Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)–LRR oligomerization interface

• Published in: Journal of allergy and clinical immunology Vol. 142; no. 6; pp. 1956 - 1967.e6
• Main Authors: Moghaddas, Fiona, Zeng, Ping, Zhang, Yuxia, Schützle, Heike, Brenner, Sebastian, Hofmann, Sigrun R., Berner, Reinhard, Zhao, Yuanbo, Lu, Bingtai, Chen, Xiaoyun, Zhang, Li, Cheng, Suyun, Winkler, Stefan, Lehmberg, Kai, Canna, Scott W., Czabotar, Peter E., Wicks, Ian P., De Nardo, Dominic, Hedrich, Christian M., Zeng, Huasong, Masters, Seth L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2018; Mosby
• Subjects: Autoinflammatory disease; IL-18; inflammasome; IPAF; macrophage activation syndrome; NLRC4; Nod-like receptor; periodic fever syndrome; Autoinflammatory disease; AIFEC; NLRC4; LRR; NLRC4-AID; CARD; ASC; NBD; periodic fever syndrome; CSF; NLRP3; T3SS; WT; CRISPR; NOD; KO; macrophage activation syndrome; IPAF; inflammasome; IL-18; IL-18BP; Nod-like receptor; HD; MAS; NAIP
• ISSN: 0091-6749; 1097-6825
• DOI: 10.1016/j.jaci.2018.04.033

Monogenic autoinflammatory disorders are characterized by dysregulation of the innate immune system, for example by gain-of-function mutations in inflammasome-forming proteins, such as NOD-like receptor family CARD-containing 4 protein (NLRC4). Here we investigate the mechanism by which a novel mutation in the leucine-rich repeat (LRR) domain of NLRC4 (c.G1965C, p.W655C) contributes to autoinflammatory disease. Methods: We studied 2 unrelated patients with early-onset macrophage activation syndrome harboring the same de novo mutation in NLRC4. In vitro inflammasome complex formation was quantified by using flow cytometric analysis of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 techniques and lentiviral transduction were used to generate THP-1 cells with either wild-type or mutant NLRC4 cDNA. Cell death and release of IL-1β/IL-18 were quantified by using flow cytometry and ELISA, respectively. The p.W655C NLRC4 mutation caused increased ASC speck formation, caspase-1–dependent cell death, and IL-1β/IL-18 production. ASC contributed to p.W655C NLRC4–mediated cytokine release but not cell death. Mutation of p.W655 activated the NLRC4 inflammasome complex by engaging with 2 interfaces on the opposing LRR domain of the oligomer. One key set of residues (p.D1010, p.D1011, p.L1012, and p.I1015) participated in LRR-LRR oligomerization when triggered by mutant NLRC4 or type 3 secretion system effector (PrgI) stimulation of the NLRC4 inflammasome complex. This is the first report of a mutation in the LRR domain of NLRC4 causing autoinflammatory disease. c.G1965C/p.W655C NLRC4 increased inflammasome activation in vitro. Data generated from various NLRC4 mutations provides evidence that the LRR-LRR interface has an important and previously unrecognized role in oligomerization of the NLRC4 inflammasome complex. [Display omitted]