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

Fiona Moghaddas; Ping Zeng; Yuxia Zhang; Heike Schützle; Sebastian Brenner; Sigrun R. Hofmann; Reinhard Berner; Yuanbo Zhao; Bingtai Lu; Xiaoyun Chen; Li Zhang; Suyun Cheng; Stefan Winkler; Kai Lehmberg; Scott W. Canna; Peter E. Czabotar; Ian P. Wicks; Dominic De Nardo; Christian M. Hedrich; Huasong Zeng; Seth L. Masters

doi:10.1016/j.jaci.2018.04.033

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

Fiona Moghaddas, Ping Zeng, Yuxia Zhang, Heike Schützle, Sebastian Brenner, Sigrun R. Hofmann, Reinhard Berner, Yuanbo Zhao, Bingtai Lu, Xiaoyun Chen, Li Zhang, Suyun Cheng, Stefan Winkler, Kai Lehmberg, Scott W. Canna, Peter E. Czabotar, Ian P. Wicks, Dominic De Nardo, Christian M. Hedrich, Huasong ZengSeth L. Masters

Research output: Contribution to journal › Article › Other › peer-review

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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.

Original language	English
Pages (from-to)	1956-1967.e6
Number of pages	18
Journal	The Journal of Allergy and Clinical Immunology
Volume	142
Issue number	6
DOIs	https://doi.org/10.1016/j.jaci.2018.04.033
Publication status	Published - 1 Dec 2018
Externally published	Yes

Keywords

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

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10.1016/j.jaci.2018.04.033

257151642-oaFinal published version, 3.86 MBLicence: CC BY

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Moghaddas, F., Zeng, P., Zhang, Y., Schützle, H., Brenner, S., Hofmann, S. R., Berner, R., Zhao, Y., Lu, B., Chen, X., Zhang, L., Cheng, S., Winkler, S., Lehmberg, K., Canna, S. W., Czabotar, P. E., Wicks, I. P., De Nardo, D., Hedrich, C. M., ... Masters, S. L. (2018). Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)–LRR oligomerization interface. The Journal of Allergy and Clinical Immunology, 142(6), 1956-1967.e6. https://doi.org/10.1016/j.jaci.2018.04.033

@article{b59f4b3c6bbb485fafc47e7c8007f3df,

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

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, inflammasome, IPAF, macrophage activation syndrome, NLRC4, Nod-like receptor, periodic fever syndrome",

author = "Fiona Moghaddas and Ping Zeng and Yuxia Zhang and Heike Sch{\"u}tzle and Sebastian Brenner and Hofmann, {Sigrun R.} and Reinhard Berner and Yuanbo Zhao and Bingtai Lu and Xiaoyun Chen and Li Zhang and Suyun Cheng and Stefan Winkler and Kai Lehmberg and Canna, {Scott W.} and Czabotar, {Peter E.} and Wicks, {Ian P.} and {De Nardo}, Dominic and Hedrich, {Christian M.} and Huasong Zeng and Masters, {Seth L.}",

year = "2018",

month = dec,

day = "1",

doi = "10.1016/j.jaci.2018.04.033",

language = "English",

volume = "142",

pages = "1956--1967.e6",

journal = "The Journal of Allergy and Clinical Immunology",

issn = "0091-6749",

publisher = "Elsevier",

number = "6",

}

Moghaddas, F, Zeng, P, Zhang, Y, Schützle, H, Brenner, S, Hofmann, SR, Berner, R, Zhao, Y, Lu, B, Chen, X, Zhang, L, Cheng, S, Winkler, S, Lehmberg, K, Canna, SW, Czabotar, PE, Wicks, IP, De Nardo, D, Hedrich, CM, Zeng, H & Masters, SL 2018, 'Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)–LRR oligomerization interface', The Journal of Allergy and Clinical Immunology, vol. 142, no. 6, pp. 1956-1967.e6. https://doi.org/10.1016/j.jaci.2018.04.033

TY - JOUR

T1 - Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)–LRR oligomerization interface

AU - Moghaddas, Fiona

AU - Zeng, Ping

AU - Zhang, Yuxia

AU - Schützle, Heike

AU - Brenner, Sebastian

AU - Hofmann, Sigrun R.

AU - Berner, Reinhard

AU - Zhao, Yuanbo

AU - Lu, Bingtai

AU - Chen, Xiaoyun

AU - Zhang, Li

AU - Cheng, Suyun

AU - Winkler, Stefan

AU - Lehmberg, Kai

AU - Canna, Scott W.

AU - Czabotar, Peter E.

AU - Wicks, Ian P.

AU - De Nardo, Dominic

AU - Hedrich, Christian M.

AU - Zeng, Huasong

AU - Masters, Seth L.

PY - 2018/12/1

Y1 - 2018/12/1

N2 - 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.

AB - 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.

KW - Autoinflammatory disease

KW - IL-18

KW - inflammasome

KW - IPAF

KW - macrophage activation syndrome

KW - NLRC4

KW - Nod-like receptor

KW - periodic fever syndrome

UR - http://www.scopus.com/inward/record.url?scp=85047923681&partnerID=8YFLogxK

U2 - 10.1016/j.jaci.2018.04.033

DO - 10.1016/j.jaci.2018.04.033

M3 - Article

AN - SCOPUS:85047923681

SN - 0091-6749

VL - 142

SP - 1956-1967.e6

JO - The Journal of Allergy and Clinical Immunology

JF - The Journal of Allergy and Clinical Immunology

IS - 6

ER -

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

Abstract

Keywords

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