Author + information
- Received April 10, 2013
- Revision received July 6, 2013
- Accepted July 9, 2013
- Published online October 8, 2013.
- Clett Erridge, PhD∗,†∗ (, )
- Jay Gracey, BA, RN∗,†,
- Peter S. Braund, MSc∗,† and
- Nilesh J. Samani, MD∗,†
- ∗Department of Cardiovascular Sciences, University of Leicester, British Heart Foundation Cardiovascular Research Centre, Glenfield Hospital, Leicester, United Kingdom
- †National Institute for Health Research Leicester Cardiovascular Biomedical Research Unit, Glenfield Hospital, Leicester, United Kingdom
- ↵∗Reprint requests and correspondence:
Dr. Clett Erridge, Department of Cardiovascular Sciences, University of Leicester, Glenfield Hospital, Leicester LE3 9QP, United Kingdom.
Objectives The study objective was to determine whether the coronary artery disease (CAD)-associated genotype at chromosome 9p21 modulates basal or induced expression of type I interferons (IFN-I).
Background The mechanism responsible for the association between common variants in chromosome 9p21.3 and CAD remains unclear. It has been reported that the CAD risk locus is rich in enhancer-like elements and that chromosome looping can lead to its physical proximity with the IFN-I gene cluster, raising the possibility that the locus influences CAD risk by modulating expression of IFN-Is.
Methods We examined whether genotype at the lead CAD-associated single nucleotide polymorphism (rs1333049) in 9p21 was associated with: 1) basal levels of IFN-I in plasma from 148 healthy male subjects; 2) induction of IFN-I by Toll-like receptor stimulants in peripheral blood mononuclear cells of 60 healthy volunteers assessed by enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, Western blot, and IFN-I bioassay; and 3) enhancer activity of predicted IFN regulatory factor 3/7 binding sites within the 9p21 CAD risk region in reporter assays.
Results No significant effects of 9p21 genotype were observed for plasma levels of IFN-α, IFN-α21, or CXCL10, or leukocyte induction of IFN-α, IFN-α21, IFN-β, CXCL10, or total IFN-I measured at the mRNA, protein, and biological activity levels. There was also no enhancement of reporter activity by predicted IFN regulatory factor 3/7 binding sites in the CAD risk locus of either genotype.
Conclusions The mechanism underlying the association between common 9p21 variants and CAD does not involve differential regulation of IFN-I responses.
Common variants in the 9p21.3 gene desert are strongly associated with the risk of coronary artery disease (CAD) (1), although the mechanisms responsible for this association remain poorly understood. Harismendy et al. (2) recently reported that chromosome looping permits physical proximity of the CAD risk locus to a region downstream of the IFNA21 gene, raising the possibility that enhancers in this locus may regulate expression of the type I interferon (IFN-I) gene cluster.
The IFN-Is, which comprise 13 interferon (IFN)-α isotypes, IFN-β, IFN-κ, IFN-ε, and IFN-ω, are expressed from a single gene cluster on chromosome 9 located approximately 800 kb from the CAD risk interval. IFN-I is co-induced in response to bacterial or viral nucleic acid motifs via pattern recognition receptors, such as endosomal Toll-like receptors (TLRs), and signal through the shared IFN-I receptor to promote expression of genes involved in antiviral defense.
The potential role played by IFN-I in atherosclerotic disease is currently debated, because both pro- and antiatherogenic properties of IFN-I have been reported (3–5). Moreover, the autoimmune disease systemic lupus erythematosus (SLE), which is thought to be driven largely by overproduction of IFN-α, is associated with an elevated risk of CAD (6). We therefore tested the hypothesis that 9p21 genotype regulates plasma levels of IFN-I or the capacity of leukocytes to produce IFN-I in response to TLR stimulants.
Subjects and samples
To assess the effect of 9p21 genotype on plasma levels of IFN-I, we analyzed stored samples from 148 healthy male subjects previously recruited into the GRAPHIC (Genetic Regulation of Arterial Blood Pressure in Humans in the Community) study (cohort 1) (7). Sixty GRAPHIC participants homozygous for rs1333049 variants were invited to donate a fresh blood sample for leukocyte IFN-I production studies (cohort 2), and 32 healthy volunteers were recruited by local advertisement with approval from the University of Leicester College of Medicine Research Ethics Committee to study IFN-I transcriptional responses (cohort 3). All subjects provided written, informed consent, and procedures were carried out in accordance with institutional guidelines and the Declaration of Helsinki. 9p21 CAD-risk genotype was determined in all subjects by genotyping the lead CAD-associated single nucleotide polymorphism (SNP) rs1333049.
Enzyme-linked immunosorbent assays, quantitative polymerase chain reactions, and bioassays for IFN-I
Cytokine and C-reactive protein levels were measured by enzyme-linked immunosorbent assay (ELISA) (R&D, Minneapolis, Minnesota; Pierce, Waltham, Massachusetts; and Caltag-Medsystems, Buckingham, United Kingdom). For stimulation experiments, peripheral blood mononuclear cells (PBMCs) (106 cells/ml) isolated from fresh venous blood were cultured with medium alone, 50 μg/ml PolyI:C, 1 μg/ml R848, or 1.5 μmol/l CpG oligodeoxynucleotide (ODN)-2216 (InvivoGen, San Diego, California), with or without 2-hour pre-treatment with 100 ng/ml IFN-γ (PeproTech, Rocky Hill, New Jersey). IFN-I bioactivity was measured at 24 h using human embryonic kidney 293 cells stably transfected with STAT2, interferon regulatory factor (IRF)-9, and an IFN stimulated response element-9 driven reporter that is sensitive to IFN-I signaling (InvivoGen Inc., San Diego, California) and compared with a recombinant IFN-α standard curve. Immunoblots were probed with anti–IFN-α21 (Sigma, St. Louis, Missouri) or antiglyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, Inc., Santa Cruz, California). PBMC cDNA was quantified using a Rotor-Gene Q real-timepolymerase chain reaction cycler (Qiagen, Venlo, the Netherlands).
Four regions of the 9p21 CAD risk locus were cloned from carriers homozygous for the risk or nonrisk rs1333049 variants into pGL3-promoter (Promega, Fitchburg, Wisconsin) and sequenced for verification of variants in high linkage disequilibrium (LD) (R2 > 0.8) with CAD-associated SNPs (2). Human embryonic kidney 293 cells were transfected with TLR4/MD2, CD14, and each reporter, and challenged with medium alone (control), 10 μg/ml PolyI:C, or 10 ng/ml lipopolysaccharide for 24 h.
The effect of genotype on plasma cytokine levels was assessed by linear regression of log-transformed responses. Stepwise regression was used to assess the effect of age, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, systolic and diastolic blood pressures, body mass index, and smoking status on cytokine levels, and those found to be significant (p < 0.05) were adjusted for when examining the effect of genotype. PBMC expression of cytokine mRNA and protein was analyzed by 2-way analysis of variance with Tukey’s post hoc test. Significance was assumed at p < 0.05.
The clinical characteristics of the 148 healthy male volunteers (cohort 1) whose plasma samples were analyzed for basal levels of cytokines, 60 subjects recruited for IFN-I secretion studies (cohort 2), and 32 healthy volunteers recruited for IFN-I transcription studies (cohort 3) are summarized in Table 1. There were no significant differences by genotype of the lead CAD-associated 9p21 SNP rs1333049 in any of the demographic, clinical, or biochemical characteristics of these subjects.
Effect of genotype on plasma levels of IFN-Is
Because of the extensive (>80%) similarity among the 13 IFN-α subtypes, total plasma IFN-α was quantified using pan-subtype reactive ELISAs. These assays revealed no effect of 9p21 genotype on plasma total IFN-α in healthy men (Fig. 1A). There was also no effect of genotype on plasma IFN-α21 or the chemokine CXCL10, which is a sensitive and stable marker of IFN-I induction, or C-reactive protein (Figs. 1B to 1D). Because ELISAs for IFN-α are known to overestimate the fraction of IFN-I that remains biologically active in plasma (8), we also measured IFN-I biological activity in plasma samples by bioassay. In confirmation of earlier studies, biologically active IFN-I was below the limit of detection (<5 pg/ml) in plasma samples from healthy subjects in any genotype group (6).
Effect of genotype on induction of IFN-α and IFN-β
We next examined the effect of 9p21 genotype on induction of IFN-α by PBMCs of 60 healthy volunteers in response to stimulants of TLR3 (PolyI:C), TLR7/8 (R848), and TLR9 (CpG ODN). Induction of total IFN-α or CXCL10 as measured by ELISA or total IFN-I measured by bioassay was not modified by genotype (Figs. 2A and 2B). Because 2 SNPs in high LD with rs1333049 disrupt an IFN-γ–responsive STAT1 binding site (2), we also examined the effects of 2-hour pre-incubation with IFN-γ on production of IFN-α. These experiments revealed no capacity of IFN-γ to prime for production of IFN-α or IFN-I biological activity (Fig. 2A). 9p21 genotype also did not specifically affect induction of IFN-α21 by CpG-ODN in cell lysates (Fig. 2C). There was also no effect of genotype on the production of IFN-β, the downstream chemokine CXCL10, or the inflammatory cytokines interleukin-1β and interleukin-6 (Figs. 3A to 3D). Examination of the transcriptional response to TLR4- or TLR9-stimulation also revealed no effect of genotype on basal levels of IFN-I mRNA or on induction of IFN-β, total IFN-α, IFN-ω, or the downstream markers of transient IFN-I production CXCL10 and ISG-54 (Figs. 3E to 3H) (and data not shown).
Enhancer activity of IRF3/7-rich regions in 9p21
Sequence analysis revealed 4 regions rich in consensus sites for the principle IFN-I–inducing transcription factors IRF3 and IRF7 (GAAANNGAAA), which are close to or contain SNPs in high LD (R2 > 0.8) with CAD-associated 9p21 SNPs (Fig. 4A). However, the selected regions of either genotype did not demonstrate appreciable enhancer activity in unstimulated cells. Moreover, activation of IRF3 and IRF7 (by PolyI:C) or of IRF3, IRF5, and nuclear factor kappa B (by lipopolysaccharide) also failed to stimulate reporter activation (Fig. 4B).
Harismendy et al. (2) recently reported that chromosome looping induced by IFN-γ permits close physical proximity between the 9p21 CAD risk interval and certain proximal genes, such as CDKN2A and CDKN2B, and also the distant IFN-I gene cluster, raising the possibility that enhancers in the CAD risk locus may modify expression of these genes in a genotype-dependent manner. However, we found no effect of genotype on plasma levels of IFN-I, the capacity of PBMC to produce IFN-α, IFN-β, or their downstream markers via the 2 major pathways of their induction (i.e., TLR3/4→IRF3 or TLR9→IRF7) by ELISA, bioassay, immunoblot, and quantitative polymerase chain reaction. As viral infections or medication could have impacted on our findings, we took care to only include subjects that were free of any current medication, disease, or infection. Moreover, although putative binding sites for IRF3 and IRF7 are present in the CAD risk region, these do not seem to possess enhancer activity basally or on TLR stimulation. It was also shown recently that IFN-γ–dependent induction of CDKN2A and CDKN2B occurs independently of 9p21 genotype (9). Our findings are supported by recent genetic evidence from studies of SLE, a disease that is driven by genetic propensity to increased IFN-α production (6,10). SLE symptoms can be triggered in humans by the administration of IFN-α, and approximately 60% of SLE-associated SNPs are linked to IFN-I-related genes (6,10). Nevertheless, despite numerous robust genome-wide association studies and meta-analyses for SLE in multiple populations, 9p21 has not emerged as a genetic risk factor for this disease (10).
It is possible that the observed lack of effect of genotype on IFN-I responses is due to limited study power. On the basis of the mean and SD of log-transformed plasma IFN-α levels in the nonrisk group, our study had an 80% power at an alpha of 5% to detect an 18.2% difference in log-plasma IFN-α in the risk genotype group (11.0% for plasma CXCL10) and 80% power to detect a difference of 8.2% or 4.5% between log-IFN-α or log-IFN-I bioactivity levels, respectively, in supernatants of CpG-ODN stimulated cultures of CC and GG genotypes. Because all the subjects studied were of European Caucasian origin, we also cannot exclude the possibility that 9p21 genotype modulates IFN-I production in populations or ethnicities distinct from those examined here.
Our findings suggest that the mechanism by which 9p21 genotype modulates cardiovascular risk does not involve regulation of IFN-I responses.
This study is part of the research portfolio supported by the Leicester National Institute for Health Research Biomedical Research Unit in Cardiovascular Disease. Jay Gracey is funded by the British Heart Foundation. Dr. Samani holds a chair supported by the British Heart Foundation. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- coronary artery disease
- enzyme-linked immunosorbent assay
- type I interferon
- interferon regulatory factor
- linkage disequilibrium
- peripheral blood mononuclear cell
- systemic lupus erythematosus
- single nucleotide polymorphism
- Toll-like receptor
- Received April 10, 2013.
- Revision received July 6, 2013.
- Accepted July 9, 2013.
- American College of Cardiology Foundation
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