Fig. 4: PAMPs and DAMPs are required to induce NF-κB–dependent IL-1β release from macrophages expressing pathogenic NLRP3 variants. | Nature Communications

Fig. 4: PAMPs and DAMPs are required to induce NF-κB–dependent IL-1β release from macrophages expressing pathogenic NLRP3 variants.

From: Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1β production

Fig. 4

A, B ELISA for IL-1β (A) and TNF-α (B) released from Nlrp3−/− immortalized macrophages (iMos) expressing the human wild type NLRP3 or the p.D303N, p.R260W, and p.T348M variants induced after 16 h treatment with doxycycline (1 µg/ml) and ATP (5 mM), MSU crystals (200 µg/ml), palmitate (1 mM), S100A9 (0.5 µg/ml), IL-6 (0.5 µg/ml), LPS (100 ng/ml) or Pam3-CSK4 (1 µg/ml), together with or without MCC950 (10 µM). C Colorimetric quantification of the levels of secreted alkaline phosphatase (SEAP) from NF-κB reporter RAW 264.7 macrophages treated for 4 h with ATP (5 mM), MSU crystals (200 µg/ml), palmitate (1 mM), S100A9 (0.5 µg/ml), IL-6 (0.5 µg/ml), LPS (0.1 µg/ml) or Pam3-CSK4 (1 µg/ml). D Percentage of ASC specking monocytes from p.A439T CAPS patients (gray bars) and healthy donors (blue bars) after whole blood treated during 6 h with Pam3CSK4 (1 µg/ml), IL-6 (0.5 µg/ml), S100A9 (0.5 µg/ml) or palmitate (1 mM). E, F ELISA for IL-1β (E) and TNF-α (F) release from healthy donors and p.A439T CAPS patients PBMCs treated as indicated in D. G Violin plot of S100A9 mRNA expression from blood cells of healthy individuals or CAPS patients with the p.G569R NLRP3 mutation during active disease or treated with anakinra (using dataset GSE57253). The median indicated by the middle line. For exact n numbers of A, B see ‘Statistics and reproducibility’ section in Methods; for C n = 3 independent experiments (each one represented by a different symbol); for D–F, each dot corresponds to a different donor (n = 3 healthy donors, n = 2 CAPS); and for G n = 5 healthy donors, n = 7 CAPS patients during active disease and the same n = 7 patients treated with anakinra; For A–F data are represented as mean ± SEM; Ordinary one-way ANOVA test for A–C, except for p.T348M mutant in B one-way Kruskal–Wallis; Wald test two-sided with Benjamini–Hochberg correction for G; significance levels are indicated as follows: *p < 0.05; ns indicates no significant difference (p > 0.05). Source data are provided as a Source Data file.

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