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 4

CARD8 with a V44I mutation exhibits reduced binding to NLRP3 and affects NLRP3 inflammasome assembly.

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CARD8 with a V44I mutation exhibits reduced binding to NLRP3 and affects...
(A) Plasmids expressing intact CARD8, CARD8 V44I mutation, or empty vector were transfected into HEK293 cells along with a plasmid expressing NLRP3. At 48 hours after transfection, cells were harvested and cell lysates were subjected to immunoprecipitation using anti-CARD8 antibody, followed with immunoblotting. (B) mDCs from proband and healthy control were stimulated with LPS (100 ng/ml, 6 hours) or LPS plus nigericin (1.2 μM, 30 minutes). Cells were lysed, and lysates were subjected to immunoprecipitation and immunoblotting. (C) HEK293 cells were transfected with a plasmid expressing NLRP3 along with Myc-tagged intact CARD8 T60, CARD8 T60 P102I, or empty vector. Cells were lysed at 48 hours, and lysates were subjected to immunoprecipitation using anti-Myc antibody, followed by immunoblotting. (D) HEK293 cells were transfected with NLRP3 plasmid along with Myc-tagged intact CARD8 T60, CARD8 T60 P102I, or empty vector. Twenty-four hours later, cells were transfected with plasmids expressing ASC, caspase-1, and pro–IL-1β to allow assembly of the NLRP3 inflammasome. Another 24 hours later, cells were stimulated with nigericin (1.2 μM, 30 minutes). The cultural supernatants were examined for IL-1β concentration by ELISA. Data are shown as mean ± SEM. *P < 0.05, 1-way ANOVA with Dunnett’s post hoc test. (E) mDCs from proband and a healthy control were stimulated with LPS (100 ng/ml, 6 hour) plus nigericin (1.2 μM, 30 minutes). Cells were prepared for Western blot as in Methods. (F) Cell lysates shown in E were prepared for Western blot as in Methods. 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.