Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease
Liming Mao, … , Ivan J. Fuss, Warren Strober
Liming Mao, … , Ivan J. Fuss, Warren Strober
Published February 6, 2018
Citation Information: J Clin Invest. 2018;128(5):1793-1806. https://doi.org/10.1172/JCI98642.
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Research Article Gastroenterology

Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

Abstract

In these studies, we evaluated the contribution of the NLRP3 inflammasome to Crohn’s disease (CD) in a kindred containing individuals having a missense mutation in CARD8, a protein known to inhibit this inflammasome. Whole exome sequencing and PCR studies identified the affected individuals as having a V44I mutation in a single allele of the T60 isoform of CARD8. The serum levels of IL-1β in the affected individuals were increased compared with those in healthy controls, and their peripheral monocytes produced increased amounts of IL-1β when stimulated by NLRP3 activators. Immunoblot studies probing the basis of these findings showed that mutated T60 CARD8 failed to downregulate the NLRP3 inflammasome because it did not bind to NLRP3 and inhibit its oligomerization. In addition, these studies showed that mutated T60 CARD8 exerted a dominant-negative effect by its capacity to bind to and form oligomers with unmutated T60 or T48 CARD8 that impeded their binding to NLRP3. Finally, inflammasome activation studies revealed that intact but not mutated CARD8 prevented NLRP3 deubiquitination and serine dephosphorylation. CD due to a CARD8 mutation was not effectively treated by anti–TNF-α, but did respond to IL-1β inhibitors. Thus, patients with anti–TNF-α–resistant CD may respond to this treatment option.

Authors

Liming Mao, Atsushi Kitani, Morgan Similuk, Andrew J. Oler, Lindsey Albenberg, Judith Kelsen, Atiye Aktay, Martha Quezado, Michael Yao, Kim Montgomery-Recht, Ivan J. Fuss, Warren Strober

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Figure 5

Mutant CARD8 exerts a dominant-negative effect on the NLRP3 inflammasome.

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Mutant CARD8 exerts a dominant-negative effect on the NLRP3 inflammasome...
(A) HEK293 cells were transfected with plasmids expressing intact and mutant CARD8 T60 alone or together (half of the amount per plasmids, to mimic the heterozygous status of the proband). After 48 hours of incubation, cell lysates were obtained and subjected to immunoprecipitation and immunoblotting to determine CARD8 binding to NLRP3. (B) HEK293 cells were transfected with NLRP3 and Flag-tagged CARD8 T48 plasmids along with intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-CARD8 antibody) and immunoblotting. (C) HEK293 cells were transfected with NLRP3 and CARD8 T48 plasmids along with intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-NLRP3 antibody) and immunoblotting. (D) HEK293 cells were transfected with NLRP3 and intact CARD8 T48 or T60 alone or together with mutant CARD8 T60 plasmids. Twenty-four hours later, T cells were transfected with ASC, caspase-1, and pro–IL-1β plasmids to allow the assembly of NLRP3 inflammasome. Twenty-four hours later, cells were stimulated with nigericin (1.2 μM) for 30 minutes. Cultural supernatants were collected for IL-1β ELISA assay. Data are shown as mean ± SEM. *P < 0.05, 1-way ANOVA with Tukey’s post hoc test. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.