Caspase-1 activation triggered by RAC2 active variants in circulating myeloid cells and primary human monocyte–derived macrophages. (A–E) Whole blood circulating leukocytes from healthy donors (Control) or patient RAC2 E62K variant were incubated with the FAM–YVAD–FLICA probe for 1 h before fluorescent antibodies staining and analyzed by flow cytometry. CD14⁺ monocytes and CD66b⁺ granulocytes were gated as well as cells with low expression of CD14, CD66b, and CD16, corresponding to lymphocytes. Cells were analyzed for (A and B) FAM–YVAD–FLICA probe MFI or (C and D) for the presence of active Caspase-1 specks using Caspase-1-A and Caspase-1-H ratio analysis. Cells with Caspase-1 specks 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. (A) Overlay of the FAM–YVAD–FLICA fluorescence intensity measured in monocytes, granulocytes, and lymphocytes of healthy donors (Control) and patient variant RAC2 E62K. (B) Quantification of the FAM–YVAD–FLICA MFI measured in monocytes (Mo), granulocytes (Gr), and lymphocytes (Ly) of healthy donors (Control) and patient variant RAC2 E62K. Control conditions were repeated at least three times, and representative data are shown. (C and D) Gating of the monocytes and granulocytes with speck in healthy donors (Control) and patient variant RAC2 E62K using Caspase-1-A and Caspase-1-H ratio. (E) Graphic representation of the percentage of monocytes and granulocytes with Caspase-1 specks defined by a high Caspase-1-A/Caspase-1-H ratio in healthy donors (Control) and patient variant RAC2 E62K. (F and G) Human monocyte–derived macrophages were cotransduced for 72 h with Vpx containing virus-like particles and HA-tagged WT and mutated RAC2 encoding lentiviral vectors. Empty HIVSFFV-HARAC2-IRES-GFP vector (EV) was used as control. LPS was added for 8 h to induce the expression of pro-IL-1β. (F and G) Supernatants were analyzed using ELISA for (F) IL-1β and (G) IL-18. Data are mean ± SEM. Statistical analyses were performed using ordinary one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are representative for n = 3. (H) Volcano plot depicting differentially expressed genes in MOLM-13 transduced with RAC2 WT or RAC2 E62K compared to MOLM-13 transduced with empty vector. Red dots represent genes expressed at higher levels while blue dots represent genes with higher expression levels. Y axis denotes −log₁₀ P values, and x axis shows log₂ fold change values. Volcano plots were generated using VolcaNoseR. Data are representative for n = 5.