Circulating Immune Cells from Early- and Late-onset Pre-eclampsia Displays Distinct Profiles with Differential Impact on Endothelial Activation.

Pre-eclampsia (PE) affects 5-8% of pregnancies and has detrimental effects on maternal-fetal health. PE is characterized by de novo hypertension after 20 wk of gestation and end-organ damage. Systemic inflammatory imbalance has been associated with PE, but its contribution to the pathology is poorly understood. Our objective was to investigate maternal systemic immune changes in early-onset PE (EOPE) and late-onset PE (LOPE) versus uncomplicated pregnancies (control [CTRL]), and their contribution to endothelial activation, hallmark of hypertension. Blood samples were analyzed by flow cytometry, multiplex assay, intracellular cytokine staining, and single-cell RNA sequencing. We performed cocultures between circulating immune cells and HUVECs to assess endothelial activation. We found that EOPE had decreased regulatory T cells (4.64±0.33, p < 0.05) and monocytes (33.92±3.08, p < 0.01), whereas LOPE had decreased regulatory T cells (4.60±0.30, p < 0.05) and Th2 cells (7.50±0.62, p < 0.01) versus CTRL. Compared to CTRL, elevated cytokines/chemokines, and growth factors were observed in LOPE, whereas EOPE primarily showed decreased levels. Using intracellular cytokine staining, we observed more monocytes producing IL-12, TNF-α, and IL-1ß (all p < 0.05) in LOPE versus CTRL. At the transcriptomic level, we found differentially expressed genes between EOPE and CTRL, predominantly related to upregulation of immune activation pathways. Lastly, EOPE PBMCs induced heightened endothelial activation in vitro observed by increased ICAM-1 and ET-1 (p < 0.05), whereas LOPE PBMCs required LPS stimulation. Although significant proteomic changes are observed in the LOPE group, the EOPE displayed changes mostly at the transcriptomic levels and could induce endothelial activation in vitro.

Assessment of Aminoglycoside-Induced Hearing Loss Risk in the Perinatal Period.

OBJECTIVE: This study aimed to determine the prevalence and heteroplasmy level(s) of MT-RNR1 variants m.1555A > G and m.1494C > T, which are associated with aminoglycoside-induced hearing loss, in a general perinatal population. This study also aimed to characterize the association of these variants and their heteroplasmy levels with hearing loss outcomes with and without aminoglycoside exposure. STUDY DESIGN: Droplet digital polymerase chain reaction was performed on 479 maternal DNA samples from a general perinatal biobank at our institution to detect the presence and heteroplasmy levels of MT-RNR1 variants m.1555A > G and m.1494C > T. Testing of paired neonatal specimen(s) was planned for positive maternal tests. A retrospective chart review was performed to characterize the population, identify aminoglycoside exposures, and determine hearing outcomes. RESULTS: All maternal samples tested negative for MT-RNR1 variants m.1555A > G and m.1494C > T. Maternal and neonatal subjects had high rates of aminoglycoside exposure (15.9 and 13.9%, respectively). No subjects with sensorineural or mixed hearing loss had documented aminoglycoside exposure. CONCLUSION: This study demonstrated that a larger sample size is needed to establish the prevalence of these variants as no subjects tested positive. Determination of variant prevalence in the neonatal population, association of variant heteroplasmy levels with hearing outcomes, and reliability of maternal testing as a surrogate for neonatal testing are important next steps toward universal prenatal or newborn screening. KEY POINTS: · MT-RNR1 variants are associated with aminoglycoside-induced hearing loss.. · Prevalence of MT-RNR1 variants is uncertain.. · Universal screening for MT-RNR1 variants may be indicated..

Proinflammatory changes in the maternal circulation, maternal-fetal interface, and placental transcriptome in preterm birth.

BACKGROUND: Preterm birth remains a leading obstetrical complication because of the incomplete understanding of its multifaceted etiology. It is known that immune alterations toward a proinflammatory profile are observed in women with preterm birth, but therapeutic interventions are still lacking because of scarcity of evidence in the integration of maternal and placental interrelated compartments. OBJECTIVE: This study aimed to obtain an integrated view of the maternal and placental contribution to preterm birth compared with normal term pregnancies for an in-depth understanding of the immune/inflammatory involvement, intending to identify novel strategies to mitigate the negative impact of inflammation. STUDY DESIGN: We prospectively recruited 79 women with preterm or term deliveries and collected placentas for RNA sequencing, histologic analyses, and to assess levels of inflammatory mediators. Blood samples were also collected to determine the circulating immune profiles by flow cytometry and to evaluate the circulating levels of inflammatory mediators. RESULTS: Placental transcriptomic analyses revealed 102 differentially expressed genes upregulated in preterm birth, including known and novel targets, which were highly enriched for inflammatory biological processes according to gene ontology analyses. Analysis of maternal immune cells revealed distinct profiles in preterm births vs term births, including an increased percentage of CD3- cells and monocyte subsets and decreased CD3+ cells along with Th17 subsets of CD4+ lymphocytes. Supporting our bioinformatic findings, we found increases in proinflammatory mediators in the plasma, placenta, and fetal membranes (primarily the amnion) of women with preterm birth, such as interleukin-6 and tumor necrosis factor-α. These findings were not distinct between spontaneous and iatrogenic preterm births except at a molecular level where spontaneous preterm birth presented with an elevated inflammatory profile compared with iatrogenic preterm birth. Analysis of placental histology revealed increased structural and inflammatory lesions in preterm vs term births. We found that genes upregulated in placentas with inflammatory lesions have enrichment of proinflammatory pathways. CONCLUSION: This work sheds light on changes within the immune system in preterm birth on multiple levels and compartments to help identify pregnancies at high risk of preterm birth and to discover novel therapeutic targets for preterm birth.

Pharmacodynamic characterization of rytvela, a novel allosteric anti-inflammatory therapeutic, to prevent preterm birth and improve fetal and neonatal outcomes.

BACKGROUND: Preterm birth is the leading cause of neonatal morbidity and mortality. Studies have shown that interleukin 1 plays a major role in the pathophysiology of preterm birth by inducing the production of proinflammatory mediators and uterine activation proteins leading to labor. More importantly, uteroplacental inflammation, associated with preterm birth parturition pathways, is detrimental to fetal tissues and leads to long-term sequelae. Our group has developed an allosteric antagonist of the interleukin 1 receptor, rytvela, found to be potent and safe in preventing preterm birth by suppressing inflammation via the inhibition of the mitogen-activated protein kinase pathway while preserving the Nuclear factor kappa B pathway (important in immune vigilance). Rytvela has been shown to inhibit inflammatory up-regulation and uterine activation while preserving fetal development. OBJECTIVE: This study aimed to further the preclinical development of rytvela by evaluating its optimal dose and minimal duration of treatment to inhibit the inflammatory cascade, prolong gestation, and promote neonatal outcomes. STUDY DESIGN: Pregnant CD-1 mice were administered with lipopolysaccharide (10 µg, intraperitoneal administration) or interleukin 1 (1 µg/kg, intrauterine administration) on gestational day 16 to induce preterm labor. Rytvela was administered at different doses (0.1, 0.5, 1.0, 2.0, 4.0 mg/kg/d subcutaneously) from gestational days 16 to 18.5. To evaluate the minimal duration of treatment, the mice were administered with rytvela (2 mg/kg/d subcutaneously) for 24, 36, or 48 hours. The rate of prematurity (gestational day <18.5) and neonate survival and weight were evaluated. Gestational tissues were collected at gestational day 17.5 to quantify cytokines, proinflammatory mediators, and uterine activating proteins by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The neonatal lungs and intestines were collected from postnatal days 5 to 7 and analyzed by histology. RESULTS: Rytvela exhibited a dose-response profile and achieved maximum efficacy at a dose of 2 mg/kg/d by reducing 70% of lipopolysaccharide-induced preterm births and 60% of interleukin 1ß-induced preterm births. In addition, rytvela attained maximum efficacy at a dose of 1 mg/kg/d by increasing neonate survival by up to 65% in both models of preterm birth. Rytvela protected fetuses from inflammatory insult as of 24 hours, preserving lung and intestinal integrity, and prevented preterm birth and fetal mortality by 60% and 50%, respectively, as of 36 hours of treatment. CONCLUSION: The maximum efficacy of rytvela was achieved at 2 mg/kg/d with improved birth outcomes and prevented inflammatory up-regulation upon 36 hours (only) of treatment. Rytvela exhibited desirable properties for the safe prevention of preterm birth and fetal protection.

Maternal Factors and Placental Pathologies Associated with a Diagnosis of Chronic Villitis.

OBJECTIVE: To evaluate maternal risk factors associated with chronic villitis of unknown etiology (VUE) and to describe cooccurring placental pathologies. STUDY DESIGN: A retrospective case-control study was conducted using placental pathology records from deliveries ≥ 20 weeks between 2010 and 2018. Cases were placentas with documented chronic villitis without infectious cause, hereafter called VUE. Controls were placentas without this diagnosis, matched to the cases 2:1. Maternal and neonatal demographic and clinical data were collected. Descriptive statistics are reported with Fisher's exact test or a chi-squared test, as appropriate, and multivariable conditional logistic regression was conducted. RESULTS: Our study included 352 cases with VUE and 657 controls. A diagnosis of gestational diabetes (p = 0.03) and gestational hypertension (p = 0.06) was 1.5 times more likely to occur in those with a VUE diagnosis. A trend was also seen for chronic hypertension (odds ratio [OR] = 1.7, p = 0.07) and preeclampsia (OR = 1.5, p = 0.09) compared with controls. Placentas with VUE, specifically high-grade VUE, were more likely to be small for gestational age (p = 0.01), and to be diagnosed with other placental findings including lymphoplasmacytic or chronic deciduitis (p < 0.01), maternal (p < 0.01) and fetal vascular malperfusion (p = 0.02), and chorionitis (acute or chronic; p < 0.01). CONCLUSION: Gestational diabetes and hypertension were associated with a diagnosis of VUE, and overall, VUE placentas have more abnormal placental findings compared with control. Understanding VUE risk factors may facilitate prenatal care strategies and counseling to achieve the best outcomes for pregnant patients and their neonates. KEY POINTS: · VUE is a common inflammatory lesion of the placenta.. · Gestational diabetes and hypertension are associated with a VUE diagnosis.. · Findings of other placental pathologies increase in VUE..

Distinct inflammatory profile in preeclampsia and postpartum preeclampsia reveal unique mechanisms.

Preeclampsia (PE) is a poorly understood pregnancy complication. It has been suggested that changes in the maternal immune system may contribute to PE, but evidence of this remains scarce. Whilst PE is commonly experienced prepartum, it can also occur in the postpartum period (postpartum PE-PPPE), and the mechanisms involved are unknown. Our goal was to determine whether changes occur in the maternal immune system and placenta in pregnancies complicated with PE and PPPE, compared to normal term pregnancies. We prospectively recruited women and collected blood samples to determine the circulating immune profile, by flow cytometry, and assess the circulating levels of inflammatory mediators and angiogenic factors. Placentas were collected for histological analysis. Levels of alarmins in the maternal circulation showed increased uric acid in PE and elevated high-mobility group box 1 in PPPE. Analysis of maternal immune cells revealed distinct profiles in PE vs PPPE. PE had increased percentage of lymphocytes and monocytes whilst PPPE had elevated NK and NK-T cells as well. Elevated numbers of immune cells (CD45+) were detected in placentas from women that developed PPPE, and those were macrophages (CD163+). This work reveals changes within the maternal immune system in both PE and PPPE, and indicate a striking contrast in how this occurs. Importantly, elevated immune cells in the placenta of women with PPPE strongly suggest a prenatal initiation of the pathology. A better understanding of these changes will be beneficial to identify women at high risk of PPPE and to develop novel therapeutic targets.

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.

Antenatal Suppression of IL-1 Protects against Inflammation-Induced Fetal Injury and Improves Neonatal and Developmental Outcomes in Mice.

Preterm birth (PTB) is commonly accompanied by in utero fetal inflammation, and existing tocolytic drugs do not target fetal inflammatory injury. Of the candidate proinflammatory mediators, IL-1 appears central and is sufficient to trigger fetal loss. Therefore, we elucidated the effects of antenatal IL-1 exposure on postnatal development and investigated two IL-1 receptor antagonists, the competitive inhibitor anakinra (Kineret) and a potent noncompetitive inhibitor 101.10, for efficacy in blocking IL-1 actions. Antenatal exposure to IL-1ß induced Tnfa, Il6, Ccl2, Pghs2, and Mpges1 expression in placenta and fetal membranes, and it elevated amniotic fluid IL-1ß, IL-6, IL-8, and PGF2α, resulting in PTB and marked neonatal mortality. Surviving neonates had increased Il1b, Il6, Il8, Il10, Pghs2, Tnfa, and Crp expression in WBCs, elevated plasma levels of IL-1ß, IL-6, and IL-8, increased IL-1ß, IL-6, and IL-8 in fetal lung, intestine, and brain, and morphological abnormalities: e.g., disrupted lung alveolarization, atrophy of intestinal villus and colon-resident lymphoid follicle, and degeneration and atrophy of brain microvasculature with visual evoked potential anomalies. Late gestation treatment with 101.10 abolished these adverse outcomes, whereas Kineret exerted only modest effects and no benefit for gestation length, neonatal mortality, or placental inflammation. In a LPS-induced model of infection-associated PTB, 101.10 prevented PTB, neonatal mortality, and fetal brain inflammation. There was no substantive deviation in postnatal growth trajectory or adult body morphometry after antenatal 101.10 treatment. The results implicate IL-1 as an important driver of neonatal morbidity in PTB and identify 101.10 as a safe and effective candidate therapeutic.

Alarmins at the maternal-fetal interface: involvement of inflammation in placental dysfunction and pregnancy complications 1.

Inflammation is known to be associated with placental dysfunction and pregnancy complications. Infections are well known to be a cause of inflammation but they are frequently undetectable in pregnancy complications. More recently, the focus has been extended to inflammation of noninfectious origin, namely caused by endogenous mediators known as "damage-associated molecular patterns (DAMPs)" or alarmins. In this manuscript, we review the mechanism by which inflammation, sterile or infectious, can alter the placenta and its function. We discuss some classical DAMPs, such as uric acid, high mobility group box 1 (HMGB1), cell-free fetal deoxyribonucleic acid (DNA) (cffDNA), S100 proteins, heat shock protein 70 (HSP70), and adenosine triphosphate (ATP) and their impact on the placenta. We focus on the main placental cells (i.e., trophoblast and Hofbauer cells) and describe the placental response to, and release of, DAMPs. We also covered the current state of knowledge about the role of DAMPs in pregnancy complications including preeclampsia, fetal growth restriction, preterm birth, and stillbirth and possible therapeutic strategies to preserve placental function.

No association between early antiretroviral therapy during pregnancy and plasma levels of angiogenic factors: a cohort study.

BACKGROUND: Early antiretroviral therapy (ART) during pregnancy has dramatically reduced the risk of perinatal HIV transmission. However, studies have shown an association between premature delivery and the use of ART during pregnancy (particularly protease inhibitor (PI)-based therapies), which could be explained by placental dysfunction. The objective of this study was to evaluate the association of ART (class, duration of exposure and time of initiation) with placental function by using angiogenic factors placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) as biomarkers. METHODS: Clinical and biological data from 159 pregnant women living with HIV were analyzed. Levels of each biomarker were measured in the first and second trimester of pregnancy. After logarithmic transformation, we compared these using generalized estimating equations according to (a) the type of ART; (b) the duration of exposure to ART; and (c) the time of initiation of ART. RESULTS: After adjusting for variables such as ethnicity, maternal age, gestational age, body mass index, parity, smoking status, and sex of the fetus, we found no significant association between the class of ART (PI-based or not) and serum concentrations of PlGF or sFlt-1. Furthermore, no significant association was found between biomarker levels and the duration of ART exposure or the timing of ART initiation (pre- or post-conception). CONCLUSIONS: This study suggests that first and second trimester angiogenic factor levels are not significantly associated with ART, regardless of the duration or type (with or without PI). These observations seem reassuring when considering the use of ART during early pregnancy.

Delineating differential regulatory signatures of the human transcriptome in the choriodecidua and myometrium at term labor.

Preterm deliveries remain the leading cause of neonatal morbidity and mortality. Current therapies target only myometrial contractions and are largely ineffective. As labor involves multiple coordinated events across maternal and fetal tissues, identifying fundamental regulatory pathways of normal term labor is vital to understanding successful parturition and consequently labor pathologies. We aimed to identify transcriptomic signatures of human normal term labor of two tissues: in the fetal-facing choriodecidua and the maternal myometrium. Microarray transcriptomic data from choriodecidua and myometrium following term labor were analyzed for functional hierarchical networks, using Cytoscape 2.8.3. Hierarchically high candidates were analyzed for their regulatory casual relationships using Ingenuity Pathway Analysis. Selected master regulators were then chemically inhibited and effects on downstream targets were assessed using real-time quantitative PCR (RT-qPCR). Unbiased network analysis identified upstream molecular components in choriodecidua including vimentin, TLR4, and TNFSF13B. In the myometrium, candidates included metallothionein 2 (MT2A), TLR2, and RELB. These master regulators had significant differential gene expression during labor, hierarchically high centrality in community cluster networks, interactions amongst the labor gene set, and strong causal relationships with multiple downstream effects. In vitro experiments highlighted MT2A as an effective regulator of labor-associated genes. We have identified unique potential regulators of the term labor transcriptome in uterine tissues using a robust sequence of unbiased mathematical and literature-based in silico analyses. These findings encourage further investigation into the efficacy of predicted master regulators in blocking multiple pathways of labor processes across maternal and fetal tissues, and their potential as therapeutic approaches.

Lactate produced during labor modulates uterine inflammation via GPR81 (HCA1).

BACKGROUND: Uterine inflammatory processes trigger prolabor pathways and orchestrate on-time labor onset. Although essential for successful labor, inflammation needs to be regulated to avoid uncontrolled amplification and resolve postpartum. During labor, myometrial smooth muscle cells generate ATP mainly via anaerobic glycolysis, resulting in accumulation of lactate. Aside from its metabolic function, lactate has been shown to activate a G protein-coupled receptor, GPR81, reported to regulate inflammation. We therefore hypothesize that lactate produced during labor may act via GPR81 in the uterus to exert in a feedback manner antiinflammatory effects, to resolve or mitigate inflammation. OBJECTIVE: We sought to investigate the role of lactate produced during labor and its receptor, GPR81, in regulating inflammation in the uterus. STUDY DESIGN: We investigated the expression of GPR81 in the uterus and the pharmacological role of lactate acting via GPR81 during labor, using shRNA-GPR81 and GPR81-/- mice. RESULTS: (1) Uterine lactate levels increased substantially from 2 to 9 mmol/L during labor. (2) Immunohistological analysis revealed expression of GPR81 in the uterus with high expression in myometrium. (3) GPR81 expression increased during gestation, and peaked near labor. (4) In primary myometrial smooth muscle cell and ex vivo uteri from wild-type mice, lactate decreased interleukin-1ß-induced transcription of key proinflammatory Il1b, Il6, Ccl2, and Pghs2; suppressive effects of lactate were not observed in cells and tissues from GPR81-/- mice. (5) Conversely, proinflammatory gene expression was augmented in the uterus at term in GPR81-/- mice and wild-type mice treated intrauterine with lentiviral-encoded shRNA-GPR81; GPR81 silencing also induced proinflammatory gene transcription in the uterus when labor was induced by endotoxin (lipopolysaccharide). (6) Importantly, administration to pregnant mice of a metabolically stable specific GPR81 agonist, 3,5-dihydroxybenzoic acid, decreased endotoxin-induced uterine inflammation, preterm birth, and associated neonatal mortality. CONCLUSION: Collectively, our data uncover a novel link between the anaerobic glycolysis and the control of uterine inflammation wherein the high levels of lactate produced during labor act on uterine GPR81 to down-regulate key proinflammatory genes. This discovery may represent a novel feedback mechanism to regulate inflammation during labor, and conveys a potential rationale for the use of GPR81 agonists to attenuate inflammation and resulting preterm birth.

Novel Noncompetitive IL-1 Receptor-Biased Ligand Prevents Infection- and Inflammation-Induced Preterm Birth.

Preterm birth (PTB) is firmly linked to inflammation regardless of the presence of infection. Proinflammatory cytokines, including IL-1ß, are produced in gestational tissues and can locally upregulate uterine activation proteins. Premature activation of the uterus by inflammation may lead to PTB, and IL-1 has been identified as a key inducer of this condition. However, all currently available IL-1 inhibitors are large molecules that exhibit competitive antagonism properties by inhibiting all IL-1R signaling, including transcription factor NF-κB, which conveys important physiological roles. We hereby demonstrate the efficacy of a small noncompetitive (all-d peptide) IL-1R-biased ligand, termed rytvela (labeled 101.10) in delaying IL-1ß-, TLR2-, and TLR4-induced PTB in mice. The 101.10 acts without significant inhibition of NF-κB, and instead selectively inhibits IL-1R downstream stress-associated protein kinases/transcription factor c-jun and Rho GTPase/Rho-associated coiled-coil-containing protein kinase signaling pathways. The 101.10 is effective at decreasing proinflammatory and/or prolabor genes in myometrium tissue and circulating leukocytes in all PTB models independently of NF-κB, undermining NF-κB role in preterm labor. In this work, biased signaling modulation of IL-1R by 101.10 uncovers a novel strategy to prevent PTB without inhibiting NF-κB.

Uterotonic Neuromedin U Receptor 2 and Its Ligands Are Upregulated by Inflammation in Mice and Humans, and Elicit Preterm Birth.

Uterine labor requires the conversion of a quiescent (propregnancy) uterus into an activated (prolabor) uterus, with increased sensitivity to endogenous uterotonic molecules. This activation is induced by stressors, particularly inflammation in term and preterm labor. Neuromedin U (NmU) is a neuropeptide known for its uterocontractile effects in rodents. The objective of the study was to assess the expression and function of neuromedin U receptor 2 (NmU-R2) and its ligands NmU and the more potent neuromedin S (NmS) in gestational tissues, and the possible implication of inflammatory stressors in triggering this system. Our data show that NmU and NmS are uterotonic ex vivo in murine tissue, and they dose-dependently trigger labor by acting specifically via NmU-R2. Expression of NmU-R2, NmU, and NmS is detected in murine and human gestational tissues by immunoblot, and the expression of NmS in placenta and of NmU-R2 in uterus increases considerably with gestation age and labor, which is associated with amplified NmU-induced uterocontractile response in mice. NmU- and NmS-induced contraction is associated with increased NmU-R2-coupled Ca++ transients, and Akt and Erk activation in murine primary myometrial smooth muscle cells (mSMCs), which are potentiated with gestational age. NmU-R2 is upregulated in vitro in mSMCs and in vivo in uterus in response to proinflammatory interleukin 1beta (IL1beta), which is associated with increased NmU-induced uterocontractile response and Ca++ transients in murine and human mSMCs; additionally, placental NmS is markedly upregulated in vivo in response to IL1beta. In human placenta at term, immunohistological analysis revealed NmS expression primarily in cytotrophoblasts; furthermore, stimulation with lipopolysaccharide (LPS; Gram-negative endotoxin) markedly upregulates NmS expression in primary human cytotrophoblasts isolated from term placentas. Correspondingly, decidua of women with clinical signs of infection who delivered preterm display significantly higher expression of NmS compared with those without infection. Importantly, in vivo knockdown of NmU-R2 prevents LPS-triggered preterm birth in mice and the associated neonatal mortality. Altogether, our data suggest a critical role for NmU-R2 and its ligands NmU and NmS in preterm labor triggered by infection. We hereby identify NmU-R2 as a relevant target for preterm birth.

Sterile inflammation and pregnancy complications: a review.

Inflammation is essential for successful embryo implantation, pregnancy maintenance and delivery. In the last decade, important advances have been made in regard to endogenous, and therefore non-infectious, initiators of inflammation, which can act through the same receptors as pathogens. These molecules are referred to as damage-associated molecular patterns (DAMPs), and their involvement in reproduction has only recently been unraveled. Even though inflammation is necessary for successful reproduction, untimely activation of inflammatory processes can have devastating effect on pregnancy outcomes. Many DAMPs, such as uric acid, high-mobility group box 1 (HMGB1), interleukin (IL)-1 and cell-free fetal DNA, have been associated with pregnancy complications, such as miscarriages, preeclampsia and preterm birth in preclinical models and in humans. However, the specific contribution of alarmins to these conditions is still under debate, as currently there is lack of information on their mechanism of action. In this review, we discuss the role of sterile inflammation in reproduction, including early implantation and pregnancy complications. Particularly, we focus on major alarmins vastly implicated in numerous sterile inflammatory processes, such as uric acid, HMGB1, IL-1α and cell-free DNA (especially that of fetal origin) while giving an overview of the potential role of other candidate alarmins.

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.

Systemic inflammation impairs tissue reperfusion through endothelin-dependent mechanisms in cerebral ischemia.

BACKGROUND AND PURPOSE: Systemic inflammation contributes to diverse acute and chronic brain pathologies, and extensive evidence implicates inflammation in stroke susceptibility and poor outcome. Here we investigate whether systemic inflammation alters cerebral blood flow during reperfusion after experimental cerebral ischemia. METHODS: Serial diffusion and perfusion-weighted MRI was performed after reperfusion in Wistar rats given systemic (intraperitoneal) interleukin-1ß or vehicle before 60-minute transient middle cerebral artery occlusion. The expression and location of endothelin-1 was assessed by polymerase chain reaction, ELISA, and immunofluorescence. RESULTS: Systemic interleukin-1 caused a severe reduction in cerebral blood flow and increase in infarct volume compared with vehicle. Restriction in cerebral blood flow was observed alongside activation of the cerebral vasculature and upregulation of the vasoconstricting peptide endothelin-1 in the ischemic penumbra. A microthrombotic profile was also observed in the vasculature of rats receiving interleukin-1. Blockade of endothelin-1 receptors reversed this hypoperfusion, reduced tissue damage, and improved functional outcome. CONCLUSIONS: These data suggest patients with a raised inflammatory profile may have persistent deficits in perfusion after reopening of an occluded vessel. Future therapeutic strategies to interrupt the mechanism identified could lead to enhanced recovery of penumbra in patients with a heightened inflammatory burden and a better outcome after stroke.

Pregnant individuals with uncomplicated pregnancies display pro-inflammatory immune changes when exposed to the COVID-19 pandemic.

PROBLEM: The COVID-19 pandemic has been shown to have a detrimental impact on the mental health of pregnant individuals, and chronic stress can alter the immune profile. However, the effects of the COVID-19 pandemic on the immune system in pregnancy are still poorly understood. We aimed to evaluate the impact of pandemic exposure on the maternal immune profile in uncomplicated pregnancies. METHOD OF STUDY: We collected blood and placenta samples from pregnant individuals exposed and unexposed to the pandemic to compare their immune and inflammatory profiles. We performed co-culture with circulating maternal immune cells and endothelial cells to assess endothelial activation. Statistical analysis was performed using unpaired t-test, Mann-Whitney, or Fisher's exact test as appropriate. RESULTS: In exposed individuals, we observed a decreased proportion of Th2 cells (p < .0001) and Treg/Th17 ratio (p < .05), as well as an increased Th1/Th2 ratio (p < .0001). Levels of IL-1ß (p < .01) and IL-18 (p < .01) were increased in the circulation of exposed participants, whilst other mediators were significantly decreased (IFNγ, IL-8, MCP-1, amongst others). Furthermore, we observed increased production of ICAM, hallmark of endothelial activation, when we co-cultured endothelial cells with immune cells from exposed individuals. Vaccination status impacted the cellular profile with increased proportions of Th1 and B cells in vaccinated participants. CONCLUSION: Overall, we observed a pro-inflammatory bias in the circulation of pregnant individuals exposed to the COVID-19 pandemic, with otherwise uncomplicated pregnancies. Our work also supports an association between the increased risk of endothelial activation/hypertension and SARS-CoV2 infection, which might be driven in part by exposure to the pandemic and associated stressors.

Spatial proteomics reveals phenotypic and functional differences in T cell and macrophage subsets during villitis of unknown etiology.

Villitis of unknown etiology (VUE) is a prevalent inflammatory pathology of the placenta characterized by infiltration of maternal T cells and accumulation of fetal macrophages into chorionic villi. VUE is associated with a variety of adverse clinical outcomes, including fetal growth restriction and fetal demise. Evaluation of the phenotypic and functional differences between two immune cell types associated with this pathology, namely T cells and macrophages, was completed to gain a deeper understanding of the immuno-pathogenesis of VUE. GeoMx Digital Spatial Profiling was performed on placental tissue from 4 high grade VUE cases and 4 controls with no underlying pathology. Placental tissues were fluorescently labeled with CD3 and CD68 antibodies and oligo-conjugated antibodies against 48 protein targets. Overall, T cells in VUE exhibited upregulated markers of activation, memory, and antigen experience compared to controls and were altered based on placental location (villi vs. decidua). Additionally, villous macrophages in VUE upregulated costimulatory and major histocompatibility complex class I and II molecules compared to controls and macrophage subtypes in the decidua. Data herein provides new mechanistic insights into T cell and macrophage biology in VUE which contribute to this abnormal immune response to pregnancy.