Figure 2. ROS production is not required to activate NLRP3.

(A) LPS-primed WT and Nlrp3^−/− BMDMs were stimulated for 6 hrs with 10 µM rotenone, 10 µg/ml antimycin A, 10 mM 3-methyladenine (3-MA) or 1 mM H₂O₂, or treated 30 min with 0.5 µM of gramidicin. NLRP3 activation was detected by measuring the secretion of IL-1β (upper panel) and caspase-1 activation (lower panel). (B) CM-H₂DCFDA -labeled BMDMs were incubated in medium containing ROS scavengers N-acetyl-L-cysteine (NAC, 2.5 mM), N-(2-Mercaptopropionyl)glycine (MPG, 2.5 mM), ascorbic acid (AA,150 µM). The oxidation of CM-H₂DCFDA (upper panel) and the oxidation rate (lower panel) were calculated as described in Experimental Procedures. (C) The effect of ROS scavengers on signal 1 and signal 2 of NLRP3 activation induced by gramicidin was evaluated. WT BMDMs were primed with LPS for 3 hrs in the presence of ROS scavengers and subsequently stimulated with 0.5 µM gramicidin (signal 1) or primed for 3 hrs with LPS and stimulated with gramicidin in the presence of ROS scavengers (signal 2). NLRP3 activation was assessed by measuring IL-1β release (upper panel) and caspase-1 activation (lower panel). (D) BMDMs labeled with the fluorescent ROS probe CM-H₂DCFDA were stimulated with 0.5 µM gramicidin (Gra), 1 mM H₂O₂ or medium. The oxidation of CM-H₂DCFDA was monitored (upper panel) as in (B) and the oxidation rate was calculated (lower panel). (E) LPS-primed WT BMDMs were stimulated with nigericin (10µM, Nig), gramicidin (0.5 µM, Gra) or 5 mM ATP 30 min with the indicated amount of NAC and caspase-1 activation was analyzed. (F and G) LPS-primed WT (F) and Nlrp3^−/− (G) BMDMs were stimulated with nigericin (10µM, Nig), gramicidin (0.5 µM, Gra) or ATP (5 mM) for 30 min, with Salmonella (MOI 10, Sal) for 1 h or with pdAdT (5 µg/ml) for 4 hrs in the presence of the indicated amounts of DPI. Caspase-1 activation (F) and the intracellular content of K⁺ (G) were measured. Caspase-1 activation was analyzed by immunoblotting and IL-1β by ELISA. Cells were treated 30’ with the inhibitors or medium before adding the agonists. The intracellular levels of K⁺ were quantified by ICP-OES in Nlrp3^−/− cells. Values represent mean ± standard deviation (n=3–4). Results are representative of at least three separate experiments. NS, not statistically significant (p ≥ 0.05). *, p < 0.05 (stimulated vs. unstimulated).