Previous studies have shown that NLRP3 is located at the endoplasmic reticulum (ER) in the steady state and that it translocates to the mitochondria-associated ER membranes following stimulation (Zhou et al., 2011). Therefore, in order to gain further insights into ethanol-induced NLRP3 inflammasome activation, we used confocal microscopy to assess the location of NLRP3 under basal and stimulated conditions. We noted that under basal conditions, NLRP3 was located at the cytoplasm, but not within mitochondria (Figure 6A). We were unable to detect caspase-1 under the steady-state conditions. However, stimulation of astrocytes with ATP, LPS or ethanol (10 mM) led to the recruitment of caspase-1 and NLRP3 within mitochondria, as demonstrated by the overlap of caspase-1 and NLRP3 in the mitochondria (mitotracker staining). Strikingly, ATP and ethanol stimulation promoted higher caspase-1 levels within mitochondria than LPS. Inhibiting caspase-1 with Z-VAD-FMK (Figure 6B) or mROS with Mito-TEMPO (Figure 6C) mostly abolished NLRP3/caspase1 recruitment within mitochondria.
