The autoinflammation-associated NLRC4V341A mutation increases microbiota-independent IL-18 production but does not recapitulate human autoinflammatory symptoms in mice

Elien Eeckhout; Tomoko Asaoka; Hanne Van Gorp; Dieter Demon; Charlotte Girard-Guyonvarc'h; Vanessa Andries; Lars Vereecke; Cem Gabay; Mohamed Lamkanfi; Geert van Loo; Andy Wullaert

doi:10.3389/fimmu.2023.1272639

The autoinflammation-associated NLRC4^V341A mutation increases microbiota-independent IL-18 production but does not recapitulate human autoinflammatory symptoms in mice

Front Immunol. 2023 Nov 28:14:1272639. doi: 10.3389/fimmu.2023.1272639. eCollection 2023.

Authors

Elien Eeckhout^{1

2}, Tomoko Asaoka^{1

2}, Hanne Van Gorp^{1

2}, Dieter Demon^{1

2}, Charlotte Girard-Guyonvarc'h³, Vanessa Andries^{1

2}, Lars Vereecke^{1

2}, Cem Gabay³, Mohamed Lamkanfi¹, Geert van Loo^{2

4}, Andy Wullaert^{1

2

5}

Affiliations

¹ Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium.
² VIB-UGent Center for Inflammation Research, VIB, Ghent, Belgium.
³ Division of Rheumatology, Department of Medicine, University Hospital of Geneva, Department of Pathology and Immunology, University of Geneva Faculty of Medicine, Geneva, Switzerland.
⁴ Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
⁵ Laboratory of Proteinscience, Proteomics and Epigenetic Signalling (PPES), Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.

Abstract

Background: Autoinflammation with infantile enterocolitis (AIFEC) is an often fatal disease caused by gain-of-function mutations in the NLRC4 inflammasome. This inflammasomopathy is characterized by macrophage activation syndrome (MAS)-like episodes as well as neonatal-onset enterocolitis. Although elevated IL-18 levels were suggested to take part in driving AIFEC pathology, the triggers for IL-18 production and its ensuing pathogenic effects in these patients are incompletely understood.

Methods: Here, we developed and characterized a novel genetic mouse model expressing a murine version of the AIFEC-associated NLRC4^V341A mutation from its endogenous Nlrc4 genomic locus.

Results: NLRC4^V341A expression in mice recapitulated increased circulating IL-18 levels as observed in AIFEC patients. Housing NLRC4^V341A-expressing mice in germfree (GF) conditions showed that these systemic IL-18 levels were independent of the microbiota, and unmasked an additional IL-18-inducing effect of NLRC4^V341A expression in the intestines. Remarkably, elevated IL-18 levels did not provoke detectable intestinal pathologies in NLRC4^V341A-expressing mice, even not upon genetically ablating IL-18 binding protein (IL-18BP), which is an endogenous IL-18 inhibitor that has been used therapeutically in AIFEC. In addition, NLRC4^V341A expression did not alter susceptibility to the NLRC4-activating gastrointestinal pathogens Salmonella Typhimurium and Citrobacter rodentium.

Conclusion: As observed in AIFEC patients, mice expressing a murine NLRC4^V341A mutant show elevated systemic IL-18 levels, suggesting that the molecular mechanisms by which this NLRC4^V341A mutant induces excessive IL-18 production are conserved between humans and mice. However, while our GF and infection experiments argue against a role for commensal or pathogenic bacteria, identifying the triggers and mechanisms that synergize with IL-18 to drive NLRC4^V341A-associated pathologies will require further research in this NLRC4^V341A mouse model.

Keywords: Citrobacter rodentium; NLRC4 inflammasome; Salmonella Typhimurium; autoinflammation; germfree mice; inflammasomopathies; microbiota.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CARD Signaling Adaptor Proteins / metabolism
Calcium-Binding Proteins / genetics
Calcium-Binding Proteins / metabolism
Enterocolitis* / genetics
Humans
Infant, Newborn
Interleukin-18 / genetics
Interleukin-18 / metabolism
Macrophage Activation Syndrome* / genetics
Mice
Mutation

Substances

CARD Signaling Adaptor Proteins
Interleukin-18
NLRC4 protein, human
Calcium-Binding Proteins

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. Research in the AW lab is supported by the research grants 3G044718, 3G044818 and G0A3422N from the Fund for Scientific Research-Flanders, the BOF UGent grant BOF.24Y.2019.0032.01, and for this project also by an INFRAFRONTIER2020 project funded by the European Union Research and Innovation programme Horizon 2020 (Grant Agreement Number 730879). EE is a Doctoral Research Fellow supported by the BOF UGent fellowship BOFDOC2018004302. GL acknowledges funding from VIB, Ghent University (BOF23/GOA/001) and from the Research Foundation - Flanders (EOS-G0H2522N-40007505). Research in the ML lab is supported by Ghent University grant BOF23/GOA/001, grants from the Fund for Scientific Research-Flanders (GOI5722N, G017121N, G014221N) and ERC (ERC-2022-PoC 101101075). CG is supported by a grant from the Swiss National Science Foundation (310030B-201269). Both CG and AW were funded by the ERARE2 Cure-AID Grant from the European Union ERA-Net for Research Programs on Rare Diseases. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.