273 Bacterial components induce fetal membrane non-pyroptotic IL-1β production through activation of the NLRP3 inflammasome

Abstract

Bacterial infections increase the risk of chorioamnionitis and preterm birth; however the mechanisms involved are not fully understood. Inflammatory interleukin-1β (IL-1β) is an important mediator of preterm birth, and its secretion is mediated by the inflammasome that processes pro-IL-1β into its active form. This study sought to determine the fetal membrane (FM) compartment involved in IL-1β production in response to bacterial components and to evaluate the mechanism of inflammasome activation. An ex vivo human FM explant system was used to evaluate the response of the amnion, chorion, and intact FMs to media or the bacterial components lipopolysaccharide (LPS), peptidoglycan (PGN), flagellin, iE-DAP, or MDP (n=8). After 24 hours, IL-1β secretion was quantified by ELISA, the pyroptotic marker Gasdermin-D (GSDMD) evaluated by Western blot, and viability measured using a LDH assay. Inflammasome function was assessed using inhibitors to NLRP3 (MCC950), caspase-1 (Z-WEHD-FMK), Pannexin-1 (carbenoxolone), and ROS (DPI) (n=8-12). LPS, PGN, and MDP significantly increased IL-1β secretion from intact FM explants by 57.0±22.4-; 38.3±16.4-; and 28.4±11.9-fold respectively (p<0.05). Flagellin and iE-DAP had no significant effect. The chorion, not the amnion, was the source of this IL-1β response. Inhibitors to NLRP3, Pannexin-1, and ROS significantly inhibited LPS, PGN, and MDP-induced IL-1β secretion from intact FM explants (p<0.05), while the caspase-1 inhibitor significantly reduced LPS and MDP, but not PGN-induced, IL-1β (p<0.05). There was no significant difference in GSDMD activity or LDH secretion by intact FMs under all treatment conditions. The chorion is the major source of IL-1β production following FM exposure to bacterial components. Bacterial LPS, PGN, and MDP induced human FM IL-1β secretion through activation of the NLRP3 inflammasome with contributions from ATP release through Pannexin-1 and ROS signaling. Further investigation is needed to determine the role of alternate caspases and non-pyroptotic mechanisms of FM IL-1β production and release.