Y. // Arch. Biochem. Biophys. 2018. V. 640. P. 47–52. https://doi.org/10.1016/j.abb.2018.01.004
  7. Arnhold J. // Int. J. Mol. Sci. 2020. V. 21. P. 8057. https://doi.org/10.3390/ijms21218057
  8. Pattison D.I., Davies M.J. // Chem. Res. Toxicol. 2001. V. 14. P. 1453–1464. https://doi.org/10.1021/tx0155451
  9. Weiss S.J. // N. Engl J. Med. 1989. V. 320. P. 365–376. https://doi.org/10.1056/NEJM198902093200606
  10. Huan Y., Kong Q., Mou H., Yi H. // Front. Microbiol. 2020. V. 11. P. 582779. https://doi.org/10.3389/fmicb.2020.582779
  11. Lehrer R. I., Lu W. // Immunol. Rev. 2012. V. 245. P. 84–112. https://doi.org/10.1111/j.1600-065X.2011.01082.x
  12. Ridyard K.E., Overhage J. // Antibiotics (Basel). 2021. V. 10. P. 650. https://doi.org/10.3390/antibiotics10060650
  13. Ricklin D., Reis E.S., Mastellos D.C., Gros P., Lambris J.D. // Immunol. Rev. 2016. V. 274. P. 33–58. https://doi.org/10.1111/imr.12500
  14. Tack B.F., Harrison R.A., Janatova J., Thomas M.L., Prahl J.W. // Proc. Natl. Acad. Sci. USA. 1980. V. 77. P. 5764–5768. https://doi.org/10.1073/pnas.77.10.5764
  15. Law S.K., Dodds A.W. // Protein. Sci. 1997. V. 6. P. 263– 274. https://doi.org/10.1002/pro.5560060201
  16. Shokal U., Eleftherianos I. // Front. Immunol. 2017. V. 8. P. 759. https://doi.org/10.3389/fimmu.2017.00759
  17. Forneris F., Ricklin D., Wu J., Tzekou A., Wallace R.S., Lambris J.D., Gros P. // Science. 2010. V. 330. P. 1816– 1820. https://doi.org/10.1126/science.1195821
  18. Fearon D.T., Austen K.F. // J. Exp. Med. 1975. V. 142. P. 856–863. https://doi.org/10.1084/jem.142.4.856
  19. Lachmann P.J., Lay E., Seilly D.J. // FASEB J. 2018. V. 32. P. 123–129. https://doi.org/10.1096/fj.201700734
  20. Xie C.B., Jane-Wit D., Pober J.S. // Am. J. Pathol. 2020. V. 190. P. 1138–1150. https://doi.org/10.1016/j.ajpath.2020.02.006
  21. D’Autréaux B., Toledano M.B. // Nat. Rev. Mol. Cell Biol. 2007. V. 8. P. 813–824. https://doi.org/10.1038/nrm2256
  22. Ricklin D., Hajishengallis G., Yang K., Lambris J.D. // Nat. Immunol. 2010. V. 11. P. 785–797. https://doi.org/10.1038/ni.1923
  23. Hawksworth O.A., Coulthard L.G., Woodruff T.M. // Mol. Immunol. 2017. V. 84. P. 17–25. https://doi.org/10.1016/j.molimm.2016.11.010
  24. Alfadda A.A., Sallam R.M. // J. Biomed. Biotechnol. 2012. P. 936486. https://doi.org/10.1155/2012/936486
  25. Gierlikowska B., Stachura A., Gierlikowski W., Demkow U. // Front. Pharmacol. 2021. V. 12. P. 666732. https://doi.org/10.3389/fphar.2021.666732
  26. Mastellos D.C., Hajishengallis G., Lambris J.D. // Nat. Rev. Immunol. 2024. V. 24. P. 118–141. https://doi.org/10.1038/s41577-023-00926-1
  27. Goldstein I.M., Weissmann G. // J. Immunol. 1974. V. 113. P. 1583–1588. https://doi.org/10.4049/jimmunol.113.5.1583
  28. Johnson U., Ohlsson K., Olsson I. // Scand. J. Immunol. 1976. V. 5. P. 421–426. https://doi.org/10.1111/j.1365-3083.1976.tb00296.x
  29. Orr F.W., Varani J., Kreutzer D.L., Senior R.M., Ward P.A. // Am. J. Pathol. 1979. V. 94. P. 75–83.
  30. Taylor J.C., Crawford I.P., Hugli T.E. // Biochemistry. 1977. V. 16. P. 3390–3396. https://doi.org/10.1021/bi00634a016
  31. Venge P., Olsson I. // J. Immunol. 1975. V. 115. P. 1505–1508. https://doi.org/10.4049/jimmunol.115.6.1505
  32. Vogt W. // Immunobiology. 2000. V. 201. P. 470–477. https://doi.org/10.1016/S0171-2985(00)80099-6
  33. Ward P.A., Hill J.H. // J. Immunol. 1970. V. 104. P. 535– 543. https://doi.org/10.4049/jimmunol.104.3.535
  34. Кренев И.А., Берлов М.Н., Умнякова Е.С. // Иммунология. 2021. Т. 42. С. 426–433. https://doi.org/10.33029/0206-4952-2021-42-4-426-433
  35. van den Berg R.H., Faber-Krol M.C., van Wetering S., Hiemstra P.S., Daha M.R. // Blood. 1998. V. 92. P. 3898–3903. https://doi.org/10.1182/blood.V92.10.3898
  36. Groeneveld T.W., Ramwadhdoebé T.H., Trouw L.A., van den Ham D.L., van der Borden V., Drijfhout J.W., Hiemstra P.S., Daha M.R., Roos A. // Mol. Immunol. 2007. V. 44. P. 3608–3614. https://doi.org/10.1016/j.molimm.2007.03.003
  37. Nordahl E.A., Rydengård V., Nyberg P., Nitsche D.P., Mörgelin M., Malmsten M., Björck L., Schmidtchen A. // Proc. Natl. Acad. Sci. USA. 2004. V. 101. P. 16879– 16884. https://doi.org/10.1073/pnas.0406678101
  38. Sonesson A., Ringstad L., Nordahl E.A., Malmsten M., Mörgelin M., Schmidtchen A. // Biochim. Biophys. Acta. 2007. V. 1768. P. 346–353. https://doi.org/10.1016/j.bbamem.2006.10.017
  39. Pasupuleti M., Walse B., Nordahl E.A., Mörgelin M., Malmsten M., Schmidtchen A. // J. Biol. Chem. 2007. V. 282. P. 2520–2528. https://doi.org/10.1074/jbc.M607848200
  40. Shingu M., Nobunaga M. // Am. J. Pathol. 1984. V. 117. P. 201–206.
  41. Shingu M., Nonaka S., Nishimukai H., Nobunaga M., Kitamura H., Tomo-Oka K. // Clin. Exp. Immunol. 1992. V. 90. P. 72–78. https://doi.org/10.1111/j.1365-2249.1992.tb05834.x
  42. Vogt W., Damerau B., von Zabern I., Nolte R., Brunahl D. // Mol. Immunol. 1989. V. 26. P. 1133–1142. https://doi.org/10.1016/0161-5890(89)90057-6
  43. Vogt W., Zimmermann B., Hesse D., Nolte R. // Mol. Immunol. 1992. V. 29. P. 251–256. https://doi.org/10.1016/0161-5890(92)90106-8
  44. Vogt W., Hesse D. // Immunobiology. 1992. V. 184. P. 384–391. https://doi.org/10.1016/S0171-2985(11)80595-4
  45. Vogt W. // Immunobiology. 1996. V. 195. P. 334–346. https://doi.org/10.1016/S0171-2985(96)80050-7
  46. Clark R.A., Klebanoff S.J. // J. Clin. Invest. 1979. V. 64. P. 913–920. https://doi.org/10.1172/JCI109557
  47. Coble B.I., Dahlgren C., Hed J., Stendahl O. // Biochim. Biophys. Acta. 1984. V. 802. P. 501–505. https://doi.org/10.1016/0304-4165(84)90369-6
  48. Miller T.E. // J. Bacteriol. 1969. V. 98. P. 949–955. https://doi.org/10.1128/jb.98.3.949-955.1969
  49. Lichtenstein A.K., Ganz T., Selsted M.E., Lehrer R.I. // Cell. Immunol. 1988. V. 114. P. 104–116. https://doi.org/10.1016/0008-8749(88)90258-4
  50. Vissers M.C., Winterbourn C.C. // Biochem. J. 1987. V. 245. P. 277–280. https://doi.org/10.1042/bj2450277
  51. Shao B., Belaaouaj A., Verlinde C.L., Fu X., Heinecke J.W. // J. Biol. Chem. 2005. V. 280. P. 29311– 29321. https://doi.org/10.1074/jbc.M504040200
  52. Hawkins C.L., Davies M.J. // Chem. Res. Toxicol. 2005. V. 18. P. 1600–1610. https://doi.org/10.1021/tx050207b
  53. Krishna H., Avinash K., Shivakumar A., Al-Tayar N.G.S., Shrestha A.K. // Spectrochim. Acta A Mol. Biomol. Spectrosc. 2021. V. 251. P. 119358. https://doi.org/10.1016/j.saa.2020.119358
  54. Peffault de Latour R., Brodsky R.A., Ortiz S., Risitano A.M., Jang J.H., Hillmen P., Kulagin A.D., Kulasekararaj A.G., Rottinghaus S.T., Aguzzi R., Gao X., Wells R.A., Szer J. // Br. J. Haematol. 2020. V. 191. P. 476–485. https://doi.org/10.1111/bjh.16711
  55. Forman H.J., Bernardo A., Davies K.J. // Arch. Biochem. Biophys. 2016. V. 603. P. 48–53. https://doi.org/10.1016/j.abb.2016.05.005
  56. Pravda J. // Mol. Med. 2020. V. 26. P. 41. https://doi.org/10.1186/s10020-020-00165-3
  57. Lipcsey M., Bergquist M., Sirén R., Larsson A., Huss F., Pravda J., Furebring M., Sjölin J., Janols H. // Biomedicines. 2022. V. 10. P. 848. https://doi.org/10.3390/biomedicines10040848
  58. Huber-Lang M., Ekdahl K.N., Wiegner R., Fromell K., Nilsson B. // Semin. Immunopathol. 2018. V. 40. P. 87– 102. https://doi.org/10.1007/s00281-017-0646-9
  59. Weiss S.J., Peppin G., Ortiz X., Ragsdale C., Test S.T. // Science. 1985. V. 227. P. 747–749. https://doi.org/10.1126/science.2982211
  60. Peppin G.J., Weiss S.J. // Proc. Natl. Acad. Sci. USA. 1986. V. 83. P. 4322–4326. https://doi.org/10.1073/pnas.83.12.4322
  61. Fu X., Kassim S.Y., Parks W.C., Heinecke J.W. // J. Biol. Chem. 2001. V. 276. P. 41279–41287. https://doi.org/10.1074/jbc.M106958200
  62. Wang Y., Rosen H., Madtes D.K., Shao B., Martin T.R., Heinecke J.W., Fu X. // J. Biol. Chem. 2007. V. 282. P. 31826–31834. https://doi.org/10.1074/jbc.M704894200
  63. Siddiqui T., Zia M.K., Ali S.S., Rehman A.A., Ahsan H., Khan F.H. // Arch. Physiol. Biochem. 2016. V. 122. P. 1–7. https://doi.org/10.3109/13813455.2015.1115525
  64. DiScipio R.G., Smith C.A., Muller-Eberhard H.J., Hugli T.E. // J. Biol. Chem. 1983. V. 258. P. 10629–10636. https://doi.org/10.1016/S0021-9258(17)44503-0
  65. Zharkova M.S., Komlev A.S., Filatenkova T.A., Sukhareva M.S., Vladimirova E.V., Trulioff A.S., Orlov D.S., Dmitriev A.V., Afinogenova A.G., Spiridonova A.A., Shamova O.V. // Pharmaceutics. 2023. V. 15. P. 291. https://doi.org/10.3390/pharmaceutics15010291
  66. Krenev I.A., Umnyakova E.S., Eliseev I.E., Dubrovskii Y.A., Gorbunov N.P., Pozolotin V.A., Komlev A.S., Panteleev P.V., Balandin S.V., Ovchinnikova T.V., Shamova O.V., Berlov M.N. // Mar. Drugs. 2020. V. 18. P. 631. https://doi.org/10.3390/md18120631
  67. Umnyakova E.S., Gorbunov N.P., Zhakhov A.V., Krenev I.A., Ovchinnikova T.V., Kokryakov V.N., Berlov M.N. // Mar. Drugs. 2018. V. 16. P. 480. https://doi.org/10.3390/md16120480
  68. Fearon D.T., Austen K.F. // Proc. Natl. Acad. Sci. USA. 1977. V. 74. P. 1683-1687. https://doi.org/10.1073/pnas.74.4.1683
  69. Fields G.B., Noble R.L. // Int. J. Pept. Protein Res. 1990. V. 35. P. 161–214. https://doi.org/10.1111/j.1399-3011.1990.tb00939.x
  70. Pozolotin V.A., Umnyakova E.S., Kopeykin P.M., Komlev A.S., Dubrovskii Y.A., Krenev I.A. Shamova O.V., Berlov M.N. // Russ. J. Bioorg. Chem. 2021. V. 47. P. 741–748.] https://doi.org/10.1134/S1068162021030158
  71. Smith B.J. // Methods Mol. Biol. 1984. V. 1. P. 63–73. https://doi.org/10.1385/0-89603-062-8:63
  72. Berlov M.N., Korableva E.S., Andreeva Y.V., Ovchinnikova T.V., Kokryakov V.N. // Biochemistry (Moscow). 2007. V. 72. P. 445–451. https://doi.org/10.1134/S0006297907040128
  73. Świerczyńska M., Słowiński D., Michalski R., Romański J., Podsiadły R. // Molecules. 2023. V. 28. Р. 6055. https://doi.org/10.3390/molecules28166055
  74. A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, 2020. https://www.R-project.org/
  75. Wickham H. // ggplot2: Elegant Graphics for Data Analysis. New York: Springer-Verlag, 2016.
  76. Kassambara A. //ggpubr: ‘ggplot2’ Based Publication Ready Plots. R package version 0.6.0, 2023. https://rpkgs.datanovia.com/ggpubr/

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3. Fig. 1. Effect of incubation of human blood serum with H2O2 under different conditions on the hemolytic activity of serum against Errab.

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4. Fig. 2. The effect of H2O2 (a, c, d) and HOCl (b, d, e) on complement in the liquid-phase activation model. (a, b) – Effect on the production of anaphylatoxin C3a; (c, d) – effect on the production of anaphylatoxin C5a; (d, e) – effect on the hemolytic activity of serum with respect to Errab. The abscissa axis is presented in logarithmic scale.

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5. Fig. 3. The effect of HOCl on complement activation in 5% serum in the presence of Mg-EGTA during incubation at 37°C for 30 min. (a) – Effect on the production of anaphylatoxins C3a and C5a in the presence of Errab; (b) – Effect on the hemolytic activity of serum with respect to Errab; (c) – Effect on C3a production in the presence of zymosan; (d) – Effect on C5a production in the presence of zymosan; (d) – Effect on C3a production in the absence of Errab and zymosan; (e) – Effect on C5a production in the absence of Errab and zymosan. The abscissa axis is shown in logarithmic scale.

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6. Fig. 4. Effect of 16 mM HOCl on the accumulation of anaphylatoxins in the absence and presence of zymosan in 70% blood serum during incubation at 37°C for 30 min.

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7. Fig. 5. Integral representation of the interaction of AMC, AMP, and complement during the implementation of the phagocytic response, taking into account the hypothesis of the interaction of HOCl with C3 and C3b and the obtained data on the additive nature of the combined microbicidal action of HOCl and AMP. For ease of understanding, the effect of HOCl on the activity of proteases/antiproteases and lysozyme is not illustrated. Abbreviations: GP – glutathione peroxidase, MAK – membrane attack complex, MPO – myeloperoxidase, PR – peroxiredoxins, SOD – superoxide reductase, HNPs – human neutrophil peptides, MBL – mannan-binding lectin, NOX2 – NADPH oxidase 2.

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