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.

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.

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.

Endometrial responses to bacterial and viral infection: a scoping review.

BACKGROUND: The endometrium is a highly dynamic tissue that undergoes dramatic proliferation and differentiation monthly in order to prepare the uterus for implantation and pregnancy. Intrauterine infection and inflammation are being increasingly recognized as potential causes of implantation failure and miscarriage, as well as obstetric complications later in gestation. However, the mechanisms by which the cells of the endometrium respond to infection remain understudied and recent progress is slowed in part owing to similar overlapping studies being performed in different species. OBJECTIVE AND RATIONALE: The aim of this scoping review is to systematically summarize all published studies in humans and laboratory animals that have investigated the innate immune sensing and response of the endometrium to bacteria and viruses, and the signaling mechanisms involved. This will enable gaps in our knowledge to be identified to inform future studies. SEARCH METHODS: The Cochrane Library, Ovid Embase/Medline, PubMed, Scopus, Google Scholar, and Web of Science databases were searched using a combination of controlled and free text terms for uterus/endometrium, infections, and fertility to March 2022. All primary research papers that have reported on endometrial responses to bacterial and viral infections in the context of reproduction were included. To focus the scope of the current review, studies in domesticated animals, included bovine, porcine, caprine, feline, and canine species were excluded. OUTCOMES: This search identified 42 728 studies for screening and 766 full-text studies were assessed for eligibility. Data was extracted from 76 studies. The majority of studies focused on endometrial responses to Escherichia coli and Chlamydia trachomatis, with some studies of Neisseria gonorrhea, Staphylococcus aureus, and the Streptococcus family. Endometrial responses have only been studied in response to three groups of viruses thus far: HIV, Zika virus, and the herpesvirus family. For most infections, both cellular and animal models have been utilized in vitro and in vivo, focusing on endometrial production of cytokines, chemokines, and antiviral/antimicrobial factors, and the expression of innate immune signaling pathway mediators after infection. This review has identified gaps for future research in the field as well as highlighted some recent developments in organoid systems and immune cell co-cultures that offer new avenues for studying endometrial responses to infection in more physiologically relevant models that could accelerate future findings in this area. WIDER IMPLICATIONS: This scoping review provides an overarching summary and benchmark of the current state of research on endometrial innate immune responses to bacterial and viral infection. This review also highlights some exciting recent developments that enable future studies to be designed to deepen our understanding of the mechanisms utilized by the endometrium to respond to infection and their downstream effects on uterine function.

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.

The serotonin reuptake inhibitor fluoxetine induces human fetal membrane sterile inflammation through p38 MAPK activation.

Serotonin Reuptake Inhibitors (SRIs) are often used as first line therapy for depression and other psychiatric disorders. SRI use during pregnancy is associated with preterm premature rupture of membranes (PPROM) and subsequent preterm birth. The objective of this study was to investigate the mechanism(s) responsible for SRI-associated PPROM. Putative mechanisms underlying PPROM include fetal membrane (FM) inflammation, increased apoptosis, and/or accelerated senescence, the later which may be reversed by statins. Human FM explants from normal term deliveries without labor, infection, or antidepressant use were treated with or without the SRI, fluoxetine (FLX), either alone or in the presence of a p38 MAPK inhibitor or the statins, simvastatin or rosuvastatin. FMs were also collected from women either unexposed or exposed to FLX during pregnancy. FLX significantly increased FM p38 MAPK activity and secretion of inflammatory IL-6. Inhibition of p38 MAPK reduced FM IL-6 secretion in response to FLX. Statins did not reduce the SRI-induced FM IL-6 production. FMs from women exposed to FLX during pregnancy expressed elevated levels of p38 MAPK activity compared to matched unexposed women. FMs exposed to FLX did not exhibit signs of increased apoptosis and/or accelerated senescence. These results indicate that the SRI, FLX, may induce sterile FM inflammation during pregnancy through activation of the p38 MAPK pathway, and in the absence of apoptosis and senescence. These findings may better inform clinicians and patients as they weigh the risks and benefits of SRI antidepressant treatment during pregnancy.

Distinct non-coding RNA cargo of extracellular vesicles from M1 and M2 human primary macrophages.

Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) carrying functional cargo including non-coding RNAs. Macrophages can be broadly classified into M1 'classical' and M2 'alternatively-activated' macrophages. M1 macrophages have been linked with inflammation-associated pathologies, whereas a switch towards an M2 phenotype indicates resolution of inflammation and tissue regeneration. Here, we provide the first comprehensive analysis of the small RNA cargo of EVs from human M1 and M2 primary macrophages. Using small RNA sequencing, we identified several types of small non-coding RNAs in M1 and M2 macrophage EVs including miRNAs, isomiRs, tRNA fragments, piRNA, snRNA, snoRNA and Y-RNA fragments. Distinct differences were observed between M1 and M2 EVs, with higher relative abundance of miRNAs, and lower abundance of tRNA fragments in M1 compared to M2 EVs. MicroRNA-target enrichment analysis identified several gene targets involved in gene expression and inflammatory signalling pathways. EVs were also enriched in tRNA fragments, primarily originating from the 5' end or the internal region of the full length tRNAs, many of which were differentially abundant in M1 and M2 EVs. Similarly, several other small non-coding RNAs, namely snRNAs, snoRNAs and Y-RNA fragments, were differentially enriched in M1 and M2 EVs; we discuss their putative roles in macrophage EVs. In conclusion, we show that M1 and M2 macrophages release EVs with distinct RNA cargo, which has the potential to contribute to the unique effect of these cell subsets on their microenvironment.

Placental macrophage responses to viral and bacterial ligands and the influence of fetal sex.

Bacterial and viral infections of the placenta are associated with inflammation and adverse pregnancy outcomes. Hofbauer cells (HBCs) are fetal-origin macrophages in the placenta, proposed to protect the fetus from vertical pathogen transmission. We performed quantitative proteomics on term HBCs under resting conditions and following exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs), and investigated the contribution of fetal sex. Resting HBCs expressed proteins pertinent to macrophage function, including chemokines, cytokines, Toll-like receptors, and major histocompatibility complex class I and II molecules. HBCs mounted divergent responses to bacterial versus viral PAMPs but exhibited protein expression changes suggestive of a more pro-inflammatory phenotype. A comparison between male and female HBCs showed that the latter mounted a stronger and wider response. Here, we provide a comprehensive understanding of the sex-dependent responses of placental macrophages to infectious triggers, which were primarily associated with lipid metabolism in males and cytoskeleton organization in females.

Antiphospholipid Antibodies Increase Endometrial Stromal Cell Decidualization, Senescence, and Inflammation via Toll-like Receptor 4, Reactive Oxygen Species, and p38 MAPK Signaling.

OBJECTIVE: Miscarriage affects 1 in 7 pregnancies, and antiphospholipid autoantibodies (aPLs) are one of the biggest risk factors for recurrent pregnancy loss. While aPLs target the endometrial stroma, little is known about their impact. Endometrial stromal cells (EnSCs) undergo decidualization each menstrual cycle, priming the uterus to receive implanting embryos. Thus, appropriate decidualization and EnSC function is key for establishment of a successful pregnancy. This study was undertaken to explore the effects of aPL on EnSC decidualization, senescence, and inflammation. METHODS: EnSCs under decidualizing conditions were exposed to aPL or control IgG alone or in the presence of either a Toll-like receptor 4 (TLR-4) antagonist, a p38 MAPK inhibitor, a reactive oxygen species (ROS) inhibitor, low molecular weight heparin (LMWH), or acetyl salicylic acid. Secretion of decidualization markers and inflammatory interleukin-8 were quantified by enzyme-linked immunosorbent assay, and senescence-associated ß-galactosidase activity was evaluated. In a mouse model of decidualization, aPL or control IgG was administered, and uterine expression levels of decidualization and inflammatory markers were quantified by real-time quantitative polymerase chain reaction. RESULTS: Antiphospholipid antibodies increased human EnSC decidualization, senescence, and inflammation. This phenotype was recapitulated in the mouse model. The decidualization and inflammatory responses were partially mediated by TLR-4 and p38 MAPK, while the decidualization and senescence responses were ROS-dependent. LMWH, commonly used to treat aPL-positive women at risk of obstetric complications, reduced the ability of aPL to increase EnSC decidualization and inflammation. CONCLUSION: These findings shed new light on the pathogenesis of pregnancy complications in women with aPLs and underscore the benefit of heparin in preventing pregnancy loss in this high-risk population.

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.

Human fetal membrane IL-1ß production in response to bacterial components is mediated by uric-acid induced NLRP3 inflammasome activation.

Inflammatory interleukin-1ß (IL-1ß) is an important mediator of preterm birth. IL-1ß secretion is mediated by the inflammasome that processes pro-IL-1ß into its active form. However the mechanisms involved at the level of the fetal membrane (FM) are not fully understood. This study sought to determine the FM compartment involved in IL-1ß production in response to bacterial components and to evaluate the mechanism of inflammasome activation. Since IL-18 is also mediated by the inflammasome and IL-8 is a chemoattractant that contributes to neutrophil recruitment in chorioamnionitis, we also evaluated the production of these factors. A human explant system was used to evaluate the response of the chorion, amnion, and intact FMs to the bacterial components lipopolysaccharide (LPS), peptidoglycan (PGN), or muramyl dipeptide (MDP). The chorion was the major source of IL-1ß and IL-8 production in response to LPS, PGN, and MDP. LPS, PGN, and MDP induced FM IL-1ß and IL-18 secretion in a non-pyroptotic manner through activation of the NLRP3 inflammasome with contributions from ATP release through Pannexin-1, and ROS signaling. Since LPS, PGN, and MDP are not known to activate NLRP3 directly, the role of uric acid as a potential mediator was assessed. FMs produced elevated uric acid in response to LPS, PGN and MDP. FM IL-1ß secretion was inhibited by allopurinol, which blocks uric acid production, for LPS and PGN, and to a lesser degree, MDP. These findings shed light on the mechanisms by which fetal membrane inflammation and subsequent preterm birth may arise.

Antiphospholipid antibody-induced trophoblast responses are differentially modulated by viral dsRNA and viral ssRNA.

PROBLEM: Women with antiphospholipid antibodies (aPL) are at increased risk for pregnancy loss and preeclampsia. aPL target the trophoblast and induce a pro-inflammatory, anti-angiogenic and anti-migratory profile. Since infection during pregnancy can increase the risk for preeclampsia, a viral infection could further increase this in women with aPL. The goal of this study was to characterize the effect of viral components on trophoblast responses to aPL. METHOD OF STUDY: A human first trimester trophoblast cell line was treated with or without aPL or control IgG in the presence of media, viral dsRNA or viral ssRNA. Supernatants were measured for inflammatory IL-1ß and IL-8; inflammasome-associated uric acid and caspase-1 activity; and anti-angiogenic sFlt-1. Trophoblast migration was measured using a two-chamber assay. RESULTS: Viral dsRNA augmented aPL-induced trophoblast caspase-1 activity, and IL-1ß and IL-8 secretion in an additive manner. Viral ssRNA inhibited aPL-induced uric acid, IL-1ß and sFlt-1 secretion, and further exacerbated aPL-inhibition of trophoblast migration. CONCLUSION: While viral ssRNA may have some protective effects on aPL-induced inflammation and anti-angiogenic responses, viral dsRNA exacerbated aPL-mediated inflammation and viral ssRNA further limited cell migration, which could prove detrimental to placentation. Thus, viral infections may contribute to adverse pregnancy outcomes in women with aPL.

Antiphospholipid Antibodies From Women With Pregnancy Morbidity and Vascular Thrombosis Induce Endothelial Mitochondrial Dysfunction, mTOR Activation, and Autophagy.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombosis and pregnancy morbidity (PM) obstetric events together with persistent high titers of circulating antiphospholipid antibodies (aPL). Several mechanisms that explain the development of thrombosis and PM in APS include the association of aPL with alterations in the coagulation cascade and inflammatory events. Other mechanisms disturbing cellular homeostases, such as mitochondrial dysfunction, autophagy, and cell proliferation, have been described in other autoimmune diseases. Therefore, the objective of this study was to investigate the impact of aPL from different patient populations on endothelial cell mitochondrial function, activation of the mammalian target of rapamycin (mTOR) and autophagy pathways, and cellular growth. Using an in vitro model, human umbilical vein endothelial cells (HUVECs) were treated with polyclonal immunoglobulin G (IgG) purified from the serum of women with both PM and vascular thrombosis (PM/VT), with VT only (VT), or with PM and non-criteria aPL (seronegative-obstetric APS, SN-OAPS). We included IgG from women with PM without aPL (PM/aPL-) and healthy women with previous uncomplicated pregnancies (normal human serum, NHS) as control groups. Mitochondrial function, mTOR activation, autophagy, and cell proliferation were evaluated by Western blotting, flow cytometry, and functional assays. IgG from women with PM/VT increased HUVEC mitochondrial hyperpolarization and activation of the mTOR and autophagic pathways, while IgG from patients with VT induced endothelial autophagy and cell proliferation in the absence of elevated mTOR activity or mitochondrial dysfunction. IgG from the SN-OAPS patient group had no effect on any of these HUVEC responses. In conclusion, aPL from women with PM and vascular events induce cellular stress evidenced by mitochondrial hyperpolarization and increased activation of the mTOR and autophagic pathways which may play a role in the pathogenesis of obstetric APS.

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

[Figure: see text].

Visualization and Quantification of Neutrophil Extracellular Traps.

Neutrophils are innate immune cells that play important roles in many physiological and pathological processes, including immune defense and cancer metastasis. In addition to the release of proinflammatory cytokines, chemokines, and cytoplasmic granules containing digestive proteins, in recent years, neutrophils have been observed to release neutrophil extracellular traps (NETs) that consist of extracellular DNA associated with antimicrobial proteins, such as histones and myeloperoxidase. These NETs are increasingly being recognized as an important mechanism of neutrophil host defense and function. This chapter will summarize the current literature on the known processes of NET formation and describe in detail an immunofluorescence approach that can be employed to visualize and quantify NETs in vitro.

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.

Polymicrobial stimulation of human fetal membranes induce neutrophil activation and neutrophil extracellular trap release.

Preterm birth is a major contributor to neonatal mortality and morbidity. While the causes of preterm birth remain incompletely understood, infection is a major risk factor, and chorioamnionitis is commonly observed. Chorioamnionitis is characterized by inflammation and neutrophil infiltration of the fetal membranes (FM). We recently reported that human FMs which had been exposed to low levels of bacterial lipopolysaccharide (LPS) recruit neutrophils and activate them, increasing their secretion of pro-inflammatory cytokines, degranulation of myeloperoxidase (MPO), and release of neutrophil extracellular traps (NETs). Herein, we demonstrate that conditioned media (CM) from viral dsRNA (Poly(I:C))-stimulated FMs also increased neutrophil migration, and induced the secretion of inflammatory IL-8 and the release of NETs. Furthermore, CM from FMs stimulated by a combination of bacterial LPS and Poly(I:C) augmented neutrophil NET release, compared to CM from FMs stimulated with either Poly(I:C) or LPS alone. NETs induced by FMs exposed to Poly(I:C), with or without LPS, were released and degraded quicker than those induced by resting or LPS-stimulated FM-CM. These findings indicate that FMs exposed to viral dsRNA promote neutrophil recruitment, activation and NET formation, similar to FMs exposed to bacterial LPS alone. However, in response to FM polymicrobial stimulation the levels and kinetics of NET release are augmented. This work builds upon our understanding of how infections at the maternal-fetal interface may affect neutrophil function.

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.

Acetaminophen Attenuates invasion and alters the expression of extracellular matrix enzymes and vascular factors in human first trimester trophoblast cells.

Acetaminophen is one of the most common medications taken during pregnancy, considered safe for maternal health and fetal development. However, recent epidemiological studies have associated prenatal acetaminophen use with several developmental disorders in offspring. As acetaminophen can freely cross into and through the placenta, epidemiological associations with prenatal acetaminophen use may reflect direct actions on the fetus and/or the impact of altered placental functions. In the absence of rigorous mechanistic studies, our understanding of how prenatal acetaminophen exposure can cause long-term effects in offspring is limited. The objective of this study was to determine whether acetaminophen can alter key functions of a major placental cell type by utilizing immortalized human first trimester trophoblast cells. This study employed a comparative analysis with the nonsteroidal, anti-inflammatory drug aspirin, which has established effects in first trimester trophoblast cells. We report that immortalized trophoblast cells express the target proteins of acetaminophen and aspirin: cyclooxygenase (COX) -1 and -2. Unlike aspirin, acetaminophen significantly repressed the expression of angiogenesis and vascular remodeling genes in HTR-8/SVneo cells. Moreover, acetaminophen impaired trophoblast invasion by over 80%, while aspirin had no effect on invasion. Acetaminophen exposure reduced the expression of matrix metalloproteinase (MMP)-2 and -9 and increased the expression of tissue inhibitors of matrix metalloproteinases 2, leading to an imbalance in the ratio of proteolytic enzymes. Finally, a bioinformatic approach identified novel acetaminophen-responsive gene networks associated with key trophoblast functions and disease. Together these results suggest that prenatal acetaminophen use may interfere with critical trophoblast functions early in gestation, which may subsequently impact fetal development.