Figure 6. Addition of metabolites downstream of glycolysis prevents NLRP3 inflammasome activation induced by glycolytic disruption.
(A) LPS-primed BMDM were treated with GB111-NH2 for 2 hr in the presence of pyruvate (pyr; 1 mM) or cell-permeable esters of lactate (lac; 1 mM) and succinate (succ; 10 mM). Cells were fixed, stained for ASC and DAPI, and inflammasome foci/nuclei quantified. At least four fields of view were quantified per condition per experiment, ~2000 cells/condition. Error bars represent mean +/- sd of fields of view analyzed. (B) BMDM were primed with LPS and then treated with 10 μM GB111-NH2 for 2 hr in the presence of the indicated concentrations of L-glutamine or succinate. Cells were fixed, stained for ASC and DAPI, and quantified by microscopy. Four fields of view (~2000 cells) were analyzed per condition. Error bars represent mean +/- sd of separate fields of view. (C) LPS-primed BMDM were treated with the indicated compounds in the presence or absence of pyruvate and analyzed as in (A). (D) BMDM were treated as in (C) and supernatants were analyzed for IL-1β by ELISA. (E) BMDM were treated as in (C) and cell death was measured by LDH release. (F) BMDM were treated with the indicated inhibitors, stained for ASC and DAPI, and quantified by microscopy as in (B). (G) BMDM were treated with GB111-NH2 for 2 hr in the presence or absence of pyruvate (1 mM), after which cytosolic NAD+/NADH was measured. (H) BMDM were treated as in (G) and cytosolic ATP measured by ATP-coupled luminescence assay. For ELISA and LDH release data, error bars represent mean +/- sd of technical triplicate. Data were analyzed for statistical significance using an unpaired, two-tailed t test.