Go to JCI Insight
  • About
  • Editors
  • Consulting Editors
  • For authors
  • Publication ethics
  • Alerts
  • Advertising
  • Job board
  • Subscribe
  • Contact
  • Current issue
  • Past issues
  • By specialty
    • COVID-19
    • Cardiology
    • Gastroenterology
    • Immunology
    • Metabolism
    • Nephrology
    • Neuroscience
    • Oncology
    • Pulmonology
    • Vascular biology
    • All ...
  • Videos
    • Conversations with Giants in Medicine
    • Author's Takes
  • Reviews
    • View all reviews ...
    • Immune Environment in Glioblastoma (Feb 2023)
    • Korsmeyer Award 25th Anniversary Collection (Jan 2023)
    • Aging (Jul 2022)
    • Next-Generation Sequencing in Medicine (Jun 2022)
    • New Therapeutic Targets in Cardiovascular Diseases (Mar 2022)
    • Immunometabolism (Jan 2022)
    • Circadian Rhythm (Oct 2021)
    • View all review series ...
  • Viewpoint
  • Collections
    • In-Press Preview
    • Commentaries
    • Research letters
    • Letters to the editor
    • Editorials
    • Viewpoint
    • Top read articles
  • Clinical Medicine
  • JCI This Month
    • Current issue
    • Past issues

  • Current issue
  • Past issues
  • Specialties
  • Reviews
  • Review series
  • Conversations with Giants in Medicine
  • Author's Takes
  • In-Press Preview
  • Commentaries
  • Research letters
  • Letters to the editor
  • Editorials
  • Viewpoint
  • Top read articles
  • About
  • Editors
  • Consulting Editors
  • For authors
  • Publication ethics
  • Alerts
  • Advertising
  • Job board
  • Subscribe
  • Contact
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.
View: Text | PDF
Research Article Gastroenterology

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

  • Text
  • PDF
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

×

Figure 6

Mutant CARD8 T60 disrupts interaction between T48 and NLRP3.

Options: View larger image (or click on image) Download as PowerPoint
Mutant CARD8 T60 disrupts interaction between T48 and NLRP3.
(A) HEK293 ...
(A) HEK293 cells were transfected with Flag-tagged CARD8 T48 and with Myc-tagged intact or mutant CARD8 T60. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (with anti-Flag antibody), followed by immunoblotting. (B) HEK293 cells were transfected with Myc-tagged CARD8 T60 along with Flag-tagged intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-Myc antibody), followed by immunoblotting. (C) HEK293 cells were transfected with Flag-tagged intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (D) HEK293 cells were transfected with Flag-tagged intact CARD8 T60 plasmid alone or together with a mutant CARD8 T60 plasmid. After 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (E) HEK293 cells were transfected with a Flag-tagged CARD8 T48 along with intact or mutant CARD8 T60 plasmids: after 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (F) Diagram showing that mutant CARD8 T60 exhibits increased binding with intact T60 and T48 and that this binding is thought to block interaction between intact CARD8 T60 or T48 with NLRP3. All data are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Copyright © 2023 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

Sign up for email alerts