Changes in Relative Counts of Different Leukocyte Subpopulations in Peripheral and Umbilical Cord Blood of Women With Preterm Prelabor Rupture of Membranes With Respect to Intraamniotic Inflammation and Fetal Inflammatory Response Syndrome.

OBJECTIVE: The aim of this study was to evaluate changes in the relative counts of different leukocyte subsets in peripheral and umbilical cord blood in pregnancies complicated by preterm prelabor rupture of membranes (PPROM) with respect to the presence of intraamniotic inflammation (IAI) and fetal inflammatory response syndrome (FIRS). METHODS: Fifty-two women with singleton pregnancies complicated by PPROM were included in this study. From samples of peripheral and umbilical cord blood, relative counts of these leukocyte subpopulations were determined using multicolor flow cytometry: granulocytes, monocytes, lymphocytes, T cells and their subpopulations, B cells and their subpopulations, and NK cells and their subpopulations. IAI was defined as increased concentrations of interleukin 6 in the amniotic fluid. Amniotic fluid samples were obtained by transabdominal amniocentesis. RESULTS: Women with IAI had higher relative counts of monocytes (p = 0.04) in peripheral blood. There was an increased relative number of granulocytes (p = 0.003) and a decreased number of lymphocytes (p = 0.0048), helper CD4+ T cells (p = 0.019), NK cells (p = 0.0001) within leukocytes, NK cells within lymphocytes (p = 0.003) and CD16+ NK cells within NK cells (p = 0.005) in umbilical cord blood samples of women with FIRS. However, after adjusting the results for gestational age at sampling, all differences disappeared. CONCLUSIONS: The presence of IAI or FIRS is not accompanied by significant changes in the relative counts of immune cells in peripheral blood or umbilical cord blood in pregnancies complicated by PPROM.

Characterization of Inflammation/Immune-, Acute Phase-, Extracellular Matrix-, Adhesion-, and Serine Protease-Related Proteins in the Amniotic Fluid of Women With Early Preterm Prelabor Rupture of Membranes.

PROBLEM: To determine whether altered concentrations of various inflammation/immune-, acute phase-, extracellular matrix-, adhesion-, and serine protease-related proteins in the amniotic fluid (AF) are independently associated with microbial invasion of the amniotic cavity and/or intra-amniotic inflammation (MIAC/IAI), imminent spontaneous preterm delivery (SPTD; ≤7 days), and major neonatal morbidity/mortality (NMM) in women with early preterm prelabor rupture of membranes (PPROM). METHOD OF STUDY: This was a retrospective cohort study involving 111 singleton pregnant women with PPROM (24-31 weeks) undergoing amniocentesis to diagnose MIAC/IAI. The following proteins were measured in stored AF samples by enzyme-linked immunosorbent assay (ELISA): APRIL, DKK-3, Gal-3BP, IGFBP-2, IL-8, VDBP, lumican, MMP-2, MMP-8, SPARC, TGFBI, TGF-ß1, E-selectin, ICAM-5, P-selectin, haptoglobin, hepcidin, SAA1, kallistatin, and uPA. RESULTS: Multivariate logistic regression analyses revealed that (i) elevated APRIL, IL-8, MMP-8, and TGFBI levels in the AF, reduced lumican and SPARC levels in the AF, and high percentages of samples above the lower limit of quantification for AF TGF-ß1 and uPA were significantly associated with MIAC/IAI; (ii) elevated AF levels of IL-8 and MMP-8 were significantly associated with SPTD within 7 days; and (iii) elevated AF IL-6 levels were significantly associated with increased risk for major NMM, when adjusted for baseline covariates. CONCLUSION: ECM (lumican, SPRAC, TGFBI, and TGF-ß1)- and serine protease (uPA)-associated proteins in the AF are involved in the regulation of the host response to infection/inflammation in the amniotic cavity, whereas AF inflammation (IL-8, MMP-8, and IL-6)-associated mediators are implicated in the development of preterm parturition and major NMM in early PPROM.

Plasma Kallistatin and Progranulin as Predictive Biomarkers of Intraamniotic Inflammation, Microbial Invasion of the Amniotic Cavity, and Composite Neonatal Morbidity/Mortality in Women With Preterm Premature Rupture of Membranes.

PROBLEM: To explore the clinical utility of nine inflammatory immune-, adhesion-, and extracellular matrix-related mediators in the plasma for predicting intraamniotic inflammation and/or microbial invasion of the amniotic cavity (IAI/MIAC) and composite neonatal morbidity and/or mortality (CNMM) in women with preterm premature rupture of membranes (PPROM) when used alone or in combination with conventional blood-, ultrasound-, and clinical-based factors. METHODS OF STUDY: This retrospective cohort comprised 173 singleton pregnant women with PPROM (24 + 0 - 33 + 6 weeks), who underwent amniocentesis. Amniotic fluid was cultured for microorganisms and assayed for IL-6 levels. Plasma levels of AFP, CXCL14, E-selectin, Gal-3BP, kallistatin, progranulin, P-selectin, TGFBI, and VDBP were determined by ELISA. Ultrasonographic cervical length (CL) and neutrophil-to-lymphocyte ratio (NLR) were measured. RESULTS: Multivariate logistic regression analyses revealed significant associations between (i) decreased plasma kallistatin levels and IAI/MIAC and (ii) decreased plasma progranulin levels and increased CNMM risk after adjusting for baseline variables (e.g., gestational age at sampling [or delivery] and parity). Using stepwise regression analysis, noninvasive prediction models for IAI/MIAC and CNMM risks were developed, which included plasma progranulin levels, NLR, CL, and gestational age at sampling, and provided a good prediction of the corresponding endpoints (area under the curve: 0.79 and 0.87, respectively). CONCLUSIONS: Kallistatin and progranulin are potentially valuable plasma biomarkers for predicting IAI/MIAC and CNMM in women with PPROM. Particularly, the combination of these plasma biomarkers with conventional blood-, ultrasound-, and clinical-based factors can significantly support the diagnosis of IAI/MIAC and CNMM.

IL-1 and TNF mediates IL-6 signaling at the maternal-fetal interface during intrauterine inflammation.

Introduction: IL6 signaling plays an important role in triggering labor and IL6 is an established biomarker of intrauterine infection/inflammation (IUI) driven preterm labor (PTL). The biology of IL6 during IUI at the maternal-fetal interface was investigated in samples from human subjects and non-human primates (NHP). Methods: Pregnant women with histologic chorioamnionitis diagnosed by placenta histology were recruited (n=28 term, n=43 for preterm pregnancies from 26-36 completed weeks of gestation). IUI was induced in Rhesus macaque by intraamniotic injection of lipopolysachharide (LPS, n=23). IL1 signaling was blocked using Anakinra (human IL-1 receptor antagonist, n=13), and Tumor necrosis factor (TNF) signaling was blocked by anti TNF-antibody (Adalimumab n=14). The blockers were given before LPS. All animals including controls (intraamniotic injection of saline n=27), were delivered 16h after LPS/saline exposure at about 80% gestation. Results: IUI induced a robust expression of IL6 mRNAs in the fetal membranes (chorion-amnion-decidua tissue) both in humans (term and preterm) and NHP. The major sources of IL6 mRNA expression were the amnion mesenchymal cells (AMC) and decidua stroma cells. Additionally, during IUI in the NHP, ADAM17 (a protease that cleaves membrane bound IL6 receptor (IL6R) to release a soluble form) and IL6R mRNA increased in the fetal membranes, and the ratio of IL6 and soluble forms of IL6R, gp130 increased in the amniotic fluid signifying upregulation of IL6 trans-signaling. Both IL1 and TNF blockade suppressed LPS-induced IL6 mRNAs in the AMC and variably decreased elements of IL6 trans-signaling. Discussion: These data suggest that IL1 and TNF blockers may be useful anti-inflammatory agents via suppression of IL6 signaling at the maternal-fetal interface.

Palmitate and group B Streptococcus synergistically and differentially induce IL-1ß from human gestational membranes.

Introduction: Rupture of the gestational membranes often precedes major pregnancy complications, including preterm labor and preterm birth. One major cause of inflammation in the gestational membranes, chorioamnionitis (CAM) is often a result of bacterial infection. The commensal bacterium Streptococcus agalactiae, or Group B Streptococcus (GBS) is a leading infectious cause of CAM. Obesity is on the rise worldwide and roughly 1 in 4 pregnancy complications is related to obesity, and individuals with obesity are also more likely to be colonized by GBS. The gestational membranes are comprised of several distinct cell layers which are, from outermost to innermost: maternally-derived decidual stromal cells (DSCs), fetal cytotrophoblasts (CTBs), fetal mesenchymal cells, and fetal amnion epithelial cells (AECs). In addition, the gestational membranes have several immune cell populations; macrophages are the most common phagocyte. Here we characterize the effects of palmitate, the most common long-chain saturated fatty acid, on the inflammatory response of each layer of the gestational membranes when infected with GBS, using human cell lines and primary human tissue. Results: Palmitate itself slightly but significantly augments GBS proliferation. Palmitate and GBS co-stimulation synergized to induce many inflammatory proteins and cytokines, particularly IL-1ß and matrix metalloproteinase 9 from DSCs, CTBs, and macrophages, but not from AECs. Many of these findings are recapitulated when treating cells with palmitate and a TLR2 or TLR4 agonist, suggesting broad applicability of palmitate-pathogen synergy. Co-culture of macrophages with DSCs or CTBs, upon co-stimulation with GBS and palmitate, resulted in increased inflammatory responses, contrary to previous work in the absence of palmitate. In whole gestational membrane biopsies, the amnion layer appeared to dampen immune responses from the DSC and CTB layers (the choriodecidua) to GBS and palmitate co-stimulation. Addition of the monounsaturated fatty acid oleate, the most abundant monounsaturated fatty acid in circulation, dampened the proinflammatory effect of palmitate. Discussion: These studies reveal a complex interplay between the immunological response of the distinct layers of the gestational membrane to GBS infection and that such responses can be altered by exposure to long-chain saturated fatty acids. These data provide insight into how metabolic syndromes such as obesity might contribute to an increased risk for GBS disease during pregnancy.

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.

Pharmacological blockade of the interleukin-1 receptor suppressed Escherichia coli lipopolysaccharide-induced neuroinflammation in preterm fetal sheep.

BACKGROUND: Intraamniotic inflammation is associated with preterm birth, especially in cases occurring before 32 weeks' gestation, and is causally linked with an increased risk for neonatal mortality and morbidity. Targeted anti-inflammatory interventions may assist in improving the outcomes for pregnancies impacted by intrauterine inflammation. Interleukin-1 is a central upstream mediator of inflammation. Accordingly, interleukin-1 is a promising candidate target for intervention therapies and has been targeted previously using the interleukin-1 receptor antagonist, anakinra. Recent studies have shown that the novel, noncompetitive, allosteric interleukin-1 receptor inhibitor, rytvela, partially resolved inflammation associated with preterm birth and fetal injury. In this study, we used a preterm sheep model of chorioamnionitis to investigate the anti-inflammatory efficacy of rytvela and anakinra, administered in the amniotic fluid in the setting of intraamniotic Escherichia coli lipopolysaccharide exposure. OBJECTIVE: We hypothesized that both rytvela and anakinra would reduce lipopolysaccharide-induced intrauterine inflammation and protect the fetal brain. STUDY DESIGN: Ewes with a singleton fetus at 105 days of gestation (term is ∼150 days) were randomized to one of the following groups: (1) intraamniotic injections of 2 mL saline at time=0 and time=24 hours as a negative control group (saline group, n=12); (2) intraamniotic injection of 10 mg Escherichia coli lipopolysaccharide in 2 mL saline and intraamniotic injections of 2 mL saline at time=0 hours and time=24 hours as an inflammation positive control group (lipopolysaccharide group, n=11); (3) intraamniotic injection of Escherichia coli lipopolysaccharide in 2 mL saline and intraamniotic injections of 2.5 mg rytvela at time=0 hours and time=24 hours to test the anti-inflammatory efficacy of rytvela (lipopolysaccharide + rytvela group, n=10); or (4) intraamniotic injection of Escherichia coli lipopolysaccharide in 2 mL saline and intraamniotic injections of 100 mg anakinra at time=0 hours and time=24 hours to test the anti-inflammatory efficacy of anakinra (lipopolysaccharide + anakinra group, n=12). Amniotic fluid was sampled at time 0, 24, and 48 hours (ie, at each intervention and at delivery). Fetal umbilical cord blood was collected at delivery for differential blood counts and chemical studies. Inflammation was characterized by the analysis of fetal tissue cytokine and chemokine levels using quantitative polymerase chain reaction, enzyme-linked inmmunosorbent assay, and histology. The primary study outcome of interest was the assessment of anakinra and rytvela brain-protective effects in the setting of Escherichia coli lipopolysaccharide-induced intrauterine inflammation. Secondary outcomes of interest were to assess protection from fetal and intrauterine (ie, amniotic fluid, chorioamnion) inflammation. RESULTS: Intraamniotic administration of lipopolysaccharide caused inflammation of the fetal lung, brain, and chorioamnionitis in preterm fetal sheep. Relative to treatment with saline only in the setting of lipopolysaccharide exposure, intraamniotic administration of both rytvela and anakinra both significantly prevented periventricular white matter injury, microglial activation, and histologic chorioamnionitis. Anakinra showed additional efficacy in inhibiting fetal lung myeloperoxidase activity, but its use was associated with metabolic acidaemia and reduced fetal plasma insulin-like growth factor-1 levels at delivery. CONCLUSION: Intraamniotic administration of rytvela or anakinra significantly inhibited fetal brain inflammation and chorioamnionitis in preterm fetal sheep exposed to intraamniotic lipopolysaccharide. In addition, anakinra treatment was associated with potential negative impacts on the developing fetus.

Olfactomedin-4-Positive Neutrophils in Neonates: Link to Systemic Inflammation and Bronchopulmonary Dysplasia.

INTRODUCTION: Little is known about the interplay between neutrophil heterogeneity in neonates in health and disease states. Olfactomedin-4 (OLFM4) marks a subset of neutrophils that have been described in adults and pediatric patients but not neonates, and this subset is thought to play a role in modulating the host inflammatory response. METHODS: This is a prospective cohort of neonates who were born between June 2020 and December 2021 at the University of Cincinnati Medical Center NICU. Olfactomedin-4-positive (OLFM4+) neutrophils were identified in the peripheral blood using flow cytometry. RESULTS: OLFM4+ neutrophil percentage was not correlated with gestational age or developmental age. Neonates with sepsis had a higher percentage than those without the condition, 66.9% (IQR 24.3-76.9%) versus 21.5% (IQR 10.6-34.7%), respectively, p = 0.0003. At birth, a high percentage of OLFM4+ neutrophils was associated with severe chorioamnionitis at 49.1% (IQR 28.2-61.5%) compared to those without it at 13.7% (IQR 7.7-26.3%), p < 0.0001. Among neonates without sepsis, the percentages of OLFM4+ neutrophils were lower in the BPD/early death group compared to those without BPD, 11.8% (IQR 6.3-29.0%) versus 32.5% (IQR 18.5-46.1%), p = 0.003, and this retained significance in a multiple logistic regression model that included gestational age, birthweight, and race. CONCLUSION: This is the first study describing OLFM4+ neutrophils in neonates and it shows that this neutrophil subpopulation is not influenced by gestational age but is elevated in inflammatory conditions such as sepsis and severe chorioamnionitis, and lower percentage at birth is associated with developing bronchopulmonary dysplasia.

Chorioamnionitis and neonatal outcomes.

Chorioamnionitis or intrauterine inflammation is a frequent cause of preterm birth. Chorioamnionitis can affect almost every organ of the developing fetus. Multiple microbes have been implicated to cause chorioamnionitis, but "sterile" inflammation appears to be more common. Eradication of microorganisms has not been shown to prevent the morbidity and mortality associated with chorioamnionitis as inflammatory mediators account for continued fetal and maternal injury. Mounting evidence now supports the concept that the ensuing neonatal immune dysfunction reflects the effects of inflammation on immune programming during critical developmental windows, leading to chronic inflammatory disorders as well as vulnerability to infection after birth. A better understanding of microbiome alterations and inflammatory dysregulation may help develop better treatment strategies for infants born to mothers with chorioamnionitis.

The Role of Innate Immune System in the Human Amniotic Membrane and Human Amniotic Fluid in Protection Against Intra-Amniotic Infections and Inflammation.

Intra-amniotic infection and inflammation (IAI) affect fetal development and are highly associated with preterm labor and premature rupture of membranes, which often lead to adverse neonatal outcomes. Human amniotic membrane (hAM), the inner part of the amnio-chorionic membrane, protects the embryo/fetus from environmental dangers, including microbial infection. However, weakened amnio-chorionic membrane may be breached or pathogens may enter through a different route, leading to IAI. The hAM and human amniotic fluid (hAF) respond by activation of all components of the innate immune system. This includes changes in 1) hAM structure, 2) presence of immune cells, 3) pattern recognition receptors, 4) cytokines, 5) antimicrobial peptides, 6) lipid derivatives, and 7) complement system. Herein we provide a comprehensive and integrative review of the current understanding of the innate immune response in the hAM and hAF, which will aid in design of novel studies that may lead to breakthroughs in how we perceive the IAI.

Sustained peripheral immune hyper-reactivity (SPIHR): an enduring biomarker of altered inflammatory responses in adult rats after perinatal brain injury.

BACKGROUND: Chorioamnionitis (CHORIO) is a principal risk factor for preterm birth and is the most common pathological abnormality found in the placentae of preterm infants. CHORIO has a multitude of effects on the maternal-placental-fetal axis including profound inflammation. Cumulatively, these changes trigger injury in the developing immune and central nervous systems, thereby increasing susceptibility to chronic sequelae later in life. Despite this and reports of neural-immune changes in children with cerebral palsy, the extent and chronicity of the peripheral immune and neuroinflammatory changes secondary to CHORIO has not been fully characterized. METHODS: We examined the persistence and time course of peripheral immune hyper-reactivity in an established and translational model of perinatal brain injury (PBI) secondary to CHORIO. Pregnant Sprague-Dawley rats underwent laparotomy on embryonic day 18 (E18, preterm equivalent). Uterine arteries were occluded for 60 min, followed by intra-amniotic injection of lipopolysaccharide (LPS). Serum and peripheral blood mononuclear cells (PBMCs) were collected at young adult (postnatal day P60) and middle-aged equivalents (P120). Serum and PBMCs secretome chemokines and cytokines were assayed using multiplex electrochemiluminescent immunoassay. Multiparameter flow cytometry was performed to interrogate immune cell populations. RESULTS: Serum levels of interleukin-1ß (IL-1ß), IL-5, IL-6, C-X-C Motif Chemokine Ligand 1 (CXCL1), tumor necrosis factor-α (TNF-α), and C-C motif chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) were significantly higher in CHORIO animals compared to sham controls at P60. Notably, CHORIO PBMCs were primed. Specifically, they were hyper-reactive and secreted more inflammatory mediators both at baseline and when stimulated in vitro. While serum levels of cytokines normalized by P120, PBMCs remained primed, and hyper-reactive with a robust pro-inflammatory secretome concomitant with a persistent change in multiple T cell populations in CHORIO animals. CONCLUSIONS: The data indicate that an in utero inflammatory insult leads to neural-immune changes that persist through adulthood, thereby conferring vulnerability to brain and immune system injury throughout the lifespan. This unique molecular and cellular immune signature including sustained peripheral immune hyper-reactivity (SPIHR) and immune cell priming may be a viable biomarker of altered inflammatory responses following in utero insults and advances our understanding of the neuroinflammatory cascade that leads to perinatal brain injury and later neurodevelopmental disorders, including cerebral palsy.

Vaginal microbiome as a tool for prediction of chorioamnionitis in preterm labor: a pilot study.

Intra-amniotic infection (IAI) is a major cause of preterm birth with a poor perinatal prognosis. We aimed to determine whether analyzing vaginal microbiota can evaluate the risk of chorioamnionitis (CAM) in preterm labor cases. Vaginal discharge samples were collected from 83 pregnant women admitted for preterm labor. Based on Blanc's classification, the participants were divided into CAM (stage ≥ II; n = 46) and non-CAM (stage ≤ I; n = 37) groups. The 16S rDNA amplicons (V1-V2) from vaginal samples were sequenced and analyzed. Using a random forest algorithm, the bacterial species associated with CAM were identified, and a predictive CAM (PCAM) scoring method was developed. The α diversity was significantly higher in the CAM than in the non-CAM group (P < 0.001). The area under the curve was 0.849 (95% confidence interval 0.765-0.934) using the PCAM score. Among patients at < 35 weeks of gestation, the PCAM group (n = 22) had a significantly shorter extended gestational period than the non-PCAM group (n = 25; P = 0.022). Multivariate analysis revealed a significant difference in the frequency of developmental disorders in 3-year-old infants (PCAM, 28%, non-PCAM, 4%; P = 0.022). Analyzing vaginal microbiota can evaluate the risk of IAI. Future studies should establish appropriate interventions for IAI high-risk patients to improve perinatal prognosis.

Direct administration of the non-competitive interleukin-1 receptor antagonist rytvela transiently reduced intrauterine inflammation in an extremely preterm sheep model of chorioamnionitis.

BACKGROUND: Intraamniotic inflammation is associated with up to 40% of preterm births, most notably in deliveries occurring prior to 32 weeks' gestation. Despite this, there are few treatment options allowing the prevention of preterm birth and associated fetal injury. Recent studies have shown that the small, non-competitive allosteric interleukin (IL)-1 receptor inhibitor, rytvela, may be of use in resolving inflammation associated with preterm birth (PTB) and fetal injury. We aimed to use an extremely preterm sheep model of chorioamnionitis to investigate the anti-inflammatory efficacy of rytvela in response to established intra-amniotic (IA) lipopolysaccharide (LPS) exposure. We hypothesized that rytvela would reduce LPS-induced IA inflammation in amniotic fluid (AF) and fetal tissues. METHODS: Sheep with a single fetus at 95 days gestation (estimated fetal weight 1.0 kg) had surgery to place fetal jugular and IA catheters. Animals were recovered for 48 hours before being randomized to either: i) IA administration of 2 ml saline 24 hours before 2 ml IA and 2 ml fetal intravenous (IV) administration of saline (Saline Group, n = 7); ii) IA administration of 10 mg LPS in 2 ml saline 24 hours before 2 ml IA and 2 ml fetal IV saline (LPS Group, n = 10); 3) IA administration of 10 mg LPS in 2 ml saline 24 hours before 0.3 mg/fetal kg IA and 1 mg/fetal kg fetal IV rytvela in 2 ml saline, respectively (LPS + rytvela Group, n = 7). Serial AF samples were collected for 120 h. Inflammatory responses were characterized by quantitative polymerase chain reaction (qPCR), histology, fluorescent immunohistochemistry, enzyme-linked inmmunosorbent assay (ELISA), fluorescent western blotting and blood chemistry analysis. RESULTS: LPS-treated animals had endotoxin and AF monocyte chemoattractant protein (MCP)-1 concentrations that were significantly higher at 24 hours (immediately prior to rytvela administration) relative to values from Saline Group animals. Following rytvela administration, the average MCP-1 concentrations in the AF were significantly lower in the LPS + rytvela Group relative to in the LPS Group. In delivery samples, the expression of IL-1ß in fetal skin was significantly lower in the LPS + rytvela Group compared to the LPS Group. CONCLUSION: A single dose of rytvela was associated with partial, modest inhibition in the expression of a panel of cytokines/chemokines in fetal tissues undergoing an active inflammatory response.

Preterm Birth Is Associated With Immune Dysregulation Which Persists in Infants Exposed to Histologic Chorioamnionitis.

Introduction: Preterm infants are at increased risk of exposure to histologic chorioamnionitis (HCA) when compared to term-born controls, and this is associated with several neonatal morbidities involving brain, lungs and gut. Preterm infants could benefit from immunomodulatory therapies in the perinatal period, but development of rational treatment strategies requires improved characterization of the perinatal response to HCA. We had two objectives: The first, to characterize the umbilical cord blood immune profile in preterm infants compared to term-born controls; the second, to investigate the postnatal immune response in preterm infants exposed to HCA versus those who were not. Population: For objective one 59 term infants [mean gestational age (GA) 39+4 (37+3 to 42+0)] and 55 preterm infants [mean GA29+0(23+3 to 32+0)] with umbilical cord samples available were included; for objective two we studied 96 preterm infants [mean GA29+1(23+2 to 32+0)] for whom placental histology and postnatal blood samples were available. Methods: Placental histopathology was used to identify reaction patterns indicative of HCA, and a customized immunoassay of 24 inflammatory markers and trophic proteins selected to reflect the perinatal immune response was performed on umbilical cord blood in term and preterm participants and postnatal day 5 blood in the preterm group. Results: The umbilical cord blood immune profile classified gestational age category with 86% accuracy (95% CI 0.78-0.92), p-value=1.242x10-14. Pro-inflammatory proteins IL-6, MCP-1 and CRP were elevated in the cord blood of preterm infants whilst BDNF, C3, C9, IL-18, MMP-9 and RANTES were decreased, compared to infants born at term. In preterm infants, exposure to HCA was associated with elevations in 8 immune proteins on postnatal day 5 (BDNF, C3, C5a, C9, IL-8, MCP-1, MIP-1ß and MMP-9) when compared to preterm infants who were not exposed. Conclusion: Preterm birth is associated with a distinct immune profile in umbilical cord blood and preterm infants exposed to HCA with evidence of a fetal inflammatory response have specific alterations in immune function that are apparent on day 5 of postnatal life.

Expression of Immune Checkpoint Receptors in Placentae With Infectious and Non-Infectious Chronic Villitis.

Pregnancy is an immunological paradox whereby maternal immunity accepts a genetically unique fetus (or fetuses), while maintaining protective innate and adaptive responses to infectious pathogens. This close contact between the genetically diverse mother and fetus requires numerous mechanisms of immune tolerance initiated by trophoblast cell signals. However, in a placental condition known as villitis of unknown etiology (VUE), there appears to be a breakdown in this tolerance allowing maternal cytotoxic T-cells to traffic into the placenta to destroy fetal villi. VUE is associated with several gestational complications and an increased risk of recurrence in a subsequent pregnancy, making it a significant obstetrical diagnosis. The cause of VUE remains unclear, but dysfunctional signaling through immune checkpoint pathways, which have a critical role in blunting immune responses, may play an important role. Therefore, using placental tissue from normal pregnancy (n=8), VUE (n=8) and cytomegalovirus (CMV) infected placentae (n=4), we aimed to identify differences in programmed cell death 1 (PD-1), programmed death ligand-1 (PD-L1), LAG3 and CTLA4 expression between these etiologies by immunohistochemistry (IHC). Results demonstrated significantly lower expression of PD-L1 on trophoblast cells from VUE placentae compared to control and CMV infection. Additionally, we observed significantly higher counts of PD-1+ (>100 cells/image) and LAG3+ (0-120 cells/image) cells infiltrating into the villi during VUE compared to infection and control. Minimal CTLA4 staining was observed in all placentae, with only a few Hofbauer cells staining positive. Together, this suggests that a loss of tolerance through immune checkpoint signaling may be an important mechanism leading to the activation and trafficking of maternal cells into fetal villi during VUE. Further mechanistic studies are warranted to understand possible allograft rejection more clearly and in developing effective strategies to prevent this condition from occurring in utero.

Transcriptomic analysis of equine chorioallantois reveals immune networks and molecular mechanisms involved in nocardioform placentitis.

Nocardioform placentitis (NP) continues to result in episodic outbreaks of abortion and preterm birth in mares and remains a poorly understood disease. The objective of this study was to characterize the transcriptome of the chorioallantois (CA) of mares with NP. The CA were collected from mares with confirmed NP based upon histopathology, microbiological culture and PCR for Amycolatopsis spp. Samples were collected from the margin of the NP lesion (NPL, n = 4) and grossly normal region (NPN, n = 4). Additionally, CA samples were collected from normal postpartum mares (Control; CRL, n = 4). Transcriptome analysis identified 2892 differentially expressed genes (DEGs) in NPL vs. CRL and 2450 DEGs in NPL vs. NPN. Functional genomics analysis elucidated that inflammatory signaling, toll-like receptor signaling, inflammasome activation, chemotaxis, and apoptosis pathways are involved in NP. The increased leukocytic infiltration in NPL was associated with the upregulation of matrix metalloproteinase (MMP1, MMP3, and MMP8) and apoptosis-related genes, such as caspases (CASP3 and CASP7), which could explain placental separation associated with NP. Also, NP was associated with downregulation of several placenta-regulatory genes (ABCG2, GCM1, EPAS1, and NR3C1), angiogenesis-related genes (VEGFA, FLT1, KDR, and ANGPT2), and glucose transporter coding genes (GLUT1, GLUT10, and GLUT12), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could elucidate placental insufficiency accompanying NP. In conclusion, our findings revealed for the first time, the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during NP, which might lead to the development of efficacious therapies or diagnostic aids by targeting the key molecular pathways.

Postnatal inflammation in ApoE-/- mice is associated with immune training and atherosclerosis.

BACKGROUND AND AIMS: Preterm birth is associated with increased risk of cardiovascular disease (CVD). This may reflect a legacy of inflammatory exposures such as chorioamnionitis which complicate pregnancies delivering preterm, or recurrent early-life infections, which are common in preterm infants. We previously reported that experimental chorioamnionitis followed by postnatal inflammation has additive and deleterious effects on atherosclerosis in ApoE-/- mice. Here, we aimed to investigate whether innate immune training is a contributory inflammatory mechanism in this murine model of atherosclerosis. METHODS: Bone marrow-derived macrophages and peritoneal macrophages were isolated from 13-week-old ApoE-/- mice, previously exposed to prenatal intra-amniotic (experimental choriomanionitis) and/or repeated postnatal (peritoneal) lipopolysaccharide (LPS). Innate immune responses were assessed by cytokine responses following ex vivo stimulation with toll-like receptor (TLR) agonists (LPS, Pam3Cys) and RPMI for 24-h. Bone marrow progenitor populations were studied using flow cytometric analysis. RESULTS: Following postnatal LPS exposure, bone marrow-derived macrophages and peritoneal macrophages produced more pro-inflammatory cytokines following TLR stimulation than those from saline-treated controls, characteristic of a trained phenotype. Cytokine production ex vivo correlated with atherosclerosis severity in vivo. Prenatal LPS did not affect cytokine production capacity. Combined prenatal and postnatal LPS exposure was associated with a reduction in populations of myeloid progenitor cells in the bone marrow. CONCLUSIONS: Postnatal inflammation results in a trained phenotype in atherosclerosis-prone mice that is not enhanced by prenatal inflammation. If analogous mechanisms occur in humans, then there may be novel early life opportunities to reduce CVD risk in infants with early life infections.

Chorioamnionitis induces hepatic inflammation and time-dependent changes of the enterohepatic circulation in the ovine fetus.

Chorioamnionitis, inflammation of fetal membranes, is an important cause of preterm birth and a risk factor for the development of adverse neonatal outcomes including sepsis and intestinal pathologies. Intestinal bile acids (BAs) accumulation and hepatic cytokine production are involved in adverse intestinal outcomes. These findings triggered us to study the liver and enterohepatic circulation (EHC) following intra-amniotic (IA) lipopolysaccharide (LPS) exposure. An ovine chorioamnionitis model was used in which circulatory cytokines and outcomes of the liver and EHC of preterm lambs were longitudinally assessed following IA administration of 10 mg LPS at 5, 12 or 24h or 2, 4, 8 or 15d before preterm birth. Hepatic inflammation was observed, characterized by increased hepatic cytokine mRNA levels (5h - 2d post IA LPS exposure) and increased erythropoietic clusters (at 8 and 15 days post IA LPS exposure). Besides, 12h after IA LPS exposure, plasma BA levels were increased, whereas gene expression levels of several hepatic BA transporters were decreased. Initial EHC alterations normalized over time. Concluding, IA LPS exposure induces significant time-dependent changes in the fetal liver and EHC. These chorioamnionitis induced changes have potential postnatal consequences and the duration of IA LPS exposure might be essential herein.

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.