Ilias Nitsos

Pulmonary and systemic endotoxin tolerance in preterm fetal sheep exposed to chorioamnionitis.

In a model of human chorioamnionitis, fetal sheep exposed to a single injection, but not repeated injections, of intra-amniotic endotoxin develop lung injury responses. We hypothesized that repeated exposure to intra-amniotic endotoxin induces endotoxin tolerance. Fetal sheep were given intra-amniotic injections of saline (control) or Escherichia coli LPS O55:B5 (10 mg) either 2 days (2-day group, single exposure), 7 days (7-day group, single exposure), or 2 plus 7 days (2- and 7-day repeat exposure) before preterm delivery at 124 days gestation (term = 150 days). Endotoxin responses were assessed in vivo in the lung and liver, and in vitro in monocytes from the blood and the lung. Compared with the single 2-day LPS exposure group, the (2 plus 7 days) repeat LPS-exposed lambs had: 1) decreased lung neutrophil and monocyte inducible NO synthase (NOSII) expression, and 2) decreased lung cytokine and liver serum amyloid A3 mRNA expression. In the lung, serum amyloid A3 mRNA expression decreased in the airway epithelial cells but not in the lung inflammatory cells. Unlike the single 7-day LPS exposure group, peripheral blood and lung monocytes from the repeat-LPS group did not increase IL-6 secretion or hydrogen peroxide production in response to in vitro LPS. Compared with controls, TLR4 expression did not change but IL-1R-associated kinase M expression increased in the monocytes from repeat LPS-exposed lambs. These results are consistent with the novel finding of endotoxin tolerance in preterm fetal lungs exposed to intra-amniotic LPS. The findings have implications for preterm infants exposed to chorioamnionitis for both responses to lung injury and postnatal nosocomial infections.

IL-1 Mediates Pulmonary and Systemic Inflammatory Responses to Chorioamnionitis Induced by Lipopolysaccharide

RATIONALE Chorioamnionitis frequently associates with preterm delivery and increased amniotic fluid IL-1, and causes fetal lung and systemic inflammation. However, chorioamnionitis is also associated with a paradoxical reduction in the incidence of surfactant deficiency-related respiratory distress syndrome in preterm infants. OBJECTIVES To identify the role of IL-1 signaling in the mediation of pulmonary and systemic inflammation and lung maturation in a fetal sheep model of lipopolysaccharide (LPS) induced chorioamnionitis. METHODS After confirming the efficacy of recombinant human IL-1 receptor antagonist (rhIL-1ra), fetal sheep were exposed to intraamniotic (IA) injections of Escherichia coli LPS with or without prior IA injections of rhIL-1ra. Preterm lambs were delivered at 82% of term gestation. MEASUREMENTS AND MAIN RESULTS rhIL-1ra decreased IA LPS-induced lung inflammation assessed by decreased lung neutrophil and monocyte influx, inducible nitric oxide synthase expression, lung IL-6 and IL-1beta mRNA expression, and airway myeloperoxidase concentrations. rhIL-1ra inhibited IA LPS-induced fetal systemic inflammation assessed by decreased plasma IL-8, protein carbonyls, blood neutrophilia, and the expression of serum amyloid A3 mRNA in the liver. rhIL-1ra also partially blocked the lung maturational effects of IA LPS. Therefore blockade of IL-1 signaling in the amniotic compartment inhibited fetal lung and systemic inflammation and lung maturation in response to LPS-induced chorioamnionitis. CONCLUSIONS IL-1 plays a central role in the pathogenesis of chorioamnionitis-induced fetal inflammatory responses.

Chronic Exposure to Intra-Amniotic Lipopolysaccharide Affects the Ovine Fetal Brain

Objective: Fetal brain injury is associated with chorioamnionitis, which is often present without signs of overt infection of fetal compromise. We aimed to determine if prolonged exposure to intrauterine inflammation caused by intra-amniotic infusion of lipopolysaccharide (LPS) would affect the fetal brain. Methods: At 80 days of pregnancy ewes bearing singletons had osmotic pumps implanted intraamniotically to infuse Escherichia coli LPS (055:B5; n = 8) or saline (n = 7) for 28 days. At delivery (110 days), umbilical arterial blood and chorioamnion were assessed for inflammation; cytokine concentrations (interleukin [IL]-6 and IL-8) in amniotic fluid and fetal and maternal plasma were measured. The fetal cerebral hemispheres were examined for gross anatomical changes and the number of activated microglia/macrophages, astrocytes, and oligodendrocytes estimated after immunohistochemical staining. Results: Intra-amniotic administration of LPS caused chorioamnionitis, fetal leucocytosis, and a moderate to extensive infiltration of activated microglia/macrophages in the subcortical white matter in six of eight fetuses; the remaining two fetuses were less affected. Within these focal regions of damage there was an attenuation of astrocytic processes, axonal injury, and a reduction in the number of 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) immunoreactive oligodendrocytes in areas of extensive focal damage. In control fetuses there was mild (#/7) or no infiltration of activated microglia/macrophages in the subcortical white matter. Overall the infiltration of activated microglia/macrophages in the white matter was significantly greater in LPS-exposed fetuses compared to controls. In regions devoid of injury, the number of oligodendrocytes and astrocytes was not different between groups, nor was there a difference in the volume of cerebral white matter or density of blood vessels within the white matter. Amniotic fluid IL-6 and IL-6, and maternal plasma IL-8 concentrations were significantly increased by LPS infusion. Conclusions: An increase in inflammatory cells and axonal disruption in the subcortical white matter of the fetal brain can accompany chorioamnionitis induced by intra-amniotic administration of LPS, but cystic lesions do not occur. Thus, the effect on the fetal brain is milder than that reported from animal models of acute fetal/intrauterine infection.

The effects of intrauterine growth retardation on the development of neuroglia in fetal guinea pigs. An immunohistochemical and an ultrastructural study.

The effects of intrauterine growth retardation on the development of myelinating oligodendrocytes and astrocytes in the brain and spinal cord of the fetal guinea pig have been examined using immunohistochemical and ultrastructural techniques. As judged by immunoreactivity for myelin basic protein, the extent of myelination in the spinal cord, cerebral cortex, corpus cellosum and cerebellum was reduced in the growth‐retarded fetuses compared with controls at both 52 (n = 4) and 62 days (n = 5) of gestation. As assessed by immunoreactivity for glial fibrillary acidic protein, there were no marked differences between control and growth‐retarded brains in the extent or distribution of radial glial cells or astrocytes at 52 or 62 days in the cerebellum. However, in the cerebral cortex at 62 days there was a striking proliferation of astrocytes surrounding cortical blood vessels in growth‐retarded fetuses. Ultrastructural studies showed that at 52 days, myelination of the corticospinal tract had begun in the control but was virtually absent in growth‐retarded fetuses. At 62 days, the total number of myelinated fibres in growth‐retarded fetuses was significantly reduced by 56% (P < 0.01) compared with control fetuses; however, there was no difference between the groups in the total number of fibres in the corticospinal tract. Where fibres were myelinated the myelin sheath was disproportionately reduced relative to axon diameter. Thus, in intrauterine growth retardation there is a delay in the initiation and in the extent of myelination. This could be due to a reduction in the number of myelinating glia formed and the restricted capacity of those which do form to generate myelin.

Positive End-Expiratory Pressure and Tidal Volume During Initial Ventilation of Preterm Lambs

Positive end-expiratory pressure (PEEP) protects the lung from injury during sustained ventilation, but its role in protecting the lung from injury during the initiation of ventilation in the delivery room is not established. We aimed to evaluate whether PEEP and/or tidal volume (VT) within the first 15-min of ventilation are protective against lung injury. Operatively delivered preterm lambs (133 ± 1 d gestation) were randomly assigned to unventilated controls or to one of four 15 min ventilation interventions: 1) VT15 mL/kg, PEEP 0 cm H2O; 2) VT15 mL/kg, PEEP 5 cm H2O; 3) VT8 mL/kg, PEEP 0 cm H2O; and 4) VT8 mL/kg, PEEP 5 cm H2O. Each group was subsequently ventilated with VT <10 mL/kg, PEEP 5 cm H2O for 1 h 45 min. Lung function was assessed and measurements of lung injury were evaluated postmortem. After the 15 min ventilation maneuver, the VT15 groups were hypocarbic, had higher oxygenation, and required lower pressures than the VT8 groups; no consistent effect of PEEP was found. Markers of lung injury were significantly elevated in all ventilation groups compared with unventilated controls; no effect of PEEP was found. Ventilation resulted in localization of IL-6 to the small airways. Initial ventilation of preterm lambs with PEEP and/or VT of 8 mL/kg did not prevent an inflammatory injury to the lung.

Preterm birth, infection, and inflammation advances from the study of animal models.

Inflammation is a protective response mediated by both innate and adaptive arms of the immune system following exposure to a range of harmful stimuli. Although inflammation is an essential mechanism in response to challenges including tissue injury and microbiological insult, inappropriate or excessive induction of the inflammatory response is itself a well-characterized cause of morbidity and mortality in adult populations. There is currently a growing appreciation of the potential for inflammation to play an adverse role in fetal health. The expression of cytokines (notably interleukin 1β [IL-1β], IL-6, IL-8, and tumor necrosis factor α [TNF-α]) by either the fetal or maternal tissues has been demonstrated to upregulate the activity of a number of uterine and cervical factors (eg, prostaglandin hormones and their receptors, matrix metalloproteinases, vascular endothelial growth factor [VEGF]), leading to premature initiation of the parturition process. Herein, we review important developments in our understanding of the link between preterm birth and fetal inflammation subsequent to infection, gained from studies undertaken in animal models.

Intra-amniotic LPS modulation of TLR signaling in lung and blood monocytes of fetal sheep

Epidemiological studies suggest that intra-uterine exposure to inflammation may prime postnatal immune responses. In fetal sheep, intra-amniotic injection of lipopolysaccharide (LPS) induced chorioamnionitis, lung inflammation and maturation, matured lung monocytes to macrophages and initiated systemic tolerance of fetal monocytes to subsequent challenge with LPS. We hypothesized that LPS-mediated chorioamnionitis altered the response of lung and blood monocytes to Toll-like receptor (TLR) ligands such as PamCysK4 (TLR2), flagellin (TLR5), and human CpG-DNA (TLR9). Time-mated ewes were given intra-amniotic injections of LPS or saline. Blood and lung monocytes were assessed after 2 days, 7 days and 2 days and 7 days repetitive LPS injections before delivery at 124 days gestational age (term 150 days). Responsiveness of blood and lung monocytes to TLR-ligands in vitro was assessed by interleukin (IL)-6, tumor necrosis factor-α (TNF-α) and hydrogen peroxide. Monocytes from preterm controls had minimal responses. Lipopolysaccharide-mediated chorioamnionitis increased IL-6, TNF- α and hydrogen peroxide to all TLR agonists in blood and lung monocytes. Repetitive exposure to antenatal LPS reduced IL-6, TNF- α and hydrogen peroxide to TLR-ligands suggesting tolerance. Tolerance to TLR-ligands reduced IL-1 receptor associated kinase-4 expression. Thus, repeated fetal exposure to LPS induced tolerance to other TLR-ligands. These modulations of fetal innate immunity have implications for host defense and injury responses in preterm infants.

Toll-Like Receptors and Agonist Responses in the Developing Fetal Sheep Lung

Toll-like receptors (TLRs) are pattern recognition molecules that initiate innate immune responses. Intra-amniotic exposure of fetal sheep to pro-inflammatory stimuli causes pulmonary inflammation and induced lung maturation. We examined TLR ontogeny and fetal lung responsiveness to three different TLR agonists. We cloned ovine TLRs 2, 3, and 4 and found 83–88% homology between these ovine and human TLRs. Lung TLR2 and 4 mRNAs increased throughout late gestation to 50% of adult level in the term newborn lamb. Doses of 10 mg of PAMCysK4 (TLR2 agonist), poly I:C dsRNA (TLR3 agonist), or E. coli O55:B5 lipopoysaccharide (LPS) (TLR4 agonist) were given by intra-amniotic injection 2 d or 7 d before operative delivery of preterm lambs at 123 d (n = 4–7/group). The TLR4 agonist induced lung inflammation and maturation, whereas the TLR2 agonist gave less consistent responses. Intra-amniotic LPS increased TLR2 mRNA expression primarily in the inflammatory cells and TLR4 mRNA diffusely in multiple cell types. The TLR3 agonist had no effects, and TLR3 mRNA in the fetal lung did not change after LPS exposure. We conclude that TLR2 and TLR4 mRNAs increase through gestation and expression of TLR2 and TLR4 are induced by LPS in the fetal sheep lung.

Antenatal Betamethasone Changes Cord Blood Monocyte Responses to Endotoxin in Preterm Lambs

Corticosteroids are routinely administered to women at risk for preterm delivery to induce fetal lung maturation. Antenatal corticosteroids have immunomodulatory effects on fetal immune cells that are poorly understood. We hypothesized that maternal betamethasone would alter in fetal monocytes both the initiation of inflammation in response to pro-inflammatory stimuli and the resolution of inflammation by phagocytosis of apoptotic neutrophils. Preterm lambs at 124 d gestation were delivered 15 h, 1 d, 2 d, or 7 d after 0.5 mg/kg maternal intramuscular betamethasone. Monocytes from cord blood were isolated and cultured and results were compared with monocytes from preterm lambs exposed to maternal saline or monocytes from adult sheep. Phagocytosis of Escherichia coli was not changed, however, phagocytosis of apoptotic neutrophils was low in fetal monocytes but increased after 7 d exposure to maternal betamethasone to the level found in adult monocytes. Hydrogen peroxide production after endotoxin stimulus was significantly reduced to 7.1 ± 2.2 μ mol at 5 h, 8.7 ± 2.9 μ mol at 24 h, and 4.1 ± 1.9 μ mol at 48 h versus 16.4 ± 3.6 μ mol in control animals; at 7 d, the hydrogen peroxide production increased to 74.3 ± 19.7 μ mol (p < 0.05, per 106 monocytes). IL-6 production was reduced at 15 h after maternal betamethasone but at no other time point. Maternal betamethasone initially suppressed several fetal monocyte functions, however, at 7 d, measurements of initiation and resolution of inflammation were increased to levels similar to monocytes from adult sheep. The time-dependent changes in maternal betamethasone modulation of the responses of fetal monocytes may influence immune function of the preterm lamb after delivery.

Airway Injury from Initiating Ventilation in Preterm Sheep

Premature infants exposed to ventilation are at risk of developing bronchopulmonary dysplasia and persistent lung disease in childhood. We report where injury occurred within the lung after brief ventilation at birth. Preterm sheep (129 d gestation) were ventilated with an escalating tidal volume to 15 mL/kg by 15 min to injure the lungs, with the placental circulation intact (fetal) or after delivery (newborn). Fetal lambs were returned to the uterus for 2 h 45 min, whereas newborn lambs were maintained with gentle ventilatory support for the same period. The control group was not ventilated. Bronchoalveolar lavage fluid (BALF) and lung tissue were analyzed. In both fetal and newborn lambs, ventilation caused bronchial epithelial disruption in medium-sized airways. Early growth response protein 1 (Egr-1), monocyte chemotactic protein 1 (MCP-1), IL-6, and IL-1β mRNA increased in the lung tissue from fetal and newborn lambs. Egr-1, MCP-1, and IL-6 mRNA were induced in mesenchymal cells surrounding small airways, whereas IL-1β mRNA localized to the epithelium of medium/small airways. Ventilation caused loss of heat shock protein 70 (HSP70) mRNA from the bronchial epithelium, but induced mRNA in the smooth muscle surrounding large airways. HSP70 protein decreased in the lung tissue and increased in BALF with ventilation. Initiation of ventilation induced a stress response and inflammatory cytokines in small and medium-sized airways.