Human Decidual Cells Research Articles

119: The effect of 1,25-dihydroxyvitamin D3 (1,25[OH2]D3), on lipopolysacharide(LPS) induced cytokine expression by human decidual cells

ObjectiveDecidual inflammation may contribute to the cascade of events that culminate in preterm labor and delivery. The anti-inflammatory effect of vitamin D is well established. There is compelling evidence for increased frequency of spontaneous preterm birth with vitamin D deficiency. The purpose of this study was to evaluate the ability of the active form of vitamin D, 1,25[OH2]D3, to modify the inflammatory response of human decidual cells exposed to LPS.Study designDecidua was scraped from the membranes from the placentae of 4 women undergoing term prelabor cesarean section . Decidual cells were obtained through enzymatic digestion and Percoll gradient purification. Cells were cultured to confluence and pretreated with 1,25[OH2]D3 for 24 hours before LPS administration. mRNA was then extracted and real time PCR was used to compare expression of interleukin(IL)-1α, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor-α(TNF-α) and IL-1 receptor antagonist(RA) mRNA relative to GAPDH.ResultsPretreatment with 1,25[OH2]D3 decreased the relative basal expression of IL-6 and IL-8 mRNA (p<0.01). IL-8 mRNA was significantly decreased in the LPS stimulated cultures pretreated with 1,25[OH2]D3 (p<0.05) versus LPS stimulation alone. In regards to IL-1α, IL-4, IL-10, TNF-α, and IL-1RA mRNA production, trends toward reduced expression were noted in all the cultures pretreated with 1,25[OH2]D3, yet statistically significant changes were not identified.ConclusionVitamin D abrogated the ability of LPS to stimulate IL-8 expression in human cultured decidual cells. This change in expression could account for the decreased frequency of spontaneous preterm birth in patients with sufficiency of vitamin D. Future studies exploring the possible therapeutic anti-inflammatory effects of vitamin D are warranted. ObjectiveDecidual inflammation may contribute to the cascade of events that culminate in preterm labor and delivery. The anti-inflammatory effect of vitamin D is well established. There is compelling evidence for increased frequency of spontaneous preterm birth with vitamin D deficiency. The purpose of this study was to evaluate the ability of the active form of vitamin D, 1,25[OH2]D3, to modify the inflammatory response of human decidual cells exposed to LPS. Decidual inflammation may contribute to the cascade of events that culminate in preterm labor and delivery. The anti-inflammatory effect of vitamin D is well established. There is compelling evidence for increased frequency of spontaneous preterm birth with vitamin D deficiency. The purpose of this study was to evaluate the ability of the active form of vitamin D, 1,25[OH2]D3, to modify the inflammatory response of human decidual cells exposed to LPS. Study designDecidua was scraped from the membranes from the placentae of 4 women undergoing term prelabor cesarean section . Decidual cells were obtained through enzymatic digestion and Percoll gradient purification. Cells were cultured to confluence and pretreated with 1,25[OH2]D3 for 24 hours before LPS administration. mRNA was then extracted and real time PCR was used to compare expression of interleukin(IL)-1α, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor-α(TNF-α) and IL-1 receptor antagonist(RA) mRNA relative to GAPDH. Decidua was scraped from the membranes from the placentae of 4 women undergoing term prelabor cesarean section . Decidual cells were obtained through enzymatic digestion and Percoll gradient purification. Cells were cultured to confluence and pretreated with 1,25[OH2]D3 for 24 hours before LPS administration. mRNA was then extracted and real time PCR was used to compare expression of interleukin(IL)-1α, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor-α(TNF-α) and IL-1 receptor antagonist(RA) mRNA relative to GAPDH. ResultsPretreatment with 1,25[OH2]D3 decreased the relative basal expression of IL-6 and IL-8 mRNA (p<0.01). IL-8 mRNA was significantly decreased in the LPS stimulated cultures pretreated with 1,25[OH2]D3 (p<0.05) versus LPS stimulation alone. In regards to IL-1α, IL-4, IL-10, TNF-α, and IL-1RA mRNA production, trends toward reduced expression were noted in all the cultures pretreated with 1,25[OH2]D3, yet statistically significant changes were not identified. Pretreatment with 1,25[OH2]D3 decreased the relative basal expression of IL-6 and IL-8 mRNA (p<0.01). IL-8 mRNA was significantly decreased in the LPS stimulated cultures pretreated with 1,25[OH2]D3 (p<0.05) versus LPS stimulation alone. In regards to IL-1α, IL-4, IL-10, TNF-α, and IL-1RA mRNA production, trends toward reduced expression were noted in all the cultures pretreated with 1,25[OH2]D3, yet statistically significant changes were not identified. ConclusionVitamin D abrogated the ability of LPS to stimulate IL-8 expression in human cultured decidual cells. This change in expression could account for the decreased frequency of spontaneous preterm birth in patients with sufficiency of vitamin D. Future studies exploring the possible therapeutic anti-inflammatory effects of vitamin D are warranted. Vitamin D abrogated the ability of LPS to stimulate IL-8 expression in human cultured decidual cells. This change in expression could account for the decreased frequency of spontaneous preterm birth in patients with sufficiency of vitamin D. Future studies exploring the possible therapeutic anti-inflammatory effects of vitamin D are warranted.

721: Omega-3 fatty acids (n-3 FA) inhibit IL-1-induced synthesis and trafficking of prostanoid enzymes in cultured human decidual cells: A potential mechanism by Which n-3 FA inhibit spontaneous preterm birth (SPTB)

Omega-3 fatty acid (n-3 FA) supplementation during pregnancy has been shown to decrease the incidence of SPTB. We previously demonstrated that preincubation with docosahexaenoic acid (DHA) reduces prostaglandin (PG) E-2 production stimulated by IL-1β. The objective of this study was to determine effects of DHA on localization of prostanoid enzyme expression in cultured decidual cells (DC).

Human decidual stromal cells express HLA-G Effects of cytokines and decidualization

Decidual stromal cells (DSC) are the main cellular component of the decidua, the maternal tissue in close contact with fetal trophoblast. Although of mesenchymal origin, DSC exert numerous immune functions that seem to be relevant for the immunological relationship between the mother and fetus. HLA-G, an antigen preferentially expressed by trophoblast, appears to participate in the immune tolerance by the mother of the semiallogeneic fetus. We show by flow cytometry, fluorescence microscopy, western blotting and RT-PCR that DSC isolated and maintained in culture express HLA-G weakly but consistently. We also detected this antigen by flow cytometry in fresh DSC. Interleukin (IL)-10, a cytokine associated with normal pregnancy, increased the expression of HLA-G by DSC (P < 0.00001), whereas IL-2, a cytokine involved in spontaneous abortion, showed no effect. Decidualization by progesterone and cAMP also up-regulated the expression of HLA-G by DSC (P < 0.001). Interferon gamma, a cytokine implicated in the vascular remodelling of the decidua necessary for embryo implantation, also increased the expression of HLA-G by DSC (P < 0.05). Our results suggest the existence of a network in which hormones together with cytokines regulate the expression of HLA-G by DSC, and that may be of relevance in the maintenance of maternal-fetal tolerance.

Human decidual cell Toll-like receptor signaling in response to endotoxin: The effect of progestins

The purpose of this study was to determine whether progesterone, 17-alpha-hydroxyprogesterone, and 17-alpha hydroxyprogesterone caproate modulate the Toll-like receptor (TLR) pathway in the response of decidua to lipopolysaccharide. Cultured human decidual cells were incubated under control conditions, lipopolysaccharide alone, or pretreatment with each of the 3 progestins. Relative expression of 113 genes in the TLR pathway was determined by microarray. We failed to demonstrate a suppression of TLR gene pathway expression in human decidual cells in response to lipopolysaccharide when the cells are pretreated with progestins. Pretreatment with each progestin before lipopolysaccharide resulted in a relative increase in the expression of the proapoptotic molecule, CASP8. There were no differences among the progestins. Our data do not support suppression of TLR pathways as a mechanism for the benefit of 17-alpha hydroxyprogesterone caproate. Increased CASP8 gene expression raises the possibility that progestins "prime" the decidual cell to respond with a NFkappaB-mediated inflammatory response.

Effects of placental isoferritin on the mouse embryo development in vitro

To investigate the effect of placental isoferritin (PLF) on mouse embryo development in vitro, mice 2-cell embryos were co-cultured with human first trimester decidual cells at different concentrations of PLF in vitro. The following changes of the above system were observed under an invert microscope and the number of embryos were recorded and the embryos were classified. The results showed there was no significant difference in the percentage of embryos development to 4-cell, 8-cell and morula (P>0.05). PLF at the doses of 10 and 100 U/mL significantly enhanced more embryos development to the blastocyst and hatching blastocyst (P<0.05). PLF at the dose of 1000 U/mL depressed more embryos development from 2-cell to hatching blastocyst, meanwhile such phenomena as cell degeneration and irregular cleavage were observed in part of embryos, but there was no significant difference in statistics (P>0.05). It was concluded that PLF at the concentration of 10-100 U/mL had no significant effects on the early development of mice embryos, however, PLF could promote the growth, differentiation, and hatching of preimplantation blastocysts.

Regulation of C-C motif chemokine ligand 2 and its receptor in human decidual stromal cells by pregnancy-associated hormones in early gestation

Decidua is in close contact with the fetal trophoblasts, and involved in immune relationship of mother to fetus. However, the roles of decidua and decidual stromal cells (DSC) in materno-fetal immune regulation remain to be elucidated. In the present study, the expression and regulation of chemokines and their receptors in decidua and DSCs were investigated. The transcription of 18 chemokine receptors in human first-trimester decidual tissue and DSC were first analysed by RT-PCR. Among these receptors, C-C motif chemokine receptor-2 (CCR2) was highly transcribed. It was demonstrated by RT-PCR and immunostaining that both CCR2 and its major ligand, C-C motif chemokine ligand 2 (CCL2, monocyte chemoattractant protein-1), were expressed in decidua and DSC. We then detected CCL2 in the supernatant of primary cultures of DSC by enzyme-linked immunosorbent assay. It was shown that DSC secreted CCL2 spontaneously and continuously over 72 h (21.72 +/- 2.34 ng/ml), and the CCR2 antagonist RS102895 and an inhibitor of the map kinase kinase/mitogen-activated protein kinase (ERK/MAPK) signal pathway decreased significantly the CCL2 secretion of DSC (both P < 0.05). We further studied effects of the pregnancy-associated hormones, estrogen, progesterone or HCG on CCL2 secretion by DSC. CCL2 secretion by DSC was up-regulated by estrogen, progesterone or HCG. CCR2 and CCL2 are co-expressed by human first-trimester DSC and decidual tissue. CCL2 is secreted in an autocrine manner through the ERK/MAPK pathway, and is up-regulated by the pregnancy-associated hormones, estrogen, progesterone and HCG, which suggests that CCL2 may play an important role at materno-fetal interface.

Physiological Smooth Muscle Cell Apoptosis Contributes to the Uterine Vascular Remodeling in Human Early Pregnancy

See related article, pages 834–841 Although equipped with the entire machinery driving cell proliferation and cell death, the turnover of the vascular smooth muscle cell (VSMC) layer within the vascular wall is quite low throughout the adult normal body. Vascular wall remodeling with VSMC apoptosis is routinely considered as associated with vascular diseases. This includes the development and regression of vascular thickening in hypertension, in advanced atherosclerotic plaques where VSMC apoptosis promotes plaque rupture, after angioplasty-induced arterial injury, as well as in arterial aneurysm where apoptosis is responsible for excessive slackening and rupture of the vessel media.1 Furthermore, in several animal studies, early pregnancy is a physiological situation where vessel wall remodeling plays a vital role. Indeed, in the first trimester of human pregnancy, vascular wall remodeling through degenerative changes converts the uterine spiral arteries located within the decidua basilis into toneless, widely opened arteries, thus enabling abundant blood supply to the maternal-fetal exchange area within the placenta. It is known that extravillous cytotrophoblasts deriving from fetal cytotrophoblastic shell migrate into segments of the spiral artery wall, through retrograde endovascular (intravasation) and interstitial migratory pathways (extravasation) into the decidua basilis to trigger the disappearance of endothelial and VSMC. In this process, trophoblasts become buried intramurally within a fibrinoid layer, which replaces the original muscular medial layer. Such trophoblast invasion converts the ends of spiral arteries into a wide caliber vessel that can better deliver maternal blood to the expanding intervillous space of the placenta (Figure 1A). Defective or incomplete trophoblast invasion can result in pathological pregnancies, including preeclampsia and intrauterine growth restriction.2,3 Therefore, understanding the cellular mechanisms governing the interaction between intramural trophoblast and vascular wall cells is yet to be determined. Oversimplified schematic depicting the trophoblasts invading the spiral artery traveling from myometrium up to …

Thrombin regulates monocyte chemoattractant protein-1 expression in human first trimester and term decidual cells

Excess decidual macrophage infiltration has been linked to preeclampsia and a failure of endovascular trophoblast invasion. Severe preeclampsia with shallow placentation has also been linked to acquired and inherited maternal thrombophilias and recurrent decidual hemorrhage, which generates thrombin from decidual cell-expressed tissue factor. Therefore, the current study evaluated whether thrombin affects monocyte chemoattractant protein-1 (MCP-1) expression in stromal cells that are derived from cycling and gestational endometrium. Stromal cells that are isolated from cycling endometrium and first trimester and term decidua were grown to confluence, treated for 7 days with 10(-8) mol/L estradiol (E2) + 10(-7) mol/L medroxyprogesterone acetate (MPA), then switched to a serum-free medium that contained the corresponding steroids +/- thrombin. MCP-1 protein release was measured by enzyme-linked immunosorbent assay and Western blot; MCP-1 messenger RNA levels were assessed by real-time quantitative reverse transcription-polymerase chain reaction. Secreted MCP-1 levels were not significantly different in stromal or decidual cell cultures that were incubated with E2 or with E2 + MPA. Thrombin increased immunoreactive MCP-1 expression in a dose-response fashion in first trimester and term decidual cells but not in endometrial stromal cells. Thrombin-induced MCP-1 protein output was unaffected by MPA but was abrogated by incubation with the thrombin inhibitor, hirudin. Unexpectedly, thrombin-enhanced MCP-1 protein expression was unaccompanied by corresponding changes in steady state MCP-1 messenger RNA levels, which suggests its effects were posttranslational. MCP-1 protein expression is up regulated by thrombin in decidual cells across gestation, but not in stromal cells from predecidualized cycling endometrium.

Innate immune function of the human decidual cell at the maternal–fetal interface

The aim of the study was to investigate the innate immune function of decidual cells from term unlabored pregnancies by determining Toll-like receptor presence and function. Using immunohistochemistry, reverse transcriptase polymerase chain reaction and NF-κB-luciferase plasmid transfection, Toll-like receptor presence and function was determined. Decidual cells express Toll-like receptors 1, 2, 4 and 6 which respond to lipopolysaccharide and peptidoglycan stimulation producing Interleukin-8. Decidual cells from women at term have innate immune function and are capable of producing Interleukin responses to bacterial ligands.

Identification of genes regulated by interleukin-1 in human decidual cells: Elucidating mechanisms of infection-mediated preterm birth

CELLS: ELUCIDATING MECHANISMS OF INFECTION-MEDIATED PRETERM BIRTH ASHLEY ROMAN, AMY STAGG, GREG DORSAINVILLE, PETER NATHANIELSZ, New York University, Obstetrics and Gynecology, New York, New York, University of Texas Health Science Center at San Antonio, Obstetrics and Gynecology, San Antonio, Texas OBJECTIVE: IL-1b is a cytokine produced by human decidua and macrophages in response to microbial invasion and has been linked to the multiple processes leading to preterm labor and delivery. To date, the effect of IL-1b on the decidual cell transcriptome has not been studied (based on a MEDLINE search using keywords ‘‘interleukin-1’’ and ‘‘pregnancy’’). The objective of this study is to characterize the pattern of gene expression in response to IL-1b in cultured decidual cells. STUDY DESIGN: Decidua was obtained from placentas of non-laboring patients at term cesarean delivery (N=6) and cultured using standard cell culture technique. Cultures were exposed to IL-1b or vehicle for 12 hours; total RNA was harvested and prepared for microarray study. The decidual transcriptome was studied using Affymetrix hgu133plus2.0 microarrays. Expression was normalized, the average probe intensity scaled, and a detection p-value used to predict gene expression. Fold-changes were calculated from mean signals in the IL-1b group versus controls; a 2-fold change was considered significant. Quantitative RT-PCR was performed for array confirmation. Groups were compared using Student t-test; significance at p!0.05. RESULTS: The expression of over 47,000 transcripts was evaluated. 208 transcripts were significantly up-regulated; 38 were significantly down-regulated. We confirmed the up-regulation of 6 genes (VEGF, VCAM-1, mPGES, GCSF, IL-6, and phospholipase A2) with RT-PCR (p!0.05 in all cases). Specific ontologic pathways activated by IL-1b were cytokine-cytokine receptor interaction, cell cycle, prostaglandin and leukotriene metabolism, glutamate metabolism, and phospholipid degradation. CONCLUSION: This first comprehensive assessment of the impact of IL-1b on gene expression in cultured decidual cells reveals significant differences in patterns of expression in multiple ontologic pathways. The genes and pathways identified may serve as potential therapeutic targets for preventing or arresting preterm labor.

Expression of ADAMTS-5/implantin in human decidual stromal cells: regulatory effects of cytokines

The restricted expression of ADAMTS-5 (A Disintegrin And Metalloproteinase with ThromboSpondin repeats-5) to the maternal-fetal interface in mice has led to this novel metalloproteinase being assigned the trivial name 'implantin'. As a first step in determining whether ADAMTS-5 also contributes to the implantation process in humans, we have examined the spatiotemporal expression of this ADAMTS subtype in the endometrium during the menstrual cycle and pregnancy by immunohistochemical analysis. A quantitative competitive PCR (QC-PCR) strategy and western blotting were subsequently used to determine whether interleukin (IL)-1beta and transforming growth factor (TGF)-beta1, two cytokines involved in the formation of the maternal-fetal interface, were capable of regulating ADAMTS-5 messenger RNA (mRNA) and protein levels in primary cultures of stromal cells isolated from first trimester decidual tissues. ADAMTS-5 expression in the stroma of the human endometrium correlates with decidualization of this cellular compartment in vivo. IL-1beta was found to increase (P < 0.05) whereas TGF-beta1 decreased (P < 0.05) ADAMTS-5 mRNA and protein levels in decidual stromal cell cultures in a concentration- and time-dependent manner. These regulatory effects were attenuated by function-perturbing antibodies directed against either cytokine. ADAMTS-5 expression is restricted to decidualized stromal cells of the human endometrium in vivo and is subject to regulation by cytokines in vitro.

Effects of 25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 on Cytokine Production by Human Decidual Cells1

The active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) is a potent immunomodulatory seco-steroid. We have demonstrated that several components of vitamin D metabolism and signaling are strongly expressed in human uterine decidua from first trimester pregnancies, suggesting that locally produced 1,25(OH)(2)D(3) may exert immunosuppressive effects during early stages of gestation. To investigate this further, we used primary cultures of human decidual cells from first and third trimester pregnancies to demonstrate expression and activity of the enzyme that catalyzes synthesis of 1,25(OH)(2)D(3), 1alpha-hydroxylase (CYP27B1). Synthesis of 1,25(OH)(2)D(3) was higher in first trimester decidual cells (41 +/- 11.8 fmoles/h/mg protein) than in third trimester cells (8 +/- 4.4 fmoles/h/mg protein; P < 0.05). Purification of decidual cells followed by quantitative RT-PCR analysis showed that CYP27B1 was expressed by both CD10(+VE) stromal-enriched and CD10(-VE) stromal-depleted cells, with higher levels of mRNA in first trimester pregnancies. Expression of CYP27B1 correlated with TLR4 and IDO. Functional responses to 1,25(OH)(2)D(3) were studied using CD56(+VE) natural killer (NK) cells isolated from first trimester decidua. Decidual NK cells treated with 1,25(OH)(2)D(3) or precursor 25-hydroxyvitamin D(3) (25OHD(3)) for 28 h showed decreased synthesis of cytokines, such as granulocyte-macrophage colony stimulating factor 2 (CSF2), tumor necrosis factor, and interleukin 6, but increased expression of mRNA for the antimicrobial peptide cathelicidin antimicrobial peptide. These data indicate that human decidual cells are able to synthesize active 1,25(OH)(2)D(3), particularly in early gestation, and this may act in an autocrine/paracrine fashion to regulate both acquired and innate immune responses at the fetal-maternal interface.

Microarray analysis of highly purified human decidual NK cells

Microarray analysis of highly purified human decidual NK cells

Regulation of chemokine production in response to pro-inflammatory cytokines in first trimester decidual cells

Objective Chemokines initiate the immune response by controlling leukocyte migration and lymphocyte development. Macrophage infiltration of the decidua has been implicated in the genesis of recurrent miscarriage and preeclampsia. Therefore, we determined whether cultured human decidual cells produce monocyte/macrophage-recruiting chemokines in response to a potent pro-inflammatory cytokine, interleukin-1β (IL-1β), and whether decidual cell-conditioned medium contains monocyte- and macrophage-chemoattractant activity. Methods Leukocyte-free first trimester decidual cells were treated for 6 h with estradiol (E 2) and medroxyprogesterone acetate (MPA) to mimic the steroidal milieu of pregnancy, or E 2 and MPA and IL-1β (1 ng/ml) to mimic inflamed decidua. Total RNA was used for cDNA synthesis. Biotinylated cRNAs were generated and chemically fragmented for hybridization on Affymetrix HG_U133 Plus 2.0 chips followed by fluorescence labeling and optical scanning. Raw data generated from Affymetrix GCOS 1.2 (GeneChip Operating Software) were analyzed by GeneSpring 7.2 software. Subsequently microarray results were validated by real time RT-PCR and Western blotting. A functional study of monocyte migration was carried out also using conditioned media from culture. Results Five chemokines responsible for monocyte/macrophage chemoattraction and activation, including C-C motif ligand 2 (CCL2), CCL5, C-X-C motif ligand 2 (CXCL2), CXCL3 and CXCL8, were markedly elevated from 29- to 975-fold after exposure to IL-1β in cultured first trimester decidual cells. The results of real-time RT-PCR (up-regulation from 43- to 3069-fold) and Western blotting (up-regulation from 15- to 300-fold) confirmed the microarray findings. Monocyte migration was significantly induced by the conditioned medium from IL-1β-treated decidual cells. Conclusions Treatment of first trimester decidual cells with IL-1β induces secretion of monocyte/macrophage recruiting-chemokines and promotes monocyte migration. Extrapolation of these in vitro results to the milieu of implantation site suggests a mechanism whereby IL-1β could mediate excessive macrophage infiltration of the decidua.

Differential effects of interleukin-1β and transforming growth factor-β1 on the expression of the inflammation-associated protein, ADAMTS-1, in human decidual stromal cells in vitro

The pro-inflammatory cytokine, interleukin-1 beta (IL-1beta) promotes the proteolytic degradation of the extracellular matrix (ECM) of maternal decidua, a critical step in pregnancy that is counterbalanced by the expression of the anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-beta1). Recently, the inflammation-associated protein, ADAMTS-1, a member of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) gene family of metalloproteinases has been assigned a central role in the formation and organization of tissues. In view of these observations, we have hypothesized that ADAMTS-1 contributes to the cytokine-mediated remodelling of decidual ECM. The spatiotemporal expression of ADAMTS-1 in human endometrium was examined by immunohistochemistry. A quantitative-competitive (QC)-PCR strategy and western blot analysis was then employed to determine whether IL-1beta and TGF-beta1 regulate ADAMTS-1 mRNA and protein expression levels in primary cultures of stromal cells isolated from first trimester decidua. ADAMTS-1 expression is associated with decidualization of the endometrial stroma in vivo. IL-1beta increased whereas TGF-beta1 decreased ADAMTS-1 mRNA and protein levels in decidual stromal cell cultures in a concentration- and time-dependent manner. These regulatory effects were attenuated by function-perturbing antibodies specific for either cytokine. IL-1beta and TGF-beta1 differentially regulate ADAMTS-1 expression in human decidual stromal cells.

Forkhead transcription factor FOXO1A is critical for induction of human decidualization

Experiments utilizing RNA interference technology were performed to determine whether the forkhead transcription factor FOXO1A, a member of the FOXO family of proteins, plays a critical role in the induction of human uterine decidualization. Human decidual fibroblast cells were decidualized in vitro for 6 days with medroxyprogester-one, estradiol, and dibutyryl cAMP in the presence or absence of a highly specific FOXO1A small interfering RNA (siRNA) that inhibits FOXO1A mRNA and protein expression by more than 80%. RNA and proteins were extracted from the cells at 0, 2, 4, and 6 days. FOXO1A and IGFBP-1 proteins were determined by immunoblotting; and intracellular mRNA levels for several decidualization marker genes were determined by real-time PCR. Exposure of the cells to FOXO1A siRNA in five separate experiments resulted in a 40-75% inhibition of prolactin, IGFBP-1, tissue inhibitor of metalloproteinase 3 (TIMP3), somatostatin and endometrial bleeding-associated factor (EBAF) mRNAs, all of which are markedly induced during the decidualization process. In contrast, actin and GAPDH mRNA levels did not change during decidualization. The inhibition of mRNA levels was first noted at day 2 and persisted for the remainder of each experiment. Western blot analysis indicated that the FOXO1A siRNA inhibited IGFBP-1 protein expression by 60-80%. Decidual fibroblast cells exposed in an identical manner to a control RNA that had no effect on FOXO1A expression caused only a 0-15% inhibition of the marker genes and IGFBP-1 protein. Taken together, these findings strongly suggest a critical role for FOXO1A in the induction of human decidualization.

Thrombin and Interleukin-1β Regulate HOXA10 Expression in Human Term Decidual Cells: Implications for Preterm Labor

Preterm delivery is commonly caused by intraamniotic infection with expression of proinflammatory cytokines (IL-1beta) or by abruption resulting in generation of decidual thrombin. Although human parturition is not preceded by overt progesterone withdrawal, progesterone resistance likely leads to labor. The uteri of Hoxa10(-/-) mice demonstrate progesterone resistance; several genes, including prostaglandin receptors, are inappropriately regulated in response to progesterone. We hypothesized that IL-1beta or thrombin would decrease HOXA10 expression, contributing to the progestin-resistant environment. We analyzed expression of HOX genes and their regulation by IL-1beta or thrombin in decidual cells. We conducted an in vitro experiment at an academic medical center. Term decidual cells were treated with estradiol (E(2)) or E(2) plus medroxyprogesterone acetate followed by addition of thrombin or IL-1beta. HOX mRNA was evaluated by microarray and confirmed by quantitative RT-PCR. Protein expression was detected using immunohistochemistry and Western analysis. HOXA9, HOXA10, and HOXA11 were expressed in decidual cells and regulated by IL-1beta and thrombin. HOXA10 was further analyzed because of its association with progesterone responsiveness. After E(2) treatment, IL-1beta and thrombin decreased HOXA10 mRNA by 94 and 81%, respectively. After E(2) plus medroxyprogesterone acetate treatment, IL-1beta and thrombin resulted in an 86 and 72% decrease in HOXA10 mRNA, respectively. A similar decrease was noted in HOXA10 protein expression. The expression of HOXA10 protein at term indicates that it may have a role in maintaining decidual cell phenotype and pregnancy. The dramatic decrease of HOXA10 in response to IL-1beta or thrombin may contribute to progestin resistance in preterm labor, mimicking progesterone resistance seen in Hoxa10(-/-) mice.

Human Decidual NK Cells: Unique Phenotype and Functional Properties – A Review

Human decidual NK cells are massively recruited at the site of embryonic implantation (decidua basalis). They differ in many ways from their peripheral blood NK cell counterparts in terms of gene expression, phenotype and functionality. The major subpopulation of decidual NK cells is CD56(bright) whereas the minor subset is CD56(dim), contrasting with the peripheral blood NK cells whose major subpopulation is CD56(dim). Decidual NK cell cytolytic function is much reduced despite the presence of several activating receptors and the essential machinery required for lysis. Decidual NK cells produce a number of cytokines that are not normally secreted by peripheral blood NK cells. Human decidual NK cell potential functions at the maternal-fetal interface are not yet clearly established but several hypotheses are being evaluated, including control of extravillous invasion, control of uterine vascular remodeling, and local anti-viral activity.

Regulation of Monocyte Chemoattractant Protein-1 Expression by Tumor Necrosis Factor-α and Interleukin-1β in First Trimester Human Decidual Cells: Implications for Preeclampsia

The current study describes a statistically significant increase in macrophages (CD68-positive cells) in the decidua of preeclamptic patients. To elucidate the regulation of this monocyte infiltration, expression of monocyte chemoattractant protein-1 (MCP-1) was assessed in leukocyte-free first trimester decidual cells. Confluent decidual cells were primed for 7 days in either estradiol or estradiol plus medroxyprogesterone acetate to mimic the decidualizing steroidal milieu of the luteal phase and early pregnancy. The medium was exchanged for a serum-free defined medium containing corresponding steroids +/- tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta. After 24 hours, enzyme-linked immunosorbent assay measurements indicated that the addition of medroxyprogesterone acetate did not affect MCP-1 output, whereas 10 ng/ml of TNF-alpha or IL-1beta increased output by 83.5-fold +/- 20.6 and 103.1-fold +/- 14.7, respectively (mean +/- SEM, n = 8, P < 0.05). Concentration-response comparisons revealed that even 0.01 ng/ml of TNF-alpha or IL-1beta elevated MCP-1 output by more than 15-fold. Western blotting confirmed the enzyme-linked immunosorbent assay results, and quantitative reverse transcriptase-polymerase chain reaction confirmed corresponding effects on MCP-1 mRNA levels. The current study demonstrates that TNF-alpha and IL-1beta enhance MCP-1 in first trimester decidua. This finding suggests a mechanism by which recruitment of excess macrophages to the decidua impairs endovascular trophoblast invasion, the primary placental defect of preeclampsia.

Human decidual natural killer cells as a source and target of macrophage migration inhibitory factor

The human uterine mucosa of early pregnancy is largely populated by CD56(bright) natural killer (NK) cells (uterine (u) NK cells). The specific functions of these cells are still unknown, but their interaction and response to foetal trophoblasts are thought to be important for the establishment of a successful pregnancy. The study reported herein shows that uNK cells respond to, and produce, macrophage migration inhibitory factor (MIF), a cytokine highly expressed in the human placenta and in the cyclic and pregnant endometrium. Recombinant human MIF reduced in a dose-dependent manner the cytolytic activity of purified uNK cells against K562 cells. RT-PCR, Western blot analysis and ELISA demonstrated the synthesis and secretion of the cytokine by uNK cells. Double immunofluorescence staining showed the presence of MIF in uterine CD56 + cells. Finally, neutralization of the endogenous cytokine by a polyclonal antibody resulted in a sharp increase in the cytolytic activity of uNK cells. These findings indicate the existence of a previously unrevealed paracrine and autocrine action of MIF on uNK cells and support its contribution to the immune privilege at the maternal-foetal interface.