Figure 5.
The RAC2 A59S human patient mutation activates the NLRP3 inflammasome. (A) HA-RAC2 (WT), HA-RAC2 A59S, and HA-RAC2 E62K mutants were expressed for 16 h in HEK293T cells. HA-RAC2 activation level measurements were assessed by GST–PAK–RBD pull-down assays and immunoblotting (IB). (B–E) HEK293T cells were transfected for 16 h with plasmids encoding the NLRP3 inflammasome components (Inflam. mix) together with the WT or RAC2 E62K or RAC2 A59S forms. Cleaved IL-1β secretion in the culture supernatant (Sup) and protein expression in the cell lysates were assessed by immunoblotting. (C) Inhibitors of NLRP3 (MCC950), PAK (IPA3 or AZ 13711265), or pan-Caspases (Emricasan) were added 6 h before transfection. Data are representative for n = 3. (D and E) Inflammasome was reconstituted in HEK293T to evaluate the effect of the NLRP3 T659A mutation on IL-1β secretion compared to NLRP3 in cells expressing (D) RAC2 A59S or (E) RAC2 E62K. Data are representative for n = 3. Molecular weight (kD) is indicated in the figure. (F) U937 Cas9 (Cas9), NLRP3 KO, or GSDMD KO cells were cotransduced for 72 h with Vpx containing virus-like particles and HA-tagged RAC2 A59S or E62K encoding lentiviral vectors. Empty HIVSFFV-HARAC2-IRES-GFP vector was used as control. LPS was added for 8 h to induce the expression of pro-IL-1β. Supernatants were analyzed using ELISA for IL-1β. Data are mean ± SEM. Statistical analyses were performed using two-way ANOVA. ****P < 0.0001. Data are representative for n = 3. (G) LDH release was measured in the culture medium of U937 Cas9 (Cas9), NLRP3 KO, or NINJ1 KO cells expressing empty vector (Control), HA-tagged RAC2 A59S, or RAC2 E62K. The graph represents the percentage of cytotoxicity. Data are representative for n = 3. (H) Single-cell analysis for a blood sample of a patient harboring a RAC2 A59S variant and a representative control healthy donor blood sample drawn at the same time and analyzed in parallel are shown. Uniform Manifold Approximation and Projection plots of whole blood cells from control and RAC2 A59S patient donors. After alignment, joint clustering allows to detect 13 cell populations. pDC, plasmacytoid dendritic cell; mDC, myeloid dendritic cell; ISG, IFN-stimulated genes. (I) Violin plots showing the distribution of gene expression of IL-1β and NLRP3 genes in the cell clusters for both control (red) and RAC2 A59S patient (blue). NKT, natural killer T. Source data are available for this figure: SourceData F5.

The RAC2 A59S human patient mutation activates the NLRP3 inflammasome. (A) HA-RAC2 (WT), HA-RAC2 A59S, and HA-RAC2 E62K mutants were expressed for 16 h in HEK293T cells. HA-RAC2 activation level measurements were assessed by GST–PAK–RBD pull-down assays and immunoblotting (IB). (B–E) HEK293T cells were transfected for 16 h with plasmids encoding the NLRP3 inflammasome components (Inflam. mix) together with the WT or RAC2 E62K or RAC2 A59S forms. Cleaved IL-1β secretion in the culture supernatant (Sup) and protein expression in the cell lysates were assessed by immunoblotting. (C) Inhibitors of NLRP3 (MCC950), PAK (IPA3 or AZ 13711265), or pan-Caspases (Emricasan) were added 6 h before transfection. Data are representative for n = 3. (D and E) Inflammasome was reconstituted in HEK293T to evaluate the effect of the NLRP3 T659A mutation on IL-1β secretion compared to NLRP3 in cells expressing (D) RAC2 A59S or (E) RAC2 E62K. Data are representative for n = 3. Molecular weight (kD) is indicated in the figure. (F) U937 Cas9 (Cas9), NLRP3 KO, or GSDMD KO cells were cotransduced for 72 h with Vpx containing virus-like particles and HA-tagged RAC2 A59S or E62K encoding lentiviral vectors. Empty HIVSFFV-HARAC2-IRES-GFP vector was used as control. LPS was added for 8 h to induce the expression of pro-IL-1β. Supernatants were analyzed using ELISA for IL-1β. Data are mean ± SEM. Statistical analyses were performed using two-way ANOVA. ****P < 0.0001. Data are representative for n = 3. (G) LDH release was measured in the culture medium of U937 Cas9 (Cas9), NLRP3 KO, or NINJ1 KO cells expressing empty vector (Control), HA-tagged RAC2 A59S, or RAC2 E62K. The graph represents the percentage of cytotoxicity. Data are representative for n = 3. (H) Single-cell analysis for a blood sample of a patient harboring a RAC2 A59S variant and a representative control healthy donor blood sample drawn at the same time and analyzed in parallel are shown. Uniform Manifold Approximation and Projection plots of whole blood cells from control and RAC2 A59S patient donors. After alignment, joint clustering allows to detect 13 cell populations. pDC, plasmacytoid dendritic cell; mDC, myeloid dendritic cell; ISG, IFN-stimulated genes. (I) Violin plots showing the distribution of gene expression of IL-1β and NLRP3 genes in the cell clusters for both control (red) and RAC2 A59S patient (blue). NKT, natural killer T. Source data are available for this figure: SourceData F5.

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