MARÍA CRISTINA MOLINA LÓPEZ¹, DIEGO ANGOSTO BAZARRA¹, Ana Tapia Abellán², PABLO PELEGRIN VIVANCOS¹

(1) CIRUGÍA DIGESTIVA, ENDOCRINA Y TRASPLANTE DE ÓRGANOS ABDOMINALES, IMIB-Arrixaca, España
(2) Eberhard Karls Universität Tübingen, Alemania, Alemania

Cryopyrin-associated periodicsyndrome (CAPS) is a hereditary autoinflammatory disorder that results from again-of-function mutation in NLRP3 gene with overproduction of IL-1β as consequence. Different pathogen and host-related signals are required to induce and activate NLRP3 inflammasome. However, mutated NLRP3 inflammasome is formed and activated without the requirement of previous signals. Molecules, as MCC950, are specific inhibitors of NLRP3 and promise novel therapeutical strategies for CAPS. Here, we aim to study the differential activation of distinct gain-of-function mutants of NLRP3 induced by different pro-inflammatory triggers.

Nlrp3-/- immortalized mouse macrophages (iMOs) with a Tet-on inducible system were used to express different mutated NLRP3 (D303N, T348M, R260W) upon doxycycline treatment .iMOs were then treated with LPS, Pam3CKS4, IL-6, ATP or uric acid crystals using different concentrations and times in absence or presence of MCC950. Cell death and cytokines measurement assays were performed in cell supernatants.

The expression of mutant NLRP3 results in IL-1b release after LPS, Pam3CKS4 and IL-6 treatment, reaching maximum release after 16h of stimulation. Released IL-1β was significant after 4h of treatment with LPS and Pam3CKS4 and later after 8h with IL-6. However, the release of TNF-α was found after 2h of stimulation, and blocking TNF-α by a neutralizing antibody, partially decreased IL-1β release. MCC950 was able to block the release of IL-1β, IL-18, HMGB1 and IL-1α induced with the different triggers, while TNF-α release was not affected.

LPS, Pam3CKS4 and IL-6 are able to activate the release of IL-1β when mutant gain-of-function NLRP3 are expressed. Initial TNF-α production is partially involved in the amplification of mutant NLRP3 activation. MCC950 blocked D303N-NLRP3, reducing the release of IL-1β, IL-1α, HMGB1, IL-18 and pyroptosis as markers of inflammasome activation, but not TNF-α release.