Preimplantation Factor (PIF) Promotes HLA-G, -E, -F, -C Expression in JEG-3 Choriocarcinoma Cells and Endogenous Progesterone Activity

Mammalian pregnancy involves the successful transplant of a semi-allogeneic or allogeneic graft, whether originating through natural conception, donor embryo acceptance or cross-species embryo transfer. Paradoxically, the maternal immune system remains competent and active during pregnancy and does not reject the foetus, as it would any other transplant [1]. Embryo rejection, in fact, indicates a pregnancy complication. It is also noteworthy that autoimmune conditions, unless severe, improve during pregnancy only to recur post-partum, indicating the existence of a unique temporary immunological milieu specific to pregnancy [2-4]. Despite extensive investigations, an inclusive explanation as to why the foetus is offered special immunologic privilege has not been forthcoming [5, 6]. It is assumed, however, that pregnancy-specific compounds play an important role [7].

Placental trophoblast cells play a key role in maintaining tolerance to the foetus [8]. Extravillous trophoblasts (EVTs), which invade the decidual stroma (interstitial invasion) and open the uterine spiral arteries [9-11], selectively express the non-classical class Ib antigens (Ag) HLA-G, HLA-E and HLA-F, and HLA-C, a non-classical class Ia Ag [10]. However, HLA-A and -B, both T-cell related HLA ligands [12] are absent from EVT, which may prevent attack by maternal cytotoxic CD8+ T lymphocytes. Progesterone (P4) promotes trophoblast invasion [13], and it increases HLA-G expression in primary trophoblasts and JEG-3 cells [14-16]. In JEG-3 cells, P4 is able to induce heterotypic associations between HLA-G and -E and cell-surface expression of HLA-C, -E and -G [15, 17]. However, early in gestation P4 is of corpus luteum origin, and the level of circulating P4 is low [18]. Effective local steroid production is only taken over by the placenta by week 12 of gestation [19]. Thus, the role of pregnancy specific endogenous compound(s) in regulating trophoblast class I HLA molecules remains currently incomplete. Our premise is that immune modulation and embryo/foetus acceptance are specifically embryo-derived and embryo-driven, in coordination with the maternal immune response.

PreImplantation Factor (PIF), a small peptide secreted by viable embryos, is likely to play an important role in maternal recognition that leads to tolerance for the semi/allogenic embryo [20-22]. PIF is detectable as early as the two-cell stage and is associated with embryo development [23, 24]. Circulating levels of PIF during early pregnancy are associated with a favourable pregnancy outcome [25]. PIF has an essentially autotrophic effect on embryo development, which is blocked by anti-PIF antibody [23]. In the embryo, PIF targets protein-disulfide isomerase/thioredoxin and heat shock proteins (HSPs), promoting embryo development and protecting against serum toxicity [23, 24, 26]. Additionally, PIF lowers natural killer (NK) cell toxicity [27]. PIF promotes endometrial receptivity to support embryo implantation [28-30]. It regulates both systemic and local maternal immunity, creating Th2 bias while preserving an effective anti-pathogenic Th1 response [31-33]. These findings have been translated to treatment of diverse immune disorders, transplantation, and brain injury models outside of pregnancy. Recently, a Fast-Track FDA clinical trial evaluating PIF for the treatment of an autoimmune disorder has been completed satisfactorily (NCT02239562) [34-41].

PIF expression in the placenta is highest shortly post-implantation, and declines around term [20, 25, 42]. A premature decline in PIF has been associated with preeclampsia and intrauterine growth retardation, thus evidencing the peptide’s important role in maintaining effective placental function [41, 43]. PIF promotes invasion by extra villous cells (EVTs), without affecting these cells’ proliferation [42, 44]. This was shown with transformed trophoblasts and confirmed by using primary human EVTs. This in line with data showing that EVTs HLA-G+ are drivers of the immune response through their interaction with decidual immunity [45]. PIF’s effect on EVTs invasion is dependent on increased metalloproteinase 9 and reduction of its inhibitor and integrin regulation. Pathway analysis demonstrated that PIF action is dependent on the MAPK, PI3K, and JAK-STAT pathways [42]. PIF acts on, and its effect is dependent on, critical apoptosis regulating the p53 pathway [46]. Relevance of the p38 MAPK/ERK signalling pathway also in polychlorinated biphenyls -induced apoptosis of human transformed EVT was reported [47]. Presently, there is limited information on local compounds that regulate HLA expression, especially during the earliest stages of pregnancy when it is most critical. Both PIF and HLA-G are secreted by viable embryos [23, 48] and interact intimately from the earliest stages of embryo development. This interaction continues, with PIF and HLA-G present in the circulation in later pregnancy. PIF and HLA-G are expressed by the trophoblast, as PIF is expressed by the viable embryo immediately post-fertilization and by the trophoblast shortly after implantation [23, 42]. Both ligands may therefore play a local regulatory role in trophoblast HLA class I function.

HLA-G expression in different trophoblastic cells was previously examined showing that JEG-3 cytotrophoblast cells expression is higher than Bewo and Jar cells [16]. In the present study, the effect of PIF on the expression of the HLA class I molecules HLA-G, -C, -E and -F in JEG-3 cells are examined using a novel and validated co-localisation and image processing approach [15]. Results were confirmed by proteome analysis. The effect of PIF was compared to that of progesterone (P4), a known HLA-G/HLA-E regulator. Whether PIF regulates endogenous P4 activity and Th1/Th2 cytokine secretion was also determined. Herein we reveal that PIF up-regulates several HLAs, potentiates progesterone action and promotes Th1/Th2 cytokine secretion by trophoblast cells. Since PIF is being tested clinically, our observations support its use for the treatment of early pregnancy disorders.

Synthetic PIF (MVRIKPGSANKPSDD) was obtained from Biosynthesis, Lewisville, NJ USA. Peptide had >95% purity, documented by mass spectrometry before use. PIF was dissolved in PBS with 0.01% dimethyl sulfoxide (DMSO) (SIGMA, Missouri, USA). Progesterone (P4) (Sigma-Aldrich, Missouri, USA) was dissolved by using absolute ethanol. IL-17RA (Life Technologies) was dissolved in Millipore water.

HLA-G and HLA-E antibody specificity was previously validated through FACS at the Third International Conference on HLA-G (Paris, July 2003) [49] and through separation validation studies, as reported by Palmisano [50] and Zhao [51]. [52] MEM-G/09 (EXBIO Praha, Vestec, Czech Republic), the IgG1 conformational antibody against HLA-G1 and HLA-G5, previously defined for fluorescence-activated cell sorting (FACS) and immunohistochemistry (IHC) staining, was used. For Western blotting, the MEM-G/01 (IgG1) (EXBIO Praha) clone, which recognizes the denatured HLA-G heavy chain of all isoforms, was used. For detection of the HLA-E molecule using Western blotting, MEM-E/02, (IgG1) (EXBIO Praha), which reacts specifically with all denatured HLA-E molecules and does not cross-react with HLA-A, -B, -C or -G, was used. For FACS and IHC staining, MEM-E/07 (IgG1) (EXBIO Praha), which recognizes the native surface HLA-E molecules, was used; this however is reported to cross-react with the classical MHC class I molecules HLA-B7, HLA-B8, HLA-B27 and HLA-B44. Anti-HLA-F clone 3D11 (IgG1), which recognizes the native and denatured forms of HLA-F and does not cross react with any other HLA-F type, was kindly provided by Dr. Daniel Geraghty (Seattle), and used for FACS, Western blotting and IHC staining. L31 (IgG1) (Media Pharma, Chieiti, Italy) antibody is known to bind to an epitope present on all HLA-C alleles (CW1 through to CW8), and is also known to react with HLA-B alleles (HLA-B7, -B8, -B35, -B51 and others). It was used for both FACS and microscopy, whilst the HLA-C clone D-9 (Biolegend, San Diego, CA, U.S.A) was used for Western blotting.

For determining the effect of PIF and P4 on HLA class I molecule expression, JEG-3 cells were passaged and cultured at a density of 1x10⁶ cells/ml in complete medium. After 24 h, the cells were serum starved by replacing the medium with DMEM-F12 supplemented with 0.1% FCS. Cells were incubated for 6 h, after which the medium was refreshed and supplemented with PIF (0-1000 nM), added for 24-72 h, or P4 (0-1µg/ml), added for 24h, as recently described [15]. Cells without PIF or P4, or serum free cultured cells, were used as control. The collected cells were further tested for expression for class I HLA molecules by using specific monoclonal antibodies.

Treated and untreated JEG-3 cells, following exposure to test agents were detached, counted and pelleted as described previously [14]. They were immediately lysed using SDS-lysis buffer, vortexed and heated at 95°C for 5 min. Cell lysates were stored at -20°C until used for protein analysis. To quantify the final concentration of the proteins we used the Bradford assay following the previously described protocol with some modifications [15]. Briefly, bovine serum albumin (BSA), at a concentration of 4 mg/ml, was used as a calibration standard. Five µl of BSA was diluted sequentially in a 96-well microplate prefilled with PBS to produce the standard curve. Five µl of each protein sample was then diluted 1: 1 with PBS and incubated with the reading reagent at room temperature for 5 min. Finally, the protein concentration was determined using a spectrophotometer, extrapolating each absorbance value over the previously created standard curve. Samples were diluted in SDS-sample buffer, heated for 5 min at 95 °C and loaded onto a polyacrylamide gel (12% resolving gel and 4% stacking gel). The samples were run for 30 min at 30 V, following increase to 100 V until the gel finished running. A pre-stained standard protein marker (Li-Cor Bioscience) was also loaded onto the gel and run parallel to the protein samples to be analysed.

The proteins resolved using SDS-PAGE were then transferred onto a PVDF membrane (Immobilion Millipore Inc.), using a Mini Trans-Blot Cell (Bio Rad). Briefly, before transfer the SDS-PAGE gel was incubated in gel running buffer (25 mM Tris/HCl, 250 mM glycine, 0.1% SDS) for 15 min. The PVDF membrane was hydrated in absolute methanol for 10 s and immediately washed with molecular biology grade water. A stack consisting of sponge, Whatman paper soaked in transfer buffer (20mM Na₂PO₄, 2% Methanol, 0.05% SDS), the PVDF membrane, the SDS-PAGE gel, Whatman paper and sponge, was then made. This stack was placed on the electro blotter and transferred to the blotting system. The transfer was done for 1 h at 110 mA and 40 V. After transfer, the membrane was washed with molecular biology grade water and incubated in blocking buffer (0.1% Tween, 3% dried skimmed milk and PBS) for a 1 h at room temperature or overnight at 4 °C. After blocking, the membrane was washed and incubated with the primary monoclonal antibody for HLA-G, -E, -C and -F overnight at 4 ºC. The membrane was then washed three times (10 min each wash) and incubated with secondary antibody (IRDye 800CW^© Donkey anti-Mouse IgG from Li-Cor Biosciences) for 1 h at room temperature. Membranes were read using an Odyssey^© infrared imaging system (Li-Cor Biosciences). Semi-quantification of each antigen studied using this technique was attained by comparing loading control band (BSA) brightness and thickness using ImageJ software (http://imagej.net/).

For surface antigen expression analysis, cells were detached using Accutase and washed with PBS. Cells were counted and at least 1x10⁶ cells were used per sample. Each sample was blocked with 0.1% BSA in PBS for 30 min at room temperature. For intracellular staining, after detaching and washing cells, the pellet was fixed with 4% paraformaldehyde on ice. Cells were then washed with 0.1% saponin-BSA in PBS, and permeabilized for 10 min at room temperature using 0.3% saponin in PBS.

Cells were washed with PBS and incubated with saturating concentrations of primary HLA antibodies, followed by washing and labelling with a conjugated secondary antibody. Cells were then re-suspended in 500 µl of PBS and at least 10, 000 events were acquired using a BD FACS Aria I equipped with the FACS Diva software (BD Biosciences). The raw data analysis was performed using FlowJo Vx software (Tree Star Inc.).

For surface antigen quantification, a previously described protocol was followed [15]. Briefly, we used the Qifikit beads kit (Dako). The cells were prepared following the same step as for flow cytometry up to the primary HLA antibody staining stage. For detection of antibody staining cell samples, set up beads and calibration beads were all stained with FITC conjugated secondary antibody. A FACS Aria I was calibrated for the cell isotype and setup beads. A calibration curve was constructed for mean fluorescence intensity for each population of beads. The cell antigen-binding capacity was calculated by extrapolation on the calibration curve.

The effect of PIF (200nM) and IL-17 (0-100 ng/ml) on P4 secretion was tested by measuring culture supernatant P4. JEG-3 cells were cultured for 24-72 h, after which supernatants were collected and kept at -20ºC until determining P4 levels by ELISA. Cells cultured in incomplete media (DMEM/Ham’s F12) were used as controls

JEG-3 cells were seeded in 8-well Lab-Tek chambers (Thermo Fisher Scientific) at a density of 8x10³ per well. Cells were grown in complete medium up to 60% confluence, after which they were incubated in a serum-starved medium (0.1% FCS) for 24 h. The cells were then treated with PIF at a concentration of 200nM for 24 h followed by fixing with 4% PFA at 4 °C and permeation with 0.25% Triton X-100 in PBS. The reaction was then treated with 2% BSA in PBS to block non-specific binding, for 1h at room temperature. Cells were then incubated with the primary anti-human monoclonal antibodies anti-HLA-G (clone MEM-G/09) and anti-HLA-E (clone MEM-E/07) at a dilution of 2 µg/100 µl in PBS for 1 h and anti-HLA-C (clone L31) and anti-HLA-F (clone 3D11) at a dilution of 1 µg/100µl in PBS for 1 h. Cells were washed with PBS, after which they were incubated with anti-mouse IgG conjugated with Alexa Fluor 488 or Alexa Fluor 555 (Invitrogen, Carlsbad, CA, USA) at a dilution of 0.25 µg/100 µl for 1h at room temperature. Cells were washed once again and air dried. The cells were then mounted and the cell nuclei stained using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA), covered with coverslips (Chance proper LTD, West Midlands, England) and sealed with Marabu Fixogum rubber cement (Marabuwerke GmbH & Co. KG, Tamm, Germany).

Cell lysates and their protein concentration were prepared and assessed as previously described [53, 54] Frozen cell pellets were dissolved in hot lysis buffer (1% SDS, 100 mM Tris-HCl), and sonicated. Five percent of 2-mercaptoethanol was added. Samples were then dissolved in solubilisation buffer (8 M urea, 2.5 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesuflate, 50 mM DTT, 24mM spermine tetrachloride) at room temperature for 1 h. The samples were centrifuged at 12000 x g for 30 min. To concentrate the protein samples, acetone precipitation was used. Once the supernatant was discarded the protein pellets were washed with a mixture of ice cold methanol-chloroform, and re-suspended in isoelectric focusing buffer (IEF) (8 M urea, 2.5 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesuflate).

For first dimensional isoelectric focusing buffer (IEF), immobilized pH Gradient (IPG) dry strip gel pH 3-10NL (Bio-Rad), was rehydrated for 24 h at room temperature with a mixture containing 55 µg of protein lysate dissolved in IEF buffer. The IPG strips were then focused using an IPGphor system (BioRad, USA). Rehydrated filter wicks were placed between the electrodes and the IPG strip. Separation of proteins in the first dimension was carried out at 20 °C for 24 h, following the manufacturer’s protocol. After IEF, strips were rinsed in deionized water, and stored at -80 °C until the second dimensional IEF.

For second dimensional IEF, IPG strips were incubated in equilibrium buffer 1 [6 M/l urea, 20% w/v glycerol, 4% w/v SDS, 0.375 M/l Tris-HCL (pH 8.8), 5% 2-mercaptoethanol] for 15 min at room temperature. After that the strips were moved to equilibrium buffer 2 [6 M/l urea, 20% w/v glycerol, 4% w/v SDS, 0.375 M/l Tris-HCL (pH 8.8), 2.5% 2-mercaptoethanol] and incubated for another 15 min at room temperature. Strips were then rinsed in electrophoresis buffer (60.57 g Tris base, 288.27 g glycine, 20 g SDS, double distilled H₂O up to 20 L). The second dimensional analysis was carried out using a Criterion gel system (Bio-Rad). Each strip was placed into the well of a 12% SDS-PAGE gel (Bio-Rad), and sealed with agarose solution. Electrophoresis was carried out at 50 V for 30 min, and following this at 150 V for 4-5 h. The gel was then fixed overnight in fixing solution (50% methanol, 5% acetic acid, 45% water), and stained using the silver staining protocol [55, 56].

The gels were scanned and analysed using SameSpots analysis software (Nonlinear dynamics Ltd). Digitized images from 12 silver stained gels were analysed for spot detection and quantification. Image analysis included spot detection, editing, background subtraction and spot matching. A master image was then created, and all spots in the other gels were matched to this master image both manually and digitally. The size of a protein, which approximates the volume of the spot, was calculated by using the software. Spots identified as differentially present were excised and analysed using mass spectrometry (MS) analysis.

Identified spots were manually excised from the 2-DE gels using a disposable sterile scalpel spot cutter and washed in molecular biology grade water. Each spot was then subjected to in gel digestion alkylation, along with tryptic digestion so as to yield peptide fragments for MS analysis following previously described protocols [57].

Peptide digests were subjected to MS analysis at the sub-picomole level through the use of a matrix assisted laser desorption ionisation-time of flight (MALDI-TOF MS) so as to generate peptide mass fingerprinting (PMF). The MALDI-TOF analysis was carried out using a Bruker Daltonics Reflex IV instrument using a linear mode with a laser power attenuation setting at 30, as described previously [58]. Once the data were examined and calibrated using the program M/Z, the results were transferred to the MASCOT peptide search engine (Matrix Science Limited) for protein identification.

The expression data were pre-processed by mean-centring (by division and medians), and over median normalized data. A heatmap was generated via hierarchical clustering for examining protein expression and treatment (http://cran.r-project.org). The generation of a heatmap for median centred protein expression data with horizontal hierarchical clustering of different proteins utilized the median linkage agglomeration method and vertical hierarchical clustering of treatment conditions, using complete linkage agglomeration. A correlation distance metric was used for clustering data.

Fold change of PIF treated vs. control and P4 treated vs control was applied on the raw expression data. (The spot volume data used followed the assumption that spot volume correlates and is a function of the protein expression level.) The expression ratios and the corresponding p-values obtained from the statistical tests applied over the differential expression data were imported into EGAN (http://akt.ucsf.edu/EGAN/). The proteins (Hugo Symnol represented) that mapped to the Entrez Genes reference were subjected to further analysis and network visualization. Selected proteins were used for related associated nodes enrichment, thus obtaining the linked annotation; GO, REACTOME, NCI-Harvard Pathways and others.

In preliminary studies, the cytokine secretion profiles of JEG-3 and ACH-P3 trophoblastic cells were determined. Subsequently, the effect of PIF and P4 on JEG-3 cell cytokines was examined, comparing against un-stimulated cell culture supernatant. At the end of the experiments the supernatant was collected and analysed for cytokine levels. The concentrations of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, IL-8, IL-10, interferon gamma (IFN-γ), transforming growth factor beta 1 (TGF-β1) and granulocyte macrophage colony-stimulating factor (GM-CSF) were measured using ELISA kits (eBioscience), following the manufacturer’s instructions.

Statistical analysis was performed using the software SigmaPlot version 12.5 (Systat Software Inc., India). The descriptive statistics and normality test were done as a first step. If the population followed a normal distribution (Gauss’ Bell), Student’s t-test was used to compare the different groups (treated vs. non-treated, surface vs. intracellular antigen expression, etc.). If the population did not follow a normal distribution Mann-Whitney U test was used. The multiparametric comparison was performed by ANOVA. The p<0.05 value is considered significant.

PIF promotes trophoblast invasion and protects against apoptosis [46]. Whether PIF is involved in regulating HLA class I antigen expression to promote maternal tolerance is not known. The viable embryo secretes both PIF and soluble HLA-G into the culture media [16, 48]. PIF promotes invasion, regulates the immune response and has anti-apoptotic effects. Since PIF is expressed by the trophoblast shortly post-implantation its potential regulatory role on local HLA class I molecules was examined. This is relevant for understanding the development of tolerance from the earliest stages of gestation.

The effect of synthetic PIF (1–1000 nM) on HLA-G expression in JEG-3 cells was determined by using serum free media. JEG-3 cells are here shown to be superior to ACH-3P and therefore were used for all experiments in the current study [52]. This was substantiated by the following observations. 1. ACH-3P cells are comprised of two populations of cells, however only in 40% of the cells HLA-G+ is expressed [52]; 2. The expression of HLA-C, -E, -F, G is higher in JEG-3 cells than in ACH-3P cells [52].; 3. HLA-C was only expressed in JEG-3 cells. 4. IL-1α, IL-8, IL-10 and TGF-β1 levels were higher in JEG-3 cells than in ACH-3P cells [52].

To validate measurements of the mean fluorescence intensity (MFI) a calibration curve was constructed, plotting against antigen binding capacity. For validating the MFI binding target data, a double logarithmic graph was used to provide the best fit, R²=0.99 (Fig. 1). This methodology was used in all subsequent experiments.

PIF was found to increase HLA-G levels in JEG-3 cells in a dose-dependent manner (Fig. 2A). PIF promoted HLA-G expression at all concentrations tested (up to 1uM). The highest effect was noted testing PIF at 200nMconcentration. In the time course experiment, 200 nM PIF added for 24 h in culture induced the most pronounced increase in HLA-G expression (28 fold) (Fig. 2B). This supports the hypothesis that PIF’s role is to be a driver of tolerance.

HLA molecules are expressed both intracellularly and on the cell surface. The effect of PIF on intracellular HLA-G, -E, –F and -C expression by JEG-3 cells was determined (Fig. 2C). PIF increased the expression of all tested HLAs. HLA-G exhibited the highest level of expression followed by HLA-E. The increase in intracellular expression was coupled with an increase of cell surface HLA-G and HLA-E (p< 0.01) (Fig. 2D). HLA-C expression also increased on the cell surface (p< 0.05). However, PIF only minimally affected surface HLA-F expression. The time-dependent increase in HLA-F and HLA-E expression was confirmed using Western blotting, which demonstrated that the maximal increase was already attained at 24 h of culture (Fig. 3A-D) Thus, PIF activates class I HLA molecules.

JEG-3 cells cultured with 1µg/ml P4 for 24 h produced increased levels of HLA-G, -E, -F and -C, with HLA-G expression being the most pronounced (Fig. 4A). Intracellular and surface measurements demonstrated significant P4-induced HLA expression (Fig. 4B, 4C). Significant differences between intra- and extracellular HLA expression were noted. The highest increase was noted with HLA-G, followed by HLA-E and a mild effect was observed on HLA-C expression (Fig. 4C). This confirmed that the 1µg/ml dose at 24 h was most effective. This observation permitted comparison of the effect of PIF with the most effective concentration of P4.

The effect of 200nM PIF was compared with 1µg/ml P4 after incubation for 24h. Western blot analysis showed that, compared to P4, PIF induced increase was more pronounced in both HLA-G and HLA-E expression (p<0.01). Basal expression of HLA-C and HLA-F was low. However, PIF induced a higher expression of both ligands as compared to P4 (Fig. 5A). Flow cytometry analysis and the antigen quantification data confirmed the Western blot data. The PIF-induced increase in both intracellular and surface HLA expression was also more pronounced compared to the effect of P4 (Fig. 5B). The most pronounced effect noted was on HLA-G and HLA-E expression (Fig. 5C). The increase in HLA expression at the intracellular level translated to an increase at the surface level, with PIF-treated cells displaying high MFI and surface antigens in comparison to P4-treated cells.

PIF promoted the expression of IL-17F, a pro-inflammatory cytokine in the trophoblast that plays an important role in angiogenesis [46]. Using a dose-dependent design, 1-100 ng/ml IL-17 promoted P4 secretion by JEG-3 cells, as determined by ELISA. The maximal effect was noted at 10ng/ml IL-17 (p<0.01) (data not shown). Both 200nM PIF and 10ng/ ml IL-17, after 6-72 h of culture, increased P4 secretion by JEG-3 cells, observed at 6 and 24 h, respectively. (Fig. 5D). Thus, PIF directly promotes P4 secretion through an IL-17-dependent pathway.

Following culture of JEG-3 cells until confluence, the cells were washed and cultured in serum-free media in the presence of 200nM PIF. The effect of PIF on HLA-G expression was examined after 24 h of incubation. It was found that the expression of HLA-G in PIF-treated JEG-3 cells (anti-HLA-G mAb stained) increased (Fig. 6). Further, DAPI staining revealed an intact nuclear structure. This provided additional evidence that PIF promotes HLA-G expression.

To determine whether PIF also regulates the placental proteome, the effect of 200nM PIF and 1µg/ml P4 on protein expression by JEG-3 cells was analysed by 2-DE gel electrophoresis. Proteins were separated in the first dimension based on their pI, which ranged from 3-10, and, based on their size, by SDS-PAGE in the second dimension. The analysis of the master gel detected 22 spots showing differences between treatment and control cells.

A comparison of the average spot volume (mean ± SD, 4 samples/group) of the 22 spots was carried out. Significant differences between PIF-treated and untreated JEG-3 cells were found. Fourteen spots from the PIF-treated group decreased, whereas nine significantly increased. Comparison of P4-treated cells with PIF’s effect was also significant. The 22 protein spots identified by mass spectrometry analysis were validated by using a ProFound probability score of 0.99-1.00. Since not all spots could be confirmed further, bioinformatic and pathway analysis was performed only on the 19 validated proteins (Table 1).

Fig. 7 shows the heat map visualized effect of PIF on the placental proteome. GO analysis of the biological functions of the 19 significant proteins showed that they are mainly related, in terms of response to stimulus, regulation of biological process, multicellular organismal process, cell communication, developmental process and cell proliferation. The proteomic data confirmed that PIF-induced an increase in HLA-G, validating the antibody based data. FOXP3+, an activation marker of pro-tolerance Tregs, also increased. Remarkably, PIF increased the P4 receptor (PRGR, 2.5 fold) protein coupled with the increase in P4 secretion (see above) which potentiates P4 action in trophoblasts. In addition, PIF increased levels of pro-coagulation factors DCBD2 V/VIII-Homology domain, TTP, Von Willebrand Factor-Cleaving Protease and CD59, involved in complement regulation. ATP5B, which is H+ transporting, mitochondrial F1 complex and ZSC21, a potent transcriptional activator, and CLIC3 and HBB, were overexpressed as well.

In contrast, proteins involved in oxidative stress and protein misfolding were reduced, including PDIA3, protein disulfide isomerase A3, PRDX2 peroxiredoxin-2, HS74L, (HSP70-4)- and HSP71 (HSP70-8). These are prime PIF targets which are also regulated in vivo [26, 33, 37]. Mitogenic effects of EGF were reduced by lowering its receptor levels, which is likely to support trophoblastic differentiation. Also, EPCAM, calcium-independent cell adhesion and LMNA and EF2 proteins were decreased, which catalyse the GTP-dependent ribosomal translocation step. Metabolic KPYM pyruvate kinase and G3P glycerol-3-phosphate dehydrogenase-1 proteins were decreased as well. Thus PIF, in contrast to P4, has a dual regulatory and protective action.

The detected differentially expressed proteins were analysed, using hierarchical clustering of normalized protein expression and treatment conditions (PIF vs. P4) and Exploratory Gene Association Networks (EGAN) analysis to explore the involvement of PIF in differentially expressed networks. Hierarchical clustering revealed that PIF reciprocally down-regulated half the proteins compared to control cells. It also triggered up-regulation in nine cases, in contrast to P4.The top up-regulated cluster was ranging from HLAG to FOXP3. On the other hand, P4 up-regulated only five of 19 differentially expressed proteins, with the remaining down-regulated (Fig. 8).

When EGAN was applied, the CLIC3 protein differed only in terms of its expression mode, since PIF up-regulated while P4 down-regulated the same protein. This protein is a voltage-dependent chloride ion channel and participates in cell membrane potential stabilization. The increase in CLIC3 may protect against intrauterine growth retardation and preeclampsia, since PIF expression is low in this condition [46, 59]. EGAN performs a hypergeometric enrichment of linked annotations to the gene-related nodes, and in the case of PIF treatment, FOXP3 is up-regulated and linked to NFAT transcriptional control, while PDIA3 is linked to oxidative stress and calcium signalling, which is decreased. Similarly, several pathways and processes are affected by EGFR down-regulation, with few selected and represented, for example for signal transduction mediated by PTPB1, by IL1 and the related protein-protein interaction of EGFR with LMNA (increased Apoptosis related Caspases). PDIA3 (increased calreticulin based calcium signalling) and ATPB5 (decreased MHC protein binding) are also targeted by PIF [26]. Thus, proteome data confirms PIF’s promoting effect on HLA-G and the P4 receptor while reducing oxidative stress and protein misfolding.

Since PIF was effective in promoting HLA expression, particularly that of HLA-G, we examined whether such stimulation also extends to an effect on the production of cytokines needed to interact with the maternal milieu. The effect of 200 nM PIF on JEG-3 cell cytokine secretion was compared with 1 µg/ml P4 using serum-free media for 24 h. PIF increased production of both clinically relevant Th1 and Th2 type cytokines (IL-10, IL-1β, IL-8, GM-CSF and TGF-β1). In contrast, P4 only increased IL-10 secretion (Table 2). The results show that PIF induces a balanced secretion of cytokines, promoting tolerance whilst sustaining anti-pathogen protection. However, the effect of P4 is limited.

Consistent with a prior study, the expression levels of HLAs and secreted cytokines in JEG-3 cells were superior to those in ACH-3P cells and the JEG-3 cell line was herein used [14]. We demonstrate that in JEG-3 cells PIF up-regulates the pivotal pro-tolerance molecule HLA-G and the associated class I molecules HLA-C, -E and -F. Proteome analysis confirmed up-regulation of HLA-G, pro-tolerance Tregs (FoxP3+), coagulation factors and complement, and the reduced expression of proteins involved in oxidative stress and protein misfolding. Reduced EGF receptor expression may limit the proliferative potential of the trophoblast. PIF’s promoting effect on all of the HLAs and cytokines studied was more pronounced than P4’s. Evidence for PIF-induced amplification of endogenous P4 action is shown by increased P4 secretion coupled with increased steroid receptor protein levels.

HLA and cytokine expression in JEG-3 cells creates a balance between a pro-inflammatory and anti-inflammatory environment. PIF increased HLA-G, HLA-E and HLA-C expression both intracellularly and at the cell surface, as evidenced by complementary methods of analysis. HLA-G up-regulation was confirmed by using HLA-G imaging and proteome analysis. Such robust multifaceted analyses provide support for PIF’s important local regulatory role. The PIF-induced increase in HLA-E expression was of similar magnitude to HLA-G. The role of HLA-E at the feto-maternal interface can be complementary to HLA-G. Both can be co-expressed and induced by P4 in the trophectoderm of preimplantation embryos [15, 16]. Recognizing P4’s important role in HLA regulation, a direct comparison with that of PIF shown confirmed PIF’s higher efficacy in all side-by-side experiments. Both PIF and P4 increased intracellular and cell surface expression of HLA-G, -E, -F, and -C, but did not affect their relative proportion. This may be critical for a successful pregnancy; while not all HLA-G molecules need to form a heterodimer with HLA-E, the lack of heterodimer formation of HLA-E, combined with HLA-G, can lead to implantation failure [60]. Moreover, for its surface expression, HLA-E must interact with and be stabilized by a signal peptide, usually derived from other HLA class I alleles. In trophoblasts, it is derived from HLA-G and HLA-C [61]. HLA-E can also interact with uNK cells through the CD49/NKG2 receptor [62]. Together, HLA-G and HLA-E may inhibit NK cells cytotoxicity by interacting with the killer-cell immunoglobulin-like receptors (KIR)2DL4 and CD94/NKG2, respectively. Trophoblast protection from cell lysis is also achieved through interaction of HLA-G homodimers with the inhibitory NK receptors ILT2 and ILT4 [15]. Both PIF and P4 increased HLA-C expression, which also targets KIR molecules on uNK cells. KIR molecules regulate trophoblast invasion and uterine spiral artery blood flow in the inter-villous space [63]. Certain maternal KIR/HLA-C combinations can lead to defective trophoblast invasion or to an incomplete transformation of the spiral arteries, ultimately leading to pregnancy complications [60, 63]. The involvement of RAC1 and downstream b-catenin role in effective trophoblast invasion by promoting metalloproteinase 9 (MMP-9) was shown [64]. PIF also promoted MMP9 while reducing the inhibitor TIMP1 and regulating integrins expression [42]. Thus, the low PIF expression in preeclampsia and intrauterine growth retardation may lead to the low local MMP-9 expression [42, 46]. In addition, detailed proteomic analysis of syncytiotrophoblast extracellular vesicles identified differentially expressed proteins in the placenta of patients with preeclampsia among them increased pro-inflammatory S100-A8 as having a major role [65]. Also, it was shown that thrombin is an inducer of FMs-like tyrosine kinase 1 through increased ROS in transformed EVT supporting role in preeclampsia pathogenesis [66]. Importantly, we reported that in human immune cells PIF targets both thrombin and S100-A8, thus through local action PIF could mitigate such pathology [33]. PIF reduced systemic NK cells cytotoxicity and up-regulated local HLA-C expression, suggesting an integrated protective action [37]. PIF and P4 only mildly affected HLA-F expression, confirming previous observations [14]. PIF is expressed in trophoblasts during the earliest phase of gestation and is also present in maternal circulation nine days after insemination. Therefore, both endogenous and exogenous PIF may regulate trophoblastic HLA-G. Collectively, the observed potent PIF-induced up-regulation of HLA in trophoblasts reveals an essential role for PIF in promoting immune tolerance.

Our 2-DE proteome analysis demonstrated the PIF-induced increase in P4 receptor levels in JEG-3 cells, coupled with increased P4 secretion [53, 54]. This reveals PIF’s important role in endogenous P4 potentiation, which thus may facilitate the steroid’s production overtake by the placenta. The stimulatory effect of hCG on the corpus luteum as well as on endogenous (trophoblast) P4 was also reported [67]. The PIF data also confirmed the increase in HLA-G and FOXP3, a marker of Treg activation, serving to amplify the pro-tolerance effect. Circulating Tregs increase prior to implantation in response to the presence of a viable embryo [68]. Effective coagulation control (through increased TTP and DCBD factors) is crucial for placental function, since an altered coagulation cascade can lead to placental abruption. Regulation of complement activation (CD59) prevents membrane attack complex -induced C9 polymerization that promotes adverse pore production on the cell surface [69]. The embryo and early pregnancy trophoblast are highly vulnerable to an oxygen rich environment. Thus, down-regulation of peroxidases and HSPs by PIF may support a protective role, as shown in the embryo and decidua [26, 29, 32, 33, 37, 42]. This protection is amplified by the ATPB protein involved in ATP production, which, together with HBB, further protects against free oxygen and nitrogen radical species. Reduced KPYM levels prevent caspase-independent cell death. Clustering analysis indicated that beyond the central role of HLA-G and FOXP3, PIF also reduced the EGF receptor, which interacts with harmful LMNA and PDIA proteins. This is relevant, since PIF’s pro-receptive effect on the decidua was negated by EGF [30]. Hence, the proteome analysis substantiates PIF’s multifaceted role in regulating the activity of the trophoblast, acknowledging that it is a transformed cell line.

Both Th1 and Th2 type cytokines were up-regulated by PIF. The increase in IL-10, a prime Th2 cytokine, was induced by both P4 and PIF in JEG-3 cells. IL-10 may be secreted by both Th1 and Th2 type cells; its function is to balance pro- and anti-inflammatory signals [70-72]. IL-10 enhances HLA-G transcription in first trimester human trophoblast cultures [73, 74]. In the endometrium PIF and P4 create a pro-inflammatory milieu by increasing IL-1β, IL-8, GM-CSF and IFN-γ secretion to promote embryo implantation [29, 30, 36]. Elevated TGF-β could promote IL-1-induced T-cell proliferation and trophoblast invasion by up-regulating integrin expression, as shown for PIF in the endometrium, independent of P4. Thus, PIF-induced secretion of diverse cytokines, in contrast to P4, which affected only IL10, supports PIF-induced trophoblast interaction with the maternal milieu.

The current study is limited, since JEG-3 cytotrophoblastic cells and not primary trophoblastic cells were used. However, this model was validated with respect to the administration of P4, which was shown to be of physiologic value. In addition, exogenous PIF was administered. However, importantly, PIF targets the embryo and significant levels are present in the maternal circulation, which support the data generated with exogenous PIF administration [25, 26]. These are in vitro observations and therefore data using PIF in an in vivo setting would help to substantiate its potential clinical utility in treatment of pregnancy disorders. Recent data showed that in an immune intact murine model PIF administration reduces foetal death due to LPS administration [75]. Spontaneous pregnancy loss was also lowered. In addition, PIF negated oxidative stress of cultured bovine IVF embryos induced by a protein-di-sulphide isomerase inhibitor. In presence of PIF more than two-fold increase in embryos reaching the blastocyst stage was noted [76, 77].

PIF promotes the expression of HLA-G, -C, –E and mildly -F which are critical for immunological tolerance in JEG-3 choriocarcinoma cells. The effect of PIF was found to be superior to that of P4 in terms of promoting expression of the HLAs and cytokines studied. By promoting P4 secretion and receptor expression, PIF potentiates the endogenous steroid’s effect. PIF regulates the trophoblast proteome, promotes tolerance by increasing HLA-G and FoxP3+ levels and affects coagulation and complement, while it reduces the level of proteins involved in oxidative stress and protein misfolding. PIF-induced increase in Th1/ Th2 cytokine secretion favours trophoblast/maternal signaling. PIF successfully completed a Phase I FDA designated Fast-Track clinical trial for an autoimmune disease (Clinicaltrials. gov NCT02239562). Current data support comparable testing in early pregnancy disorders as well.

Professor I. L. Sargent for providing the JEG-3 cell line, Dr. G. Desoye for providing the ACH-3P cell line, and Dr. D. Geraghty for providing the anti-HLA-F clone 3D11. We thank Amy Carter, Stephanie Zinn and Amanda Roma for editorial assistance.

ERB is BioIncept LLC CSO and PIF is patent protected. Other authors have no competing interests.

E.R. Barnea and N. Fernandez contributed equally to this work.