4-octyl itaconate reduces human NLRP3 inflammasome constitutive activation with the cryopyrin-associated periodic syndrome p.R262W, p.D305N and p.T350M variants

Abstract

Cryopyrin-associated periodic syndrome (CAPS) is a condition characterized by dominant genetic variants in the NLRP3 gene, which lead to the formation of constitutively active inflammasomes. These inflammasomes play a crucial role in CAPS patients' inflammatory episodes, these being primarily driven by the production of interleukin (IL)-1b. Although treatment with IL-1 blockers is effective for CAPS, some patients develop refractory responses and adverse reactions to these therapies. Consequently, there is a need for novel treatments for CAPS patients. Promising candidates are the derivatives of itaconate, which have been shown to impair NLRP3 inflammasome activation and IL-1β release in blood mononuclear cells from CAPS patients. In this study, we provide insight into the inhibitory mechanisms by which the itaconate derivative 4-octyl itaconate (4-OI) acts on NLRP3 that has different gain-of-function mutations (p.R262W, p.D305N and p.T350M) associated with CAPS. Notably, 4-OI effectively blocks the basal auto-activation of the inflammasome formed by NLRP3 p.R262W, p.D305N and p.T350M variants, which in turn reduces caspase-1 activation, gasdermin D processing, and IL-18 release. Furthermore, after lipopolysaccharide priming of macrophages, 4-OI also decreases IL-1β gene expression and release. Overall, 4-OI impairs CAPS-associated inflammasome function at multiple levels, meaning that therapeutic agents based on itaconate could be a promising therapeutic approach to managing inflammatory episodes in CAPS patients carrying p.R262W, p.D305N or p.T350M variants.

Keywords: Autoinflammatory disease; CAPS; Inflammasome; Itaconate; NLRP3.

Fig. 1

4-octyl itaconate blocks basal IL-18 release from macrophages expressing CAPS-associated NLRP3 variants. A Box plot of Acod1 mRNA expression from Nlrp3^−/− immortalized macrophages (iMos) treated for 16 h with or without doxycycline (1 µg/ml) to induce the expression of either the human wild type NLRP3 or the p.D305N variant, in the absence or presence of MCC950 (10 µM). The data were represented relative to each untreated condition. B Heatmap of gene expression profile of Nlrp3^−/− iMos treated as in A. Each column represents independent experiments. Gene expression values were normalized using Z-score per row (gene-wise normalization). C ELISA for IL-18 released from Nlrp3^−/− iMos expressing human wild-type NLRP3 or human NLRP3 p.D305N, p.R262W, and p.T350M variants treated as in A, with 4-OI (100 µM). For canonical NLRP3 wild-type activation, iMos were then treated with nigericin (10 µM) 30 min. D ELISA for IL-18 released from Nlrp3^−/− iMos expressing human NLRP3 p.R262W variants induced after treatment for 16 h with doxycycline (1 µg/ml) in the absence/presence of MCC950 (10 µM), 4-OI (50, 100 and 200 µM) or LPS (100 ng/ml). E ELISA for IL-18 released from Nlrp3^−/− iMos expressing human NLRP3 p.D305N variant treated as in C, at different times (0, 2, 4, 8, and 16 h). For A–D, graphics are representative of n = 3–5 independent experiments; For A, the middle line depicts the mean, error bars represent minimum and maximum, and bounds of box represent the 25 th to 75 th percentile respectively; for C–E, the data are represented as mean ± SEM; For A, B, data is from GEO dataset #GSE246713. An ordinary one-way ANOVA test was used for A–D; t test was used in E; *p < 0.05; **p < 0.0021; ***p < 0.0002; ****p < 0.0001; ns, no significant difference (p > 0.05)

Fig. 2

4-octyl itaconate inhibits basal caspase-1 activation, GSDMD cleavage and pyroptosis. A Western blot for NLRP3, GSDMD, IL-1β, caspase 1 and β-actin in cell lysates and supernatants from Nlrp3^−/− immortalized macrophages expressing human NLRP3 p.D305N variant treated with doxycycline (1 µg/ml) in the absence/presence of MCC950 (10 µM), 4-OI (100 µM) or LPS (100 ng/ml) for 16 h. B Lactate dehydrogenase (LDH) released from cells treated as in A. Graphic is representative of n = 3 independent experiments and data are shown as mean ± SEM; Western blots are representative of n = 3 independent experiments; an ordinary one-way ANOVA test was used in B; *p < 0.05; ns, no significant difference (p > 0.05)

Fig. 3

4-octyl itaconate affect the expression of Il1b, Tnf and Il6, but not the expression of Il18. A Gene expression (2^−ΔCt) for Tnf, Il1b, Il18 and Il6 from Nlrp3^−/− immortalized macrophages (iMos) expressing human NLRP3 p.D305N variant induced after treatment for 16 h with doxycycline (1 μg/ml) in the absence/presence of MCC950 (10 µM), 4-OI (100 µM) or LPS (100 ng/ml). B, C Seahorse analysis of glycolysis in Nlrp3^−/− iMos treated for 16 h with or without doxycycline (1 µg/ml) to induce the expression of the human NLRP3 p.D305N variant treated as in A. For A data are representative of n = 3 independent experiments. For B, C data are derived from n = 4 biological replicates, which are representative of n = 3 independent experiments. Graphics are represented as mean ± SEM; an ordinary one-way ANOVA test was used; *p < 0.05; **p < 0.0021; ns, no significant difference (p > 0.05)

Fig. 4

4-octyl itaconate inhibits the ASC-oligomerization induced by NLRP3 p.D305N variant. A Representative fluorescence images of HEK293T cells stably expressing wild-type or p.D305N YFP-NLRP3 treated for 24 h with MCC950 (10 µM) or 4-OI (100 µM); NLRP3 is shown in green. Images are representative of n = 3 independent experiments. B Percentage of HEK293T cells with a puncta distribution of cells treated as in A. C BRET signal recorded from HEK293T cells expressing wild-type or p.D305N YFP-NLRP3-Luc, treated for 16 h with MCC950 (10 µM) or 4-OI (100 µM). D Percentage of ASC specking HEK293T cells transfected with ASC-RFP and NLRP3-YFP wild type (WT) or p.D305N, treated for 16 h with MCC950 (10 µM) or 4-OI (100 µM). Graphics are representative of at least n = 3 independent experiments and data are represented as mean ± SEM; an ordinary one-way ANOVA test was used in B, C, t test was used in D; *p < 0.05; **p < 0.0021; ns, no significant difference (p > 0.05)