Problem-Women with antiphospholipid antibodies (aPL) are at risk for recurrent miscarriage, preeclampsia and preterm labor. aPL target the placenta directly by binding to Beta 2 -Glycoprotein I (β 2 GPI) expressed on the surface of trophoblast cells. The objective of this study was to determine the effects of aPL on trophoblast function and the mechanisms involved.Method of study-First trimester trophoblast were treated with anti-β 2 GPI monoclonal antibodies and patient-derived aPL, after which cell survival and function was evaluated.Results-We report that anti-β 2 GPI antibodies trigger an inflammatory response in trophoblast, characterized by increased secretion of IL-8, MCP-1, GRO-α and IL-1β, and that this occurs in a TLR-4/MyD88-dependent manner. At high concentrations, these antibodies also induce caspasemediated cell death. This was attenuated upon disabling of the MyD88 pathway, suggesting that anti-β 2 GPI-induced inflammatory mediators compromise trophoblast survival by acting in an autocrine/ paracrine manner. Enhanced IL-8, GRO-α and IL-1β secretion also occured when trophoblast were incubated with antibodies from patients with antiphospholipid syndrome. Heparin, which acts as a pro-survival factor in human trophoblast, attenuated the anti-β 2 GPI antibody-mediated cell death, and also the pro-inflammatory response, but only at high concentrations.Conclusions-These findings demonstrate that aPL triggers a placental inflammatory response via the TLR-4/MyD88 pathway, which in turn compromises trophoblast survival. Thus, the TLR-4/ MyD88 pathway may provide a new therapeutic target to improve pregnancy outcome in antiphospholipid syndrome patients.

Women with antiphospholipid syndrome (APS) are at risk of recurrent pregnancy loss and obstetrical disorders, such as preeclampsia and intrauterine growth restriction (IUGR). Antiphospholipid antibodies (aPL) directly target the placenta by binding beta2-glycoprotein I (β2GPI) expressed on the trophoblast. We recently demonstrated in human first trimester trophoblast cells that anti-β2GPI antibodies (Abs) induce the secretion of IL-1β in a Toll-like receptor 4 (TLR4)-dependent manner. IL-1β secretion requires processing of pro-IL-1β and this is mediated by the inflammasome, a complex of Nalp3, apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1. The objective of this study was to determine if aPL induce IL-1β production in trophoblast via the inflammasome. Using a human first trimester trophoblast cell line, we demonstrated that a mouse anti-β2GPI mAb and human polyclonal aPL-IgG induce IL-1β processing and secretion, which was partially blocked upon caspase-1 inhibition. Nalp3 and ASC knockdown also attenuated anti-β2GPI Ab-induced IL-1β secretion. Furthermore, aPL stimulated the production of uric acid in a TLR4-dependent manner; and inhibition of uric acid prevented aPL-induced IL-1β production by the trophoblast. These findings demonstrate that aPL, via TLR4 activation, induce a uric acid response in human trophoblast, which in turn activates the Nalp3/ASC inflammasome leading to IL-1β processing and secretion. This novel mechanism may account for the inflammation at the maternal-fetal interface, which causes placental dysfunction and increases the risk of adverse pregnancy outcome in patients with APS.

Problem-Preeclampsia is associated with hyperuricemia, which correlates with the disease severity. Levels of circulating uric acid increase before the clinical manifestations, suggesting they may be causally related. Uric acid, or monosodium urate (MSU), activates the Nod-like receptor, Nalp3, leading to inflammasome activation and IL-1β processing. Since preeclampsia is associated with placental immune/inflammatory dysregulation, we sought to determine in the trophoblast, the presence of the Nalp3 inflammasome, and the effect of MSU on its activation.Method of Study-Isolated first and third trimester trophoblast were assessed for expression of the inflammasome components, Nalp1, Nalp3 and ASC. First trimester trophoblast cells were incubated with or without MSU, after which, IL-1β secretion and processing and caspase-1 activation was determined.Results-Trophoblast cells expressed Nalp1, Nalp3 and ASC under basal conditions. Following incubation with MSU, first trimester trophoblast IL-1β secretion was upregulated. This correlated with increased expression levels of active IL-1β and active caspase-1. ASC knockdown reduced MSU-induced IL-1β secretion.Conclusion-These findings demonstrate that uric acid activates the inflammasome in the trophoblast, leading to IL-1β production. This may provide a novel mechanism for the induction of inflammation at the maternal-fetal interface leading to placental dysfunction and adverse pregnancy outcome, including preeclampsia.

PROBLEM  Women with antiphospholipid antibodies (aPL) are at risk of miscarriage and pre-eclampsia, obstetrical disorders associated with reduced trophoblast invasion and spiral artery transformation. aPL target the placenta by binding beta(2) -glycoprotein I (β(2) GPI) on the trophoblast. In this study, we determined whether aPL alter the trophoblast secretion of angiogenic factors and evaluated the effect of low molecular weight heparin (LMWH) on this response. METHOD OF STUDY  First-trimester trophoblast was treated with anti-β(2) GPI antibodies with or without LMWH. Angiogenic factor secretion was measured by enzyme-linked immunosorbent assay. RESULTS  Trophoblast cells produced more vascular endothelial growth factor (VEGF), placenta growth factor (PlGF), and soluble endoglin following exposure to anti-β(2) GPI Abs, and this occurred in both a MyD88-dependent and MyD88-independent manner. LMWH was unable to reverse the effects of the anti-β(2) GPI Abs on trophoblast VEGF secretion, but enhanced PlGF. Strikingly, LMWH upregulated soluble fms-like tyrosine kinase receptor-1 (sFlt-1) secretion independently of aPL. CONCLUSION  This study demonstrates that aPL perturb the secretion of trophoblast angiogenic factors. LMWH does not reverse this effect but exacerbates sFlt-1 secretion, a potent anti-angiogenic factor. These findings may help to explain why women with antiphospholipid syndrome, who are treated with heparin to prevent early pregnancy loss, remain at increased risk of developing late obstetrical complications, such as pre-eclampsia.

Problem Diabetes confers an increased risk of preeclampsia, but its pathogenic role in preeclampsia is poorly understood. The objective of this study was to elucidate the effects of excess glucose on trophoblast function and whether any changes could be reversed by metformin. Method of Study The human first trimester trophoblast cell line (Sw.71) was treated with glucose at 5mM, 10mM, 25mM and 50mM, in the presence and absence of metformin. Trophoblast migration was quantified and supernatant cytokine, chemokine, and angiogenic factors measured. Results Increasing concentrations of glucose significantly increased trophoblast secretion of the inflammatory cytokines/chemokines: IL-1β, IL-6, IL-8, GRO-α, RANTES and G-CSF; significantly increased trophoblast secretion of the anti-angiogenic factors sFlt-1 and sEndoglin; and significantly decreased trophoblast migration. Excess glucose-induced trophoblast IL-1β production was inhibited by disabling the Nalp3/ASC inflammasome. Metformin partially reduced the glucose-induced inflammatory response, but had no effect on the anti-angiogenic or anti-migratory response. Conclusion Excess glucose induced a pro-inflammatory, anti-angiogenic, and anti-migratory state in first trimester trophoblast cells. Glucose-induced trophoblast IL-1β secretion was mediated by the inflammasome. Glucose-induced inflammation was partially reversed by metformin. These findings demonstrate the pleiotropic effects of hyperglycemia on the trophoblast, providing potential explanations for the strong link between diabetes and preeclampsia.