Down-regulation of RORA gene expression in the blood of multiple sclerosis patients

1. Introduction

Multiple sclerosis (MS) is a chronic inflammatory condition of the central nervous system (CNS) [1] characterized by demyelination and loss of oligodendrocytes and axons which leads to physical and cognitive problems [2]. The etiology and clinical behavior of MS is unknown, but according tocurrent data, the pathological features of MS are caused by various subsets of CD4 + T cells [3]. The CD4 + T cells differentiate into three helper lineages, Th1, Th2 and Th17 cells. Th1 and Th17 cells have a central role in the initiation of autoimmune CNS inflammation [4, 5]. The IL-17 cytokine family includes six members: IL-17A, B, C, D, E (IL-25) and F. IL-17A are critical for Th17 differentiation [6]. IL-17A play key regulatory roles in the pathology of several autoimmune diseases [7].

The retinoid acid-related orphan receptor (ROR) subfamily of orphan nuclear receptors consists of ROR α , - β , and - γ . They are expressed in several tissues and involved in the maintenance of the circadian rhythm, cellular metabolism, lymph nodes development and immune response [8, 9]. RORA is induced by activation of the STAT3 pathway by IL-6 [10] and is expressed in Th17 cells and promotes Th17 differentiation [11]. So RORA deficiency in mouse T cells results in reduced IL-17 expression in vitro and in vivo [12]. ROR α 1–4, are four isoforms that are transcribed from the RORA gene that located at 15q22.2 [13]. These isoforms are produced through alternative splicing [14]. The RORA protein structure consists of four functional groups: an N-terminal (A/B) domain, a conserved DNA-binding domain containing two zinc fingers, a hinge domain, and a C-terminal ligand-binding domain [15, 16]. The purpose of our study was to investigate the expression of RORA in the whole blood of relapsing-remitting multiple sclerosis (RR-MS) patients, the most common form of MS, compared to normal individuals. Moreover, we analyzed the correlation between expression of RORA and Kurtzke Expanded Disability Status Scale (EDSS) and disease duration in patients.

4. Discussion

Studies both in MS patients and in the experimental autoimmune encephalomyelitis (EAE, the animal model for multiple sclerosis) supportedthat Th17 cells have an important role in the pathogenesis of MS [22, 23, 24]. Results of EAE studies suggest that targeting the Th17 response may have a beneficial effect in MS patients [25]. In the present study, we investigated the expression levels of RORA gene in 50 RRMS patients who were treated with IFN- β in comparison with 50 healthy controls. Our results demonstrated that the RORA expression level was significantly down-regulated in MS patient versus controls. In downstream of STAT3, ROR α and ROR γ synergically mediate Th17 differentiation and IL-17A production [12, 26]. IL-17A have protective roles in host defense and inflammatory diseases [27, 28]. In addition, IL-17 family cytokines are involved in many autoimmune diseases including rheumatoid arthritis (RA), inflammatory bowel disease and MS [29, 30]. CAstro et al. conducted different approaches to determine signature genes for RORA and they have reported that IL-17A was the most important gene amongst all signature genes [11]. Moreover the results of the Yang study showed that ROR α deficiency significantly reduced IL-17 expression in vitro and in vivo and attenuated pathologic effects of Th17 in EAE model [12]. So, RORA is one of the main molecules in the pathogenesis of MS. The presence of RORA in peripheral blood may reflect pathological events in the CNS. Our findings showed that the expression of RORA in MS patients was significantly lower than that in the healthy control group. The varying results between the mentioned studies and our results illustrate the complexity of the factors involved in MS.

All of the patients in this study were in the remission phase and clinically responsive to IFN- β and took Cinnovex™ as treatment. IFN- β is widely used over the past 15 years as first-line treatment for RRMS, however its immunomodulatory mechanisms are incompletely understood. Several studies have shown that IFN- β mostly oppose the pathological processes in MS. For instance, IFN- β increases anti-inflammatory cytokines such as interleukin (IL)-10 [31], induces production of proinflammatory cytokines [32], reduces leukocyte migration across the blood brain barrier [33], reduces the activity of T cell matrix metalloproteinases [34], may assist in CNS repair and recovery [35, 36], increases the number of CD56 bright natural killer cells [37] and influences known immunomodulatory and apoptosis-promoting genes [38]. In vivo studies have demonstrated that IFN- β inhibits IL-17A, IL-17F secretion in patients with RRMS in comparison with the control subjects [39]. In addition, several studies indicate that RORA enhances IL-17A production [26] and promotes Th17 differentiation [11]. Therefore, RORA reduced expression in the patient group maybe resulted from IFN- β and this indirectly attenuates Th17 differentiation and could affect the clinical course of disease in RRMS patients. Our finding raises a new hypothesis that RORA downregulation might be a predictor of response to IFN- β . However, this study was the first work to report the lower expression of RORA in MS disease so its clinical value needs confirmation. Further validation in larger samples including RRMS patients who are not treated with INF- β is still needed to examine the results of the current study and to better understand the effect of IFN- β on RORA gene expression in MS.

5. Conclusion

Our data indicates that RORA was down-regulated in RRMS who were responsive to IFN- β . There was no correlation between RORA and EDSS. Our data possibly reflect a new mechanism associated with response to IFN- β treatment.