Evaluation of the T helper 17 axis in ankylosing spondylitis

Rheumatol Int (2012) 32:2511–2515 DOI 10.1007/s00296-011-1995-7 ORIGINAL ARTICLE Evaluation of the T helper 17 axis in ankylosing spondylitis Ali Taylan • Ismail Sari • Didem L. Kozaci • Arif Yuksel • Safak Bilge • Yasar Yildiz • Gulten Sop • Isil Coker • Necati Gunay • Nurullah Akkoc Received: 22 December 2010 / Accepted: 3 July 2011 / Published online: 16 July 2011 Ó Springer-Verlag 2011 Abstract To evaluate the T helper 17 (Th17) axis and its relation to tumor necrosis factor (TNF) alpha blockage and disease activity in ankylosing spondylitis (AS). The study included 127 AS patients (100M/27F) and 38 (27M/11F) controls. Spinal mobility was assessed by the bath ankylosing spondylitis metrology index (BASMI). Patients were also evaluated with the bath ankylosing spondylitis functional (BASFI) and bath ankylosing spondylitis disease A. Taylan (&) Department of Rheumatology, Izmir Tepecik Training and Research Hospital, Yenisehir, Izmir, Turkey e-mail: taylanally@yahoo.com I. Sari Department of Rheumatology, Izmir Bozyaka Training cand Research Hospital, Izmir, Turkey D. L. Kozaci Department of Biochemistry, Adnan Menderes University School of Medicine, Aydin, Turkey D. L. Kozaci
N. Gunay Bilim ve Teknoloji Araştırma ve Uygulama Merkezi (ADU-BILTEM), Adnan Menderes University, Aydin, Turkey A. Yuksel
Y. Yildiz
G. Sop Department of Internal Medicine, Izmir Bozyaka Training and Research Hospital, Izmir, Turkey S. Bilge Department of Physical Medicine and Rehabilitation, Izmir Tepecik Training and Research Hospital, Izmir, Turkey I. Coker Department of Biochemistry, Izmir Tepecik Training and Research Hospital, Izmir, Turkey N. Akkoc Medical Faculty, Department of Rheumatology, Dokuz Eylul University, Izmir, Turkey activity index. Cytokines including IL-6, IL-12, TGF-b, IL17A, and IL-23 were measured in serum sample using commercially available ELISA kits. Cytokines including IL-6, IL-12, TGF-b, IL-17, and IL-23 were significantly higher in the AS patients than the controls (P \ 0.05). The Th-17-related cytokines were not different between patients treated with anti-TNF and conventional therapies (P [ 0.05). Cytokines were also similar between patients with active and inactive disease (P [ 0.05). On correlation analysis, IL-17 was correlated with IL-23 and IL-12 (P \ 0.05) and IL-23 showed correlations with IL-12 and BASMI (P \ 0.05). We found serum levels of Th-17related cytokines to be significantly increased in the sera of AS patients. Disease activity and treatment type did not affect the level of these cytokines. Keywords Ankylosing spondylitis
Interleukin-6
Interleukin-12
TGF-beta
Interleukin-17
Interleukin-23 Introduction Ankylosing spondylitis (AS) is a chronic inflammatory disorder that primarily affects the axial skeleton. Although the etiology of AS is unknown, data strongly suggest genetic factors play an important role in an individual’s susceptibility to the disease. Recent studies suggest interleukin-23 receptor (IL-23R) is one of the major generic factors involved in one’s susceptibility to AS [1–4]. In accordance with this observation, recent studies focused on the T helper 17 (Th17) axis in patients with AS or spondyloarthropathy (SpA) [5–11]. However, the available data is still limited and additional information is needed. Our primary objective in this study was to evaluate the Th-17 axis in AS patients. Our secondary aim was to 123 2512 evaluate the effect of tumor necrosis factor (TNF) alpha blockage and disease activity on the related cytokines. Patients and methods This study included 127 patients with a diagnosis of AS, according to the Modified New York criteria, and 38 healthy individuals in the control group. AS patients, with no history of other chronic diseases, were recruited from the rheumatology outpatient clinics of the Bozyaka and Tepecik training hospitals. Healthy volunteers were the relatives of the health professionals and blood donors. The local ethical committee approved the study, and written informed consent was obtained from each subject. Spinal mobility was assessed by the bath ankylosing spondylitis metrology index (BASMI) [12]. Patients were also evaluated with the bath ankylosing spondylitis functional index (BASFI) [13] and the bath ankylosing spondylitis disease activity index (BASDAI) [14]. Patients with a BASDAI C 4 were defined as having active disease. Conventional treatment refers to treatment with non-steroidal anti-inflammatory drugs (NSAIDs) and/or sulfasalazine, but not anti-TNF-a drugs. Laboratory evaluation Following an overnight fasting, morning (8:00–9:00 A.M.) venous blood samples were collected for the laboratory tests. Serum and plasma were isolated and stored at -80°C until analysis. The turbidimetric method was used to assess standard C-reactive protein (CRP) levels (Tokyo Boeiki, Prestige 24i, Tokyo, Japan). Serum sample measurement of cytokines including IL-6, IL-12, TGF-b, IL-17A, and IL-23 were performed using commercially available ELISA kits (BenderMed Systems, Vienna, Austria, catalog numbers: BMS213/2CE, BMS238CE, BMS8249FF, BMS2017, and BMS2023/2, respectively) according to the manufacturer’s instructions. ELISA plates were coated with specific antihuman antibodies. Color was developed with 3.30 50 5-tetramethylbenzidine, and absorbance was measured at 450 nm against standard curves. The sensitivity and the intra and inter-assay coefficient of variations were as follows: IL-6, 0.5 pg/mL, 3.4 and 5.2%, respectively; IL-12, 10 pg/mL, 3 and 4.8%, respectively; TGF-b, 10 pg/mL, \10 and \15%, respectively; IL-17A, 0.5 pg/mL, 7.1 and 9.1%, respectively; and IL-23, 10 pg/mL, 5.9 and 8.2%, respectively. Statistical analysis The Kolmogorov–Smirnov normality test was used to determine the distribution pattern of the variables. The 123 Rheumatol Int (2012) 32:2511–2515 majority of the parameters including CRP, IL-6, IL-12, IL17, and IL-23 showed non-normal distribution, and we used non-parametric tests for the statistical analysis. Data were presented as the median with minimum and maximum values. Comparison between groups of continuous variables was performed using the Mann–Whitney U test. Differences between categorical variables were analyzed with the Chi-square test. Relationships between variables were analyzed using Spearman’s rank correlation coefficients. Statistical analysis was carried out by using statistical package for social science (SPSS), version 13.0 (SPSS Inc., Chicago, IL). A P value of \0.05 was considered statistically significant. Results The mean age of the 127 AS patients (100M/27F) was 38 (16–64) years, and the mean age of the 38 healthy controls (27M/11F) was also 38 (23–56) years. Age, sex distribution, and body mass index (BMI) were similar between patients and controls (P [ 0.05, Table 1). The mean disease duration of the AS patients was 10 (1–35) years. Values for BASDAI, BASFI, and BASMI were 4.4 (0–9.2), 3.6 (0–9.5), and 2 (1–9), respectively. All the individuals in the study had no personal or family history of psoriasis and inflammatory bowel disease. A history of peripheral arthritis was present in 34% of the patients. There were no subjects with active arthritis at the time of the study. CRP levels were significantly higher in the patient group compared with controls (7.5 [0.27–119.1] vs. 1.15 [0.19-6.4] mg/L, P \ 0.001). Similarly, cytokine concentrations, including IL-6, IL-12, IL-17, and IL-23, were significantly increased in the AS patients group compared with healthy controls (3.06 [0.73–339.4] vs.1.42 [0.74–4] pg/mL, P \ 0.001; 4.65 [3.4–41.1] vs. 4.27 [3.47–57.8] pg/mL, P = 0.04; 0.72 [0–31.6] vs. 0.15 [0–3.85] pg/mL, P \ 0.001; and 98 (38–206) vs. 57 (36–160) pg/mL, P = 0.001; respectively). The clinical and laboratory characteristics of the groups are shown in Table 1. Comparison of AS subjects with respect to treatment type Seventy patients were on conventional drugs (NSAIDS and/or sulfasalazine), and 57 were on anti-TNFa blocking drugs (27 etanercept, 18 infliximab, and 12 adalimumab) with mean treatment duration of 24 (1–96) months. All subjects treated with TNFa targeting agents received their treatment regularly. None of the patients treated with conventional drugs received anti-TNFa agents in their medical history. A comparison of the patients who were on Rheumatol Int (2012) 32:2511–2515 Table 1 Clinical and laboratory characteristics of the patients and controls Data are presented as the median with minimum and maximum values BASFI bath ankylosing spondylitis functional index, BASDAI bath ankylosing spondylitis disease activity index, BASMI bath ankylosing spondylitis metrology index 2513 AS patients (n = 127) 38 (16–64) 38 (23–56) 0.94 Sex (M/F) 100/27 27/11 0.38 BMI (kg/m2) 25.9 (16.3–36.7) 25.3 (18.2–34.3) 0.28 Disease duration (years) 10 (1–35) – – BASFI 3.6 (0–9.5) – – BASDAI 4.4 (0–9.2) – – BASMI 2 (1–9) – – CRP (mg/L) 7.5 (0.27–119.1) 1.15 (0.19–6.4) \0.001 IL-6 (pg/mL) 3.06 (0.73–339.4) 1.42 (0.74–4) \0.001 IL-12 (pg/mL) 4.65 (3.4–41.1) 4.27 (3.47–57.8) TGF-b (pg/mL) 26.4 (5.2–87) 22.1 (0.4–39) 0.01 IL-17 (pg/mL) 0.72 (0–31.6) 0.15 (0–3.85) \0.001 IL-23 (pg/mL) 98 (38–206) 57 (36–160) Comparison of AS subjects with respect to disease activity Seventy-three AS patients had active disease (BASDAI C 4), and 54 were inactive. A comparison of active and inactive patients showed BASDAI, BASFI, and BASMI were significantly higher in the active AS patients (6.1 [4–9.2] vs. 2.16 [0–3.8], P \ 0.001; 5.37 [0.7–9.5] vs. 0.9 [0–7.4], P \ 0.001; 3 [0–9] vs. 1 [0–8], P = 0.002; respectively). Disease duration, CRP, IL-6, IL-12, TGF-b, IL-17, and IL-23 were similar between the groups Data are presented as the median with minimum and maximum values BASFI bath ankylosing spondylitis functional index, BASDAI bath ankylosing spondylitis disease activity index, BASMI bath ankylosing spondylitis metrology index P value Age (years) anti-TNFa and conventional drug therapies revealed that disease duration and BASMI were significantly higher in the patients receiving anti-TNFa drugs (15 [2–31] vs. 9 [1–35] years; P \ 0.001 and 3 [0–9] vs. 2 [0–9]; P = 0.03, respectively). Other parameters including BASFI, BASDAI, CRP, IL-6, IL-12, TGF-b, IL-17, and IL-23 were not different between the groups (P [ 0.05). Table 2 summarizes the comparison between the anti-TNF and conventional treated groups. Table 2 Comparison of AS subjects with respect to treatment type Controls (n = 38) 0.04 0.001 (P [ 0.05). Data from patients with active and inactive disease are shown in Table 3. Correlation analysis On correlation analysis, IL-17 significantly correlated with IL-23 and IL-12 (P \ 0.05; r = 0.6 and 0.6, respectively). However, BASFI, BASDAI, BASMI, disease duration, CRP, and being on anti-TNFa treatment did not correlate with IL-17 concentrations (P [ 0.05). IL-23 showed significant correlations with IL-12 and BASMI (P \ 0.05; r = 0.6 and 0.3, respectively), but not with other parameters. Discussion Th-17 cells have been implicated in the pathogenesis of several inflammatory diseases including rheumatoid arthritis (RA) [15], psoriasis [16], and inflammatory bowel diseases [17]. In this study, we found serum levels of Anti-TNF treatment (n = 57) Conventional treatment (n = 70) P value Disease duration (years) 15 (2–31) 9 (1–35) \0.001 BASFI 4.2 (0–8.5) 3.1 (0–9.5) 0.22 BASDAI 4.6 (0.6–8.6) 4.3 (0–9.2) 0.96 2 (1–9) 0.03 BASMI 3 (1–9) CRP (mg/L) 3.9 (0.34–109.1) 8.37 (0.27–119.1) 0.08 IL-6 (pg/mL) 2.17 (0.73–330.2) 3.57 (0.85–339.3) 0.14 IL-12 (pg/mL) 4.7 (3.4–41) 4.63 (3.5–14.25) 0.66 TGF-b (pg/mL) 26.5 (6.9–62.1) 25.4 (5.2–87) 0.38 IL-17 (pg/mL) 0.78 (0–31.6) 0.67 (0–3.06) 0.21 IL-23 (pg/mL) 102 (38–206) 80 (42–188) 0.4 123 2514 Rheumatol Int (2012) 32:2511–2515 Table 3 Comparison of AS subjects with respect to disease activity Active patients (n = 73) Disease duration (years) BASFI BASDAI Data are presented as the median with minimum and maximum values BASFI bath ankylosing spondylitis functional index, BASDAI bath ankylosing spondylitis disease activity index, BASMI bath ankylosing spondylitis metrology index BASMI P value 11 (1–30) 10 (1–35) 0.93 5.37 (0.7–9.5) 0.9 (0–7.4) \0.001 6.1 (4–9.2) 2.16 (0–3.8) \0.001 3 (1–9) 1 (1–8) 6 (0.3–109.1) 0.002 CRP (mg/L) 8.6 (0.27–119.1) IL-6 (pg/mL) 3.29 (0.85–330.2) IL-12 (pg/mL) 4.8 (3.4–41.05) TGF-b (pg/mL) 26 (5.2–87) 26.6 (6.9–53) 0.97 IL-17 (pg/mL) 0.79 (0–10.1) 0.68 (0–31.6) 0.09 IL-23 (pg/mL) 102 (38–188) 86 (42–206) 0.39 cytokines related to the Th-17 to be significantly increased in the peripheral blood of AS patients. The result supports recent studies that suggest a role for Th-17 in the pathogenesis of SpA. Wendling et al. [6] and Wang et al. [10] included 28 and 57 AS patients, respectively, in two different studies and found increased serum IL-17 levels in AS patients compared with controls. However, another study by Melis et al. [11] failed to demonstrate any difference in the serum IL-17 levels of SpA patients and healthy controls. Synovial fluid concentrations of IL-17 were increased in SpA compared to RA [5, 11]. Moreover, some recent studies reported that the number of IL-17 producing T cells was significantly increased in the peripheral blood of patients with psoriatic arthritis [7] and AS [7, 9]. In this study, we found significantly increased serum IL-17 levels in AS patients compared with healthy controls. Another cytokine related to the Th-17 pathway is IL-23. IL-23 is responsible for the expansion and survival of Th17 cells, which are a subset of CD4? cells [18]. IL-23 is a heterodimeric cytokine that is composed of p40 and p19 subunits. The p40 subunit of IL-23 is also shared by cytokine IL-12, which is another heterodimeric molecule [19]. There are a limited number of studies on IL-23 levels in AS. A recent study found increased IL-23 in AS [10], but other studies reported contradicting results [8, 11]. In this study, the concentrations of both IL-12 and IL-23 were significantly higher in AS patients; we also found AS patients had significantly higher TGF-b and IL-6 concentrations, which are responsible for the differentiation of Th-17 cells [18]. Collectively, our finding demonstrated that serum levels of cytokines related to the Th-17 axis were significantly increased in AS patients. We also evaluated the impact of disease activity (according to BASDAI) on serum levels of Th-17 cytokines. The groups with active and inactive disease had similar serum IL-6, 12, 17, and 23 concentrations. Correlation analysis also did not show an association between 123 Inactive patients (n = 54) 2.98 (0.73–339.4) 4.5 (3.65–8.77) 0.12 0.96 0.18 BASDAI, BASFI, and CRP with IL-17 and IL-23. On the other hand, BASMI, a composite index of spinal mobility reflecting disease severity [12], showed a positive weak association with IL-23 but not with IL-17. Our results are in agreement with the findings of previous studies that found no correlation between IL-17, IL-23, p40 subunit levels, and the laboratory and clinical indices of disease activity and function in AS and SpA patients [6, 8, 11]. Subgroup analysis of AS patients with respect to treatment type revealed Th-17-related cytokines were not different between groups treated with anti-TNF and conventional therapy. Although our study was a crosssectional study and may not reflect the actual effects of anti-TNF agents on these cytokines, recent reports conducted in SpA patients also reported no change in the concentrations of IL-17, IL-23 [11], and p40 subunits after treatment with TNF-a targeting agents [8]. In conclusion, we extensively evaluated the serum levels of Th-17-related cytokines in a large group of AS patients and found significantly increased concentrations compared with healthy controls. Advances in the technology allow targeting of mediators of inflammatory pathways, and the development of the biological drugs allows many inflammatory rheumatic diseases to be controlled. Unlike RA, therapeutic options for AS are limited and new agents are needed for the management of the disease. Targeting the cytokines related to the Th-17 axis may be a reasonable approach in AS patients who do not respond to other therapies. Conflict of interest authors. 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