Extended Data Fig. 2 |. Cytosolic LPS induces NLRP3-independent IL-18 release from human cells.

a, b, WT or NLRP3-deficient THP1 monocytes were primed with Pam3CSK4, or left unprimed, and electroporated with LPS (or PBS) in presence or absence of MCC950 and LDH and IL-18 release into supernatant was quantified after 2 h. c, ELISA analysis of purified pro-IL-18 and mature IL-18. Mature IL-18 was generated by cleavage of pro-IL-18 with recombinant caspase and complete cleavage was confirmed by immunoblot. Immunoblot and ELISA results are representative of two experiments. d, e, WT or NLRP3-deficient THP1 monocytes electroporated with LPS (or PBS) in the presence of absence of MCC950. IL-18 from cell culture supernatants was immunoprecipitated and analyzed by immunoblot. f, g, Pam3CSK4-primed WT or NLRP3-deficient THP1 monocytes were electroporated with LPS (or PBS) in the presence of absence of MCC950. IL-1β was immunoprecipitated from supernatants and analyzed by immunoblot. h, Immunoblot analysis of THP1 cells in which expression of caspase-1 or caspase-4 was disrupted by CRISPR/Cas9. i, j, LPS-primed THP1 macrophages deficient for caspase-1, caspase-4 or treated with a non-target sgRNA (NT) were electroporated with LPS (or PBS) and LDH release and IL-18 levels in supernatants were quantified after 2 h. IL-1β from supernatants was immunoprecipitated and analyzed by immunoblot. Immunoblot is representative of three biological replicates. k, Immunoblot analysis of LPS-primed caspase-1 or caspase-4-deficient THP1 macrophages compared to caspase-4-deficient THP1 cells reconstituted with caspase-5 by retroviral transduction. Cells were differentiated into macrophages and stimulated with LPS for 4 h. l, Immunoblot analysis of caspase-4-deficient THP1 cells reconstituted with caspase-4 or caspase-5 by retroviral transduction. m, Caspase-4-deficient THP1 macrophages expressing GFP only, caspase-4 or caspase-5 were primed with LPS before delivery of LPS into the cytosol by electroporation. LDH and IL-18 release into supernatants was quantified after 2 h. n, WT or caspase-4-deficient THP1 macrophages were infected with a flagellin-deficient strain of Salmonella and LDH and IL-18 release was quantified after 24 h. Immunoblots are representative of three (d, e, f, g, h, j) or two (h, l, k, c) biological replicates. Bars and error bars represent mean ± SEM of three biological replicates. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test: ns = not significant (p > 0.05). For gel source data, see Supplementary Figure 1.