Sergey Shirshev’s scientific contributions

During physiological pregnancy, the number of low-density neutrophils in the peripheral blood increases, but their phenotypic and functional features have not been practically studied. Therefore, the aim of this work is to study the expression of molecules characterizing cytotoxicity (CD16, CD107a), glucose transport (GLUT-1) and the state of mitochondria (by the inclusion of the MitoSpyGreen FM dye) by low-density granulocytes and in the general pool of granulocytes during physiologically proceeding pregnancy. The object of the study was the peripheral blood of conditionally healthy women in the first and third trimesters of physiological pregnancy. The comparison group consisted of conditionally healthy non-pregnant women in the follicular phase of the menstrual cycle. Low-density granulocytes were analyzed as a percentage of CD3-CD16+ cells in mononuclear leukocytes isolated on a ficolla-urographin density gradient (1.077 g/ml). The total granulocyte pool was studied as the percentage of CD3-CD16+ cells in the gate of peripheral blood granulocytes after removal of erythrocytes. Among granulocytes, subpopulations with high CD16 (CD16hi) and reduced CD16int expression were isolated. It was found that in non-pregnant women CD16hi granulocytes dominate both in the general pool and among low-density granulocytes. During pregnancy, the number of low-density granulocytes increases and CD16int granulocytes become the dominant subpopulation both in the general pool of granulocytes and among low-density granulocytes. In non-pregnant and pregnant women in the first trimester, CD16hi- and CD16int-granulocytes of low density and total pool are comparable in expression of GLUT-1, CD107a, mass and volume of mitochondria. In the third trimester, CD107a expression on low-density CD16int granulocytes and in the general pool decreases compared to non-pregnant ones, and the mass and volume of mitochondria increase in both CD16hi and CD16int granulocytes of the general pool only compared to the first trimester. The results obtained expand our understanding of the mechanisms regulating granulocyte functions during physiological pregnancy.

РЕЗЮМЕ Цель-оценить метаболическую активность разных субпопуляций натуральных киллеров (NK-клеток) периферической крови в I и в III триместрах физиологически протекающей беремен-ности (ФПБ). Материал и методы. В исследование включены здоровые женщины в I или III триместрах ФПБ. Группу сравнения составили условно-здоровые небеременные женщины в фолликулярной фазе менструального цикла. Объектом исследования являлась периферическая кровь. Метаболическую активность оценивали по экспрессии белка-транспортера глюкозы Glut-1 и включению митохондриального зонда Mito-Spy Green, отражающего массу и объем митохондриального компартмента, методом проточной цитофлюориметрии на регуляторных (CD56 bright CD16-), цитотоксических (CD56 dim CD16 +), минорных цитотоксических (CD56-CD16 hi) NK-клетках. Результаты и обсуждение. У небеременных количество метаболически активных клеток, экспрес-сирующих Glut-1 и имеющих большую массу и объем митохондрий (Mito +), значительно выше в субпопуляции цитотоксических CD56-CD16 hi NK-клеток, чем среди регуляторных CD56 bright CD16-и цитотоксических CD56 dim CD16 + NK-клеток. В I тримеcтре беременности уровень Glut-1 + Mito + ре-гуляторных CD56 bright CD16-и цитотоксических CD56 dim CD16 + NK-клеток значительно нарастает по сравнению с таковым у небеременных, тогда как процент цитотоксических Glut-1 + Mito + CD56-CD16 hi NK-клеток остается высоким, но не меняется. В III триместре количество Glut-1 + Mito + регу-ляторных CD56 bright CD16-NK-клеток остается выше, чем у небеременных. Количество цитотокси-ческих Glut-1 + Mito + CD56 dim CD16 + NK-клеток снижается по сравнению с таковым у небеременных, а Glut-1 + Mito + CD56-CD16 hi NK-по отношению к I триместру. Заключение. При ФПБ увеличивается количество метаболически активных цитотоксических и ре-гуляторных NK-клеток периферической крови. Ключевые слова: натуральные киллеры, беременность, Glut-1, масса митохондрий.

Objective: Congenital heart diseases (CHD) are often associated with thymus development disorders and immune dysfunctions. However, the features of thymocyte differentiation in cyanotic and acyanotic CHD remain unknown. Materials and Methods: We have analyzed the main thymocyte subsets depending on CD4 and CD8 co-expression, and the number of natural regulatory T-cells (nTreg) and invariant natural killer T-cells (iNKT) precursors in the co-cultures of thymocytes with thymic plasmacytoid (p) dendritic cells (DCs) in vitro, isolated from the thymus of children with acyanotic (without hypoxia) and cyanotic (with severe hypoxia) CHD. Results: In the thymocyte co-cultures with pDCs in cyanotic CHD, compared to acyanotic CHD, a decreased number of thymocytes expressing αβ chains of the T-cell receptor with CD4 and CD8 lower levels (CD4loCD8loαβTCR+), but the increased numbers of CD4hiCD8-/loαβTCR+ cells were detected. The numbers of CD4-CD8-αβTCR+, CD4hiCD8hiαβTCR+, CD4-/loCD8hiαβTCR+ cells did not differ between cyanotic or acyanotic CHD. In cyanotic CHD in the thymocyte co-cultures with pDCs, the decreased number of CD4+CD25+FOXP3+ cells, nTreg precursors, was detected in comparison with acyanotic CHD. In cyanotic CHD, the number of CD3hiVα24Jα18+ cells, iNKT precursors, in the thymocyte co-cultures with pDCs did not differ in comparison with acyanotic CHD. Hypoxia in cyanotic CHD increased the resistance to apoptosis of thymocytes in co-cultures with pDCs in comparison with acyanotic CHD. Conclusion: Thus, hypoxia affected the main CD4+ and CD8+ αβTCR T-cell subsets and the number of CD4+CD25+FOXP3+ cells in the thymocyte co-cultures with pDCs isolated from thymus of children with CHD.

Hypothalamic hormone kisspeptin is also produced by placental syncytiotrophoblast. Blood leukocytes express a specific membrane receptor of kisspeptin (KISS-1R). Since kisspeptin-54 enters the bloodstream during pregnancy, the hormone has a systemic effect on the leukocytes only in this period. Earlier we demonstrated that kisspeptin-54 enhances the generation of regulatory T (Treg) cells and suppresses the IL-17-producing T helper (Th17) cells. NK cells specialize into a regulatory subtype with reduced cytotoxic activity against fetal cells under the hormone influence. Kisspeptin-54 also regulates the maturation of thymic dendritic cells (DC). However, kisspeptin-54 effect on leukocytes functional activity has not been studied. Although it is known that these cells play an important role in ensuring fetus survival. The aim of the study was investigation of kisspeptin-54 effect in concentrations comparable to its level during physiological pregnancy on neutrophils and monocytes functional activity. Cells were isolated from peripheral blood of healthy non-pregnant women and cultured with kisspeptin-54 for 1 h. The monocytes and neutrophils phagocytic activity was determined by reducing bacterial bioluminescence. The production of reactive oxygen species (ROS) was evaluated in a luminol-dependent chemiluminescence test. The activity of myeloperoxidase (MPO) and indolamine-2,3-dioxygenase (IDO) was determined spectrophotometrically. It was revealed that kisspeptin-54 inhibits phagocytosis, MPO and ROS of neutrophils. On the contrary, the hormone activates phagocytosis, MPO activity, ROS production and IDO activity of monocytes. Thus, received data demonstrate that kisspeptin-54 is an important reproductive hormone that regulates leukocytes functional activity. The action of kisspeptin-54 depends on the type of cells expressing KISS-1 R.