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.

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.

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.

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.

Predictive biomarkers and initial analysis of maternal immune alterations in postpartum preeclampsia reveal an immune-driven pathology.

Introduction: Postpartum preeclampsia (PPPE) is an under-diagnosed condition, developing within 48 hours to 6 weeks following an uncomplicated pregnancy. The etiology of PPPE is still unknown, leaving patients vulnerable and making the identification and treatment of patients requiring postpartum care an unmet need. We aimed to understand the immune contribution to PPPE at the time of diagnosis, as well as uncover the predictive potential of perinatal biomarkers for the early postnatal identification of high-risk patients. Methods: Placentas were collected at delivery from uncomplicated pregnancies (CTL) and PPPE patients for immunohistochemistry analysis. In this initial study, blood samples in PPPE patients were collected at the time of PPPE diagnosis (48h-25 days postpartum; mean 7.4 days) and compared to CTL blood samples taken 24h after delivery. Single-cell transcriptomics, flow cytometry, intracellular cytokine staining, and the circulating levels of inflammatory mediators were evaluated in the blood. Results: Placental CD163+ cells and 1st trimester blood pressures can be valuable non-invasive and predictive biomarkers of PPPE with strong clinical application prospects. Furthermore, changes in immune cell populations, as well as cytokine production by CD14+, CD4+, and CD8+ cells, suggested a dampened response with an exhausted phenotype including decreased IL1ß, IL12, and IFNγ as well as elevated IL10. Discussion: Understanding maternal immune changes at the time of diagnosis and prenatally within the placenta in our sizable cohort will serve as groundwork for pre-clinical and clinical research, as well as guiding clinical practice for example in the development of immune-targeted therapies, and early postnatal identification of patients who would benefit from more thorough follow-ups and risk education in the weeks following an uncomplicated pregnancy.

Placenta analysis of Hofbauer cell profile according to the class of antiretroviral therapy used during pregnancy in people living with HIV.

INTRODUCTION: The use of antiretroviral therapy drastically reduces vertical transmission of Human Immunodeficiency Virus. However, recent studies demonstrate associations between ART use during pregnancy and placental inflammation, particularly within protease inhibitor (PI)-based regimens. We sought to characterize placental macrophages, namely Hofbauer cells, according to the class of ART used during pregnancy. METHODS: Using immunofluorescence and immunohistochemistry, placentas from 79 pregnant people living with HIV (PPLWH) and 29 HIV-uninfected people were analyzed to quantify the numbers and frequencies of leukocytes (CD45+) and Hofbauer cells (CD68+ and/or CD163+). PPLWH were stratified into three groups based on class of ART: non-nucleoside reverse transcriptase inhibitor (NNRTI)-based, integrase strand-transfer inhibitor (INSTI)-based, and PI-based regimens. RESULTS: Placentas of PPLWH contained significantly more leukocytes and Hofbauer cells than controls. Multivariable analyses revealed that this increase in immune cells was associated with a predominantly CD163+ profile in all ART subgroups compared to the HIV-negative group. This was characterized by an increase in total CD163+ cells in the PI and INSTI subgroups, and a higher frequency of CD163+ cells and CD163+/CD68+ ratio in the NNRTI and PI subgroups. DISCUSSION: Placentas of PPLWH treated with any ART regimen during their entire pregnancy displayed a selection for CD163+ cells compared to the HIV-negative group, regardless of class of ART, suggesting that class of ART does not intrinsically affect selection of CD163+ and CD68+ Hofbauer cells. Further investigations into the role of Hofbauer cells in ART-associated placental inflammation are warranted to identify the mechanisms behind their potential involvement in maternal-fetal tolerance maintenance.

Prenatal administration of IL-1Ra attenuate the neurodevelopmental impacts following non-pathogenic inflammation during pregnancy.

Prenatal inflammation negatively affects placental function, subsequently altering fetal development. Pathogen-associated molecular patterns (PAMPs) are used to mimics infections in preclinical models but rarely detected during pregnancy. Our group previously developed an animal model of prenatal exposure to uric acid (endogenous mediator), leading to growth restriction alongside IL-1-driven placental inflammation (Brien et al. in J Immunol 198(1):443-451, 2017). Unlike PAMPs, the postnatal impact of prenatal non-pathogenic inflammation is still poorly understood. Therefore, we investigated the effects of prenatal uric acid exposure on postnatal neurodevelopment and the therapeutic potential of the IL-1 receptor antagonist; IL-1Ra. Uric acid induced growth restriction and placental inflammation, which IL-1Ra protected against. Postnatal evaluation of both structural and functional aspects of the brain revealed developmental changes. Both astrogliosis and microgliosis were observed in the hippocampus and white matter at postnatal day (PND)7 with IL-1Ra being protective. Decreased myelin density was observed at PND21, and reduced amount of neuronal precursor cells was observed in the Dentate Gyrus at PND35. Functionally, motor impairments were observed as evaluated with the increased time to fully turn upward (180 degrees) on the inclined plane and the pups were weaker on the grip strength test. Prenatal exposure to sterile inflammation, mimicking most clinical situation, induced growth restriction with negative impact on neurodevelopment. Targeted anti-inflammatory intervention prenatally could offer a strategy to protect brain development during pregnancy.

A Systematic Review of the Safety of Blocking the IL-1 System in Human Pregnancy.

Blockade of the interleukin-1 (IL-1) pathway has been used therapeutically in several inflammatory diseases including arthritis and cryopyrin-associated periodic syndrome (CAPS). These conditions frequently affect women of childbearing age and continued usage of IL-1 specific treatments throughout pregnancy has been reported. IL-1 is involved in pregnancy complications and its blockade could have therapeutic potential. We systematically reviewed all reported cases of IL-1 blockade in human pregnancy to assess safety and perinatal outcomes. We searched several databases to find reports of specific blockade of the IL-1 pathway at any stage of pregnancy, excluding broad spectrum or non-specific anti-inflammatory intervention. Our literature search generated 2439 references of which 22 studies included, following extensive review. From these, 88 different pregnancies were assessed. Most (64.8%) resulted in healthy term deliveries without any obstetrical/neonatal complications. Including pregnancy exposed to Anakinra or Canakinumab, 12 (15.0%) resulted in preterm birth and one stillbirth occurred. Regarding neonatal complications, 2 cases of renal agenesis (2.5%) were observed, and 6 infants were diagnosed with CAPS (7.5%). In conclusion, this systematic review describes that IL-1 blockade during pregnancy is not associated with increased adverse perinatal outcomes, considering that treated women all presented an inflammatory disease associated with elevated risk of pregnancy complications.

Pandemic stress and SARS-CoV-2 infection are associated with pathological changes at the maternal-fetal interface.

INTRODUCTION: The reported effects of SARS-CoV-2 on pregnancy outcomes are conflicting; studies frequently overlook the placenta, which is critical for the health of the mother and infant(s). This study aimed to determine the effect of pandemic stress ± SARS CoV-2 infection on placental histopathology. METHODS: Women were recruited in Canada (n = 69); France (n = 21) or in the UK (n = 25), between March and October 2020. Historic controls (N = 20) were also included. Placenta and fetal membrane samples were collected rapidly after delivery and were fixed and stained for histopathological analysis. Maternal demographical data and obstetric outcomes were recorded. RESULTS: Over 80% of the placentas from SARS-CoV-2+ pregnancies had histopathological abnormalities: predominantly structural (71-86%) or inflammatory (9-22%), depending on geographical location. Excessive fibrin was seen in all sites, whereas deciduitis (Canada), calcifications (UK), agglutinations and chorangiosis (France) predominated in different locations. The frequency of abnormalities was significantly higher than in SARS-CoV-2 negative women (50%, p < 0.05). Demographic and obstetric data were similar in the SARS-CoV-2+ women across all sites - characterised by predominantly Black/Middle Eastern women, and women with elevated body mass index. DISCUSSION: Overall, the frequency of placental abnormalities is increased in SARS-CoV-2+ women, but the incidence of placental abnormalities is also higher in SARS-CoV-2- women that gave birth during the pandemic, which highlights the importance of appropriate control groups to ascertain the roles of pandemic stress and SARS-CoV-2 infection on the placenta and pregnancy outcomes.

Specific inflammatory profile in each pregnancy complication: A comparative study.

PROBLEM: Pre-eclampsia (PE), preterm birth (PTB) and intra-uterine growth restriction (IUGR) affect 5%-12% of pregnancies. They have been associated with placental inflammation, although the detection of inflammatory mediators in the maternal circulation is still controversial. Our goal was to determine the inflammatory changes occurring in the second part of pregnancy to identify profiles distinguishing pathological pregnancies from each other. METHOD OF STUDY: We performed a nested case-control study of 200 women randomly selected from a cohort recruited at the CHU de Quebec-Universite Laval, Quebec, Canada. Women with uncomplicated term pregnancy (CTRL); PE (severe or not); PTB or IUGR (N = 50/each) were included. Plasma samples, obtained from the late second trimester and at delivery, were analysed for over 30 selected mediators (including cytokines/alarmins), by multiplex, ELISA or specific assays. Demographic and obstetrical information were obtained for classification. RESULTS: In CTRL, we observed significant differences between 2nd trimester and delivery, with increased levels of inflammatory mediators (ex. MCP-1, IL-6), supporting an inflammatory profile towards term. Increased levels of IL-6, CXCL10 and CRP were observed in PE as compared to CTRL. In PTB, we observed increased CXCL9 in 2nd trimester and decreased progesterone at delivery. In IUGR, increased HMGB1 and IL-1α were observed only in the 2nd trimester. CONCLUSIONS: Our work showed significant inflammatory changes in uncomplicated pregnancies towards delivery, supporting that normal delivery is pro-inflammatory, although not to the same extent as in pathological pregnancies. Inflammatory profiles are specific to each pregnancy complication which may help to understand the contribution of inflammation to the clinical presentation of these conditions.

Differential effect of LPS and IL-1ß in term placental explants.

INTRODUCTION: Inflammation is an important cause of placental dysfunction often associated with pregnancy complications. One well-known cause of inflammation is infection, through conserved "pathogen-associated molecular patterns" (PAMPs). Endogenous inducers of inflammation, known as "damage-associated molecular patterns" (DAMPs), have also been associated with pathological pregnancies and could contribute to the observed placental inflammation. Although both stimuli (i.e. PAMPs/DAMPs) can induce inflammation, they have yet to be studied together to compare their inflammatory effects on the placenta. METHODS: We used a model of term placental explants to compare the effects of a classical PAMP, bacterial lipopolysaccharide (LPS), and a DAMP, the pro-inflammatory cytokine interleukin (IL)-1. Gene and protein expression of several cytokines were analysed by qPCR and ELISAs and immunohistochemistry performed to study placental resident immune cells and apoptosis. RESULTS: LPS induced pro-inflammatory mediators (IL-6, IL-1ß/α, TNF-α) whereas IL-1ß induced only IL-6. Furthermore, LPS but not IL-1 exposure, led to elevated IL-10 and IL-1Ra secretion. Blocking the IL-1 signalling pathway abrogated the pro-inflammatory actions of LPS, whilst anti-inflammatory effects were preserved. The number of CD45 + immune cells was elevated in explants treated with LPS only. A subpopulation of CD45 + cells were positive for PCNA indicating proliferation of tissue resident macrophages. DISCUSSION: We conclude that LPS, a classical PAMP, and IL-1, a DAMP, have shared and distinct actions with pro-inflammatory effects mediated through IL-1 but anti-inflammatory actions having a distinct pathway. Identification of an inflammatory mediator (i.e. IL-1) common to multiple stimuli could be a therapeutic target to preserve the placenta.