Fig. 6: The NLRP3–casp8/3 cleavage cascade leads to GSDME pores for IL-1α release.
From: Human TH17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation
a, Differential gene expression determined by transcriptome analysis of TH17 cells treated as in Fig. 2c (n = 3 individual healthy blood donors). b, RT–qPCR analysis of anti-CD3 and anti-CD28 monoclonal antibody-stimulated, naive T cells in polarizing cytokine conditions (n = 4, one-way ANOVA with Dunnett’s multiple-comparison test). c, Immunoblot analysis of cell lysates from TH17 cells stimulated with anti-CD3 and anti-CD28 monoclonal antibodies for different durations. The data represent three experiments. d, ELISA of cell culture supernatants from TH17 cells with and without deletion of GSDME (left) or GSDMD (right) by CRISPR–Cas9 technology. Individual experiments were normalized to the first time point of analysis on day 2 (n = 3 individual biological samples, two-way ANOVA with Bonferroni’s multiple-comparison test). e, Immunoblot analysis of cell lysates from TH17 cells stimulated with anti-CD3 and anti-CD28 monoclonal antibodies for the indicated time points and of CD14+ monocytes stimulated for 24 h with LPS and 30 min with nigericin. Casp, Caspase. f, Lane view of electropherograms obtained with a Jess Simple Western System for cell lysates of TH17 cells stimulated for 5 d as in e in the presence or absence of the indicated inhibitors. It is a representative experiment. g, Cumulative data of f (one-sample Student’s t-test). AUC, area under the curve. h, Luminex assay of the supernatants of TH17 cells stimulated with plate-bound anti-CD3 (1 μg ml−1, TR66) and phorbol-12,13-dibutyrate for 8 h on day 4 of culture (n = 3 individual biological samples, two-tailed, paired Student’s t-test). i, ELISA of supernatants of TH17 cells stimulated as in f. Each circle indicates an independent blood donor in h and i (n = 4 individual biological samples; two-tailed, paired Student’s t-test).
