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 3

The CARD8 mutation affects NLRP3 and AIM2 inflammasome, but not pyrin and NLRC4 inflammasome, activation.

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The CARD8 mutation affects NLRP3 and AIM2 inflammasome, but not pyrin an...
mDCs from the proband and a healthy control were primed with LPS (100 ng/ml, 6 hours) and then stimulated with ATP (5 mM, A), poly(dA:dT) (1 μg/ml, 2 hours, B), TcdB (1 μg/ml, 2 hours, C), or flagellin (1 μg/ml, 2 hours, D) to activate the NLRP3, AIM2, pyrin, or NLRC4 inflammasomes, respectively. Culture supernatants were collected and subjected to IL-1β (A–D) and IL-6 (E) assay by ELISA. Primary mDCs from proband and healthy control were treated with or without LPS (100 ng/ml) for 6 hours, after which cells were harvested and subjected to RNA extraction. Quantitative reverse-transcriptase PCR (qRT-PCR) of the extracted RNA was performed to determine the expression of ASC (F), caspase-1 (G), NLRP3 (H), and pro–IL-1β (I). Data are shown as mean ± SEM. n = 3. **P < 0.01; *P < 0.05, 2-tailed Student’s t test. All data are representative of 3 independent experiments.