Several inflammatory mediators, including cytokines, cause mononuclear cell infiltration into the synovium and cartilage and bone destruction. These cytokines affect the interaction between cellular, immunological and biochemical mediators of inflammation at many levels.^[23] Proinflammatory cytokines such as TNF-α, IL-1β, IL-17, and IFN-γ play a critical role in the pathogenesis of RA.^[24]

The involvement of TNF-α and IL-1, which are interrelated in regulatory processes through a positive feedback, is crucial as they cause most of the pathological changes. These cytokines are the major inducers of fibroblast proliferation in pannus that produce collagenase and other proteolytic enzymes, which in turn cause cartilage destruction. They also activate osteoclasts for bone demineralization and stimulate angiogenesis, which cause systemic symptoms such as malaise, fatigue, and increased acute phase reactants in serum. The IL-23/IL-17 axis has been shown to play an essential role in the pathogenesis of autoimmune processes, such as experimental autoimmune encephalomyelitis, type 1 diabetes mellitus, and uveitis. In RA, IL-17 is detected in the synovium, and this cytokine has been shown to synergize with IL-1 to induce production of IL-6 by synovial fibroblasts and enhance the expression of specific chemokines in connective tissue. IL-17 is produced by Th17 and other cells^{[23, 25]}; it stimulates the secretion of TNF-α, IL-1β, and chemokines by macrophages and other cell types. Transforming growth factor-β (TGF-β) and IL-6 induce the differentiation of Th17 cells, while IL-23 secreted by the antigen-presenting cells (APCs) facilitates the expansion and maintenance of this subset of T cells.^[23]

IL-17 induces many chemokines and cytokines, in part by activating NF-κB via the classical pathway and exhibits pronounced synergism with TNF-α. Th1 cells are primarily responsible for cell-mediated immunity and Th2 cells for humoral immunity. An additional helper T-cell type called Th17 has been identified after labeling its cytokine IL-17, suggesting the involvement of Th17 and IL-17 in the pathogenesis of RA. The effect of IL-17 is significantly enhanced by synergism with TNF-α, which is produced by T cells and activated macrophages. Activated macrophages also produce IL-6 and IL-1. Interleukin 6, in some cases IL-1, TNF-α, and IL-17, in addition to TLR-2 and -4 ligands, directly or indirectly leads to the expression of RANK ligand (RANKL) on osteoblast stromal cells and synoviocytes, and RANKL is the central mediator of osteoclastogenesis that is essential for the function of mature osteoclasts. Th17 cells can directly stimulate this process, as only this T-helper class preferentially expresses RANKL. In addition, IL-17 regulates osteoprotegerin, the natural antagonist of RANKL. The increased ratio of RANKL to osteoprotegerin ensures the generation of osteoclasts from monocyte precursors and the continued activation and maintenance of mature osteoclasts that erode bone and thus participate in pathological processes in RA IL-1 and TNF-α, also directly contributing to a differential of osteoclasts and their activation. Neutrophils activated by IL-17-induced chemokines also further contribute to tissue destruction. Proinflammatory cytokines, including TNF-α, IL-1, and IL-17, induce the classical NF-κB activation pathway. Th17 cells produce IL-17A (also known as IL-17), as well as IL-17F, which is thought to have the same biological activity as IL-17, although it has a lower affinity for the IL-17 receptor.^[25] Interleukin 17 has been implicated in the pathogenesis of numerous autoimmune diseases including RA.

Ninety-one participants were included in the study. The patient group consisted of 47 patients diagnosed with RA (22% men and 78% women) with a mean age of 59±12, hospitalized in the Clinic of Rheumatology of Kaspela University Hospital and the Rheumatology Department of COH Hospital. The control group included 44 healthy subjects (32% men and 68% women) with a mean age of 49±13 years.

Patients with RA were diagnosed according to the Classification Criteria of ACR/EULAR 2010. The average duration of the disease was 8 years. Disease activity was assessed according to the accepted DAS 28 disease activity scoring system. According to this scale, patients were assessed with high disease activity (DAS 28>5.1), moderate disease activity (5.1<DAS 28>3.2), low disease activity (3.2<DAS 28>2.6), and remission (DAS 28<2.6).

The inclusion criteria for the patient group were the following: adult patients (18 – all -19-82, RA 31-82, HC 19-79 years); diagnosed RA according to ACR/EULAR 2010 criteria; a stable dose of DMARDs for a period of three months prior to enrollment; lack of other rheumatic disease. The inclusion criteria for the control group were age ≥18 years old, and no evidence of rheumatic disease.

All patients were on stable drug modifying anti-rheumatic drugs (DMARDs) therapy for at least three months at the time of blood sampling. All the patients received DMARDs – conventional synthetic DMARDs (csDMARDs) (methotrexate, leflunomide) (n=47), biologic agents (bDMARDs) (n=3), and combination of csDMARDs with bDMARDs (n=14).

The study was conducted according to the requirements of Good Clinical Practice and in compliance with the Declaration of Helsinki. All participants signed informed consent prior to enrollment in the study. The study was approved by the Local Ethics Commission.

The immunological tests were performed in the Department of Medical Biochemistry, Faculty of Pharmacy, in the Medical University of Plovdiv. Anti-MCV antibody – an ELISA Kit from ORGENTEC Diagnostika, Mainz – Germany was used to test IgG class autoantibodies against mutated citrullinated vimentin (MCV). Anti-CCP antibody – anti-CCPhs (high-sensitive) from ORGENTEC Diagnostika, Mainz – Germany were used to test autoantibodies against cyclic citrullinated peptides (CCP) IgG class. Human IL-17A A – human IL-17A ELISA kit, Diaclone, France, was used to test human IL-17A. Anti-CarP antibodies – human anti-carbamylated protein antibody (ACPAb) ELISA Kit from Sincere Biotech, China was used to test anti-carbamylated antibodies.

Data analysis was performed using IBM SPSS, version 24 (2016). The serum levels of IL-17 in the RA patients and healthy controls were compared using the Mann-Whitney U test. Associations between serum levels of IL-17 with anti-CCP, anti-MCV, and anti-CarP were established using Spearman rank-order correlation. Receiver operating characteristic (ROC) curve was used to examine the ability of IL-17 to distinguish between anti-CarP (+/−) cases. The results were interpreted as statistically significant at p<0.05.

IL-17 levels were assessed in 47 RA patients and 44 healthy controls (Table 1). In patients with RA, the median serum level of IL-17 was higher (7.0 pg/m) than in healthy controls (3.9 pg/m) with no statistically significant difference (p=0.11).

Spearman rho correlation analysis was used for this analysis because association was sought between data encoded on a dichotomous scale (0-1) – anti-CCP, anti-MCV, and anti-CarP and others measured by continuous scale – (IL-17).

The correlation coefficients are interpreted in relation to Cohen reference values (Cohen, 1988) as follows:

The correlation coefficients are presented in Table 2. No significant correlation was observed between anti-CCP (+/−) and IL-17 (r_s=0.162, p=0.380). There was no significant correlation between anti-MCV (+/−) and IL-17 (r_s=0.157, p=0.340). А significant positive correlation of moderate value was found between anti-CarP (+/−) and IL-17 (r_s=0.388, p=0.015).

The association between anti-CarP and IL-17 serum levels is illustrated by the ROC curve (Fig. 1), showing a significant ability of IL-17 to distinguish anti-CarP positive from negative cases, AUC = 0.748 (95% CI 0.56–0.88, p=0.005). The optimal criterion IL-17 level was established as >3.61 pg/ml, with sensitivity of 72% and specificity of 80%.