MORPHO-FUNCTIONAL CHANGES OF THYMUS AND CONTENTS OF BLOOD LYMPHOCYTE SUBPOPULATIONS IN FEMALE WISTAR RATS WITH DIFFERENT RESISTANCE TO HYPOXIA IN SYSTEMIC INFLAMMATORY RESPONSE

Hypoxia and immune reactions are closely interrelated at molecular, cellular and organism levels, and the individuals differ in resistance to oxygen deficiency. Animals with high and low resistance to hypoxia have different adaptive capabilities and predisposition to the development of inflammatory diseases. Data on the individual characteristics of hypoxia resistance in female laboratory animals and humans, and its relationship to immune system reactions in both normal conditions and inflammatory diseases are not available in the literature. It is known, however, that acute infectious and inflammatory diseases develop at lesser rates and are less severe in women and female laboratory animals than in males, which can be explained by higher resistance of females to hypoxia. The aim of our study is to reveal the features of morpho-functional thymus changes, and subpopulation of peripheral blood lymphocytes in systemic inflammatory response induced by LPS administration to female Wistar rats with different resistance to hypoxia. 

Resistance of mature female Wistar rats to hypoxia was determined as a survival period in a ventilated lowpressure chamber simulating high altitude condition (11 500 m). The rats with a lifetime “at high altitude” of > 180 s have been classified as highly resistant to hypoxia, and the animals surviving for < 20 seconds were designated low-resistant. One month after determining the hypoxia resistance, the females were injected intraperitoneally with E. coli O26:B6 lipopolysaccharide (LPS) at a dose of 1.5 mg/kg during the dioestrus phase. The animals were withdrawn from the experiment by i/m Zoletyl injection (15 mg/kg) one day after LPS administration. The relative volume fractions of thymic cortex and medulla were evaluated; the areas of necrosis were determined in the liver, and the number of neutrophils in the interalveolar septa was counted in the lungs. The serum contents of corticosterone, testosterone, TGF-β were determined. A flow cytometry evaluation of the relative and absolute numbers was performed for major subpopulations of lymphocytes in peripheral blood. The number of apoptotically dying cells of the thymus was assessed. For statistical processing of the obtained data, the Statistica 8.0 software was applied, using criteria of multiple comparisons by Kruskal–Wallis and Dann. The differences were considered statistically significant at p < 0.05.

In both high- and low-resistant to hypoxia females, the development of a systemic inflammatory -response was accompanied by a moderately severe thymic involution, apoptosis of thymocytes, an increase in the absolute number of NK, and rise of testosterone and corticosterone contents. LPS injection into low-resistant rats, if compared to females highly resistant to hypoxia, led to more severe manifestations of systemic inflammation, i.e., a pronounced inflammatory reaction in the lungs and a more extensive liver necrotic area accompanied by increased absolute numbers of regulatory T lymphocytes and T helper cells, and more pronounced thymic accidental involution with apoptotic death of thymocytes. Systemic manifestations of inflammation were less pronounced in hypoxia-resistant female rats, which was apparently associated with activation of lymphocyte  migration from the thymus and blood to the inflammation focus, and development of more effective immune response.

Conclusion: immune reactions in the systemic inflammatory response induced by LPS in female Wistar rats depend on individual resistance to hypoxia. These data should be used to develop approaches to personalized therapy of infectious and inflammatory diseases in women.

1. Shalova I.N., Lim J.Y., Chittezhath M., Zinkernagel A.S., Beasley F., Hernández-Jiménez E., Toledano V., Cubillos-Zapata C., Rapisarda A., Chen J., Duan K., Yang H., Poidinger M., Melillo G., Nizet V., Arnalich F., López-Collazo E., Biswas S.K. Human monocytes undergo functional re-programming during sepsis mediated by hypoxiainducible factor-1α. Immunity, 2015, Vol. 42, no. 3, pp. 484-498.

2. Avrushchenko M.S., Ostrova I.V., Grechko A.V. Gender peculiarities of postresuscitation in the expression of brain-derived neurotrophic factor (BDNF). Obshchaya reanimatologiya = General Reanimatology, 2017, Vol. 13, no. 5, pp. 44-57. (In Russ.)

3. Tatura R., Zeschnigk M., Adamzik M., Probst-Kepper M., Buer J., Kehrmann J. Quantification of regulatory T cells in septic patients by real-time PCR-based methylation assay and flow cytometry. PLoS ONE, 2012, Vol. 7, e49962. doi: 10.1371/journal.pone.0049962.

4. Gao Yu., Postovalova E.A., Dobrynina M.T., Makarova O.V. Sex-related differences in morphological changes and immune disorders during experimental acute ulcerative colitis. Klinicheskaya i eksperimentalnaya morfologiya = Clinical and Experimental Morphology, 2016, Vol. 17, no. 1, pp. 37-43. (In Russ.)

5. Tatura R., Zeschnigk M., Hansen W., Steinmann J., Vidigal P.G., Hutzler M., Pastille E., Westendorf A.M., Buer J., Kehrmann J. Relevance of Foxp3+ regulatory T cells for early and late phases of murine sepsis. Immunology, 2015, Vol. 146, no. 1, pp. 144-156.

6. Dzhalilova D.Sh., Diatroptov M.E., Tsvetkov I.S., Makarova O.V., Kuznetsov S.L. Expression of hif-1a, nf-kb and vegf genes in the liver and the content of HIF-1α, erythropoietin, VEGF, TGF-β, 8-isoprostane and corticosterone in the blood serum in tolerant and susceptible to hypoxia Wistar rats. Byulleten eksperimentalnoy biologii i meditsiny = Bulletin of Experimental Biology and Medicine, 2018, Vol. 165, no. 6, pp. 742-747. (In Russ.)

7. Vázquez-Martínez E.R., García-Gómez E., Camacho-Arroyo I., González-Pedrajo B. Sexual dimorphism in bacterial infections. Biol. Sex Differ., 2018, Vol. 9, no. 1, p. 27.

8. Kirova Yu.I., Germanova E.L., Lukyanova L.D. Phenotypic features of the dynamics of HIF-1α levels in rat neocortex in different hypoxia regimens. Byulleten eksperimentalnoy biologii i meditsiny = Bulletin of Experimental Biology and Medicine, 2012, Vol. 154, no. 12, pp. 681-686. (In Russ.)

9. Venet F., Chung C.S., Kherouf H., Geeraert A., Malcus C., Poitevin F., Bohe J., Lepape A., Avala A., Monneret G. Increased circulating regulatory T cells (CD4+ CD25+ CD127-) contribute to lymphocyte anergy in septic shock patients. Intensive Care Med., 2009, Vol. 35, no. 4, pp. 678-686.

10. Kosyreva A.M., Simonova E.Yu., Makarova O.V. Gender differences in pulmonary and immune response in acute expeimental endotoxicosis. Byulleten eksperimentalnoy biologii i meditsiny = Bulletin of Experimental Biology and Medicine, 2012, Vol. 153, no. 3, pp. 318-321. (In Russ.)

11. Yang L., Pang Y., Moses H.L. TGF-beta and immune cells: an important regulatory axis in the tumor microenvironment and progression. Trends Immunol., 2010, Vol. 31, pp. 220-227.

12. Kosyreva A.M., Diatroptov M.E. Morphological development of systemic inflammatory response syndrome in the liver and lung of Wistar rats in the different stages of estrous cycle. Immunologiya = Immunology, 2013, Vol. 34, no. 2, pp. 111-114. (In Russ.)

13.

14. Lukyanova L.D., Kirova Yu.I. Effect of hypoxic preconditioning on free radical processes in tissues of rats with different resistance to hypoxia. Byulleten eksperimentalnoy biologii i meditsiny = Bulletin of Experimental Biology and Medicine, 2011, Vol. 151, no. 3, pp. 263- 268. (In Russ.)

15. Pisarev V.B., Novochadov V.V., Bogomolova N.V. Bacterial endotoxicosis: pathologist’s view. Volgograd, 2008. 308 p.

16. Ceyran A.B., Şenol S., Güzelmeriç F., Tunçer E., Tongut A., Özbek B., Şavluk O., Aydın A., Ceyran H. Effects of hypoxia and its relationship with apoptosis, stem cells, and angiogenesis on the thymus of children with congenital heart defects: a morphological and immunohistochemical study. Int. J. Clin. Exp. Pathol., 2015, Vol. 8, no. 7, pp. 8038-8047.

17. Clambey E.T., McNamee E.N., Westrich J.A., Glover L.E., Campbell E.L., Jedlicka P., de Zoeten E.F., Cambier J.C., Stenmark K.R., Colgan S.P., Eltzschig H.K. Hypoxia-inducible factor-1-dependent induction of FoxP3 drives regulatory T-cell abundance and function during inflammatory hypoxia of the mucosa. Proc. Natl. Acad. Sci. USA, 2012, Vol. 109, pp. E2784-E2793.

18. Cinel I., Opal S.M. Molecular biology of inflammation and sepsis: a primer. Crit. Care Med., 2009, Vol. 37, no. 1, pp. 291-304.

19. Cummins E.P., Berra E., Comerford K.M., Ginouves A., Fitzgerald K.T., Seeballuck F., Godson C., Nielsen J.E., Moynagh P., Pouyssegur J., Taylor C.T. Prolyl hydroxylase-1 negatively regulates IkappaB kinase-beta, giving insight into hypoxia-induced NFkappaB activity. Proc. Natl. Acad. Sci. USA, 2006, Vol. 103, no. 48, pp. 18154-18159.

20. Jain K., Suryakumar G., Prasad R., Ganju L. Upregulation of cytoprotective defense mechanisms and hypoxia-responsive proteins imparts tolerance to acute hypobaric hypoxia. High Alt. Med. Biol., 2013, Vol. 14, no. 1, pp. 65-77.

21. Kiers H.D., Scheffer G.J., van der Hoeven J.G., Eltzschig H.K., Pickkers P., Kox M. Immunologic consequences of hypoxia during critical illness. Anesthesiology, 2016, Vol. 125, no. 1, pp. 237-249.

22. Krzywinska E., Stockmann C. Hypoxia, Metabolism and immune cell function. Biomedicines, 2018, Vol. 6, no. 2, p. 56.

23. Kubiczkova L., Sedlarikova L., Hajek R., Sevcikova S. TGF-β – an excellent servant but a bad master. J. Transl. Med., 2012, Vol. 10, p. 183.

24. Oliver K.M., Taylor C.T., Cummins E.P. Hypoxia. Regulation of NFkappaB signalling during inflammation: the role of hydroxylases. Arthritis Res. Ther., 2009, Vol. 11, no. 1, p. 215.

25. Peyssonnaux C., Cejudo-Martin P., Doedens A., Zinkernagel A.S., Johnson R.S., Nizet V. Cutting edge: Essential role of hypoxia inducible factor-1alpha in development of lipopolysaccharide-induced sepsis. J. Immunol., 2007, Vol. 178, no. 12, pp. 7516-7519.

26. Rubtsov Y.P., Rasmussen J.P., Chi E.Y., Fontenot J., Castelli L., Ye X., Treuting P., Siewe L., Roers A., Henderson W.R., Muller W., Rudensky A.Y. Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces. Immunity, 2008, Vol. 28, no. 4, pp. 546-558.

27. Sceneay J., Chow M.T., Chen A., Halse H.M., Wong C.S.F., Andrews D.M., Sloan E.K., Parker B.S., Bowtell D.D., Smyth M.J., Möller A. Primary tumor hypoxia recruits CD11b+/Ly6Cmed/Ly6G+ immune suppressor cells and compromises NK cell cytotoxicity in the premetastatic niche. Cancer Res., 2012, Vol. 72, no. 16, pp. 3906- 3911.

28. Shalova I.N., Lim J.Y., Chittezhath M., Zinkernagel A.S., Beasley F., Hernández-Jiménez E., Toledano V., Cubillos-Zapata C., Rapisarda A., Chen J., Duan K., Yang H., Poidinger M., Melillo G., Nizet V., Arnalich F., López-Collazo E., Biswas S.K. Human monocytes undergo functional re-programming during sepsis mediated by hypoxiainducible factor-1α. Immunity, 2015, Vol. 42, no. 3, pp. 484-498.

29. Tatura R., Zeschnigk M., Adamzik M., Probst-Kepper M., Buer J., Kehrmann J. Quantification of regulatory T cells in septic patients by real-time PCR-based methylation assay and flow cytometry. PLoS ONE, 2012, Vol. 7, e49962. doi: 10.1371/journal.pone.0049962.

30. Tatura R., Zeschnigk M., Hansen W., Steinmann J., Vidigal P.G., Hutzler M., Pastille E., Westendorf A.M., Buer J., Kehrmann J. Relevance of Foxp3+ regulatory T cells for early and late phases of murine sepsis. Immunology, 2015, Vol. 146, no. 1, pp. 144-156.

31. Vázquez-Martínez E.R., García-Gómez E., Camacho-Arroyo I., González-Pedrajo B. Sexual dimorphism in bacterial infections. Biol. Sex Differ., 2018, Vol. 9, no. 1, p. 27.

32. Venet F., Chung C.S., Kherouf H., Geeraert A., Malcus C., Poitevin F., Bohe J., Lepape A., Avala A., Monneret G. Increased circulating regulatory T cells (CD4+ CD25+ CD127-) contribute to lymphocyte anergy in septic shock patients. Intensive Care Med., 2009, Vol. 35, no. 4, pp. 678-686.

33. Yang L., Pang Y., Moses H.L. TGF-beta and immune cells: an important regulatory axis in the tumor microenvironment and progression. Trends Immunol., 2010, Vol. 31, pp. 220-227.

34.