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J Allergy Clin Immunol. 2018 Dec;142(6):1956-1967.e6. doi: 10.1016/j.jaci.2018.04.033. Epub 2018 May 17.

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

Author information

1
Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia; Department of Medical Biology, University of Melbourne, Parkville, Australia.
2
Department of Rheumatology, Guangzhou Women and Children's Medical Centre, Guangzhou, China.
3
Immunology Laboratory, Guangzhou Institute of Paediatrics, Guangzhou, China.
4
Department of Pediatrics, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany.
5
Immunology Laboratory, Guangzhou Institute of Paediatrics, Guangzhou, China; Department of Chemical Biology, Guizhou Medical University, Guiyang, China.
6
Division of Pediatric Stem Cell Transplantation and Immunology, University Medical Center Hamburg Eppendorf, Hamburg, Germany.
7
Pediatric Rheumatology/RK Mellon Institute, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, Pa.
8
Department of Medical Biology, University of Melbourne, Parkville, Australia; Structural Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.
9
Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia; Department of Medical Biology, University of Melbourne, Parkville, Australia; Rheumatology Department, Royal Melbourne Hospital, Parkville, Australia.
10
Department of Pediatrics, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany; Department of Women's & Children's Health, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom; Department of Paediatric Rheumatology, Alder Hey Children's NHS Foundation Trust Hospital, Liverpool, United Kingdom.
11
Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia; Department of Medical Biology, University of Melbourne, Parkville, Australia; Immunology Laboratory, Guangzhou Institute of Paediatrics, Guangzhou, China. Electronic address: masters@wehi.edu.au.

Abstract

BACKGROUND:

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).

OBJECTIVE:

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.

RESULTS:

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.

CONCLUSION:

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.

KEYWORDS:

Autoinflammatory disease; IL-18; IPAF; NLRC4; Nod-like receptor; inflammasome; macrophage activation syndrome; periodic fever syndrome

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