Maternal selection of human embryos in early gestation: Insights from recurrent miscarriage.

Compared to most mammals, human pregnancy is unusual in that it involves chromosomally diverse embryos, cyclical breakdown and regeneration of the uterine mucosa, and intimate integration of fetal and maternal cells at the uteroplacental interface. Not surprisingly, pregnancy often falters in early gestation. Whether these losses result in clinical miscarriages depends on the origins and impacts of chromosomal errors on fetal development and the ability of the decidualizing endometrium to engage in embryo biosensing and selection. Aneuploidy originating in oocytes during meiosis drives the age-related risk of miscarriage. By contrast, the frequency of endometrial cycles with an impaired decidual response may account for the stepwise increase in miscarriage rates with each pregnancy loss independently of maternal age. Additional physiological mechanisms operate in early gestation to ensure that most failing pregnancies are lost before vascular maternal-fetal connections are established by the end of the first trimester. Here, we summarise how investigations into the mechanisms that cause miscarriage led to new insights into the processes that govern maternal selection of human embryos in early gestation.

TLR8-activating miR-146a-3p is an intermediate signal contributing to fetal membrane inflammation in response to bacterial LPS.

Preterm birth is the largest contributor to neonatal morbidity and is often associated with chorioamnionitis, defined as inflammation/infection of the fetal membranes (FMs). Chorioamnionitis is characterised by neutrophil infiltration of the FMs and is associated with elevated levels of the neutrophil chemoattractant, interleukin (IL)-8 and the proinflammatory cytokine, IL-1ß. While FMs can respond to infections through innate immune sensors, such as toll-like receptors (TLRs), the downstream mechanisms by which chorioamnionitis arises are not fully understood. A novel group of non-classical microRNAs (miR-21a, miR-29a, miR-146a-3p, Let-7b) function as endogenous danger signals by activating the ssRNA viral sensors TLR7 and TLR8. In this study, the pro-inflammatory roles of TLR7/TLR8-activating miRs were examined as mediators of FM inflammation in response to bacterial lipopolysaccharide (LPS) using an in vitro human FM explant system, an in vivo mouse model of pregnancy, and human clinical samples. Following LPS exposure, miR-146a-3p was significantly increased in both human FM explants and wild-type mouse FMs. Expression of miR-146a-3p was also significantly elevated in FMs from women with preterm birth and chorioamnionitis. FM IL-8 and inflammasome-mediated IL-1ß production in response to LPS was dependent on miR-146a-3p and TLR8 downstream of TLR4 activation. In wild-type mice, LPS exposure increased FM IL-8 and IL-1ß production and induced preterm birth. In TLR7-/-/TLR8-/- mice, LPS exposure was able to initiate but not sustain preterm birth, and FM inflammation was reduced. Together, we demonstrate a novel signalling mechanism at the maternal-fetal interface in which TLR8-activating miR-146a-3p acts as an intermediate danger signal to drive FM inflammasome-dependent and -independent mechanisms of inflammation and, thus, may play a role in chorioamnionitis and subsequent preterm birth.

Protein Phosphatase 2A Activation Via ApoER2 in Trophoblasts Drives Preeclampsia in a Mouse Model of the Antiphospholipid Syndrome.

[Figure: see text].

Activated Neutrophils Propagate Fetal Membrane Inflammation and Weakening through ERK and Neutrophil Extracellular Trap-Induced TLR-9 Signaling.

Preterm birth is associated with significant neonatal mortality and morbidity worldwide. Chorioamnionitis, inflammation of the fetal membranes (FMs), is a major risk factor and is characterized by neutrophil infiltration. However, the role of neutrophils at the FMs remains unclear. We recently reported that FMs exposed to bacterial LPS recruited more neutrophils compared with resting FMs and activated them to degranulate and release reactive oxygen species, chemokines/cytokines, and neutrophil extracellular traps. We posit that under resting conditions, neutrophils play a protective surveillance role, whereas during infection/inflammation, they induce FM tissue injury. To test this, human FM explants were exposed to neutrophil conditioned media (CM). We demonstrate that CM from neutrophils exposed to resting FM-CM did not affect FM viability or function. Conversely, CM from neutrophils activated by LPS-stimulated FM-CM significantly increased FM secretion of inflammatory IL-6, IL-8, GRO-α, and the markers of membrane weakening, MMP-9 and PGE2 This FM response was partially mediated by ERK signaling and neutrophil extracellular traps through the activation of the DNA sensor, TLR-9. Thus, neutrophils recruited by FMs during infection can propagate FM inflammation and weakening, acting in a feed-forward mechanism to propagate tissue injury at the maternal-fetal interface, increasing the risk of premature FM rupture and preterm birth in women with intrauterine infection.

Recurrent miscarriage: evidence to accelerate action.

Women who have had repeated miscarriages often have uncertainties about the cause, the likelihood of recurrence, the investigations they need, and the treatments that might help. Health-care policy makers and providers have uncertainties about the optimal ways to organise and provide care. For this Series paper, we have developed recommendations for practice from literature reviews, appraisal of guidelines, and a UK-wide consensus conference that was held in December, 2019. Caregivers should individualise care according to the clinical needs and preferences of women and their partners. We define a minimum set of investigations and treatments to be offered to couples who have had recurrent miscarriages, and urge health-care policy makers and providers to make them universally available. The essential investigations include measurements of lupus anticoagulant, anticardiolipin antibodies, thyroid function, and a transvaginal pelvic ultrasound scan. The key treatments to consider are first trimester progesterone administration, levothyroxine in women with subclinical hypothyroidism, and the combination of aspirin and heparin in women with antiphospholipid antibodies. Appropriate screening and care for mental health issues and future obstetric risks, particularly preterm birth, fetal growth restriction, and stillbirth, will need to be incorporated into the care pathway for couples with a history of recurrent miscarriage. We suggest health-care services structure care using a graded model in which women are offered online health-care advice and support, care in a nurse or midwifery-led clinic, and care in a medical consultant-led clinic, according to clinical needs.

Lipopolysaccharide-Stimulated Human Fetal Membranes Induce Neutrophil Activation and Release of Vital Neutrophil Extracellular Traps.

Preterm birth is a major contributor to neonatal mortality and morbidity, and infection is a major risk factor. Chorioamnionitis, inflammation of the placenta, and fetal membranes (FMs) are commonly observed in preterm birth and are characterized by neutrophil infiltration. However, interactions between FMs and neutrophils remain incompletely understood. The objectives of this study were to determine how FMs, with or without bacterial LPS stimulation, affect neutrophil recruitment, activation, and the formation of neutrophil extracellular traps (NETs) and to elucidate the signaling mechanisms involved. Using a combination of in vitro, ex vivo, and in vivo approaches, we show that human resting FMs can directly recruit neutrophils and induce them to produce proinflammatory factors. Furthermore, neutrophils release vital NETs in response to FM-derived factors. LPS-stimulated FMs further augmented neutrophil recruitment, inflammatory cytokine/chemokine secretion, and vital NET release and also induced reactive oxygen species production and degranulation. We demonstrate a role for FM-derived TNF-α in mediating these effects through activation of neutrophil p38 MAPK. We propose that, during infection, neutrophil recruitment and activation may neutralize pathogens, vital NET formation, and prolonged neutrophil viability, and in combination with degranulation, reactive oxygen species production and inflammatory chemokine/cytokine production may contribute to tissue injury at the maternal/fetal interface.

Allopurinol inhibits excess glucose-induced trophoblast IL-1ß and ROS production.

Pre-gestational diabetes is a risk factor for preeclampsia, a condition associated with inflammatory markers, a dysregulated angiogenic profile, and impaired placentation. Using an in vitro model, we previously reported that hyperglycemic levels of glucose induced a pro-inflammatory (IL-1ß, IL-8, RANTES, GRO-α), anti-angiogenic (sFlt-1) and anti-migratory profile in a human trophoblast cell line. The IL-1ß response to excess glucose was mediated by uric acid-induced activation of the NLRP3 inflammasome. Allopurinol is a xanthine oxidase inhibitor that inhibits uric acid and reactive oxygen species (ROS) production. Thus, we sought to test the effects of allopurinol on the IL-1ß and other inflammatory, angiogenic and migratory responses that are triggered in the trophoblast by excess glucose. Under excess glucose conditions, allopurinol significantly inhibited trophoblast secretion of inflammatory IL-1ß; caspase-1 activity; IL-8; RANTES; and GRO-α. Allopurinol also significantly inhibited excess glucose-induced trophoblast secretion of anti-angiogenic sFlt-1. The presence of IL1Ra significantly inhibited excess glucose-induced trophoblast IL-8 and GRO-α secretion but had no effect on RANTES or sFlt-1. Conversely, DPI, a ROS inhibitor, significantly inhibited excess glucose-induced trophoblast GRO-α and sFlt-1 secretion, but had no effect on IL-8 or RANTES. Together, our findings indicate that the xanthine oxidase inhibitor allopurinol inhibited excess glucose-induced trophoblast IL-1ß secretion. Additionally, through its inhibition of both IL-1ß and ROS production by the trophoblast, allopurinol reduced the additional pro-inflammatory and anti-angiogenic responses to excess glucose. Thus, allopurinol may be a candidate medication to prevent placental dysfunction and adverse pregnancy outcomes, such as preeclampsia, in pregnant women with diabetes.

Role of NOD2 in antiphospholipid antibody-induced and bacterial MDP amplification of trophoblast inflammation.

Women with antiphospholipid antibodies (aPL) are at high risk for pregnancy complications, such as preeclampsia. We previously demonstrated that aPL recognizing ß2GPI promote an extravillous trophoblast pro-inflammatory, anti-migratory and anti-angiogenic profile similar to that seen in preeclampsia. Since preeclampsia in the absence of aPL may have an underlying infectious element, women with aPL may be at increased risk for preeclampsia or other adverse outcomes if an infection is present. Our objective was to determine the impact the common bacterial component, muramyl dipeptide (MDP), has on trophoblast responses to aPL. Herein, we report that bacterial MDP amplifies trophoblast IL-1ß expression, processing, and secretion in the presence of aPL through activation of NOD2. In the absence of MDP, NOD2 also mediates anti- ß2GPI antibody-induced trophoblast IL-1ß and VEGF secretion. Additionally, we report a role for extravillous trophoblast vimentin as a novel danger signal that contributes to the aPL-induced trophoblast IL-1ß production. Together our data indicate that NOD2 mediates trophoblast inflammatory and angiogenic responses to aPL alone, and mediates trophoblast inflammation in the presence of bacterial MDP. These findings suggest that a bacterial infection at the maternal-fetal interface may exacerbate the impact aPL have on trophoblast inflammation and, thus, on pregnancy outcome.

Viral Infection Sensitizes Human Fetal Membranes to Bacterial Lipopolysaccharide by MERTK Inhibition and Inflammasome Activation.

Chorioamnionitis, premature rupture of fetal membranes (FMs), and subsequent preterm birth are associated with local infection and inflammation, particularly IL-1ß production. Although bacterial infections are commonly identified, other microorganisms may play a role in the pathogenesis. Because viral pandemics, such as influenza, Ebola, and Zika, are becoming more common, and pregnant women are at increased risk for associated complications, this study evaluated the impact that viral infection had on human FM innate immune responses. This study shows that a herpes viral infection of FMs sensitizes the tissue to low levels of bacterial LPS, giving rise to an exaggerated IL-1ß response. Using an ex vivo human FM explant system and an in vivo mouse model of pregnancy, we report that the mechanism by which this aggravated inflammation arises is through the inhibition of the TAM receptor, MERTK, and activation of the inflammasome. The TAM receptor ligand, growth arrest specific 6, re-establishes the normal FM response to LPS by restoring and augmenting TAM receptor and ligand expression, as well as by preventing the exacerbated IL-1ß processing and secretion. These findings indicate a novel mechanism by which viruses alter normal FM immune responses to bacteria, potentially giving rise to adverse pregnancy outcomes.

Uric Acid Crystals Induce Placental Inflammation and Alter Trophoblast Function via an IL-1-Dependent Pathway: Implications for Fetal Growth Restriction.

Excessive placental inflammation is associated with several pathological conditions, including stillbirth and fetal growth restriction. Although infection is a known cause of inflammation, a significant proportion of pregnancies have evidence of inflammation without any detectable infection. Inflammation can also be triggered by endogenous mediators, called damage associated molecular patterns or alarmins. One of these damage-associated molecular patterns, uric acid, is increased in the maternal circulation in pathological pregnancies and is a known agonist of the Nlrp3 inflammasome and inducer of inflammation. However, its effects within the placenta and on pregnancy outcomes remain largely unknown. We found that uric acid (monosodium urate [MSU]) crystals induce a proinflammatory profile in isolated human term cytotrophoblast cells, with a predominant secretion of IL-1ß and IL-6, a result confirmed in human term placental explants. The proinflammatory effects of MSU crystals were shown to be IL-1-dependent using a caspase-1 inhibitor (inhibits IL-1 maturation) and IL-1Ra (inhibits IL-1 signaling). The proinflammatory effect of MSU crystals was accompanied by trophoblast apoptosis and decreased syncytialization. Correspondingly, administration of MSU crystals to rats during late gestation induced placental inflammation and was associated with fetal growth restriction. These results make a strong case for an active proinflammatory role of MSU crystals at the maternal-fetal interface in pathological pregnancies, and highlight a key mediating role of IL-1. Furthermore, our study describes a novel in vivo animal model of noninfectious inflammation during pregnancy, which is triggered by MSU crystals and leads to reduced fetal growth.

Immunological effects of placental extracellular vesicles.

Extracellular vesicles (EVs) extruded by the human placenta are increasingly being recognized as an essential mode of feto-maternal communication. In the past two decades, there has been an explosion of research into the roles that placental EVs play in modulating the maternal immune and cardiovascular systems during healthy pregnancies, as well as how this communication is altered in obstetric diseases. This review aims to introduce readers to the processes of placental EV formation and the cargos they carry, and also to collate and summarize the published literature that investigates the immunological effects of placental EVs throughout human pregnancy.

Antiphospholipid antibody-induced miR-146a-3p drives trophoblast interleukin-8 secretion through activation of Toll-like receptor 8.

STUDY QUESTION: What is the role of microRNAs (miRs) in antiphospholipid antibody (aPL)-induced trophoblast inflammation? SUMMARY ANSWER: aPL-induced up-regulation of trophoblast miR-146a-3p is mediated by Toll-like receptor 4 (TLR4), and miR-146a-3p in turn drives the cells to secrete interleukin (IL)-8 by activating the RNA sensor, TLR8. WHAT IS KNOWN ALREADY: Obstetric antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating aPL and an increased risk of pregnancy complications. We previously showed that aPL recognizing beta2 glycoprotein I (ß2GPI) elicit human first trimester trophoblast secretion of IL-8 by activating TLR4. Since some miRs control TLR responses, their regulation in trophoblast cells by aPL and functional role in the aPL-mediated inflammatory response was investigated. miRs can be released from cells via exosomes, and therefore, miR exosome expression was also examined. A panel of miRs was selected based on their involvement with TLR signaling: miR-9; miR-146a-5p and its isomiR, miR-146a-3p; miR-155, miR-210; and Let-7c. Since certain miRs can activate the RNA sensor, TLR8, this was also investigated. STUDY DESIGN, SIZE, DURATION: For in vitro studies, the human first trimester extravillous trophoblast cell line, HTR8 was studied. HTR8 cells transfected to express a TLR8 dominant negative (DN) were also used. Plasma was evaluated from pregnant women who have aPL, either with or without systemic lupus erythematous (SLE) (n = 39); SLE patients without aPL (n = 30); and healthy pregnant controls (n = 20). PARTICIPANTS/MATERIALS, SETTING, METHODS: Trophoblast HTR8 wildtype and TLR8-DN cells were incubated with or without aPL (mouse anti-human ß2GPI mAb) for 48-72 h. HTR8 cells were also treated with or without aPL in the presence and the absence of a TLR4 antagonist (lipopolysaccharide from Rhodobacter sphaeroides; LPS-RS), specific miR inhibitors or specific miR mimics. miR expression levels in trophoblast cells, trophoblast-derived exosomes and exosomes isolated from patient plasma were measured by qPCR. Trophoblast IL-8 secretion was measured by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: aPL significantly increased trophoblast cellular and exosome expression of miR-146a-5p, miR-146a-3p, miR-155 and miR-210. aPL-induced up-regulation of trophoblast miR-146a-5p, miR-146a-3p and miR-210, but not miR-155, was inhibited by the TLR4 antagonist, LPS-RS. While inhibition or overexpression of miR-146a-5p had no effect on aPL-induced trophoblast IL-8 secretion, miR-146a-3p inhibition significantly reduced this response. aPL-induced trophoblast IL-8 secretion was inhibited by the presence of the TLR8-DN. In the absence of aPL, transfection of trophoblast cells with a miR-146a-3p mimic significantly increased IL-8 secretion and this was inhibited by the presence of the TLR8-DN. Patients with aPL and adverse pregnancy outcomes (APOs) expressed significantly higher levels of circulating miR-146a-3p compared with healthy pregnant controls with no pregnancy complications (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: While the enrichment of miR-146a-3p in trophoblast-derived exosomes support the role of this miR acting in a paracrine or endocrine manner through exosome delivery, this has not been demonstrated. However, miR-146a-3p may also exert its pro-inflammatory effect intracellularly within the same trophoblast cell targeted by aPL. WIDER IMPLICATIONS OF THE FINDINGS: These findings provide a novel mechanism of trophoblast inflammation through miRs activating RNA-sensing receptors. Furthermore, circulating exosomal-associated miR-146a-3p in APS patients may serve clinically as a biomarker for related APOs. STUDY FUNDING/COMPETING INTERESTS: This study was supported in part by grants from the American Heart Association (#10GRNT3640032 to V.M.A.), the March of Dimes Foundation (Gene Discovery and Translational Research Grant #6-FY12-255 to V.M.A.), NICHD, NIH (R01HD049446 to V.M.A.), the Gina M. Finzi Memorial Student Summer Fellowship from the Lupus Foundation of America (to S.M.G.), and the Yale University School of Medicine Medical Student Fellowship (to S.M.G.). The authors declare no competing financial interests. TRIAL REGISTRATION NUMBER: N/A.

The role of anti-phospholipid antibodies in autoimmune reproductive failure.

Anti-phospholipid antibodies (aPL) are autoantibodies that are associated with thrombosis and a range of pregnancy complications including recurrent pregnancy loss and pre-eclampsia. The three clinically relevant, well-characterized aPL are anti-cardiolipin antibodies, lupus anticoagulant and anti-beta-2-glycoprotein I (ß2GPI) antibodies. aPL do not bind directly to phospholipids but instead bind to a plasma-binding 'cofactor'. The most extensively studied cofactor is ß2GPI, whose role in pregnancy is not fully elucidated. Although the pathogenicity of aPL in recurrent pregnancy loss is well established in humans and animal models, the association of aPL with infertility does not appear to be causative. aPL may exert their detrimental effects during pregnancy by directly binding trophoblast cells of the placenta, altering trophoblast signalling, proliferation, invasion and secretion of hormones and cytokines, and by increasing apoptosis. Heparin is commonly used to treat pregnant women with aPL; however, as thrombotic events do not occur in the placentae of all women with aPL, it may exert a protective effect by preventing the binding of aPL to ß2GPI or by acting through non-thrombotic pathways. The aim of this review is to present evidence summarizing the current understanding of this field.

Placental origins of adverse pregnancy outcomes: potential molecular targets: an Executive Workshop Summary of the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

Although much progress is being made in understanding the molecular pathways in the placenta that are involved in the pathophysiology of pregnancy-related disorders, a significant gap exists in the utilization of this information for the development of new drug therapies to improve pregnancy outcome. On March 5-6, 2015, the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health sponsored a 2-day workshop titled Placental Origins of Adverse Pregnancy Outcomes: Potential Molecular Targets to begin to address this gap. Particular emphasis was given to the identification of important molecular pathways that could serve as drug targets and the advantages and disadvantages of targeting these particular pathways. This article is a summary of the proceedings of that workshop. A broad number of topics were covered that ranged from basic placental biology to clinical trials. This included research in the basic biology of placentation, such as trophoblast migration and spiral artery remodeling, and trophoblast sensing and response to infectious and noninfectious agents. Research findings in these areas will be critical for the formulation of the development of future treatments and the development of therapies for the prevention of a number of pregnancy disorders of placental origin that include preeclampsia, fetal growth restriction, and uterine inflammation. Research was also presented that summarized ongoing clinical efforts in the United States and in Europe that has tested novel interventions for preeclampsia and fetal growth restriction, including agents such as oral arginine supplementation, sildenafil, pravastatin, gene therapy with virally delivered vascular endothelial growth factor, and oxygen supplementation therapy. Strategies were also proposed to improve fetal growth by the enhancement of nutrient transport to the fetus by modulation of their placental transporters and the targeting of placental mitochondrial dysfunction and oxidative stress to improve placental health. The roles of microRNAs and placental-derived exosomes, as well as messenger RNAs, were also discussed in the context of their use for diagnostics and as drug targets. The workshop discussed the aspect of safety and pharmacokinetic profiles of potential existing and new therapeutics that will need to be determined, especially in the context of the unique pharmacokinetic properties of pregnancy and the hurdles and pitfalls of the translation of research findings into practice. The workshop also discussed novel methods of drug delivery and targeting during pregnancy with the use of macromolecular carriers, such as nanoparticles and biopolymers, to minimize placental drug transfer and hence fetal drug exposure. In closing, a major theme that developed from the workshop was that the scientific community must change their thinking of the pregnant woman and her fetus as a vulnerable patient population for which drug development should be avoided, but rather be thought of as a deprived population in need of more effective therapeutic interventions.

Viral single stranded RNA induces a trophoblast pro-inflammatory and antiviral response in a TLR8-dependent and -independent manner.

Interest is growing in the role of viral infections and their association with adverse pregnancy outcomes. The trophoblast is permissive to viruses, but little is known about their impact on the placenta. We previously established that viral single stranded RNA (ssRNA), a Toll-like receptor 8 (TLR8) agonist, induces a restricted trophoblast pro-inflammatory cytokine/chemokine response by upregulating the secretion of interleukin (IL)-6 and IL-8. In parallel, the type I interferon, IFNbeta, is produced and acts back on the cell in an autocrine/paracrine manner to trigger caspase-3-dependent apoptosis. In this study, we sought to extend these findings by determining the mechanisms involved, examining whether viral ssRNA could induce a trophoblast antiviral response, and evaluating the influence of viral ssRNA on pregnancy outcome using a mouse model. Viral ssRNA induced human first-trimester trophoblast inflammation, type I interferon production, an antiviral response, and apoptosis in both a TLR8/MyD88-dependent and -independent manner. Furthermore, administration of viral ssRNA to pregnant mice induced placental caspase-3 activation, a pro-inflammatory cytokine/chemokine, type I interferon, and antiviral response as well as immune cell infiltration. Thus, ssRNA viral infections may compromise pregnancy by altering placental trophoblast survival and function through both TLR8 and non-TLR8 signaling pathways, leading to immune changes at the maternal-fetal interface.

Human fetal membranes generate distinct cytokine profiles in response to bacterial Toll-like receptor and nod-like receptor agonists.

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.

Antiphospholipid-exposed trophoblast-derived extracellular vesicles express elevated levels of TLR7/8-activating microRNAs and induce endometrial endothelial activation, in part, through TLR7.

Women with antiphospholipid syndrome (APS) are at high risk for miscarriage and preeclampsia. Unlike pro-thrombotic systemic APS, obstetric APS is associated with insufficient placentation, as well as inflammation and vascular dysfunction at the maternal-fetal interface. Antiphospholipid antibodies (aPL) can target the placental trophoblast and induce inflammation. We reported that aPL trigger trophoblast cells to produce elevated levels of IL-8 through activation of Toll-like receptor 4 (TLR4). Downstream of TLR4, we found this IL-8 response is mediated by a TLR8-activating microRNA (miR), miR-146a-3p, which is also released by the trophoblast via extracellular vesicles (EVs). Since endothelial dysfunction is a feature of obstetric APS, we sought to determine if other miRs that can activate the RNA sensors, TLR7 and/or TLR8, are released by the trophoblast via EVs after exposure to aPL, and if these EVs can activate human endometrial endothelial cells (HEECs). Using a human first trimester extravillous trophoblast cell line we found that aPL elevated their release of small EVs (<150 nm). These extracellular vesicles released from trophoblast cells exposed to aPL expressed elevated levels of TLR7/8-activating miR-21a and miR-29a, in addition to the previously reported miR-146a-3p. Extracellular vesicles from aPL-exposed human trophoblast cells triggered human endometrial endothelial cells to generate an inflammatory IL-8 response, in part through TLR7. This study highlights EVs as a mode of communication between the placenta and the maternal vasculature, as well as a potential role for TLR7/8-activating miRs in contributing to inflammation at the maternal-fetal interface in obstetric APS.

Nod1 activation by bacterial iE-DAP induces maternal-fetal inflammation and preterm labor.

There is a strong association between infection and prematurity; however, the underlying mechanisms remain largely unknown. Nod1 and Nod2 are intracellular pattern recognition receptors that are activated by bacterial peptides and mediate innate immunity. We previously demonstrated that human first-trimester trophoblasts express Nod1 and Nod2, which trigger inflammation upon stimulation. This study sought to determine the expression and function of Nod1 and Nod2 in third-trimester trophoblasts, and to characterize the in vivo effects of Nod1 activation on pregnancy outcome. Human term placental tissues and isolated term trophoblast expressed Nod1, but not Nod2. Activation of Nod1 by its agonist, bacterial γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), in term trophoblast cultures induced a proinflammatory cytokine profile, characterized by elevated levels of secreted IL-6, GRO-α, and MCP-1, when compared with the control. However, these cytokines were not upregulated in response to Nod2 stimulation with bacterial MDP. Administration of high-dose bacterial iE-DAP to pregnant C57BL/6J mice on embryonic day 14.5 triggered preterm delivery within 24 h. iE-DAP at a lower dose that did not induce prematurity, reduced fetal weight, altered the cytokine profile at the maternal-fetal interface, and induced fetal inflammation. Thus, functional Nod1 is expressed by trophoblast cells across gestation and may have a role in mediating infection-associated inflammation and prematurity. This study demonstrates that pattern recognition receptors, other than the TLRs, may be implicated or involved in infection-associated preterm labor.

Cell-free fetal DNA impairs trophoblast migration in a TLR9-dependent manner and can be reversed by hydroxychloroquine.

Growing evidence suggests a relationship between elevated circulating placental-derived cell-free fetal DNA (cffDNA) and preeclampsia. Hypomethylation of CpG motifs, a hallmark of cffDNA, allows it to activate Toll-like receptor 9 (TLR9). Using an in vitro human first trimester extravillous trophoblast cell model, we sought to determine if trophoblast-derived cffDNA and ODN 2216, a synthetic unmethylated CpG oligodeoxynucleotide, directly impacted spontaneous trophoblast migration. The role of the DNA sensors TLR9, AIM2, and cGAS was assessed using the inhibitor A151. To test whether any effects could be reversed by therapeutic agents, trophoblasts were treated with or without cffDNA or ODN 2216 with or without aspirin (ASA; a known cGAS inhibitor), aspirin-triggered lipoxin (ATL), or hydroxychloroquine (HCQ; a known TLR9 inhibitor). Trophoblast-derived cffDNA and ODN 2216 reduced trophoblast migration without affecting cell viability. Reduced trophoblast migration in response to cffDNA or ODN 2216 was reversed by A151. cffDNA inhibition of trophoblast migration was reversed by HCQ, while ASA or ATL had no effect. In contrast ODN 2216 inhibition of trophoblast migration was reversed by ASA, ATL and HCQ. Our findings suggest that cffDNA can exert a local effect on placental function by impairing trophoblast migration through activation of innate immune DNA sensors. HCQ, a known TLR9 inhibitor, reversed the effects of cffDNA on trophoblast migration. Greater insights into the molecular underpinnings of how cffDNA impacts placentation can aid in our understanding of the pathogenesis of preeclampsia, and in the development of novel therapeutic approaches for preeclampsia therapy.

Decidualization dampens toll-like receptor mediated inflammatory responses in human endometrial stromal cells by upregulating IκBα.

Endometrial stromal cells (EnSCs) are the major cell type of the human endometrium and they undergo dramatic differentiation, termed decidualization, every month that enables them to be receptive to implantation. Appropriate decidualization and EnSC function is key for a successful pregnancy. EnSC function may be affected when the uterus is exposed to bacterial and viral infection. However, how human EnSCs respond to viral and bacterial components have not been well-studied and it remains unclear whether uterine innate immune responses change during decidualization. This study demonstrated that viral double-stranded RNA [Poly(I:C)] and bacterial lipopolysaccharide (LPS) upregulated undecidualized human EnSC production of a large array of proinflammatory cytokines and chemokines, and revealed that these immune responses were significantly dampened during decidualization in vitro and in vivo. This dampened response was associated with increased NFKBIA transcription during decidualization that leads to the accumulation of this negative regulator in decidualizing EnSCs that can bind to NFκB p65 and prevents its nuclear translocation and downstream Toll-like receptor signaling. These findings highlight that endometrial responses to infection may vary at different stages of the menstrual cycle which may be important for preparing the endometrium to support the growth of the semi-allogenic blastocyst. This work emphasizes the need to consider menstrual cycle stage, sex hormone levels and the differentiation status of cells when examining inflammatory responses in the future.