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

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

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.

Publication types

  • Case Reports
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CARD Signaling Adaptor Proteins / chemistry
  • CARD Signaling Adaptor Proteins / genetics*
  • CARD Signaling Adaptor Proteins / immunology
  • Calcium-Binding Proteins / chemistry
  • Calcium-Binding Proteins / genetics*
  • Calcium-Binding Proteins / immunology
  • Female
  • HEK293 Cells
  • Humans
  • Infant
  • Infant, Newborn
  • Inflammasomes / chemistry
  • Inflammasomes / genetics*
  • Inflammasomes / immunology
  • Leucine*
  • Macrophage Activation
  • Male
  • Protein Domains
  • Syndrome
  • THP-1 Cells

Substances

  • CARD Signaling Adaptor Proteins
  • Calcium-Binding Proteins
  • Inflammasomes
  • NLRC4 protein, human
  • Leucine