Decreased number of myeloid-derived suppressor cells in the placental trophoblast of gestational diabetes mellitus. Possible role of leptin

Decreased number of myeloid-derived suppressor cells in the placental trophoblast of gestational diabetes mellitus. Possible role of leptin Malika Tami a,1 , Lourdes Hontecillas-Prieto a,b,1 , Daniel García-Domínguez a,b , Rocío Flores-Campos a , Teresa Vilari˜ no-García a , Flora S´ anchez-Jim´ enez a , Pilar Guadix c , Jos´ e L. Due˜ nas c , Carlos Jim´ enez-Cortegana a , Luis de la Cruz-Merino b,d , Antonio P´ erez-P´ erez a , Víctor S´ anchez-Margalet a,b,* a Department of Medical Biochemistry and Molecular Biology, and Immunology, Medical, School. Virgen Macarena University Hospital, University of Seville, Spain b Institute of Biomedicine of Seville, Virgen Macarena University Hospital, CSIC, University of Seville, Seville, Spain c Service of Obstetrics and Gynecology, Virgen Macarena University Hospital. Medical, School, University of Seville, Spain d Medical Oncology Service, Department of Medicine, Medical School, Virgen Macarena, University Hospital, University of Seville, Spain ARTICLE INFO Editor name: Banu Bayram Keywords: MDSC Leptin Placenta Gestational diabetes mellitus ABSTRACT Gestational diabetes mellitus (GDM) is the most common complication of pregnancy and significantly increases both maternal and fetal morbidity and mortality. Inflammation is a hallmark of GDM, and placental inflammation may play a key role in the pathophysiology of the disease. Myeloid-derived suppressor cells (MDSCs), which are innate immunosuppressive, are thought to contribute to feto-maternal tolerance. In normal pregnancies, elevated levels of MDSCs have been observed in both peripheral and umbilical cord blood. Our hypothesis postulates that trophoblasts from placentas belonging to women with GDM may have lower levels of MDSCs compared to trophoblasts from placentas originating from healthy pregnancies. Furthermore, since leptin is overexpressed in the placenta of GDM patients, we hypothesized that leptin might contribute to the reduction of MDSCs. To test this, we investigated the in vitro effects of leptin on MDSC levels in isolated peripheral blood leukocytes after 24 h of incubation. Our findings indicate that trophoblasts from placentas from women with GDM contain a lower percentage of MDSCs compared to trophoblasts from healthy pregnancies. In addition, in vitro studies demonstrated that leptin reduces the number of MDSCs in peripheral blood leukocytes. In conclusion, MDSCs are decreased in placentas from pregnancies with GDM, and leptin appears to reduce the number of MDSCs in leukocytes isolated in vitro. Increased leptin expression in trophoblasts from placentas of women with GDM may contribute to the lower levels of MDSCs, potentially playing a role in placental inflammation. However, further investigations are required to fully elucidate this mechanism. 1. Introduction Gestational diabetes mellitus (GDM) is the most common complication of pregnancy and a major cause of increased morbidity and mortality for both mother and fetus. GDM is associated with an increased risk of various pregnancy complications, including fetal macrosomia (Buchanan et al., 2012). The pathophysiology of GDM is not completely understood, but the involvement of immune cells and inflammation has been proposed. However, further research is needed to definitively establish the role of the immune system in GDM (Pantham et al., 2015; De Luccia et al., 2020). Recently, it has been hypothesized that myeloid-derived suppressor cells (MDSCs) may be involved in GDM (Hua et al., 2023). MDSCs are immature myeloid cells within the innate immune system that exert an immunosuppressive effect on T lymphocytes (Gabrilovich and Nagaraj, 2009). These cells play a role in the pathophysiology of numerous diseases (Zhang et al., 2015; Jimenez-Cortegana et al., 2021a) including cancer (Tcyganov et al., 2018; Sanchez-Leon et al., 2023; Jimenez-Cortegana et al., 2021b). Elevated levels of MDSCs have been observed in both peripheral and * Corresponding author at: Department of Medical Biochemistry and Molecular Biology, and Immunology, Medical, School Virgen Macarena University Hospital, University of Seville, Av. Dr. Fedriani 3, 41009 Seville, Spain. E-mail address: [email protected] (V. S´ anchez-Margalet). 1 These authors should be considered as first author Contents lists available at ScienceDirect Immunobiology journal homepage: www.elsevier.com/locate/imbio https://doi.org/10.1016/j.imbio.2025.152897 Received 19 November 2024; Received in revised form 8 March 2025; Accepted 24 March 2025 Immunobiology 230 (2025) 152897 Available online 25 March 2025 0171-2985/© 2025 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). umbilical cord blood have previously been observed in women with normal pregnancies (K¨ ostlin et al., 2014; Bizymi et al., 2022). In fact, increasing evidence shows that MDSCs play a key role as immunomodulator cells in the maintenance of maternal-fetal immune tolerance (He et al., 2018; K¨ ostlin et al., 2018), and are even involved in supporting fetal development (Shi et al., 2021). In fact, pregnancy presents a unique immunological challenge, as the maternal immune system must tolerate the presence of the fetus, which expresses paternal antigens. This tolerance is achieved through multiple mechanisms, including the presence of MDSCs (Ahmadi et al., 2019). It has also been found that the induction of MDSCs during pregnancy can reduce the incidence of preeclampsia and miscarriage (Verma et al., 2019; Pan et al., 2016a). Therefore, the increased circulating MDSCs during healthy pregnancy are crucial for ensure maternal–fetal immune tolerance, adequate placentation, and fetal growth (K¨ ostlin-Gille and Gille, 2020). In contrast, the absence or reduction of MDSCs has been associated with pregnancy complications such as preeclampsia, preterm birth, fetal death and recurrent miscarriage (Shah et al., 2023). In this context, human trophoblasts have been shown to induce MDSCs from peripheral blood CD14+myelomonocytic cells via elevated levels of CCL2 (Zhang et al., 2016). In addition, other studies have shown that monocyte reprogramming is mediated by placental-derived exosomes (Bai et al., 2022). MDSCs have been reported to localize mainly in the decidua and interventricular space, where they play a role in the polarization of CD4+T cells toward a Th2 cytokine response (K¨ ostlin et al., 2016; Bartmann et al., 2016). Leptin is a hormone secreted mainly by adipocytes, and by other organs such as the placenta. In particular, it has been described that trophoblast cells are able to secrete it exerting both autocrine and paracrine effects (Schanton et al., 2018). Thus, leptin plays an important role in energy homeostasis, immune function, and reproduction (Park and Ahima, 2015; Behnes et al., 2012; Perez-Perez et al., 2015). Throughout human gestation, placental trophoblasts produce leptin and express leptin receptors (LepR), suggesting that this hormone plays a role in placental development (Henson and Castracane, 2006; Henson et al., 1998) in both normal and pathological pregnancies (Perez-Perez et al., 2018). In addition, leptin has also been shown to play a key role as an immune modulator. Thus, leptin exhibits pleiotropic effects on the immune system, including promotion of inflammation and modulation of innate and adaptive immunity (Barrientos et al., 2015). Within its role as an immunomodulator, leptin has been suggested to be involved in the induction and accumulation of MDSCs in cancer (Clements et al., 2018) as well as an induction of the differentiation and accumulation of MDSCs in the tumor microenvironment (Jimenez-Cortegana et al., 2024). Furthermore, in obese individuals, leptin promotes the accumulation of MDSCs, whereas MDSCs down-regulate leptin production (Clements et al., 2018). However, the modulation of immune cells by leptin is an under-researched aspect in the context of pregnancy. Therefore, and given that we have previously demonstrated an elevated expression of leptin and its receptor in the placental trophoblast in women with GDM (P´ erez-P´ erez et al., 2013), in this study we have analyzed the relationship of placental trophoblasts from women with GDM with the levels of MDSCs in comparison, as well as the contribution of leptin in this reduction. 2. Materials and methods 2.1. Patients characteristics and ethics approval Placentas were collected at term from non-twin pregnancies by programmed cesarean section from women without pregnancy-related pathology (n =11) and placentas from women with GDM, all of whom also had programmed cesarean sections (n =5). All placentas were obtained from the Hospital Universitario Virgen Macarena (Seville, Spain). All women who participated in the study had a mean age at delivery (26.0 years ±7.0). This study was conducted in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects, the Declaration of Helsinki, good clinical practice guidelines, and local laws. The research protocol was approved by the Local Research Ethics Committee of the Hospital Universitario Virgen Macarena, and written consent was obtained from the patients and healthy donors. 2.2. Isolation of placental cells samples and processing All human placentas obtained were immediately immersed in icecold phosphate buffered saline (PBS) in order to avoid tissue degradation. Once in the laboratory they were washed three times with sterile PBS at room temperature to remove excess blood. Subsequently, samples were taken from the villous tissue which did not contain infarcted areas, calcifications or visible hematomas. Five cotyledons per placenta were then removed at an intermediate distance between the chorionic and basal plates and washed with cold DMEN/F12 culture medium (Merck D9785) without fetal bovine serum (FBS) (FBS11A Capricorn). The central parts of the cotyledons were mechanically disaggregated in 0 % FBS DMEN/F12 and the cell suspension was filtered through filters with a pore size of 0.8 mm (Merck AABG03700). The filtered cell suspension was then centrifuged at 1200 rpm for 5 min. The isolated cells were used for the assays described below. 2.3. MDSC analysis The cells isolated previously were incubated with antibodies against various cell markers, labeled with fluorophores for flow cytometric analysis of myeloid-derived suppressor cells (MDSCs), including both monocytic (M-MDSC, defined as CD14+) and granulocytic (G-MDSC, defined as CD15+) subsets. MDSCs were identified based on the expression of CD45+, CD33+, CD11b+, HLA-DR-, CD14+, and CD15+. The total leukocyte count was defined by the number of CD45+cells. Antibodies were obtained from Becton Dickinson Immunocytometry Systems (BDIS, San Jose, CA, USA) and were used at the manufacturer’s recommended concentrations to identify MDSCs: PerCP-Cy5.5 Mouse Anti-Human CD 45 (ref no. 564105), APC-Cy7 Rat Anti-CD11b (ref no. 557657), PE Mouse Anti-Human CD 33 (ref no. 555450), PECy7 Mouse Anti-Human HLA-DR (ref no. 560651), FITC Mouse Anti-Human CD 14 (ref no. 555397) and APC Mouse Anti-Human CD 15 (ref no. 551376). Cells were analyzed by flow cytometry using the FACSCanto II flow cytometry system (Becton Dickinson), with FSC-SSC to check that dead cells (necrotic or apoptotic) had not appeared and CD45-SSC gating to select alive non-apoptotic leukocytes. They were further gated to identify MDSC as previously described (Jimenez-Cortegana et al., 2021b), and described in the supplementary Figure. 2.4. Whole blood samples and leptin treatment MDSC were studied in peripheral blood from healthy donors (n = 17), who were healthy women, not pregnant at the time of the study and of reproductive age (19–39 years). Blood samples were collected in EDTA-K3 tubes to isolate leukocytes. Total leukocytes were obtained by dextran-based sedimentation (Boyum, 1968). Isolated leukocytes were cultured in 6-well plates for 24 h at 37◦and 5 % CO2 atmosphere, using RPMI-1640 (P0417500 PAN Biotech) with 1 % FBS. Samples were incubated in the absence or presence of leptin (10 nM or 100 nM concentration), and cells were analyzed by labeling and flow cytometry as described before. Selection of myeloid suppressor cell subpopulations was performed with the sequential gating strategy (Supplementary Fig. 1). Lyophilized leptin (Leptin Human L4146 Sigma Aldrich) is resuspended in sterile water and allowed to reach a stock concentration of 10 −4 M which is frozen at −80 ◦C. M. Tami et al. Immunobiology 230 (2025) 152897 2 2.5. Statistical analysis Non-parametric t-test and Kruskal-Wallis test were used to evaluate differences between two or more groups, respectively. All statistical analyses in the study were performed using the software GraphPad Prism (6.01). The average of samples with SD is presented in all experiments. For all analyses, p-values of ≤0.05 were considered statistically significant. 3. Results 3.1. Decreased number of MDSCs in the trophoblast from placenta from GDM Since MDSCs play an important role in the maintenance of fetomaternal tolerance and placentas from GDM patients exhibit trophoblast cell dysfunction, we first analyzed the levels of MDSCs population of trophoblasts isolated from placentas of healthy pregnant women and women with GDM. Total MDSCs levels were significantly lower in trophoblasts from placentas belonging to women with GDM (mean 2.2) than in healthy women (mean 17.5) (Fig. 1A). The different subpopulations of MDSCs were then analyzed. The results showed that both M-MDSCs and G-MDSCs were also significantly decreased in the trophoblasts of placentas from GDM placentas (M-MDSCs mean 1.3 and GMDSCs mean 1.0) compared with those from healthy women (M-MDSCs mean 15.2 and G-MDSC mean 2.4) (Fig. 1B and C). Our results show that trophoblasts in placentas from GDM pregnancies contain a lower level of MDSCs compared to those from healthy pregnancies. 3.2. Leptin decreases the MDSC in vitro To learn about some of the factors that might be involved in the reduction of MDSCs in placentas of GDM patients, we evaluated the in vitro effect of leptin on MDSCs levels in isolated peripheral blood leukocytes. Leptin was observed to decrease the percentage of total MDSCs in a dose-dependent manner, with the 100 nM concentration showing a statistically significant decrease with respect to the control (approximately 28.9 %) (Fig. 2A). Similarly, the same results were obtained in the M-MDSCs and G-MDSCs subpopulations (Fig. 2B and C). A reduction of 26.6 % in the M-MDSCs subpopulation and 47.7 % in the GMDSCs subpopulation was observed with leptin treatment at 100 nM with respect to the control (Fig. 2B and C). In vitro studies demonstrated that leptin reduces the percentage of MDSCs in peripheral blood leukocytes. 4. Discussion Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with potent immunosuppressive properties (Gabrilovich and Nagaraj, 2009). Initially described in the context of cancer, where they promote tumor immune evasion (Sinha et al., 2005; Talmadge, 2007), MDSCs have since been recognized to play important roles in other contexts, such as infection, autoimmunity, and pregnancy (Ostrand-Rosenberg et al., 2023). During pregnancy, maternal immune tolerance toward the semi-allogenic fetus is critical, and MDSCs are believed to be central in mediating this immune tolerance (Zhang et al., 2022). In recent years, there is increasing evidence that MDSCs contribute to fetal-maternal immune regulation by suppressing the activity of maternal immune cells that might otherwise reject the fetus (Ahmadi et al., 2019; K¨ ostlin-Gille and Gille, 2020). MDSCs are also found at the maternal-fetal interface, specifically in the decidua and intervillous space. Here, they contribute to the immunologically privileged environment by interacting with decidual immune cells, including T cells and NK cells, and promoting a shift toward a tolerogenic immune environment (Pang et al., 2023). Placental MDSCs have been shown to polarize CD4+T cells toward a Th2 phenotype, which promotes tolerance and suppresses the pro-inflammatory Th1 response that could threaten pregnancy (Zhao et al., 2016). In addition to maintaining immune tolerance, MDSCs also contribute to fetal development. By modulating immune responses, MDSCs contribute to proper placentation and vascular remodeling in the placenta, which is essential for the exchange of nutrients and oxygen between mother and fetus (Aplin et al., 2020). Furthermore, an interrelationship between trophoblast and MDSCs has been suggested, as in Fig. 1. Placental trophoblasts from GDM patients have low levels of MDSC. Analysis of total MDSC (A), M-MDSC (B) and G-MDSC (C) levels of trophoblasts from healthy pregnant women and women with GDM. The average of samples with SD is presented in all experiments. For all the analyses, *P ≤0.05, **P ≤0.01, ***P ≤0.001 and ****P ≤0.0001. ns, not significant. M. Tami et al. Immunobiology 230 (2025) 152897 3 vitro studies with human trophoblast cells induced MDSCs from peripheral blood monocytes (Zhang et al., 2016). Thus, the placenta itself can modulate the levels and function of MDSCs. As for the possible mechanisms involved, estrogen and progesterone, both of which are elevated during pregnancy, have been shown to promote the expansion and suppressive activity of MDSCs. These hormones may contribute to the increased levels of circulating MDSCs observed in pregnancy and enhance their immunosuppressive capacity (Pan et al., 2016b; Dong et al., 2015). In the present work, we have provided data regarding the presence of MDSCs in the trophoblast of placentas from control pregnancies. Therefore, given that MDSCs can enhance trophoblast cell activity (Pang et al., 2023), the presence of MDSCs in placenta trophoblast further supports the suggested positive feedback loop of trophoblast implantation and MDSCs recruitment at the maternal–fetal interface. The immunosuppressive function of MDSCs is vital for normal pregnancy outcomes, and dysregulation of MDSC activity has been associated with pregnancy complications (Shah et al., 2023; Jiang et al., 2021). For instance, pre-eclampsia, a pregnancy disorder characterized by hypertension and organ damage, is associated with abnormal placentation and systemic inflammation. Studies have reported reduced levels of MDSCs in the blood and placenta of women with pre-eclampsia, suggesting that inadequate MDSC-mediated immune suppression may contribute to the elevated inflammatory state seen in this condition (Wang et al., 2018). Reduced MDSCs activity may lead to excessive maternal immune activation, contributing to endothelial dysfunction and the development of preeclampsia (K¨ ostlin et al., 2014). Furthermore, the possible role of MDSCs in GDM has been proposed as a plausible hypothesis, taking into account the existing knowledge on MDSCs and pregnancy pathology (Hua et al., 2023). In this context, we aimed to assess the difference in the number of MDSCs in GDM placental trophoblast compared with control pregnancy placental trophoblast. We observed that the GDM placental trophoblast has a lower number of MDSCs, which may contribute to the increased inflammatory state previously described in this pregnancy complication (Ray et al., 2024). Possible mechanisms for the lower number of MDSCs in the GDM placenta may be increased adipokines such as leptin. The immunomodulatory effects of leptin have been previously described both by our group and by other investigators. Thus, leptin activates human monocytes, promoting the Th1 response (P´ erez-P´ erez et al., 2013; SantosAlvarez et al., 1999; Sanchez-Margalet and Martin-Romero, 2001; Najib and S´ anchez-Margalet, 2002; S´ anchez-Margalet et al., 2003; MartínRomero et al., 2000; P´ erez-P´ erez et al., 2017; Gabay et al., 2001; Zarkesh-Esfahani et al., 2001; Lord et al., 1998). Also, it has been shown that leptin, at a concentration typical of the second half of pregnancy (trimesters II-III), enhances the differentiation of Th17 lymphocytes and reduces the formation of regulatory T lymphocytes (Orlova and Shirshev, 2017) (Behnes et al., 2012). In addition, leptin has been found to have effects on the maturation and activity of dendritic cells generated from peripheral blood monocytes of women (Orlova et al., 2019). Furthermore, other groups have suggested that leptin may increase MDSCs in mice models of obesity (Clements et al., 2018). However, those results were obtained in in vivo model of obesity, whereas we have tested the in vitro effect of leptin on blood leukocytes to evaluate its effect on MDSCs for the first time. We have found that high doses of leptin decreased the number of MDSCs of blood leukocytes cultured in vitro for 24 h. The high doses used are consistent with the high expression of leptin in the trophoblast, especially in GDM. Therefore, overexpression of leptin in the placenta of pregnant women with GDM may at least contribute to the reduction in the number of MDSCs observed in the trophoblast, and this effect may contribute to the proinflammatory state in the placenta of pregnant women with GDM. A limitation of the study is the lack of mechanistic explanation of the Fig. 2. MDSC in peripheral blood decrease with leptin treatment. Effect of leptin on the percentage of total MDSC (A), M-MDSC (B) and G-MDSC (C) in peripheral blood. The average of samples with SD is presented in all experiments. For all the analyses, *P ≤0.05, **P ≤0.01, ***P ≤0.001 and ****P ≤0.0001. ns, not significant. M. Tami et al. Immunobiology 230 (2025) 152897 4 effect of leptin on MDSC. We do not know whether leptin is acting directly on MDSC or indirectly acting on other cell population. Besides, we have identified MDSCs by gating HLA-DR negative cells and a possible explanation of the lower number of MDSC may be an increase in HLA-DR expression on these cells as previously found in other cell populations (Santos-Alvarez et al., 1999; Martín-Romero et al., 2000). In this context, the number of MDSC expressing HLA-DR have been previously found to be a positive prognostic factor in high risk neuroblastoma (Gowda et al., 2013). Another limitation of the study is the small number of samples, and therefore, further studies should be carried out to confirm these results. Given the role of MDSCs in maintaining immune tolerance, these immunosuppressive cells have been proposed as a potential therapeutic target in other pregnancy-related complications (Ostrand-Rosenberg et al., 2017), being GDM another example. In addition, therapies that target the enhancement of the function or expansion of MDSCs could be promising therapies for treating pregnancy disorders characterized by excessive inflammation, such as GDM. In conclusion, MDSCs are decreased in the trophoblast of placentas from women with GDM, which may contribute to the immune activation of inflammatory cells in this pathophysiological condition. We have also found that leptin decreases the number of MDSC in leukocytes from healthy donors in vitro. Since leptin is known to be highly expressed in the trophoblast of placentas from GDM, this hormone may contribute to the lower number of MDSC found in the placentas of these patients. Supplementary data to this article can be found online at https://doi. org/10.1016/j.imbio.2025.152897. CRediT authorship contribution statement Malika Tami: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Lourdes Hontecillas-Prieto: Writing – review & editing, Writing – original draft, Investigation, Formal analysis. Daniel García-Domínguez: Data curation. Rocío Flores-Campos: Writing – review & editing, Writing – original draft, Validation, Supervision. Teresa Vilari˜ no-García: Writing – review & editing, Supervision, Methodology, Formal analysis. Flora S´ anchezJim´ enez: Writing – review & editing, Formal analysis. Pilar Guadix: Writing – review & editing, Methodology, Data curation. Jos´ e L. Due˜ nas: Writing – review & editing, Methodology, Investigation. Carlos Jim´ enez-Cortegana: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Luis de la Cruz-Merino: Writing – review & editing, Resources, Investigation. Antonio P´ erezP´ erez: Writing – review & editing, Investigation, Funding acquisition, Formal analysis. Víctor S´ anchez-Margalet: Writing – review & editing, Writing – original draft, Supervision, Resources, Methodology, Investigation, Funding acquisition, Conceptualization. Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Victor Sanchez-Margalet reports financial support and equipment, drugs, or supplies were provided by Carlos III Health Institute. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This research was funded by a grant from Instituto de Salud Carlos III (ISCIII) (PI19/01741), funded in part by FEDER Funds to VS-M. L.H-P. is supported by Miguel Servet research fellow at Instituto de Salud Carlos III. D.J.G-D. is supported by the VII Plan Propio de Investigaci´ on y Transferencia of Universidad de Sevilla [Contrato de Acceso (II.4)/VII PPIT-US]. Data availability Data will be made available on request. References Ahmadi, M., Mohammadi, M., Ali-Hassanzadeh, M., Zare, M., Gharesi-Fard, B., 2019. MDSCs in pregnancy: critical players for a balanced immune system at the fetomaternal interface. Cell. Immunol. 346. Aplin, J.D., Myers, J.E., Timms, K., Westwood, M., 2020. Tracking placental development in health and disease. Nat. Rev. Endocrinol. 16 (9), 479–494. Bai, K.F., Lee, C.L., Liu, X.F., Li, J.L., Cao, D.D., Zhang, L., et al., 2022. Human placental exosomes induce maternal systemic immune tolerance by reprogramming circulating monocytes. J Nanobiotechnol. 20 (1). Barrientos, G., Toro, A., Moschansky, P., Cohen, M., Garcia, M.G., Rose, M., et al., 2015. Leptin promotes HLA-G expression on placental trophoblasts via the MEK/Erk and PI3K signaling pathways. Placenta 36 (4), 419–426. Bartmann, C., Junker, M., Segerer, S.E., H¨ ausler, S.F., Krockenberger, M., K¨ ammerer, U., 2016. CD33/HLA-DR and CD33/HLA-DR Cells: rare Populations in the Human Decidua with Characteristics of MDSC. Am. J. Reprod. Immunol. 75 (5), 539–556. Behnes, M., Brueckmann, M., Lang, S., Putensen, C., Saur, J., Borggrefe, M., et al., 2012. Alterations of leptin in the course of inflammation and severe sepsis. BMC Infect. Dis. 12, 217. Bizymi, N., Georgopoulou, A., Mastrogamvraki, N., Matheakakis, A., Gontika, I., Fragiadaki, I., et al., 2022. Myeloid-Derived Suppressor Cells (MDSC) in the Umbilical Cord Blood: Biological significance and possible Therapeutic applications. Journal of. Clin. Med. 11 (3). Boyum, A., 1968. Isolation of Mononuclear Cells and Granulocytes from Human Blood - Isolation of Mononuclear Cells by one Centrifugation and of Granulocytes by Combining Centrifugation and Sedimentation at 1 G. Scand J Clin Lab Inv. S 21, 77. Buchanan, T.A., Xiang, A.H., Page, K.A., 2012. Gestational diabetes mellitus: risks and management during and after pregnancy. Nat. Rev. Endocrinol. 8 (11), 639–649. Clements, V.K., Long, T., Long, R., Figley, C., Smith, D.M.C., Ostrand-Rosenberg, S., 2018. Frontline Science: High fat diet and leptin promote tumor progression by inducing myeloid-derived suppressor cells. J. Leukoc. Biol. 103 (3), 395–407. De Luccia, T.P.B., Pendeloski, K.P.T., Ono, E., Mattar, R., Pares, D.B.S., Sun, S.Y., et al., 2020. Unveiling the pathophysiology of gestational diabetes: Studies on local and peripheral immune cells. Scand. J. Immunol. 91 (4). Dong, G.J., You, M., Fan, H.Y., Ji, J.J., Ding, L., Li, P.F., et al., 2015. 17β-estradiol contributes to the accumulation of myeloid-derived suppressor cells in blood by promoting TNFα secretion. Acta Bioch Bioph Sin. 47 (8), 620–629. Gabay, C., Dreyer, M.G., Pellegrinelli, N., Chicheportiche, R., Meier, C.A., 2001. Leptin directly induces the secretion of interleukin 1 receptor antagonist in human monocytes. J Clin Endocr Metab. 86 (2), 783–791. Gabrilovich, D.I., Nagaraj, S., 2009. Myeloid-derived suppressor cells as regulators of the immune system. Nat. Rev. Immunol. 9 (3), 162–174. Gowda, M., Payne, K.K., Godder, K., Manjili, M.H., 2013. HLA-DR expression on myeloid cells is a potential prognostic factor in patients with high-risk neuroblastoma. Oncoimmunology 2 (10), e26616. He, Y.M., Li, X., Perego, M., Nefedova, Y., Kossenkov, A.V., Jensen, E.A., et al., 2018. Transitory presence of myeloid-derived suppressor cells in neonates is critical for control of inflammation. Nat. Med. 24(2):224. Henson, M.C., Castracane, V.D., 2006. Leptin in pregnancy: an update. Biol. Reprod. 74 (2), 218–229. Henson, M.C., Swan, K.F., O’Neil, J.S., 1998. Expression of placental leptin and leptin receptor transcripts in early pregnancy and at term. Obstet. Gynecol. 92 (6), 1020–1028. Hua, S.Y., Wang, S.S., Cai, J.Y., Wu, L.M., Cao, Y., 2023. Myeloid-derived suppressor cells: are they involved in gestational diabetes mellitus? Am. J. Reprod. Immunol. 90 (1). Jiang, H.H., Zhu, M.T., Guo, P.P., Bi, K.H., Lu, Z.M., Li, C.H., et al., 2021. Impaired myeloid-derived suppressor cells are associated with recurrent implantation failure: a case-control study. J. Reprod. Immunol. 145. Jimenez-Cortegana, C., Sanchez-Jimenez, F., Perez-Perez, A., Alvarez, N., Sousa, A., Canton-Bulnes, L., et al., 2021a. Low Levels of Granulocytic Myeloid-Derived Suppressor Cells May be a Good Marker of Survival in the Follow-up of patients with Severe COVID-19. Front. Immunol. 12, 801410. Jimenez-Cortegana, C., Palazon-Carrion, N., Martin Garcia-Sancho, A., NogalesFernandez, E., Carnicero-Gonzalez, F., Rios-Herranz, E., et al., 2021b. Circulating myeloid-derived suppressor cells and regulatory T cells as immunological biomarkers in refractory/relapsed diffuse large B-cell lymphoma: translational results from the R2-GDP-GOTEL trial. J. Immunother. Cancer 9 (6). Jimenez-Cortegana, C., Gutierrez-Garcia, C., Sanchez-Jimenez, F., Vilarino-Garcia, T., Flores-Campos, R., Perez-Perez, A., et al., 2024. Impact of obesity-associated myeloid-derived suppressor cells on cancer risk and progression (Review). Int. J. Oncol. 65 (2). K¨ ostlin, N., Kugel, H., Spring, B., Leiber, A., Marm´ e, A., Henes, M., et al., 2014. Granulocytic myeloid derived suppressor cells expand in human pregnancy and modulate T-cell responses. Eur. J. Immunol. 44 (9), 2582–2591. K¨ ostlin, N., Hofst¨ adter, K., Ostermeir, A.L., Spring, B., Leiber, A., Haen, S., et al., 2016. Granulocytic Myeloid-Derived Suppressor Cells Accumulate in Human Placenta and Polarize toward a Th2 Phenotype. J. Immunol. 196 (3), 1132–1145. K¨ ostlin, N., Schoetensack, C., Schwarz, J., Spring, B., Marm´ e, A., Goelz, R., et al., 2018. Granulocytic Myeloid-Derived Suppressor Cells (GR-MDSC) in Breast Milk (BM); GRM. Tami et al. Immunobiology 230 (2025) 152897 5 MDSC Accumulate in Human BM and Modulate T-Cell and Monocyte Function. Front. Immunol. 9. K¨ ostlin-Gille, N., Gille, C., 2020. Myeloid-Derived Suppressor Cells in Pregnancy and the Neonatal Period. Front. Immunol. 11. Lord, G.M., Matarese, G., Howard, L.K., Baker, R.J., Bloom, S.R., Lechler, R.I., 1998. Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression. Nature 394 (6696), 897–901. Martín-Romero, C., Santos-Alvarez, J., Goberna, R., S´ anchez-Margalet, V., 2000. Human leptin enhances activation and proliferation of human circulating T lymphocytes. Cell. Immunol. 199 (1), 15–24. Najib, S., S´ anchez-Margalet, V., 2002. Human leptin promotes survival of human circulating blood monocytes prone to apoptosis by activation of p42/44 MAPK pathway. Cell. Immunol. 220 (2), 143–149. Orlova, E.G., Shirshev, S.V., 2017. Role of PKA and PI3K in Leptin and Ghrelin Regulation of Adaptive Subpopulations of Regulatory CD4+T-Lymphocyte Formation. Biochemistry (Mosc.) 82 (9), 1061–1072. Orlova, E.G., Shirshev, S.V., Loginova, O.A., 2019. Mechanisms of Leptin and Ghrelin Action on Maturation and Functions of Dendritic Cells. Biochemistry (Mosc.) 84 (1), 1–10. Ostrand-Rosenberg, S., Sinha, P., Figley, C., Long, R., Park, D., Carter, D., et al., 2017. Frontline Science: Myeloid-derived suppressor cells (MDSCs) facilitate maternal-fetal tolerance in mice. J. Leukoc. Biol. 101 (5), 1091–1101. Ostrand-Rosenberg, S., Lamb, T.J., Pawelec, G., 2023. Here, there, and everywhere: Myeloid-Derived Suppressor Cells in Immunology. J. Immunol. 210 (9), 1183–1197. Pan, T., Liu, Y.F., Zhong, L.M., Shi, M.H., Duan, X.B., Wu, K., et al., 2016a. Myeloidderived suppressor cells are essential for maintaining feto-maternal immunotolerance via STAT3 signaling in mice. J. Leukoc. Biol. 100 (3), 499–511. Pan, T., Zhong, L., Wu, S., Cao, Y., Yang, Q., Cai, Z., et al., 2016b. 17β-Oestradiol enhances the expansion and activation of myeloid-derived suppressor cells via signal transducer and activator of transcription (STAT)-3 signalling in human pregnancy. Clin. Exp. Immunol. 185 (1), 86–97. Pang, B., Hu, C., Li, H.M., Nie, X.Y., Wang, K.Q., Zhou, C., et al., 2023. Myeloidderived suppressor cells: escorts at the maternal-fetal interface. Front. Immunol. 14. Pantham, P., Aye, I.L.M.H., Powell, T.L., 2015. Inflammation in maternal obesity and gestational diabetes mellitus. Placenta 36 (7), 709–715. Park, H.K., Ahima, R.S., 2015. Physiology of leptin: energy homeostasis, neuroendocrine function and metabolism. Metabolism 64 (1), 24–34. P´ erez-P´ erez, A., Maym´ o, J.L., Gambino, Y.P., Guadix, P., Due˜ nas, J.L., Varone, C.L., et al., 2013. Activated translation Signaling in Placenta from Pregnant Women with Gestational Diabetes Mellitus: possible Role of Leptin. Horm. Metab. Res. 45 (6), 436–442. Perez-Perez, A., Sanchez-Jimenez, F., Maymo, J., Duenas, J.L., Varone, C., SanchezMargalet, V., 2015. Role of leptin in female reproduction. Clin. Chem. Lab. Med. 53 (1), 15–28. P´ erez-P´ erez, A., Vilari˜ no-García, T., Fern´ andez-Riejos, P., Martín-Gonz´ alez, J., SeguraEgea, J.J., S´ anchez-Margalet, V., 2017. Role of leptin as a link between metabolism and the immune system. Cytokine Growth F R. 35, 71–84. Perez-Perez, A., Toro, A., Vilarino-Garcia, T., Maymo, J., Guadix, P., Duenas, J.L., et al., 2018. Leptin action in normal and pathological pregnancies. J. Cell. Mol. Med. 22 (2), 716–727. Ray, G.W., Zeng, Q.L., Kusi, P., Zhang, H.L., Shao, T.T., Yang, T.L., et al., 2024. Genetic and inflammatory factors underlying gestational diabetes mellitus: a review. Front. Endocrinol. 15. Sanchez-Leon, M.L., Jimenez-Cortegana, C., Silva Romeiro, S., Garnacho, C., de la CruzMerino, L., Garcia-Dominguez, D.J., et al., 2023. Defining the Emergence of New Immunotherapy Approaches in Breast Cancer: Role of Myeloid-Derived Suppressor Cells. Int. J. Mol. Sci. 24 (6). Sanchez-Margalet, V., Martin-Romero, C., 2001. Human leptin signaling in human peripheral blood mononuclear cells: Activation of the JAK-STAT pathway. Cell. Immunol. 211 (1), 30–36. S´ anchez-Margalet V, Martín-Romero C, Santos-Alvarez J, Goberna R, Najib S, GonzalezYanes C. Role of leptin as an immunomodulator of blood mononuclear cells:: mechanisms of action. Clin. Exp. Immunol. 2003;133(1):11–9. Santos-Alvarez, J., Goberna, R., S´ anchez-Margalet, V., 1999. Human leptin stimulates proliferation and activation of human circulating monocytes. Cell. Immunol. 194 (1), 6–11. Schanton, M., Maym´ o, J.L., P´ erez-P´ erez, A., S´ anchez-Margalet, V., Varone, C.L., 2018. Involvement of leptin in the molecular physiology of the placenta. Reproduction 155 (1), R1–R12. Shah, N.K., Xu, P., Shan, Y.H., Chen, C., Xie, M., Li, Y., et al., 2023. MDSCs in pregnancy and pregnancy-related complications: an update. Biol. Reprod. 108 (3), 382–392. Shi, M.Y., Chen, Z.Y., Chen, M.Q., Liu, J.P., Li, J., Xing, Z., et al., 2021. Continuous activation of polymorphonuclear myeloid-derived suppressor cells during pregnancy is critical for fetal development. Cell. Mol. Immunol. 18 (7), 1692–1707. Sinha, P., Clements, V.K., Ostrand-Rosenberg, S., 2005. Reduction of myeloid-derived suppressor cells and induction of M1 macrophages facilitate the rejection of established metastatic disease. J. Immunol. 174 (2), 636–645. Talmadge, J.E., 2007. Pathways mediating the expansion and immunosuppressive activity of myeloid-derived suppressor cells and their relevance to cancer therapy. Clin. Cancer Res. 13 (18), 5243–5248. Tcyganov, E., Mastio, J., Chen, E., Gabrilovich, D.I., 2018. Plasticity of myeloid-derived suppressor cells in cancer. Curr. Opin. Immunol. 51, 76–82. Verma, P., Nair, R.R., Singh, S., Rajender, S., Khanna, A., Jha, R.K., et al., 2019. High Level of APOA1 in Blood and Maternal Fetal Interface is Associated with early Miscarriage. Reprod. Sci. 26 (5), 649–656. Wang, Y.A., Liu, Y.H., Shu, C., Wan, J.Y., Shan, Y.H., Zhi, X.Y., et al., 2018. Inhibition of pregnancy-associated granulocytic myeloid-derived suppressor cell expansion and arginase-1 production in preeclampsia. J. Reprod. Immunol. 127, 48–54. Zarkesh-Esfahani, H., Pockley, G., Metcalfe, R.A., Bidlingmaier, M., Wu, Z.D., Ajami, A., et al., 2001. High-dose leptin activates human leukocytes via receptor expression on monocytes. J. Immunol. 167 (8), 4593–4599. Zhang, Q., Fujino, M., Xu, J.H., Li, X.K., 2015. The Role and potential Therapeutic Application of Myeloid-Derived Suppressor Cells in Alloand Autoimmunity. Mediators Inflamm. 2015. Zhang, Y., Qu, D.W., Sun, J.T., Zhao, L., Wang, Q.J., Shao, Q.Q., et al., 2016. Human trophoblast cells induced MDSCs from peripheral blood CD14 myelomonocytic cells via elevated levels of CCL2. Cell. Mol. Immunol. 13 (5), 615–627. Zhang, Y., Wang, X.Y., Zhang, R.C., Wang, X., Fu, H.Y., Yang, W., 2022. MDSCs interactions with other immune cells and their role in maternal-fetal tolerance. Int. Rev. Immunol. 41 (5), 534–551. Zhao, A.M., Xu, H.J., Kang, X.M., Zhao, A.M., Lu, L.M., 2016. New insights into myeloidderived suppressor cells and their roles in feto-maternal immune cross-talk. J. Reprod. Immunol. 113, 35–41. M. Tami et al. Immunobiology 230 (2025) 152897 6