Intermediate Signaling Mechanisms Regulating Human Fetal Membrane Responses to Gram-Positive Bacterial Peptidoglycan

Abstract

Problem

Chorioamnionitis and preterm birth are leading causes of neonatal morbidity and mortality. Despite ongoing research, the signaling pathways involved in the pathogenesis of chorioamnionitis—inflammation of the fetal membranes (FM)—are not well understood. Previously, we reported that FMs utilize miR-146a-3p as an endogenously produced danger signal to sequentially activate Toll-like receptor (TLR) 8 and subsequent inflammation following lipopolysaccharide stimulation of TLR4. In this current study, following stimulation of fetal membrane explants by the TLR2 agonist peptidoglycan (PDG), we investigated sequential microRNA-activation of TLR8, intermediate signaling pathways NFκB and MAPK (p38, ERK), and their effects on inflammation and mediators of membrane weakening.

Method of Study

Human FMs explants were treated with or without PDG in the presence or absence of inhibitors to TLR7, TLR8, p65 NFκB, p38 MAPK, or ERK. Culture supernatants were measured for secreted factors by ELISA, tissue RNA was measured for TLR7/8-activating miRs by RT-qPCR, and tissue protein was measured for phosphorylated proteins by Western blot.

Results

PDG-treated FMs produced elevated levels of TLR8-activating miR-146a-3p in a p65 NFκB-dependent manner. PDG-treated FMs produced elevated levels of the pro-inflammatory cytokine IL-1β, the neutrophil recruiting chemokine IL-8, and membrane weakening MMP1, MMP9, and PGE₂ in a TLR8-dependent manner. Except for MMP9, this inflammatory and membrane weakening response to PDG was dependent upon p65 NFκB, p38 MAPK, and ERK signaling.

Conclusions

This study gives new insight into the molecular mechanisms involved in FM responses to Gram-positive bacteria and into the pathogenesis of chorioamnionitis.