Systemic Inflammatory Responses to Repeated and Increasing Endotoxin Challenges in Fetal Sheep

Abstract

Introduction: Perinatal infections caused by bacteria lead to fetal systemic inflammation, which is associated with a greater incidence of prematurity and adverse neonatal outcomes. Chronic exposure to subacute lipopolysaccharide (LPS) during gestation serves as an effective means of modeling of chorioamnionitis in sheep. Studies show that repeated low-dose administration of LPS, intravenously in fetus, intra-amniotically and intraperitoneally in mother, attenuates the fetal response to LPS. Our study aims to develop a fetal inflammatory response syndrome (FIRS) model that consistently and effectively elicits a marked physiological response to increasing LPS doses.Hypothesis: Repeated increasing doses of LPS when given intravenously to the fetus will overcome tachyphylaxis to LPS exposure and produce a reproducible systemic inflammatory response in fetal sheep. Methods: LPS boluses were administered intravenously to fetal sheep (n=5; 0.83 of gestation) with surgically placed indwelling vascular catheters. Four LPS boluses were given with doses of 0.3µg, 1.5µg, 3µg, and 15µg on days 1, 3, 4, and 5, respectively. To assess the physiological responses, we measured blood gases, pH, glucose, and lactate levels at 0, 1, 3, and 5 hours relative to the LPS bolus. Peripheral blood mononuclear cells (PBMCs) were isolated on days 1, 4, and 5 prior to the daily LPS bolus. Data were analyzed with mixed-model ANOVA and post hoc Tukey’s test. Results: Repeated LPS challenges increased (p < 0.01) lactate concentrations and pCO2 levels and decreased (p < 0.01) pH, pO2, and oxygen saturation at 3 and 5-hours. Cortisol concentrations (p <0.05) increase at 3 and 5 hours. Notably, no interaction between day (increasing LPS doses) and hour (LPS response to each dose) was found. PBMCs showed a consecutive increase with each LPS dose (p<0.001, from day0 to day5). Transcriptional analysis on PBMCs shows enrichment of inflammatory pathways. mRNA expression using qPCR show elevation in pro inflammatory cytokines (TNF-α, CRP, IL-6, IL1-A, IL-10) in liver (p<0.05), (IL-6) in lung (p<0.05) and decrease in (TNF-α, IL1-B and IL-10) in spleen (p<0.01). Conclusion: Changes in physiological indices collectively show both respiratory and metabolic acidosis and impaired oxygen transfer. Cortisol concentrations also increased with successive LPS challenges. PBMCs increase significantly by Day 4, which further demonstrates an inflammatory response to LPS. With increasing LPS doses, the magnitude of the fetal responses was maintained demonstrating robust systemic response despite tachyphylaxis to LPS in fetal sheep. Transcriptional analysis along with increased mRNA expression of pro inflammatory cytokine in multiple tissues further highlights that repeated increasing LPS boluses were able to produce a robust and sustained physiological response over time.