Session Type: Abstract Submissions (ACR)
Background/Purpose: 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 cytokine IL-1β. Some investigators have proposed a model of NLRP3-inflammasome containing CARD8, another adaptor protein, but the role of CARD8 in the NLRP3-inflammasome system remains obscure. In a previous ACR meeting, we presented some data suggesting that CARD8 normally plays a role as a negative regulator of NLRP3-inflammasome. In the assay system using HEK293 cells transiently transfected with genes for inflammasome components, CARD8 interacted with wild-type NLRP3 resulting in suppression of IL-1β secretion. In contrast, CARD8 did not interact with CAPS-associated mutant NLRP3. Current study aimed to extend the previous study to obtain more convincing data using also cells endogenously expressing components of NLRP3 inflammasome and CARD8.
Methods: HEK293 cells were transiently transfected with ASC, CARD8, procaspase-1, proIL-1β, and NLRP3. The interaction of CARD8 and NLRP3 was studied by immunoprecipitation using lysates of peripheral blood mononuclear cells (PBMCs). Human monocyte-derived macrophages (HMDMs) were obtained by culture of monocytes from peripheral blood cells with GM-CSF for 7 days. Control or specific siRNA against CARD8 was transfected to HMDMs and the knockdown efficacy was evaluated by quantitative RT-PCR. After priming with LPS, HMDMs were stimulated by ATP, and secreted IL-1β was measured by ELISA.
Results: When HEK293 cells were transfected with every inflammasome components, large amounts of IL-1β were secreted spontaneously, which was partially but significantly reduced by coexpression of CARD8. On the other hand, CARD8 did not suppress IL-1β secretion from the cells transfected with CAPS-associated mutant NLRP3 (R260W, D303N, H312P, or N477K). In immunoprecipitation experiments using resting PBMCs from healthy subjects, NLRP3 was precipitated with CARD8, but not with ASC. When PBMCs were primed with LPS followed by ATP stimulation, however, NLRP3 was precipitated with ASC instead of CARD8. By stimulating HMDMs with LPS and ATP, HMDMs secreted IL-1β, and it was further increased by CARD8 knockdown.
Conclusion: These results support our hypothesis that CARD8 normally associates with NLRP3 to prevent unnecessary activation of the inflammasome by subtle stimuli. Taken together with previous data which showed CARD8 was unable to bind to NLRP3 with CAPS-related mutation, one of the mechanisms of hypersensitivity of the NLRP3 inflammasome in CAPS patients might be the escape from CARD8 restriction.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-possible-mechanism-of-nlrp3-inflammasome-hypersensitivity-in-cryopyrin-associated-periodic-syndrome/