Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Mutated NLRP3 in Cryopyrin-Associated Periodic Syndromes Is Released from Restriction by CARD8.
Ito2, Sayaka, Hara3, Yukichi, Shima1, Yukio, Kubota3, Tetsuo
NLRP3 in monocytes and other cell types plays a role in innate immunity as one of the intracellular pathogen recognition receptors, and its mutation is responsible for cryopyrin-associated periodic syndromes (CAPS). Once activated, NLRP3 forms a protein complex called the inflammasome with procaspase-1 and an adaptor protein ASC, leading to activation of caspase-1 and production of mature proinflammatory cytokines IL-1b and IL-18. Some experiments using cells expressing truncated NLRP3 indicated interaction between the NACHT domain of the NLRP3 and the FIIND domain of another adaptor protein CARD8, but involvement of CARD8 in NLRP3 inflammasome remains controversial. We herein aimed to clarify the role of CARD8 in NLRP3 inflammasome and its relevance to the pathophysiology of CAPS.
HEK 293 cells were transiently transfected with ASC, CARD8, procaspase-1, proIL-1b, and NLRP3 in wildtype or with CAPS-associated mutation. 24h later, whole cell lysates were provided for immunoprecipitation and western blotting. 48h after transfection, IL-1b in the culture supernatant was measured by ELISA.
Wild type full-length NLRP3 was immunoprecipitated with CARD8, but interestingly, this interaction was inhibited by coexpression of ASC in a dose-dependent manner, suggesting that affinity between NLRP3 and ASC is higher than that between NLRP3 and CARD8. In contrast, NLRP3 with CAPS-associated mutation R260W or H312P was unable to interact with CARD8. Cells expressing wildtype NLRP3, ASC, procaspase-1 and proIL-b spontaneously produced IL-b, and it was significantly suppressed by cotransfection with full-length CARD8. Cotransfection of the FIIND domain of CARD8 showed less, but still significant, suppressive effect on the IL-1b production.
CARD8 interacts with full-length wildtype NLRP3, and may prevent the NLRP3 inflammasome from unristricted activation. Disability of CARD8 to interact with mutated NLRP3 may explain continuous release of IL-1b by monocytes from CAPS patients.
To cite this abstract, please use the following information:
Ito, Sayaka, Hara, Yukichi, Shima, Yukio, Kubota, Tetsuo; Mutated NLRP3 in Cryopyrin-Associated Periodic Syndromes Is Released from Restriction by CARD8. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :2228