Fig. 3. Cholesterol crystals activate the NLRP3 inflammasome by inducing lysosomal damage.

a, IL-1β ELISA of supernatants from LPS-primed murine macrophages stimulated with cholesterol crystals or nigericin in the presence or absence of cytochalasin D. b, c, Combined confocal fluorescence and reflection microscopy of murine macrophages incubated with DQ ovalbumin alone (b, left) or together with cholesterol crystals (125 µg/ml) (b, right; c) for 2h and plasma membrane was stained with A647-conjugated choleratoxin B (b, c). c, Cells were fixed, permeabilized (0.05% saponin) and internal membranes additionally stained with A555-conjugated choleratoxin B. Nuclei were stained with Hoechst dye. d, Murine macrophages stimulated with cholesterol crystals for 6h, stained with acridine orange and analyzed by FACS. e, f, IL-1β ELISA of supernatants from LPS-primed, murine macrophages stimulated with cholesterol crystals in the presence or absence of bafilomycin A1 (e) or from LPS-primed wild-type, cathepsin B or L deficient macrophages stimulated with cholesterol crystals (f). g, h, Murine macrophages stimulated with 100 µg/ml oxLDL for the indicated times and fluorescent dextran (g, 20h) were imaged by combined confocal fluorescence and reflection microscopy i, Unprimed murine macrophages were incubated with oxidized LDL as indicated for 24 h and IL-1β in supernatants was measured by ELISA. (a, d-f, i) One out of three independent experiments are shown; means and s.d. (a, e, f); Representative images of five (b, c) or two (g, h) independent experiments.