Fig. 4

Activation of the macrophage NLRP3 inflammasome by ceramides from HBV-transfected hepatocytes. A, B Two million of murine peritoneal macrophages were stimulated with 5 μg/ml of HBV proteins alone, or addition of 0, 5, 10 or 20 μM of C6-ceramide for 24 h. 0 μM represents the medium containing 0.1% DMSO (solvent for C6-ceramide). A Bar graphs (mean ± SEM) show the amounts of secreted (in supernatant) IL-1β, IL-18 and IL-23 determined in three independent experiments. The differences were compared with paired two-tailed Student’s t-test. *P < 0.05, **P < 0.01 and ***P< 0.001 between the cells stimulated with specified concentration of C6-ceramide alone (empty columns) and those with addition of the HBV proteins. B The cells treated with the HBV proteins alone (HBV protein + solvent) or addition of 20 μM C6-ceramide (HBV protein + ceramide) were collected for analysis of NLRP3, procaspase-1 and cleaved caspase-1 (p20) generation. The immunoblot image shows one representative of three independent experiments, and the bar graph shows the relative amount of indicated proteins. C, D As described in Fig. 3D, the medium from differently treated HepG2 cells was collected and mixed with same volume of completed DMEM respectively as conditioned medium (CM) (depicted in Additional file 1: Fig. S8). Two million macrophages were treated with different CM for 72 h. C Bar graphs (mean ± SEM) show the macrophage generation of indicated cytokines determined by qRT-PCR (upper panel), or secretion in supernatant quantified by ELISA (low panel), from three independent experiments. The differences were compared with one-way ANOVA. ^$P < 0.05, ^$$P < 0.01, ^$$$P < 0.001 between Ref-HBV and empty-vector; *P < 0.05, **P < 0.01, ***P < 0.001 between Ref-HBV and mtPreS1 or mtPreS2; ^#P < 0.05 between mtPreS1 and mtPreS2. D Immunoblot analysis of NLRP3 expression and activation in the differently treated macrophages. Bar graphs show the relative amount of specified proteins of the experiment