THE ROLE OF GENETICALLY DETERMINED BODY RESISTANCE OF WARM-BLOODED ANIMALS TO HYPOXIA IN THE REALIZATION OF EFFECTOR FUNCTIONS OF NEUTROPHILS IN THE MODEL OF ADJUVANT-INDUCED RHEUMATOID ARTHRITIS

Abstract

Introduction: Hypoxia can be both a cause and a consequence of the pathogenetic mechanisms of infectious, autoinflammatory, and autoimmune processes. Given that human and animal populations are genetically heterogeneous in terms of the body's resistance to oxygen deficiency, the modern approach to predicting and treating diseases associated with impaired immune regulation requires taking into account the role of hypoxia in the implementation of pathogenetic mechanisms of inflammation. The purpose of the study: to evaluate the peculiarities of the realization of the effector functions of neutrophils in normal conditions and against the background of an induced inflammatory process in animals with genetically predetermined high and low resistance to hypoxia. The material for the study was 8-month-old male rats with genetically determined tolerance to hypoxia (highly resistant HR/SmY line; low-resistant LR/SmY line), weighing 400-450 g. Rats from the experimental groups of the HR/SmY and LR/SmY lines were induced to develop immune responses in a model of rheumatoid arthritis (RA). After 35 days, the blood taken from the animals was incubated with a carcass suspension (1:10). The smears recorded in formalin vapors were stained with 0.5% methylene blue solution and analyzed at 400x magnification of a microscope. The phagocytic index (PHI), phagocytic number (PN), and the number of suicidal netosis with partial and complete chromatin decondensation were calculated. The significance of differences in the groups was assessed by the Mann-Whitney test and the Student's t-test. Results: In the control groups of LR/SmY and HR/SmY animals, PHI and PN of neutrophils did not significantly differ. (52,5%/49%; 1,68/1,80 respectively). In the experimental groups, the PHI of neutrophils of the LR/SmY line (66%, p<0.05) exceeded that of the HR/SmY line (56%). With systemic inflammation, the PHI of neutrophils in the group of low-tolerance rats increased by 1.26 (p<0.05) times, and the number of neutrophils capturing 2-8 particles increased by 1.3 (p<0.01) times in healthy rats. In the experimental HR/SmY group, where 1.81% of more effective neutrophils capable of absorbing from 9 to 12 particles (p<0.01) were recorded, the PHI was 1.2 times lower than that of the LR/SmY line. In healthy HR/SmY rats, 1.4 (p<0.05) times more neutrophils are involved in the process of suicidal netosis than in the similar group of the LR/SmY line. Systemic inflammation in the LR/SmY group caused a twofold increase in NETs (19.67%), which was 1.7 times higher than in the experimental HR/SmY group. Conclusion: Organisms with genetically determined low resistance to hypoxia against the background of the inflammatory process have a greater stress on the cell-mediated link of immunity in the process of phlogogen elimination compared to highly resistant ones, which may cause them to develop more severe pathogenetic forms of inflammatory and autoimmune diseases.

1. Бочкарева Л.А., Недосугова Л.В., Петунина Н.А., Тельнова М.Э., Гончарова Е.В. Некоторые механизмы развития воспаления при сахарном диабете 2 типа. Сахарный диабет. 2021; 24(4):334-341. Bochkareva LA, Nedosugova LV, Petunina NA, Telnova ME, Goncharova EV. Some mechanisms of inflammation development in type 2 diabetes mellitus. Diabetes Mellitus. 2021;24(4):334-341 doi: 10.14341/DM12746

2. Громыко М.В., Грицук А.И. Экспериментальные модели ревматоидного артрита. Проблемы здоровья и экологии. 2012;(2):115-118. Gromyko M.V., Gritsuk A.I. Experimental models of rheumatoid arthritis. Health and Ecology Issues. 2012;(2):115-118. (In Russ.)

3.

4. https://doi.org/10.51523/2708-6011.2012-9-2-22

5. Донина Ж.А., Баранова Е.В., Александрова Н.П. Сравнительная оценка влияния основных медиаторов острофазового ответа (ИЛ-1, ФНО-α и ИЛ-6) на паттерн дыхания и выживаемость крыс при острой нарастающей гипоксии. Российский физиологический журнал им. И.М. Сеченова. 2021;107(8):996-1006. Donina J.A., Baranova E.V., Alexandrova N.P.Comparative Assessment of the Effect of the Main Mediators of Acute Phase Response (IL-1, TNF-α and IL-6) on Breathing Pattern and Survival in Rats with Acute Progressive Hypoxia. I.M. Sechenov Russian Journal of Physiology. 2021;107(8):996-1006. https://doi.org/10.31857/S086981392108004

6. Жапаралиева Ч.О., Мухамедова И.П., Вишневский А. А. Изменения мембран эритроцитов и некоторых морфофункциональных особенностей головного мозга в условиях гипоксической гипоксии в группах крыс с различной устойчивостью к гипоксии // Ульяновский медико-биологический журнал. 2012. №1. -morfofunktsionalnyh-osobennostey-golovnogo-mozga-v-usloviyah-gipoksicheskoy-gipoksii-v (дата обращения: 02.04.2025). Japaralieva Ch.O., Muhamedova I.P., A.A., Vishnevskii I.P.Changes of membranes of erythrocytes and some morphological and functional features of the brain in the conditions of a hypoxemic hypoxemia in groups of rats with different resistance to hypoxia // Ulyanovsk Medical and Biological Journal. 2012. №1. URL: https://cyberleninka.ru/article/n/izmeneniya-membran-eritrotsitov-i-nekotoryh

7. Зарубина И. В. Молекулярные механизмы индивидуальной устойчивости к гипоксии // Обзоры по клинич. фармакол. и лек. терапии. 2005.№1. Zarubina I. V. Molecular mechanisms of individual resistance to hypoxia // Reviews on clinical. pharmacol. and lek. therapy. 2005. №1.e to hypoxia URL: https://cyberleninka.ru/article/n/molekulyarnye-mehanizmy-individualnoy-ustoychivosti-k-gipoksii (дата обращения: 11.03.2025).

8. Литвицкий П. Ф. Гипоксия. Вопросы современной педиатрии. 2016; 15 (1): 45-58. Litvitsky P.F. Hypoxia. Current Pediatrics. 2016;15(1):45-58. (In Russ.) https://doi.org/10.15690/vsp.v15i1.1499

9. Лукьянова Л. Д., Дудченко А. М., Белоусова В. В. Влияние различных концентраций кислорода на содержание АТФ в изолированных гепатоцитах адаптированных и неадаптированных к гипоксии крыс // Бюл. эксперим. биол. и мед. - 1994. - Т. 118, №12. -С. 576—58 Vliianie razlichnykh kontsentratsiĭ kisloroda na soderzhanie ATF v izolirovannykh gepatotsitakh adaptirovannykh i neadaptirovannykh k gipoksii krys [The effect of various concentration of oxygen on ATP level in isolated hepatocytes in rats adapted and not adapted to hypoxia]. Biull Eksp Biol Med. 1994 Dec;118(12):576-81. Russian. PMID: 7703447.

10. Медведев, А. Н. Способ исследования поглотительной фазы фагоцитоза / А. Н. Медведев, А. Н. Маянский, В. В. Чаленко // Лабораторное дело. – 1991. – №2. – с. 19-20с. – EDN ZAVONB. Medvedev, A. N. Method of studying the absorption phase of phagocytosis / A. N. Medvedev, A. N. Mayansky, V. V. Chalenko // Laboratory business. – 1991. – No. 2. – pp. 19-20. – EDN ZAVONB.

11. Нестерова И.В., Чудилова Г.А., Ломтатидзе Л.В. Ковалева С.В, Чапурина В.Н., Тетерин Ю.В. Расчетный индекс нейтрофильных гранулоцитов в дифференциальной диагностике степени тяжести бактериальных инфекционно-воспалительных заболеваний. Эффективная фармакотерапия. 2024; 20 (38): 34–44. I.V. Nesterova, MD, PhD, Prof., G.A. Chudilova, DBS, L.V. Lomtatidze, PhD, S.V. Kovaleva, MD, PhD, V.N. Chapurina, PhD, Yu.V. Teterin Nesterova I.V., Chudilova G.A., Lomtatidze L.V. and others. The calculated index of neutrophilic granulocytes in the differential diagnosis of the severity of bacterial infectious and inflammatory diseases. Effective pharmacotherapy. 2024; 20 (38): 34–44. DOI 10.33978/2307-3586-2024-20-38-34-44

12. Овсепян Л.М., Карагезян К.Г., Мелкумян А.В, Захарян Г.В. Взаимосвязь окислительного фосфорилирования и процесса перекисного окисления липидов в митохондриальной фракции головного мозга при гипоксии.// Биохимия. – 2006. – Т. 34. – С. 76–79. Hovsepyan L.M., Karagezyan K.G., Melkumyan A.V., Zakharyan G.V. The relationship between oxidative phosphorylation and lipid peroxidation in the mitochondrial fraction of the brain during hypoxia.// Biochemistry. - 2006. – Vol. 34. – pp. 76-79. http://elib.sci.am/2006_4/10/10r.htm

13. Руководство по проведению доклинических исследований лекарственных средств. Часть первая / под редакцией А. Н. Миронова – М.: Гриф и К, 2012. — 944 с. – С. 68.

14. Guidelines for conducting preclinical studies of medicines. Part one / edited by A. N. Mironov, Moscow: Grif and K, 2012. 944 p. – p. 68.

15. Cramer T, Yamanishi Y, Clausen BE, Förster I, Pawlinski R, Mackman N, Haase VH, Jaenisch R, Corr M, Nizet V, Firestein GS, Gerber HP, Ferrara N, Johnson RS. HIF-1alpha is essential for myeloid cell-mediated inflammation. Cell. 2003 Mar 7;112(5):645-57. - doi: 10.1016/s0092-8674(03)00154-5.

16. Devraj G, Beerlage C, Brüne B, Kempf VA. Hypoxia and HIF-1 activation in bacterial infections. Microbes Infect. 2017;19(3):144–156. - doi: 10.1016/j.micinf.2016.11.003.

17. .Fensterheim BA, Guo Y, Sherwood ER, Bohannon JK. The Cytokine Response to Lipopolysaccharide Does Not Predict the Host Response to Infection. J Immunol. 2017 Apr 15;198(8):3264-3273. - doi: 10.4049/jimmunol.1602106

18. Gierlikowska B, Stachura A, Gierlikowski W and Demkow U (2021) Phagocytosis, Degranulation and Extracellular Traps Release by Neutrophils—The Current Knowledge, Pharmacological Modulation and Future Prospects. Front. Pharmacol. 12:666732. - doi: 10.3389/fphar.2021.666732

19. Greer SN, Metcalf JL, Wang Y, Ohh M. The updated biology of hypoxia-inducible factor. EMBO J. 2012 May 30;31(11):2448-60. - doi: 10.1038/emboj.2012.125.

20. Hirota K. Involvement of hypoxia-inducible factors in the dysregulation of oxygen homeostasis in sepsis. Cardiovasc Hematol Disord Drug Targets. 2015;15(1):29-40. - doi: 10.2174/1871529x15666150108115553.

21. Hochachka PW. Mechanism and evolution of hypoxia-tolerance in humans. J Exp Biol. 1998 Apr;201(Pt 8):1243-54. - doi: 10.1242/jeb.201.8.1243.

22. Jantsch J, Wiese M, Schödel J, Castiglione K, Gläsner J, Kolbe S, Mole D, Schleicher U, Eckardt KU, Hensel M, Lang R, Bogdan C, Schnare M, Willam C. Toll-like receptor activation and hypoxia use distinct signaling pathways to stabilize hypoxia-inducible factor 1α (HIF1A) and result in differential HIF1A-dependent gene expression. J Leukoc Biol. 2011 Sep;90(3):551-62. - doi: 10.1189/jlb.1210683.

23. Kiers H.D., Scheffer G.-J., van der Hoeven J.G., Eltzschig H.K., et al. Immunologic Consequences of hypoxia during critical illness. Anesthesiology. 2016; 125 (1): 237-49. - doi: 10.1097/ ALN.0000000000001163

24. Peyssonnaux C, Datta V, Cramer T, Doedens A, Theodorakis EA, Gallo RL, Hurtado-Ziola N, Nizet V, Johnson RS. HIF-1alpha expression regulates the bactericidal capacity of phagocytes. J Clin Invest. 2005 Jul;115(7):1806-15. - doi: 10.1172/JCI23865.

25. Song D, Li LS, Arsenault PR, Tan Q, Bigham AW, Heaton-Johnson KJ, Master SR, Lee FS. Defective Tibetan PHD2 binding to p23 links high altitude adaption to altered oxygen sensing. J Biol Chem. 2014 May 23;289(21):14656-65. - doi: 10.1074/jbc.M113.541227.

26. Uribe-Querol E and Rosales C (2020) Phagocytosis: Our Current Understanding of a Universal Biological Process. Front. Immunol. 11:1066. - doi: 10.3389/fimmu.2020.01066

27. Wang JS, Chiu YT. Systemic hypoxia enhances exercise-mediated bactericidal and subsequent apoptotic responses in human neutrophils. J Appl Physiol (1985). 2009 Oct;107(4):1213-22. - doi: 10.1152/japplphysiol.00316.2009.

28. Xiu Q, Kong C, Gao Y, Gao Y, Sha J, Cui N, Zhu D. Hypoxia regulates IL-17A secretion from nasal polyp epithelial cells. Oncotarget. 2017 Oct 31;8(60):102097-102109. - doi: 10.18632/oncotarget.22189

29. Zagórska A, Dulak J. HIF-1: the knowns and unknowns of hypoxia sensing. Acta Biochim Pol. 2004;51(3):563-85. - PMID: 15448722.