Inflammation underlies the pathology of numerous diseases. It can be initiated by macrophages through the secretion of pro-inflammatory cytokines, such as IL-18 and IL-1β, following the activation of a molecular complex called the inflammasome. Inflammasomes are a protein scaffolding complex consisting of three known components: a sensory NLR, such as NLRP3, ASC, and caspase-1. The NLRP3 inflammasome is activated by a diverse array of stimuli, including crystals, ATP, pore-forming toxins, such as tetanolysin O (TLO), and the potassium ionophore nigericin. Here, we have explored the mechanism of NLRP3 inflammasome activation using nigericin, TLO and ATP. We found that nigericin induced NLRP3 inflammasome activation in bone-marrow derived macrophages (BMDM) results in inflammasome dependent lysosomal membrane permeabilization (LMP), mitochondrial membrane permeabilization (MMP), and the processing and secretion of IL-1β. All of these events required mitochondrial reactive oxygen species (ROS). Through combining bulk biochemical assays with live cell analysis of individual cells, we provide a kinetic analysis and sequence of events resulting from nigericin stimulation of LPS primed BMDM. We recapitulated NLRP3 inflammasome activation in a dendritic cell line, using the novel system of D2SC-1 transduced with ASC. These transduced cells undergo a similar sequence of events as macrophages, confirming that this mechanism is a general result of NLRP3 inflammasome activation applicable to multiple cell types. Furthermore, we have found that ATP and TLO stimulation of BMDM results in a similar sequence of NLRP3 dependent events. ATP, but not nigericin, requires P2X7 for activation of the NLRP3 inflammasome. Surprisingly, inhibition of P2X4 blocked nigericin, but not ATP, induced, NLRP3-dependent IL-1β secretion in BMDM. Our work has demonstrated a central, common role for mitochondrial ROS in NLRP3 inflammasome activation and determined the kinetics of organelle crosstalk during inflammasome activation. These data place both the mitochondria and lysosomes in a critical position controlling NLRP3 inflammasome activation. Based on these results, we suggest mitochondrial ROS as a potential therapeutic target for treating NLRP3 inflammasome related diseases. Inhibition or scavenging of mitochondrial ROS would not only prevent the pro-inflammatory effects of IL-1β secretion in these patients, but also NLRP3 dependent organelle damage and the resulting cell death
