Studies on redox modulation of nlrp3 inflammasome

NLRP3 inflammasome activation provides an innate host defence against pathogens. Two steps of inflammasome complex activation are recognised: Signal 1 or priming (expression of NLRP3 protein, pro-IL-1β, pro-IL-18 and pro-caspase-1), followed by Signal 2 or activation (expression of active IL-1β and IL-18). Additionally, caspase 5 may be involved via a non-canonical pathway. The purpose of this study was to investigate the possible role of cytosolic superoxide and slow-release hydrogen sulfide generation in mitochondria, in activating the NLRP3 inflammasome in THP-1 and HUVEC and EA.hy926 endothelial cells. Cells were differentiated with PMA (5 ng/ml) for 1-3 days. Subsequently, LPS treatment (5 μg/ml) was administered for 24 h for Signal 1 activation followed by bzATP (300 μM) for 1 hour for Signal 2. Signal 1 and Signal 2 of the NLRP3 inflammasome were up-regulated in the human macrophage cell model. Both endothelial cell types showed evidence of low level Signal 1 activation. Despite HUVEC cells showing priming of the NLRP3 inflammasome, there was no evidence for Signal 2 activation. Surprisingly, EA.hy926 cells showed low levels of active IL-1β, when exposed to PMA, suggesting other endpoints of an active NLRP3 inflammasome such as HMGB1, which was expressed in EA.hy926 cells, are worthy of investigation. Subsequently, the effects of intracellular generation of superoxide (mitoparaquat and paraquat at 1 and 5 μM) were investigated in differentiated THP-1 cells before and after LPS. In our hands, intracellular superoxide generation was unable to enhance Signal 1 NLRP3 inflammasome in PMA-differentiated THP-1 cells. However, the higher concentration of mitoparaquat was able to increase Signal 2. Non-canonical pathway was not detected in THP-1 cells. Interestingly, slow-release hydrogen sulfide donors (GYY4137 and AP39) reduced Signal 2 NLRP3 inflammasome suggesting anti-inflammatory properties. These data support a role for redox active oxygen and sulfur species in modulating inflammatory IL-1β and IL-18 synthesis in human monocytes with pro- and anti-inflammatory activities respectively.