(19) Murine Chorioamnionitis Alters the Regulation of Toll-interacting Protein

Background: Chorioamnionitis is infection and/or inflammation of the fetal membranes and the placenta and is an independent risk factor for many different neonatal morbidities, including neonatal sepsis, necrotizing enterocolitis, and bronchopulmonary dysplasia. Despite the significant risks associated with chorioamnionitis, the underlying immune mechanisms of this exposure remain poorly understood.

Objectives: Our goal is to define immune alterations in neonatal macrophages following exposure to chorioamnionitis. We previously demonstrated that murine chorioamnionitis-exposed F4/80+ neonatal macrophages display a hyporesponsive phenotype to heat-killed bacterial pathogens ex vivo. This is associated with decreased systemic inflammation and lower mortality during neonatal bacterial sepsis, despite equivalent expression of toll-like receptor (TLR) 2 and TLR4. Therefore, we hypothesized that chorioamnionitis-exposed neonatal macrophages would display alterations in regulators of TLR2 and TLR4 signaling, including toll-interacting protein (Tollip). Tollip acts as a negative regulator of TLR2 and TLR4 signaling to prevent excessive inflammation.

Design/Methods: Genetically homogenous C57BL/6 dams were bred. Chorioamnionitis was induced on E17.5 via IP injection of dams with 100 µg/kg lipopolysaccharide (LPS) or PBS control. Pups were born naturally, and on postnatal days 3-5 (P3-5), pups were euthanized and spleens were harvested. Splenic macrophages were isolated using an F4/80+ isolation kit and were analyzed via assay for transposase accessible chromatin with sequencing (ATAC-seq), qPCR for mRNA expression of Tollip, or western blot for protein expression of Tollip.

Results: Chorioamnionitis-exposed male neonatal macrophages demonstrated increased chromatin accessibility at the promoter of Tollip (Figure 1; p=0.013, FDR = 0.039), which corresponded to increased mRNA expression (Figure 2; p=0.042). However, chorioamnionitis-exposed male neonatal macrophages demonstrated a trend towards decreased protein expression of Tollip (Figure 3; p=0.058).

Conclusions: Murine chorioamnionitis alters the regulation of Tollip in male neonatal macrophages, which may contribute to their bacterial hyporesponsiveness and differential outcomes during sepsis.