Steffen Kunzmann

Thymic changes after chorioamnionitis induced by intraamniotic lipopolysaccharide in fetal sheep

OBJECTIVE Regulatory T lymphocytes mediate homeostasis of the immune system and differentiate under the control of the transcription factor FoxP3 in the fetal thymus. We asked whether fetal inflammation caused by chorioamnionitis would modulate thymus development. STUDY DESIGN Fetal sheep were exposed to an intraamniotic injection of 10 mg lipopolysaccharide at 5 hours, 1 day, 2 days, or 5 days before delivery at 123 gestation days. Cord blood lymphocytes, plasma cortisol, and thymus weight were measured. Glucocorticoid receptor-, activated caspase-3-, Ki-67-, proliferating cell nuclear antigen-, nuclear factor-kappaB-, and FoxP3-positive cells were immunohistochemically evaluated in thymus. RESULTS Intraamniotic lipopolysaccharide exposure decreased the number of circulating lymphocytes by 40% after 1 day. Thymus-to-body weight ratios were reduced in all lipopolysaccharide groups by a maximum of 40% at 5 days. Lipopolysaccharide exposure modestly increased plasma cortisol concentration, increased nuclear factor-kappaB immunostaining in fetal thymus and reduced the number of FoxP3-positive cells by 40% at 1 day. CONCLUSION Intraamniotic exposure to lipopolysaccharide induced thymic changes and influenced thymic FoxP3 expression.

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.

Intravenous lipopolysaccharide-induced pulmonary maturation and structural changes in fetal sheep

BACKGROUND Antenatal pulmonary inflammation is associated with reduced risk for respiratory distress syndrome but with an increased risk for bronchopulmonary dysplasia (BPD) with impaired alveogenesis. OBJECTIVE We hypothesized that fetal systemic inflammation induced by intravenous (IV) lipopolysaccharide (LPS) would affect lung development in utero. STUDY DESIGN Twenty-one fetal sheep were instrumented (107 days gestational age). Control fetuses received saline (N = 12) and 9 in the study group received 100 ng of LPS IV 3 days after surgery. Animals were assessed for lung maturation and structure after 3 (N = 5) and 7 (N = 4) days. RESULTS Interleukin-6 concentration increased in the bronchoalveolar lavage more than 40-fold 3 days after LPS IV. Processing of pro-surfactant protein (SP)-B to mature SP-B and increased SP-B concentrations were shown 7 days after LPS IV. Deposition of elastin fibers at sites of septation was disturbed within 3 days after LPS IV. CONCLUSION Lung maturation and disturbed lung structure occurred after short-term exposure to fetal inflammation and suggests new targeted therapies for BPD.

Surfactant proteins A and D in pulmonary diseases of preterm infants

Immaturity of the pulmonary and immune systems represents an important risk factor for increased morbidity and mortality in neonates. Surfactant protein (SP)-A and SP-D, linking molecules between these two systems, are critical for lung homeostasis as they regulate surfactant metabolism and host immunodefense activities in innate and adaptive immunity. Preterm neonates with respiratory distress syndrome showed lower concentrations of SP-A and SP-D, and the administration of exogenous surfactant was found to strengthen the secretion of SPs. Low levels of SP-A and SP-D also correlated with a higher risk of infection and development of bronchopulmonary dysplasia. Moreover, SP-A- and SP-D-enriched surfactant formulations were more resistant to the inhibitory action of the plasmatic proteins in animal models. Based on these assumptions, new-generation surfactants, enriched with SP-A and/or SP-D, may enhance the function of immune system and lungs in neonates, potentially improving the clinical outcome.

Antenatal Inflammation Reduces Expression of Caveolin-1 and Influences Multiple Signaling Pathways in Preterm Fetal Lungs

Bronchopulmonary dysplasia (BPD), associated with chorioamnionitis, results from the simultaneous effects of disrupted lung development, lung injury, and repair superimposed on the developing lung. Caveolins (Cavs) are implicated as major modulators of lung injury and remodeling by multiple signaling pathways, although Cavs have been minimally studied in the injured developing lung. We hypothesized that chorioamnionitis-associated antenatal lung inflammation would decrease the expression of Cav-1 in preterm fetal lungs. We tested whether changes occurred in the transcription factors Smad2/3, Smad1/5, Stat3, and Stat1, and we also studied the activation of acid-sphingomyelinase (a-SMase) with the generation of ceramide, along with changes in the expression of heme oxygenase-1 (HO-1) as indicators of possible Cav-1-mediated effects. Fetal sheep were exposed to 10 mg of intra-amniotic endotoxin or saline for 2, 7, or 2 + 7 days before preterm delivery at 124 days of gestation. The expression of Cav-1 and HO-1 and the phosphorylation of Smad and Stat were evaluated by real-time PCR, Western blotting, and/or immunohistochemistry. The activity of a-SMase and the concentrations of ceramide were measured. Intra-amniotic endotoxin decreased Cav-1 mRNA and protein expression in the lungs, with a maximum reduction of Cav-1 mRNA to 50% ± 7% of the control value (P < 0.05), and of Cav-1 protein expression to 20% ± 5% of the control value (P < 0.05). Decreased concentrations of Cav-1 were associated with the elevated phosphorylation of Smad2/3, Stat3, and Stat1, but not of Smad1/5. The expression of HO-1, a-SMase activity, and ceramide increased. Antenatal inflammation decreased the expression of Cav-1 in the preterm fetal lung. The decreased expression of Cav-1 was associated with the activation of the Smad2/3, Stat, and a-SMase/ceramide pathways, and with the increased expression of HO-1. The decreased concentrations of Cav-1 and changes in other signaling pathways may contribute to BPD.

Glucocorticoids potentiate IL-6-induced SP-B expression in H441 cells by enhancing the JAK-STAT signaling pathway

The respiratory distress syndrome (RDS) contributes to perinatal morbidity and mortality associated with preterm birth. Surfactant protein B (SP-B) is decreased in RDS. Both maternal antenatal steroid administration and chorioamnionitis reduce the incidence and severity of RDS. An important mediator in chorioamnionitis is IL-6 using the JAK-STAT signaling pathway for signal transduction. We hypothesized that the steroids, betamethasone (BTM) and dexamethasone (DXM), and IL-6 had synergistic effects on SP-B gene expression and STAT3 phosphorylation in H441 cells. DXM and BTM increased SP-B mRNA levels by 16.5 (13.3)-fold and IL-6 alone by 2.3-fold. After 48-h exposure of cells to DXM or BTM, IL-6 caused a significantly greater increase in SP-B mRNA levels (28.1-fold) than IL-6 or glucocorticoids alone. Whereas IL-6 stimulated tyrosine phosphorylation of STAT3 in a time- and dose-dependent way, DXM and BTM had no effect on STAT3 phosphorylation. Both DXM and BTM could potentiate IL-6-induced phosphorylation of STAT3. The synergism of glucocorticoids and IL-6 on SP-B gene expression and the effect of glucocorticoids on IL-6-induced STAT3 phosphorylation could be blocked by a JAK inhibitor. Expression level analysis showed that glucocorticoids increased the expression of the IL-6-binding α-subunit receptor (IL-6R) on mRNA and protein level. Our findings could represent an example of a pulmonary regulation system in which one role of glucocorticoids is to increase the effect of a cytokine by upregulation of its receptor. The described in vitro interaction of IL-6 and glucocorticoids could help explain the clinical observation that prenatal inflammation in preterm babies with antenatal steroid administration can attenuate severity of RDS.

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.

Immunomodulatory properties of surfactant preparations

Surfactant replacement significantly decreased acute pulmonary morbidity and mortality among preterm neonates with respiratory distress syndrome. Besides improving lung function and oxygenation, surfactant is also a key modulator of pulmonary innate and acquired immunity regulating lung inflammatory processes. In this review, we describe the immunomodulatory features of surfactant preparations. Various surfactant preparations decrease the proinflammatory cytokine and chemokine release, the oxidative burst activity, and the nitric oxide production in lung inflammatory cells such as alveolar neutrophils, monocytes and macrophages; they also affect lymphocyte proliferative response and immunoglobulin production, as well as natural killer and lymphokine-activated killer cell activity. In addition, surfactant preparations are involved in airway remodeling, as they decrease lung fibroblast proliferation capacity and the release of mediators involved in remodeling. Moreover, they increase cell transepithelial resistance and VEGF synthesis in lung epithelial cells. A number of different signaling pathways and molecules are involved in these processes. Because the inhibition of local immune response may decrease lung injury, surfactant therapeutic efficacy may be related not only to its biophysical characteristics but, at least in part, to its anti-inflammatory features and its effects on remodeling processes. However, further studies are required to identify which surfactant preparation ensures the highest anti-inflammatory activity, thereby potentially decreasing the inflammatory process underlying respiratory distress syndrome. In perspective, detailed characterization of these anti-inflammatory effects could help to improve the next generation of surfactant preparations.

Myocardial response in preterm fetal sheep exposed to systemic endotoxinaemia.

Exposure of the fetus to antenatal inflammation can occur from chorioamnionitis, which may progress to a fetal inflammatory response syndrome (FIRS) and to fetal sepsis. We tested whether the fetal myocardium responded to systemic Gram-negative endotoxinaemia. We hypothesized that the myocardium would respond to inflammation by changes in hypoxia-inducible factor-α (HIF-1α), inducible NO-synthase (iNOS), Toll-like receptors 2 and 4 (TLR2 and TLR4), IL-6, and phosphorylated signal transducer and activator of transcription-3 (pSTAT3). To model systemic endotoxinaemia, fetal sheep were exposed to Gram-negative endotoxin or saline i.v. 3 d before preterm delivery at 113 d of gestation (term = 147 d). All endotoxin-exposed animals developed cardiac dysfunction within these 72 h. Cardiac mRNA and protein levels of HIF-1α and TLR2 and TLR4 mRNA increased, whereas STAT3 phosphorylation decreased significantly. IL-6 and iNOS mRNA remained unchanged. Fetal systemic endotoxinaemia induced myocardial inflammation by activating TLR2 and 4. The following cardiac dysfunction seems not to be mediated via cardiac iNOS.