Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)–LRR oligomerization interface - 06/12/18

Doi : 10.1016/j.jaci.2018.04.033

Fiona Moghaddas, MBBS ^a,^b,^{^{∗
These authors contributed equally to this work.}}, Ping Zeng, MD ^c,^{^{∗
These authors contributed equally to this work.}}, Yuxia Zhang, PhD ^d, Heike Schützle, MD ^e, Sebastian Brenner, MD, PhD ^e, Sigrun R. Hofmann, MD, PhD ^e, Reinhard Berner, MD ^e, Yuanbo Zhao, PhD ^d,^f, Bingtai Lu, PhD ^d, Xiaoyun Chen, MSc ^d, Li Zhang, MD ^d, Suyun Cheng, MD ^c, Stefan Winkler, MD ^e, Kai Lehmberg, MD ^g, Scott W. Canna, MD ^h, Peter E. Czabotar, PhD ^b,ⁱ, Ian P. Wicks, MBBS, PhD ^a,^b,^j, Dominic De Nardo, PhD ^a,^b, Christian M. Hedrich, MD, PhD ^e,^k,^l,^{‡
These authors contributed equally to this work.}, Huasong Zeng, MD, PhD ^c,^{‡
These authors contributed equally to this work.}, Seth L. Masters, PhD ^a,^b,^d,^⁎,^{‡
These authors contributed equally to this work.}
^a Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia
ⁱ Structural Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia
^b Department of Medical Biology, University of Melbourne, Parkville, Australia
^c Department of Rheumatology, Guangzhou Women and Children's Medical Centre, Guangzhou, China
^d Immunology Laboratory, Guangzhou Institute of Paediatrics, Guangzhou, China
^e Department of Pediatrics, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany
^f Department of Chemical Biology, Guizhou Medical University, Guiyang, China
^g Division of Pediatric Stem Cell Transplantation and Immunology, University Medical Center Hamburg Eppendorf, Hamburg, Germany
^h Pediatric Rheumatology/RK Mellon Institute, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, Pa
^j Rheumatology Department, Royal Melbourne Hospital, Parkville, Australia
^k Department of Women's & Children's Health, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom
^l Department of Paediatric Rheumatology, Alder Hey Children's NHS Foundation Trust Hospital, Liverpool, United Kingdom

^∗Corresponding author: Seth L. Masters, PhD, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3052, Australia.Walter and Eliza Hall Institute of Medical Research1G Royal ParadeParkville3052Australia

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.

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Graphical abstract

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Key words : Autoinflammatory disease, periodic fever syndrome, NLRC4, macrophage activation syndrome, inflammasome, Nod-like receptor, IPAF, IL-18

Abbreviations used : AIFEC, ASC, CARD, CRISPR, CSF, HD, IL-18BP, KO, LRR, MAS, NAIP, NBD, NLRC4, NLRC4-AID, NLRP3, NOD, T3SS, WT

Plan

Methods

Patient and study approval

Genetic analysis

Serum cytokine analysis

Generation of NLRC4-deficient cells

Generation of lentiviral constructs

Site-directed mutagenesis

Cell-culture procedures

Transduction of KO cell lines

Cell stimulation

Cytokine quantification from cell-culture supernatants

Western blot analysis

Time-of-flight inflammasome evaluation

p.W655 is located in the LRR domain of NLRC4

p.W655C NLRC4 results in increased ASC speck formation

p.W655C NLRC4 causes increased IL-1β and IL-18 release and pyroptosis

Increased IL-18 secretion, but not cell death, is dependent on ASC

Increased IL-1β and IL-18 secretion and cell death are not dependent on caspase-8 or NLRP3

p.W655 does not tolerate substitution

The LRR interface is important for inflammasome assembly

Discussion

Funding for this work was provided by a Australian National Health and Medical Research Council Program (NHMRC) project grant (1113577; to I.P.W.); NHMRC projects grants (1099262 and 1081299), a Viertel Fellowship, the Howard Hughes Medical Institute, and GlaxoSmithKline (to S.L.M.); the National Natural Science Foundation of China (31770978) and Children's Medical Centre Startup Fund (5001-3001032; to Y.Z.); the intramural MeDDrive program of TU Dresden and the Fritz-Thyssen Foundation (to C.M.H.); internal funding of the Paediatric Institute of Guangzu and a Guangzhou Women and Children's Medical Centre project grant (0160001; to P.Z.); and the German Research Foundation (KF0249, HO4510/1-2; to S.R.H.).

Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

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Vol 142 - N° 6

P. 1956 - décembre 2018 Retour au numéro

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Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)–LRR oligomerization interface - 06/12/18

Abstract

Background

Objective

Results

Conclusion

Graphical abstract

Plan

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