243 - Modulation of NLRP3 inflammasome activation in human macrophages by slow release hydrogen sulfide compounds

Abstract

The NLRP3 inflammasome complex undergoes priming and activation leading to pro-inflammatory IL-1β and IL-18 synthesis. This study investigated the potential role of mitochondrial redox modulation in activation of the NLRP3 inflammasome in human cells. THP-1 cells were differentiated to macrophages with PMA for 24 hours and the NLRP3 inflammasome primed with LPS (0.1µg/ml) followed by activation with bzATP. NLRP3 and pro-IL1β were detected by Western blotting and ASC speck formation by immunofluorescence as markers of NLRP3 inflammasome priming. IL-1β and IL-18 in cell media were quantified by ELISA as markers of inflammasome activation. Under the conditions employed, intracellular oxidant generation (mitoparaquat and paraquat; Pq) assessed by mitoSOX fluorescence, was not sufficient to prime macrophages and did not affect priming mediated by LPS. At a relatively high extracellular concentration (5 µM) mitoPq, but not Pq, was able to increase IL-1β production in LPS-primed macrophages, suggesting promotion of inflammatory responses; IL-18 production was not affected. In LPS-primed and bzATP activated cells, the non-targeted slow release H2S compound, GYY4137, slightly reduced IL-1β production but was able to reduce IL-18 synthesis by approximately 50%. Although the mitochondria-targeted, slow release H2S compound AP39 did not affect inflammasome priming, at 300 nM (final media concentration) it reduced IL-1β and IL-18 synthesis in primed and activated cells. These data support a role for redox active oxygen and sulfur species, particularly when targeted to mitochondria, in modulating inflammatory cytokine synthesis in human macrophages with pro- and anti-inflammatory activities respectively.