Reconsidering the Role of Melatonin in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an inflammatory joint disorder characterized by synovial proliferation and inflammation, with eventual joint destruction if inadequately treated. Modern therapies approved for RA target the proinflammatory cytokines or Janus kinases that mediate the initiation and progression of the disease. However, these agents fail to benefit all patients with RA, and many lose therapeutic responsiveness over time. More effective or adjuvant treatments are needed. Melatonin has shown beneficial activity in several animal models and clinical trials of inflammatory autoimmune diseases, but the role of melatonin is controversial in RA. Some research suggests that melatonin enhances proinflammatory activities and thus promotes disease activity in RA, while other work has documented substantial anti-inflammatory and immunoregulatory properties of melatonin in preclinical models of arthritis. In addition, disturbance of the circadian rhythm is associated with RA development and melatonin has been found to affect clock gene expression in joints of RA. This review summarizes current understanding about the immunopathogenic characteristics of melatonin in RA disease. Comprehensive consideration is required by clinical rheumatologists to balance the contradictory effects.


Introduction
Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by synovial proliferation and inflammatory responses, the presence of autoantibodies including rheumatoid factor and anti-citrullinated protein antibodies (ACPA) in sera, cartilage, and bone erosion with deformity, and co-occurring health conditions such as cardiovascular disease events, pulmonary, psychological, and metabolic bone disorders [1]. Proinflammatory cytokines that mediate the progression of RA disease include tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and IL-6. Current international guidelines for patients with early RA recommend starting disease-modifying antirheumatic drugs (DMARDs) as soon as possible, with methotrexate being the preferred choice [2]. Methotrexate is usually supplemented with short-term, low-dose oral or intra-articular glucocorticoids (GCs) for fast relief of pain and swelling and for arresting the inflammatory process. GCs must be carefully managed to prevent their inappropriate use and tapered as soon as possible to avoid long-term adverse effects [2]. The highly efficacious biologic DMARDs targeting the proinflammatory cytokines and Janus kinase inhibitors are intended for patients with persistently active and bone differentiation [23]. Moreover, while RORα is not a receptor for melatonin or its metabolites, the constitutive activity of RORα may be modulated by membrane melatonin receptors [21,23].
Melatonin also displays antioxidant and anti-inflammatory activity, depending on the cellular state [24,25]. Evidence suggests that melatonin serves as a link between circadian rhythms and joint diseases, including RA and osteoarthritis (OA) [26,27]. For instance, oxidative stress induced by RA is reduced by melatonin and/or its metabolites, which not only neutralize the reactive oxygen (ROS) and reactive nitrogen species (RNS), but also upregulate levels of glutathione and antioxidant enzyme expression and activity [28,29]. In RA and OA, melatonin and its metabolites modulate several molecular signaling pathways including those governing inflammation, proliferation, and apoptosis [28,29].

A Role for Melatonin in Rheumatoid Arthritis Therapy?
In investigations involving melatonin in animal models of inflammatory autoimmune diseases (multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, and type 1 diabetes), melatonin has demonstrated beneficial effects in these diseases, including prophylactic and therapeutic effects in rats with adjuvant-induced arthritis (AA), in which melatonin dose-dependently repressed the inflammatory response and enhanced proliferation of thymocytes and secretion of IL-2 [30]. In addition, melatonin decreased the elevated level of cyclic 3 ,5 -AMP (cAMP) induced by forskolin. The drop in thymocyte proliferation induced by injection of Freund's complete adjuvant was highly correlated with a decrease in the levels of Met-enkephalin (Met-Enk) in the thymocytes, which were strikingly augmented by melatonin; this effect was blocked by the Ca 2+ channel antagonist, nifedipine. The anti-inflammatory and immunoregulatory actions of melatonin involved a G protein-adenyl cyclase-cAMP transmembrane signal and Met-Enk release by thymocytes [30].

Modulation of the Circadian Clock by Melatonin in RA
Importantly, circadian rhythms exist in almost all cells of the body, and are regulated by circadian clock gene expression [7,15]. Any disruption in these circadian clocks is associated with the onset of inflammatory-related disease states and joint diseases, including RA [7,15]. Patients with RA exhibit abnormal clock gene expression, with disturbances in the hypothalamic-pituitary-adrenal axis influencing changes in circadian rhythms of circulating serum levels of melatonin, IL-6, cortisol and in chronic fatigue [15]. Melatonin exerts its effects in RA by modulating clock gene expression, including the Cry1 gene [7,15]. By attenuating the expression of the Cry1 gene, melatonin upregulates levels of cAMP production and increases activation of protein kinase A (PKA) and nuclear factor kappa B (NF-κB), which increases CIA severity in rats [31,32]. As detailed earlier, the diurnal secretion of melatonin is also closely related to the production of IL-12 and NO among RA synovial macrophages and human monocytic myeloid THP-1 cells [33].
A positive correlation has been reported between elevated early morning serum melatonin concentrations and disease activity scores as well as erythrocyte sedimentation rate (ESR) levels in patients with juvenile rheumatoid arthritis, although higher melatonin concentrations did not correlate with disease severity [34], echoing the findings of Forrest and colleagues reported earlier, who noted that elevated ESR and neopterin concentrations following melatonin treatment did not worsen the severity of RA disease [35]. El-Awady and colleagues suggested that melatonin may promote the activity of RA disease, rather than its severity [34]. However, as reported above, Akfhamizadeh and colleagues found no link between elevated morning serum melatonin concentrations and RA disease activity or other disease characteristics, despite also observing significantly higher melatonin values in newly diagnosed RA patients compared with those who had established RA disease [36].
There also appears to be a relationship between melatonin and the Bmal1 and ROR clock genes [15]. It is speculated that high melatonin concentrations in RA patients may modulate ROR activation [15]. ROR acts as a negative regulator of inflammation via the NF-κB signaling pathway and is essential in the activity of both melatonin and the clock gene Bmal1, which helps to maintain 24-h rhythms and regulate immune responses [15,37]. Moreover, ROR proteins bind into the promoter region and drive Bmal1 gene expression [38]. This activity at the binding site is inhibited by reverse-eritroblastosis viruses (REV-ERBs), which may contribute to Bmal1 suppression and exacerbation of RA [15].

Adverse Effects of Melatonin in RA
Evidence suggests that melatonin is not beneficial in RA. For instance, the development of collagen-induced arthritis (CIA) in DBA/1 mice is exacerbated by constant darkness [39] and by daily exogenous administration of melatonin 1 mg/kg [40]. Hansson and colleagues then investigated the effects of surgical pinealectomy in DBA/1 and NFR/N mice with collagen-induced arthritis (CIA) [41]. Serum melatonin levels were reduced in the pinealectomized mice to around 30% of levels in normal or sham-operated controls [41]. In both mouse strains, pinealectomy was associated with a delay in onset of arthritic disease, less severe arthritis (lower clinical scores), and lower serum anti-CII levels compared with sham-operated animals [41]. The researchers interpreted these findings as showing that high physiological levels of melatonin stimulate the immune system and worsen CIA, while inhibiting the release of melatonin is beneficial [41]. Their speculation was supported by observations from mice subjected to 30 days of Bacillus Calmette-Guérin (BCG) inoculations into the left hind paw, inducing chronic granulomatous inflammation [42]. Higher vascular permeability was seen around the granulomatous lesions at midnight than at midday; this rhythmic variation was eliminated by pinealectomy and restored by nocturnal replacement of melatonin [42].
This ability of melatonin to modulate immune response was further illustrated by experiments in which the production of IL-12 and nitric oxide (NO) was significantly increased in the media of melatonin-stimulated RA synovial macrophages and cultured THP-1 cells compared with RPMI-treated synovial macrophage controls [33]. Unexpectedly, the opposite effects in IL-12 and NO levels were seen when RA synovial macrophages were pretreated with lipopolysaccharide (LPS) prior to melatonin, as compared with synovial macrophages treated with LPS alone [33]. This study explained the possible mechanism of joint morning stiffness in relation to diurnal rhythmicity of neuroendocrine pathways [33].
These conclusions are supported by later evidence from in vitro and in vivo studies, as well as clinical investigations, showing how melatonin stimulates the production of NO, T helper type 1 (Th1)-type and other inflammatory cytokines besides IL-12 (IL-1, IL-2, IL-4, IL-5, IL-6, TNF-α, granulocyte-macrophage colony-stimulating factor [GM-CSF], and transforming growth factor [TGF]-β, interferon [IFN]-γ), and enhances both cell-mediated and humoral responses [43][44][45]. In the early morning, patients with RA exhibit high serum levels of proinflammatory cytokines, especially TNF-α and IL-6, when melatonin serum concentrations are also higher [6,43]. The effects of these circadian rhythms are thought to promote the joint pain and morning stiffness that characterizes RA [6]. Animal studies have shown that melatonin treatment (10 mg/kg) dysregulates circadian clock genes, which may promote the progression of RA [31]. Intriguingly, a dual effect of melatonin as a proinflammatory agent and antioxidant has been observed in CIA rats [32]. In that study, a lower dosage of melatonin (30 µg/kg) increased anti-collagen antibodies, IL-1β, and IL-6 levels in the serum and joints of arthritic rats, worsening the severity of joint damage, while simultaneously lowering oxidative markers nitrite/nitrate and lipid peroxidation in serum, but not in joints [32].

Neutral or Beneficial Effects of Melatonin in RA
Notably, a cross-sectional study from Iran has reported finding significantly higher morning serum levels of melatonin in patients with RA compared with healthy controls, but no correlation between melatonin and RA disease activity score or other disease characteristics, including age, disease duration, medications, gender, or season of sampling [36]. The study also reported finding higher serum melatonin values in newly diagnosed patients compared with patients with established RA, which needs further investigation [36].
Matrix metalloproteinases (MMPs) are a family of endopeptidases primarily responsible for catalyzing the degradation of the extracellular matrix (ECM) [46]. MMPs play important roles in RA. Elevated levels of circulating MMP-3, MMP-8 and MMP-9 are associated with disease progression in RA [47]. In particular, MMP-2 and MMP-9 are expressed in synoviocytes, CD34 + endothelial cells, monocytes and macrophages of rheumatoid synovium, indicating that both molecules are critical to pannus formation and invasion in RA progression [47]. Interestingly, melatonin reportedly directly inhibits secreted MMP-9 by binding to the active site and significantly reducing the catalytic activity of MMP-9 in both in vitro and cultured cells, in a dose-and time-dependent manner [46]. Thus, melatonin could have an important role in the prevention of joint destruction in RA.
Research demonstrating that melatonin dose-dependently inhibits the proliferation of RA fibroblast-like synoviocytes (FLS) through the activation of the ERK/P21/P27 pathway suggests that inhibiting the invasion of RA FLS through cartilage and into bone may have important implications in the treatment of RA [48]. Blocking NF-κB signaling appears to be the way in which melatonin protects cells from oxidative stress [28] and largely explains how melatonin suppresses proinflammatory cytokines such as IL-1β and TNF-α [20]. Other pathways and molecules associated with inflammation that are modulated by melatonin include the mitogen-activated protein kinase (MAPK) and nuclear erythroid 2-related factor 2 (Nrf2) pathways, as well as Toll-like receptors [15].
In a clinical trial involving RA patients, six months of melatonin treatment (10 mg/day) was associated with a general decrease from baseline in concentrations of peroxidation markers [35]. Conversely, ESR and neopterin concentrations were increased from baseline with melatonin and significantly higher at six months than concentrations in the placebo-treated cohort, which experienced a significant downward trend in these inflammatory indicators during the trial [35]. Paradoxically, neither the elevations in ESR and neopterin concentrations nor the decrease in tissue peroxidation associated with melatonin translated into significant differences from the placebo group in terms of patients' symptoms, or in the concentrations of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) [35]. The study researchers concluded that melatonin does not appear to be beneficial in RA [35].
The findings of Forrest et al. are discussed by Maestroni et al., who argue that because melatonin enhances the production of Th1-type and inflammatory cytokines in RA, upregulates cell-mediated and humoral responses, and also exacerbates CIA in mice, melatonin likely promotes RA disease and is inappropriate for therapeutic use [49]. Maestroni et al. also emphasized the high blood melatonin concentrations (280 pg/mL) observed 12 h after dosing in the RA cohort [49], in relation to the notion that higher blood melatonin concentrations, especially in the early morning, may be responsible for morning stiffness and joint swelling experienced by patients with arthritis [6,43]. Maestroni et al. conjecture that autoreactive T cells in RA patients synthesize and release melatonin, thereby worsening the disease process [49].
Nevertheless, Korkmaz [50] has defended Forrest et al., pointing out that melatonin has shown strong anti-inflammatory activity in studies using known inflammatory agents, such as zymosan [51], lipopolysaccharide [52,53], and carrageenan [54]. Korkmaz speculated that the high blood melatonin concentrations in the RA cohort may have been a compensatory response to RA inflammation and were comparable to the high levels of melatonin in cerebrospinal fluid and its metabolites in meningitis populations and in pediatric patients with epilepsy [50]. Korkmaz maintained that melatonin is an appropriate adjunctive therapy for RA [50].
Melatonin appears to play an important role in microRNA (miRNA) expression in RA. miRNAs are small, non-coding RNAs that post-transcriptionally mediate protein expression by targeting protein-coding genes implicated in cancer cell proliferation, differentiation, apoptosis, and migration [55]. A recent study found that melatonin appears to inhibit miR-590-3p expression and induce apoptosis in human osteoblasts [55]. In another study, melatonin treatment effectively downregulated TNF-α and IL-1β production in human RA synovial fibroblasts (the MH7A cell line) by suppressing PI3K/AKT, ERK, and NF-κB signaling and upregulating miR-3150a-3p expression [20]. Those investigations confirmed that the MT 1 receptor mediates the anti-inflammatory effects of melatonin and that melatonin not only inhibits inflammatory cytokine release in mice with CIA-induced arthritis, but also attenuates CIA-induced cartilage degradation and bone erosion [20]. This evidence suggests that melatonin targets miRNAs, which could be explored in clinical trials examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.    examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.  examining the efficacy of melatonin in the treatment of RA. The following figure ( Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.

Summary
Various research suggests that melatonin has disease-promoting effects in RA and that it could increase the severity of RA, in contradiction to the beneficial effects of melatonin in other autoimmune inflammatory diseases. However, in the past decade, some studies have demonstrated that melatonin can alleviate RA through the inhibition of RA synovial fibroblast proliferation, TNF-α and IL-1β expression, as well as MMP-9 activity. The anti-inflammatory character of melatonin in RA is associated with regulation of microRNAs (such as miR-3150a-3p). More investigations are therefore warranted to explore the possible double-edged effects of melatonin in RA. The use of melatonin in patients with RA needs thorough consideration by clinical physicians.

Conflicts of Interest:
The authors declare no conflict of interest.