Protective Role of the Interleukin 33 rs3939286 Gene Polymorphism in the Development of Subclinical Atherosclerosis in Rheumatoid Arthritis Patients

Objectives To determine whether the interleukin-33 (IL-33)-interleukin-1 receptor like 1 (IL-1RL1) signaling pathway is implicated in the risk of subclinical atherosclerosis in patients with rheumatoid arthritis (RA). Methods A total of 576 Spanish RA patients from Northern Spain were genotyped for 6 well-known IL33-IL1RL1 polymorphisms (IL33 rs3939286, IL33 rs7025417, IL33 rs7044343, IL1RL1 rs2058660, IL1RL1 rs2310173 and IL1RL1 rs13015714) by TaqMan genotyping assay. The presence of subclinical atherosclerosis was determined by the assessment of carotid intima-media thickness (cIMT) by carotid ultrasound (US). Results RA patients carrying the TT genotype of the IL33 rs3939286 polymorphism had lower cIMT values than those homozygous for the CC genotype (mean ± standard deviation (SD): 0.71 ± 0.14 mm versus 0.76 ± 0.16 mm, respectively) while patients carrying the CT genotype had intermediate cIMT values (mean ± SD: 0.73 ± 0.17 mm). Moreover, RA patients carrying the mutant allele T of the IL33 rs3939286 polymorphism exhibited significantly lower cIMT values than those carrying the wild allele C (mean ± SD: 0.72 ± 0.16 mm versus 0.75 ± 0.18 mm respectively; p = 0.04). The association of both genotype and allele frequencies of IL33 rs3939286 and cIMT levels remained statistically significant after adjustment for sex, age at the time of US study, follow-up and center (p = 0.006 and p = 0.0023, respectively), evidencing that the potential effect conferred by IL33 rs3939286 may be independent of confounder factors. No association with other IL33-IL1RL1 genetic variants was observed. Conclusions In conclusion, our results may suggest a potential protective effect of the IL33 rs3939286 allele T in the risk of subclinical atherosclerosis in patients with RA.


Introduction
Rheumatoid arthritis (RA) is a complex autoimmune disease associated with progressive disability, systemic complications and early death [1]. Mortality is higher among RA patients than in the general population, and cardiovascular (CV) complications remain a major challenge [2]. The mechanisms leading to accelerated atherosclerosis in RA are complex, including not only the effect of traditional CV risk factors and chronic inflammation [2][3]. In this regard, several pieces of evidence indicate that genetic polymorphisms located inside and outside of the human leukocyte antigen (HLA) region also influence the risk of CV disease in RA [2,[4][5]. Subclinical atherosclerosis has been observed in RA patients, even in those without traditional CV risk factors, and abnormally high values of carotid intima-media thickness (cIMT) have been found to predict the risk of CV events in these patients [6].
Interleukin-33 (IL-33) is a newly characterized cytokine that belongs to the IL-1 family [7]. This cytokine is constitutively expressed by tissue barrier cells (such as the epithelial and endothelial cells of many organs), but it is also expressed by some innate immune cells (such as macrophages and dendritic cells). IL-33 displays both nuclear and extracellular effects and mediates its biological function by interacting with its receptor (IL-1 receptor like 1 (IL-1RL1) also called ST2) and coreceptor (IL-1 receptor accessory protein (IL-1RAcP)). Since this binding exerts relevant immunomodulatory functions producing chemokines and pro-inflammatory and Th2-associated cytokines [7], IL-33-IL-1RL1 has been considered as a key pathway involved in inflammatory diseases. In accordance with that, a pathogenic role of IL-33 has been found in RA, where its levels are significantly elevated both in synovial fluid and serum [8]. Moreover, a relationship between baseline detectable IL-33 concentrations and the development of severe subclinical atherosclerosis in patients diagnosed with RA has been described [9].
Regarding genetic studies, polymorphisms located both in IL33 and IL1RL1 have been associated with autoimmunity. An association between IL33 rs3939286 and some immune-mediated diseases such as inflammatory bowel disease (IBD) has been described [10]. Additionally, IL33 rs7025417 and IL33 rs4742170, which is in high linkage disequilibrium with IL33 rs7025417, have been related to coronary artery disease (CAD) and ischemic stroke in nonrheumatic Chinese individuals, respectively [11,12]. Also in the Chinese population, the IL33 rs7044343 polymorphism has been associated with RA [13]. Regarding IL1RL1, a relevant role of IL1RL1 rs2058660, IL1RL1 rs2310173 and IL1RL1 rs13015714 in the development of several inflammatory conditions such as IBD and ankylosing spondylitis has been proposed [10,14].
Taking into account all these considerations, in the present study we aimed to determine, for the first time, whether 6 genetic variants at IL33 and IL1RL1, previously associated with immune-mediated diseases, are involved in the risk of subclinical atherosclerosis in Spanish patients with RA.

Patients and Study Protocol
A set of 576 Spanish patients with RA were included in the present study. Blood samples were obtained from patients recruited from Hospital Lucus Augusti (Lugo) and Hospital Marqués de Valdecilla (Santander) in Northern Spain. A subject's written consent was obtained according to the declaration of Helsinki, and the study was approved by the Ethics Committees of clinical research of Galicia (CAEI) for Hospital Lucus Augusti in Lugo and Cantabria (CEIC) for Hospital Universitario Marqués de Valdecilla in Santander.
All the patients fulfilled the 2010 classification criteria for RA [15]. In all the cases, patients were assessed for 3 polymorphisms within IL33 (rs3939286, rs7025417 and rs7044343) and 3 genetic variants located within IL1RL1 (rs2058660, rs2310173, rs13015714). Also, cIMT was determined by carotid ultrasound (US) technology. Information on the main demographic data, clinical characteristics, CV risk factors and CV events of the patients enrolled in the study is shown in Table 1. Definitions of CV events (ischemic heart disease, heart failure, cerebrovascular accident or peripheral arteriopathy) and those for traditional CV risk factors were established as previously described [2,6].

Genotyping
Genomic deoxyribonucleic acid (DNA) from all patients was extracted from peripheral white blood cells using standard procedures.
The selection of single-nucleotide polymorphisms (SNPs) was based on their position with regards to the IL33-IL1RL1 region and their previously reported associations with several inflammatory diseases. Following these criteria, we performed a tagging using data from Hap-Map project (http://www.hapmap.org) and Haploview software version 4.2 and considering r 2 >0.8, haplotype frequency >5%, minor allele frequency >10%) (data in S1 and S2 figs). After that, we identified 6 polymorphisms, 3 in the IL33 region (rs3939286 (C___2762168_10), rs7025417 (C__31940410_20) and rs7044343 (C__29340326_10), located in different haplotype blocks of this locus, and 3 in the IL1RL1 region (rs2058660 (C__11487892_10), rs2310173 (C___2676437_10) and rs13015714 (C__31439507_10). Although these polymorphisms seem to be apparently non-coding variants, they map and tag both IL33 and IL1RL1 regions. Regarding IL33 region, rs7025417 and rs7044343 are located within IL33 gene (specifically in the promoter and in the intron 5, respectively) (data in S1 fig). By contrast, rs3939286 is situated in an intergenic region close to IL33 gene (data in S1 fig). With respect to IL1RL1 region, whereas rs13015714 is downstream of IL1RL1 gene, both r2058660 and rs2310173 are placed close to IL1RL1 gene (data in S2 fig).
All genetic variants were genotyped with TaqMan predesigned single nucleotide polymorphism genotyping assays in a 7900 HT Real-Time polymerase chain reaction (PCR) system, according to the conditions recommended by the manufacturer (Applied Biosystems, Foster City, CA, USA).
Negative controls and duplicate samples were included to check the accuracy of genotyping.

Carotid US Examination
The measurement of the cIMT was performed by carotid US. Patients from Santander were assessed using a commercially available scanner, Mylab 70, Esaote (Genoa, Italy) equipped with 7-12 MHz linear transducer and the automated software guided technique radiofrequency-Quality Intima Media Thickness in real-time (QIMT, Esaote, Maastricht, Holland)- [16]. Patients from Lugo were assessed using high-resolution B-mode ultrasound, Hewlett Packard SONOS 5500, with a 10-MHz linear transducer as previously reported [17]. cIMT was measured at the far wall of the right and left common carotid arteries, 10 mm from the carotid bifurcation, over the proximal 15 mm-long segment. cIMT was determined as the average of three measurements in each common carotid artery. The final cIMT was the largest average cIMT (left or right). Agreement between the two US methods in patients with RA was previously reported [18]. Experts with high experience and close collaboration in the assessment of subclinical atherosclerosis in RA from Lugo and Santander performed the studies.

Statistical Analysis
All genotype data were checked for deviation from Hardy-Weinberg equilibrium (HWE) using http://ihg.gsf.de/cgi-bin/hw/hwa1.pl. cIMT values were displayed as mean ± standard deviation (SD). The association between genotypes and alleles frequencies of the IL33 rs3939286, IL33 rs7025417, IL33 rs7044343, IL1RL1 rs2058660, IL1RL1 rs2310173 and IL1RL1 rs13015714 polymorphisms and cIMT values was tested using unpaired t test to compare between 2 groups and one-way analysis of variance (ANOVA) to compare among more than two groups. Comparisons of means were adjusted for sex, age at the time of US study, follow-up time and center as potential confounders using analysis of covariance (ANCOVA). Statistical significance was defined as p<0.05. All analyses were performed with STATA statistical software 12/SE (Stata Corp., College Station, TX, USA).

Results
All genotype distributions were in Hardy-Weinberg equilibrium. The genotyping success was greater than 97% in all the cases.
Since sex, age at the time of US study, follow-up time and center may act as potential confounders of the results derived from the US assessment; adjustment for these potential confounders was performed by an ANCOVA model. Interestingly, even after adjustment for potential confounders, both genotype and allele frequencies of the IL33 rs3939286 polymorphism were statistically significant (p = 0.006 and p = 0.0023, respectively) evidencing that the potential effect conferred by IL33 rs3939286 polymorphism may be independent of confounder factors (Table 2). Nevertheless, no significant association between IL33 rs7025417, IL33 rs7044343, IL1RL1 rs2058660, IL1RL1 rs2310173 and IL1RL1 rs13015714 and cIMT values was observed (Table 2).

Discussion
CV disease is the most common cause of premature mortality in patients with RA being a consequence of accelerated atherosclerosis [1]. In the last years, several studies have been focused on the assessment of biologic mechanisms that influence the risk of CV disease in RA. Accordingly, several genetic markers associated with the risk of subclinical atherosclerosis and CV disease in RA have recently been identified [2,[4][5].
Outside the HLA region, cytokine pathway genes, which have critical modulatory effects on innate and adaptive immunity, have been shown to represent a key component of the genetic network associated with immune-mediated processes.
IL-33-IL-1RL1 pathway is a key proinflammatory mediator that may play a pathogenic role in RA [8]. Moreover, detectable IL-33 plasma concentrations at the time of disease diagnosis were found to predict the presence of severe subclinical atherosclerosis in the extended followup of patients with RA [9]. Taking into account these considerations and the implication of 6 genetic IL33-IL1RL1 variants in the susceptibility to several inflammatory diseases [10][11][12][13][14], we aimed to determine the potential association of these genetic polymorphisms with subclinical atherosclerosis in RA. Interestingly, our results suggest a potential protection effect of the mutant IL33 rs3939286T allele in the risk of subclinical atherosclerosis, established by the assessment of cIMT, in patients with RA. This association remained statistically significant after adjustment for potential confounders, evidencing that this potential effect may be independent of sex, age at the time of the carotid US study, follow-up time and center. It is worth noting that according to the public database RegulomeDB the IL33 rs3939286 genetic variant appears to be a regulatory DNA element [19]. Consequently, it could be plausible to think that the mutant IL33 rs3939286T allele might influence the development of subclinical atherosclerosis by regulating the expression of IL33 and, at last, by decreasing IL-33 levels. Despite having a cohort that might be considered relatively small, our study encompassed the largest series of RA patients with cIMT data ever assessed for genetic studies.
In keeping with our findings, recent studies have described a protective effect of the mutant allele of another IL33 gene polymorphism, rs7025417, in the risk of CAD in a Chinese population and also in the development of giant cell arteritis in Europeans [11,20]. Since IL33 rs3939286 and IL33 rs7025417 genetic variants are located in different haplotype blocks and present a low linkage disequilibrium (LD) (D´= 0.13 and r 2 = 0.01), we speculate that either they are independent susceptibility factors for the different phenotypes or they might be tagging the real causal variant. The results obtained in the present study provide additional evidence on the potential role that genetic factors may play in the development of CV disease in RA. The search for genetic markers associated with CV disease in RA may be important for a better characterization of RA patients at risk of CV disease. They may be useful to predict disease outcome at diagnosis of RA and to establish future therapeutic targets to decrease the risk of CV disease in RA patients.
In conclusion, our results may suggest a potential protective effect of the IL33 rs3939286 allele T in the risk of subclinical atherosclerosis in patients with RA.