Fig 2: A secondary stimulus is required for the secretion of NLRP3-dependent IL-1ß in the human GEN2.2 pDC cell line. GEN2.2 cells were pretreated with 1 µM CpG-A, 1 µM CpG-B or 5 µg/mL IMQ for 3 h, then 20 µM nigericin was added to the cells in a time-dependent manner (A). In parallel experiments, cells were incubated in the presence of 1 µM MCC950 (specific NLRP3 inhibitor) for the last 30 min of TLR stimulation prior to nigericin exposure (A). In similar experiments, GEN2.2 cells were primed with live E. coli (MOI 10), Bacillus subtilis (MOI 10), Lactobacillus rhamnosus (MOI 10) and Candida albicans (MOI 0.01), then nigericin was added to the cells in a time-dependent manner (B). Similarly, in a parallel experiment, cells were incubated with MCC950 for the last 30 min of the microbial stimulation prior to nigericin treatment (B). IL-1 ß secretion was measured by ELISA (A,B). Data are represented as mean ± SD of 6 individual experiments and were analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test. ** p < 0.01, *** p < 0.0001, **** p < 0.0001 vs. control (ctrl), # p < 0.05, ## p < 0.01, ### p < 0.0001, #### p < 0.0001. ND: not determined, IMQ: imiquimod, NIG: nigericin, EC: Escherichia coli, BS: Bacillus subtilis, LR: Lactobacillus rhamnosus, CA: Candida albicans.

Fig 3: Inhibition of NLRP3 resulted in down-regulation of the expression of inflammasomes and pyroptosis-related proteins. THP-1 macrophages were pre-treated with NLRP3 inhibitor MCC950 10 µM for 2 h, followed by BCG infection for 24 h (A,B). Compared to the BCG group, the expression of the inflammasome-related proteins (NLRP3, Caspase-1 p48, ASC) was downregulated in the MCC950+BCG group. (A,C) Compared to the BCG group, the expression of the pyroptosis-related proteins (GSDMD-N, IL-1 p17, IL-18 p22) was down-regulated in the MCC950+BCG group. Data are from mean ± SD of three independent experiments, ns p > 0.05, ** p < 0.01, *** p < 0.001.