Serum level of interleukin-17 and its relation to interleukin-6 and tumor necrosis factor alpha level in patients with rheumatoid arthritis – Nguyen Huy Thong

Journal of military pharmaco-medicine n o 6-2019 154 SERUM LEVEL OF INTERLEUKIN-17 AND ITS RELATION TO INTERLEUKIN-6 AND TUMOR NECROSIS FACTOR ALPHA LEVEL IN PATIENTS WITH RHEUMATOID ARTHRITIS Nguyen Huy Thong1; Nguyen Dang Dung2; Quyen Dang Tuyen1 SUMMARY Objectives: To investigate serum level of interleukin-17 in rheumatoid arthritis patients and to assess the relationship of this cytokine with serum levels of IL-6 and tumor necrosis factor α. Subjects and methods: 82 patients with rheumatoid arthritis and 30 healthy volunteers were enrolled in the study. Disease activity was determined by disease activity score (DAS28) in patients with rheumatoid arthritis. Serum levels of IL-6, IL-17 and TNF-α were measured by fluorescence covalent microbead immunosorbent assay. Results: Serum level of IL-17 in rheumatoid arthritis patients and controls were 0.59 ± 0.92 and 0.62 ± 0.94 pg/mL, respectively. There was no difference in serum levels of IL-17 in RA patients compared to that in controls (p > 0.05). Serum IL-17 level, however, seemed to be correlated with changes in serum levels of IL-6 and TNF-α in rheumatoid arthritis patients: in patients with elevated serum levels of IL-17, the IL-6 and TNF-α were higher compared to those in patients with normal serum level of IL-17. Conclusions: Serum level of IL-17 in patients with rheumatoid arthritis did not differ from that in healthy people. Higher serum level of IL-17 correlated with higher serum levels of of IL-6 and TNF-α. * Keywords: Rheumatoid arthritis; IL-6; IL-17; TNF-α. INTRODUCTION Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint swelling, joint tenderness, and destruction of synovium, leading to severe disability and premature mortality of patients [1]. Cytokine networks play a fundamental role in the processes that cause inflammation, articular destruction of RA [2]. IL-17 possesses properties of a pro- inflammatory cytokine, and plays very important roles in pathogenesis of RA [3]. IL-17 is a cytokine that stimulates the production of a variety of inflammatory mediators, and plays a leading role in regulating the relationship between pro- inflammatory cytokines. In this role, IL-17 activates not only B cells to produce antibodies, but also macrophages, synovial fibroblasts, chondrocytes to secret cytokines, such as IL-1, IL-6, TNF-α, and matrix metalloproteinase (MMPs) [4, 5].That is the reason why serum IL-17 may be related to serum IL-6 and TNF-α in RA patients. Thus, the aim of this study was: To evaluate serum levels of IL-17 and its relation serum IL-6 and TNF-α levels in RA patients. 1. 103 Military Hospital 2. Vietnam Military Medical University Corresponding author: Nguyen Dang Dung (dzungmd@yahoo.com) Date received: 26/06/2019 Date accepted: 06/08/2019 Journal of military pharmaco-medicine n o 6-2019 155 SUBJECTS AND METHODS 1. Subjects. * Patients: This study was carried out at Department of Rheumatology and Endocrinology of 103 Military Hospital between May 2012 and June 2015. Eighty two patients, 71 women and 11 men, with the diagnosis of RA fulfilled the ACR/EULAR 2010 RA classification criteria [1]. Before entering study, 43 and 4 patients were taken glucocorticoids and conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs), respectively. Patients with other concomitant rheumatic diseases, severe infections, chronic autoimmune diseases, and/or taking bio-DMARDs which may influence laboratory and cytokine profile were excluded from the study. * Healthy subject population: thirty sex- matched healthy controls (mean age 41.60 ± 4.57; range, 35 - 50 years, 26 women and 4 men) were included in the study. 2. Methods. * Clinical assessment: Disease activity was assessed by the 28-joint disease activity score C-reactive protein (DAS28 CRP) [6] in RA patients. Patient global assessment of disease activity and provider global assessment of disease activity were evaluated using Visual Analog Scale Formats for assessment of disease activity [7]. Erythrocyte Sedimentation Rate (ESR) and C-reactive protein (CRP) were also recorded. * Laboratory analysis: Blood samples of patients and controls were collected and put in a sterile plain tube and stored frozen at -80oC until analysis. We used commercially available human Fluorescence covalent microbead immunesorbent assay (FCMIA) kits for IL- 6, IL-17 and TNF-α (R&D systems MN, USA). The assay was performed according to the instructions provided by the manufacturer. Serum levels of cytokines were reported as pg/mL. The cut-off values of serum IL-6, IL-17 and TNF-α were determined by ROC (Receiver Operating Curve). * Statistical analysis: All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) for Windows, version 18.0 (SPSS, Chicago, IL, USA). Continuous variables are presented as mean ± standard deviation or median. The normality of the distribution for all variables was assessed by Kolmogorov- Smirnov test. Intergroup comparisons were made using the student’s t-test for normally distributed variables and Mann- Whitney U test for non-parametric variables. Difference with p value being less than 0.05 was considered statistically significant. Journal of military pharmaco-medicine n o 6-2019 156 RESULTS 1. Patients and demographic, clinical characteristics. Table 1: Demographic and clinical characteristics of RA patients and controls. RA group (n = 82) Control group (n = 30) Mean age ± SD (min - max) (years) 53.44 ± 7.30; 35 - 66 41.60 ± 4.57; 35 - 50 Sex, n (female/male) 75/11 26/4 Mean disease duration ± SD (years) 4.29 ± 5.34 Mean tender joint count ± SD (range 0 - 28) 14.13 ± 9.08; 13.00 Mean swollen joint count ± SD (range 0 - 28) 10.52 ± 7.38; 9.0 Mean morning stiffness ± SD (minutes) 37.25 ± 33.82; 30.00 Mean patient global assessment of disease activity ± SD (cm) 7.16 ± 2.25 Mean provider global assessment of disease activity ± SD (cm) 5.65 ± 1.92 Mean ESR ± SD (mm/h) 79.68 ± 44.37 7.63 ± 3.92 Mean plasma CRP ± SD (mg/L) 68.37 ± 47.24 0.52 ± 0.36 Mean, DAS28 CRP 6.19 ± 1.36; 2.81 - 8.50 Low or moderate (n, %) 17; 20.5 DAS28 CRP High (n, %) 66; 79.5 Glucorticoids (n, %) 43 (50.6) Pre-study treatment DMARDs (n, %) 4 (4.7) (DAS28 (CRP) is missing in three patients) (Anti-CCP: Anti-cyclic citrulinated peptide; CRP: C-reative protein; DAS28: Disease Activity Score; ESR: Erythrocyte Sedimentation Rate) Patients and healthy people in the control group did not significantly differ in sex distribution. The mean age of controls was considerably lower than that in RA patients. The mean disease duration in RA patients was 4.29 ± 5.34 years. The mean DAS28 CRP was 6.19 ± 1.36 (range, 2.81 - 8.50). The proportion of patients had low or moderate and high disease activity based on DAS28 CRP was 20.5% (17/83 patients) and 79.5% (66/86 patients), respectively. Journal of military pharmaco-medicine n o 6-2019 157 2. Changes in serum levels of IL-17, IL-6, TNF-α and relations between serum IL-17 and serum IL-6, TNF-α in RA patients. Table 2: The comparison of serum IL-6, IL-17 and TNF-α levels of patients and controls. Patients Controls Serum cytokines (pg/mL) n = 82 n = 30 p X ± SD 19.06 ± 22.94 9.19 ± 8.43 Median 10.49 7.18 IL-6 IQR 3.69 - 25.55 2.84 - 11.41 < 0.05* X ± SD 0.59 ± 0.92 0.62 ± 0.94 Median 0.30 0.27 IL-17 IQR 0.00 - 0.07 0.00 - 1.07 > 0.05 X ± SD 2.37 ± 2.69 3.87 ± 2.11 Median 1.68 3.69 TNF-α IQR 0.48 - 2.82 2.42 - 4.84 < 0.001* (*: Test Mann - Whitney, IQR: Interquartile Range) Statistics shows that the median of serum IL-6 of patients was considerably higher than that in controls (p < 0.05). There was no significant difference in the median of serum IL-17 levels between patients and controls. The median of serum TNF-α of patients was significantly lower than that in controls (p < 0.001). Chart 1: The distributions of RA according to serum IL-17 levels. The percentage of patients with elevated serum cytokine levels (elevated serum levels of 1, 2 or 3 cytokines) was 73.20%, among which the percentage of patients with elevated serum IL-17 (either single elevation of IL-17 level, or elevated IL-17 in combination with IL-6 and/or serum TNF-α) was 56.10%; meanwhile, only 17.10% of RA patients had elevated IL-6 and/or TNF-α level. Journal of military pharmaco-medicine n o 6-2019 158 Table 3: The comparison of serum IL-6 and serum TNF-α based on serum IL-17 groups. Normal Elevated Serum IL-17 n = 36 n = 46 p X ± SD 16.70 ± 23.19 20.68 ± 22.86 Median 6.81 12.87 Serum IL-6 (pg/mL) IQR 2.94 - 20.19 6.30 - 26.28 0.068* X ± SD 1.81 ± 2.53 2.81 ± 2.76 Median 1.06 2.24 Serum TNF-α (pg/mL) IQR 0.48 - 2.24 0.89 - 3.50 < 0.05* (*: Test Mann - Whitney; IQR: Interquartile Range) Assessing the change of serum IL-6, TNF-α according to serum IL-17 groups of RA patients, the results showed that the median of serum TNF-α of patient group with elevated serum IL-17 was higher than that of healthy group (p < 0.05). The median serum IL-6 had an increased trend in patients with elevated serum IL-17 compared to healthy group (p = 0.068). DISCUSSION In the present study, serum levels of IL-17 as well as IL-6 and TNF-α cytokine were evaluated in patients with RA. Additionally, the relationships between serum IL-17 and serum IL-6, TNF-α were also assessed. Our results showed that there was no significant difference in median value of serum IL-17 level of the patients compared to that of the controls (p = 0.879, by Mann-Whitney test) (table 2). However, the percentage of RA patients having elevated serum IL-17 level was 56.10%, which was higher than that of IL-6 and TNF-α (chart 1). In contrast, it was reported that in RA patients, serum level of IL-17 was significantly higher than that in healthy people [8, 9, 10], as well as that of patients with osteoarthritis [11]. IL-17 level was not only elevated in serum, but also in synovial fluid of RA patients at early stage of the disease without treatment, and the level of IL-17 in synovial fluid was proportionally correlated with that in serum [9, 12]. The results of current study showed that serum IL-17 levels in RA patients were not elevated compared to that in healthy people. This was probably because in RA patients at the clinical period of the disease, serum IL-17 level might be lower than that before the disease onset, which was in accordance with remarks by Kokkonen H et al (2010), in which the authors found that median value of serum IL-17 levels of RA patients before disease onset was 28.7 pg/mL and then it decreased to 6.0 pg/mL during the illness period of the disease, which was lower than that in healthy people at the same age and gender distribution (being Journal of military pharmaco-medicine n o 6-2019 159 21.1 pg/mL). The difference in serum IL- 17 level of the RA patients and of the controls was significant with p value being 6.1 x 10-5 [13]. Additionally, it was reported that the production and secretion of most of Th1 cytokines (IL-1β, IL-2, IL-3, IL-6, TNF, interferon-γ) and Th17 cytokines (IL-17, GM-CSF) were down-regulated by corticoids [14]. In this study, 50.6% of RA patients were treated by corticoids before having serum cytokines testing (table 1), and it might be one possible cause of the low level of serum IL-17 we have observed. However, when analyzing data, the results of this study indicated that there was no significant difference between median values of serum IL-17 levels of the RA patients who underwent corticoid treatment compared to that of treatment-naùve ones. Furthermore, in this study, the change in serum IL-17 level seemed to influence the change in serum IL-6 and TNF-α levels. The results of chart 1 and table 3 indicated that serum IL-17 levels of RA patients were increased along with elevation of serum IL-6 and TNF-α levels. These findings were in accordance with a report by Miossec P (2007), in that IL-17 seemed to play a central role in pathogenesis of RA by stimulating synovial macrophages, fibroblasts and dendritic cells to produce and secrete pro-inflammatory cytokines, including IL-6 and TNF-α [4], and in the meantime, IL-17 was the "conductor" that regulated the interaction between cytokines [5]. Our study has some limitations. The sample size of patients was relatively small, many patients were on medication treatment, including glucocorticoids as well as DMARDs, before enrolment in this research. Treatment regimens might influence the serum levels of cytokines. On the other hand, this study was designed as a cross-sectional one, and cytokines profile was not evaluated in comparison with that in treatment-naive RA patients. Furthermore, patients of this study were mainly in an established period of RA disease. 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