Abstract

NLRP3-associated autoinflammatory disease (NLRP3-AID or CAPS) is an heterogenous group of monogenic autoinflammations associated with NLRP3 gain-of-function mutations. The poor functional characterization of most NLRP3 variants is a barrier to diagnosis although patients can be efficiently treated with anti-IL-1 approaches. In addition, while NLRP3 inflammasome is controlled by coordinated priming and activation signals, gain-of-functions of NLRP3 variants have been only investigated in response to priming. Here, we functionally characterize 34 NLRP3 variants in vitro by determining their activity in response to induction, priming and/or activation signals, and their sensitivity to inhibitors. We highlight the functional diversity of the gain-of-function mutants and describe four groups based on their profile of signals required for their activation, that correlate partly with the symptoms severities. We identify a new group of NLRP3 mutants responding to the activation signal without priming, with patients often misdiagnosed. Our results identify key NLRP3 residues controlling the inflammasome activity and sensitivity to inhibitors. The comparison of four inhibitors on the 34 variants identifies inhibitory mechanisms with broader efficiency for future drug design. Altogether, our results provide new insights on NLRP3 activation and an explanatory mechanism for NLRP3-AID heterogeneity, and original tools for NLRP3-AID diagnosis and anti-inflammatory disease drug development. eTOC SummaryFunctional characterization of 34 CAPS-associated NLRP3 variants identifies polymorphisms versus gain-of-function pathogenic mutants, and highlights diversities in the signals controlling their activation and in their sensitivity to inhibitors. This study provides tools for CAPS diagnosis and anti-inflammation drug development and insights on NLRP3 control mechanisms.