Regulation of iron metabolism in HEC‐1A endometrium cells by macrophage‐derived factors and fractalkine

Macrophages in the endometrium promote receptivity and implantation by secreting proinflammatory cytokines and other factors like fractalkine (FKN). Macrophages are closely linked to regulating iron homeostasis and can modulate iron availability in the tissue microenvironment. It has been revealed that the iron metabolism of the mother is crucial in fertility. Iron metabolism is strictly controlled by hepcidin, the principal iron regulatory protein. The inflammatory cytokines can modulate hepcidin synthesis and, therefore, the iron metabolism of the endometrium. It was proven recently that FKN, a unique chemokine, is implicated in maternal–fetal communication and may contribute to endometrial receptivity and implantation. In the present study, we investigated the effect of activated THP‐1 macrophages and FKN on the iron metabolism of the HEC‐1A endometrial cells. We established a noncontact coculture with or without recombinant human FKN supplementation to study the impact of the macrophage‐derived factors and FKN on the regulation of hepcidin synthesis and iron release and storage of endometrial cells. Based on our findings, the conditioned medium of the activated macrophages could modify hepcidin synthesis via the nuclear factor kappa‐light‐chain‐enhancer of activated B cells, the signal transducer and activator of transcription 3, and the transferrin receptor 2/bone morphogenetic protein 6/suppressor of mothers against decapentaplegic 1/5/8 signaling pathways, and FKN could alter this effect on the endometrial cells. It was also revealed that the conditioned macrophage medium and FKN modulated the iron release and storage of HEC‐1A cells. FKN signaling may be involved in the management of iron trafficking of the endometrium by the regulation of hepcidin. It can contribute to the iron supply for fetal development at the early stage of the pregnancy.


| INTRODUCTION
Endometrial receptivity and embryo implantation are complex and multifactorial processes in which the immune system has an essential role (Robertson et al., 2022).The immune-related factors contribute to the development of endometrial receptivity (Li et al., 2022).
Macrophages are closely linked to the regulation of iron homeostasis, and they can modulate iron availability in the tissue microenvironment (Recalcati & Cairo, 2021;Winn et al., 2020).Iron metabolism of the mother, including the endometrium, is crucial in fertility (Cerami, 2017;Sangkhae et al., 2020).Iron deficiency can decrease the chance of fertilization (Chavarro et al., 2006;Kopeć et al., 2021).Moreover, iron availability acts as a regulatory factor of endometrial receptivity as well as embryo implantation.Recent findings underlie the importance of iron in the expression of receptivity-related genes (Pandur et al., 2023).
Iron metabolism is strictly controlled by hepcidin, the major iron regulatory hormone (Nemeth & Ganz, 2023).Hepcidin decreases iron release from the cells by acting on its iron-exporter receptor, ferroportin (FP) (Nemeth & Ganz, 2021).Plasma hepcidin levels are reduced during the second trimester of pregnancy to provide iron to the growing fetus (Nemeth & Ganz, 2023).Before the development of the placenta, the endometrium is responsible for iron transport towards the embryo, in which FP may play a crucial role (Pandur et al., 2021).
The hepcidin (HAMP) gene encodes hepcidin, and it is transcribed as preprohepcidin.The preprohepcidin messenger RNA (mRNA) is translated into preprohepcidin protein and cleaved at the signal sequence to form prohepcidin (Nemeth & Ganz, 2023).Next, prohepcidin further matures into hepcidin by the action of prohormone convertase such as furin (Valore & Ganz, 2008).The posttranslational maturation of hepcidin is regulated by alpha 1-antitrypsin (A1AT), a serine protease inhibitor that can bind to prohepcidin and cover the cleavage site of the propeptide (Lechowicz et al., 2020).At the transcriptional level, iron metabolism is controlled by factors like inflammatory molecules, iron availability, hypoxia, and erythropoiesis (Ganz, 2021).The inflammatory cytokines produced by endometrial macrophages or endometrial cells can modulate hepcidin synthesis and, therefore, the iron metabolism of the endometrium.The primary regulatory pathway is the Janus kinase/ signal transducer and activator of transcription (JAK/STAT) signaling, which triggers the mRNA expression of hepcidin by inducing the transcription of the HAMP, the hepcidin encoding gene (Ganz, 2021).
The nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway also works as an altering factor of hepcidin synthesis, mediated by inflammation (Kowdley et al., 2021).
It has been recently revealed that fractalkine (CX3CL1, FKN), the sole member of the CX3C chemokine subfamily, is implicated in maternal-fetal communication (Du et al., 2014;Kervancioglu Demirci et al., 2016).FKN is expressed in two forms.The membrane-bound form is mainly responsible for cell-cell interactions, while the soluble form may act as an inflammatory molecule and regulate chemotaxis (Garton et al., 2001;Jones et al., 2010).Both forms can interact with the CX3CR1.FKN is synthesized by the endometrial cells as well as macrophages, and its receptor CX3CR1 is expressed by the trophoblast cells, the endometrial cells, and macrophages (Du et al., 2014;Li et al., 2021).
The present study investigated the relationship between the various factors influencing iron metabolism in the implantation process.Adequate iron levels during pregnancy have been analyzed in many aspects, but the amount of detectable iron during implantation has been little studied.The role of inflammatory cytokines in receptivity and the induction of implantation is already known, but their influence on endometrial iron metabolism has yet to be considered.This is compounded by the fact that the relationship between FKN and iron metabolism has been demonstrated.Still, the interaction of these three components (cytokines, FKN, iron levels) on the endometrium has yet to be investigated.Our main objective was to examine the temporal and concentration-dependent relationship of these three factors.We established a noncontact coculture, in which only the culture medium of the phorbol 12-myristate 13-acetate (PMA)-activated THP-1 macrophages was added to the HEC-1A cells with or without recombinant human FKN to reveal the impact of the and the THP-1 cells were cultured in RPMI-1640 culture medium (Lonza Ltd.)Both culture media were supplemented with 10% fetal bovine serum (EuroClone S.p.A) and 1% penicillin/streptomycin (Lonza Ltd.).The cells were cultured in a humified atmosphere with 5% CO 2 at 37°C.We followed the good practice guidelines for cell lines.PMA was purchased from Merck (Merck Life Science Kft.).The PMA was solved in dimethyl sulfoxide (Merck Life Science Kft.), making a 1 mg/mL stock solution.
The THP-1 cells were treated with 100 ng/mL of PMA for 24 h.The effect of PMA and the activation of the macrophages were monitored by real-time polymerase chain reaction (PCR) analysis of the proinflammatory cytokines .The mRNA expression levels of the proinflammatory cytokines can be seen in Supporting Information S1: Figure 1.After the PMA treatment, the culture medium was removed, and the THP-1 cells were washed twice with 1X phosphate buffer saline (PBS; Lonza Ltd.) to eliminate PMA.The activated THP-1 cells were incubated in freshly added complete RPMI-1640 medium for 24 or 48 h.After the incubation, the conditioned medium was transferred to HEC-1A endometrial cells.The endometrium cell cultures were separated into two groups.In the first group, the cells were cultured in the THP-1 conditioned medium for 24 h.In the second group, the cells were cultured in the conditioned THP-1 medium supplemented with 10 or 20 ng/mL of recombinant human FKN (Shenandoah Biotechnology Inc.) for 24 h.In each experiment, the control HEC-1A cells were cultured in complete RPMI-1640 medium obtained from untreated THP-1 cell cultures after a 24 or 48 h incubation period.The experimental procedure can be found in

| Real-time PCR
The THP-1 cells were seeded onto six-well plates (Biologix Europe) using 5 × 10 5 cells/well and were treated with PMA as described above.The HEC-1A endometrial cells were placed onto six-well plates using 5 × 10 5 cells/well.The endometrial cell cultures were treated with the conditioned THP-1 medium as it was described earlier.After incubation, the HEC-1A cells were collected by trypsinization and centrifugation.The cell pellets were washed once with 1X PBS.Total RNA was isolated by Aurum Total RNA Mini Kit (Bio-Rad Inc.).The RNA concentration of the samples was determined using a MultiSkan GO spectrophotometer (Thermo Fisher Scientific Inc.).The complementary DNA (cDNA) synthesis was performed using the iScript Select cDNA Synthesis Kit (Bio-Rad Inc.).
The real-time PCR reactions were carried out in a CFX96 Opus Real-Time PCR System (Bio-Rad Inc.) using iTaq Universal SYBR Green Reagent Mix (Bio-Rad Inc.) in a 20 µL of total volume.A melting curve was generated after each run to make sure that the reaction was specific and that only one product was synthesized in each reaction.
The relative mRNA expression levels (fold change) of the target genes were calculated by the Livak (ΔΔC t ) method using the Bio-Rad CFX Maestro 2.3.software (Bio-Rad Inc.).Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene was used to normalize the real-time PCR reactions.The mRNA expression levels of the target genes in the control cells were regarded as one.The control cells were maintained in a nonactivated macrophage-derived culture medium.Real-time PCR analyses were carried out in triplicate in three independent experiments.The primer sequences used for the real-time PCR are presented in Table 1.

| Western blot
The THP-1 cells were placed onto 6 cm wide cell culture dishes (Biologix Europe) using 10 6 cells/dish and were treated with PMA as described above.The HEC-1A cells were cultured in 6 cm wide cell F I G U R E 1 Description of the experimental protocol.The THP-1 cells were differentiated into macrophages by PMA treatments.After exchanging the culture medium, the cells were incubated for 24 or 48 h to release macrophage factors such as proinflammatory cytokines.Then, the culture medium was added to HEC-1A endometrial cells with or without FKN supplementation.FKN, fractalkine; PMA, phorbol 12-myristate 13-acetate.The protein content of each sample was determined using the DC Protein Assay Kit (Bio-Rad Laboratories) and the MultiSkan GO spectrophotometer (Thermo Fisher Scientific Inc.) at 750 nm.The protein separation was performed in sodium dodecyl sulfatepolyacrylamide gels using the Mini Protean Tetra Cell equipment (Bio-Rad Laboratories).Then, the proteins were blotted onto nitrocellulose membranes (Amersham Biosciences, GE Healthcare).
The membranes were blocked with a TBST buffer containing 5% (wt/vol) nonfat dry milk at room temperature for 1 h.In the case of the examination of p50 and p65 proteins, the cells were fractionated with Subcellular Protein Fractionation Kit for cultured cells (Thermo Fisher Scientific Inc.), and the chromatin-bound protein fraction was used for the Western blots.GAPDH was used as a loading control from the cytosolic protein fraction in this case.We used the following primary antibodies in the Western blots: anti-FP immunoglobulin G (IgG) (1:1000, 1 h, room temperature; Bio-Techne); anti-phospho-

| Enzyme-linked immunosorbent assay (ELISA)
The supernatants of the control and differently treated HEC-1A cells

| Iron measurements and determination of heme concentration
The THP-1 cells were placed onto 6 cm wide cell culture dishes using 10 6 cells/dish and were treated with PMA as described above.The HEC-1A cells were cultured in 6 cm wide cell culture dishes using 10 6 cells/dish.The endometrial cell cultures were treated with the conditioned THP-1 medium as it was described earlier.The HEC-1A cells were lysed in 50 mM NaOH for 2 h with gentle shaking at room temperature.An equal volume of 10 mM HCl was added to the samples to neutralize them.After neutralization, the samples were mixed with acidic KMnO 4 and incubated at 60°C for 2 h to release protein-bound iron.Reduction of iron and complex formation with ferrozine was carried out in the aqueous solution of 6.5 mM neocuproine, 1 M ascorbic acid, 2.5 M ammonium-acetate, and 6.5 mM ferrozine at room temperature for 30 min.The purple ferrozine-iron complexes are detectable at 550 nm.The absorbance was measured using a MultiSkan GO spectrophotometer (Thermo Fisher Scientific Inc.).The concentration of the samples was calculated against the FeCl 3 standard curve and was expressed as mM iron/mg protein.
The HEC-1A endometrial cells were harvested and lysed in 100 µL of distilled water using gentle sonication for the heme concentration measurements.The heme concentration measurements were performed using a Heme Assay Kit (Merck Life Science Kft.).The samples were mixed with a fourfold Heme Reagent and incubated for 5 min at room temperature.The optical density values were measured at 400 nm.The results were expressed as micrometers.All measurements were carried out in triplicate in three independent experiments.
T A B L E 1 Real-time PCR primer list.

| Statistical analysis
Statistical analysis was performed using SPSS software version 24.0 (IBM Corporation).Depending on the quantity of data collected, the normality of our data set was assessed using either the Kolmogorov-Smirnov or the Shapiro-Wilk test.Owing to the verification of normal distribution in the obtained data, a parametric test was consequently utilized.Our analysis employed a two-way analysis of variance due to the presence of multiple variables.
Subsequently, Scheffe's post hoc test was selected for its conservative nature, which diminishes the likelihood of type I errors (false positives).This approach is particularly advantageous in scenarios where it is imperative to reduce the probability of erroneously identifying significant differences.Data are shown as the mean ± standard deviation and were considered statistically significant if the p-value was lower than .05.The protein level of the iron exporter FP decreased significantly using 24 h conditioned medium and by the addition of 10 ng/mL of FKN but increased back to the control level using 20 ng/mL of FKN supplementation, suggesting a concentration-dependent effect of FKN on the iron export of HEC-1A cells (Figure 2a,b).In treating the HEC-1A cells with the 48 h conditioned THP-1 medium, the FP level was significantly reduced, and the FKN supplementation reversed the effect of the macrophage-derived factors.In treating the endometrial cells with 20 ng/mL of FKN, the FP level was raised to the control level (Figure 2a,b).These observations suggest that the THP-1derived factors in the culture medium alter the iron transport of the endometrial cells to the iron retention route.The presence of FKN contributes to the changes in iron metabolism, but its effect seems to be concentration-dependent.
The Western blot analysis of FTH presented a significant augmentation using the 24 h conditioned medium complemented with FKN (Figure 2a,c).At the same time, the 48 h conditioned medium was only successful in the elevation of FTH protein level when it was utilized together with the 20 ng/mL of FKN (Figure 2a,c).
In the case of FTMT, both the 24 and 48 h conditioned media induced the FTMT protein expression (Figure 2a,d).The 24 h culture medium supplementation with FKN successfully increased the FTMT level (Figure 2a,d).Treatment of the HEC-1A cells with 48 h culture medium and FKN slightly elevated the FTMT level compared to the medium treatment alone (Figure 2a,d).
These observations propose the increasing iron storage capacity of the endometrial cells and suggest iron transport between the cytosol and the mitochondria.
Haptoglobin synthesis, which is responsible for heme transport, showed significant elevation when the endometrial cells were treated with the 24 h conditioned THP-1 medium (Figure 2e).The addition of FKN to the endometrial cells resulted in a significant elevation of haptoglobin production compared to the 24 h conditioned medium treatment (Figure 2e).Considering the different treatments, the 10 ng/mL of FKN showed the highest effect on haptoglobin secretion (Figure 2e).When the 48 h conditioned medium supplemented with FKN was added to the endometrial cells, significantly higher haptoglobin secretion occurred.Still, these values were lower than the 24 h conditioned medium treatment (Figure 2e).
3.2 | Variations in the total iron content and heme concentration in the endometrial cell treated with conditioned THP-1 medium and FKN These parameters were determined to see if the changes in the iron metabolism of the HEC-1A endometrial cells impact the iron and heme contents of the cells.Interestingly, the 24 h conditioned medium significantly augmented the total iron content of the endometrial cells compared to the control (Figure 3a).Still, the 48 h conditioned medium did not affect the iron content.Regarding FKN complementation, only the 48 h conditioned medium caused a significant increase in the total iron amount of the endometrial cells compared to the control and the conditioned medium-treated cells (Figure 3a).
Examining the heme concentration of the endometrial cells, the addition of FKN to the 24 h conditioned medium-treated cells significantly reduced the heme levels (Figure 3b), while using the 48 h conditioned medium, the higher concentration of FKN treatment resulted in a remarkable increment of heme content compared to the control and the conditioned medium-treated HEC-1A cells (Figure 3b).F I G U R E 2 Western blot (WB) analysis (a) of FP (b), ferritin heavy chain (c), and mitochondrial ferritin (d) and concentration measurement of haptoglobin (e) of the HEC-1A cells.For the WBs, the same amount of protein from each sample was separated into SDS-polyacrylamide gels.After blotting, the antibodies were used according to the manufacturer's protocol.GAPDH was used as the loading control.The analysis of the blots was performed using the ImageJ software.The ELISA measurement was done using the Human Haptoglobin Quantikine ELISA Kit according to the manufacturer's protocol.The columns represent the mean ± SD of three independent experiments (n = 3).For (b-d), the asterisk shows p < .05compared to the control, the cross shows p < .05compared to the cells treated with culture medium of PMA-activated THP-1 cells, and the double-cross means p < .05compared to the 10 ng/mL of FKN-supplemented cells.In (e), the bullet shows p < .05compared to Ctrli, the asterisk means p < .05compared to the Ctrl, the cross signs p < .05compared to PMAi, and the double-cross means p < .05compared to PMA.Ctrli, initial concentration of the protein in the control culture medium; ELISA, enzyme-linked immunosorbent assay; F10, fractalkine 10 ng/mL; F20, fractalkine 20 ng/mL; FKN, fractalkine; FP, ferroportin; FTH, ferritin heavy chain; FTMT, mitochondrial ferritin; GAPDH-glyceraldehyde 3-phosphate dehydrogenase; PMA, phorbol 12-myristate 13-acetate; PMAi, initial concentration of the protein in the culture medium of PMA-treated THP-1 cells; SDS, sodium dodecyl sulfate.The experiments were repeated three times (n = 3), using triplicates from each sample.The ELISA measurement was carried out using the Human Hepcidin Quantikine ELISA Kit according to the manufacturer's protocol.For WB, the same amount of protein from each sample was separated into SDS-polyacrylamide gels.After blotting, the A1AT antibody was used according to the manufacturer's protocol.GAPDH was used as the loading control.The blots in this figure are representative images.The columns show the mean values ± SD.In (a) and (c), the asterisk indicates p < .05compared to the control, and cross signs p < .05compared to the cells treated with culture medium of PMA-activated THP-1 cells.In (b), the bullet shows p < .05compared to Ctrli, the asterisk means p < .05compared to the Ctrl, the cross signs p < .05compared to PMAi, and the double-cross means p < .05compared to PMA.A1AT-alpha 1-antitrypsin; Ctrli, initial concentration of the protein in the control culture medium; ELISA, enzyme-linked immunosorbent assay; F10, fractalkine 10 ng/mL; F20, fractalkine 20 ng/mL; FKN, fractalkine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HAMP, hepcidin gene; mRNA, messenger RNA; PMA, phorbol 12-myristate 13-acetate; PMAi, initial concentration of the protein in the culture medium of PMA-treated THP-1 cells; SDS, sodium dodecyl sulfate.
The macrophages synthesize and secrete hepcidin, too; therefore, we also measured the hepcidin levels of the THP-1 culture media before adding them to the endometrial cells (Ctrli and PMAi in Figure 4b).Based on the above results it can be decided whether the endometrium cells secrete hepcidin or utilize the macrophageoriginated hepcidin for regulating FP.The endometrial cells significantly increased the hepcidin secretion using the 24 h conditioned medium and FKN compared to both Ctrl and conditioned mediumtreated endometrial cells (Figure 4b).Despite this result, the treatment of the HEC-1A cells with the 48 h conditioned medium and FKN significantly reduced the hepcidin secretion (Figure 4b).The latter observation suggests that the THP-1-derived hepcidin may activate the internalization of FP or plug up the transport channel of FP.
The mature hepcidin secretion of the cells is influenced by the rate of hepcidin maturation, which means the cleavage of prohepcidin into hepcidin intracellularly.The maturation process is regulated by A1AT, the inhibitor of prohepcidin cleavage.The expression of the A1AT protein reflects the levels of the secreted hepcidin (Figure 4c,d).A1AT level decreased when the hepcidin secretion was elevated, and it was upregulated when the hepcidin level increased in the culture medium of the 48 h conditioned mediumtreated and FKN-supplemented HEC-1A cells (Figure 4c,d).These results propose that macrophage-derived factors, as well as FKN, alter both the transcriptional and posttranslational regulation of hepcidin.

| Transcriptional regulation of HAMP by FKN/ CX3CR1 axis in the treated endometrium cells
We examined whether macrophage-derived factors with/without FKN alter the major HAMP-regulating signaling pathways.First, we focused on the FKN/CX3CR1 axis-mediated signal transduction, the NFκB, and the STAT3 transcriptional regulators (Figure 5a).
The protein level of CX3CR1 was elevated in the HEC-1A cells after treatment with 24 h conditioned THP-1 medium, but the supplementation with FKN did not promote the CX3CR1 level over the conditioned medium-treated cells (Figure 5a,b).Using the 48 h conditioned medium on the HEC-1A cells, the CX3CR1 protein levels significantly decreased, suggesting the desensitization of the endometrium cells to FKN (Figure 5a,b).This phenomenon may be due to the combined effects of macrophage-secreted and added recombinant FKN (Figure 5f).Interestingly, in the case of the NFκB transcription factors, p50 and p65, the 24 conditioned medium-treated HEC-1A cells, as well as the FKN supplementation, were able to increase their protein level compared to the controls (Figure 3a,c,d).Moreover, FKN was capable of further raising the p50 protein level.However, we did not find a difference between the 48 h conditioned medium treated and FKNsupplemented cells in the case of p50 and p65 levels (Figure 5a,c,d).
The above results may be due to the action of the macrophagederived TNF-α proinflammatory cytokine on the NFκB signaling pathway, which can contribute to the transcriptional activation of HAMP (Figure 5g).The Western blot analysis of p50, p65, and P-STAT3 revealed the downregulation of these transcription factors (Figure 6b-e).
Interestingly, the mRNA expression level of HAMP did not decrease, suggesting that an additional signaling pathway maintains HAMP transcription when the FKN-mediated pathways are downregulated (Figure 6a).
The level of the secreted BMP6 protein significantly increased both using 24 or 48 h conditioned media, and the addition of FKN further elevated the BMP6 concentration in the culture medium of the HEC-1A cells (Figure 7a).The phosphorylation of SMAD transcription factor followed the changes in the BMP6 protein levels proving the contribution of this signaling pathway in the transcriptional activation of HAMP (Figure 7a,b,d).
The iron sensor TfR2 showed slight elevation upon conditioned medium treatments.The administration of FKN significantly increased the TfR2 protein level when it was utilized together with the 24 h conditioned macrophage medium (Figure 7a We examined whether the iron-mediated pathway is altered by the inhibition of the FKN/CX3CR1-mediated signaling pathway.It was revealed that the BMP6 secretion of the HEC-1A cell was higher upon ACHP pretreatment of the cells, even in the controls (Figure 8a).The utilization of the conditioned medium and FKN supplementation were able to raise BMP6 synthesis (Figure 8a).The level of the phosphorylated SMAD factors was significantly elevated in the treated HEC-1A cells compared to the controls, suggesting the role of BMP6/SMAD signaling pathway in the maintenance of the HAMP expression after ACHP treatment (Figure 8b,d).
The protein level of TfR2 was also heightened using the conditioned medium with or without FKN supplementation, suggesting that TfR2 actively contributes to the transcriptional activation of HAMP (Figure 8b,c).
Maternal body iron status can influence fertility and receptivity and implantation of the embryo (Cerami, 2017;Sangkhae et al., 2020).
It has been described recently that FKN, a special chemokine acting on the CX3CR1 FKN receptor, has regulatory functions in endometrial receptivity, embryo implantation, as well as in iron metabolism of endometrial cells and trophoblasts (Pandur et al., 2021;Pap et al., 2020).FKN can alter the expression of hepcidin, the master regulator of iron homeostasis (Pandur et al., 2019), in the BV-2 microglia.Moreover, FKN can modify the proinflammatory cytokine production of macrophages (Pandur et al., 2022), which establishes receptivity and implantation (Craciunas et al., 2019).
The endometrial immune status is associated with reproduction (Zhang et al., 2017).The contribution of the immune system to the maintenance of the pregnancy is critical in regulating immune tolerance against the fetus (Abu-Raya et al., 2020).FKN, secreted by the endometrial cells, may be involved in the macrophage infiltration into the endometrium to help prepare the uterus for the attachment of the blastocyst (Ma et al., 2022).Also, the elevated TNF-α, MIP1B, and GROα levels produced by the macrophages in the proliferative phase favor pregnancy by preparing the endometrium for implantation (Ma et al., 2022).
Inflammatory molecules like TNF-α, IL-6, or IL-1β function as regulators of the cellular iron metabolism by regulating the JAK/STAT, NFκB, and MAPK signaling pathways (Ganz, 2021;Kanamori et al., 2017;Kowdley et al., 2021).In this manner, the macrophages can support the iron metabolism of the endometrium.Iron availability in the cells also works as a potent regulator of cellular iron metabolism (Nemeth & Ganz, 2023).The HFE/TfR2 system can help maintain the basal transcriptional level of hepcidin, the iron metabolism regulator hormone (D'Alessio et al., 2012).TfR2 can bind to the BMP/BMPR complex, regulating the SMAD1/5/8 transcription factors acting as positive regulators of hepcidin transcription (Nemeth & Ganz, 2009).
In the present study, we focused on the action of macrophagederived factors and FKN on regulating hepcidin and endometrial iron metabolism.The THP-1 monocyte/macrophage cells were differentiated into macrophages.Then, they were incubated into a fresh culture medium that was added to cultured HEC-1A endometrial cells, forming a noncontact coculture.
Inflammatory molecules can increase cellular iron storage by upregulating the transcription FTH via the NFκB signaling pathway ) and (e) phospho-STAT3 transcription factors and concentration determinations of fractalkine (f) and TNF-α (g).After the treatments, the endometrial cells were collected and lysed or fractionated.The exact amount of protein from each sample was separated into SDS-polyacrylamide gels.After blotting, the antibodies were used according to the manufacturer's protocol.GAPDH was used as the loading control.The analysis was performed using the ImageJ software.Secreted TNF-α concentrations in the culture medium.The columns represent the mean ± SD of three independent experiments (n = 3).In (b-e), the asterisk shows p < .05compared to the control, the cross shows p < .05compared to the cells treated with culture medium of PMA-activated THP-1 cells, and the double-cross means p < .05compared to the 10 ng/mL of FKN-supplemented cells.In (f) and (g), the bullet shows p < .05compared to Ctrli, the asterisk means p < .05compared to the Ctrl, the cross signs p < .05compared to PMAi, and the double-cross means p < .05compared to PMA.Ctrli, initial concentration of the protein in the control culture medium; CX3CR1, fractalkine receptor; F10, fractalkine 10 ng/mL; F20, fractalkine 20 ng/mL; FKN, fractalkine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NFκB, nuclear factor kappa-lightchain-enhancer of activated B cells; PMA, phorbol 12-myristate 13-acetate; PMAi, initial concentration of the protein in the culture medium of PMA-treated THP-1 cells; P-STAT3, phospho-signal transducer and activator of transcription 3; SDS, sodium dodecyl sulfate; TNF-α, tumor necrosis factor α.
Besides the cytosolic iron storage protein FTH, the expression of FTMT, the mitochondrial ferritin, was also monitored in HEC-1A cells.
The FTMT level does not hinge on the cellular iron status, although its level increases in case of iron accumulation.FTMT is pivotal in iron sequestration and traffic between the cytosol and the mitochondria (Gao & Chang, 2014;Levi et al., 2021).According to our results, conditioned-medium treatment and FKN supplementation of the endometrial cells strongly affect the FTMT protein level.The elevated expression of FTMT suggests that the treatments alter intracellular iron trafficking and initiate iron redistribution in the HEC-1A cells.Considering the effects of the two FKN concentrations on the iron metabolism of the HEC-1A cells, it can be concluded that the lower FKN concentration decreases the FP level, proposing that iron trafficking is shifted toward the heme efflux.Meanwhile, the higher FKN concentration increases FP level, mediating iron release through the iron exporter protein.
It has been proposed that haptoglobin is involved in the iron transport between endometrium cells and trophoblasts (Pandur et al., 2021).Inflammatory mediators like IL-6 induce haptoglobin synthesis and transport free hemoglobin/heme (Thomsen et al., 2013).
The haptoglobin/heme complex is eliminated from the environment by the CD163 receptor, which is expressed by the trophoblast cells surrounding the blastocyst (Kristiansen et al., 2001;Li et al., 2016).
Haptoglobin secretion of HEC-1A endometrial cells was induced by conditioned macrophage medium and FKN, suggesting that haptoglobin supports heme transport originating from the endometrial cells.
The total intracellular iron content and the heme concentration measurements support the hypothesis that the endometrium cells modify the release of iron in the presence of macrophage-derived factors and FKN chemokine.The heme levels decreased upon FKN treatment in parallel with the lower iron content and the elevated haptoglobin secretion.The iron content of the HEC-1A cells also demonstrates the alterations in iron distribution.The treatment of the HEC-1A cells with the macrophage medium incubated for a longer time on the macrophages and the addition of FKN reduced FTH level, suggesting the elevated concentration of the labile iron pool that later can be released from the endometrial cells or can be utilized by the mitochondria for heme synthesis.
Our findings proved that macrophage factors and FKN significantly induce hepcidin transcription in HEC-1A cells.On the other hand, macrophages also secrete hepcidin, which may act on the iron metabolism of the endometrial cells.In the first part of the experiments, when the endometrial cells were treated with the 24 h conditioned macrophage medium, the hepcidin secretion was significantly raised at FKN treatment.On the contrary, treating the endometrial cells with the macrophage medium, which was incubated for a longer time on the active immune cells and FKN together, FKN decreases hepcidin secretion, increasing iron release and availability for the patient.Interestingly, the conditioned medium alone did not change the hepcidin secretion of the endometrial cells.
The downregulation of hepcidin secretion of the HEC-1A cells may be caused by the changes in the A1AT protein level, which regulates the cleavage of prohepcidin into the mature hepcidin form (Lechowicz et al., 2020).We found that the A1AT level decreased when the hepcidin secretion increased from the cells, and its protein level was elevated when the hepcidin secretion dropped, suggesting that the macrophage-derived factors and FKN also influence the A1AT levels.
The inflammatory cytokines are known positive regulators of hepcidin transcription acting through the JAK/STAT3 signaling pathway (Verga Falzacappa et al., 2007).FKN can also modulate the JAK/STAT3 pathway directly or via MAPK signal transduction (Cormican & Griffin, 2021)  The expression of the target gene was considered one in the control cells.The control cells were maintained in a nonactivated macrophagederived culture medium.The experiments were repeated three times (n = 3), using triplicates from each sample.For WBs, the endometrial cells were collected and lysed or fractionated.The same amount of protein from each sample was separated into SDS-polyacrylamide gels.After blotting, the antibodies were used according to the manufacturer's protocol.GAPDH was used as the loading control.The analysis was performed using the ImageJ software.The ELISA measurement was carried out using the Human BMP6 ELISA Kit according to the manufacturer's protocol.For the WBs, the cells were collected and lysed.The same amount of protein from each sample was separated into SDS-polyacrylamide gels.After blotting, the antibodies were used according to the manufacturer's protocol.GAPDH was used as the loading control.The blots in this figure are representative images.The analysis was performed using the ImageJ software.The columns show the mean values ± SD.In (a), the bullet shows p < .05compared to Ctrli, the asterisk means p < .05compared to the Ctrl, the cross signs p < .05compared to PMAi, and the double-cross means p < .05compared to PMA.In (c) and (d), the asterisk shows p < .05compared to the control, cross signs p < .05compared to the cells treated with culture medium of PMA-activated THP-1 cells.BMP6, bone morphogenetic protein 6; Ctrli, initial concentration of the protein in the control culture medium; ELISA, enzyme-linked immunosorbent assay; F10, fractalkine 10 ng/mL; F20, fractalkine 20 ng/mL; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PMA, phorbol 12-myristate 13-acetate; PMAi, initial concentration of the protein in the culture medium of PMA-treated THP-1 cells; SDS, sodium dodecyl sulfate; SMAD1/5/8, suppressor of mothers against decapentaplegic 1/5/8; TfR2, transferrin receptor 2.
influenced by the presence of inflammatory molecules.Besides FP, FTH expression is also controlled by inflammation in addition to iron uptake and release.Although the total iron content decreased in the FKN experiments, presumably due to heme shedding, the FTH and FTMT protein levels were elevated.The observed elevation of the iron storage proteins' levels may also be due to enhanced iron uptake by endometrial cells, which may also be modified by FKN and inflammatory processes.Based on the results, the development of absolute iron retention was observed at longer incubation times with the conditioned THP-1 medium and FKN.
macrophage-derived factors and FKN on the hepcidin synthesis and regulation, the iron release, and storage.Based on the findings, the conditioned medium of the activated macrophages could modify the hepcidin synthesis via the STAT3 and the NFκB pathways.Still, FKN could alter this effect on the endometrial cells.If the FKN-CX3CR1 axis-regulated signal transduction is inhibited, the bone morphogenetic protein 6 (BMP6)/suppressor of mothers against decapentaplegic (SMAD)1/5/8, and transferrin receptor 2 (TfR2) signaling maintain hepcidin transcription.It was also revealed that the conditioned macrophage medium and FKN modulated the FP level and the synthesis of the iron storage proteins.The observed changes in the endometrial iron metabolism may contribute to regulating iron availability in the embryo at the early stage of pregnancy.2 | MATERIALS AND METHODS 2.1 | Cell culture experiments The HEC-1A human endometrial cell line (HT-112; ATCC) and the THP-1 monocyte/macrophage cell line (TIB-202; ATCC) were purchased from ATCC (LGC Standards Sp. z.o.o.).The HEC-1A cells were maintained in McCoy's 5A medium with Ishikawa and Grace modification (Corning Inc.)
10 6 cells/dish.The endometrial cell cultures were treated with the conditioned THP-1 medium as it was described earlier.After the treatments, the cells were harvested by centrifugation.They were lysed in 200 µL of ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, pH 7.4).The samples were supplemented with a Complete Mini protease inhibitor cocktail (Roche Ltd.) and a PhosStop inhibitor for phosphatases (Roche Ltd.).
were stored at −80°C until the ELISA measurements.The supernatants of the untreated and PMA-treated THP-1 cells were used to determine the initial concentration of the target protein secreted by the THP-1 cells.The secreted hepcidin concentration was measured by the Human Hepcidin Quantikine ELISA Kit (Bio-Techne), and the haptoglobin concentration was determined with a Human Haptoglobin Quantikine ELISA Kit (Bio-Techne) according to the protocols of the manufacturer.The BMP6 protein level was determined using the Human BMP6 ELISA Kit (Merck Life Science Kft.).The TNF-α proinflammatory cytokine concentration was estimated by the Human TNF-α ELISA Kit (Thermo Fisher Scientific Inc.), and the FKN level was measured using the Human Fractalkine ELISA Kit (Thermo Fisher Scientific Inc.).The measurements were performed in triplicate in three independent experiments.
3 | RESULTS3.1 | The effects of PMA-activated macrophages and FKN on the expression of FP iron exporter and iron storage proteins of endometrial cells Differentiation of THP-1 monocytes into macrophages activates the secretion of several molecules, like proinflammatory cytokines, which can alter the iron metabolism of the HEC-1A endometrial cells.First, we focused on the iron release and retention of the endometrial cells, which can be monitored by the protein levels of FP iron exporter and the iron storage proteins FTH and mitochondrial ferritin (FTMT).

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These findings predispose the modulation of iron metabolism via altering the iron transport, utilization, and release of the endometrial cells mediated by the macrophage-derived factors and the FKN/ CX3CR1 axis.3.3 | Alterations of HAMP expression and hepcidin secretion of HEC-1A endometrial cells after treatments with activated THP-1 medium and FKNHepcidin is the master regulator of iron metabolism by controlling the iron release of the cells via the iron exporter FP.Hepcidin may indirectly affect heme synthesis and iron-sulfur cluster synthesis, thus affecting the overall function of the cell.Therefore, modification PANDUR ET AL.I G U R E 2 (See caption on next page).C Cell ell B Biology iology I International nternational PANDUR ET AL.F I G U R E 3 Total intracellular iron content (a) and heme concentration (b) of the HEC-1A endometrial cells.The HEC-1A cells were treated with the culture medium of PMA-activated THP-1 cells or with the culture medium supplemented with 10 or 20 ng/mL of FKN.Total iron content was determined using a ferrozine-based colorimetric method.The iron content was normalized to the protein concentration of the samples and was expressed as µM iron/mg protein.The heme concentration was measured by the Heme Assay Kit.The measurements were carried out in triplicates from three independent experiments (n = 3).The columns represent the mean value ± SD.The asterisk shows p < .05compared to the control, and cross signs p < .05compared to the cells treated with culture medium of PMA-activated THP-1 cells.F10, fractalkine 10 ng/mL; F20, fractalkine 20 ng/mL; FKN, fractalkine; PMA, phorbol 12-myristate 13-acetate.
of the endometrial cells may alter the iron release, affecting the iron availability of the embryo during pregnancy.The influence of the THP-1 macrophages on the HAMP expression of the endometrial cells depends on the incubation time of the conditioned medium of the THP-1 cells.The 24 h conditioned medium of THP-1 cells significantly increased the HAMP mRNA level of the HEC-1A cells (Figure4a).Supplementation with FKN significantly upregulated HAMP expression when it was used together with the 24 h conditioned THP-1 medium (Figure4a).Meanwhile, FKN administration together with the 48 h conditioned THP-1 medium resulted in the downregulation of HAMP expression compared to the treatment using a conditioned THP-1 culture medium alone (Figure4a).These results may reflect that the signaling pathways mediated by macrophage-derived cytokines or other factors and the FKN/CX3CR1 signaling together modify the response of the endometrial cells on the transcriptional regulation of HAMP.F I G U R E 4 (a)The mRNA expression of HAMP and (b) secretion of hepcidin of HEC-1A cells and the (c, d) Western blot (WB) analysis of A1AT.The HEC-1A cells were treated with the culture medium of PMA-activated THP-1 cells or with the culture medium supplemented with 10 or 20 ng/mL of FKN.The real-time PCR was performed using a SYBR Green protocol and GAPDH as a normalization gene.The expression of the target gene was considered one in the control cells.The control cells were maintained in a nonactivated macrophage-derived culture medium.
Treatment of the endometrial cells only with conditioned THP-1 medium significantly decreased STAT3 phosphorylation compared to the controls (Figure5a,e).The treatment of the endometrial cells with the conditioned medium supplemented with FKN provided the opposite effect, and it triggered STAT3 phosphorylation in the HEC-1A cells compared to the 24 h conditioned medium treated cells (Figure5a,e).Despite this, adding the 48 h conditioned macrophage medium or with 10 ng/mL FKN supplementation, the STAT3 phosphorylation significantly reduced compared to the control cells, which corresponds to the changes in the CX3CR1 level (Figure5a,b,e).

3. 5 |
Downregulation of the NFκB and P-STAT3 does not affect HAMP transcription in HEC-1A cells To examine the regulatory mechanisms of FKN in HAMP transcription of HEC-1A cells, the downstream signaling pathways of the FKN/CX3CR1 were inhibited by ACHP, which acts as the inhibitor of the NFκB pathway by blocking the translocation of the transcription factors into the nucleus.ACHP also works as an inhibitor of STAT3 phosphorylation.

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,b).On the other hand, in the case of 48 h conditioned medium treatment, only the lower FKN concentration further elevated the TfR2 level I G U R E 5 (See caption on next page).Downregulation of the NFκB and P-STAT3 pathways increases BMP6 secretion and triggers TfR2 and SMAD expression

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Western blot analysis (a) of fractalkine receptor (CX3CR1) (b) and the downstream signaling pathways mediated by the NFκB1 ([c]) p50 and [d] p65 (a) Representative Western blots of the target proteins.CX3CR1 and P-STAT3 were examined in the total cell lysates.The p50 and p65 proteins were analyzed in the chromatin-bound protein fraction.The upper GAPDH blot originated from the whole cell lysates; the lower GAPDH blot is from the cytosolic fraction of the HEC-1A cells.(b) Analysis of the protein level of CX3CR1.(c) Analysis of the protein levels of p50.(d) Analysis of the levels of p65 protein.(e) Analysis of the phospho-STAT3 transcription factor.(f) Secreted and recombinant FKN concentrations of the culture medium of the THP-1 (Ctrli and PMAi) and HEC-1A cells.(g) U R E 6 (See caption on next page).
and activate the NFκB pathway.The macrophage-derived factors, including soluble FKN and the recombinant FKN treatment, can both act on the expression of the CX3CR1, F I G U R E 6 The mRNA expression of HAMP (a) and (b) Western blot (WB) analysis of the NFκB1 (p50 [c] and p65 [d]) and (e) phospho-STAT3 transcription factors using ACHP inhibitor.The real-time PCR was performed using a SYBR Green protocol and GAPDH as a normalization gene.
(a)  Representative WBs of the target proteins.CX3CR1 and P-STAT3 were examined in the total cell lysates.The p50 and p65 proteins were analyzed in the chromatin-bound protein fraction.The upper GAPDH blot originated from the total cell lysates; the lower GAPDH blot is from the cytosolic fraction of the HEC-1A cells.(b) Analysis of the protein level of CX3CR1.(c) Analysis of the protein levels of p50.(d) Analysis of the levels of p65 protein.(e) Analysis of the phospho-STAT3 transcription factor.The columns represent the mean ± SD of three independent experiments (n = 3).The asterisk shows p < .05compared to the control, the cross shows p < .05compared to the cells treated with culture medium of PMA-activated THP-1 cells, and the double-cross means p < .05compared to the 10 ng/mL of FKN-supplemented cells.ACHP, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(4-piperidinyl)-3-pyridinecarbonitrile; CX3CR1, fractalkine receptor; F10, fractalkine 10 ng/mL; F20, fractalkine 20 ng/mL; FKN, fractalkine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HAMP, hepcidin gene; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate; P-STAT3, phospho-signal transducer and activator of transcription 3; SDS, sodium dodecyl sulfate.phenomenon of STAT3 activation.The levels of p50 and p65 were triggered upon macrophage-derived molecules and FKN, but using the 48 h conditioned medium, the activating effect of FKN was diminished.It can be supposed that the macrophage-derived TNF-α also influences the NFκB pathway and is evolved in the hepcidin regulation.Interestingly, the conditioned medium of the activated THP-1 cells and FKN increased the activity of the BMP6/SMAD1/5/8 signaling pathway.Moreover, when the FKN/CX3CR1-mediated signaling pathways, the NFκB and STAT3 were downregulated by ACHP treatment of the HEC-1A cells, the endometrial cells raised the BMP6 level and SMAD phosphorylation, and the protein level of TfR2 was also elevated upon conditioned medium therapy with or without FKN supplementation.These changes in the signaling pathways can contribute to elevated HAMP mRNA expression even if the aforementioned regulatory pathways are inhibited.Taken together, the alterations in the hepcidin-FP axis, the iron storage and release, and the iron and heme contents of the endometrium cells suggest that regulating the iron metabolism of the HEC-1A cells is a complex mechanism.Although the hepcidin level was elevated in the culture medium, which should activate the internalization of FP, FKN affected the outcome, which may be F I G U R E 7 ELISA measurements of (a) BMP6 and (b) Western blot (WB) analysis of (c) TfR2 and (d) phospho-SMAD1/5/8 transcriptional regulator.
Based on our results, macrophages, and FKN act as regulators of endometrial iron metabolism by modulating hepcidin synthesis.HEC-1A-derived hepcidin does not work only in an autocrine way on FP.The incubation time of activated THP-1 cells, possibly by the different levels of the secreted macrophage factors, influences the effect of FKN on the HEC-1A cells.A more complex model is necessary to elucidate the role of hepcidin in regulating overall endometrial iron metabolism.FKN signaling may be involved in the direction of the iron trafficking from the endometrium toward the embryo, implementing the reasonable iron supply for fetal development at the early stage of the pregnancy.

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I G U R E 8 ELISA measurements of BMP6 (a) and Western blot analysis (b) of TfR2 (c) and phospho-SMAD1/5/8 transcriptional regulator (d) using ACHP inhibitor.The ELISA measurement was carried out using the Human BMP6 ELISA Kit according to the manufacturer's protocol.For the WBs, the same amount of protein from each sample was separated into SDS-polyacrylamide gels.After blotting, the antibodies were used according to the manufacturer's protocol.GAPDH was used as the loading control.The blots in this figure are representative images.The analysis was performed using the ImageJ software.The columns show the mean values ± SD.In (a), the bullet shows p < .05compared to Ctrli, the asterisk means p < .05compared to the Ctrl, the cross signs p < .05compared to PMAi, and the double-cross means p < .05compared to PMA.In (c) and (d), the asterisk shows p < .05compared to the control, cross signs p < .05compared to the cells treated with culture medium of PMA-activated THP-1 cells.ACHP, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(4-piperidinyl)-3-pyridinecarbonitrile; BMP6, bone morphogenetic protein 6; Ctrli, initial concentration of the protein in the control culture medium; ELISA, enzyme-linked immunosorbent assay; F10, fractalkine 10 ng/mL; F20, fractalkine 20 ng/mL; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PMA, phorbol 12-myristate 13-acetate; PMAi, initial concentration of the protein in the culture medium of PMA-treated THP-1 cells; SDS, sodium dodecyl sulfate; SMAD1/ 5/8, suppressor of mothers against decapentaplegic; TfR2, transferrin receptor 2observations, we could get a better insight into the regulation of endometrial iron metabolism by macrophagederived factors and FKN, which are both involved in establishing endometrial receptivity.Although the results underlie the regulating effects of activated THP-1 macrophages and FKN on the iron metabolism of HEC-1A endometrial cells, which are the significant limitations of the study, further investigations are necessary for verifying the revealed alterations in a coculture or in vivo systems.5 | CONCLUSIONSThe study aimed to investigate the relationship between the various factors influencing iron metabolism of the endometrium and, therefore, the receptivity and implantation.The relationship between FKN and iron metabolism has been demonstrated, but the interaction of cytokines, FKN, and iron levels in the endometrium has yet to be investigated.The observations support our hypothesis that FKN and the factors secreted by the activated macrophages are implicated in the regulation of the iron metabolism of the endometrial cells.The FKN/CX3CR1 axis and the macrophages affect hepcidin synthesis and impact the endometrial cells' iron utilization.It is supposed that both macrophage-derived and recombinant FKN evolved to control the iron transport from the endometrium, and the proper concentration of FKN is essential in maintaining iron homeostasis.Macrophage-derived factors and FKN are crucial in the iron support of the growing fetus before the development of the placenta.The utilization of the conditioned medium of the THP-1 cells provides us a deeper insight into the action of the secreted molecules, including FKN, on the endometrial cells and can add new aspects to the importance of the proper FKN concentration in the regulation of endometrial iron metabolism, receptivity, and the implantation.