METABOLIC ACTIVITY AND ENERGY SUPPLY OF PERIPHERAL BLOOD LYMPHOCYTES IN RESIDENTS OF THE EUROPEAN NORTH OF RUSSIA

Abstract

Introduction: T cells are able 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. The aim: to determine the metabolic activity and energy supply of peripheral blood lymphocytes in practically healthy northerners by measuring the intracellular content of HIF-1α, SIRT3 and ATP. Methods: 39 volunteers, residents of the Arkhangelsk region (23 women and 16 men, 23-62 years old), were examined. We determined 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 using enzyme immunoassay, the concentration of ATP using the luciferin-luciferase reaction. Statistical analysis was carried out 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: it was found that in the examined volunteers, the metabolic activity of lymphocytes associated with HIF-1α regulation differs statistically significantly, while in the group with a lower total number of lymphocytes and their phenotypes (CD3+, CD4+, CD8+, CD71+) there is a predominant glycolytic orientation of metabolism and a higher level of cells energy supply. Conclusion: metabolic activity, which can be judged by the HIF-1α/SIRT3 ratio, has a significant impact on the differentiation, proliferation, functioning, and fate of T cells.

Full Text

Introduction. Metabolic activity with the prevalence of certain pathways of ATP production (aerobic glycolysis, glutaminolysis, oxidative phosphorylation) determines the functioning and fate of T-cells, ensures the development of all stages of the T cell adaptive response: from response to the formation of memory cells and the exhaustion of the cells pool. To meet T-cell increased energy needs during the period of their differentiation and active proliferation, the so-called metabolic reprogramming occurs [4], which is characterized by increased absorption of nutrients, a high level of aerobic glycolysis, an increase in glutaminolysis, division and fusion of mitochondria [1, 8, 15]. During T cell expansion, redistribution of mitochondria into immune synapses and a local increase in ATP concentration are observed [2]. It is known that in order to maintain their functional state with minimal nutrient uptake, metabolically resting naive T cells mainly use the oxidative phosphorylation pathway (OXPHOS) in mitochondria, which is highly efficient in terms of ATP production [8]. In contrast, antigen-activated cells switch to maximum nutrient uptake with the activation of aerobic glycolysis [18], which is a faster, but less efficient than OXPHOS, way of producing ATP. Despite its less involvement in energy production, the role of mitochondria in activated T cells remains significant for ensuring the increased anabolic processes required during the period of growth and proliferation (synthesis of lipids, proteins, elements of the building blocks of macromolecules), for maintaining calcium homeostasis, and other [6, 20]. In addition, mitochondria are involved in metabolic rearrangement and coordinate T cell differentiation through key metabolites and metabolic by-products. Thus, the proliferative activity of T cells is stimulated by reactive oxygen species (ROS) formed during tissue respiration [7]. An increase in the mitochondrial pool is characteristic of memory T cells, which require high mitochondrial activity to function [14, 29].

The metabolic activity of T cells is controlled by intracellular signaling mechanisms. Among them are the regulation of the process of glycolysis with the help of hypoxia inducible factor 1 (HIF1) and the control of the work of mitochondria with the participation of the protein with deacetylase activity sirtuin 3 (SIRT3). For example, SIRT3 affects the permeability of the mitochondrial membrane, stimulates the operation of the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC), and increases the efficiency of ОХРНОS [9]. The HIF1 signaling contributes to an increase in the expression of proteins genes involved in glycolysis: membrane glucose transporters (Glut 1, 4) [24], many of glycolytic enzymes [13], monocarboxylate transporter 4 [28], which leads to an increase in the intensity of the glycolytic flow due to an increase of glucose in the cell, the rate of its oxidation to pyruvate, the conversion of pyruvate to lactate, and, finally, the release of lactate from the cell. With a negative effect on mitochondrial ATP production during OXPHOS, HIF1 has a positive effect on the plastic role of mitochondria during the proliferative activity of T cells [19, 27]. The mechanisms of regulation and variants of interaction of intracellular signaling pathways, the role of key metabolites and molecular sensors are currently being actively studied.

The aim of the study was to determine the metabolic activity and energy supply of peripheral blood lymphocytes in practically healthy northerners by measuring the intracellular content of HIF-1α, SIRT3 and ATP.

Materials and methods. The study participants were 39 volunteers, practically healthy residents of Arkhangelsk and the region, 16 men and 23 women (age from 23 to 62 years). All volunteers gave their voluntary consent to participate in the survey conducted in accordance with the requirements of the Helsinki Declaration of the World Medical Association on the ethical principles of medical research (2000). Volunteers venous blood was taken in the morning on an empty stomach, then the lymphocytic fraction of the blood was isolated, the content of differentiation antigens (CD) and the concentration of ATP, HIF-1α, SIRT3 in lymphocytes were determined.

CD typing of lymphocytes (CD3+, CD4+, CD8+, CD71+) was performed by indirect immunoperoxidase reaction (reagents from Sorbent LLC, Russia). The content of HIF-1α and SIRT3 was determined in the cell lysate of lymphocytes by the enzyme immunoassay method on an Evolis automatic analyzer manufactured by Bio-RAD (Germany). The ATP concentration was measured on a luminometer using the luciferin-luciferase reaction (reagent kit “Lumtek”, Russia).

Statistical processing of the study results was carried out using the Statistica 10.0 program (StatSoft, USA). Cluster groups were identified using the "K means" method in the "multivariate exploratory analysis" module. In the "descriptive statistics" module, mean values ​​(M), standard deviation (SD) were calculated. The Kolmogorov-Smirnov and Lilliefors normality test was used to check the data for normal distribution. With a distribution close to normal, the Student's t-test was calculated to compare the results, the differences were considered significant at p < 0.05. In the "graphics" module, 3D plots of surfaces (in the XYZ axes) and sequential 2D plots for several indicators were drawn.

 Results. The nature of fluctuations in the values of the determined indicators was defined with the help of 2D plots constructed sequentially and without overlap including all the determined cases (Fig. 1). The result of the overlay graphs shown in Figure 1 reflects the unidirectional fluctuations in the CD3+, CD4+, CD8+, CD71+ cells content and the concentrations of intracellular regulators (HIF-1α, SIRT3). The dependence of change in the number of T-cells with CD3, CD4, CD8, CD71 receptors on the level of metabolic regulators is visualized using statistical 3D plots of surfaces plotted in the XYZ axes (where X is the concentration of SIRT3, Y is the concentration of HIF-1α and Z is content of T cells, Fig. 2a–d). As can be seen from the graphs, the largest numbers of T cells with each of the defined differentiating antigens are observed at the highest concentrations of metabolic regulators, and the graph reflecting the content of CD4+ cells (Fig. 2b) has a shift in the height of the surface area towards the Y axis (HIF-1α), which indicates a pronounced dependence on the glycolytic pathway of metabolism.

The cluster analysis using the “K means” method made it possible to distinguish two groups among the examined people (the first group included 24, the second - 15 people), which had statistically significant differences in all determined immune parameters and HIF-1α, while no such differences were found for SIRT3 (table). It should be noted that in group 1, with statistically lower quantitative indicators of the lymphocyte pool compared to group 2, the calculated ratio of HIF-1α to SIRT3 was 1.9 times higher, as was the level of intracellular ATP. The latter averaged 3.71 (1.319) and 1.25 (0.387) µmol/106 cells (p < 0.0001) in groups 1 and 2, respectively. The relationship between the ratio of HIF-1α/SIRT3 and ATP is displayed as proportions on a pie chart. The analyzed values are displayed ​​on the second chart for accentuated visualization of the differences obtained between groups (Fig. 3). The illustration shows that the amount of ATP changes in accordance with a change in the ratio of HIF-1α/ IRT3 (increases or decreases).

The discussion of the results. The transcription factor HIF1 is the central regulator of the metabolic rearrangement which is the base to the formation of T-cell subpopulations [26]. HIF-1a is an oxygen-dependent subunit of HIF1 that is highly expressed and remains stable during hypoxia. The destruction of HIF-1a occurs in proteasomes under normoxic conditions because with enough oxygen, proline amino acid residues in the HIF-1a polypeptide chain are hydroxylated under the action of oxygen-sensitive prolyl-4-hydroxylase domain (PHD) and this contributes to ubiquitin-mediated proteasomal degradation of HIF-1a [12]. However, for immune cells, due to the specifics of their metabolism, hypoxia-independent production and stabilization of HIF-1a or the so-called “pseudo-hypoxia” is possible [19]. To varying degrees, HIF-1a expression is facilitated by stimulation of the T cell receptor (TCR), activation of the kinase cascade PI-3K/Akt/mTOR, STAT3 signaling, transcription factors NF-kB, AP-4, c-Myc. [10, 22, 25]. For example, mTOR regulates the translation of HIF-1a mRNA [21]. Stabilization of HIF-1a in activated T cells can be mediated through the metabolic regulation of PHD by succinate [16] and ROS [5]. In particular, the activity of the anaplerotic way of formation of succinate, which serves as an inhibitor of PHD, is increased in response to TCR stimulation. Also, succinate can be produced in a reaction catalyzed by PHD using α-ketoglutarate and inhibit PHD by the principle of negative feedback [12]. ROS can lead to PHD inactivation by oxidizing the iron atom in its active site [3]. ROS are very important for the reprogramming of activated T cells. Decrease in ROS or disruption of ROS signaling impairs T cell activation and clonal expansion [23], while ROS production stimulates T cell proliferation [7]. It has been established that, in concert with an increase in the level of ROS, the expression of HIF-1a increases, which is extremely important for metabolically active effector T cells that intensively use aerobic glycolysis [17, 26]

Enzymatic acetylation/deacetylation of proteins is one of the common ways of post-translational modification and influence on cellular metabolism. Deacetylases of the sirtuin family (SIRTs) are involved in the regulation of metabolism and contribute to the functioning and survival of cells [11]. SIRT3 positively regulates mitochondrial biogenesis through the activation of proliferation factors PPAR-α and PGC-1α; stimulates the TCA cycle by increasing the enzymatic activity of acetyl-CoA synthetase 2 and isocitrate dehydrogenase; increases OXPHOS, activating the work of complexes I, II, III ETS; promotes glutaminolysis, positively affecting glutamate dehydrogenase [9, 11]. At the same time, SIRT3 acts as a suppressor of the transcription factor HIF-1a, suppressing ROS-mediated stabilization of HIF-1a [9].

Our data show that the concentration of SIRT3 in peripheral blood lymphocytes did not have statistically significant differences in the groups identified by cluster analysis, while the content of HIF-1a differed statistically significantly. This reflects the difference in the metabolic activity of lymphocytes, namely, in the severity of glycolysis. An interesting fact is that a higher level of HIF-1a was observed in the group with lower values ​​of the lymphocyte pool, in particular cells with CD3, CD4, CD8, CD71 surface antigens (CD3 is a common T-cell activation marker associated with the antigen-recognition receptor , CD4 is a marker of helper cells, CD8 is a marker of cytotoxic cells, CD71 is a marker of activated cells, a transferrin receptor that ensures the entry of iron ions into the cell without which proliferation of T cells is impossible). An increase in the level of HIF-1a can be considered as a response to a decrease in the number of T cells, for which HIF1 acts as the main moderator of the metabolic shift upon their activation. The severity of glycolytic activity can be judged by an increase in the ratio of HIF-1a/SIRT3 which leads to an increase in the amount of ATP, providing T cells with the necessary energy level.

Conclusion. As a result of the study, it was found that even in a group of practically healthy people, intragroup statistically significant differences can be observed in the levels of interrelated indicators of the lymphocyte pool and lymphocytes metabolic activity. In particular, it was found that the group of examined patients with a lower total number of lymphocytes and the content of T-cells with CD3, CD4, CD8, CD71 receptors compared to the other group was characterized by a statistically significant higher level of the glycolysis regulator HIF-1α and did not have statistically significant differences by the level of SIRT3, which controls the work of mitochondria. The transcription factor HIF1 plays a very significant role for certain subpopulations of T cells, especially effector cells, facilitating metabolism with a predominance of glycolysis. Important in assessing the direction of metabolism is the change in the ratio of HIF-1α/SIRT3, the increase of which indicates the increase in glycolytic activity. The dominance of glycolysis in cell metabolism which is reflected in the level of cells energy supply has a significant impact on the differentiation, proliferation, functioning, and fate of T cells.

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About the authors

Olga Zubatkina

Author for correspondence.
Email: ozbiochem@gmail.com

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