Fig. 4: Acute INPP5D inhibition results in inflammasome activation and an increase in the secretion of IL-1ß and IL-18 in iMG cultures.
From: INPP5D regulates inflammasome activation in human microglia

a Fold change of secreted cytokines from iMGs treated with LPS (100 ng/mL) or 3AC (1.25 μM) for 6 h across iMGs derived from four iPSC lines, assayed by ELISA. Fold change was calculated compared to vehicle-treated cells. b Levels of IL1B following 6 h treatment with vehicle, LPS (100 ng/mL), or 3AC (1.25 μM, 2.5 μM) treatment as measured by quantitative real-time PCR (qPCR). Data are normalized to GAPDH expression then values were normalized to vehicle treatment within each experiment. n = 3 differentiations with well-level data shown as dots. One-way ANOVA with Dunnett’s T3 multiple comparisons test. c Secreted levels of IL-1ß measured for the same experiments as (b) following 6 h treatment as measured by ELISA. IL-1ß levels were normalized to 1.25 μM 3AC-treated conditions within each experiment. n = 3 differentiations with well-level data shown as dots. d Representative western blot of protein expression in iMGs with biallelic loss-of-function mutations generated with CRISPR targeting. Loss of INPP5D protein observed repeatedly over 3 differentiations. e, f WT INPP5D iMGs and KO INPP5D iMGs were treated with 3AC for 6 h and the levels of IL-1ß and IL-18 were measured by ELISA. Multiple two-sided t-tests, Two-stage step-up (Benjamini, Krieger, and Yekutieli), **q < 0.01; ***q < 0.005; ****q < 0.001; LLOD= lower limit of detection. n = 3 biological replicates. g, h Treatment of iMGs with either vehicle or 3AC (5 μM) with either VX-765 (25 μM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 measured via ELISA following treatments and normalized to 3AC-treated samples in each experiment. One-way ANOVA with Sidak’s multiple comparison test. 3 differentiations, n = 10 per condition. i, j iMGs were treated with either vehicle, primed with LPS (100 ng/mL) for 3 h and then treated with nigericin (10 μM) for 1 h, or treated with 3AC (1.25 μM or 5 μM) for 2 h. Cells were immunostained for ASC and IBA1, DNA is stained with DAPI (i) and imaged using confocal microscopy. Scale bars = 100 μm. j Quantification of ASC specks. Number of cells analyzed: n = 226 (vehicle); 151 (1.25 μM 3AC); 173 (5.0 μM 3AC) over three experiments and 10 images per condition. Kruskal–Wallis test to determine significance. k, l Treatment of iMGs with either vehicle or 3AC (5 μM) with either MCC950 (10 μM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 in the media were measured and normalized to 3AC-treated samples in each experiment. One-way ANOVA with Sidak’s multiple comparison test. n = 4 differentiations with well-level data shown as dots. m A summary figure of experiments performed to interrogate inflammasome activation, created using BioRender.com. For all graphs, data are presented as mean values ± SEM. For b, c, g, h, j, l: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Cell lines used for experiments are detailed in Supplementary Data 8. See also Supplementary Figs. 7 and 8.