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Problem: Women with antiphospholipid syndrome (APS) are at increased risk of recurrent pregnancy loss (RPL) and preeclampsia. Antiphospholipid antibodies (aPL) directly alter trophoblast function. Treatment with low molecular weight heparin (LMWH) reduces the risk of RPL but not preeclampsia. Moreover, LMWH stimulates trophoblast sFlt-1 release, an anti-angiogenic factor associated with preeclampsia. Since vitamin D deficiency is associated with APS and preeclampsia, this study sought to determine the effect of vitamin D on trophoblast function in the setting of aPL and LMWH., Method of Study: A human first trimester trophoblast cell line (HTR8) and primary trophoblast cultures were treated with or without aPL in the presence and absence of vitamin D, LMWH or both. Trophoblast secretion of inflammatory cytokines and angiogenic factors were measured by ELISA., Results: Vitamin D alone or in combination with LMWH attenuated the aPL-induced trophoblast inflammatory response in the HTR8 cells and primary cultures. While vitamin D did not have any impact on aPL-mediated modulation of angiogenic factors in the primary trophoblast, it significantly inhibited LMWH-induced sFlt-1 release., Conclusion: LMWH in combination with vitamin D may be more beneficial than single-agent therapy by preventing aPL-induced trophoblast inflammation and reversing LMWH-induced sFlt-1 secretion., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Problem: Inflammation during pregnancy has devastating consequences for the placenta and fetus. These events are incompletely understood, thereby hampering screening and treatment., Method of Study: The inflammatory profile of villous tissue was studied in pregnancies at high-risk of placental dysfunction and compared to uncomplicated pregnancies. The systemic inflammatory profile was assessed in matched maternal serum samples in cases of reduced fetal movements (RFM)., Results: Placentas from RFM pregnancies had a unique inflammatory profile characterized by increased interleukin (IL)-1 receptor antagonist and decreased IL-10 expression, concomitant with increased numbers of placental macrophages. This aberrant cytokine profile was evident in maternal serum in RFM, as were increased levels of alarmins (uric acid, HMGB1, cell-free fetal DNA)., Conclusion: This distinct inflammatory profile at the maternal-fetal interface, mirrored in maternal serum, could represent biomarkers of placental inflammation and could offer novel therapeutic options to protect the placenta and fetus from an adverse maternal environment., (© 2014 The Authors. American Journal of Reproductive Immunology Published by John Wiley & Sons Ltd.)

Bacterial infection-associated inflammation is thought to be a major cause of preterm premature rupture of membranes. Proinflammatory cytokines, such as interleukin 1B (IL1B), can weaken fetal membranes (FM) by upregulating matrix metalloproteinases and inducing apoptosis. The mechanism by which infection leads to inflammation at the maternal-fetal interface and subsequent preterm birth is thought to involve innate immune pattern recognition receptors (PRR), such as the Toll-like receptors (TLR) and Nod-like receptors (NLR), which recognize pathogen-associated molecular patterns (PAMPs). The objective of this study was to determine the cytokine profile generated by FMs in response to the bacterial TLR and NLR agonists peptidoglycan (PDG; TLR2), lipopolysaccharide (LPS; TLR4), flagellin (TLR5), CpG ODN (TLR9), iE-DAP (Nod1), and MDP (Nod2). PDG, LPS, flagellin, iE-DAP, and MDP triggered FMs to generate an inflammatory response, but the cytokine profiles were distinct for each TLR and NLR agonist, and only IL1B and RANTES were commonly upregulated in response to all five PAMPs. CpG ODN, in contrast, had a mild stimulatory effect only on MCP-1 and primarily downregulated basal FM cytokine production. IL1B secretion induced by PDG, LPS, flagellin, iE-DAP, and MDP was associated with its processing. Furthermore, FM IL1B secretion in response to TLR2, TLR4, and TLR5 activation was caspase 1-dependent, whereas Nod1 and Nod2 induced IL1B secretion independent of caspase 1. These findings demonstrate that FMs respond to different bacterial TLR and NLR PAMPs by generating distinct inflammatory cytokine profiles through distinct mechanisms that are specific to the innate immune PRR activated.

Normal pregnancy is associated with the presence of circulating placental microvesicles (MVs). Increased MV shedding and altered immune activation are seen in patients with preeclampsia, suggesting that placental MVs may play a role in the pathophysiology of this disease. Therefore, the aim of this study was to investigate the activation of peripheral blood mononuclear cells (PBMCs) by MVs shed by first-trimester, normal term, and preeclamptic term placenta. First-trimester and preeclamptic term, but not normal term, placental-derived MVs activated PBMCs, as evidenced by elevated IL1B. Significant changes were also seen with several other cytokines and chemokines, and in general when compared to normal term MVs, preeclamptic MVs induced a greater pro-inflammatory response in PBMCs. Pretreatment of PBMCs with first-trimester or normal term placental MVs resulted in a dampened IL1B response to a subsequent lipopolysaccharide (LPS) challenge. In contrast, treatment of PBMCs with preeclamptic term placental MVs exacerbated the LPS response. This was also the case for several other cytokines and chemokines. These studies suggest that placental MVs can modulate basal peripheral immune cell activation and responsiveness to LPS during normal pregnancy, and that in preeclampsia this effect is exacerbated.

Problem: Intrauterine bacterial infection during pregnancy may lead to adverse outcome. The objective of this study was to assess whether peptidoglycan (PGN) derived from Gram-positive bacteria induces trophoblast stem (TS) cell death or alters TS cell cytokine production., Method of Study: Toll-like receptor (TLR) transcript expression was assessed by RT-PCR. Protein expression was determined by confocal microscopy or flow cytometry. 7-Aminoactinomycin D (7-AAD) staining was used to assess TS cell death. Morphological features of cell death were evaluated by transmission electron microscopy. The presence of cleaved caspase-3 and high mobility group box 1 (HMGB1) protein was examined by Western blot. Cytokine levels in cell supernatants were determined using a mouse cytokine 23-plex panel., Results: Toll-like receptor 2 and TLR4 protein was expressed from the 1-cell stage through the blastocyst stage of murine embryo development. Murine TS cells expressed TLR2 and TLR6 but not TLR1 or TLR4 RNA. Only TLR2 protein was detected at the plasma membrane of TS cells. PGN induced TS cell death by a caspase-3-independent mechanism. The cell death pathway induced by PGN was morphologically consistent with necrosis. Finally, PGN induced HMGB1 release and increased MIP-1β secretion while inhibiting the constitutive release of RANTES., Conclusion: Peptidoglycan-induced TS cell necrosis and the subsequent release of HMGB1 and MIP-1β may regulate an infection-induced inflammatory response at the maternal-fetal interface and thus may play a role in the pathogenesis of infection-associated pregnancy complications., (© 2011 John Wiley & Sons A/S.)

Problem: Infection during pregnancy represents a significant cause of mobility and mortality. While viruses pose a major threat, little is known about their effect on early pregnancy, or the mechanisms involved. The objective of this study was to characterize the trophoblast response following exposure to viral ssRNA., Method of Study: First trimester trophoblast cells were treated with or without viral ssRNA. Cytokine production was measured using multiplex analysis and ELISA. Apoptosis was determined using Hoechst staining, cell viability, and caspase activity assays., Results: Treatment of trophoblasts with viral ssRNA increased their secretion of IL-8, IL-6, and IFNbeta. However, the ssRNA also induced trophoblast apoptosis. To test whether the viral ssRNA-induced inflammatory response was responsible for this induction of apoptosis, conditioned media (CM) from trophoblasts were added to a fresh culture of cells. The CM from viral ssRNA-treated induced higher levels of trophoblast apoptosis than the control CM. Moreover, recombinant IFNbeta induced trophoblast apoptosis., Conclusion: We demonstrate that viral ssRNA induces a pro-inflammatory and type I interferon response in the trophoblast and this inflammatory process may indirectly induce trophoblast apoptosis. These results provide a novel mechanism by which certain viral infections might compromise placental integrity and function, and therefore, pregnancy outcome.

It is well established that intrauterine infections can pose a threat to pregnancy by gaining access to the placenta and fetus, and clinical studies have strongly linked bacterial infections with preterm labor. Although Chlamydia trachomatis (Ct) can infect the placenta and decidua, little is known about its effects on trophoblast cell immune function. We have demonstrated that Ct infects trophoblast cells to form inclusions and completes the life cycle within these cells by generating infectious elementary bodies. Moreover, infection with Ct leads to differential modulation of the trophoblast cell's production of cytokines and chemokines. Using two human first trimester trophoblast cell lines, Sw.71 and H8, the most striking feature we found was that Ct infection results in a strong induction of IL-1beta secretion and a concomitant reduction in MCP-1 (CCL2) production in both cell lines. In addition, we have found that Ct infection of the trophoblast results in the cleavage and degradation of NF-kappaB p65. These findings suggest that the effect of a Chlamydia infection on trophoblast secretion of chemokines and cytokines involves both activation of innate immune receptors expressed by the trophoblast and virulence factors secreted into the trophoblast by the bacteria. Such altered trophoblast innate immune responses may have a profound impact on the microenvironment of the maternal-fetal interface and this could influence pregnancy outcome.

Problem: The placental syncytium is a differentiated cell type on the surface of the villus that has the potential to release cytokines directly to maternal blood. Responsiveness of this cell type to inflammatory compounds remains largely unelucidated., Method of Study: Response to a pro-inflammatory (lipopolysaccharide, LPS) and an anti-inflammatory (dexamethasone, DEX) compound was studied in primary cultures of syncytiotrophoblasts (SCTs). Cells were incubated with and without LPS and DEX. Cytokine levels in conditioned media were determined by enzyme-linked immunosorbent assay and proteome arrays., Results: LPS treatment induced a fourfold increase in interleukin-8 (IL-8) levels in SCTs. LPS enhanced the expression of both pro- and anti-inflammatory cytokines in SCTs. DEX treatment reduced IL-8 levels in control and LPS-treated cultures by 70-90%., Conclusion: Cytokine expression in SCTs was enhanced by LPS treatment and this effect was suppressed by glucocorticoid treatment. This suggests that inflammatory compounds may alter cytokine expression in the syncytium throughout gestation.

Problem: Inflammasomes are signaling platforms that, upon sensing pathogens and sterile stressors, mediate the release of mature forms of interleukin (IL)‐1β and IL‐18. The aims of this study were to determine (i) the expression of major inflammasome components in the chorioamniotic membranes in spontaneous labor at term, (ii) whether there are changes in the inflammasome components associated with the activation of caspase‐1 and caspase‐4, and (iii) whether these events are associated with the release of the mature forms of IL‐1β and IL‐18. Method of study: Chorioamniotic membranes were collected from women at term with and without spontaneous labor. mRNA abundance and protein concentrations of inflammasome components, nucleotide‐binding oligomerization domain‐containing (NOD)1 and NOD2 proteins, caspase‐1, caspase‐4, IL‐1β, and IL‐18 were quantified by qRT‐PCR (n = 28–29 each), ELISA (n = 10 each) or immunoblotting (n = 8 each), and immunohistochemistry (n = 10 each). Active caspase‐1 and caspase‐4, as well as mature IL‐18, were determined by immunoblotting (n = 4 each), and pro‐ and mature forms of IL‐1β were determined by ELISA (n = 4–7 each). Results: Inflammasome components and NOD proteins were expressed in the chorioamniotic membranes obtained from women at term. The chorioamniotic membranes from women who underwent labor had (i) higher concentrations of NLRP3 (NOD‐like receptor family, pyrin domain‐containing protein 3) and NOD1 protein, (ii) greater immunoreactivity for caspase‐1 and caspase‐4, (iii) a greater quantity of the active form of caspase‐1 (p20), and (iv) higher mRNA abundance and protein concentrations of pro‐ and mature IL‐1β. However, mRNA abundance and protein concentrations of the mature form of IL‐18 were not increased in tissues from women who underwent labor at term. Conclusions: Spontaneous labor at term is characterized by the expression of inflammasome components, which may participate in the activation of caspase‐1 and lead to the cleavage and release of mature IL‐1β by the chorioamniotic membranes. These results support the participation of the inflammasome in the mechanisms responsible for spontaneous parturition at term. [ABSTRACT FROM AUTHOR]

Inflammasomes are cytosolic multiprotein complexes that orchestrate inflammation in response to pathogens and endogenous danger signals. Herein, we determined whether the chorioamniotic membranes from women in spontaneous preterm labor with acute histologic chorioamnionitis (1) express major inflammasome components; (2) express caspase (CASP)-1 and CASP-4 as well as their active forms; (3) exhibit apoptosis-associated speck-like protein containing a CARD (ASC)/CASP-1 complex formation; and (4) release the mature forms of interleukin (IL)-1β and IL-18. We utilized quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunoblotting, and immunohistochemistry to determine the messenger RNA (mRNA) and protein expression of major inflammasome components, nucleotide-binding oligomerization domain (NOD) proteins, and the pro- and mature/active forms of CASP-1, CASP-4, IL-1β, and IL-18. The ASC/CASP-1 complex formation was determined using an in situ proximity ligation assay. When comparing the chorioamniotic membranes from women in spontaneous preterm labor with acute histologic chorioamnionitis to those without this placental lesion, we found that (1) the mRNA of NLR family pyrin domain-containing protein (NLRP)1, NLRP3, NLR family CARD domain-containing protein 4 (NLRC4), and NOD2 were higher; (2) the NLRP3 protein was increased; (3) the mRNA and active form (p10) of CASP-1 were greater; (4) the mRNA and active form of CASP-4 were increased; (5) the mRNA and mature form of IL-1β were higher; (6) the mature form of IL-18 was elevated; and (7) ASC/CASP-1 complex formation was increased. In conclusion, spontaneous preterm labor with acute histologic chorioamnionitis is characterized by an upregulation of NLRP3 and the active form of CASP-4, as well as increased ASC/CASP-1 complex formation, which may participate in the activation of CASP-1 and the maturation of IL-1β and IL-18 in the chorioamniotic membranes. These findings provide the first evidence that supports a role for the inflammasome in the pathological inflammation implicated in spontaneous preterm labor with acute histologic chorioamnionitis. [ABSTRACT FROM AUTHOR]

Problem Women with antiphospholipid syndrome ( APS) are at risk for pregnancy complications. Antiphospholipid antibodies (a PL) alter trophoblast function by triggering an inflammatory cytokine response; modulating angiogenic factor secretion; and inhibiting migration. While patients with APS are often treated with hydroxychloroquine ( HCQ), its effect on trophoblast function is poorly understood. Method of study A human first trimester trophoblast cell line was treated with or without antihuman β2 GPI m Abs in the presence or absence of HCQ. Supernatants were analyzed by ELISA. Cell migration was measured using a colormetric assay. Results Antiphospholipid antibodies-induced trophoblast IL-8, IL-1 β, PlGF, and s Endoglin secretion were not altered by HCQ. a PL-induced inhibition of trophoblast migration was partially reversed by HCQ, even though HCQ significantly increased secretion of pro-migratory IL-6 to greater than baseline. a PL-induced upregulation of TIMP2 appears to inhibit trophoblast migration; the inability of HCQ to prevent a PL-induced TIMP2 may explain why migration was only partially restored. Conclusion Hydroxychloroquine reversed the a PL-inhibition of trophoblast IL-6 secretion and partially limited a PL-inhibition of cell migration. Thus, some form of combination therapy that includes HCQ may be beneficial to pregnant APS patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Envelope glycoproteins of human endogenous retrovirus (HERV), such as syncytin 1 (HERV-W), are highly expressed in the placenta and some family members have immunomodulatory properties. Placental microvesicles (MV), which are shed into the maternal circulation during pregnancy, have been demonstrated to induce immune cell activation. Therefore, the aim of this study was to investigate the immunological properties of the highly expressed placental HERV-W protein, syncytin 1, and its potential involvement in placental MV modulation of immune cell activity. The MV shed from first trimester, normal term and pre-eclamptic term placentas, and from the BeWo trophoblast cell line, all contain syncytin 1. Recombinant syncytin 1 and syncytin 1-positive BeWo trophoblast MV both induced peripheral blood mononuclear cell (PBMC) activation, indicated through production of cytokines and chemokines. Reducing syncytin 1 content in BeWo MV inhibited PBMC activation. Recombinant syncytin 1 and syncytin-1-positive BeWo MV dampened PBMC responses to lipopolysaccharide challenge. Our findings suggest that syncytin 1 is shed from the placenta into the maternal circulation in association with MV, and modulates immune cell activation and the responses of immune cells to subsequent lipopolysaccharide stimulation. These studies implicate placental MV-associated HERV in fetal regulation of the maternal immune system. [ABSTRACT FROM AUTHOR]

Problem: The cytoplasmic pattern recognition receptors, Nod1 and Nod2, are thought to be important for detecting intracellular bacteria. We have previously reported that first trimester trophoblast cells express Nod1 and Nod2, and that trophoblast Nod2 activation triggers an inflammatory response. The objectives of this study were to characterize the effects of Nod1 stimulation, and to determine the regulation of Nod1 and Nod2, in the trophoblast. Method of Study: The effect of Nod1 activation on trophoblast cells was determined by analyzing the cytokine response following treatment with γ-D-glutamyl- meso-diaminopimelic acid (iE-DAP). The regulation of Nod1 and Nod2 expression by trophoblast cells was evaluated by RT-PCR. Results: Treatment of trophoblast cells with iE-DAP significantly increased their production of cytokines and chemokines. In addition, Nod1 and Nod2 mRNA expression was upregulated following treatment of trophoblast cells with lipopolysaccharide (LPS), and this was significantly reduced by the presence of a NFκB inhibitor and a TLR4-dominant negative (DN). Conclusion: This study demonstrates that LPS, through TLR4, increases trophoblast expression of Nod1 and Nod2 via the NFκB pathway; and that Nod1 is functional in the trophoblast. These findings suggest that extracellular recognition of bacterial LPS by TLR4 may prime the trophoblast in preparation for its cytoplasmic recognition of, and response to, bacterial peptides through the Nod proteins. [ABSTRACT FROM AUTHOR]

Problem Through the expression of pattern recognition receptors, the trophoblast can recognize and respond to infectious microorganisms and, therefore, participate in the control of pathogens that may compromise fetal well-being. We hypothesize that the trophoblast has the ability to sense invasive intracellular bacteria through the cytoplasmic-based nucleotide-binding oligomerization domain (NOD) proteins. The aim of this study was to characterize the expression and function of NOD proteins in first trimester trophoblast cells. Method of study NOD1 and NOD2 expressions by first trimester trophoblast cells were evaluated by immunohistochemistry, Western blot analysis and reverse transcription-polymerase chain reaction. The effect of NOD2 activation on trophoblast cells was determined by analyzing the cytokine response following treatment with muramyl dipeptide (MDP). Results Both NOD1 and NOD2 were expressed by first trimester placental villi and localized to trophoblast cells. Moreover, NOD1, NOD2 and the signaling effector protein, RIP-like interacting CLARP kinase (RICK), were all expressed by isolated trophoblast cells. Following exposure to the NOD2 ligand, MDP, trophoblast cells generated a pro-inflammatory cytokine response. This response was confirmed to be specific, as an NOD2-deficient trophoblast cell line failed to respond to MDP unless transfected with NOD2. Conclusion These findings suggest that, through the expression and function of NOD proteins, first trimester trophoblast cells are able to recognize and respond to invasive intracellular pathogens that may have evaded other forms of pattern recognition. [ABSTRACT FROM AUTHOR]

Problem The placental syncytium is a differentiated cell type on the surface of the villus that has the potential to release cytokines directly to maternal blood. Responsiveness of this cell type to inflammatory compounds remains largely unelucidated. Method of study Response to a pro-inflammatory (lipopolysaccharide, LPS) and an anti-inflammatory (dexamethasone, DEX) compound was studied in primary cultures of syncytiotrophoblasts (SCTs). Cells were incubated with and without LPS and DEX. Cytokine levels in conditioned media were determined by enzyme-linked immunosorbent assay and proteome arrays. Results LPS treatment induced a fourfold increase in interleukin-8 (IL-8) levels in SCTs. LPS enhanced the expression of both pro- and anti-inflammatory cytokines in SCTs. DEX treatment reduced IL-8 levels in control and LPS-treated cultures by 70–90%. Conclusion Cytokine expression in SCTs was enhanced by LPS treatment and this effect was suppressed by glucocorticoid treatment. This suggests that inflammatory compounds may alter cytokine expression in the syncytium throughout gestation. [ABSTRACT FROM AUTHOR]

Abrahams VM, Kim YM, Straszewski SL, Romero R, Mor G. Macrophages and apoptotic cell clearance during pregnancy. AJRI 2004; 51:275–282 © Blackwell Munksgaard, 2004 During implantation, apoptosis is critical for the appropriate tissue remodeling of the maternal decidua and invasion of the developing embryo, yet the regulation of apoptosis is also imperative for a successful pregnancy. The quick and effective removal of apoptotic cells by tissue macrophages represents an essential process, which prevents the release of self-antigens, and in the case of pregnancy, paternal alloantigens. Recent studies have shown that the process of apoptotic cell clearance is not a neutral event, but rather an active one that induces macrophage production of anti-inflammatory cytokines and survival factors. Apoptotic cell clearance is, therefore, necessary for the resolution of inflammatory conditions, which during pregnancy could have lethal consequences. The function of the maternal immune system during implantation and throughout pregnancy is, therefore, an important area of investigation. This review will discuss the role of decidual macrophages in apoptotic cell clearance during pregnancy. [ABSTRACT FROM AUTHOR]