Interaction of the inflammasome genes CARD8 and NLRP3 in abdominal aortic aneurysms

doi:10.1016/j.atherosclerosis.2011.04.043

Atherosclerosis

Volume 218, Issue 1, September 2011, Pages 123-126

https://doi.org/10.1016/j.atherosclerosis.2011.04.043 Get rights and content

Abstract

Objective

Cholesterol crystals have been shown to cause inflammation, and ultimately atherosclerotic lesions through the activation of the NLRP3 inflammasome. As cholesterol crystals have also been found in the walls of patients with abdominal aortic aneurysms (AAA), it is possible that the NLRP3 inflammasome is involved in AAA and genetic variability within this protein complex could alter disease risk. The primary objective of this study was to assess whether there is genetic evidence for a role of the NLRP3 inflammasome in AAA by testing for association of AAA with functional single nucleotide polymorphisms (SNPs) in the CARD8 and NLRP3 genes.

Methods

AAA patients (n = 1151) and controls (n = 727) were genotyped for CARD8 SNP rs2043211 and NLRP3 SNP rs35829419 using TaqMan SNP assays. IL1-β, C-reactive protein (CRP), and lipoprotein (a) [Lp(a)] were measured in the plasma of a subset of study participants. The Kruskal–Wallis Rank test was conducted to test for differences in mean concentration of IL1-β, CRP and Lp(a). Logistic regression was used to test for interaction between CARD8 and NLRP3.

Results

Significantly higher mean concentration of plasma IL1-β was observed in study participants who were homozygous for the common C allele of NLRP3 rs35829419 (p = 0.010). Interaction between rs2043211 and rs35829419 was observed in this dataset (χ² = 6.22; p = 0.044), which strengthened when adjusted for age, gender, smoking, diabetes, hypertension, and dyslipidemia (χ² = 14.75; p = 0.012); and separately for NOD2 genotype (χ² = 14.06; p = 0.015).

Conclusion

Our finding suggests genetic variability within the NLRP3 inflammasome may be important in the pathophysiology of AAA.

Introduction

Abdominal aortic aneurysms (AAA) manifest as localised and pronounced dilations of the abdominal aorta which are caused by an adventitial and transmural infiltrate comprising macrophages, lymphocytes, dendritic cells and plasma cells [1]. The large amounts of IL-1β, TNFalpha, IL-6, and other proinflammatory cytokines released by this lymphomonocytic infiltrate leads to enhanced expression of matrix metalloproteinases, which digest the elastin and collagen fibres that form the extracelluar matrix of the aortic wall [2]. Inflammation and degradation of elastin are considered responsible for aortic expansion and aneurysm formation, whereas loss of collagen, which is the major source of tensile strength, is believed to be the event that ultimately causes rupture in some AAA [1], [2].

Inter-individual variation in susceptibility to AAA has been shown to be 70% genetic and 30% environmental [3]. High dietary cholesterol is a well-recognised environmental risk factor [4] and cholesterol crystals have been reported within the aortic wall of patients with AAA [5]. Moreover, the mechanism by which cholesterol likely contributes to vascular disease has emerged from a recent study investigating the development of atherosclerosis in Apo-E-deficient mice [6]. In that study, cholesterol crystals were found to trigger peritoneal inflammation and ultimately atherosclerotic lesions by entering macrophages where they ruptured lysosomes. The ensuing release of the proteolytic content from these lysosomes resulted in activation of the NOD-like receptor pyrin containing 3 (NLRP3) inflammasome [6]. This multi-protein complex, comprising the NLRP3 polypeptide, caspase recruitment domain-containing protein 8 (CARD8), the PYD and CARD domain-containing protein (PYCARD), and caspase 1 [7] forms when pattern-recognition receptors on innate immune cells detect endogenous and exogenous ‘danger signals’ (e.g. bacterial lipopolysaccharide, ATP, or monosodium urate crystals) and activate the NFκB-mediated transcription of pro-IL-1β. Once assembled the inflammasome processes the pro-interleukin (IL)-1β to the mature pro-inflammatory cytokine IL-1β ready for secretion [7].

NLRP3 inflammasome-mediated production of IL-1β has previously been shown to be important in the pathogenesis of gout, systemic onset juvenile arthritis, Crohn's disease (CD), Blau syndrome, Familial Mediterranean fever, and cryopyrin-associated periodic syndromes [8]. Given that cholesterol crystals activate this inflammasome [6], and a diet high in cholesterol is a risk factor for AAA [4], [9], it seems plausible that the NLRP3 inflammasome may also play a role in this inflammatory disease. However, as yet there is no data to support this suggestion.

Mice whose bone marrow-derived cells lacked components of the NLRP3 inflammasome have been found to be markedly resistant to the development of cholesterol-induced atherosclerosis, compared to mice with wildtype bone marrow [6]. This observation introduces the possibility that genetic variability which affects NLRP3 inflammasome functioning may alter susceptibility to chronic inflammatory diseases. Indeed, the discovery and subsequent replication of a synergistic interaction between functional SNPs within CARD8 and NLRP3 that alters susceptibility to Crohn's disease (CD) [10], [11] and rheumatoid arthritis (RA) [12] supports this assertion. Specifically, the minor (T) allele of CARD8 rs2043211 was found to be a significant modifier of CD or RA risk when it occurred in combination with the minor (A) allele of NLRP3 rs35829419. Schoultz et al. [11] proposed that the T allele of CARD8 rs2043211 was unable to inhibit NFκB activation, resulting in constitutive over-production of pro-IL-1β which the putative ‘gain-of-function’ A allele of NLRP3 rs35829419 then cleaved, culminating in the secretion of large amounts of IL-1β.

AAA is a multifactorial disease and although elevated cholesterol is a risk factor for both atherosclerosis and AAA, it is widely accepted that AAA is not the result of a strictly atherosclerotic process. As a result, involvement of the NLRP3 inflammasome in atherosclerosis does not automatically imply this inflammasome is also involved in AAA. Therefore, the primary objective of this study was to assess whether there is genetic evidence for a role of the NLRP3 inflammasome in AAA pathogenesis. To do this, we tested for association of CARD8 rs2043211 and NLRP3 rs35829419 with AAA. The secondary aim of this study was to determine whether differences in plasma concentrations of IL-1β (as the direct product of the NLRP3 inflammasome), lipoprotein a (Lp(a)), an independent lipid risk factor for AAA [13] and c-reactive protein (CRP, as a global marker of inflammation) correlated with CARD8 rs2043211 and NLRP3 rs35829419 genotype.

Section snippets

Study participants

Basic demographic and clinical characteristics of the study participants are listed in Table 1. Controls and AAA cases were all recruited from the Otago and Southland regions of New Zealand as previously described [13]. AAA cases were selected as a consecutive series from the Vascular Research Consortium of New Zealand research database. Controls were aged >55 years and confirmed to be free of AAA by ultrasound. All AAA patients had infra-renal aneurysms of >30 mm in maximum antero-posterior

Results

Genotyping for the CARD8 SNP rs2043211 and the NLRP3 SNP rs35829419 was successful in 97% and 96% of AAA patients and 99% and 95% of controls, respectively. No deviations from Hardy–Weinberg equilibrium were observed for either SNP across cases or controls (p > 0.05). The frequency of the minor (T) allele of rs2043211 did not differ significantly between controls (32.0%, 463/1448) and AAA patients (30.4%, 727/2394) (p = 0.296; OR = 0.93, 95% CI [0.81.07]). In contrast, CARD8 SNP rs2043211 genotype

Discussion

The primary goal of the current study was to investigate whether there is genetic evidence to support a role for the NLRP3 inflammasome in AAA. We evaluated this possibility by testing whether inflammasome SNPs, previously associated with CD and RA susceptibility, also exhibited association with AAA. In our dataset we found that the minor allele of CARD8 SNP rs2043211 conferred protection against AAA. This is consistent with the significant protective effect this minor allele conferred against

Acknowledgements

This study was supported by a program grant from the Health Research Council (HRC) of New Zealand and by funding from the New Zealand Lottery Grants Board. R.L.R. is the recipient of a Sir Charles Hercus Health Research Fellowship (HRC).

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Interaction of the inflammasome genes CARD8 and NLRP3 in abdominal aortic aneurysms

Abstract

Objective

Methods

Results

Conclusion

Introduction

Section snippets

Study participants

Results

Discussion

Acknowledgements

Lancet

J Vasc Surg

Gastroenterology

Gastroenterology

Inflammation, metalloproteinases, and increased proteolysis: an emerging pathophysiological paradigm in aortic aneurysm

Circulation

Pathophysiology and epidemiology of abdominal aortic aneurysms

Nat Rev Cardiol

Mineral–cholesterol concentrations of the aortic aneurysmatic wall

Pol J Pathol

NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals

Nature

The inflammasomes: guardians of the body

Annu Rev Immunol