Associations between interleukin and interleukin receptor gene polymorphisms and risk of gout

Gout is a self-limiting, auto-inflammatory arthritis induced by the deposition of monosodium urate crystals in the synovial fluid and periarticular tissues. The aim of this study was to investigate the associations between genetic variants in the interleukin (IL) and interleukin receptor (ILR) genes IL-33, IL-1RL1, IL-23R, and signal transducer and activator of transcription 4 (STAT4) and susceptibility to gout in Chinese Han male individuals. The genetic distributions of rs3939286 in IL-33, rs13015714 in IL-1RL1, rs10889677 in IL-23R, and rs7574865 in STAT4 were detected in 1100 men with gout and 1227 ethnically matched controls, using Taqman allelic discrimination real-time polymerase chain reaction (PCR). Differences in these polymorphisms between the groups were investigated using χ2 tests. The genotype-phenotype relationship among gout patients was tested by analysis of variance. There was a significant difference in genotypic frequencies of IL-23R rs10889677 between gout patients and controls (χ2 = 81.386, P < 0.001). However, there were no significant differences in distributions of the other polymorphisms between the groups. Our results revealed that the rs10889677 variant in IL-23R may be involved in the development of gout in Chinese Han male individuals. However, further studies in other ethnic groups are needed to confirm these results.

Scientific RepoRts | 5:13887 | DOi: 10.1038/srep13887 The IL-33/IL-1RL1 axis plays a critical role in several autoimmune and inflammatory disorders. IL-23 is a member of the IL-12 family and also a pro-inflammatory cytokine, comprising a p19 subunit and a p40 subunit of IL-12B. After binding to the IL-23 receptor (IL-23R), IL-23 stimulates the secretion of a variety of inflammatory factors such as IL-1, IL-6, IL-8, and TNF-α by activated CD4 + Th17 cells, which in turn drive gouty inflammation 13 . In addition, signal transducer and activator of transcription 4 (STAT4), encoded by STAT4 mapped to chromosome 2q33, regulates the IL-23-related inflammatory response by transmitting signals in response to several cytokines such as IL-23 and IL-12 14 . STAT4 is also essential for the expansion of Th17 cells activated by IL-23, which contributes to the development of many autoimmune diseases 15 . The production and function of cytokines may be affected by polymorphisms in the functional regions of their genes, suggesting that IL-33, IL-1RL1, IL-23R, and STAT4 may be candidate genes for the inflammatory pathogenesis of gout. The current study therefore aimed to investigate the associations between the IL-33 rs3939286 A/G, IL-1RL1 rs13015714 G/T, IL-23R rs10889677 A/C, and STAT4 rs7574865 G/T single nucleotide polymorphisms (SNPs) and the risk of gout.
Genotype-phenotype analysis of the rs10889677 polymorphism. As shown in Table 4, a detailed genotype-phenotype analysis of IL-23R rs10889677 was conducted among gout patients in relation to clinical factors including demographic characteristics and serum biochemical parameters, as well as tophi and previous medical history (hypertension, diabetes mellitus, and obesity). When gout patients were divided into AA, AC, and CC groups, there were significant differences in hypertension history between the three groups (P = 0.020) and between the AA and AC + CC groups (P = 0.009). However, there were no significant differences between the three groups in any other parameters, including BMI,

Discussion
Gouty inflammation is a paradigm of innate immunity and the IL-1β /IL-1R pathway plays a key role in acute gout attack 16 . Phagocytosis of MSU by macrophages can activate the NLRP3 inflammasome, which is considered to play a critical role in gouty arthritis. Caspase-1 is in turn activated by the NLRP3 inflammasome and can lead to the conversion of pre-IL-1β and pre-IL-18 to mature IL-1β and IL-18 and secretion of the latter. Binding of IL-1β to the IL-1β receptor in endothelial cells and resident macrophages, together with other pro-inflammatory cytokines and chemokines such as IL-6, IL-8, and TNF-α , can finally lead to gouty inflammation 17 . Genetic factors are known to play key roles in the pathogenesis of gout, and genetic variants in genes for cytokines involved in gouty inflammatory pathways, such as IL-1β , IL-1R, IL-6, IL-8, and IL-18, have been associated with susceptibility to gout 17 . We previously suggested that the IL-8 251T/A and IL-12B 1188A/C polymorphisms may be relevant host susceptibility factors for the development of gout 18 . In addition, a case-control study in 400 gout patients and 582 gout-free controls revealed that the IL-23R rs7517847 G/T polymorphism might be associated with gout in Chinese Han males 19 . Moreover, the analysis of linkage disequilibrium between rs7517847 and rs10889677 in IL-23R was done further, and the D' value was 0.653, while the r 2 value was 0.157, which indicated that there was no linkage disequilibrium between these two SNPs. Sites rs10889677 and rs7517847 of IL-23R were independent to each other and this may demonstrated IL-23R as an important candidate gene for gout susceptibility. However, Tsai et al. 20 found no significant association between four polymorphisms in the IL-6 gene promoter, including -597G/A, -572C/G, -373A/T, and -174G/C and gout.
As an IL-1 family member, IL-33 is synthesized as a 30-kDa peptide and then cleaved by caspase-1 to form an active 18-kDa mature peptide, which is widely expressed in tissues, specifically in epithelial linings and smooth muscle cells 21 . Through binding to the IL-33R encoded by IL-1RL1, IL-33 can activate mast cells and Th2 cells as well as Th17 cells, leading to the production of pro-inflammatory cytokines and chemokines 21 . IL-23/IL-23R represents a novel pathway involved in inflammatory responses. The effect of IL-23 depends on its binding to IL-23R, a heterodimer of the IL-12RB1 subunit 22 , which was identified as an additional candidate gene in some autoimmune and inflammatory diseases such as inflammatory bowel disease, psoriasis, Graves' ophthalmopathy, ankylosing spondylitis, and multiple sclerosis 23,24 . A genome-wide association study in Japanese subjects reported that the rs12119179 variant in IL-23R was associated with Behcet's disease 25,26 . As noted above, IL-23 may be associated with the production of IL-17A, IL-17F, IL-6, and TNF-α by active Th17 cells, which are important effector cytokines during gouty inflammation. In return, the engagement of IL-17 and/or IL-17F with endothelial cells promotes the expression of IL-1, IL-6, IL-8, TNF-α , and ICAM-1, which are critical factors driving the inflammatory response 13 . Moreover, the ability of IL-23 to induce IL-17A, IL-17F, and other pro-inflammatory   factors appears important for neutrophil recruitment 27 . STAT4 transmits signals induced by several cytokines such as IL-12 and IL-23, which are key cytokines in the development of autoimmune diseases 14 . Furthermore, STAT4 plays a key role in IL-12-induced T-cell differentiation into Th1 cells 14 and in IL-23-mediated production of IL-17 by activated Th17 cells 15,28 . The functions of these inflammatory cytokines suggest that they may play important roles in the inflammatory pathogenesis of gout. We therefore propose IL-33, IL-1RL1, IL-23R, and STAT4 as potential candidate susceptibility genes for gout.
To the best of our knowledge, this is the first study to investigate the association between these SNPs in IL-1-related cytokine genes and the risk of gout. We found that IL-23R rs10889677 was significantly associated with susceptibility to gout in Chinese Han male individuals. The genotypic frequency differed significantly between gout patients and controls, and patients with the AC or CC genotype were more likely to develop gout than patients with the AA genotype. It found that significant differences exist in patients with hypertension history between the AA and AC + CC groups and between the three groups when doing an analysis on the genotype-phenotype of IL-23R rs10889677, which showed C allele may be a risk allele for gout patients to develop hypertension, however, it may need further research as C allele frequency of rs10889677 is smaller than A allele. rs10889677 is located in the 3′ -UTR of IL-23R, suggesting that it might regulate the expression and function of IL23R mRNA. The other three tested SNPs showed no significant association with gout risk. However, Carriere et al. and Oboki et al. reported that IL-33 rs3939286 and IL-1RL1 rs13015714 were associated with other autoimmune diseases such as Crohn's disease, rheumatoid arthritis (RA), asthma, and Alzheimer's disease 29,30 . Hayakawa et al. also found that blocking IL-1RL1 receptor signaling could prevent arthritis development and airway inflammation 31,32 . A relevant study in western Algeria indicated that the STAT4 rs7574865 polymorphism was clearly associated with the risk of RA in an Algerian population 33 , while a meta-analysis by Jiang et al. also indicated a significant association between the rs7574865 polymorphism and a decreased RA risk in an Asian population 34 . In addition, Liu et al. found that rs7574865 in STAT4 was significantly associated with increased susceptibility to and severity of ankylosing spondylitis in a Chinese Han Population.
There were some limitations to this study. First, all the subjects were Chinese Han individuals from Shandong Province, and further studies including participants from other nationalities and regions are therefore needed to determine the general applicability of the results, given the important roles of both genetic and environmental factors in the development of gout. Furthermore, other important SNPs exist on these genes, and the contributions of these to gout susceptibility should also be investigated. However, despite these limitations, our study demonstrated an association between IL-23R and susceptibility to gout in Chinese Han male individuals.

Methods
Populations and clinical data. A total of 1100 men with gout who visited the Department of Gout, the Affiliated Hospital of Qingdao University, China between January 2009 and December 2014 were recruited to this study. Gout diagnosis followed the criteria published by the American College of Rheumatology in 1997 35 . No patients had any history of cancer, hematopathy, nephropathy, or other autoimmune diseases. Some metabolic diseases such as hypertension, obesity, diabetes and so on were not excluded from gout patients because there were no researches have indicated relationships between the four SNPs and these diseases. A further 1227 gout-free male controls were enrolled. All the participants were Chinese Han men. Blood samples were collected into anticoagulant-coated tubes and transferred immediately to the laboratory for DNA extraction and genetic analysis. Demographic and laboratory parameters, and medical histories were recorded by experienced endocrinologist physicians, including serum uric acid, glucose, TG, TC and creatinine levels, blood pressure, and BMI, as well as age of onset, tophi, and medical history among gout patients. This study was approved by the Ethics Committee of the Affiliated Hospital, Qingdao University and conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki.
Scientific RepoRts | 5:13887 | DOi: 10.1038/srep13887 Statistical analysis. Statistical analyses were carried out using the Statistical Package for Social Sciences version 22.0 (SPSS Inc., Chicago, IL, USA). The Hardy-Weinberg equilibrium of the control group was tested using the goodness-of-fit χ 2 test. Differences in genotypes and alleles between cases and controls were analyzed using the Pearson's χ 2 test (or Fisher's exact test if the expected values were < 5), and P-values < 0.013 were considered significant when Bonferroni's correction was made. Differences in demographic and clinical indexes between the two groups were compared using the Student's t-test. Differences between genotyping and clinical characteristics in gout patients, including clinical and biochemical parameters, tophi, and past medical history were assessed by analysis of variance. The strength of the relationships in allelic distributions between gout patients and controls was assessed by OR and 95% CI. All statistical tests were two-sided with a significance level of 0.05, and differences were deemed to be significant at P < 0.01.