Lipopolysaccharide/adenosine triphosphate-mediated signal transduction in the regulation of NLRP3 protein expression and caspase-1-mediated interleukin-1β secretion

Reactive oxygen species (ROS) plays a critical role in the regulation of NLRP3 inflammasome activation. However, the ROS-mediated signaling pathways controlling NLRP3 inflammasome activation are not well defined. Using lipopolysaccharide (LPS) and adenosine triphosphate (ATP) activated murine macrophages as the testing model, cytokine release and protein expression were quantified by enzyme-linked immunosorbent assay and Western blot, respectively. ROS was scavenged by N-acetyl cysteine; NADPH oxidase, the major source of ROS, was inhibited by diphenyliodonium, apocynin or gp91-phox siRNA transfection; and protein kinase was inhibited by its specific inhibitor. LPS-induced NLRP3 protein expression was regulated through the NADPH oxidase/ROS/NF-kappa B-dependent, JAK2/PI3-kinase/AKT/NF-kappa B-dependent, and MAPK-dependent pathways, while ATP-induced caspase-1 activation was regulated through the NADPH oxidase/ROS-dependent pathway. These results demonstrate that ROS regulates not only the priming stage, but also the activation stage, of NLRP3 inflammasome activation in LPS + ATP-activated macrophages.