Ekologiya cheloveka (Human Ecology)Ekologiya cheloveka (Human Ecology)Экология человека1728-08692949-1444Eco-Vector10936310.17816/humeco109363ORIGINAL STUDY ARTICLESОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯResearch ArticleAssessment of metabolic activity and energy supply of peripheral blood lymphocytesОценка метаболической активности и энергетической обеспеченности лимфоцитов периферической кровиhttps://orcid.org/0000-0002-5039-22201581-5178ZubatkinaOl'ga V.ЗубаткинаОльга Владимировна
Russian Federation

MD, Dr. Sci. (Biol.), professor

д.б.н., профессор

ozbiochem@gmail.comhttps://orcid.org/0000-0001-5080-65024518-6925DobrodeevaLilija K.ДобродееваЛилия Константиновна
Russian Federation
dobrodeevalk@mail.ruhttps://orcid.org/0000-0001-9835-80836469-0408SamodovaAnna V.СамодоваАнна Васильевна
Russian Federation
annapoletaeva2008@yandex.ruhttps://orcid.org/0000-0002-4085-409X2532-9912KruglovSergej D.КругловСергей Дмитриевич
Russian Federation
stees67@yandex.ruN. Laverov Federal Center for Integrated Arctic ResearchФедеральный исследовательский центр комплексного изучения Арктики имени академика Н.П. Лавёрова161220222912202229128558631507202225112022Copyright ©; 2022, Eco-VectorCopyright ©; 2022, Эко-Вектор2022Eco-VectorЭко-Векторhttps://creativecommons.org/licenses/by-nc-nd/4.0https://hum-ecol.ru/1728-0869/article/view/109363

BACKGROUND: T-cells have the capability to change their metabolism in response to activation signals. Resting T-cells primarily use the oxidation of higher fatty acids and oxidative phosphorylation in mitochondria for their energy needs, whereas activated T-cells switch to aerobic glycolysis and glutaminolysis, using glucose, and glutamine as substrates, respectively.

AIM: To determine the metabolic activity and energy supply of peripheral blood lymphocytes in predominantly healthy northerners by measuring the intracellular content of HIF-1α (hypoxia-induced factor 1α), SIRT3 (sirtuin 3), and ATP (adenosine triphosphate).

MATERIALS AND METHODS: 39 volunteers, residents of the Arkhangelsk region (23 women and 16 men, 23–62 years old), were selected, and examined for this experiment. We established the total number of peripheral blood lymphocytes with CD-typing of lymphocytes (CD3+, CD4+, CD8+, CD71+) by indirect immunoperoxidase reactions, the content of HIF-1α and SIRT3 in the lymphocyte lysate employing enzyme immunoassay, the concentration of ATP by luminescent analysis through luciferin-luciferase reaction. Statistical analysis was conducted in "Statistica 10.0", cluster analysis was applied using the k-means method. Mean values (M) and standard deviations (SD) were calculated, the normal distribution was tested by the Kolmogorov–Smirnov and Lilliefors criterion. Student's t-test was calculated, and the differences were considered statistically significant at p <0.05.

RESULTS: The study revealed that the metabolic activity of lymphocytes associated with HIF-1α regulation differs significantly in the examined volunteers,, while in the group with a lower total number of lymphocytes and their subpopulations (CD3+, CD4+, CD8+, CD71+) there is a predominant glycolytic orientation of metabolism with proliferated cells energy supply.

CONCLUSION: Metabolic activity which can be determined by the HIF-1α/SIRT3 ratio, and the energy supply of lymphocytes have a substantial impact on their differentiation, proliferation, and functioning.

Обоснование. Т-клетки способны изменять свой метаболизм в ответ на активационные сигналы. В состоянии покоя Т-клетки преимущественно используют для своих энергетических нужд окисление высших жирных кислот и окислительное фосфорилирование в митохондриях, а после активации переходят на аэробный гликолиз и глутаминолиз, используя в качестве субстратов глюкозу и глутамин соответственно.

Цель. Определение метаболической активности и энергообеспеченности лимфоцитов периферической крови у практически здоровых северян путём установления внутриклеточного содержания HIF-1α (гипоксией индуцируемого фактора 1-α), SIRT3 (сиртуина 3) и АТФ (аденозинтрифосфата).

Материалы и методы. Обследованы 39 волонтёров — жителей Архангельской области (23 женщины и 16 мужчин, возраст от 23 до 62 лет), у которых определяли общее количество лимфоцитов в периферической крови с проведением CD-типирования лимфоцитов (CD3+, CD4+, CD8+, CD71+) методом непрямой иммунопероксидазной реакции, в лизате лимфоцитов определяли содержание HIF-1α и SIRT3 методом иммуноферментного анализа, концентрацию АТФ методом люминесцентного анализа с использованием люциферин-люциферазной реакции. Статистическую обработку результатов исследования проводили в программе Statistica 10.0, применяли кластерный анализ с использованием метода k-средних, вычисляли средние значения (M), стандартное отклонение (SD); нормальность распределения оценивали по критерию Колмогорова–Смирнова и Лиллиефорса, вычисляли t-критерий Стьюдента, различия считали статистически значимыми при p <0,05.

Результаты. Установлено, что у обследованных волонтёров метаболическая активность лимфоцитов, связанная с HIF-1α-регуляцией, статистически значимо различается, при этом в группе с более низким общим количеством лимфоцитов и их субпопуляций (CD3+, CD4+, CD8+, CD71+) наблюдается преимущественная гликолитическая направленность метаболизма и более высокий уровень энергообеспеченности клеток.

Заключение. Метаболическая активность, о которой можно судить по соотношению HIF-1α/SIRT3, и энергетическая обеспеченность лимфоцитов оказывают существенное влияние на их дифференцировку, пролиферацию и функционирование.

lymphocytesimmunometabolismsirtuin 3hypoxia-induced factor 1αadenosine triphosphateлимфоцитыиммунометаболизмсиртуин 3гипоксией индуцируемый фактор 1αаденозинтрифосфатФИЦКИА имени академика Н.П. Лавёрова Уро РАНN. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences122011300377-5Chapman NM, Chi H. Hallmarks of T-cell exit from quiescence. Cancer Immunol Res. 2018;6(5):502–508. doi: 10.1158/2326-6066.CIR-17-0605Almeida L., Lochner M., Berod L., Sparwasser T. Metabolic pathways in T cell activation and lineage differentiation // Semin Immunol. 2016. Vol. 28, N 5. P. 514–524. doi: 10.1016/j.smim.2016.10.009Almeida L, Lochner M, Berod L, Sparwasser T. Metabolic pathways in T cell activation and lineage differentiation. Semin Immunol. 2016;28(5):514–524. doi: 10.1016/j.smim.2016.10.009Baixauli F., Martín-Cófreces N.B., Morlino G., et al. The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse // EMBO J. 2011. Vol. 30, N 7. P. 1238–1250. doi: 10.1038/emboj.2011.25Dimeloe S, Burgener AV, Grehlert J, Hess C. T-cell metabolism governing activation, proliferation and differentiation; a modular view. Immunology. 2017;150(1):35–44. doi: 10.1111/imm.12655Chandel N.S., McClintock D.S., Feliciano C.E., et al. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia inducible factor-1alpha during hypoxia: a mechanism of O2 sensing // J Biol Chem. 2000. Vol. 275, N 33. P. 25130–25138. doi: 10.1074/jbc.M001914200Maciolek JA, Pasternak JA, Wilson HL. Metabolism of activated T lymphocytes. Curr Opin Immunol. 2014;27:60–74. doi: 10.1016/j.coi.2014.01.006Chapman N.M., Chi H. Hallmarks of T-cell exit from quiescence // Cancer Immunol Res. 2018. Vol. 6, N 5. P. 502–508. doi: 10.1158/2326-6066.CIR-17-0605Baixauli F, Martín-Cófreces NB, Morlino G, et al. The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse. EMBO J. 2011;30(7):1238–1250. doi: 10.1038/emboj.2011.25Chua Y.L., Dufour E., Dassa E.P., et al. Stabilization of hypoxia-inducible factor-1alpha protein in hypoxia occurs independently of mitochondrial reactive oxygen species production // J Biol Chem. 2010. Vol. 285, N 41. P. 31277–31284. doi: 10.1074/jbc.M110.158485Palmer CS, Ostrowski M, Balderson B, et al. Glucose metabolism regulates T cell activation, differentiation, and functions. Front Immunol. 2015;6:1. doi: 10.3389/fimmu.2015.00001Desdín-Micó G., Soto-Heredero G., Mittelbrunn M. Mitochondrial activity in T cell // Mitochondrion. 2018. Vol. 41. P. 51–57. doi: 10.1016/j.mito.2017.10.006Desdín-Micó G, Soto-Heredero G, Mittelbrunn M. Mitochondrial activity in T cell. Mitochondrion. 2018;41:51–57. doi: 10.1016/j.mito.2017.10.006Diebold L., Chandel N.S. Mitochondrial ROS regulation of proliferating cells // Free Radic Biol Med. 2016. Vol. 100. P. 86–93. doi: 10.1016/j.freeradbiomed.2016.04.198Ron-Harel N, Santos D, Ghergurovich JM, et al. Mitochondrial biogenesis and proteome remodeling promote one-carbon metabolism for T cell activation. Cell Metab. 2016;24(1):104–117. doi: 10.1016/j.cmet.2016.06.007Dimeloe S., Burgener A.V., Grehlert J., Hess C. T-cell metabolism governing activation, proliferation and differentiation; a modular view // Immunology. 2017. Vol. 150, N 1. P. 35–44. doi: 10.1111/imm.12655Diebold L, Chandel NS. Mitochondrial ROS regulation of proliferating cells. Free Radic Biol Med. 2016;100:86–93. doi: 10.1016/j.freeradbiomed.2016.04.198Giralt A., Villarroya F. SIRT3, a pivotal actor in mitochondrial functions: metabolism, cell death and aging // Biochem J. 2012. Vol. 444, N 1. P. 1–10. doi: 10.1042/BJ20120030Liesa M, Shirihai OS. Mitochondrial networking in T cell memory. Cell. 2016;166(1):9–10. doi: 10.1016/j.cell.2016.06.035Gnanaprakasam J.N.R., Sherman J.W., Wang R. MYC and HIF in shaping immune response and immune metabolism // Cytokine Growth Factor Rev. 2017. Vol. 35. P. 63–67. doi: 10.1016/j.cytogfr.2017.03.004van der Windt GJ, Everts B, Chang CH, et al. Mitochondrial respiratory capacity is a critical regulator of CD8+T cell memory development. Immunity. 2012;36(1):68–78. doi: 10.1016/j.immuni.2011.12.007Houtkooper R.H., Pirinen E., Auwerx J. Sirtuins as regulators of metabolism and healthspan // Nat Rev Mol Cell Biol. 2016. Vol. 13, N 4. P. 225–238. doi: 10.1038/nrm3293Giralt A, Villarroya F. SIRT3, a pivotal actor in mitochondrial functions: metabolism, cell death and aging. Biochem J. 2012;444(1):1–10. doi: 10.1042/BJ20120030Jaakkola P., Mole D.R., Tian Y.M., et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation // Science. 2001. Vol. 292, N 5516. P. 468–472. doi: 10.1126/science.1059796Shi LZ, Wang R, Huang G, et al. HIF-1alpha dependent glycolytic pathway orchestrates a metabolic checkpoint for the differentiation of TH17 and Treg cells. J Exp Med. 2011;208(7):1367–1376. doi: 10.1084/jem.20110278Kim J.W., Tchernyshyov I., Semenza G.L., Dang C.V. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia // Cell Metab. 2006. Vol. 3, N 3. P. 177–185. doi: 10.1016/j.cmet.2006.02.002Kim JW, Tchernyshyov I, Semenza GL, Dang CV. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab. 2006;3(3):177–185. doi: 10.1016/j.cmet.2006.02.002Liesa M., Shirihai O.S. Mitochondrial networking in T cell memory // Cell. 2016. Vol. 166, N 1. P. 9–10. doi: 10.1016/j.cell.2016.06.035Ullah MS, Davies AJ, Halestrap AP. The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1alpha-dependent mechanism. J Biol Chem. 2006;281(14):9030–9037. doi: 10.1074/jbc.M511397200Maciolek J.A., Pasternak J.A., Wilson H.L. Metabolism of activated T lymphocytes // Curr Opin Immunol. 2014. Vol. 27. P. 60–74. doi: 10.1016/j.coi.2014.01.006Pugha CW, Ratcliffe PJ. New horizons in hypoxia signaling pathways. Exp Cell Res. 2017;356(2):116–121. doi: 10.1016/j.yexcr.2017.03.008Mills E., O'Neill L.A. Succinate: a metabolic signal in inflammation // Trends Cell Biol. 2014. Vol. 24, N 5. P. 313–320. doi: 10.1016/j.tcb.2013.11.008Thomas LW, Ashcroft M. Exploring the molecular interface between hypoxia-inducible factor signalling and mitochondria. Cell Mol Life Sci. 2019;76(9):1759–1777. doi: 10.1007/s00018-019-03039-yPalmer C.S., Hussain T., Duette G. Regulators of glucose metabolism in CD4+ and CD8+ T cells // Int Rev Immunol. 2016. Vol. 35, N 6. P. 477–488. doi: 10.3109/08830185.2015.1082178Tao JH, Barbi J, Pan F. Hypoxia-inducible factors in T lymphocyte differentiation and function. Am J Physiol Cell Physiol. 2015;309(9):C580–C589. doi: 10.1152/ajpcell.00204.2015Palmer C.S., Ostrowski M., Balderson B., et al. Glucose metabolism regulates T cell activation, differentiation, and functions // Front Immunol. 2015. Vol. 6. P. 1. doi: 10.3389/fimmu.2015.00001Jaakkola P, Mole DR, Tian YM, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 2001;292(5516):468–472. doi: 10.1126/science.1059796Pugha C.W., Ratcliffe P.J. New horizons in hypoxia signaling pathways // Exp Cell Research. 2017. Vol. 356, N 2. P. 116–121. doi: 10.1016/j.yexcr.2017.03.008Gnanaprakasam JNR, Sherman JW, Wang R. MYC and HIF in shaping immune response and immune metabolism. Cytokine Growth Factor Rev. 2017;35:63–67. doi: 10.1016/j.cytogfr.2017.03.004Ron-Harel N., Santos D., Ghergurovich J.M., et al. Mitochondrial biogenesis and proteome remodeling promote one-carbon metabolism for T cell activation // Cell Metab. 2016. Vol. 24, N 1. P. 104–117. doi: 10.1016/j.cmet.2016.06.007Saravia J, Raynor JL, Chapman NM, et al. Signaling networks in immunometabolism. Cell Res. 2020;30(4):328–342. doi: 10.1038/s41422-020-0301-1Salmond R.J. mTOR regulation of glycolytic metabolism in T cells // Front Cell Dev Biol. 2018. Vol. 6. P. 122. doi: 10.3389/fcell.2018.00122Tan H, Yang K, Li Y, et al. Integrative proteomics and phosphoproteomics profiling reveals dynamic signaling networks and bioenergetics pathways underlying T cell activation. Immunity. 2017;46(3):488–503. doi: 10.1016/j.immuni.2017.02.010Saravia J., Raynor J.L., Chapman N.M., et al. Signaling networks in immunometabolism // Cell Res. 2020. Vol. 30, N 4. P. 328–342. doi: 10.1038/s41422-020-0301-1Salmond RJ. mTOR regulation of glycolytic metabolism in T cells. Front Cell Dev Biol. 2018:6;122. doi: 10.3389/fcell.2018.00122Sena L.A., Li S., Jairaman A., et al. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling // Immunity. 2013. Vol. 38, N 2. P. 225–236. doi: 10.1016/j.immuni.2012.10.020Mills E, O'Neill LA. Succinate: a metabolic signal in inflammation. Trends Cell Biol. 2014;24(5):313–320. doi: 10.1016/j.tcb.2013.11.008Shi L.Z., Wang R., Huang G., et al. HIF-1alpha dependent glycolytic pathway orchestrates a metabolic checkpoint for the differentiation of TH17 and Treg cells // J Exp Med. 2011. Vol. 208, N 7. P. 1367–1376. doi: 10.1084/jem.20110278Chua YL, Dufour E, Dassa EP, et al. Stabilization of hypoxia-inducible factor-1alpha protein in hypoxia occurs independently of mitochondrial reactive oxygen species production. J Biol Chem. 2010;285(41):31277–31284. doi: 10.1074/jbc.M110.158485Tan H., Yang K., Li Y., et al. Integrative proteomics and phosphoproteomics profiling reveals dynamic signaling networks and bioenergetics pathways underlying T cell activation // Immunity. 2017. Vol. 46, N 3. P. 488–503. doi: 10.1016/j.immuni.2017.02.010Chandel NS, McClintock DS, Feliciano CE, et al. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia inducible factor-1alpha during hypoxia: a mechanism of O2 sensing. J Biol Chem. 2000;275(33):25130–25138. doi: 10.1074/jbc.M001914200Tao J.H., Barbi J., Pan F. Hypoxia-inducible factors in T lymphocyte differentiation and function // Am J Physiol Cell Physiol. 2015. Vol. 309, N 9. P. C580–C589. doi: 10.1152/ajpcell.00204.2015Sena LA, Li S, Jairaman A, et al. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling. Immunity. 2013;38(2):225–236. doi: 10.1016/j.immuni.2012.10.020Thomas L.W., Ashcroft M. Exploring the molecular interface between hypoxia-inducible factor signalling and mitochondria // Cell Mol Life Sci. 2019. Vol. 76, N 9. P. 1759–1777. doi: 10.1007/s00018-019-03039-yPalmer CS, Hussain T, Duette G. Regulators of glucose metabolism in CD4+ and CD8+ T Cells. Int Rev Immunol. 2016;35(6):477–488. doi: 10.3109/08830185.2015.1082178Ullah M.S., Davies A.J., Halestrap A.P. The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1alpha-dependent mechanism // J Biol Chem. 2006. Vol. 281, N 14. P. 9030–9037. doi: 10.1074/jbc.M511397200Houtkooper RH, Pirinen E, Auwerx J. Sirtuins as regulators of metabolism and healthspan. Nat Rev Mol Cell Biol. 2016;13(4):225–238. doi: 10.1038/nrm3293van der Windt G.J., Everts B., Chang C.H, et al. Mitochondrial respiratory capacity is a critical regulator of CD8+T cell memory development // Immunity. 2012. Vol. 36, N 1. P. 68–78. doi: 10.1016/j.immuni.2011.12.007