S. Joseph Huang

Preeclampsia-Related Inflammatory Cytokines Regulate Interleukin-6 Expression in Human Decidual Cells

Preeclampsia, a common pregnancy disorder associated with an increase in systemic inflammation, is the leading cause of maternal and fetal morbidity and mortality throughout the world. It is associated with shallow extravillous trophoblast invasion of the decidua, leading to uteroplacental blood flow that is inadequate for the developing fetal-placental unit. In preeclamptic women, interleukin-6 (IL-6) levels in plasma, but not placenta, are elevated, prompting evaluation of the decidua as a potential source of this excess, circulating IL-6. The current study found significantly higher immunohistochemical staining for IL-6 in decidual cells from preeclamptic versus preterm, gestational age-matched control placentas. Pro-inflammatory cytokines associated with the genesis of preeclampsia (i.e., tumor necrosis factor-alpha and interleukin-1beta) enhanced IL-6 mRNA levels and increased secreted IL-6 levels in first trimester leukocyte-free decidual cell incubations, as measured by real time quantitative RT-PCR, ELISA, and Western blotting. Therefore, decidual cell-derived IL-6 may contribute to excess circulating IL-6 levels that can promote both endothelial cell dysfunction (and subsequent vascular dysfunction) and the pathogenesis of preeclampsia whereas locally elevated IL-6 levels may contribute to an excess of decidual macrophages implicated in shallow extravillous trophoblast invasion of the decidua.

Matrix Metalloproteinase 9 (MMP9) Expression in Preeclamptic Decidua and MMP9 Induction by Tumor Necrosis Factor Alpha and Interleukin 1 Beta in Human First Trimester Decidual Cells

Extravillous trophoblasts (EVTs) invade human decidua via sequential integrin-mediated binding and proteolysis of basement membrane proteins in the extracellular matrix (ECM). In preeclampsia, shallow EVT invasion impairs spiral artery and arteriole remodeling to reduce uteroplacental blood flow. Excess decidual cell-expressed matrix metalloproteinases (MMPs) 2 and 9, in response to preeclampsia-related interleukin 1 beta (IL1B) and tumor necrosis factor alpha (TNF), may inappropriately degrade these basement membrane proteins and impede EVT invasion. This study found significantly higher immunohistochemical MMP9 levels in decidual cells and adjacent interstitial trophoblasts in placental sections of preeclamptic versus gestational age-matched control women. In contrast, immunostaining for MMP2 and tissue inhibitor of matrix metalloproteinases 1 and 2 (TIMP1 and TIMP2) were similar in preeclamptic and control groups. First-trimester decidual cells were incubated with estradiol (E(2)) or E(2) + medroxyprogesterone acetate (MPA), with or without TNF or IL1B. As measured by ELISA, both cytokines elicited concentration-dependent increases in secreted MMP9 levels that were unaffected by MPA. In contrast, secreted levels of MMP2, TIMP1, and TIMP2 were unchanged in all treatment groups. Substrate gel zymography and Western blotting confirmed that each cytokine increased secreted levels of MMP9 but not MMP2. Similarly, quantitative RT-PCR found that TNF and IL1B enhanced MMP9, but not MMP2, mRNA levels. At the implantation site, inflammatory cytokine-enhanced MMP9 may promote preeclampsia by disrupting the decidual ECM to interfere with normal stepwise EVT invasion.

A genomic and proteomic investigation of the impact of preimplantation factor on human decidual cells.

OBJECTIVEnPreimplantation factor (PIF) is a novel, 15 amino acid peptide, secreted by viable embryos. This study aims to elucidate PIFs effects in human endometrial stromal cells (HESC) decidualized by estrogen and progestin, which mimics the preimplantation milieu, and in first-trimester decidua cultures (FTDC).nnnSTUDY DESIGNnHESC or FTDC were incubated with 100 nmol/L synthetic PIF or vehicle control. Global gene expression was analyzed using microarray and pathway analysis. Proteins were analyzed using quantitative mass spectrometry, and PIF binding by protein array.nnnRESULTSnGene and proteomic analysis demonstrate that PIF affects immune, adhesion, and apoptotic pathways. Significant up-regulation in HESC (fold change) include: nuclear factor-k-beta activation via interleukin-1 receptor-associated kinase binding protein 1 (53); Toll-like receptor 5 (9); FK506 binding protein 15, 133kDa protein (2.3); and Down syndrome cell adhesion molecule like 1 (16). B-cell lymphoma protein 2 was down-regulated in HESC (21.1) and FTDC (27.1). Protein array demonstrates PIF interaction with intracellular targets insulin-degrading enzyme and beta-K+ channels.nnnCONCLUSIONnPIF displays essential multitargeted effects, of regulating immunity, promoting embryo-decidual adhesion, and regulating adaptive apoptotic processes.

High levels of Hsp90 cochaperone p23 promote tumor progression and poor prognosis in breast cancer by increasing lymph node metastases and drug resistance.

p23 is a heat shock protein 90 (Hsp90) cochaperone located in both the cytoplasm and nucleus that stabilizes unliganded steroid receptors, controls the catalytic activity of certain kinases, regulates protein-DNA dynamics, and is upregulated in several cancers. We had previously shown that p23-overexpressing MCF-7 cells (MCF-7+p23) exhibit increased invasion without affecting the estrogen-dependent proliferative response, which suggests that p23 differentially regulates genes controlling processes linked to breast tumor metastasis. To gain a comprehensive view of the effects of p23 on estrogen receptor (ER)-dependent and -independent gene expression, we profiled mRNA expression from control versus MCF-7+p23 cells in the absence and presence of estrogen. A number of p23-sensitive target genes involved in metastasis and drug resistance were identified. Most striking is that many of these genes are also misregulated in invasive breast cancers, including PMP22, ABCC3, AGR2, Sox3, TM4SF1, and p8 (NUPR1). Upregulation of the ATP-dependent transporter ABCC3 by p23 conferred resistance to the chemotherapeutic agents etoposide and doxorubicin in MCF-7+p23 cells. MCF-7+p23 cells also displayed higher levels of activated Akt and an expanded phosphoproteome relative to control cells, suggesting that elevated p23 also enhances cytoplasmic signaling pathways. For breast cancer patients, tumor stage together with high cytoplasmic p23 expression more accurately predicted disease recurrence and mortality than did stage alone. High nuclear p23 was found to be associated with high cytoplasmic p23, therefore both may promote tumor progression and poor prognosis by increasing metastatic potential and drug resistance in breast cancer patients.

Regulation of Interleukin-6 Expression in Human Decidual Cells and Its Potential Role in Chorioamnionitis

Chorioamnionitis frequently precedes both genital tract and placental inflammation and is both a primary cause of maternal morbidity and a major antecedent of preterm premature rupture of the membranes (PPROM) as well as preterm delivery (PTD). In most cases of chorioamnionitis, neutrophils dominate the decidua. In a subset of these cases, a predominance of monocytes is uniquely associated with both neonatal intraventricular hemorrhage and death. The multifunctional cytokine, interleukin-6, promotes local monocyte dominance via several mechanisms. In this study, immunostaining of placental sections revealed significantly higher interleukin-6 HSCOREs in decidual cells (DCs) but not in interstitial trophoblasts, in chorioamnionitis versus gestational age-matched control placentas (P < 0.05). In confluent leukocyte-free term DCs, secreted interleukin-6 levels in incubations with estradiol-17β were increased 2500-fold by IL-1β (P < 0.05). This up-regulation was inhibited by more than 50% in parallel incubations that included medroxyprogesterone acetate (n = 12, P < 0.05). Western blotting data confirmed these enzyme-linked immunosorbent assay results; quantitative RT-PCR findings demonstrated corresponding changes in interleukin-6 mRNA levels. Specific inhibitors of signaling for both nuclear factor-κB activation and p38-mitogen-activated protein kinase, but not for protein kinase C, significantly decreased IL-1β-enhanced interleukin-6 expression levels in cultured DCs. In conclusion, in situ and in vitro results indicate that significantly enhanced interleukin-6 expression levels in DCs during chorioamnionitis could be pivotal in skewing decidual monocyte differentiation to macrophages.

Preimplantation factor promotes first trimester trophoblast invasion.

OBJECTIVEnPreimplantation factor is a novel embryo-derived peptide that influences key processes in early pregnancy implantation, including immunity, adhesion, remodeling, and apoptosis. Herein, we explore the effects of synthetic preimplantation factor on trophoblast invasion.nnnSTUDY DESIGNnInvasion patterns of immortalized cultured HTR-8 trophoblast cells were analyzed through Matrigel extracellular matrix ± synthetic preimplantation factor (25-100 nM) in a transwell assay. Effects were compared with epidermal growth factor 10 μg/mL, scrambled aminoacid sequence of preimplantation factor, or media alone as controls.nnnRESULTSnSynthetic preimplantation factor enhances trophoblast invasion at physiologic doses (at 50 nM, 260%; 95% confidence interval [CI], 174-346%; P = .05; 100 nM ,178%; 95% CI, 170-184%; P < .02), compared with scrambled amnioacid sequence preimplantation factor or control media. Epidermal growth factor added to synthetic preimplantation factor does not further enhance trophoblast invasion (synthetic preimplantation factor 50 nM + epidermal growth factor, 238%; 95% CI, 237-239%; P < .03; synthetic preimplantation factor 100 nM + epidermal growth factor 269%; 95% CI, 265-273%; P < .04).nnnCONCLUSIONnPreimplantation factor should be further investigated as it shows a potential preventative or therapeutic role for pregnancy complications associated with inadequate trophoblast invasion.

NFκB and JNK/MAPK Activation Mediates the Production of Major Macrophage- or Dendritic Cell-Recruiting Chemokine in Human First Trimester Decidual Cells in Response to Proinflammatory Stimuli

CONTEXTnPreeclampsia is associated with elevated levels of proinflammatory cytokines, excess decidual macrophages, and dendritic cells. IL-1β- or TNF-α-stimulated leukocyte-free first trimester decidual cells produced abundant macrophage- and dendritic cell-recruiting chemokines identified in preeclamptic decidua.nnnOBJECTIVEnThe relative potency of IL-1β- or TNF-α-induced first trimester decidual cell-secreted chemokines in chemoattracting macrophages or dendritic cells and the signaling pathways involved in the expression of these chemokines were evaluated.nnnINTERVENTIONS AND MAIN OUTCOME MEASURESnFirst trimester decidual cells were treated with estradiol + medroxyprogesterone acetate ± IL-1β or TNF-α. The chemotaxis assay was performed by incubating conditioned medium from first trimester decidual cells with neutralizing antibody for six chemokines. The activation of each signaling pathway was examined by Western blotting, flow cytometry, confocal microscopy, and ELISA with or without kinase and nuclear factor κB (NFκB) inhibitors.nnnRESULTSnNeutralization of CCL2 and CCL5 significantly reduced chemotaxis of monocyte and dendritic cells up to 50 and 36%, respectively. NFκB and MAPK (MAPK kinase, JUN NH₂-terminal kinase, p38 kinase) pathways were activated by IL-1β or TNF-α in first trimester decidual cells. In IL-1β- or TNF-α-stimulated first trimester decidual cells, NFκB inhibitor suppressed production of all six chemokines; JUN NH₂-terminal kinase inhibitor inhibited secretion of CCL2, CCL4, and CCL5; and MAPK kinase and p38 inhibitor decreased production of CXCL8.nnnCONCLUSIONSnUp-regulation of CCL2 and CCL5 by first trimester decidual cells in response to proinflammatory stimuli may account for the accumulation of macrophages and dendritic cells in preeclamptic decidua. These chemokines and underlying IL-1β- or TNF-α-induced signaling molecules are potential diagnostic and therapeutic targets for preeclampsia.

The Implication of Aberrant GM-CSF Expression in Decidual Cells in the Pathogenesis of Preeclampsia

Preeclampsia is characterized by an exaggerated systemic inflammatory state as well as shallow placentation. In the decidual implantation site, preeclampsia is accompanied by an excessive number of both macrophages and dendritic cells as well as their recruiting chemokines, which have been implicated in the impairment of endovascular trophoblast invasion. Granulocyte-macrophage colony-stimulating factor is known to regulate the differentiation of both macrophages and dendritic cells, prompting both in vivo and in vitro evaluation of granulocyte-macrophage colony-stimulating factor expression in human decidua as well as in a mouse model of preeclampsia. This study revealed increased granulocyte-macrophage colony-stimulating factor expression levels in preeclamptic decidua. Moreover, both tumor necrosis factor-α and interleukin-1 β, cytokines that are implicated in the genesis of preeclampsia, markedly up-regulated granulocyte-macrophage colony-stimulating factor production in cultured first-trimester human decidual cells. The conditioned media of these cultures promoted the differentiation of both macrophages and dendritic cells from a monocyte precursor. Evaluation of a murine model of preeclampsia revealed that the decidua of affected animals displayed higher levels of immunoreactive granulocyte-macrophage colony-stimulating factor as well as increased numbers of both macrophages and dendritic cells when compared to control animals. Because granulocyte-macrophage colony-stimulating factor is a potent inducer of differentiation and activation of both macrophages and dendritic cells, these findings suggest that this factor plays a crucial role in the pathogenesis of preeclampsia.

Interferon-γ protects first-trimester decidual cells against aberrant matrix metalloproteinases 1, 3, and 9 expression in preeclampsia.

Human extravillous trophoblast (EVT) invades the decidua via integrin receptors and subsequently degrades extracellular matrix proteins. In preeclampsia (PE), shallow EVT invasion elicits incomplete spiral artery remodeling, causing reduced uteroplacental blood flow. Previous studies show that preeclamptic decidual cells, but not interstitial EVTs, display higher levels of extracellular matrix-degrading matrix metalloproteinase (MMP)-9, but not MMP-2. Herein, we extend our previous PE-related assessment of MMP-2 and MMP-9 to include MMP-1, which preferentially degrades fibrillar collagens, and MMP-3, which can initiate a local proteolytic cascade. In human first-trimester decidual cells incubated with estradiol, tumor necrosis factor-α (TNF-α) significantly enhanced MMP-1, MMP-3, and MMP-9 mRNA and protein levels and activity measured by real-time quantitative RT-PCR, ELISA, immunoblotting, and zymography, respectively. In contrast, interferon γ (IFN-γ) reversed these effects and medroxyprogesterone acetate elicited further reversal. Immunoblotting revealed that p38 mitogen-activated protein kinase signaling mediated TNF-α enhancement of MMP-1, MMP-3, and MMP-9, whereas IFN-γ inhibited p38 mitogen-activated protein kinase phosphorylation. Unlike highly regulated MMP-1, MMP-3, and MMP-9, MMP-2 mRNA and protein expression was constitutive in decidual cells. Because inflammation underlies PE-associated shallow EVT invasion, these results suggest that excess macrophage-derived TNF-α augments expression of MMP-1, MMP-3, and MMP-9 in decidual cells toxa0interfere with normal stepwise EVT invasion of the decidua. In contrast, decidual natural killer cell-derived IFN-γ reverses such TNF-α-induced MMPs to protect against PE.

Decidual Cell Regulation of Natural Killer Cell–Recruiting Chemokines: Implications for the Pathogenesis and Prediction of Preeclampsia

First trimester human decidua is composed of decidual cells, CD56(bright)CD16(-) decidual natural killer (dNK) cells, and macrophages. Decidual cells incubated with NK cell-derived IFN-γ and either macrophage-derived TNF-α or IL-1β synergistically enhanced mRNA and protein expression of IP-10 and I-TAC. Both chemokines recruit CXCR3-expressing NK cells. This synergy required IFN-γ receptor 1 and 2 mediation via JAK/STAT and NFκB signaling pathways. However, synergy was not observed on neutrophil, monocyte, and NK cell-recruiting chemokines. Immunostaining of first trimester decidua localized IP-10, I-TAC, IFN-γR1, and -R2 to vimentin-positive decidual cells versus cytokeratin-positive interstitial trophoblasts. Flow cytometry identified high CXCR3 levels on dNK cells and minority peripheral CD56(bright)CD16(-) pNK cells and intermediate CXCR3 levels on the majority of CD56(dim)CD16(+) pNK cells. Incubation of pNK cells with either IP-10 or I-TAC elicited concentration-dependent enhanced CXCR3 levels and migration of both pNK cell subsets that peaked at 10 ng/mL, whereas each chemokine at a concentration of 50 ng/mL inhibited CXCR3 expression and pNK cell migration. Deciduae from women with preeclampsia, a leading cause of maternal and fetal morbidity and mortality, displayed significantly lower dNK cell numbers and higher IP-10 and I-TAC levels versus gestational age-matched controls. Significantly elevated IP-10 levels in first trimester sera from women eventually developing preeclampsia compared with controls, identifying IP-10 as a novel, robust early predictor of preeclampsia.