Plasma interleukin-21 levels and genetic variants are associated with susceptibility to rheumatoid arthritis

Two hundred eleven rheumatoid arthritis patients (156 females and 55 males) were recruited in the present study from January 2018 to December 2019. All patients visited or admitted in the Department of Rehabilitation, Shanghai Putuo People’s Hospital rheumatology division of the hospital and fulfilled the 2010 criteria for American College of Rheumatology/European League against Rheumatism criteria for the classification of RA [24] were enrolled in the study. The mean age of patients was 42.9 ± 13.5 years, and the duration of diseases was 18.3 ± 9.4 months. The exclusion criteria included hypo or hyperthyroidism, diabetes, other autoimmune disorders, chronic liver failure, acute/chronic diarrhea, and congestive heart failure. Three hundred three healthy controls hailing from similar geographical areas, with a mean age of 46.1 ± 18.3 years, were included in the study. RA patients’ various clinical data, such as numbers of swollen and tender joints, disease activity score (DAS 28), and swollen joints count (SJC) were collected from medical records. Further, based on DAS28 scores, patients were sub-grouped into low (DAS 28, < 3.2), intermediate (DAS 28, 3.2–5.1), and high (DAS 28, > 5.1), as per the classification criteria for disease activity by European League against Rheumatism (EULAR) [25]. Different biochemical parameters such as C-reactive protein (CRP), rheumatoid factor (RF), erythrocytic sedimentation rate (ESR), and antibodies to cyclic citrullinated peptides (anti-CCP antibodies) were also examined. All patients were treated with disease-modifying anti-rheumatic drugs (DMARDs) alone or combined with glucocorticoids (GCs). Details of treatments are shown in Table 2. The study was carried out according to the Declaration of Helsinki on ethical principles for medical research involving human subjects [26]. The study protocol was approved by the Institutional Human Ethical Committee of Shanghai Putuo People’s Hospital (PTRMYY20200826), and written informed consent was obtained from each participant.

About 4 mL of intravenous blood was collected from each participant with anti-coagulant at the time of enrollment. Plasma was separated after centrifuging blood at 2500 rpm for 15 min and stored at − 20 °C until further use.

According to the manufacturer’s instructions, total genomic DNA was isolated from 200 μL of whole blood by using GenElute Blood Genomic DNA Kit (Merck). In brief, about 200 μL of whole blood samples were lysed with lysing solution with proteinase K at 55 °C for 10 min. The lysed cells were loaded in the DNA isolation column and centrifuged at 6500 g for 1 min. Subsequently, the column was washed twice with wash buffer. The membrane-bound DNA was eluted with elution buffer after centrifugation at 6500 g for 1 min. The isolated genomic DNA was stored at − 20 degrees until further use.

A total of four SNPs (rs907715, rs2221903, rs2055979, and rs6822844) were typed by TaqMan SNPs genotyping method. Predesigned SNP genotyping assays kit were procured from Thermo Fisher Scientific and used to assess enrolled subjects’ genotype. Details of probes are mentioned in Table 1. In brief, a total of 10 μL of the reaction mixture was prepared with 1X TaqMan Genotyping master mix, 1X custom SNP genotyping assay, and 20 ng of DNA from each participant. The reaction cycle was carried out in three steps as follows: step-1, initial heating at 50 °C for 2 min; step-2, heating at 95 °C for 10 min to activate AmpliTaq gold polymerase; step-3, 40 cycles of denaturation at 94 °C for 15 s followed by annealing and extension at 62 °C for 1 min. The fluorescence was read using the allele discrimination program of Applied Biosystems Real-time PCR system (7900HT).

Plasma level IL-21 was measured in patients and controls using human IL-21 Duo Set ELISA kit (R&D Systems, Inc., USA) according to the manufacturer’s instructions. All plasma samples were measured in duplicate, and the average absorbance value was recorded for a study subject. Furthermore, various autoantibodies such as Anti-cyclic citrullinated peptide (Anti-CCP: Euroimmune, Germany), anti rheumatoid factors (IgG and IgM: Abnova, Germany) were quantified by ELISA according to the instructions of the manufacturer’s.

The statistics analysis was performed by GraphPad Prism version 8.3.0 (GraphPad Software, Inc., La Jolla, CA, USA). The distribution of variables was tested by the D’Agostino-Pearson omnibus normality test. Based on the normality test result, differences in IL-21 levels in RA patients and HC were compared by Mann Whitney U test. Other comparisons with more than two groups were performed with analysis of variance (ANOVA) followed by Tukey’s post-test. Further, the relationship between the IL-21 and DAS 28 scores was conducted by Spearman’s correlation test. Genotype and allele frequency in RA patients and healthy controls were compared by Chi-square (χ²) test. A p-value of less than 0.05 was considered statistically significant.

Baseline characteristics of rheumatoid arthritis patients and healthy controls are shown in Table 2. As demonstrated earlier, the RA is most frequent in females compared to males. In our studied cohort, female patients were 2.83 folds higher chance of having RA compared to males. Biochemicals parameters such as ESR and CRP levels were significantly elevated in RA patients compared to healthy controls. Significantly, results for subgrouping of patients based on DAS 28 score revealed that 30.3% of subjects had low disease activity (DAS 28, < 3.2), whereas, 36.9% of subjects had medium (DAS 28, 3.2–5.1) and the remaining 32.8% patients showed a high disease activity (DAS 28, > 5.1). On screening of RA patients’ rheumatoid factors, about 63% of patients were found positive for RF, and 62% of patients had antibodies to cyclic citrullinated peptides (CCP).

Plasma levels of IL-21 in RA patients and healthy controls were quantified by ELISA, and results are shown in Fig. 1. RA patients (19.6 ± 0.79 ng/mL) displayed significantly higher levels of plasma IL-21 compared to healthy controls (2.12 ± 0.08 ng/mL) (p < 0.0001).

As the DAS28 scores represent the disease severity of rheumatoid arthritis patients, we hypothesized a possible correlation between DAS28 scores and plasma levels of IL-21. Spearman rank coefficient analysis revealed a significant positive correlation between plasma IL-21 levels and DAS28 scores (spearman r = 0.61, P < 0.0001) (Fig. 2a).

RA patients were further categorized into four subgroups based on DAS28 scores. As shown in Fig. 2b, RA patients with higher disease activity scores (DAS28 > 5.1) had higher mean plasma IL-21 levels compared to those with medium (p < 0.0001) and low disease activity scores (p < 0.0001) and remission (p < 0.0001). Furthermore, a significant difference in mean levels of plasma IL-21 was observed among the lower and intermediate disease activity group (p < 0.0001) (Fig. 2b).

IL-21 has been linked with increased follicular T cells, elevated B cell activation, proliferation, and production of antibodies [27]. Further, IL-21 levels are positively correlated with disease activity scores in rheumatoid arthritis patients [27]. However, no significant correction was observed among IL-21 and anti-CCP, RF-IgG, and RF-IgM levels in the present study (data not shown).

A total of 303 healthy Chinese subjects were genotyped for four common SNPs (rs907715, rs2221903, rs2055979, and rs6822844) TaqMan genotyping method. All subjects were having major genotype (GG) for rs6822844 polymorphism [17]. As shown in Table 3, heterozygous mutants were more frequent in rs907715 and rs2055979 polymorphism, followed by wild type and homozygous mutant. Further, for rs2221903 polymorphism, the wildtype remained highly prevalent compared to heterozygous (23%) and homozygous mutant (2%). Distribution of genotypes for three SNPs were in Hardy-Weinberg Equilibrium (HWE) (rs907715: χ² = 0.01, p = 0.90, rs2221903: χ² = 0.04, p = 0.82, rs2055979: χ² = 0.18, p = 0.66).

To test whether common genetic variants in the IL-21 gene are associated with predisposition to rheumatoid arthritis development, we genotyped rs907715, rs2221903 rs2055979 polymorphism in 211 RA patients and 303 healthy controls. As shown in Table 3, the prevalence of homozygous mutant (AA) of rs2055979 polymorphism was significantly higher in RA patients compared to healthy controls (p < 0.0001, χ² = 34.73, OR = 4.342). The frequency of mutants (CA + AA) was also higher in RA than in controls (p = 0.001, χ² = 10.71, OR = 1.901). Furthermore, the mutant allele (A) was even more frequent in patients than healthy controls (p < 0.0001, χ² = 35.53, OR = 2.149), indicating an essential genetic susceptible factor on predisposition to RA development.

Plasma levels of IL-21 in RA patients and healthy controls were analyzed among different genotypes of IL-21 polymorphisms (rs907715, rs2221903, and rs2055979) to investigate the possible association plasma IL-21 levels. As shown in Fig. 3a, the AA genotype of rs2055979 polymorphisms had higher plasma levels of IL-21 than other genotypes, i.e., CA demonstrated intermediate levels, and CC had the lowest levels of plasma IL-21. Interestingly, similar observations were noticed when the association of IL-21 rs2055979 polymorphism was analyzed in RA patients (Fig. 3b) and healthy controls (Fig. 3c). For other studied SNPs (rs907715 and rs2221903), no significant association between genotypes and plasma levels of IL-21 was observed (data not shown).

As DAS 28 and plasma levels of IL-21 were correlated; further, we analyzed the possible association of IL-21 polymorphisms with DAS28 scores. As shown in Fig. 4c, we observed a significant association between IL-21 rs2055979 polymorphism with DAS28 scores: subjects with AA genotyped had higher DAS28 scores than CA and CC genotypes. However, such association was not observed in rs907715 and rs2221903 polymorphisms (Fig. 4a and b).