Behcet's is an autoinflammatory disease that is defined by Hulusi Behcet in 1937 as an inflammatory process of unknown etiology, characterized by recurrent aphthous stomatitis, uveitis, genital ulcers, and skin lesions.1 Although Behçet's disease (BD) is an extensive and worldwide disease in different parts of the world, it has remarkable local differences, with the maximum of incidences in the Mediterranean, the Middle East, and the Far East which was locally called the Silk Road. Most prevalence of Behçet's disease has been reported in Turkey including 421 people per 105.2 Among all genetic factors, HLA-B51 has been confirmed as the strongest risk factor for BD, which was verified in various populations. More recently studies have disclosed the association of many non-HLA genes with the BD, such as chemokine receptor 1 (CCR1),3 Vitamin D Receptor (VDR),4 interleukin-23 (IL-23),5 IL-23R and IL-12RB2,3,6 fork head box P3 (Foxp3),7 IL-2, IL-4, transforming growth factor (TGF)-beta,8 IL-279 and IL-610 for BD.

IL-6 is a multifunctional cytokine with a wide-range of biologic activities such as the regulation of the acute-phase response to infection.11 Dysregulation and dysfunction of IL-6 gene expression contributes to the inception of numerous diseases, such as several types of cancer, autoimmune disease for example rheumatoid arthritis (RA), type 1 diabetes (T1D), inflammatory bowel disease (IBD), multiple sclerosis (MS), and autoinflammatory disorders such as Behçet's disease (BD).11,12 According to previous studies, it was found that IL-6 exerts its action via the activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAPK (mitogenactivated protein kinase) cascades.11,13

Previous studies have found four types of epigenetic mechanisms, which are responsible for regulating the expression of genes including: DNA methylation, histone modification, non-coding RNAs (nc RNAs) and chromatin remodeling.14–16 DNA methylation is an epigenetic alteration that efficiently controls gene expression via gene silencing, and may significantly contribute to the risks of many multiplex diseases, including cancer, autoimmune disease, and metabolic disorders.16–18 Recent researches of cytokine's gene expression have focused on the epigenetic alterations, because the DNA methylation status at CpG sites is often related with cytokine expression changes.19 Decreasing of methylation is related with chromatin's open position, but increasing of methylation corresponds to chromatin's closed status.20 Then, the extent to which CpG islands methylation is associated with the level of cytokine expression. Thus, we questioned whether DNA methylation of IL-6 is reduced in BD T cells with modifying transcription factor recruitment.

Proinflammatory and anti-inflammatory agents are involved in the beginning and development of BD; thus, a comprehensive survey of the regulatory mechanism for the production of cytokines during the progression of BD is the most importance challenge. IL-6 produced mainly by monocytes, B cells, T helper type 2 (Th2) and endothelial cells and plays an essential role in encouraging inflammation and stimulating the immune response.21 Also, the production of IL-6 in the joint tissues is usually done in response to interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) and is mostly performed by chondrocytes, osteoblasts, macrophages, and adipocytes.22,23 Also, IL-6 is an important cytokine in BD pathogenesis as well as it plays a key role in differentiation of CD4+ T cells to Th17 cells.24 IL-6 is an inflammatory cytokine that has been associated in several immune-mediated inflammatory diseases.25 IL-6 can encourage inflammation by increasing the production of inflammatory cytokines such as TNF-α, IL-1β and other agents. In addition, this cytokine plays a key role by inhibiting the anti-inflammatory cytokines and interleukin-1 receptor antagonists (IL-1Ra) production.26,27

Evidence has indicated that IL-6 is involved in the pathogenesis of autoimmune disease. In this study, we analyzed the IL-6 expression in patient with BD in comparison with healthy groups. Also, we evaluated the methylation level of interleukin-6 in both healthy and patient groups.

Behcet's disease (BD) is an autoinflammatory disease of unknown etiology. Cytokines involved can be classified as Th1, Th2, Th17, Treg types, chemokines and others proinflammatory agents. The cytokine network plays an important role in the pathogenesis of diseases, especially autoimmune disorders, as well in the formation and progression of lesions of the organism and may be blocked by one of the basic links of cytokines, which in turn may obtain the critical clues to view the target therapy program of cytokine in BD.30

Chronic inflammation and responses creates a main mechanism for autoimmune disease. The definite pathways by which the inflammation causes autoimmune are not completely recognized. IL-6 is a multifunctional cytokine produced and released by inflammatory cells and can trigger intracellular transcription factors leading to gene expression, cell division, apoptosis processes and inflammation response.25,31,32 Persistent differences in the production of IL-6 have been identified among different individuals in various populations with polymorphism at the end of 5′ region33 and the genetic associations of IL-6 polymorphism have been identified with rates of infections, inflammation and immune diseases. This suggested that the production rate of this cytokine is a risk factor for most diseases and their condition.34 However, to date, the role of epigenetic variable has not been reported in IL-6 gene on Behçet's disease.

There are some findings indicated that IL-6 may stimulate immune response through the effect on the expression of effective genes by altering patterns of DNA methylation.35 On the other hand, IL-6 production is regulated by methylation of the IL-6 gene promoter.35 DNA demethylation is a property of transcriptionally active genes, and our results are fit with the view that the level of methylation is essential in the regulation of the IL-6 production in patients with BD and could participate in production of this cytokine as seen in BD and other autoimmune disease.35,36

In a study of 51 BD patients aged 16–60y, we examined the level of promoter methylation of IL-6 in association with demographic and clinical risk factors such as gender and severe BD as well as inflammation markers. As the results showed, the gene expression level of IL-6 was significantly different in phlebitis section. IL-6 gene expression was also significantly increased in individuals with positive phlebitis. Furthermore, in order to compare the methylation status of IL-6 in two groups of BD and healthy individuals, the results showed that there was a statistical significant difference between both groups in terms of methylation rate of IL-6 gene (p-value <0.001). According to our results, there was no relationship between the gene expression and its methylation in different parts of the clinical profile. One of the possible reasons for these results is that our population sample size was small, and if we selected a larger population size, our results may be more meaningful.

Epigenetic differences, especially the level of IL-6 methylation between BD patients and healthy group, can be an initial manifestation suggested an increased risk of BD prevalence with the low methylation level of IL-6 promoter. On the other hand, this difference can be considered as a main manipulation for BD prevention or treatment.

Scientists from China reported in previous studies that a significantly higher level of IL-6 in cerebrospinal fluid (CSF) had been detected in Chinese patients with active BD with central nervous system (CNS) involvement.37 Also, in a previous study about the methylation level of the IL-6 promoter in patients with RA in comparison with healthy groups, the results indicated that a single CpG site at -1,181 was considerably less methylated in subjects with rheumatoid arthritis (RA).35 Some pioneering studies demonstrated that dysregulation of immune system is related to aging. Furthermore, these results revealed that the level of IL-6 expression was intensified with age in animals model such as mice.38 It has also been shown that the level of IL-6 are much higher in serum, synovial fluid and synovial tissue in patients with severe RA compared with subjects without inflammatory arthritis which indicates that it is related to the activity of disease.39 Some contradictory results reported in previous studies. A decrease or no effect of age on IL-6 production had been shown already.40 According to the results of this clinical study, significant difference could not be seen between patients when dichotomized by age. These diverse results may be caused by the differences in experimental conditions and the health status of subjects in various investigations.

These results recommend that the level of methylation in promoter region of gene may be a key mechanism for regulation of IL-6 gene expression in BD and IL-6 CpG island hypomethylation might be involved in the progression of BD. Besides, according to the results, molecular methods such as the analysis of DNA methylation could recognize in early detection by changes in the genotype and genome sequencing. This may be used for early diagnosis and initiation of rapid therapies. Therefore, IL-6 can be used as a molecular marker for the diagnosis of Behcet's patients.

Regarding the results of expression and methylation of IL-6 in this study, it may be considered that methylation may be as one of the regulatory mechanisms involved in regulating gene expression. Generally, epigenetic alteration such as hypermethylation of IL-6 gene may be a potent therapeutic approach in controlling BD.

The authors declare that they have no conflicts of interest.