Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

Progestin-only long-acting reversible-contraceptive (pLARC)-exposed endometria displays decidualized human endometrial stromal cells (HESCs) and hyperdilated thin-walled fragile microvessels. The combination of fragile microvessels and enhanced tissue factor levels in decidualized HESCs generates excess thrombin, which contributes to abnormal uterine bleeding (AUB) by inducing inflammation, aberrant angiogenesis, and proteolysis. The- zinc finger and BTB domain containing 16 (ZBTB16) has been reported as an essential regulator of decidualization. Microarray studies have demonstrated that ZBTB16 levels are induced by medroxyprogesterone acetate (MPA) and etonogestrel (ETO) in cultured HESCs. We hypothesized that pLARC-induced ZBTB16 expression contributes to HESC decidualization, whereas prolonged enhancement of ZBTB16 levels triggers an inflammatory milieu by inducing pro-inflammatory gene expression and tissue-factor-mediated thrombin generation in decidualized HESCs. Thus, ZBTB16 immunostaining was performed in paired endometria from pre- and post-depo-MPA (DMPA)-administrated women and oophorectomized guinea pigs exposed to the vehicle, estradiol (E2), MPA, or E2 + MPA. The effect of progestins including MPA, ETO, and levonorgestrel (LNG) and estradiol + MPA + cyclic-AMP (E2 + MPA + cAMP) on ZBTB16 levels were measured in HESC cultures by qPCR and immunoblotting. The regulation of ZBTB16 levels by MPA was evaluated in glucocorticoid-receptor-silenced HESC cultures. ZBTB16 was overexpressed in cultured HESCs for 72 h followed by a ± 1 IU/mL thrombin treatment for 6 h. DMPA administration in women and MPA treatment in guinea pigs enhanced ZBTB16 immunostaining in endometrial stromal and glandular epithelial cells. The in vitro findings indicated that: (1) ZBTB16 levels were significantly elevated by all progestin treatments; (2) MPA exerted the greatest effect on ZBTB16 levels; (3) MPA-induced ZBTB16 expression was inhibited in glucocorticoid-receptor-silenced HESCs. Moreover, ZBTB16 overexpression in HESCs significantly enhanced prolactin (PRL), insulin-like growth factor binding protein 1 (IGFBP1), and tissue factor (F3) levels. Thrombin-induced interleukin 8 (IL-8) and prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA levels in control-vector-transfected HESCs were further increased by ZBTB16 overexpression. In conclusion, these results supported that ZBTB16 is enhanced during decidualization, and long-term induction of ZBTB16 expression by pLARCs contributes to thrombin generation through enhancing tissue factor expression and inflammation by enhancing IL-8 and PTGS2 levels in decidualized HESCs.

Leptin-Mediated Induction of IL-6 Expression in Hofbauer Cells Contributes to Preeclampsia Pathogenesis.

Leptin plays a crucial role in regulating energy homoeostasis, neuroendocrine function, metabolism, and immune and inflammatory responses. The adipose tissue is a main source of leptin, but during pregnancy, leptin is also secreted primarily by the placenta. Circulating leptin levels peak during the second trimester of human pregnancy and fall after labor. Several studies indicated a strong association between elevated placental leptin levels and preeclampsia (PE) pathogenesis and elevated serum interleukin-6 (IL-6) levels in PE patients. Therefore, we hypothesized that a local increase in placental leptin production induces IL-6 production in Hofbauer cells (HBCs) to contribute to PE-associated inflammation. We first investigated HBCs-specific IL-6 and leptin receptor (LEPR) expression and compared their immunoreactivity in PE vs. gestational age-matched control placentas. Subsequently, we examined the in vitro regulation of IL-6 as well as the phosphorylation levels of intracellular signaling proteins STAT3, STAT5, NF-κB, and ERK1/2 by increasing recombinant human leptin concentrations (10 to 1000 ng/mL) in primary cultured HBCs. Lastly, HBC cultures were incubated with leptin ± specific inhibitors of STAT3 or STAT5, or p65 NF-κB or ERK1/2 MAPK signaling cascades to determine relevant cascade(s) involved in leptin-mediated IL-6 regulation. Immunohistochemistry revealed ~three- and ~five-fold increases in IL-6 and LEPR expression, respectively, in HBCs from PE placentas. In vitro analysis indicated that leptin treatment in HBCs stimulate IL-6 in a concentration-dependent manner both at the transcriptional and secretory levels (p < 0.05). Moreover, leptin-treated HBC cultures displayed significantly increased phosphorylation levels of STAT5, p65 NF-κB, and ERK1/2 MAPK and pre-incubation of HBCs with a specific ERK1/2 MAPK inhibitor blocked leptin-induced IL-6 expression. Our in situ results show that HBCs contribute to the pathogenesis of PE by elevating IL-6 expression, and in vitro results indicate that induction of IL-6 expression in HBCs is primarily leptin-mediated. While HBCs display an anti-inflammatory phenotype in normal placentas, elevated levels of leptin may transform HBCs into a pro-inflammatory phenotype by activating ERK1/2 MAPK to augment IL-6 expression.

Preeclampsia is Associated With Reduced ISG15 Levels Impairing Extravillous Trophoblast Invasion.

Among several interleukin (IL)-6 family members, only IL-6 and IL-11 require a gp130 protein homodimer for intracellular signaling due to lack of intracellular signaling domain in the IL-6 receptor (IL-6R) and IL-11R. We previously reported enhanced decidual IL-6 and IL-11 levels at the maternal-fetal interface with significantly higher peri-membranous IL-6 immunostaining in adjacent interstitial trophoblasts in preeclampsia (PE) vs. gestational age (GA)-matched controls. This led us to hypothesize that competitive binding of these cytokines to the gp130 impairs extravillous trophoblast (EVT) differentiation, proliferation and/or invasion. Using global microarray analysis, the current study identified inhibition of interferon-stimulated gene 15 (ISG15) as the only gene affected by both IL-6 plus IL-11 vs. control or IL-6 or IL-11 treatment of primary human cytotrophoblast cultures. ISG15 immunostaining was specific to EVTs among other trophoblast types in the first and third trimester placental specimens, and significantly lower ISG15 levels were observed in EVT from PE vs. GA-matched control placentae (p = 0.006). Induction of primary trophoblastic stem cell cultures toward EVT linage increased ISG15 mRNA levels by 7.8-fold (p = 0.004). ISG15 silencing in HTR8/SVneo cultures, a first trimester EVT cell line, inhibited invasion, proliferation, expression of ITGB1 (a cell migration receptor) and filamentous actin while increasing expression of ITGB4 (a receptor for hemi-desmosomal adhesion). Moreover, ISG15 silencing further enhanced levels of IL-1ß-induced pro-inflammatory cytokines (CXCL8, IL-6 and CCL2) in HTR8/SVneo cells. Collectively, these results indicate that ISG15 acts as a critical regulator of EVT morphology and function and that diminished ISG15 expression is associated with PE, potentially mediating reduced interstitial trophoblast invasion and enhancing local inflammation at the maternal-fetal interface. Thus, agents inducing ISG15 expression may provide a novel therapeutic approach in PE.

Decidual cell FKBP51-progesterone receptor binding mediates maternal stress-induced preterm birth.

Depression and posttraumatic stress disorder increase the risk of idiopathic preterm birth (iPTB); however, the exact molecular mechanism is unknown. Depression and stress-related disorders are linked to increased FK506-binding protein 51 (FKBP51) expression levels in the brain and/or FKBP5 gene polymorphisms. Fkbp5-deficient (Fkbp5-/-) mice resist stress-induced depressive and anxiety-like behaviors. FKBP51 binding to progesterone (P4) receptors (PRs) inhibits PR function. Moreover, reduced PR activity and/or expression stimulates human labor. We report enhanced in situ FKBP51 expression and increased nuclear FKBP51-PR binding in decidual cells of women with iPTB versus gestational age-matched controls. In Fkbp5+/+ mice, maternal restraint stress did not accelerate systemic P4 withdrawal but increased Fkbp5, decreased PR, and elevated AKR1C18 expression in uteri at E17.25 followed by reduced P4 levels and increased oxytocin receptor (Oxtr) expression at 18.25 in uteri resulting in PTB. These changes correlate with inhibition of uterine PR function by maternal stress-induced FKBP51. In contrast, Fkbp5-/- mice exhibit prolonged gestation and are completely resistant to maternal stress-induced PTB and labor-inducing uterine changes detected in stressed Fkbp5+/+ mice. Collectively, these results uncover a functional P4 withdrawal mechanism mediated by maternal stress-induced enhanced uterine FKBP51 expression and FKPB51-PR binding, resulting in iPTB.

Zika Virus-Infected Decidual Cells Elicit a Gestational Age-Dependent Innate Immune Response and Exaggerate Trophoblast Zika Permissiveness: Implication for Vertical Transmission.

Vertical transmission of the Zika virus (ZIKV) causes severe fetal defects, but the exact pathogenic mechanism is unclear. We identified up to a 10,480-fold higher expression of viral attachment factors AXL, GAS6, and PROS1 and a 3880-fold increase in ZIKV infectiousness/propagation in human term decidual stromal cells versus trophoblasts. Moreover, levels of viral attachment factors and ZIKV are significantly increased, whereas expression of innate immune response genes are significantly decreased, in human first trimester versus term decidual cells. ZIKV-infected decidual cell supernatants increased cytotrophoblasts infection up to 252-fold compared with directly infected cytotrophoblasts. Tizoxanide treatment efficiently inhibited Zika infection in both maternal and fetal cells. We conclude that ZIKV permissiveness, as well as innate immune responsiveness of human decidual cells, are gestational age dependent, and decidual cells augment ZIKV infection of primary human cytotrophoblast cultures, which are otherwise ZIKV resistant. Human decidual cells may act as reservoirs for trimester-dependent placental transmission of ZIKV, accounting for the higher Zika infection susceptibility and more severe fetal sequelae observed in early versus late pregnancy. Moreover, tizoxanide is a promising agent in preventing perinatal Zika transmission as well as other RNA viruses such as coronavirus.

Thrombin-Induced Decidual Colony-Stimulating Factor-2 Promotes Abruption-Related Preterm Birth by Weakening Fetal Membranes.

Preterm premature rupture of membranes (PPROM) and thrombin generation by decidual cell-expressed tissue factor often accompany abruptions. Underlying mechanisms remain unclear. We hypothesized that thrombin-induced colony-stimulating factor-2 (CSF-2) in decidual cells triggers paracrine signaling via its receptor (CSF2R) in trophoblasts, promoting fetal membrane weakening and abruption-associated PPROM. Decidua basalis sections from term (n = 10), idiopathic preterm birth (PTB; n = 8), and abruption-complicated pregnancies (n = 8) were immunostained for CSF-2. Real-time quantitative PCR measured CSF2 and CSF2R mRNA levels. Term decidual cell (TDC) monolayers were treated with 10-8 mol/L estradiol ± 10-7 mol/L medroxyprogesterone acetate (MPA) ± 1 IU/mL thrombin pretreatment for 4 hours, washed, and then incubated in control medium with estradiol ± MPA. TDC-derived conditioned media supernatant effects on fetal membrane weakening were analyzed. Immunostaining localized CSF-2 primarily to decidual cell cytoplasm and cytotrophoblast cell membranes. CSF-2 immunoreactivity was higher in abruption-complicated or idiopathic PTB specimens versus normal term specimens (P < 0.001). CSF2 mRNA was higher in TDCs versus cytotrophoblasts (P < 0.05), whereas CSF2R mRNA was 1.3 × 104-fold higher in cytotrophoblasts versus TDCs (P < 0.001). Thrombin enhanced CSF-2 secretion in TDC cultures fourfold (P < 0.05); MPA reduced this effect. Thrombin-pretreated TDC-derived conditioned media supernatant weakened fetal membranes (P < 0.05), which MPA inhibited. TDC-derived CSF-2, acting via trophoblast-expressed CSFR2, contributes to thrombin-induced fetal membrane weakening, eliciting abruption-related PPROM and PTB.

The extracellular signal-regulated kinase 1/2 triggers angiogenesis in human ectopic endometrial implants by inducing angioblast differentiation and proliferation.

PROBLEM: The role of extracellular signal-regulated kinase (ERK)1/2-mediated angiogenesis during endometriotic nidation is unknown. We posit that ERK1/2-induced angioblast differentiation and proliferation promotes ectopic endometrial angiogenesis. METHODS OF STUDY: Human eutopic and ectopic endometria were immunostained for total- (T-) or phosphorylated- (P-) ERK1/2 or double-immunostained for P-ERK1/2-CD34 and PCNA-CD34. Estradiol (E2 ), cytokines, normal peritoneal fluid (NPF) or endometriotic peritoneal fluid (EPF) ±PD98059, an ERK1/2 inhibitor, treaded primary human endometrial endothelial cells (HEECs) were evaluated by T-/P-ERK1/2 immunoblotting, MTT viability and tube formation assays. RESULTS: HEECs exhibited higher endothelial P-ERK1/2 immunoreactivity in ectopic vs eutopic endometria. Double-immunostained ectopic endometria displayed abundant CD34-positive angioblasts exhibiting strong P-ERK1/2 and PCNA immunoreactivity. EPF and vascular growth factor (VEGF)-A significantly increased HEEC proliferation and P-ERK1/2 levels. PD98059 reduced basal, EPF, and VEGF-induced HEEC proliferation and promoted vascular stabilization following tube formation. CONCLUSION: Enhanced ERK1/2 activity in angioblasts by such peritoneal factors as VEGF, E2 induces proliferation to trigger ectopic endometrial angiogenesis.

Thrombin impairs human endometrial endothelial angiogenesis; implications for progestin-only contraceptive-induced abnormal uterine bleeding.

OBJECTIVE: Progestin-only contraceptives induce abnormal uterine bleeding, accompanied by prothrombin leakage from dilated endometrial microvessels and increased thrombin generation by human endometrial stromal cell (HESC)-expressed tissue factor. Initial studies of the thrombin-treated HESC secretome identified elevated levels of cleaved chondroitin sulfate proteoglycan 4 (CSPG4), impairing pericyte-endothelial interactions. Thus, we investigated direct and CSPG4-mediated effects of thrombin in eliciting abnormal uterine bleeding by disrupting endometrial angiogenesis. STUDY DESIGN: Liquid chromatography/tandem mass spectrometry, enzyme-linked immunosorbent assay (ELISA) and quantitative real-time-polymerase chain reaction (PCR) evaluated conditioned medium supernatant and cell lysates from control versus thrombin-treated HESCs. Pre- and post-Depo medroxyprogesterone acetate (DMPA)-administered endometria were immunostained for CSPG4. Proliferation, apoptosis and tube formation were assessed in human endometrial endothelial cells (HEECs) incubated with recombinant human (rh)-CSPG4 or thrombin or both. RESULTS: Thrombin induced CSPG4 protein expression in cultured HESCs as detected by mass spectrometry and ELISA (p<.02, n=3). Compared to pre-DMPA endometria (n=5), stromal cells in post-DMPA endometria (n=5) displayed stronger CSPG4 immunostaining. In HEEC cultures (n=3), total tube-formed mesh area was significantly higher in rh-CSPG4 versus control (p<.05). However, thrombin disrupted HEEC tube formation by a concentration- and time-dependent reduction of angiogenic parameters (p<.05), whereas CSPG4 co-treatment did not reverse these thrombin-mediated effects. CONCLUSION: These results suggest that disruption of HEEC tube formation by thrombin induces aberrant angiogenesis and abnormal uterine bleeding in DMPA users. IMPLICATIONS: Mass spectrometry analysis identified several HESC-secreted proteins regulated by thrombin. Therapeutic agents blocking angiogenic effects of thrombin in HESCs can prevent or minimize progestin-only contraceptive-induced abnormal uterine bleeding.

Endoplasmic Reticulum Stress and Homeostasis in Reproductive Physiology and Pathology.

The endoplasmic reticulum (ER), comprises 60% of the total cell membrane and interacts directly or indirectly with several cell organelles i.e., Golgi bodies, mitochondria and proteasomes. The ER is usually associated with large numbers of attached ribosomes. During evolution, ER developed as the specific cellular site of synthesis, folding, modification and trafficking of secretory and cell-surface proteins. The ER is also the major intracellular calcium storage compartment that maintains cellular calcium homeostasis. During the production of functionally effective proteins, several ER-specific molecular steps sense quantity and quality of synthesized proteins as well as proper folding into their native structures. During this process, excess accumulation of unfolded/misfolded proteins in the ER lumen results in ER stress, the homeostatic coping mechanism that activates an ER-specific adaptation program, (the unfolded protein response; UPR) to increase ER-associated degradation of structurally and/or functionally defective proteins, thus sustaining ER homeostasis. Impaired ER homeostasis results in aberrant cellular responses, contributing to the pathogenesis of various diseases. Both female and male reproductive tissues undergo highly dynamic cellular, molecular and genetic changes such as oogenesis and spermatogenesis starting in prenatal life, mainly controlled by sex-steroids but also cytokines and growth factors throughout reproductive life. These reproductive changes require ER to provide extensive protein synthesis, folding, maturation and then their trafficking to appropriate cellular location as well as destroying unfolded/misfolded proteins via activating ER-associated degradation mediated proteasomes. Many studies have now shown roles for ER stress/UPR signaling cascades in the endometrial menstrual cycle, ovarian folliculogenesis and oocyte maturation, spermatogenesis, fertilization, pre-implantation embryo development and pregnancy and parturition. Conversely, the contribution of impaired ER homeostasis by severe/prolong ER stress-mediated UPR signaling pathways to several reproductive tissue pathologies including endometriosis, cancers, recurrent pregnancy loss and pregnancy complications associated with pre-term birth have been reported. This review focuses on ER stress and UPR signaling mechanisms, and their potential roles in female and male reproductive physiopathology involving in menstrual cycle changes, gametogenesis, preimplantation embryo development, implantation and placentation, labor, endometriosis, pregnancy complications and preterm birth as well as reproductive system tumorigenesis.

Mechanisms of chorioamnionitis-associated preterm birth: interleukin-1ß inhibits progesterone receptor expression in decidual cells.

In chorioamnionitis (CAM), a major cause of preterm birth (PTB), maternal-fetal inflammation of the decidua and amniochorion cause the release of cytokines that elicit cervical ripening, fetal membrane rupture and myometrial activation. We posit that this inflammatory milieu triggers PTB by inhibiting progesterone receptor (PR) expression and increasing decidual prostaglandin (PG) production. Immunohistochemical staining of decidua detected significantly lower PR levels in decidual cells (DCs) from CAM-complicated PTB. Incubation of DCs with IL-1ß decreased PR expression and significantly increased PGE2 and PGF2α production and COX-2 expression. The addition of PGF2α to DC cultures also suppressed PR expression. However, the COX inhibitor, indomethacin, did not reverse IL-1ß suppression of PR expression in DC cultures. Although IL-1ß treatment activated the NF-KB, ERK1/2 and p38 MAPK signalling cascades in DCs, inhibition of ERK1/2 MAPK signalling alone was sufficient to completely reverse the suppression of PR levels by IL-1ß. These findings suggest that CAM-associated PTB is induced at least in part by IL-1ß-mediated functional progesterone withdrawal.

Plasmid-based system for high-level gene expression and antisense gene knockdown in Bartonella henselae.

Six broad-host-range plasmid vectors were developed to study gene expression in Bartonella henselae. The vectors were used to express a beta-galactosidase reporter gene in B. henselae and to generate antisense RNA for gene knockdown. When applied to ompR, a putative transcription response regulator of B. henselae, this antisense RNA gene knockdown strategy reduced bacterial invasion of human endothelial cells by over 60%.

Cannabinoid receptors and T helper cells.

We have reported that injection of marijuana cannabinoids, such as Delta(9)-tetrahydrocannabinol (THC), into mice, followed by infection with Legionella pneumophila (Lp), suppresses the development of cell-mediated immunity T helper 1 (Th1) activity. These effects are accompanied by suppression of interleukin (IL)-12 and interferon (IFN) gamma production and enhancement of IL-4 production suggesting THC-induced T helper cell biasing. In the current report, other T helper cell biasing mechanisms were studied. Mice were injected with THC followed 18 h later by a challenge infection with Lp. Two-hour post-infection, spleens were removed and analyzed for mRNA to either IL-12Rbeta2 or GATA3 gene products. The results showed that THC suppressed IL-12Rbeta2 but increased GATA3. Receptor antagonists for CB1 (SR141716A, SR1) and CB2 (SR144528, SR2) were also injected to analyze the involvement of cannabinoid receptors. It was determined that SR1 attenuated the THC suppression of IL-12Rbeta2, while SR2 attenuated the increase in GATA3 mRNA. These results suggest that THC suppresses Th1 biasing activity such as IL-12Rbeta2 by a CB1 mediated mechanism and enhances the Th2 biasing activity, GATA3, by a CB2 mechanism. This dichotomy of receptor involvement might result from differential expression and/or signaling function of CB1 and CB2 on Th1 and Th2 cells.

The cannabinoid system and immune modulation.

Studies on the effects of marijuana smoking have evolved into the discovery and description of the endocannabinoid system. To date, this system is composed of two receptors, CB1 and CB2, and endogenous ligands including anandamide, 2-arachidonoyl glycerol, and others. CB1 receptors and ligands are found in the brain as well as immune and other peripheral tissues. Conversely, CB2 receptors and ligands are found primarily in the periphery, especially in immune cells. Cannabinoid receptors are G protein-coupled receptors, and they have been linked to signaling pathways and gene activities in common with this receptor family. In addition, cannabinoids have been shown to modulate a variety of immune cell functions in humans and animals and more recently, have been shown to modulate T helper cell development, chemotaxis, and tumor development. Many of these drug effects occur through cannabinoid receptor signaling mechanisms and the modulation of cytokines and other gene products. It appears the immunocannabinoid system is involved in regulating the brain-immune axis and might be exploited in future therapies for chronic diseases and immune deficiency.