Figure 5. Hexokinase Dissociation from Mitochondria Is Sufficient to Activate the NLRP3 Inflammasome.

(A) LPS-primed BMDMs were treated with cell-permeable hexokinase dissociation peptide (HKVBD) or scrambled control peptides (Ctl) fused to TAT peptide (20 μM) for the indicated times, and the amount of hexokinase in the cytosolic fraction was determined by immunoblot. Control lysate was included on each gel (Antibody Specificy Ctl) to confirm antibody staining for each marker (non-continuous lane from the same gel). UT, untreated.

(B) LPS-primed BMDMs expressing HK2-GFP and mitochondria-localized DsRed (Mito-DsRed) were imaged following treatment with HKVBD and Ctl peptides fused to TAT (20 μM) to assess hexokinase redistribution.

(C and D) LPS-primed BMDMs were treated with HKVBD or control peptides fused to cell-permeable antennapedia peptide; IL-1β (C) and IL-18 (D) were measured by ELISA after 2 hr.

(E) LPS-primed BMDMs from wild-type and Nlrp3^−/−mice were stimulated with 5 mM ATP, pdA:dT, HKVBD or control peptides fused to antennapedia peptide, and IL-1β was measured in the supernatant after 2 hr.

(F and G) LPS-primed BMDMs were treated with HKVBD or control peptides fused to TAT peptide; (F) cleaved IL-1β and caspase-1 were detected by immunoblot at 2 hr, and (G) inflammasome assembly was observed by NLRP3 and caspase-1 p10 proximity ligation (PLA,red). Nucleiwere stained withDAPI(blue). Sup, supernatant. (H and I) Indicated mice were injected i.p. with 500 μl of PBS (n = 6), 240 μM HKVBD (n = 7), or control peptide fused to TAT peptide (n = 6); peritoneal cavities were lavaged after 4 hr; total cells were counted; and neutrophil content was determined by flow cytometry (both experiments were each done 1×). ns, not significant. Error bars indicate SD. *p % 0.05; **p % 0.01; ***p % 0.001, Student’s t test (C–E and H) and one-way ANOVA and Newman-Keuls multiple comparison test (G).

See also Figure S5.