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. 2023 Oct 31;4(11):101245. doi: 10.1016/j.xcrm.2023.101245

Figure 2.

Figure 2

Protective adjuvants LMQ and SQ trigger different innate pathways in vitro

(A) Summary of the experimental protocol.

(B) IL-1β and TNF-α secretion in supernatants after stimulation of WT BMDMs with indicated amounts of adjuvants was measured by ELISA. LDH release was measured using a colorimetric assay and is depicted as a percentage of lysed positive control (representative data from three independent experiments; cells are stimulated in triplicates; mean ± SEM are shown).

(C) BMDMs derived from C57BL/6 WT and Nlrp3−/− mice were stimulated with adjuvants (1:20 dilution). Cytokines and LDH were measured as described in (B). LPS/nigericin (100 ng/mL LPS for 6 h with 5 μM nigericin for the last 1 h) stimulation was used as positive control (pooled data from three independent experiments; cytokines were normalized to LPS/nigericin control set to 1; cell death/LDH was normalized to maximal cell death control, set to 100%; cells were stimulated in triplicates; mean ± SEM are shown).

(D) Representative western blots for pro-caspase-1 (p46), cleaved caspase-1 (p20), NLRP3, and GAPDH in cell lysates and supernatants from cultures of WT and Nlrp3−/− BMDMs stimulated with adjuvants (1:20 dilution) or LPS/nigericin (representative data from two independent experiments).

(E) Summary of the experimental protocol.

(F) HMDMs were stimulated for 6 h with adjuvants (1:20 dil.) in the presence or absence of R21 (given at 1/5 of mouse dose to preserve the ratio of Ag:adjuvant given in vivo), in the presence or absence of NLRP3 inhibitor MCC950 (10 μM) added 0.5 h before the adjuvants. IL-1β and TNF-α secretion in supernatants was measured by ELISA. LDH release was measured using a colorimetric assay. LPS/nigericin served as a positive control (100 ng/mL LPS for 6 h with 10 μM nigericin for the last 2 h) (pooled data from three independent experiments/donors for IL-1β and LDH and two independent experiments/donors for TNF-α; cells are stimulated in triplicates; cytokines were normalized to LPS/nigericin control set to 1; cell death/LDH was normalized to maximal cell death lysis control, set to 100%; mean ± SEM are shown).

(G) Representative western blots for pro-caspase-1 (p46), cleaved caspase-1 (p20), NLRP3, and GAPDH in cell lysates and supernatants from HMDMs, stimulated as in (F) (representative data from two independent experiments/donors are shown). All statistical analyses were done using two-way ANOVA with Bonferroni’s correction for multiple comparisons; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. In (B), significant adjuvant effect is reported. In (C) and (F), significant genotype effect is reported.