Fig 1. H. capsulatum induces NLRP3-dependent inflammasome activation.

(A, B and C) BMDCs from wild type mice were stimulated with live H. capsulatum at (A and B) different MOI and (C) at MOI of 20 for 6, 12, 18 and 24 h. (D) BMDCs were pretreated with caspase-1 inhibitor (Z-YVAD-FMK) at different concentrations before stimulation with H. capsulatum for another 18 h. (E, G and H) BMDCs and (F) sorted MHCII⁺CD11c⁺ splenic DCs from wild type and NLRP3-deficient (Nlrp3^-/-) mice were stimulated with H. capsulatum at MOI of 10 and 20 for 18 h. (B and G) Cell-free supernatants and cell lysates were analyzed by Western blotting with indicated antibodies. (H) ASC pyroptosome and inflammasome components were analyzed in cell pellets and cell lysates, respectively. Stimulation with LPS (500 ng/ml, 6 h) plus ATP (5 mM, 30 minutes) (L+A) was used as a positive control for NLRP3-dependent IL-1β induction. (I and J) Wild type and NLRP3-deficient mice were intravenously infected with H. capsulatum (1 × 10⁷). (I) Fungal burden in the spleen was determined on day 11 after infection. It is shown as CFU per organ (n = 6). (J) Survival was analyzed by log-rank test. (K) Wild type and NLRP3-deficient mice were intravenously infected with 2.5 × 10⁵ of H. capsulatum. MHCII⁺CD11c⁺ splenic DCs were sorted from mice on day 5 after infection and stimulated with live H. capsulatum at MOI of 1 for 18 h. (A, C, D, E, F and K) IL-1β in cell-free supernatants were quantified by ELISA (n = 3). One representative of three (A, B, C, D and E) or two (F, G, H, I and J) independent experiments is presented. ND, not detectable. CM, complete medium. * p < 0.05, ** p < 0.01, *** p < 0.001 [one-way ANOVA with Tuckey post-hoc analysis (A, B and D); 2-tailed t-test (E, F, I and K); log-rank test (J)].