Journal: Nature Communications
Article Title: The fungal peptide toxin Candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes
Figure Lengend Snippet: Potassium-dependent and actin-dependent inflammasome activation. a IL-1β and IL-8 levels measured by ELISA in culture supernatants of LPS-primed hMDMs treated with synthetic Candidalysin or Nigericin for 5 h. Selected samples were pre-treated with the actin cytoskeleton inhibitor Cytochalasin D or the inhibitor solute control DMSO 1 h prior to administration of Candidalysin. b IL-1β release measured by ELISA in culture supernatants of LPS-primed hMDMs that were infected with C . albicans Wt, ece1 Δ/Δ, efg1 Δ/Δ/ cph1 Δ/Δ, hgc1 Δ/Δ mutant strains, or heat-killed Wt (MOI 10) in presence or absence of synthetic Candidalysin for 5 h. c – f IL-1β and c IL-8 or d , e TNF levels measured by ELISA in culture supernatants of LPS-primed c , f hMDMs d mBMDMs, and e mBMDCs. Phagocytes were treated with synthetic Candidalysin or Nigericin for 5 or 4 h (BMDCs). Selected samples were pre-treated with the following inhibitors 1 h prior to administration of Candidalysin: c – e the potassium channel inhibitor glibenclamide or inhibitor solute control DMSO, KCl was added after LPS priming, f (2R,4 R)-4-aminopyrrolidine-2,4-dicarboxylate (PDTC). g Intracellular ROS production in hMDMs pre-loaded with 20 μM H2DCF-DA for 30 min and infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 10) or treated with H 2 O 2 (positive control) for 5 h. Fluorescence (Ex 485/Em 535) measured immediately after infection was subtracted from fluorescence (Ex 485/Em 535) measured after 5 h. h Total ROS production in hMDMs subjected to synthetic Candidalysin or PMA (positive control) was monitored by Luminol-enhanced chemiluminescence. Relative luminescence units (RLU) were recorded for 60 min and the difference between maximum and minimum luminescence values was calculated. Data are represented as scatterplot and median of at least three different donors ( n ≥ 3) or independent experiments. For statistical analysis, a one-way ANOVA with Dunnett’s multiple comparison test was used. For analysis of the different C . albicans mutants, a two-way ANOVA with Sidak’s multiple comparison test was applied. *** p ≤ 0.001, * p ≤ 0.05, n/a not applicable, nd not detectable
Article Snippet: For inhibitor studies, the following compounds were added 1 h prior to infection: the caspase-1-inhibitor Z-YVAD-FMK (88.9 µM; Merck) or Ac-YVAD-cmk (20 µM, Invivogen), the caspase-1-inhibitor VX-765 (50 µg/mL, Invivogen) or vehicle control, the actin cytoskeleton inhibitor Cytochalasin D (10 µM; Sigma Aldrich), the V-ATPase inhibitor Bafilomycin A1 (50–500 nM; Sigma Aldrich), the ROS inhibitor 4-Aminopyrrolidine-2,4-dicarboxylate (PDTC) (100, 500 µM; Enzo Life Sciences), the potassium channel inhibitor glibenclamide (25 µM; Sigma Aldrich) or the RIP1-kinase inhibitor Necrostatin-1 (12.5–50 µM; Biomol).
Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Infection, Mutagenesis, Positive Control, Fluorescence