Association of IL10RA, IL10RB, and IL22RA Polymorphisms/Haplotypes with Susceptibility to and Clinical Manifestations of SLE

Systemic lupus erythematosus (SLE) is characterized by an imbalance between proinflammatory and anti-inflammatory mediators. Single-nucleotide polymorphisms (SNPs) in genes coding IL10RA, IL10RB, and IL22RA could affect their expression or function and disrupt immune homeostasis. We aimed to analyze the associations of IL10RA, IL10RB, and IL22RA polymorphisms/haplotypes with patients’ susceptibility to and clinical manifestations of SLE. Our study included 103 SLE patients and 99 healthy controls. The genotypes of the selected polymorphisms within IL10RA (rs10892202, rs4252270, rs3135932, rs2228055, rs2229113, and rs9610), IL10RB (rs999788, rs2834167, and rs1058867), and IL22RA (rs3795299 and rs16829204) genes were determined by TaqMan® Assays. IL10RB rs1058867 G allele carriers were significantly more frequent among the controls than among the SLE patients (76.8% vs. 61.2%; p = 0.017, OR = 0.477, 95% CI: 0.258–0.879). The IL10RB CAA haplotype was more frequent among the SLE patients than in the control group (42.7% vs. 30.7%; p = 0.027). The IL22RA rs3795299 C allele and rs16829204 CC genotype were associated with Hashimoto thyroiditis in the SLE patients (n = 103; p = 0.002 and p = 0.026, respectively), and in all the included participants (n = 202, p < 0.000 and p = 0.007, respectively), and the IL22RA CC haplotype was more frequent in the SLE patients with Hashimoto thyroiditis (p = 0.047) and in the overall participants with Hashimoto thyroiditis (n = 32, p = 0.004). The IL10RA, IL10RB, and IL22RA polymorphisms/haplotypes could be associated with SLE susceptibility and various clinical manifestations, and the IL22RA CC haplotype could be associated with Hashimoto thyroiditis.


Introduction
Systemic lupus erythematosus (SLE) is a chronic, multisystemic autoimmune disease with heterogeneous clinical manifestations associated with autoantibody production, immune complex formation and deposition, and other immune processes [1,2]. There is a significantly higher prevalence in woman, and the female to male ratio goes up to 15:1 for women at reproductive age [3]. Its etiology is not fully elucidated, but it is well established that this multifactorial disease results from a complex interaction between genetic and environmental factors [2,4]. Genetic factors could predetermine subtle changes in immune system cell signaling and disrupt the balance between pro-inflammatory and anti-inflammatory pathways [4]. Interleukin 10 (IL10) is a potent anti-inflammatory cytokine which plays an important role in immunoregulation. It has also been shown that IL10 promotes B lymphocyte differentiation, proliferation, and class switching [5].
IL10 exerts its effect via the IL10 receptor (IL10R). This receptor is a heterotetramer composed of two IL10R A subunits (IL10RA) and two IL10R B subunits (IL10RB). Both molecules are members of the class II cytokine receptor family (CRF2) [6,7]. After IL10 binds to IL10RA, this complex changes conformation and allows for the binding of the IL10rb subunit of the receptor [6]. As a result, the JAK1-TIK2-STAT3 signaling cascade is triggered [8,9]. Although STAT3 is the main downstream transcription factor, IL10 also activates STAT1 and STAT5 [10]. Except for the suppression of proinflammatory cytokine production, this pathway leads to decreased macrophage activity, the inhibition of monocyte differentiation to dendritic cells, and the downregulation of costimulatory molecules on antigen-presenting cells [5,9]. Meanwhile, B lymphocyte stimulation by IL-10 results in pro-inflammatory behavior [11][12][13].
While the IL10RA subunit is present mainly in leukocytes and is specific for IL10RA, the IL10R B subunit is ubiquitous and participates in the building of receptor complexes for IL22, IL26, IL28a, IL28b, and IL29 [8,14]. Single-nucleotide polymorphisms (SNPs) in gene-coding IL10R subunits could affect their interaction as well as signaling through this pathway and could disrupt immune homeostasis. As cytokines play a crucial role in the regulation of our immune system, we aimed to assess the associations between selected polymorphisms in the IL10RA, IL10RB, and IL22RA genes with SLE susceptibility and its clinical manifestations.

Demographic and Clinical Data
The demographic data and clinical manifestations in the control group and the SLE patients are shown in Tables 1 and 2. The median disease duration in our SLE patients was five years (0.0-42.0). Among the SLE patients, 41 (39.8%) had high disease activity with a clinical SLEDAI score above 4. Corticosteroid therapy was used by 90 (87.4%) of the patients with a median daily prednisone dosage of 12.5 mg (0-80), 86 (83.5%) patients used hydroxychloroquine, 30 (29.1%) had had immunosuppressants (AZA, MTX, MMF) during treatment, and pulse cyclophosphamide and/or glucocorticoids therapy was administered in 13 (12.7%) patients. Secondary Sjogren syndrome was present in 39 (37.9%) of the SLE patients while secondary APS was observed in 9 (8.7%) of the SLE patients. The damage accumulated since the onset of lupus was assessed using the SLICC/ACR damage index, and in our patients, it ranged between 0.0 and 6.0 with a median value of 0.0.

Genotype Analysis
The frequencies of the genotypes/alleles for all of the investigated polymorphisms within the IL10RA, IL10RB, and IL22RA genes are presented in Table 3. The genotype distributions of the studied polymorphisms did not deviate from the Hardy-Weinberg equilibrium in the SLE patients and the control group. IL10RB rs1058867 G allele carriers were significantly more frequent among the controls than among the SLE patients (76.8% vs. 61.2%; p = 0.038, OR = 0.477, 95% CI: 0.258-0.879). This result was significant after using a logistic regression analysis adjusted for age and gender.

IL22RA Haplotypes
Haplotype blocks were not observed between the analyzed IL22RA polymorphisms (rs16829204 and rs3795299; D' = 0.8, r 2 = 0.07). The GC, CC, GT, and CT haplotypes were present in the SLE patients with a frequency of 49.3%, 32.2%, 16.2%, and 2.2%, respectively, while in the control group, the frequencies of the GC, CC, and GT haplotypes were 48%, 39.4%, and 12.6%, respectively. The haplotype frequencies of the patients and the control group did not differ significantly.

IL22RA Haplotypes
Haplotype blocks were not observed between the analyzed IL22RA polymorphisms (rs16829204 and rs3795299; D' = 0.8, r 2 = 0.07). The GC, CC, GT, and CT haplotypes were present in the SLE patients with a frequency of 49.3%, 32.2%, 16.2%, and 2.2%, respectively, while in the control group, the frequencies of the GC, CC, and GT haplotypes were 48%, 39.4%, and 12.6%, respectively. The haplotype frequencies of the patients and the control group did not differ significantly.

Genotype/Haplotype Analysis and Clinical Manifestation in SLE Patients
The association of the analyzed polymorphisms and the clinical manifestations in the SLE patients are shown in Table 4. Additionally, we included both the controls and SLE patients (n = 202) and analyzed the association between rs16829204, rs3795299, and Hashimoto thyroiditis. We confirmed that among the participants with the rs16829204 CC genotype, Hashimoto thyroiditis was more frequent than in those with T allele carriers (20.4% vs. 5.1%; p = 0.007, OR = 4.79, 95% CI: 1.399-16.407), and that among the rs3795299 C allele carriers, Hashimoto thyroiditis was more frequent than in those with GG genotype carriers (23.5% vs. 4.9%; p < 0.000, OR = 6, 95% CI: 2.016-17.853). This result remains significant after a multivariate logistic regression. An additional analysis showed that the IL22RA CC haplotype was more frequent (48.4% vs. 33%; p = 0.004) in the participants with Hashimoto thyroiditis (n = 32).
The haplotype analysis showed that the SLE patients without Sjogren syndrome had the IL10RA GCGAAG haplotype significantly more frequently than the patients with Sjogren syndrome (21.9% vs. 7%, p = 0.007). The patients without Hashimoto thyroiditis had IL10RA GCAAGA and IL22RA GT haplotypes significantly more frequently than the patients with Hashimoto thyroiditis (50.6% vs. 29.4%; p = 0.024 and 19.6% vs. 0%; 0.016, respectively). At the same time, the IL22RA CC haplotype was more frequent in the SLE patients with Hashimoto thyroiditis (44.1% vs. 29.6%; p = 0.047). The patients without a malar rash had the IL10RA CTAAGG, IL10RB TGG, and IL22RA GC haplotypes more frequently than the patients with a malar rash (p = 0.009, p = 0.031, p = 0.049, respectively).
The IL10RA GCAAGA haplotype was more frequent in patients with than in patients without alopecia (56.2% vs. 41.3%; p = 0.036). The IL10RA GCAGGG haplotype was more frequent in patients with pathological urine sediment than in patients with normal findings (16.7% vs. 4.3%; p = 0.025). The IL10RB CAA haplotype was more frequent in patients with oral ulcerations than in patients without this classification criteria (54.3% vs 38.9%; p = 0.038). The IL10RB CGA haplotype was more frequent in patients with thrombocytopenia and lupus nephritis than in patients without these manifestations (12.5% vs. 4.3%; p = 0.045 and 13% vs. 4.2%; p = 0.030, respectively), while this haplotype was less frequent in patients with than in patients without arthritis (20% vs. 4.8%; p = 0.008).

Discussion
SLE is a multifactorial disease with a genetic basis and a pathogenesis that has not fully been elucidated yet [4]. Numerous gene variants are associated with SLE susceptibility [15]. It is known that SLE is characterized by an imbalance between the production of pro-inflammatory and anti-inflammatory cytokines [1,2]. IL10 was singled out as one of the essential immunomodulatory cytokines [16]. It has been known for years that polymorphisms located in the IL10 gene promoter (rs1800896, rs1800871, and rs1800872) affect gene expression [17,18]. Previous studies have shown that the IL10 levels are higher in the blood of patients with SLE than in healthy individuals. The IL10 levels correlate well with the disease activity estimated through the SLEDAI score [17,[19][20][21]. Meanwhile, only a few studies have examined the influence of polymorphisms in gene-coding IL10 receptors on patients' susceptibility to and clinical course of SLE [14,22].
According to our research, only one study has analyzed the association of rs3135932 and rs2229113 IL10RA polymorphisms and SLE, and one study has analyzed the rs2834167 IL10RB polymorphism and SLE [14,22]. Hermann et al. showed that both the rs3135932 (S138G) and rs2229113 (G330R) polymorphisms are loss-of-function variants and lead to reduced IL10RA signal transduction. In this study, rs2229113 was associated with RA and SLE susceptibility, while the influence of rs3135932 was not significant [22].
We included six IL10RA polymorphisms: rs10892202, rs4252270, rs3135932, rs2228055, rs2229113, and rs9610. These polymorphisms and the IL10RA haplotypes did not show an association with the patients' SLE susceptibility. At the same time, we investigated the association of IL10RA polymorphisms with clinical manifestations in SLE patients. Skin manifestations were among the most common in our patients. The malar rash was associated with rs10892202, rs4252270, and their GC haplotype. The IL10RA rs2229113 polymorphism GG genotype was associated with photosensitivity. Although rs10892202 was initially associated with discoid manifestations, a combined genotype and allele analysis did not confirm its influence. Arthritis was associated with the rs9610 G allele. According to Peng et al., in the Chinese population, the rs9610 A allele is associated with RA susceptibility [23]. These two results are difficult to compare due to the different pathogenesis of erosive arthritis in RA and non-erosive arthritis in SLE, as well as the difference in the genetic basis of the two populations, which can be seen through the distribution of the rs9610 allele (MAF(G) = 0.45 in East Asia; MAF(A) = 0.45 in Europe) [24]. Patients with hemolytic anemia carry the rs10892202 C allele and rs4252270 T allele more often than controls. Due to the small number of patients with hemolytic anemia, this result should be confirmed on a larger number of patients. These alleles had a protective effect on the early onset of IBD [25].
Peng et al. associated the rs2834167 allele A with SLE susceptibility [14]. In our study, this polymorphism was not significant in this manner, and this could be the result of the different genetic backgrounds of the Chinese Han and European populations. According to the 1000-genome study, allele A has significantly different frequencies in East Asian and European populations (0.44 vs. 0.73) [26]. On the other hand, the IL10RB CAA haplotype (rs999788, rs2834167, and rs1058867) was more frequent in the SLE patients than in the control group. In our study, rs1058867 allele A was significantly associated with SLE, and this polymorphism could carry the significance of the entire haplotype. Still, we must consider the possibility that the remaining two polymorphisms have a subtle influence that could not be seen due to the small number of study participants. Our study is the first to assess the IL10RB polymorphism's influence on the clinical manifestation of this disease. In our study, the rs2834167 AA genotype was associated with malar rashes, the same as the rs999788 CC genotype. Hikami et al. associated the rs999788 CC genotype and rs2834167 AA genotype with systemic sclerosis. These two polymorphisms showed strong LD in Hikami et al.'s study and in our study [27]. Allele A (47lys) showed an association with a lower IL10RB expression in Frodshman et al.'s research, possibly due to strong LD with rs999788, located in the NF-κβ binding site [27,28]. This binding site may be compromised in presence of C allele [27]. Polymorphism rs1058867 is located in the 3 UTR region and according to our study, it is not strongly linked with rs999788 and rs2834167. Polymorphisms in the 3 UTR region could affect protein expression through changes in mRNA stability and its interaction with regulatory proteins, and miRNA [29]. After a variant in the non-coding region affects the receptor expression or transduction, changes in the production of IL10, as seen in SLE, could be a response to receptor availability or malfunction. Consequently, the control of pro-inflammatory cytokines by IL10 would be disrupted. Such an imbalance could result in an increased tendency towards an altered immune response and the development of autoimmune diseases such as SLE.
An additional haplotype analysis revealed an association of the IL10RA and IL10RB haplotypes with the clinical course of disease. As a multisystemic disease, SLE is characterized by heterogenous manifestations frequently followed by the development of secondary autoimmune entities, such as secondary antiphospholipid disease, Sjogren syndrome, and thyroiditis [30]. Our study showed that the IL10RA GCGAAG haplotype was more frequent in the SLE patients without secondary Sjogren syndrome, and the GCAAGA haplotype was more frequent in the SLE patients without secondary Hashimoto thyroiditis. These haplotypes could be protective for the development of secondary entities in SLE patients. Interestingly, some of haplotypes, such as the IL10RB CGA haplotype, acted protectively toward arthritis occurrence, and at the same time, this haplotype was more frequent in the patients with thrombocytopenia and nephritis.
Our study showed that the IL22RA rs3795299 C allele and rs16829204 CC genotype are associated with Hashimoto thyroiditis in SLE patients and in all included participants, and we confirmed these results in our haplotype analysis. The IL22RA gene rs3795299 (c.1552C > G, p.Arg518Gly) and rs16829204 (c.613G > A p.Val205ILe) polymorphisms are both missense variants, and this can explain their effect on IL22RA function [26,31,32]. This is the first study that analyzed the effect of IL22RA polymorphisms on the development and clinical manifestation of autoimmune diseases. Niu et al. showed that the rs3795299 G allele protects preeclampsia development in Chinese Han women [32]. Dinarte et al. found no significant association between rs16829204 and chronic rhinosinusitis [31].
The limitation of our study that needs to be acknowledged was the relatively small number of patients, which reflects the number of inhabitants of our country. We would like to emphasize that this is the first study that has examined the variants within the IL10RA, IL10RB, and IL22RA genes and their association with SLE susceptibility and the disease's manifestations. We would like for this study to be the basis for further research that includes a larger number of patients and contributes to the elucidation of the enigmatic role of anti-inflammatory pathways in the development of autoimmune diseases.

Patients and Controls
All 103 SLE patients in the study fulfilled the American College of Rheumatology (ACR) 1997 classification criteria for SLE [33]. All patients were diagnosed, treated, and prospectively followed up with at the Clinic for Allergology and Immunology University Clinical Center of Serbia and the Institute of Rheumatology, Belgrade between June 2020 and November 2021. Disease activity was assessed using the SLEDAI 2K scale, and Physician Global Assessment (PGA) [34,35]. Demographic, clinical, and laboratory data were collected from each patient at the time of their enrollment in the study. We collected the following data on the presence of antibodies in each patient: antinuclear (ANA), anti-dsDNA antibodies, anti-Sm, anti-SSA, anti-cardiolipin antibodies (aCL), anti-beta2-glycoprotein I (anti-β2-GPI) IgG and IgM, anticardiolipin IgG/IgM, anti-β2-GPI IgG/IgM, anti-SSA and anti-Sm, and rheumatoid factor (RF). ANA titer above 1:80 was considered positive. Serum concentrations of C3, C4, IgG, IgM, and IgA were recorded. The exclusion criteria for both study cohorts were those who were under 18, who were inable to cooperate, and who had malignancies. Patients with significant comorbidities (severe cardiac, pulmonary, and psychiatric diseases or dementia) were excluded from the study. All patients gave written informed consent to participate in the study. The study was carried out according to the Declaration of Helsinki and approved by the Ethical Board of the Faculty of Medicine, University of Belgrade (No. 1550/IX-14). The control group included 99 participants who did not suffer from any autoimmune rheumatology disease with negative family history of these diseases.

Data Analysis
Depending on the data distribution, numerical data are described with the arithmetic mean and standard deviation or median and range. Normal distribution was evaluated by mathematical (Kolmogorov-Smirnov and Shapiro-Wilk test, skewness and kurtosis, and coefficient of variation) and graphical methods (histogram, box plot, and Q-Q diagram). Categorical variables were presented as an absolute and relative numbers in the form of n (%). Student's t-test for two independent samples or Mann-Whitney U test were used to compare numerical data between study cohorts, depending of the data distribution. The Chi-square test was used to test the difference in the distribution of categories of two independent samples.
In order to evaluate all possible factors influencing one's probability of having systematic lupus erythematosus, univariate logistic regression analysis was performed first, with multivariate analysis thereafter with significant variables from the previous analysis in the model. VIF multicollinearity testing method and correlation coefficients were used. All variables with VIF > 5 were eliminated from the multivariate model. moreover, according to the covariance matrix and correlation coefficients, one of two correlated variables with a lower p-value in univariate logistic regression was eliminated. Forward (Wald) regression method was applied, and only the last step was presented in the results. Odds ratio (OR), 95% confidence interval of odds ratio (95% CI OR), and p-value were reported. The sample size was calculated by G Power 3.1.9.2 for the major outcomes. According to previously known data about the IL10RB rs1058867 polymorphism's allele distribution given by 1000 Genomes Project Consortium for a medium effect size of 0.3, α of 0.05, power of 80%, and df = 1, we obtained a total sample size per group of 88 [24].
Haplotype analysis was performed using HaploView 4.2 software [37]. Haplotype blocks were evaluated using the "Confidence Intervals LD" method.

Conclusions
Our study showed that the polymorphisms/haplotypes in the genes coding IL10R could contribute to SLE development. Additionally, the result of this study indicates the possibility that specific genotypes/haplotypes can direct the course of the disease towards specific clinical manifestations. Such knowledge could help in the development of personalized medicine, providing a valuable tool for monitoring high-risk patients with a goal of the early detection of the first signs of the development of severe disease manifestations.  Data Availability Statement: Data are available upon reasonable request. Please contact the corresponding author. They are not publicly available to ensure the strict confidentiality of the patients' data.

Conflicts of Interest:
The authors declare no conflict of interest.