Elke Kuypers

New Surfactant with SP-B and C Analogs Gives Survival Benefit after Inactivation in Preterm Lambs

Background Respiratory distress syndrome in preterm babies is caused by a pulmonary surfactant deficiency, but also by its inactivation due to various conditions, including plasma protein leakage. Surfactant replacement therapy is well established, but clinical observations and in vitro experiments suggested that its efficacy may be impaired by inactivation. A new synthetic surfactant (CHF 5633), containing synthetic surfactant protein B and C analogs, has shown comparable effects on oxygenation in ventilated preterm rabbits versus Poractant alfa, but superior resistance against inactivation in vitro. We hypothesized that CHF 5633 is also resistant to inactivation by serum albumin in vivo. Methodology/Principal Findings Nineteen preterm lambs of 127 days gestational age (term = 150 days) received CHF 5633 or Poractant alfa and were ventilated for 48 hours. Ninety minutes after birth, the animals received albumin with CHF 5633 or Poractant alfa. Animals received additional surfactant if PaO2 dropped below 100 mmHg. A pressure volume curve was done post mortem and markers of pulmonary inflammation, surfactant content and biophysiology, and lung histology were assessed. CHF 5633 treatment resulted in improved arterial pH, oxygenation and ventilation efficiency index. The survival rate was significantly higher after CHF 5633 treatment (5/7) than after Poractant alfa (1/8) after 48 hours of ventilation. Biophysical examination of the surfactant recovered from bronchoalveolar lavages revealed that films formed by CHF 5633-treated animals reached low surface tensions in a wider range of compression rates than films from Poractant alfa-treated animals. Conclusions For the first time a synthetic surfactant containing both surfactant protein B and C analogs showed significant benefit over animal derived surfactant in an in vivo model of surfactant inactivation in premature lambs.

Intra-amniotic LPS and antenatal betamethasone: inflammation and maturation in preterm lamb lungs

The proinflammatory stimulus of chorioamnionitis is commonly associated with preterm delivery. Women at risk of preterm delivery receive antenatal glucocorticoids to functionally mature the fetal lung. However, the effects of the combined exposures of chorioamnionitis and antenatal glucocorticoids on the fetus are poorly understood. Time-mated ewes with singleton fetuses received an intra-amniotic injection of lipopolysaccharide (LPS) either preceding or following maternal intramuscular betamethasone 7 or 14 days before delivery, and the fetuses were delivered at 120 days gestational age (GA) (term = 150 days GA). Gestation matched controls received intra-amniotic and maternal intramuscular saline. Compared with saline controls, intra-amniotic LPS increased inflammatory cells in the bronchoalveolar lavage and myeloperoxidase, Toll-like receptor 2 and 4 mRNA, PU.1, CD3, and Foxp3-positive cells in the fetal lung. LPS-induced lung maturation measured as increased airway surfactant and improved lung gas volumes. Intra-amniotic LPS-induced inflammation persisted until 14 days after exposure. Betamethasone treatment alone induced modest lung maturation but, when administered before intra-amniotic LPS, suppressed lung inflammation. Interestingly, betamethasone treatment after LPS did not counteract inflammation but enhanced lung maturation. We conclude that the order of exposures of intra-amniotic LPS or maternal betamethasone had large effects on fetal lung inflammation and maturation.

Mesenchymal stem cells induce T-cell tolerance and protect the preterm brain after global hypoxia-ischemia.

Hypoxic-ischemic encephalopathy (HIE) in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC) in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI) was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 106 MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI), in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE.

Cerebral inflammation and mobilization of the peripheral immune system following global hypoxia-ischemia in preterm sheep

BackgroundHypoxic-ischemic encephalopathy (HIE) is one of the most important causes of brain injury in preterm infants. Preterm HIE is predominantly caused by global hypoxia-ischemia (HI). In contrast, focal ischemia is most common in the adult brain and known to result in cerebral inflammation and activation of the peripheral immune system. These inflammatory responses are considered to play an important role in the adverse outcomes following brain ischemia. In this study, we hypothesize that cerebral and peripheral immune activation is also involved in preterm brain injury after global HI.MethodsPreterm instrumented fetal sheep were exposed to 25 minutes of umbilical cord occlusion (UCO) (n = 8) at 0.7 gestation. Sham-treated animals (n = 8) were used as a control group. Brain sections were stained for ionized calcium binding adaptor molecule 1 (IBA-1) to investigate microglial proliferation and activation. The peripheral immune system was studied by assessment of circulating white blood cell counts, cellular changes of the spleen and influx of peripheral immune cells (MPO-positive neutrophils) into the brain. Pre-oligodendrocytes (preOLs) and myelin basic protein (MBP) were detected to determine white matter injury. Electro-encephalography (EEG) was recorded to assess functional impairment by interburst interval (IBI) length analysis.ResultsGlobal HI resulted in profound activation and proliferation of microglia in the hippocampus, periventricular and subcortical white matter. In addition, non-preferential mobilization of white blood cells into the circulation was observed within 1 day after global HI and a significant influx of neutrophils into the brain was detected 7 days after the global HI insult. Furthermore, global HI resulted in marked involution of the spleen, which could not be explained by increased splenic apoptosis. In concordance with cerebral inflammation, global HI induced severe brain atrophy, region-specific preOL vulnerability, hypomyelination and persistent suppressed brain function.ConclusionsOur data provided evidence that global HI in preterm ovine fetuses resulted in profound cerebral inflammation and mobilization of the peripheral innate immune system. These inflammatory responses were paralleled by marked injury and functional loss of the preterm brain. Further understanding of the interplay between preterm brain inflammation and activation of the peripheral immune system following global HI will contribute to the development of future therapeutic interventions in preterm HIE.

Thrown off balance: the effect of antenatal inflammation on the developing lung and immune system

In recent years, translational research with various animal models has been helpful to answer basic questions about the effect of antenatal inflammation on maturation and development of the fetal lung and immune system. The fetal lung and immune systems are very plastic and their development can be conditioned and influenced by both endogenous and/or exogenous factors. Antenatal inflammation can induce pulmonary inflammation, leading to lung injury and remodeling in the fetal lung. Exposure to antenatal inflammation can induce interleukin-1α production, which enhances surfactant protein and lipid synthesis thereby promoting lung maturation. Interleukin-1α is therefore a candidate for the link between lung inflammation and lung maturation, preventing respiratory distress syndrome in preterm infants. Antenatal inflammation can, however, cause structural changes in the fetal lung and affect the expression of growth factors, such as transforming growth factor-beta, connective tissue growth factor, fibroblast growth factor-10, or bone morphogenetic protein-4, which are essential for branching morphogenesis. These alterations cause alveolar and microvascular simplification resembling the histology of bronchopulmonary dysplasia. Antenatal inflammation may also affect neonatal outcome by modulating the responsiveness of the immune system. Lipopolysaccharide-tolerance (endotoxin hyporesponsiveness/immunoparalysis), induced by exposure to inflammation in utero, may prevent fetal lung damage, but increases susceptibility to postnatal infections. Moreover, prenatal exposure to inflammation appears to be a predisposition for the development of adverse neonatal outcomes, like bronchopulmonary dysplasia, if the preterm infant is exposed to a second postnatal hit, such as mechanical ventilation oxygen exposure, infections, or steroids.

White matter injury following fetal inflammatory response syndrome induced by chorioamnionitis and fetal sepsis: Lessons from experimental ovine models☆

Chorioamnionitis and fetal sepsis can induce a fetal inflammatory response syndrome (FIRS) which is closely related to the development of white matter injury in the fetal brain. Large epidemiological studies support the link between FIRS and fetal brain injury with a clear association between the presence of in utero inflammation and neurodevelopmental complications such as cerebral palsy, autism and cognitive impairments later in life. Translational animal models of chorioamnionitis and fetal sepsis are essential in understanding the underlying pathophysiological mechanisms of fetal brain injury after exposure to intra-uterine inflammation. Concerning this aspect, ovine models have high translational value since neurodevelopment in sheep closely resembles the human situation. In this article, we will review clinical and experimental evidence for the link between FIRS and white matter injury in the fetal brain. With respect to experimental findings, we will particularly focus on the lessons learned from ovine models of chorioamnionitis and fetal sepsis. We also highlight two key players implied in the pathophysiology of white matter injury after in utero exposure to inflammation.

LPS-induced chorioamnionitis and antenatal corticosteroids modulate Shh signaling in the ovine fetal lung

Chorioamnionitis and antenatal corticosteroids mature the fetal lung functionally but disrupt late-gestation lung development. Because Sonic Hedgehog (Shh) signaling is a major pathway directing lung development, we hypothesized that chorioamnionitis and antenatal corticosteroids modulated Shh signaling, resulting in an altered fetal lung structure. Time-mated ewes with singleton ovine fetuses received an intra-amniotic injection of lipopolysaccharide (LPS) and/or maternal intramuscular betamethasone 7 and/or 14 days before delivery at 120 days gestational age (GA) (term = 150 days GA). Intra-amniotic LPS exposure decreased Shh mRNA levels and Gli1 protein expression, which was counteracted by both betamethasone pre- or posttreatment. mRNA and protein levels of fibroblast growth factor 10 and bone morphogenetic protein 4, which are important mediators of lung development, increased 2-fold and 3.5-fold, respectively, 14 days after LPS exposure. Both 7-day and 14-day exposure to LPS changed the mRNA levels of elastin (ELN) and collagen type I alpha 1 (Col1A1) and 2 (Col1A2), which resulted in fewer elastin foci and increased collagen type I deposition in the alveolar septa. Corticosteroid posttreatment prevented the decrease in ELN mRNA and increased elastin foci and decreased collagen type I deposition in the fetal lung. In conclusion, fetal lung exposure to LPS was accompanied by changes in key modulators of lung development resulting in abnormal lung structure. Betamethasone treatment partially prevented the changes in developmental processes and lung structure. This study provides new insights into clinically relevant prenatal exposures and fetal lung development.

Effects of Intra-Amniotic Lipopolysaccharide and Maternal Betamethasone on Brain Inflammation in Fetal Sheep

Rationale Chorioamnionitis and antenatal glucocorticoids are common exposures for preterm infants and can affect the fetal brain, contributing to cognitive and motor deficits in preterm infants. The effects of antenatal glucocorticoids on the brain in the setting of chorioamnionitis are unknown. We hypothesized that antenatal glucocorticoids would modulate inflammation in the brain and prevent hippocampal and white matter injury after intra-amniotic lipopolysaccharide (LPS) exposure. Methods Time-mated ewes received saline (control), an intra-amniotic injection of 10 mg LPS at 106d GA or 113d GA, maternal intra-muscular betamethasone (0.5 mg/kg maternal weight) alone at 113d GA, betamethasone at 106d GA before LPS or betamethasone at 113d GA after LPS. Animals were delivered at 120d GA (term=150d). Brain structure volumes were measured on T2-weighted MRI images. The subcortical white matter (SCWM), periventricular white matter (PVWM) and hippocampus were analyzed for microglia, astrocytes, apoptosis, proliferation, myelin and pre-synaptic vesicles. Results LPS and/or betamethasone exposure at different time-points during gestation did not alter brain structure volumes on MRI. Betamethasone alone did not alter any of the measurements. Intra-amniotic LPS at 106d or 113d GA induced inflammation as indicated by increased microglial and astrocyte recruitment which was paralleled by increased apoptosis and hypomyelination in the SCWM and decreased synaptophysin density in the hippocampus. Betamethasone before the LPS exposure at 113d GA prevented microglial activation and the decrease in synaptophysin. Betamethasone after LPS exposure increased microglial infiltration and apoptosis. Conclusion Intra-uterine LPS exposure for 7d or 14d before delivery induced inflammation and injury in the fetal white matter and hippocampus. Antenatal glucocorticoids aggravated the inflammatory changes in the brain caused by pre-existing intra-amniotic inflammation. Antenatal glucocorticoids prior to LPS reduced the effects of intra-uterine inflammation on the brain. The timing of glucocorticoid administration in the setting of chorioamnionitis can alter outcomes for the fetal brain.

Antenatal glucocorticoids counteract LPS changes in TGF-β pathway and caveolin-1 in ovine fetal lung

Inflammation and antenatal glucocorticoids, the latter given to mothers at risk for preterm birth, affect lung development and may contribute to the development of bronchopulmonary dysplasia (BPD). The effects of the combined exposures on inflammation and antenatal glucocorticoids on transforming growth factor (TGF)-β signaling are unknown. TGF-β and its downstream mediators are implicated in the etiology of BPD. Therefore, we asked whether glucocorticoids altered intra-amniotic lipopolysaccharide (LPS) effects on TGF-β expression, its signaling molecule phosphorylated sma and mothers against decapentaplegic homolog 2 (pSmad2), and the downstream mediators connective tissue growth factor (CTGF) and caveolin-1 (Cav-1). Ovine singleton fetuses were randomized to receive either an intra-amniotic injection of LPS and/or maternal betamethasone (BTM) intramuscularly 7 and/or 14 days before delivery at 120 days gestational age (GA; term = 150 days GA). Saline was used for controls. Protein levels of TGF-β1 and -β2 were measured by ELISA. Smad2 phosphorylation was assessed by immunohistochemistry and Western blot. CTGF and Cav-1 mRNA and protein levels were determined by RT-PCR and Western blot. Free TGF-β1 and -β2 and total TGF-β1 levels were unchanged after LPS and/or BTM exposure, although total TGF-β2 increased in animals exposed to BTM 7 days before LPS. pSmad2 immunostaining increased 7 days after LPS exposure although pSmad2 protein expression did not increase. Similarly, CTGF mRNA and protein levels increased 7 days after LPS exposure as Cav-1 mRNA and protein levels decreased. BTM exposure before LPS prevented CTGF induction and Cav-1 downregulation. This study demonstrated that the intrauterine inflammation-induced TGF-β signaling can be inhibited by antenatal glucocorticoids in fetal lungs.

Ovine Fetal Thymus Response to Lipopolysaccharide-Induced Chorioamnionitis and Antenatal Corticosteroids

Rationale Chorioamnionitis is associated with preterm delivery and involution of the fetal thymus. Women at risk of preterm delivery receive antenatal corticosteroids which accelerate fetal lung maturation and improve neonatal outcome. However, the effects of antenatal corticosteroids on the fetal thymus in the settings of chorioamnionitis are largely unknown. We hypothesized that intra-amniotic exposure to lipopolysaccharide (LPS) causes involution of the fetal thymus resulting in persistent effects on thymic structure and cell populations. We also hypothesized that antenatal corticosteroids may modulate the effects of LPS on thymic development. Methods Time-mated ewes with singleton fetuses received an intra-amniotic injection of LPS 7 or 14 days before preterm delivery at 120 days gestational age (term = 150 days). LPS and corticosteroid treatment groups received intra-amniotic LPS either preceding or following maternal intra-muscular betamethasone. Gestation matched controls received intra-amniotic and maternal intra-muscular saline. The fetal intra-thoracic thymus was evaluated. Results Intra-amniotic LPS decreased the cortico-medullary (C/M) ratio of the thymus and increased Toll-like receptor (TLR) 4 mRNA and CD3 expression indicating involution and activation of the fetal thymus. Increased TLR4 and CD3 expression persisted for 14 days but Foxp3 expression decreased suggesting a change in regulatory T-cells. Sonic hedgehog and bone morphogenetic protein 4 mRNA, which are negative regulators of T-cell development, decreased in response to intra-amniotic LPS. Betamethasone treatment before LPS exposure attenuated some of the LPS-induced thymic responses but increased cleaved caspase-3 expression and decreased the C/M ratio. Betamethasone treatment after LPS exposure did not prevent the LPS-induced thymic changes. Conclusion Intra-amniotic exposure to LPS activated the fetal thymus which was accompanied by structural changes. Treatment with antenatal corticosteroids before LPS partially attenuated the LPS-induced effects but increased apoptosis in the fetal thymus. Corticosteroid administration after the inflammatory stimulus did not inhibit the LPS effects on the fetal thymus.