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.

Effects of multiple pro-inflammatory stimuli in utero on the ileum of extremely premature ovine fetuses.

Introduction: Chorioamnionitis is common in preterm birth and associated with a higher risk of intestinal inflammation and necrotizing enterocolitis. The intestinal inflammation influences the enteric nervous system development. We hypothesized that inflammation and innervation in the fetal ileum may be modified by chorioamnionitis induced by repeated challenge with lipopolysaccharide and/or preexisting Ureaplasma parvum infection at very low gestational age equivalent to 60% of term. Materials and methods: Time mated ovine fetuses were exposed by intraamniotic injections to chronic Ureaplasma parvum for 24 days and/or lipopolysaccharide for 7 days, 2 days, or 7 & 2 days before delivery at 94 +/-2 days of gestational age (term at approximately 150 days). Intestinal inflammation as well as structural changes of the enteric nervous system were assessed. Results: Lipopolysaccharide exposure increased CD3 and myeloperoxidase-positive cells (p < 0.05). Repetitive exposure to lipopolysaccharide or combined Ureaplasma parvum & lipopolysaccharide exposure increased intestinal inflammation (p < 0.05). The reduction of nuclei of neurons was most significant with repetitive lipopolysaccharide exposures but could be detected in all other intervention groups compared to the control group. Astrocyte-like glial cells increased if exposure to lipopolysaccharide was only 2 days before delivery or chronic exposure to Ureaplasma parvum existed beforehand (p < 0.05). Discussion: After exposure to chorioamnionitis induced by Ureaplasma parvum and/or lipopolysaccharide, inflammatory responses as well as structural changes of the enteric nervous system were more pronounced the longer and the more frequent the exposure to pro-inflammatory stimuli before birth. These changes may cause functional effects of clinical importance.

Tdap vaccination during pregnancy and risk of chorioamnionitis and related infant outcomes.

INTRODUCTION: An increased risk of chorioamnionitis in people receiving tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (Tdap) vaccine during pregnancy has been reported. The importance of this association is unclear as additional study has not demonstrated increased adverse infant outcomes associated with Tdap vaccination in pregnancy. METHODS: We conducted a retrospective observational cohort study of pregnant people ages 15-49 years with singleton pregnancies ending in live birth who were members of 8 Vaccine Safety Datalink (VSD) sites during October 2016-September 2018. We used a time-dependent covariate Cox model with stabilized inverse probability weights applied to evaluate associations between Tdap vaccination during pregnancy and chorioamnionitis and preterm birth outcomes. We used Poisson regression with robust variance with stabilized inverse probability weights applied to evaluate the association of Tdap vaccination with adverse infant outcomes. We performed medical record reviews on a random sample of patients with ICD-10-CM-diagnosed chorioamnionitis to determine positive predictive values (PPV) of coded chorioamnionitisfor "probable clinical chorioamnionitis," "possible clinical chorioamnionitis," or "histologic chorioamnionitis." RESULTS: We included 118,211 pregnant people; 103,258 (87%) received Tdap vaccine during pregnancy; 8098 (7%) were diagnosed with chorioamnionitis. The adjusted hazard ratio for chorioamnionitis in the Tdap vaccine-exposed group compared to unexposed was 0.96 (95% CI 0.90-1.03). There was no association between Tdap vaccine and preterm birth or adverse infant outcomes associated with chorioamnionitis. Chart reviews were performed for 528 pregnant people with chorioamnionitis. The PPV for clinical (probable or possible clinical chorioamnionitis) was 48% and 59% for histologic chorioamnionitis. The PPV for the combined outcome of clinical or histologic chorioamnionitis was 81%. CONCLUSIONS AND RELEVANCE: Tdap vaccine exposure during pregnancy was not associated with chorioamnionitis, preterm birth, or adverse infant outcomes. ICD-10 codes for chorioamnionitis lack specificity for clinical chorioamnionitis and should be a recognized limitation when interpreting results.

Inflammatory blockade prevents injury to the developing pulmonary gas exchange surface in preterm primates.

Perinatal inflammatory stress is associated with early life morbidity and lifelong consequences for pulmonary health. Chorioamnionitis, an inflammatory condition affecting the placenta and fluid surrounding the developing fetus, affects 25 to 40% of preterm births. Severe chorioamnionitis with preterm birth is associated with significantly increased risk of pulmonary disease and secondary infections in childhood, suggesting that fetal inflammation may markedly alter the development of the lung. Here, we used intra-amniotic lipopolysaccharide (LPS) challenge to induce experimental chorioamnionitis in a prenatal rhesus macaque (Macaca mulatta) model that mirrors structural and temporal aspects of human lung development. Inflammatory injury directly disrupted the developing gas exchange surface of the primate lung, with extensive damage to alveolar structure, particularly the close association and coordinated differentiation of alveolar type 1 pneumocytes and specialized alveolar capillary endothelium. Single-cell RNA sequencing analysis defined a multicellular alveolar signaling niche driving alveologenesis that was extensively disrupted by perinatal inflammation, leading to a loss of gas exchange surface and alveolar simplification, with notable resemblance to chronic lung disease in newborns. Blockade of the inflammatory cytokines interleukin-1ß and tumor necrosis factor-α ameliorated LPS-induced inflammatory lung injury by blunting stromal responses to inflammation and modulating innate immune activation in myeloid cells, restoring structural integrity and key signaling networks in the developing alveolus. These data provide new insight into the pathophysiology of developmental lung injury and suggest that modulating inflammation is a promising therapeutic approach to prevent fetal consequences of chorioamnionitis.

Differential Brain and Cerebrospinal Fluid Proteomic Responses to Acute Prenatal Endotoxin Exposure.

Chorioamnionitis (CA) is a risk factor for preterm birth and is associated with neurodevelopmental delay and cognitive disorders. Prenatal inflammation-induced brain injury may resolve during the immediate postnatal period when rapid brain remodeling occurs. Cerebrospinal fluid (CSF) collected at birth may be a critical source of predictive biomarkers. Using pigs as a model of preterm infants exposed to CA, we hypothesized that prenatal lipopolysaccharide (LPS) exposure induces proteome changes in the CSF and brain at birth and postnatally. Fetal piglets (103 days gestation of full-term at 117 days) were administered intra-amniotic (IA) lipopolysaccharide (LPS) 3 days before preterm delivery by caesarian section. CSF and brain tissue were collected on postnatal Days 1 and 5 (P1 and P5). CSF and hippocampal proteins were profiled by LC-MS-based quantitative proteomics. Neuroinflammatory responses in the cerebral cortex, periventricular white matter and hippocampus were evaluated by immunohistochemistry, and gene expression was evaluated by qPCR. Pigs exposed to LPS in utero showed changes in CSF protein levels at birth but not at P5. Complement protein C3, hemopexin, vasoactive intestinal peptide, carboxypeptidase N subunit 2, ITIH1, and plasminogen expression were upregulated in the CSF, while proteins associated with axon growth and synaptic functions (FGFR1, BASP1, HSPD1, UBER2N, and RCN2), adhesion (talin1), and neuronal survival (Atox1) were downregulated. Microglia, but not astrocytes, were activated by LPS at P5 in the hippocampus but not in other brain regions. At this time, marginal increases in complement protein C3, LBP, HIF1a, Basp1, Minpp1, and FGFR1 transcription indicated hippocampal proinflammatory responses. In conclusion, few days exposure to endotoxin prenatally induce proteome changes in the CSF and brain at birth, but most changes resolve a few days later. The developing hippocampus has high neuronal plasticity in response to perinatal inflammation. Changes in CSF protein expression at birth may predict later structural brain damage in preterm infants exposed to variable types and durations of CA-related inflammation in utero.

A rodent model of intra-amniotic inflammation/infection, induced by the administration of inflammatory agent in a gestational sac, associated with preterm delivery: a systematic review.

BACKGROUND: Rodents are the most commonly used animals in the study of spontaneous preterm delivery (PTD). Intra-amniotic inflammation/infection is a frequent and important cause of PTD. Intraperitoneal and intrauterine administrations of inflammatory agents are traditional methods to establish a rodent model of PTD associated with inflammation and infection. The intra-amniotic administration of inflammatory or infectious triggering agents to rodents can be useful to study not only intra-amniotic inflammatory response but also PTD associated with intra-amniotic inflammation/infection. OBJECTIVE: This systematic review aimed mainly to assess and analyze all described methods of intra-amniotic administration of infectious and/or inflammatory agents to create a rodent model of intra-amniotic inflammation associated with PTD. METHODS: A literature search through two electronic databases from their earliest entries to February 2019 was performed. The selection criteria were as follows: (1) rodents as model animals, (2) a model of intra-amniotic inflammation/infection associated with PTD, and (3) intra-amniotic administration of triggering agents. Data extraction included specification of the study (author and year of publication), characteristics of study animals (species, strain, and number of animals), characteristics of intervention (timing and used technique), substance used for induction of intra-amniotic inflammation/infection, and outcome assessment. RESULTS: The search identified a total of 4673 articles, of which 118 were selected for full-text reading, but only 13 studies were included in the review. Intra-amniotic administration was used only in the articles that were published beyond 2004. Two different approaches were identified: (1) open surgery with direct puncture of the amniotic sacs and (2) transabdominal ultrasound-guided puncture of the gestational sacs. Live microorganisms (Ureaplasma parvum), bacterial products (extracellular membrane vesicles), and pathogen-associated (lipopolysaccharide) and damage-associated molecular patterns (high mobility group box-1, S100B, and surfactant protein A) were used to simulate intra-amniotic inflammation/infection. Differences in the effect on intra-amniotic inflammation/infection associated with PTD in the mouse model were identified among triggering agents. Intra-amniotic application of lipopolysaccharide in the rat model caused intra-amniotic inflammation, but it did not lead to PTD. CONCLUSION: The intra-amniotic administration of the triggering agents can be used to study intra-amniotic inflammatory response and intra-amniotic inflammation/infection in the rodents model.

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.

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.

Betamethasone as a potential treatment for preterm birth associated with sterile intra-amniotic inflammation: a murine study.

OBJECTIVES: Preterm birth remains the leading cause of perinatal morbidity and mortality worldwide. Preterm birth is preceded by spontaneous preterm labor, which is commonly associated with sterile intra-amniotic inflammation; yet, no approved treatment exists for this clinical condition. Corticosteroids are the standard of care to improve neonatal outcomes in women at risk of preterm birth. Herein, we first validated our model of alarmin-induced preterm birth. Next, we investigated whether treatment with betamethasone could prevent preterm birth resulting from sterile intra-amniotic inflammation in mice. METHODS: Under ultrasound guidance, the first cohort of dams received an intra-amniotic injection of the alarmin high-mobility group box-1 (HMGB1, n=10) or phosphate-buffered saline (PBS, n=9) as controls. A second cohort of dams received HMGB1 intra-amniotically and were subcutaneously treated with betamethasone (n=15) or vehicle (n=15). Dams were observed until delivery, and perinatal outcomes were observed. RESULTS: Intra-amniotic HMGB1 reduced gestational length (p=0.04), inducing preterm birth in 40% (4/10) of cases, of which 100% (4/4) were categorized as late preterm births. Importantly, treatment with betamethasone extended the gestational length (p=0.02), thereby reducing the rate of preterm birth by 26.6% (from 33.3% [5/15] to 6.7% [1/15]). Treatment with betamethasone did not worsen the rate of neonatal mortality induced by HMGB1 or alter weight gain in the first three weeks of life. CONCLUSIONS: Treatment with betamethasone prevents preterm birth induced by the alarmin HMGB1. This study supports the potential utility of betamethasone for treating women with sterile intra-amniotic inflammation.

Intestinal Goblet Cell Loss during Chorioamnionitis in Fetal Lambs: Mechanistic Insights and Postnatal Implications.

Chorioamnionitis, an important cause of preterm birth, is linked to necrotizing enterocolitis (NEC). NEC is characterized by a disrupted mucus barrier, goblet cell loss, and endoplasmic reticulum (ER) stress of the intestinal epithelium. These findings prompted us to investigate the mechanisms underlying goblet cell alterations over time in an ovine chorioamnionitis model. Fetal lambs were intra-amniotically (IA) exposed to lipopolysaccharides (LPS) for 5, 12, or 24 h, or 2, 4, 8, or 15 d before premature delivery at 125 d gestational age (GA). Gut inflammation, the number, distribution, and differentiation of goblet cells, ER stress, and apoptosis were measured. We found a biphasic reduction in goblet cell numbers 24 h-2 d after, and 15 d after IA LPS exposure. The second decrease of goblet cell numbers was preceded by intestinal inflammation, apoptosis, and crypt ER stress, and increased SAM-pointed domain-containing ETS transcription factor (SPDEF)-positive cell counts. Our combined findings indicated that ER stress drives apoptosis of maturating goblet cells during chorioamnionitis, ultimately reducing goblet cell numbers. As similar changes have been described in patients suffering from NEC, these findings are considered to be clinically important for understanding the predecessors of NEC, and targeting ER stress in this context is interesting for future therapeutics.

Budesonide with surfactant decreases systemic responses in mechanically ventilated preterm lambs exposed to fetal intra-amniotic lipopolysaccharide.

BACKGROUND: Chorioamnionitis is associated with increased rates of bronchopulmonary dysplasia (BPD) in ventilated preterm infants. Budesonide when added to surfactant decreased lung and systemic inflammation from mechanical ventilation in preterm lambs and decreased the rates and severity of BPD in preterm infants. We hypothesized that the addition of budesonide to surfactant will decrease the injury from mechanical ventilation in preterm lambs exposed to intra-amniotic (IA) lipopolysaccharide (LPS). METHODS: Lambs at 126 ± 1 day GA received LPS 10 mg IA 48 h prior to injurious mechanical ventilation. After 15 min, lambs received either surfactant mixed with: (1) saline or (2) Budesonide 0.25 mg/kg, then ventilated with normal tidal volumes for 4 h. Injury markers in the lung, liver, and brain were compared. RESULTS: Compared with surfactant alone, the addition of budesonide improved blood pressures, dynamic compliance, and ventilation, while decreasing mRNA for pro-inflammatory cytokines in the lung, liver, and multiple areas of the brain. LPS caused neuronal activation and structural changes in the brain that were not altered by budesonide. Budesonide was not retained within the lung beyond 4 h. CONCLUSIONS: In preterm lambs exposed to IA LPS, the addition of budesonide to surfactant improved physiology and markers of lung and systemic inflammation. IMPACT: The addition of budesonide to surfactant decreases the lung and systemic responses to injurious mechanical ventilation preterm lambs exposed to fetal LPS. Budesonide was present in the plasma by 15 min and the majority of the budesonide is no longer in the lung at 4 h of ventilation. IA LPS and mechanical ventilation caused structural changes in the brain that were not altered by short-term exposure to budesonide. The budesonide dose of 0.25 mg/kg being used clinically seems likely to decrease lung inflammation in preterm infants with chorioamnionitis.

Evaluation of leucine-rich alpha-2 glycoprotein as a biomarker of fetal infection.

This study aimed to determine the association between umbilical cord leucine-rich alpha-2 glycoprotein (LRG) and fetal infection and investigate the underlying mechanism of LRG elevation in fetuses. We retrospectively reviewed the medical records of patients who delivered at Osaka University Hospital between 2012 and 2017 and selected those with histologically confirmed chorioamnionitis (CAM), which is a common pregnancy complication that may cause neonatal infection. The participants were divided into two groups: CAM with fetal infection (CAM-f[+] group, n = 14) and CAM without fetal infection (CAM-f[-] group, n = 31). Fetal infection was defined by the histological evidence of funisitis. We also selected 50 cases without clinical signs of CAM to serve as the control. LRG concentrations in sera obtained from the umbilical cord were unaffected by gestational age at delivery, neonatal birth weight, nor the presence of noninfectious obstetric complications (all, p > 0.05). Meanwhile, the LRG levels (median, Interquartile range [IQR]) were significantly higher in the CAM-f(+) group (10.37 [5.21-13.7] µg/ml) than in the CAM-f(-) (3.61 [2.71-4.65] µg/ml) or control group (3.39 [2.81-3.93] µg/ml; p < 0.01). The area under the receiver operating characteristic (ROC) curve of LRG for recognizing fetal infection was 0.92 (optimal cutoff, 5.08 µg/ml; sensitivity, 86%; specificity, 88%). In a mouse CAM model established by lipopolysaccharide administration, the fetal LRG protein in sera and LRG mRNA in the liver were significantly higher than those in phosphate-buffered saline (PBS)-administered control mice (p < 0.01). In vitro experiments using a fetal liver-derived cell line (WRL68) showed that the expression of LRG mRNA was significantly increased after interleukin (IL)-6, IL-1ß, and tumor necrosis factor- alpha (TNF-α) stimulation (p < 0.01); the induction was considerably stronger following IL-6 and TNF-α stimulation (p < 0.01). In conclusion, LRG is an effective biomarker of fetal infection, and fetal hepatocytes stimulated with inflammatory cytokines may be the primary source of LRG production in utero.

Immunohistochemical analysis of protective effects of maternal fingolimod on the placenta and fetal lung and brain in chorioamnionitis-induced preterm birth rat model.

OBJECTIVES: Fingolimod (FIN) is used for multiple sclerosis treatment and has potential antiapoptotic and anti-inflammatory effects. We aimed at expanding our knowledge on various immunohistochemical markers for elucidating the possible mechanisms of action of fingolimod in the placenta and fetal lung and brain. METHODS: Sixteen pregnant rats were divided into four groups. On gestational day 17, lipopolysaccharide (LPS) was injected intraperitoneally to induce preterm fetal injury followed by intraperitoneal injection of fingolimod. Hysterotomy for preterm delivery was performed 6 h after fingolimod was injected. The study groups included (1) control, (2) LPS (1 mg/kg), (3) FIN (4 mg/kg), and (4) FIN + LPS. Fetal brain and lung and placenta samples were collected for histopathological examination. Moreover, fetal lungs (surfactant protein-A (SP-A), SP-B, SP-D, caspase-3, and caspase-8), fetal brains (interleukin-10, interleukin-1ß, TNF-α, caspase-8, glial fibrillary acidic protein, vimentin, myelin basic protein, and receptor activator of nuclear factor kappa), and placenta tissues (interleukin-10, interleukin-1ß, TNF-α, caspase-3, and caspase-8) were immunohistochemically evaluated. RESULTS: Maternal fingolimod treatment led to attenuation of LPS-induced fetal brain, lung, and placental injury, as indicated by lower immunoexpression of inflammatory markers compared to LPS group (p < .0001 for all comparisons). CONCLUSION: The findings of the present study confirm the neuroprotective effects of antenatally administered fingolimod, which also significantly improved preterm fetal lung injury and placental inflammation in LPS-exposed preterm pregnancies by possible antiapoptotic and anti-inflammatory effects.

Immune Cells in the Placental Villi Contribute to Intra-amniotic Inflammation.

Intra-amniotic (IA) inflammation is associated with significant morbidities for both the mother and the fetus. Prior studies have illustrated many of the effects of IA inflammation on the uterine lining (decidua) and membranous layers of the placenta at the fetal-maternal interface. However, much less is known about the immunological response occurring within the villous placenta. Using a rhesus macaque model of lipopolysaccharide (LPS)-induced IA inflammation, we showed that pregnancy-matched choriodecidua and villi have distinct immunological profiles in rhesus pregnancies. In the choriodecidua, we show that the abundance of neutrophils, multiple populations of antigen-presenting cells, and two populations of natural killer (NK) cells changes with prenatal IA LPS exposure. In contrast, in immune cells within the villous placenta we observed alterations in the abundance of B cells, monocytes, and CD8 T cells. Prior work has illustrated that IA inflammation leads to an increase in tumor necrosis factor alpha (TNFα) at the fetal-maternal interface. In this study, pretreatment with a TNFα blockade partially reversed inflammation in the placental villi. Furthermore, we report that immune cells in the villous placenta sensed LPS during our experimental window, and subsequently activated T cells to produce proinflammatory cytokines. Moreover, this study is the first report of memory T cells in third-trimester non-human primate placental villi and provides evidence that manipulation of immune cells in the villi at the fetal-maternal interface should be considered as a potential therapeutic target for IA inflammation.

Chronic Intra-Uterine Ureaplasma parvum Infection Induces Injury of the Enteric Nervous System in Ovine Fetuses.

Background: Chorioamnionitis, inflammation of the fetal membranes during pregnancy, is often caused by intra-amniotic (IA) infection with single or multiple microbes. Chorioamnionitis can be either acute or chronic and is associated with adverse postnatal outcomes of the intestine, including necrotizing enterocolitis (NEC). Neonates with NEC have structural and functional damage to the intestinal mucosa and the enteric nervous system (ENS), with loss of enteric neurons and glial cells. Yet, the impact of acute, chronic, or repetitive antenatal inflammatory stimuli on the development of the intestinal mucosa and ENS has not been studied. The aim of this study was therefore to investigate the effect of acute, chronic, and repetitive microbial exposure on the intestinal mucosa, submucosa and ENS in premature lambs. Materials and Methods: A sheep model of pregnancy was used in which the ileal mucosa, submucosa, and ENS were assessed following IA exposure to lipopolysaccharide (LPS) for 2 or 7 days (acute), Ureaplasma parvum (UP) for 42 days (chronic), or repetitive microbial exposure (42 days UP with 2 or 7 days LPS). Results: IA LPS exposure for 7 days or IA UP exposure for 42 days caused intestinal injury and inflammation in the mucosal and submucosal layers of the gut. Repetitive microbial exposure did not further aggravate injury of the terminal ileum. Chronic IA UP exposure caused significant structural ENS alterations characterized by loss of PGP9.5 and S100ß immunoreactivity, whereas these changes were not found after re-exposure of chronic UP-exposed fetuses to LPS for 2 or 7 days. Conclusion: The in utero loss of PGP9.5 and S100ß immunoreactivity following chronic UP exposure corresponds with intestinal changes in neonates with NEC and may therefore form a novel mechanistic explanation for the association of chorioamnionitis and NEC.

TNF-Signaling Modulates Neutrophil-Mediated Immunity at the Feto-Maternal Interface During LPS-Induced Intrauterine Inflammation.

Accumulation of activated neutrophils at the feto-maternal interface is a defining hallmark of intrauterine inflammation (IUI) that might trigger an excessive immune response during pregnancy. Mechanisms responsible of this massive neutrophil recruitment are poorly investigated. We have previously showed that intraamniotic injection of LPS in rhesus macaques induced a neutrophil predominant inflammatory response similar to that seen in human IUI. Here, we demonstrate that anti-TNF antibody (Adalimumab) inhibited ~80% of genes induced by LPS involved in inflammatory signaling and innate immunity in chorio-decidua neutrophils. Consistent with the gene expression data, TNF-blockade decreased LPS-induced neutrophil accumulation and activation at the feto-maternal interface. We also observed a reduction in IL-6 and other pro-inflammatory cytokines but not prostaglandins concentrations in the amniotic fluid. Moreover, TNF-blockade decreased mRNA expression of inflammatory cytokines in the chorio-decidua but not in the uterus, suggesting that inhibition of TNF-signaling decreased the inflammation in a tissue-specific manner within the uterine compartment. Taken together, our results demonstrate a predominant role for TNF-signaling in modulating the neutrophilic infiltration at the feto-maternal interface during IUI and suggest that blockade of TNF-signaling could be considered as a therapeutic approach for IUI, the major leading cause of preterm birth.

Prenatal inflammation suppresses blood Th1 polarization and gene clusters related to cellular energy metabolism in preterm newborns.

Chorioamnionitis (CA, fetal membrane inflammation) predisposes to preterm birth and is associated with increased neonatal infection risk, but the separate effects of prematurity, CA, and postnatal adaptations on this risk are unclear. Using pigs as models for infants, we examined the systemic immune-metabolic status in cesarean-delivered preterm pigs, with and without CA induced by intra-amniotic (IA) LPS exposure. At birth, cord blood of preterm pigs showed neutropenia and low expressions of innate and adaptive immune genes, relative to term pigs. IA LPS induced CA and fetal systemic innate immune activation via complement and neutrophil-related pathways. These were mainly modulated via cellular regulations rather than granulopoiesis, as validated by the in vitro LPS stimulation of cord blood. After birth, IA LPS-exposed preterm pigs did not follow normal immune-metabolic ontogenies found in fetuses or newborns without prenatal insults, but showed consistently high levels of Treg, impaired Th1 polarization, and reduced expressions of multiple genes related to cellular oxidative phosphorylation and ribosomal activities. In conclusion, our results provide cellular and molecular evidence for CA-induced distinct neonatal immune-metabolic status with increased disease tolerance strategy, suggesting mechanisms for the clinical observation of elevated sepsis risks in immune-compromised preterm infants born with CA.

Gut and immune effects of bioactive milk factors in preterm pigs exposed to prenatal inflammation.

Prenatal inflammation may predispose to preterm birth and postnatal inflammatory disorders such as necrotizing enterocolitis (NEC). Bioactive milk ingredients may help to support gut maturation in such neonates, but mother's milk is often insufficient after preterm birth. We hypothesized that supplementation with bioactive ingredients from bovine milk [osteopontin (OPN), caseinoglycomacropeptide (CGMP), colostrum (COL)] supports gut, immunity, and NEC resistance in neonates born preterm after gram-negative infection before birth. Using preterm pigs as a model for preterm infants, fetal pigs were given intraamniotic injections of lipopolysaccharide (LPS; 1 mg/fetus) and delivered 3 days later (90% gestation). For 5 days, groups of LPS-exposed pigs were fed formula (FOR), bovine colostrum (COL), or formula enriched with OPN or CGMP. LPS induced intraamniotic inflammation and postnatal systemic inflammation but limited effects on postnatal gut parameters and NEC. Relative to FOR, COL feeding to LPS-exposed pigs showed less diarrhea, NEC severity, reduced gut IL-1ß and IL-8 levels, greater gut goblet cell density and digestive enzyme activities, and blood helper T-cell fraction. CGMP improved neonatal arousal and gut lactase activities and reduced LPS-induced IL-8 secretion in intestinal epithelial cells (IECs) in vitro. Finally, OPN tended to reduce diarrhea and stimulated IEC proliferation in vitro. No effects on villus morphology, circulating cytokines, or colonic microbiota were observed among groups. In conclusion, bioactive milk ingredients exerted only modest effects on gut and systemic immune parameters in preterm pigs exposed to prenatal inflammation. Short-term, prenatal exposure to inflammation may render the gut less sensitive to immune-modulatory milk effects. NEW & NOTEWORTHY Prenatal inflammation is a risk factor for preterm birth and postnatal complications including infections. However, from clinical studies, it is difficult to separate the effects of only prenatal inflammation from preterm birth. Using cesarean-delivered preterm pigs with prenatal inflammation, we documented some beneficial gut effects of bioactive milk diets relative to formula, but prenatal inflammation appeared to decrease the sensitivity of enteral feeding. Special treatments and diets may be required for this neonatal population.

Prenatal Intra-Amniotic Endotoxin Induces Fetal Gut and Lung Immune Responses and Postnatal Systemic Inflammation in Preterm Pigs.

Prenatal inflammation is a major risk for preterm birth and neonatal morbidity, but its effects on postnatal immunity and organ functions remain unclear. Using preterm pigs as a model for preterm infants, we investigated whether prenatal intra-amniotic (IA) inflammation modulates postnatal systemic immune status and organ functions. Preterm pigs exposed to IA lipopolysaccharide (LPS) for 3 days were compared with controls at birth and postnatal day 5 after formula feeding. IA LPS induced mild chorioamnionitis but extensive intra-amniotic inflammation. There were minor systemic effects at birth (increased blood neutrophil counts), but a few days later, prenatal LPS induced delayed neonatal arousal, systemic inflammation (increased blood leukocytes, plasma cytokines, and splenic bacterial counts), altered serum biochemistry (lower albumin and cholesterol and higher iron and glucose values), and increased urinary protein and sodium excretion. In the gut and lungs, IA LPS-induced inflammatory responses were observed mainly at birth (increased LPS, CXCL8, and IL-1ß levels and myeloperoxidase-positive cell density, multiple increases in innate immune gene expressions, and reduced villus heights), but not on postnatal day 5 (except elevated lung CXCL8 and diarrhea symptoms). Finally, IA LPS did not affect postnatal gut brush-border enzymes, hexose absorption, permeability, or sensitivity to necrotizing enterocolitis on day 5. Short-term IA LPS exposure predisposes preterm pigs to postnatal systemic inflammation after acute fetal gut and lung inflammatory responses.