Monocytes and macrophages isolated from patients harboring the RAC2 E62K or RAC2 A59S mutations have an abnormal NLRP3 inflammasome activation. (A) PBMCs isolated from healthy donors (Control) or patients with RAC2 mutation (RAC2 A59S and RAC2 E62K) were treated for 1 h with FAM–YVAD–FLICA before CD14 labeling and then analyzed by flow cytometry. Cells with high expression of CD14, corresponding to monocytes, were gated and then analyzed for the presence of Caspase-1 speck using FITC-Caspase-1-A and FITC-Caspase-1-H. Monocytes with Caspase-1 speck were defined by a high Caspase-1-A/Caspase-1-H ratio. Control conditions were repeated at least three times, and representative data are shown. (B and C) Graphic representation of percentage of cells with Caspase-1 specks. Control conditions are expressed as the mean ± SEM of at least three independent experiments. (D) Monocyte-derived macrophages isolated from control healthy donors or patients presenting RAC2 A59S or RAC2 E62K variants were stained with anti-ASC (green), anti-NLRP3 (red), Hoechst 33342 for nuclei (blue), and phalloidin-Alexa 647 for actin filaments (magenta). The arrow indicates ASC speck colocalization with NLRP3 staining. Scale bar represents 10 µm. (E and F) Monocyte-derived macrophages isolated from patient RAC2 A59S (E) or a RAC2 E62K (F) variants were treated with LPS alone or LPS and MCC950 for 8 h to induce the expression of IL-1β in the absence or presence of NLRP3 inhibition, respectively. Non-treated cells (NT) were used as control. IL-1β secretion was measured using ELISA. The data were reused in Fig. S1, A and B, to illustrate the gating strategy. Data are mean ± SEM. Data are representative for n = 2.