Genetic Association of Interkeukin-21 and Interleukin-23 Receptor Polymorphisms With Systemic Lupus Erythematosus in Iranian Population

Objective: T helper 17 (Th17) is believed to play a crucial role in pathophysiology of systemic lupus erythematosus (SLE). Our study aimed to detect the potential association of IL-21 and IL-23R polymorphisms with susceptibility to SLE. Method: Two hundreds and ten patients with SLE and the same number of matched controls were genotyped by polymerase chain reaction to detect two polymorphisms of IL-21 and one polymorphism of IL-23 receptor (IL-23R). Clinical data was collected from patients’ archives. Results: IL-21 polymorphisms (rs4833837 C/T and rs2055979 G/T) did not show any association with the disease susceptibility (p= 0.54 and p= 0.49, respectively). Comparison of patients with rs2055979GG and T allele carriers showed higher frequency of rs2055979GG genotype in patients with pleurisy compared to the ones without pleurisy (p=0.004). In addition, categorization of IL-23R rs10889677 to GG and T allele carriers (TT+TG) showed an association between TT+TG genotypes and susceptibility to SLE (p=0.0002). Moreover, a signicant association was found between the inheritance of rs10889677GG+TG genotypes and presence of mucosal ulcers in SLE patients (p=0.01). Discussion: The results showed that the lower IL-21 producer rs2055979GG genotype was associated to pleurisy. Lower IL-21 serum levels in patients with rs2055979GG genotype may lead to more frequent pulmonary infections and an increased risk of pleurisy. Association of high+ intermediate IL-23R producer genotypes (rs10889677TT+TG) with SLE may be explained by possible overactivation of Th17 cells. Higher activity cells


Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease involving various organs in the body with a signi cant mortality and morbidity. SLE is characterized by a relapsing remitting course and production of several autoantibodies against host cell's nuclear components. Still not clearly understood, the disease etiology in believed to be the outcome of impaired interaction between genes and environment (1). Abundant studies have con rmed the role of genetic background in SLE development.
The disease does not follow a simple Mendelian inheritance rule; thus a polygenic inheritance model has been suggested (2). Due to the in ammatory nature of SLE, current studies are focusing on nding abnormal immune responses to the triggers. Cytokines are of the major actors of in ammation which regulate the proliferation, maintenance, differentiation and apoptosis of immune cells (3). IL-21 is one of the newly investigated cytokines in pathogenesis of autoimmune diseases. Mostly produced by CD4 + cells, IL-21 has various immune regulatory functions including augmenting T cells proliferation, increasing the activity of natural killer cells and induction of B cell differentiation to plasma and memory cells resulting in increased antibody production (4). According to previous studies, some polymorphisms of IL-21 predispose the individual to autoimmunity (5). Of interest, patients with active SLE have higher serum concentration of IL-21 and blockade of IL-21 receptor in murine models results in decreased disease activity (6-7). Therefore, single nucleotide polymorphisms (SNPs) in the IL-21 gene may be associated with susceptibility to SLE.
Another interesting cytokine in the pathogenesis of autoimmune diseases is IL-23. This cytokine is produced by the activated dendritic cells and its main function is to stimulate the production of IL-17 by Th17 cells. The release of IL-17 embarks an in ammatory response by production of several other proin ammatory mediators (8). The IL-23/IL-17 axis is a well-known pathway necessary for maintaining chronic in ammatory states such as bacterial infections and autoimmune disorders. Over expression of IL-23R has been reported in some autoimmune diseases and allergic diseases (9,10). In addition, several IL-23R gene polymorphisms have been associated with increased risk of various autoimmune diseases (11)(12).
Considering the importance of IL-21 and IL-23 in Th17-related immune responses and development of SLE (13), the association of the two IL-21 gene polymorphisms (rs4833837 C/T and rs2055979 G/T) as well as rs10889677 G/T polymorphism in the IL-23R gene with SLE was investigated in the present study.

Methods
Patients and controls SLE patients were enrolled from Shiraz Medical School related rheumatology clinics including Hafez and Motahari centers. Two hundred and ten patients and the same number of healthy controls were selected. Patients were con rmed cases of SLE by the laboratory tests and certi ed physicians and all met the new SLICC criteria (14). Patients were reviewed based on the most recent follow up and their self-reported questionnaires and notes of certi ed rheumatologists. Healthy controls were recruited from blood donors of blood transfusion units. Control cases had no history of known autoimmune, in ammatory diseases, and cancer. Patients and controls were matched for age, gender and ethnicity. The female/male ratio was 11.35/1 in both groups. All subjects signed an inform consent regarding blood sampling and access to medical records before enrolling in the study. The study was approved by the ethics committee of Shiraz University of Medical Sciences.

Genotyping
Genotyping was performed on genomic DNA isolated from peripheral leukocytes. DNA was extracted by using the salting-out method in Autoimmune Diseases Research Centre of Shiraz University of Medical Sciences and was stored at -21 ℃ until use. The international HapMap project databank was used to get access to the nucleotide sequence of the DNA segments containing the desired SNPs and the appropriate primers were designed by the Primer 3 software and were ordered for the PCR process (Table 1). To genotype IL-21 rs2055979 G/T, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used. The ampli cation reaction was done by using 2.5 µl of PCR buffer (1.8 mM MgCl2), 1 unit of Taq polymerase, 2 µl of 2.5 mmol/L dNTPs and 20 pmol/L of primers with 50 ng of DNA. The PCR program was designed as following, 5 minutes heating at 94°C, denaturation for 30 cycles of 30 seconds at 94°C, annealing for 30 seconds at 61°C and elongation for 30 seconds at 72°C. The extension time lasted for 5 minutes at 72°C at the end of the cycles. The next step, we added the restriction enzyme Hin1II to the PCR products for digestion. The digestion process lasted for 16 hours at 37°C. The digested PCR products were stained by 2% ethidium bromide and were run on 1.5% agarose gel at 100 V for 70 minutes. Results were interpreted using a UV illuminator machine. Genotypes were determined by comparing the DNA segment sizes ( Fig. 1-A). To genotype IL-21 rs4833837 C/T and IL-23R rs10889677 G/T we used allele speci c PCR method. The rst round PCR program was set as previously stated. After the ampli cation, 2 µl of a 1:50 dilution of the rst round PCR mixture was used for the second PCR round, which needed two tubes for the ampli cation of wild and variant type alleles using different primers (Table 1.). The PCR products are shown in Fig. 1B Allele speci c PCR PCR-RFLP using Hin1II as restriction enzyme TTT TAG CCA TTC  TTC TGC CTc-3'   5'-TTT TAG CCA TTC  TTC TGC CTa-3'   5'-TTT TAG CCA TTC  TTC TGC CTa-3' Allele speci c PCR

Statistical Analysis
Genotypes and alleles frequencies were compared between patients and controls and between patients classi ed based on clinical manifestations using the chi-square test. Association of genotypes with continuous parameters (such as age at disease onset) was analyzed by ANOVA. All the tests were performed two-tailed with con dence interval (CI) and a p-value < 0.05 was regarded as the level of statistical signi cance. SPSS version 11.5 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.

Results
Two SNPs within IL-21 gene were genotyped in 210 SLE patients and matched controls. None of the selected SNPs showed signi cant association with susceptibility to the disease in our study ( Table 2).
The minor allele frequency of the rs4833837C/T was 20.2% in patients compared to 18.6% in matched controls (p = 0.54) while for rs2055979G/T was 46.9% in patients and 49.3% in controls (p = 0.49%).
Of interest, analysis of the rs10889677G/T genotypes in IL-23R gene showed a signi cant association with susceptibility to the disease (p < 0.0001; Table 3). After classi cation of cases and controls based on their gender, the difference remains signi cant in females (Table 3). Of interest, after categorization of females into rs10889677GG genotype and rs10889677T allele carriers (TT + TG genotypes), it was revealed that part of the observed difference in female patients and controls is due to the higher frequency of rs10889677T allele carriers in female patients compared to controls (84.4% and 67.9%, respectively; p < 0.0001; Table 3). Of interest, the frequency of heterozygote rs10889677TG genotype was also signi cantly higher in female patients compared to female controls (55.4% and 34.2%, respectively; p < 0.0001). The same analysis for IL-23R rs10889677G/T showed a signi cant association between this polymorphism and the presence of mucosal ulcers (p = 0.001). In further analysis, patients were categorized into TT and TG + GG or TG and TT + GG genotypes. The results showed that the association between rs10889677 and mucosal ulcers is a result of higher expression of rs10889677TT genotype in patients without mucosal ulcers compared to those patients with mucosal ulcers (40.5% and 10.5%, respectively; p = 0.01) or lower frequency of rs10889677TG genotype in patients without mucosal ulcers compared to ones with mucosal ulcers (38.8% and 84.2%, respectively; p = 0.0002; Table 4). Furthermore, we found a trend towards signi cant association between rs10889677 genotype distribution and neurological symptoms in SLE patients (p = 0.064; Table 4). After patients classi cation based on their allele inheritance it has been cleared that rs10889677TT genotype was associated with resistance to neurological involvement in the patients (p = 0.025; Table 4).   (17,18). However, neither of the rs4833837 alleles was signi cantly associated with higher serum levels of IL-21 (17,18). Previously, Hughes, et al. reported that the C allele was the risk allele in SLE patients of European descent but such association was not seen in African American patients (19). In our study, the minor allele (C) frequency of rs4833837 was 20.2% in south Iranian patients and 18.6% in matched controls and no statically remarkable association of this SNP with SLE was found (p = 0.54). The inconsistent ndings might result from differences in genetic make-up between African American and Iranian population as well as the complicated interaction of various genes and environmental triggers in pathogenesis of SLE. Lan, et al. reported a signi cant association between rs2055979T allele and increased levels of IL-21 and susceptibility to SLE in Chinese population (20). However, this association was not repeated in a study on 1318 American patients (21). Also, in the current study no signi cant association was found between the inheritance of rs2055979T allele and development of SLE (p = 0.49). However, classi cation of patients into rs2055979GG genotype holders and T allele carriers (GT + TT) showed an increased frequency of GG genotype in patients with pleurisy compared to the ones without it (100% vs 24.4%, respectively; p = 0.004). This nding is in favor of the risk role of the G allele or the protective role of T allele in developing pleurisy. The base of this association is not clear at the present time. However, given the fact that infections are among the most important inducers of pleurisy and IL-21 plays an important role in antibody production, it can be hypothesized that SLE patients with low IL-21 producer genotype (rs2055979GG) may be prone to pulmonary infections and pleurisy due to the their lower ability of IL-21 production.
IL-23 is a pro-in ammatory cytokine which embarks a cascade of in ammatory factors via activation of IL-17, a well-known mediator of in ammation (8). Consequently, the association of IL-23R gene variations with disorders of the immune system has been studied in several studies (22)(23). So far, none of the SNPs in the IL-23R gene was reported to be associated with the susceptibility to SLE in the Hungarian, Korean and Chinese patients (24)(25)(26). rs10889677 is located in the 3'-UTR region of the IL-23R gene. 3'-UTR region is not translated to proteins but has regulatory functions and in uences gene expression and mRNA stability (27). It has been reported that rs10889677T allele increases the mRNA production and results in an overall increase in IL-23R production (28). Of interest, in contrast to previous studies, the result of the present study showed an association between rs10889677T allele carriers (GT + TT genotypes) and susceptibility to SLE (p = 0.0002), which is consistent with the enhancing effect of rs10889677T allele on IL-23R mRNA production. To our surprise, this association was only detected in the studied females. This gender biased association may be related to the difference in hormonal factors between various genders. However, it should be considered that the number of the male patients in the present study was rather small (a total of 16 males) and further studies are needed before taking any conclusion. In addition, the results of the present study showed a signi cant association between inheritance of high producer rs10889677T allele carriers (GT + TT) and the presence of mucosal ulcers in SLE patients (p = 0.01). Inheritance of rs10889677T allele carriers may predispose patients to the development of mucosal lesions through enhancing IL17/IL23 axis activity. This in ammatory axis is profoundly active during active phases of SLE (29), when the patients experience more oropharyngeal lesions. Unexpectedly, our results showed an increased frequency of rs10889677TT genotype in patients without neurologic involvement compared to the ones with neurologic manifestations (41.5% vs 15%, respectively; p = 0.025). Considering the importance of Th17-related cytokines in the development of immune-associated neurological diseases (30), the basis for this association is not clear to us and indicates the need for further researches in this area.

Conclusions
The result of the present study showed that rs2055979 in the IL-21 gene is associated with pleurisy development in SLE patients while this SNP is not associated with SLE development. Furthermore, we found that rs10889677 in the IL-23R gene is associated with susceptibility to SLE as well as some clinical manifestations in the studied SLE patients. It is worth mentioning that these preliminary ndings need further studies on larger SLE populations to come into a conclusive conclusion.

Declarations
Ethics approval and consent to participate The study involving a human participant to report was approved by the ethics committee of the department of Immunology at Shiraz University of Medical Sciences. Written consent was obtained from the patients.

Consent for publication
Written informed consent was obtained from the patients for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Availability of data and material
Not applicable.

Competing interests
The authors declare no con ict of interest.

Funding
The Work was funded by Shiraz University of Medical Sciences.
Authors' contributions MAN, EL and SSH were the clinical rheumatologists who followed the patients and gathered the clinical data. MRM was the immunology PHD candidate who performed the laboratory tests. EKS was the professor of immunology who supervised the study in all stages. NA was the student of the research committee and took part in data analysis and preparation of the manuscript. All authors reviewed the paper and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.