Orsolya Oravecz1,2
Roberto Romero3,4,5,6,7
Eszter Tóth1
Judit Kapitány1Máté Posta1,8Dahiana M. Gallo3,9,10Simona W. Rossi11
Adi L. Tarca3,6,9,11Offer Erez3,9,11,12Zoltán Papp13,14
János Matkó1
Nándor Gábor Than1,3,11,13,14*
Andrea Balogh1- 1Systems Biology of Reproduction Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
- 2Doctoral School of Biology, Institute of Biology, ELTE Eötvös Loránd University, Budapest, Hungary
- 3Perinatology Research Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, United States Department of Health and Human Services, Detroit, MI, United States
- 4Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, United States
- 5Department of Epidemiology and Biostatistics, Michigan State University, East Lansing, MI, United States
- 6Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, United States
- 7Detroit Medical Center, Detroit, MI, United States
- 8Károly Rácz Doctoral School of Clinical Medicine, Semmelweis University, Budapest, Hungary
- 9Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, United States
- 10Department of Obstetrics and Gynecology, Universidad Del Valle, Cali, Colombia
- 11Genesis Theranostix Group, Budapest, Hungary
- 12Department of Obstetrics and Gynecology, Soroka University Medical Center, Beer Sheva, Israel
- 13Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary
- 14Maternity Private Clinic of Obstetrics and Gynecology, Budapest, Hungary
Introduction: Galectins are master regulators of maternal immune responses and placentation in pregnancy. Galectin-13 (gal-13) and galectin-14 (gal-14) are expressed solely by the placenta and contribute to maternal-fetal immune tolerance by inducing the apoptosis of activated T lymphocytes and the polarization of neutrophils toward an immune-regulatory phenotype.Furthermore, their decreased placental expression is associated with pregnancy complications, such as preeclampsia and miscarriage. Yet, our knowledge of the immunoregulatory role of placental galectins is incomplete.
Methods: This study aimed to investigate the effects of recombinant gal-13 and gal-14 on cell viability, apoptosis, and cytokine production of peripheral blood mononuclear cells (PBMCs) and the signaling pathways involved.
Results: Herein, we show that gal-13 and gal-14 bind to the surface of non-activated PBMCs (monocytes, natural killer cells, B cells, and T cells) and increase their viability while decreasing the rate of their apoptosis without promoting cell proliferation. We also demonstrate that gal-13 and gal-14 induce the production of interleukin (IL)-8, IL-10, and interferon-gamma cytokines in a concentration-dependent manner in PBMCs. The parallel activation of Erk1/2, p38, and NF-ĸB signaling evidenced by kinase phosphorylation in PBMCs suggests the involvement of these pathways in the regulation of the galectin-affected immune cell functions.
Discussion: These findings provide further evidence on how placenta-specific galectins assist in the establishment and maintenance of a proper immune environment during a healthy pregnancy.
Introduction
The interactions of maternal, fetal, and placental immune responses during pregnancy as well as the mechanisms that maintain maternal immune tolerance to the semi-allogeneic fetus, while guarding against microbial infection, are of great interest in reproductive medicine, even though these topics have been only partially explored (1–12). Investigators have described that time- and site-specific modifications are characteristic of these immune interactions, such as the pro-inflammatory milieu at the time of implantation, the anti-inflammatory state during the second trimester, and local and systemic inflammation in the third trimester, preceding delivery (13–17). The mediators of this complex network of maternal-fetal-placental immune interactions involve a large array of cellular, vesicular, and soluble components (8, 18–29). In the past decade, an increasing body of evidence has been collected on the key contribution of galectins to immune interactions in pregnancy (30–43).
Galectins are glycan-binding proteins belonging to the lectin subfamily, with the common property of specifically binding to glycoconjugates containing β-galactoside carbohydrates (9, 20, 44). Of the 19 galectins found in mammals, 13 are expressed in human tissues (20, 44–46). These human galectins affect a variety of cellular processes when they contact immune cells, e.g., apoptosis, cell proliferation, cell adhesion, chemotaxis, cytokine production, or degranulation (31, 39, 41, 47–52). In pregnancy, several galectins are expressed at the maternal-fetal interface and assist in regulating placental and fetal development, local and systemic inflammation as well as the establishment and maintenance of maternal immune tolerance toward the fetus (9, 38, 46, 48, 49, 53–58).
In humans, three galectins (gal-13, gal-14, and gal-16) are expressed solely by the placenta (20, 46, 57, 59). The genes encoding these galectins are located within a cluster of galectin genes and pseudogenes on chromosome 19, which arose in anthropoid primates through birth-and-death evolution (20, 46, 59, 60). This evolutionary gene duplication process led to neofunctionalization, the acquisition of a new function(s) in the duplicated genes, as measured by the differing carbohydrate-binding capacity of these placental galectins (59). At the RNA and protein levels, these are primarily expressed in the multinucleated syncytiotrophoblast layer of the placenta, formed by the fusion of mononucleated cytotrophoblast cells, but not in the underlying progenitor cytotrophoblasts (59, 61). In comparison, expression of these galectins is less abundant in the extravillous trophoblasts, amniotic epithelium, and fetal endothelium (53, 59, 60, 62).
During a healthy pregnancy, maternal blood gal-13 concentrations increase with advancing gestation and then diminish after delivery (61, 63). Notably, gal-13 is secreted into the maternal circulation from the syncytiotrophoblast (62–65), and low gal-13 concentrations were found in the maternal circulation during the first trimester in women who later developed preterm preeclampsia (66–70), a severe obstetrical syndrome with a strong systemic immune dysregulation (19, 71–76). In line with these findings, we have also demonstrated that the placental expression of gal-13 and gal-14 is down-regulated in preterm preeclampsia (60, 62, 64, 77) and in miscarriage (78).
Although several galectins have been studied extensively regarding their effects on maternal immune cells (79–85), placenta-specific galectins have been less studied in this context. We and others have explored that gal-13 and gal-14 treatment induces apoptosis of activated T cells (59, 78) and alters the cell surface expression of T-cell activation markers (78). On the other hand, gal-13 induces the production of interleukin (IL)-1ɑ and IL-6 in peripheral blood mononuclear cells (PBMC) (86) and the production of IL-8 in non-activated T cells (78), but decreases tumor necrosis factor-alpha expression in neutrophils (87). Overall, gal-13 may have a critical role in the maintenance of the physiological immune balance at the maternal-fetal interface and its lower expression may contribute to immune dysregulation in pregnancy complications such as preeclampsia, intrauterine growth restriction, and miscarriage. This phenomenon was also suggested for other galectins expressed at the maternal-fetal interface such as gal-1 and gal-3, highlighting their importance in proper placental functions (20, 36, 43, 55, 86, 88–91).
To further characterize the role of placenta-specific galectins in the regulation of maternal-fetal immune interactions, we aimed to investigate the effects of recombinant gal-13 and gal-14 on the viability, apoptosis, cytokine production, and cell signaling of PBMCs.
Materials and methods
Expression and purification of recombinant gal-13 and gal-14
Recombinant gal-13 and gal-14 were expressed in ClearColi BL21(DE3) (Lucigen, Middleton, WI, USA) bacterial strains with slight modifications, as previously described by Balogh et al. (78). All purification steps were carried out in the presence of 1mM dithiothreitol (DTT). Gal-13 and gal-14 were aliquoted in phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1 mM DTT and stored at −80°C until use.
Isolation of peripheral blood mononuclear cells
Buffy coats, obtained from healthy, non-pregnant human females (n=3-5, depending on the experiment), were purchased from the Hungarian National Blood Transfusion Service (Budapest, Hungary). PBMCs were isolated by Ficoll-Paque PLUS (GE Healthcare Life Sciences, Chicago, IL, USA) density gradient centrifugation. Isolated cells were washed with PBS before the experiments. PBMCs were placed into RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and gentamicin (Thermo Fisher Scientific). Cell counts were determined with trypan blue stain and a TC20 automated cell counter (Bio-Rad, Hercules, CA, USA). Isolated PBMCs were cultured for 24–72 hours depending on the experiment. To obtain experimental replicates, all assays were repeated with multiple donors.
Binding of recombinant gal-13 and gal-14 to peripheral blood mononuclear cells
Recombinant gal-13 or gal-14, conjugated with CF488 (Mix-n-Stain CF488 Kit, Biotium, Fremont, CA, USA), according to the manufacturer’s instructions, was added to 5×105 PBMCs in PBS containing 0.5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) and 0.05% sodium azide (flow cytometry wash buffer), and incubated on ice for 45 minutes. Samples were washed with wash buffer at 500g for 5 minutes at 4°C. To reduce nonspecific antibody binding, Fc receptors were blocked with human FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 5 minutes on ice. Population marker antibodies (Supplementary Table 1) were added to the cells, then incubated for 20 minutes on ice in the dark. Cells were then washed twice and resuspended in wash buffer. Flow cytofluorimetric measurements were carried out on a CytoFLEX device and with CytExpert software (Beckman Coulter, Brea, CA, USA) for data acquisition. Data were collected from 50,000 cells/sample and analyzed with FlowJo v10 software (FlowJo LLC, Ashland, OR, USA).
Cell viability assay
PBMCs (2×105 cells/well) were treated with 0.04 µM, 0.4 µM, or 4 µM recombinant gal-13, gal-14, or vehicle (PBS supplemented with 1 mM DTT in the same dilution) and incubated on 96-well plates in RPMI-1640 medium supplemented with 10% FBS and gentamicin for 68 hours (at 37°C, 5% CO2). CCK-8 solution (Sigma-Aldrich) was added to the wells, and the plates were incubated for 4 hours at 37°C. Absorbance at 450 nm was measured by using an EnSpire microplate reader (Perkin Elmer, Waltham, MA, USA).
Apoptosis assay
PBMCs (4×105 cells/well) were treated with 4 µM of recombinant gal-13 or gal-14. Galectin-treated and vehicle-treated cells were incubated for 24 hours on 24-well tissue culture plates (Eppendorf, Hamburg, Germany) in RPMI-1640 medium supplemented with 10% FBS and gentamicin. PBMCs were transferred to flow cytometry tubes and washed with wash buffer at 350g for 10 minutes at 4°C. Then, cells were stained with population marker antibodies (Supplementary Table 1) to discriminate between PBMC populations. After incubation on ice for 20 minutes in the dark, cells were washed twice in wash buffer. To differentiate between early-stage and late-stage apoptotic cells, phycoerythrin-conjugated annexin V (annexin V-PE) and 7-aminoactinomycin D (7-AAD) were applied in the presence of annexin binding buffer (Annexin-V/7-AAD Apoptosis Detection Kit, BioLegend, San Diego, CA, USA). After incubation for 15 minutes in the dark at room temperature, annexin binding buffer was added, and samples were measured immediately on a CytoFLEX flow cytometer. Data were collected from 30,000 cells/sample and analyzed by using FlowJo v10 software (FlowJo, LLC).
Measurement of cell proliferation by flow cytometry
PBMCs (4×106 cells/ml) were incubated with carboxyfluorescein succinimidyl ester (CFSE) cell staining dye (BioLegend) for 20 minutes at room temperature in the dark. Next, cells were incubated for 5 minutes at room temperature in RPMI-1640 medium supplemented with 10% FBS and gentamicin. Cells were then centrifuged twice with medium at 300g for 10 minutes at 20°C. Cell concentration was determined by trypan blue staining (Bio-Rad). Overall, 5×105 cells were plated in 48-well cell culture plates (Eppendorf) in completed RPMI-1640 medium and treated with 4 μM of gal-13, gal-14, or vehicle for 72 hours (37°C, 5% CO2). Then, cells were centrifuged with PBS (350 g, 4°C, 8 minutes) and incubated for 20 minutes with Zombie Violet vitality dye (BioLegend) at room temperature. After centrifugation in wash buffer, PBMC populations were labeled with cell-specific antibodies (Supplementary Table 1). Cells were incubated for 20 minutes in the dark on ice, followed by the addition of wash buffer and centrifugation twice. Subsequently, cells were resuspended in wash buffer and measured with a CytoFLEX cytofluorimeter. Data were collected from 100,000 cells/sample, and the results were analyzed with FlowJo v10 software (FlowJo, LLC).
Measurement of cytokine production by enzyme-linked immunosorbent assay
Commercially available Human ELISA Kits for IL-8, IL-10, and interferon-gamma (IFN-γ) (Biolegend) were used to estimate the concentration of cytokines in the culture medium (RPMI-1640 supplemented with 10% FBS and gentamicin) of PBMCs treated with 0.04 µM, 0.4 µM, or 4 µM of recombinant gal-13, gal-14, or vehicle for 24 hours. The procedure was carried out by following the manufacturer’s protocol. The reaction was stopped with 4N sulfuric acid and then the absorbance was measured at 450 nm (reference filter: 620 nm) with an EnSpire microplate reader (Perkin Elmer).
Western blot for phospho-protein detection
PBMCs (6×105 cells/sample) were incubated with recombinant gal-13 (4 µM), gal-14 (4 µM), vehicle, or cell culture medium (absolute control). Samples were incubated for 10, 20, or 30 minutes at 37° C in a thermoblock (Grant Instruments Ltd., Shepreth, Cambridgeshire, United Kingdom) at 300 rpm. At the end of the treatment, samples were centrifuged at 20,000g for 30 seconds, supernatants were discarded, and cell pellets were frozen in liquid nitrogen. Then, a mixture of PBS, reducing 2× Laemmli sample buffer (Bio-Rad), protease, and phosphatase inhibitors (Thermo Fisher Scientific), was prepared and added to the frozen samples that were then heat-denatured at 95°C for 5 minutes. The electrophoretic separation was performed on 4-20% SDS-polyacrylamide gel (Thermo Fisher Scientific) in Tris-glycine buffer. Following SDS-PAGE, samples were blotted onto nitrocellulose membranes (Bio-Rad), which were blocked for 1 hour in Tris-buffered saline supplemented with 0.05% of Tween-20 (Sigma-Aldrich) (TBST) and 5% of BSA. Next, membranes were incubated overnight with rabbit anti-human primary antibodies specific to phosphorylated proteins, such as extracellular signal-regulated kinase 1/2 (Erk1/2), mitogen-activated protein kinase (MAPK) p38, or nuclear factor kappa B (NF-κB) (Cell Signaling Technology, Danvers, MA, USA) at 4°C in TBST containing 0.5% of BSA (Supplementary Table 2). The next day, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody for 1 hour in TBST containing 0.5% of BSA (Sigma-Aldrich). β-actin-specific antibody was used as a loading control. Membranes were placed in an iBind Western blot device (Thermo Fisher Scientific) and incubated for 2 hours and 30 minutes with mouse anti-human β-actin (Thermo Fisher Scientific) primary antibody and HRP-conjugated rabbit anti-mouse secondary antibody (Life Technologies, Waltham, MA, USA), according to the manufacturer’s protocol (Supplementary Table 2). SuperSignal Western Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) was used to visualize the protein bands, and the ChemiDoc imaging device (Bio-Rad) was utilized for band detection. The relative signal intensities of phospho-proteins and β-actin were analyzed with ImageLab software (Bio-Rad).
Statistical analysis
The one-way analysis of variance test with Tukey’s post-hoc test was used to compare cell viability/proliferation, apoptosis, and cytokine production of various galectin- and vehicle-treated groups. The one-sample t-test was used to analyze Western blot experiments. All analyses were performed with GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). To ensure the concentration-dependent effect of recombinant galectins, we performed ordered factor analysis in R environment, using linear models, in case of cell viability and cytokine production experiments. Results were considered statistically significant at p<0.05.
Results
Gal-13 and gal-14 bind differentially to T and B lymphocytes, natural killer cells, and monocytes
The binding of gal-13 and gal-14 to helper and cytotoxic T cells as well as to neutrophil granulocytes has already been demonstrated by flow cytometry (78, 87). Herein, we extended these observations by examining the binding of fluorescent gal-13 and gal-14 to other PBMC populations as well, including B cells, natural killer (NK) cells, and monocytes (Figures 1A–Q). Recombinant gal-13 and gal-14 bound to the investigated PBMC populations to a different extent. The strongest binding of both galectins to monocytes (Figures 1F–H, R, S) was observed (47-95%), followed by NK cells (27-83%) and B cells (11-51%) (Figures 1I–K, L–N, R, S, respectively). Gal-13 and gal-14 bound the least to T cells (2-12%) (Figures 1O–Q, R, S).
Figure 1 Binding of gal-13 and gal-14 to the peripheral blood mononuclear cells. Detection of gal-13 or gal-14 binding to human PBMCs was achieved by flow cytometry. Cells were incubated with recombinant gal-13-CF488 or gal-14-CF488. PBMCs were also stained with anti-CD3-APC, or anti-CD19-PerCP, anti-CD14-APC, and anti-CD56-APC/Fire750. Gating strategy (A–Q). Graphs show the percentage of cells to which gal-13 (R) or gal-14 (S) bound (mean ± SEM, n = 4). FMO, Fluorescence minus one; PBMC, peripheral blood mononuclear cells; SEM, standard error of the mean.
Gal-13 and gal-14 increase cell viability of immune cells through decreased apoptosis
Next, we examined the effect of recombinant gal-13 and gal-14 on the viability of PBMCs after 24 and 72 hours of treatment. Interestingly, gal-13 and gal-14 increased PBMC viability after 24 hours of treatment by 62–82% (gal-13) and 58–83% (gal-14), depending on the applied galectin concentration (Figure 2A). Ordered factor analysis revealed the significant concentration-dependent effect of both galectins (gal-13: p<0.01; gal-14: p<0.05).
Figure 2 Gal-13 or gal-14 treatment increases the viability of immune cells. PBMCs were treated with gal-13, gal-14, or the vehicle for 24 and 72 hours. CCK-8 cell counting kit was used to examine cell viability. Graphs show the mean ± SEM of the optical density (450 nm) of viable cells after 24 hours (A) or 72 hours (B) of treatment (n=5). One-way ANOVA and Tukey post-hoc tests were used. Statistical significance was set at *p<0.05, **p<0.01, or ***p<0.001. PBMC: peripheral blood mononuclear cell, SEM: standard error of the mean.
After 72 hours of gal-13 and gal-14 administration, the viability of PBMCs showed even more of an increase than that of cells treated with the vehicle (gal-13: 123–153% increase; gal-14: 136–168% increase, depending on the galectin concentration) (Figure 2B). Herein, we also observed the significant concentration-dependent effect of both galectins (gal-13: p<10-4; gal-14: p<10-5).
To examine whether the increased cell viability could be attributed to altered apoptosis or to proliferation, the impact of gal-13 and gal-14 on the apoptosis of immune cells was then explored (Supplementary Figure 1 and Figure 3). In non-activated conditions, gal-13 and gal-14 reduced the proportion of early apoptotic T cells (gal-13: 19.6%; gal-14: 16.9%), B cells (gal-13: 13.6%; gal-14: 12.3%), NK cells (gal-13: 22.5%; gal-14: 21.7%), and monocytes (gal-13: 6.2%; gal-14: 5.9%) compared to vehicle-treated cells (Figures 3A–D). The proportion of late apoptotic B cells (Figure 3B) and monocytes (Figure 3D) was also reduced after gal-13 (by 4.2% and 12.1%, respectively) or gal-14 treatment (by 3.3% and 12.4%, respectively); however, the proportion of late apoptotic T cells and NK cells did not change upon treatment with placental galectins.
Figure 3 Gal-13 or gal-14 treatment reduces the apoptosis of PBMCs. PBMCs were incubated for 24 hours with gal-13, gal-14, or the vehicle. Graphs show the mean ± SEM of the percent of single positive (Annexin V +/7-AAD-, black bars) and double positive (Annexin V +/7-AAD +, grey bars) T lymphocytes, (A) B lymphocytes (B), NK cells (C), and monocytes (D) (n=4). One-way ANOVA and Tukey post-hoc tests were used. Statistical significance was set at *p<0.05, **p<0.01, or *** p<0.001. PBMC, peripheral blood mononuclear cell, SEM, standard error of the mean.
We also examined how gal-13 and gal-14 affect the proliferation of immune cell populations after 72 hours of treatment. Our results demonstrated that gal-13 and gal-14 had no impact on the proliferation of the PBMC populations under investigation (Supplementary Figure 2).
Gal-13 and gal-14 treatment induce IL-8, IL-10, and IFN-γ production by immune cells
Based on our previous results concerning IL-8 production of T cells (78), we were interested in how placental galectins may regulate the production of an anti-inflammatory cytokine (IL-10) and a mainly pro-inflammatory cytokine (IFN-γ), both of which are important at the maternal-fetal interface (92–94). Therefore, PBMCs were treated with three different concentrations of gal-13 or gal-14 for 24 hours or for 72 hours. In comparison to the vehicle (mean: 0.137 ng/ml), IL-8 production increased after 24 hours of gal-13 or gal-14 treatment in a dose-dependent manner, ranging from 33.89 to 150.3 ng/ml (Figures 4A, B). Furthermore, a concentration-dependent effect of galectin treatment was also detected for IL-10 secretion into cell culture supernatants, ranging from 2.361 to 5.173 ng/ml (vehicle mean: 0.003 ng/ml) (Figures 4C, D). Twenty-four hours of gal-13 or gal-14 treatment increased IFN-γ secretion by immune cells as well (vehicle mean: 3.62 pg/ml, gal-13/gal-14 mean: from 112.1 to 392.5 pg/ml), although not as robustly as seen in IL-8 and IL-10 (Figures 4E, F). Ordered factor analysis revealed the significant concentration-dependent effect of both galectins in all comparisons (gal-13: IL-8: p<10-5; IL-10: p<10-5; IFN-γ: p<10-5; gal-14: IL-8: p<10-5; IL-10: p<10-5; IFN-γ: p<0.01). Similar outcomes, but with fewer significant values, were observed after the 72-hour galectin treatment (Supplementary Figure 3). Again, we observed significant concentration-dependent effect of both galectins in all comparisons (gal-13: IL-8: p<10-5; IL-10: p<10-5; IFN-γ: p<0.01; gal-14: IL-8: p<10-5; IL-10: p<10-5; IFN-γ: p<0.01). Overall, these results showed that treatment with either gal-13 or gal-14 increased the production of certain cytokines in immune cells to various extents—IL-8 production the most and IFN-γ production the least.
Figure 4 Gal-13 and gal-14 enhance the secretion of cytokines (IL-8, IL-10, IFN-γ) by immune cells. Cells were treated for 24 hours with different concentrations of gal-13 or gal-14. Cytokine production was measured by ELISA. Graphs show the mean ± SEM of IL-8 (A, B), IL-10 (C, D), and IFN-γ (E, F) cytokine concentrations (n=4). One-way ANOVA and Tukey post-hoc tests were used. Statistical significance was set at *p<0.05, **p<0.01, or ***p<0.001. IFN, interferon; IL, interleukin; SEM, standard error of the mean.
Gal-13 and gal-14 cause phosphorylation of Erk1/2 and p38 MAPKs and of NF-ĸB in immune cells
To identify signaling pathways upon gal-13 or gal-14 stimuli in PBMCs, the phosphorylation of two MAPKs (Erk1/2 and p38) and a transcription factor (NF-κB), all of which are crucial in immune cell functions, was examined. We found that 10–30 minutes of gal-13 or gal-14 treatment significantly phosphorylated Erk1/2 (gal-13: 4.40–13.85-fold; gal-14: 25.79–31.07-fold) (Supplementary Figures 4A, B, and Figures 5A, B) and p38 (gal-13: 3.05–3.77-fold; gal-14: 5.53–6.45-fold) (Supplementary Figures 4C, D, and Figures 5C, D). Furthermore, the phosphorylation of NF-κB was also increased after stimuli with recombinant galectins at all time points (gal-13: 2.47–2.69-fold; gal-14: 2.50–3.39-fold) (Supplementary Figures 4E, F, and Figures 5E, F).
Figure 5 Galectin-13 and galectin-14 induce phosphorylation of Erk1/2, p38, and NF-κB in immune cells. The phosphorylation level of the mitogen-activated protein kinases Erk1/2 and p38, as well as transcription factor NF-κB was examined by Western blot. Freshly isolated PBMCs were incubated with the vehicle, gal-13, or gal-14 for 10, 20, and 30 minutes. Serum-free medium (RPMI-1640) was used as absolute control. Representative membrane strips for Erk1/2 (A), p38 (C), and NF-κB (E), as well as for β-actin as a loading control. Graphs show the mean value ± SEM of densitometry results for Erk1/2 (B), p38 (D), and NF-κB (F) (n=4). One-sample t-test was used. Statistical significance was set at *p<0.05, **p<0.01, or ***p<0.001. Erk1/2, extracellular signal-regulated kinase 1/2; NF-κB, nuclear factor kappa B; SEM, standard error of the mean.
Discussion
Principal findings of the study
The current study provides the following evidence: 1) Gal-13 and gal-14 bind to all investigated PBMC populations: the greatest extent to monocytes, followed by NK cells and B cells, and the least extent to T cells; 2) Gal-13 and gal-14 enhance the viability of PBMCs; 3) Gal-13 and gal-14 decrease the apoptosis of T cells, B cells, NK cells, and monocytes but have no effect on the proliferation of lymphocytic populations under the experimental conditions; 4) Gal-13 and gal-14 increase the production of cytokines in PBMCs, to the greatest extent in the pro-angiogenic and granulocyte chemoattractant IL-8, to a lesser extent in the main anti-inflammatory cytokine IL-10, and to the least extent in the pro-inflammatory and Th17 differentiation inhibiting IFN-γ production; and 5) Gal-13 and gal-14 induce Erk1/2 and p38 MAPKs and persistent NF-κB phosphorylation, which may contribute to the aforementioned effects by modulating the production of anti-apoptotic proteins and cytokines. These findings provide insights into the causal relationship between placenta-specific galectins and maternal-fetal immune regulation, which is further discussed below.
Gal-13 and gal-14 increase cell viability via decreasing the apoptosis of immune cells
Several galectins affect immune responses by regulating cell viability, apoptosis, and proliferation, resulting in a shift in innate and adaptive immune responses at the maternal-fetal interfaces (30, 49, 50, 52). For instance, gal-9 is highly expressed by fetal cells (95) and suppresses cytotoxic T-cell functions by the induction of their apoptosis (96). In this study, we found that placental galectins increase the viability of the examined PBMC populations. Since viability assay alone could not determine whether reduced cell death or increased proliferation resulted in increased viability, we examined how gal-13 and gal-14 affected the apoptosis and proliferation of PBMCs. Both galectins reduced the early apoptosis of T and B lymphocytes, NK cells, and monocytes, and the late apoptosis of B cells and monocytes, in line with the results of the viability test. This is in accord with our recent study showing that gal-13 reduces the apoptosis of purified neutrophil granulocytes, thus promoting the polarization toward a placenta-growth-permissive phenotype, which contributes to trophoblast invasion and normal placentation during pregnancy (87). Our previous study found that gal-13 or gal-14 treatment increased the apoptosis of T cells (57, 60, 78). It is likely that the varying experimental conditions contributed to these differences since earlier studies (57, 60, 78) examined the effect of these galectins on the apoptosis of activated T lymphocytes or lymphocytes already kept in culture for several days, whereas, herein non-activated cells were used for the experiments after a short incubation period. These differences might have affected cell surface glycosylation events, which are known to significantly influence the overall effect of galectins on cell survival (97–99). Additionally, the effect of the microenvironment and surrounding immune cell populations in the mixed cell culture may have also impacted the overall anti-apoptotic activity of gal-13 and gal-14.
An important difference might be that apoptosis-inducing or -inhibiting effects of galectins are highly influenced by the cell-surface glycosylation status of target cells (98). This is tightly regulated by their activation status, e.g., resting, activated, and various differentiation states, determined by the local microenvironment and the set of stimuli reaching the cell. For example, activated Th1 and Th17 cells express a repertoire of glycans required for gal-1 binding and are susceptible to gal-1-induced apoptosis. For example, it was demonstrated that activated T cells and Th1 cells express a repertoire of glycans required for gal-1 binding, which makes them susceptible to gal-1-induced apoptosis, whereas Th2 cells are less responsive to gal-1 binding and gal-1-induced apoptosis due to their 2,6-sialylation on the cell-surface glycoproteins (99). Activated T cells, Th1 cells, and Th2 cells, however, displayed comparable levels of gal-3-induced apoptosis, proving that Th2 cells’ resistance to gal-1-induced death is specific and does not signify a generally higher resistance to apoptosis (99). Another study by Kopcow et al. found that peripheral and decidual T cells have different glycophenotypes, thus a distinct gal-1-binding capacity compatible with higher sensitivity to gal-1 of decidual T cells (40). This finding is supported by the substantial proportion of apoptotic T cells in the decidua due to T-cell death mediated by local gal-1 (48). As neither gal-13 nor gal-14 had an effect on the proliferation of T and B lymphocytes as well as NK cells, we concluded that the anti-apoptotic activity of these placental galectins is responsible for the observed increase in PBMC viability.
Gal-13 and gal-14 induce the production of IL-8, IL-10, and IFN-γ cytokines in immune cells
Earlier, gal-13 was described to increase the production of IL-1ɑ and IL-6 in PBMCs (86) as well as gal-13 and gal-14 to enhance IL-8 production in T cells (78). Herein, we were interested in determining whether gal-13 and gal-14 may influence the production of other cytokines in PBMCs as well. Our experimental conditions differed from the study applied by Kliman et al. (86), also by using DTT and higher galectin concentrations, which might have led to the observation of galectin-induced production of IL-8, IL-10, and IFN-γ in PBMCs in our study. IL-8 can contribute to the development of blood supply at the maternal-fetal interface through its pro-angiogenic effect on endothelial cells (100, 101). IL-8 is also a chemoattractant for neutrophils, so by attracting these cells to the maternal-fetal interface and promoting their phenotypic change, gal-13 and gal-14 may contribute to placentation and the formation of a proper immune environment (87). The increased production of IL-10, which can be produced by several immune cells, may contribute to the shift toward a tolerogenic immune environment in pregnancy (94). Previous publications described that gal-9 increased the production of IL-4, IL-6, and IL-10 by PBMCs (102) and that gal-1 also increased the production of IL-10 but decreased the production of IFN-γ by T lymphocytes (103). Since IFN-γ is produced by NK cells, inhibits Th17 differentiation (104), and promotes decidual NK cell differentiation and placental development (93, 105), a modest IFN-γ production upon gal-13 and gal-14 treatment may also show profound effects at the maternal-fetal interface. Decidual T cells express a high level of IFN-γ besides IL-4 (106), which may also relate to local effects of progesterone (107). Therefore, in the future, it would be interesting to study whether placental galectins can contribute to the phenotypic changes of these immune cells in the decidua.
Gal-13 and gal-14 regulate immune cells by the activation of Erk1/2, p38, and NF-κB signaling pathways
Galectins were found to influence immune cell functions via key signaling pathways, e.g., the MAPK and NF-κB pathways (108–113). However, the interaction partners and receptors of gal-13 and gal-14 expressed on immune cells, and the effects of these galectins on signal transduction, have not yet been revealed. Herein, we showed that gal-13 and gal-14 treatment induced the phosphorylation of Erk1/2 and p38 MAPKs and the persistent phosphorylation of the p65 subunit of the NF-κB transcription factor. These signaling molecules are essential for the generation of immune responses, such as inflammation, pathogen recognition, T- and B-cell responses, and lymphocyte longevity (15, 114–117), and several galectins, e.g., gal-1, gal-8, gal-9, have been shown to induce or inhibit their activation (108, 110, 118, 119). For example, it was found that gal-8 induced apoptosis in Jurkat T cells by triggering an Erk1/2-dependent death pathway (108). In another study, human monocytes upregulated FcγRI and downregulated MHC-II surface expression in an Erk1/2-dependent manner upon gal-1 treatment (110). It was also demonstrated that gal-9 increased the phosphorylation of Akt, c-Jun N-terminal kinase, and Erk1/2—the key kinases involved in NK cell function—in peripheral TIM-3+ NK cells, which led to their suppressed activation (118). Furthermore, gal-14 was found to regulate cell function through the interaction with the transcription factor c-Rel, a subunit of NF-ĸB, which has many functions in pathways related to inflammation, apoptosis, cell growth, and cell differentiation, among others (119). However, in the current study, gal-14 was overexpressed in a HeLa (cervical cancer) cell line, in which gal-14 is normally not expressed, so the results may not be physiologically relevant (119). In light of these findings, it will be valuable to investigate additional signaling pathways that may be regulated by gal-13 and gal-14.
Of significance, while intracellular galectins can bind to proteins, extracellular galectins bind to glycan sidechains of glycoproteins at cell surfaces. Therefore, various glycoproteins with the same carbohydrate moieties can be considered as receptors for placental galectins, similar to other galectins. Indeed, we have shown previously that gal-13 differentially bound to red blood cells due to its varying affinity for blood-group AB0 glycans. For example, gal-13 binds blood-group AB erythrocytes with the strongest while blood-group B erythrocytes with the weakest affinity (120). The affinity of gal-13 and gal-14 to certain mono- or disaccharide ligands was also revealed (53, 59, 62). It is an interesting question whether placental galectins can bind to the glycan chains on the surface of extracellular vesicles secreted or deported from the placenta, which would add another layer of signaling events between the fetus and the mother. Earlier studies showed that gal-13 is indeed localized in/on extracellular vesicles, but the ways of this binding require further examination (62, 65). Future studies also need to investigate the cell surface glycoprotein receptors of these galectins on white blood cells and how the glycosylation pattern of these cells may determine the strength of galectin binding and galectin-induced signaling events.
Strengths and limitations of the study
Strengths: 1) A large set of functional assays was applied to examine the effects of gal-13 and gal-14 on immune cells obtained from non-pregnant female donors; 2) we used a mixed PBMC population instead of purified cell populations to better mimic the physiological conditions in human circulation. However, future investigations of isolated immune cell populations shall reveal more insights into the effects of gal-13 and gal-14 on specific immune cell types; and 3) recombinant galectins were expressed in a modified bacterial vector to avoid lipopolysaccharide contamination and biased immune cell activation. Limitations: 1) The applied quantities of gal-13 and gal-14 were supraphysiologic relative to that found in human circulation. However, these concentrations were similar to those previously used by us and others (10 to 100 µg/mL) to study the functional effects of a large set of galectins (121, 122). Furthermore, galectin blood concentrations do not represent their effective tissue or cell surface concentrations, where galectin-glycan lattices present a high local galectin concentration (123–126). Another technical rationale for using larger galectin concentrations was to prevent subunit dissociation in the DTT-containing solvent, which could lead to functional changes (127); 2) due to the use of mixed PBMC populations, we cannot exclude the effects of immune cell populations on each other; and 3) immune cells were isolated from non-pregnant female donors, which do not represent a perfect match for those cells that could be isolated from pregnant women. However, this method was the only option to avoid pre-exposure of immune cells to gal-13/gal-14 binding, which could have biased our studies. Nevertheless, future studies shall also characterize the effects of gal-13 and gal-14 on peripheral blood and decidual leukocytes isolated from pregnant women, since pregnancy hormones may strongly impact the glycosylation pattern and the galectin-binding capacity of these cells.
Conclusions
We demonstrate herein that gal-13 and gal-14 bind to non-activated PBMC populations, increase their viability by reducing their apoptosis, and induce the production of IL-8, IL-10, and IFN-γ, probably by the involvement of Erk1/2, p38, and NF-ĸB signaling pathways (Figure 6). Our findings suggest that these placental galectins regulate immune cells and their decreased expression in obstetrical syndromes may play a role in the severe immunopathology of these syndromes.
Figure 6 The effects of placental galectins on leukocyte functions. Gal-13 and gal-14 bind to the surface of immune cell populations where they activate Erk1/2, p38 MAPK, and NF-κB signaling pathways. These signaling effects of placenta-specific galectins increase the viability of leukocytes, decrease the apoptosis of immune cells without influencing the proliferation of cells, and induce the production of IL-8, IL-10, and IFN-γ in a different manner. Erk1/2, extracellular signal-regulated kinase 1/2; IFN, interferon; IL, interleukin; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B.
Data availability statement
The raw data supporting the conclusions of this article willbe made available by the authors, without undue reservation.
Ethics statement
Ethical review and approval were not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.
Author contributions
OO, JM, NGT, and AB conceptualized the study and designed research. OO, ET, JK, and AB performed research. RR, NGT, and AB contributed new reagents/analytic tools/clinical specimens. OO, RR, JK, MP, DG, SR, AT, OE, ZP, JM, NGT, and AB analyzed and interpreted data. All authors contributed to the article and approved the submitted version.
Funding
This research was funded, in part, by the Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, USA Department of Health and Human Services (NICHD/NIH/DHHS); in part, with Federal funds from NICHD/NIH/DHHS under Contract No. HHSN275201300006C (RR); by the Hungarian Academy of Sciences Momentum Grant No. LP2014-7/2014 (NGT) and Premium Postdoc_2019-436 Grant (AB); and by the Ministry of Innovation and Technology of Hungary from the National Research, Development Innovation Fund financed under the OTKA K124862 (NGT) and FIEK_16-1-2016- 0005 (NGT) funding schemes.
Acknowledgments
We thank Judit Baunoch (Research Centre for Natural Sciences) for her technical assistance and Maureen McGerty (Wayne State University) for her critical reading of the manuscript.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2022.1088024/full#supplementary-material
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Keywords: danger signal, evolution, glycomics, leukocyte, obstetrical syndrome, PP13, primate, trophoblast
Citation: Oravecz O, Romero R, Tóth E, Kapitány J, Posta M, Gallo DM, Rossi SW, Tarca AL, Erez O, Papp Z, Matkó J, Than NG and Balogh A (2022) Placental galectins regulate innate and adaptive immune responses in pregnancy. Front. Immunol. 13:1088024. doi: 10.3389/fimmu.2022.1088024
Received: 02 November 2022; Accepted: 05 December 2022;
Published: 28 December 2022.
Edited by:
Kirsty Pringle, The University of Newcastle, AustraliaReviewed by:
Laszlo Szereday, University of Pécs, HungaryVadim V. Sumbayev, University of Kent, United Kingdom
Hamutal Meiri, ASPRE-T consortium and TeleMarpe LTD, Israel
Copyright © 2022 Oravecz, Romero, Tóth, Kapitány, Posta, Gallo, Rossi, Tarca, Erez, Papp, Matkó, Than and Balogh. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Nándor Gábor Than, than.gabor@ttk.hu