РОЛЬ ГЛИКОДЕЛИНА В РЕГУЛЯЦИИ ДИФФЕРЕНЦИРОВКИ МИЕЛОИДНЫХ СУПРЕССОРНЫХ КЛЕТОК Заморина

. Glycodelin (PP14, PAEP, alpha-2-microglobulin, dimeric glycoprotein with molecular weight of 42 to 56 kDa) is considered as a reproductive tissue receptivity marker. Despite that glycodelin immunosuppressive effects are well-known there still remains uncovered its role in myeloid suppressor cell (MDSC) regulation. MDSC represent the heterogeneous population of immature myeloid cells that acquire suppressor phenotype while inhibiting the immune response under the pathological states. MDSC are known to play an essential role in supporting the immune tolerance in pregnancy and at transplantation. Our hypothesis suggests that glycodelin is capable of inducing the MDSC formation as the level of these cells is elevated during the successful pregnancy, whereas the spontaneous abortion and progression of eclampsia are associated with low circulating glycodelin. Therefore, the aim of the work was to analyze the role of recombinant glycodelin in physiological concentrations in regulation of MDSC differentiation. Peripheral blood mononuclear cells of donor volunteers were separated via centrifugation on density gradient of 1,077 g/cm 3 (Ficoll-Hypaque, Sigma-Aldrich) to obtain MDSC generation in vitro . Then cells obtained were cultured in 24-well plate at a concentration of 1 × 10 6 cell/ ml in complete medium with cytokines IL-6 (20 ng/ml), GM-CSF (40 ng/ ml) therein for 14 days at 37 °C and 5% CO 2 . Medium replacement was made by 7 th day in culture followed by cytokine re-introduction, and on the 11 th day recombinant glycodelin in physiological concentrations (0,2; 2 mkg/ml) was applied while the pharmacological concentration was 50 mkg/ml. The M-MDSC (Lin HLA- DR - CD33 + CD11b + CD14 + CD66b - ) and PMN-MDSC (Lin - HLA-DR - CD33 + CD11b + CD14 - CD66b + ) level was not render significant effect on MDSC differentiation. Meanwhile, glycodelin in concentrations correspanding the healthy pregnancy (0,2; 2 mkg/ml) was stated to increase the MDSC percentage in induced cultures of human mononuclear cells. When analyzing the subsets it was disclosed that this effect was conditioned by the increase in PMN-MDSC level while the M-MDSC level remained significantly unchanged. This result could be interpreted as glycodelin fetoprotective effect as the increase of the PMN-MDSC level is associated with the suppression of the immune response to paternal antigens. The PMN-MDSC level is known to be elevated in peripheral blood of healthy pregnant women at all the stages of pregnancy as compared to non-pregnant subjects whereas the M-MDSC amount remains unaltered. Meanwhile, patients with miscarriage demonstrated more that by 30% lowering in the MDSC amount in blood and endometrium and in I trimester, in particular. During the physiological pregnancy PMN-MDSC accumulate in placenta, but at spontaneous abortion their number is found to be declined. Placental PMN-MDSC efficiently suppress the T-cell response while concurrently polarizing the CD4 + lymphocytes in Th2 phenotype. PMN-MDSC are suggested to play an essential role in inducing and supporting the tolerance to fetal antigens that allows considering these as promising target of therapeutical manipulation in pregnancy complications. As a whole, we have originally demonstrated the GdA effect on MDSC differentiation.

В то же время у пациенток с выкидышем более чем на 30% снижено количество MDSC в крови и эндометрии, особенно в первом триместре. В период физиологической беременности PMN-MDSC накапливаются в плаценте, но при спонтанном аборте их количество уменьшается. Плацентарные PMN-MDSC эффективно подавляют Т-клеточный ответ, одновременно поляризуя CD4 + -лимфоциты в фенотип Th2. Вероятно, PMN-MDSC играют важную роль в индуцировании и поддержании толерантности к антигенам плода, что позволяет рассматривать их как перспективную мишень терапевтического манипулирования при осложнениях беременности. В целом, мы впервые продемонстрировали влияние GdA на процесс дифференцировки MDSC. Abstract. Glycodelin (PP14, PAEP, alpha-2-microglobulin, dimeric glycoprotein with molecular weight of 42 to 56 kDa) is considered as a reproductive tissue receptivity marker. Despite that glycodelin immunosuppressive effects are well-known there still remains uncovered its role in myeloid suppressor cell (MDSC) regulation. MDSC represent the heterogeneous population of immature myeloid cells that acquire suppressor phenotype while inhibiting the immune response under the pathological states. MDSC are known to play an essential role in supporting the immune tolerance in pregnancy and at transplantation. Our hypothesis suggests that glycodelin is capable of inducing the MDSC formation as the level of these cells is elevated during the successful pregnancy, whereas the spontaneous abortion and progression of eclampsia are associated with low circulating glycodelin. Therefore, the aim of the work was to analyze the role of recombinant glycodelin in physiological concentrations in regulation of MDSC differentiation. Peripheral blood mononuclear cells of donor volunteers were separated via centrifugation on density gradient of 1,077 g/cm 3 (Ficoll-Hypaque, Sigma-Aldrich) to obtain MDSC generation in vitro. Then cells obtained were cultured in 24-well plate at a concentration of 1 × 10 6 cell/ ml in complete medium with cytokines IL-6 (20 ng/ml), GM-CSF (40 ng/ ml) therein for 14 days at 37 °C and 5% CO 2 . Medium replacement was made by 7 th day in culture followed by cytokine re-introduction, and on the 11 th day recombinant glycodelin in physiological concentrations (0,2; 2 mkg/ml) was applied while the pharmacological concentration was 50 mkg/ml. The M-MDSC (Lin -HLA-DR -CD33 + CD11b + CD14 + CD66b -) and PMN-MDSC (Lin -HLA-DR -CD33 + CD11b + CD14 -CD66b + ) level was evaluated in cultures using flow cytometry (СytoFlexS (Beckman Coulter)) and "R&D Systems" antibodies according to standard protocol. Statistical data processing was realized with GraphPad Prizm software using Friedman test. It was found that glycodelin did not significantly affect cell viability being assessed with flow cytometry (PI). It was revealed that high GdA concentration (50 mkg/ml) being pharmacological did not render significant effect on MDSC differentiation. Meanwhile, glycodelin in concentrations correspanding the healthy pregnancy (0,2; 2 mkg/ml) was stated to increase the MDSC percentage in induced cultures of human mononuclear cells. When analyzing the subsets it was disclosed that this effect was conditioned by the increase in PMN-MDSC level while the M-MDSC level remained significantly unchanged. This result could be interpreted as glycodelin fetoprotective effect as the increase of the PMN-MDSC level is associated with the suppression of the immune response to paternal antigens. The PMN-MDSC level is known to be elevated in peripheral blood of healthy pregnant women at all the stages of pregnancy as compared to nonpregnant subjects whereas the M-MDSC amount remains unaltered. Meanwhile, patients with miscarriage demonstrated more that by 30% lowering in the MDSC amount in blood and endometrium and in I trimester, in particular. During the physiological pregnancy PMN-MDSC accumulate in placenta, but at spontaneous abortion their number is found to be declined. Placental PMN-MDSC efficiently suppress the T-cell response while concurrently polarizing the CD4 + lymphocytes in Th2 phenotype. PMN-MDSC are suggested to play an essential role in inducing and supporting the tolerance to fetal antigens that allows considering these as

Introduction
In 1976, D.D. Petrunin, Ju.S. Tatarinov et al. initially isolated and identified glycodelin as a novel placental antigen that was called chorionic α2-microglobulin [8]. Subsequently, while accumulating new data on the protein location and properties, its name changed from placental α2-microglobulin, fertility α2-microglobulin (AMGF), and finally to specific α2-microglobulin.
Glycodelin (GdA) is considered as the receptivity marker for reproductive tissue. In 2018, glycodelin was found to prevent experimental graft rejection [2] that favors its pharmacological application. Schneider et al. [9,10] suggested a possible usage of glycodelin in lung transplantation. Despite that, immunosuppressive ef fects of glycodelin are well-known [1]. Its role in the regulation of cell functions shaping immune tolerance against allogenic graft remains unclear.
In particular, the role of glycodelin in the regulation of key (with regard to evolutionary biology) cell population myeloid-derived suppressor cells (MDSC) is still uncertain. MDSC represents a heterogeneous population of immature myeloid cells acquiring suppressor phenotype under the pathological states and repressing the immune response [3].
Recent research works demonstrated the MDSC key role in supporting immune tolerance in transplantation [12]. There is a hypothesis that glycodelin might be capable of inducing the MDSC formation because their level is elevated in successful pregnancy. In contrast, spontaneous abortion and preeclampsia progression are associated with low circulating glycodelin level. Therefore, this work was aimed at examining human recombinant GdA role in MDSC formation and regulation playing an important role in allograft tolerance induction .

Materials and methods
The study was conducted according to the requirements of the Declaration of Helsinki WMA (2000) and protocol to the European Council Convention on Human Rights and Biomedicine (1999); the permission from the Ethics Committee of the IEGM UB RAS of 30.08.2019 (IRB00010009) was received.
Glycodelin was used in physiological range because it corresponds to its female peripheral blood level during pregnancy. GdA peaks at 2.0 µg/ml by the end of the trimester I reaching as high as 0.2 µg/ml in the trimester II, but declining to the minimal value or even not detected in the trimester III.
A complete medium was replaced on day 7, followed by three days of exposure to recombinant glycodelin (GdA, "MyBioSource. Inc.", E. coli; USA) at physiological concentrations (0.2; 2.0 µg/ ml) corresponding to its level in peripheral blood of pregnant women ; pharmacological concentration was equal to 50 µg/ml [7]. Thereafter, cells were cultured for another three days followed by collection using the "Accutase" according to the manufacturer's recommendations (Capricorn Scientific, Germany).
Then cells were stained to detect viability with propidium iodide (PI) (Biolegend, USA) according to the manufacturer's protocol. The percentage of live (PI -) cells in cultures (using a gate of living cells on FSC/SSC plot) varied within 85,5-92,1%.
Statistical data processing was realized with GraphPad Prism 8 and the non-parametric Wilcoxon test.

Results and discussion
Experiments demonstrated that GdA resulted in elevated MDSC percentage in cytokine-induced human mononuclear cell cultures that was significant only at its low concentrations (0.2 and 2.0 µg/ ml) that fits its pregnancy-related range. While analyzing the cell subsets, it was demonstrated that the rise in the MDSC level treated with GdA (0.2 and 2.0 µg/ml) resulted in significant PMN-MDSC increase, whereas the M-MDSC level was changed insignificantly. Therefore, GdA did not reliably affect the M-MDSC (Table 1). It was determined that high GdA concentration (50 µg/ml) corresponding to the pharmacological level conferred no significant effect on MDSC differentiation.
Cell viability in cultures was assessed following the propidium iodide staining. It was revealed that GdA at all concentrations did not affect cell viability in culture. Gating strategy according to light scattering parameters in FSC/SSC plot was applied to analyze solely live cells. Therefore, the effects obtained are related indeed to GdA direct influence on MDSC differentiation.
More recently, it became evident that the elevation of MDSC level also accompanied pregnancy. In 2014, it was found that the PMN-MDSC level was increased in peripheral blood of healthy pregnant women at all the stages of pregnancy as compared to non-pregnant subjects, while the M-MDSC amount remained unchanged [4]. Around the same time, it was revealed that patients with miscarriage had count of blood and endometrial MDSCs by 30% lower particularly in trimester I [6].
PMN-MDSC are known to accumulate in placenta of healthy pregnant women, while their number declines in spontaneous abortion. Placental PMN-MDSCs effectively suppress T-cell response with subsequent polarization of CD4 + -lymphocytes to Th2-phenotype [4]. PMN-MDSC are suggested to play an essential role in inducing and supporting the tolerance to fetal antigens allowing to consider them as a promising target for therapeutic manipulation under the pregnancy complications.
Meanwhile, the question about exact nature of receptor mediating GdA effects remains open. Subsequent analysis of GdA mode of action showed that the surface L-selectin molecule on monocytemacrophage lineage cells is a binding protein for GdA. Thus, the antibody against L-selectin reduced the GdA binding and GdA-induced IL-6 production [5]. In particular, Vijayan et al. in 2018 found potential receptors for GdA on human peripheral blood monocyte membrane. SIGLEC-7 (sialic acid-binding immunoglobulinlike lectin 7, CD328) -receptor to GdA was identified using co-immunoprecipitation and flow cytometry. GdA was found under culture conditions to improve the expression of decidual macrophage markers IDO-1 and CD209 on GdA-polarized macrophages. SIGLEC-7 receptor blocks GdA-related biological effects on monocyte differentiation [11]. However, it is not clear through which receptor this occurred, and we can only speculate.
Therefore, we have initially demonstrated that GdA can regulate the MDSC differentiation primarily by increasing its polymorphonuclear subset (PMN-MDSC). Potentially, GdA possesses the potential to manipulate the MDSC level that, in turn, serve as the immune response suppressors.