Effect of statins as modulators of CD39+ tregs in patients with rheumatoid arthritis who were unsuccessfully treated with methotrexate

Objective The aim of this study was to determine the effects of combined atorvastatin (AV) with etanercept (ETA) in patients with active rheumatoid arthritis (RA), who were nonresponders to methotrexate (MTX) therapy, and its effect on disease activity and CD39+ regulatory T-cell (Tregs). Patients and methods This study included 50 patients with active RA. Patients with RA were divided into two groups. Group I (n=25) received MTX therapy plus ETA (50 mg/week) (ETA+MTX) and group II (n=25) received MTX and ETA plus AV therapy (20 mg/day) (ETA+MTX+AV). In addition, 25 healthy volunteers were used as controls. DAS-28, erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, lipid profile, interleukin-6, CD39+ Tregs, ultrasonography 7 score (US7), carotid intima–media thickness, and flow-mediated dilatation (FMD) of the brachial artery were measured before and after 6 months of treatment. Results After 6 months of treatment, statin therapy combined with MTX and ETA significantly decreased disease activity variables, interleukin-6 and US7 synovitis, and tenosynovitis sum score. In addition, FMD% and CD39+ Tregs were significantly elevated. The increase in CD39+ Tregs was correlated with DAS-28 (P<0.001), FMD% (P<0.05), and US7 synovitis and tenosynovitis sum score (P<0.001). Conclusion Combination therapy with AV and ETA provides an added immunomodulatory benefit through enhancement of the immune suppression mediated by CD39+ Treg cells. Therefore, statins can be used safely with antitumor necrosis factor drugs to control disease activity and atherosclerotic changes in patients with RA, who are treated unsuccessfully with MTX.


Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory disorder caused by breakdown of immune tolerance with autoreactive T cells and/or suppressive T-cell deficiency. An imbalance between effector T-cell and regulatory T-cell (Treg) activities is known contribute to RA pathogenesis [1].
CD39 is an ectoenzyme with anti-inflammatory action through hydrolyzation of ATP and ADP to AMP. Extracellular ATP has a number of proinflammatory effects in human cells through the release of interleukin (IL)-6, IL-1β, and IL-18: cytokines that are linked to arthritis and chronic multisystem inflammatory and immune disorders [2].
Statins are 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors shown to possess anti-inflammatory and immune modulatory properties, which contribute to cholesterol reduction. Therefore, they may benefit patients with RA [3].

Aim
The aim of this study was to determine the effects of combined atorvastatin (AV) with etanercept (ETA) in patients with active RA, who were nonresponders to methotrexate (MTX) therapy as well as its effect on disease activity and CD39 + Tregs.

Patients and methods
A total of 50 patients with RA were selected from the outpatient Rheumatology Clinic. They fulfilled the 2010 ACR/European League Against Rheumatism (EULAR) classification criteria for RA [4] and had active disease, were nonresponders to MTX therapy, defined as more than or equal to six swollen joints or more than or equal to six tender joints, and had either an erythrocyte sedimentation rate more than or equal to 28 mm/h or a global health assessment score more than or equal to 20 on a 100-mm visual analogue scale. In addition, 25 healthy volunteers matched for age and sex were recruited for participation as controls.
All the patients had received MTX for at least 16 weeks with a stable dose of 10-25 mg/week and had taken no other disease-modifying antirheumatic drugs or any intraarticular or systemic corticosteroid injections within 4 weeks. All patients were naive to treatment with biological disease modifying antirheumatic drugs (DMARDs) before their inclusion. In addition, we excluded patients with a recent history of significant infection and those who suffered from conditions that affect the lipid profile, endothelial dysfunction and arterial stiffness, such as diabetes mellitus, hypothyroidism, liver or kidney disease, obesity (BMI >30), current smokers, familial dyslipidemia, history of myocardial infarction during the last 6 months, malignancy, and those using lipid-lowering medication.
Approval was obtained from the Local Research Ethics Committee and written informed consent was obtained from each participant. The procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975, as revised in 2000.

Study design
Patients with RA were randomly divided into two groups and the treatment was given in nonblinded fashion. Group I (n=25) received MTX therapy plus ETA (subcutaneous injection of 50 mg/week) (ETA +MTX) and group II (n=25) received MTX and ETA with the same previous doses plus AV therapy (20 mg/ day) (ETA+MTX+AV).
The clinical response was evaluated according to EULAR criteria, which compares the DAS-28 from one patient on two different time points [6].

Laboratory investigations
Overnight fasting-state venous blood samples were taken from the controls and patients with RA before and after the recommended lines of treatment, transferred slowly into a dry sterile centrifuge tube, centrifuged as soon as possible at 2000g for 10 min at 4°C, and aliquots of serum were immediately stored at −70°C until the time of analysis.

Routine laboratory investigations
These include complete blood count, erythrocyte sedimentation rate, serum C-reactive protein, rheumatoid factor, and lipid profile.

Specific laboratory investigations
These include the following: (1) IL-6 concentrations were determined by ELISA (Roche Diagnostics GmbH, Mannheim, Germany We recorded adverse events throughout the study. We performed hematology and biochemical screening for creatine kinase, liver function, and renal function at baseline and 6 months later.

Musculoskeletal ultrasonography
The ultrasonography 7 score (US7) was applied at screening and baseline using a 5-13 MHz linear scanner (UGEO H60 Samsung Medison Ultrasound System; Samsung Medison). The assessment was carried out by a single rheumatologist practicing in the US who was unaware of the clinical, laboratory, and radiographic findings and who was not involved in the treatment decisions.
This score includes US evaluation of wrist, meta carpophalangeal II and III, proximal interphalangeal II and III, and metatarsophalangeal II and V joints. Synovitis and synovial or tenosynovial vascularity are scored semi-quantitatively (grade 0-3) using grey scale (GSUS) and power Doppler (PDUS) and tenosynovitis as well as erosions for their presence (0/1) according to EULAR criteria and Outcome measures in Rheumatology definition [8].
Ultrasound assessment of intima-media thickness of carotid artery Common carotid arteries were assessed using B-mode ultrasound with a linear transducer (mid frequency, 10 MHz). Measurement of carotid intima-media thickness (cIMT) was performed 1 cm proximal to the carotid bifurcation. Images were obtained in longitudinal and axial projections. In longitudinal projection, the sound beam was placed perpendicular to the far wall of the common carotid artery, obtaining two parallel echogenic lines corresponding to the lumen or intima and media or adventitia interfaces. The distance between these two parallel lines corresponded to the cIMT. Values were expressed in millimeters [9].

Ultrasound examination of brachial artery flow-mediated dilation
Participants underwent noninvasive examination of endothelium-dependent vasodilation [flow-mediated dilation (FMD)] and endothelium-independent vasodilation (nitroglycerine-mediated dilation) of the brachial artery in the nondominant arm, according to the International Brachial Artery Reactive Task Force guidelines. FMD was expressed as the relative increase in brachial artery diameter using hyperemia, and defined as (posthyperemic diameter−basal diameter)/ basal diameter×100. The maximum FMD and nitroglycerine-mediated dilation diameters were calculated as the average of the three consecutive measurements and the percent changes in the diameters compared with baseline resting diameter and expressed as percent diameter variation. FMD was performed under conditions of being blinded to the treatment given [10].
After screening, study visits were scheduled for 0 and 6 months.

Statistical analysis
Data were analyzed using SPSS software (version 11; SPSS Inc., Chicago, Illinois, USA). Baseline characteristics are presented as mean±SD for the continuous variables, and as frequency and percentage for the discrete ones. Baseline comparisons between groups were conducted using analysis of variance and Fisher least significant difference test. Analysis of covariance was used for comparisons of the effect of drugs between groups. Correlation between variables was examined using the Pearson's correlation coefficient. P value less than 0.05 was considered statistically significant. Multiple linear regression analysis was performed to investigate the independent association of clinical and ultrasonography parameters with changes in CD39 + Tregs [11].

Baseline characteristics of patients with rheumatoid arthritis
A total of 50 patients with active RA completed the study.
The baseline characteristics of patients with RA and controls are shown in Table 1.
Patients with RA exhibited mild dyslipidemia compared with controls. In patients with RA, the peripheral blood CD4 + CD39 + , CD4 + CD39 + FoxP3 + , and CD4 + CD39 + FoxP3 − lymphocytes were significantly decreased when compared with those in controls (P<0.001). In addition, we studied the expression of CD39 on Tregs (CD39 + Tregs), defined by the expression of FoxP3 on CD4 + CD25 + lymphocytes, which was significantly decreased in patients with RA compared with controls.
Endothelial function was determined using postocclusion flow-mediated vasodilatation of the brachial artery (FMD), which was significantly lower in patients with RA compared with controls. Moreover, there was a significant increase in serum IL-6 and cIMT in patients with RA compared with controls (P<0.001).
Clinical, laboratory and ultrasonography parameters under various therapies over 6 months There was a significant decrease in the disease activity variables after 6 months of treatment (Tables 2  and 3). The reduction was more pronounced in group II treated with ETA+MTX+AV than in group I treated with ETA+MTX (P<0.001). The levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and atherogenic ratios did not change significantly after treatment in group I (ETA+MTX). However, in group    II, AV therapy produced a significant reduction in TC, LDL-C, TG, and atherogenic ratio levels with a significant increase in HDL-C.
IL-6 was significantly reduced in the two groups after treatment compared with the baseline with no significant difference between the two studied groups after treatment.
Carotid IMT measurement was not significantly affected after treatment in the two studied groups. However, the percentage of FMD was significantly increased after treatment in group II (ETA+MTX +AV) (P<0.001) without significant change in group I (ETA+MTX).
US7 synovitis and tenosynovitis sum score in GSUS as well as PDUS decreased significantly after treatment in the two studied groups. In addition, the US7 synovitis and tenosynovitis sum score were significantly dropped in group II patients with RA (ETA+MTX+AV) after treatment compared with group I patients with RA (ETA+MTX) (P<0.001) (Figs. 2 and 3).
Moderate or good DAS-28 responses (EULAR criteria) were achieved in 20 out of 25 (80%) group II patients with RA (ETA+MTX+AV) compared with 16 out of 25 (64%) in group I patients with RA (ETA +MTX) after 6 months of treatment.
We examined correlations with change over time in patients receiving AV (group II); the increased percentage of CD39 + Tregs after treatment was found to correlate with the improvement of FMD (r=0.45, P<0.05) and with the reduction of DAS-28 (r=−0.78, P<0.001) and US7 synovitis and tenosynovitis sum score in GSUS as well as PDUS (r=−0.75, P<0.001), (r=−0.76, P<0.001), (r=−0.48, P<0.05) and (r=−0.53, P<0.001).In addition, multiple linear regression showed that the change in percentage of CD39 + Tregs was statistically significantly associated with the change in DAS-28 (B=0.14; P=0.034) and FMD (B=0.09; P<0.001).   AV was well tolerated in the study population. Adverse events (mild gastrointestinal upset) arose with similar frequency in the two studied groups. No significant liver function or muscle abnormality was detected in those given AV.

Discussion
RA is an inflammatory arthritis that affects nearly 1% of the world's adults. It is characterized by symmetric polyarthritis. This inflammation results in pain and stiffness and, if not controlled, it can lead to progressive joint damage resulting in deformities and loss of function. In addition, chronic inflammation secondary to RA can lead to an increased risk for cardiovascular disease and changes in bone metabolism [12].
In this study, all patients had active RA (DAS-28 and US7 scores) and were nonresponders to MTX therapy. They exhibited mild dyslipidemia, which is highly prevalent in patients with RA. Park et al. [13] reported that oxidized LDL was increased in inflamed synovial fluid. Moreover, Bartels et al. [14] documented that both intracellular and extracellular oxidized LDL were detected in the rheumatoid synovium.
Recently, a common genetic predisposition for the synchronicity of RA and dyslipidemia was discovered [15,16]. As a result, dyslipidemia was linked to the pathogenesis of RA.
Levels of Tregs in our RA cases were significantly decreased compared to the controls. Peres et al. [17] found that MTX unresponsiveness in RA was associated with low expression of CD39 on Tregs. MTX mediated its anti-inflammatory role in RA through the 5-aminoimidazole-4-carboxamide ribo nucleotide (AICAR) pathway, inhibiting the enzyme AICAR transformilase. As a consequence of the inhibition of this enzyme, there was an increase in adenosine concentration, which is not only directly anti-inflammatory but can also induce more Tregs in a feedback-amplification manner. This may partly explain the markedly elevated Treg population in the RA patients responding to MTX.
CD39 (ectonucleoside triphosphate diphosphohydrolase 1), a transmembrane protein, has its expression driven by the Treg-specific transcription factor Foxp3 and its catalytic activity is strongly enhanced by T-cell receptor ligation. Tregs suppress the activation, proliferation, and effector functions of a wide range of immune cells. Moreover, CD39 identified on B-lymphoid cells with potent nucleotidase activity [18]. CD39 hydrolyzes ATP and ADP to provide the substrate for generation of the anti-inflammatory and antithrombotic mediator adenosine. The purinergic signaling system, with CD39 at its center, plays an important role in modulating vascular homeostasis and the response to vascular injury [19].
After 6 months of treatment, we found that there was a significant decrease in the disease activity that was measured clinically and radiologically using the US7 score. The reduction was more pronounced in group two, which was receiving AV therapy. This result coincided with those of many authors who found that AV 20 mg was a safe and well-tolerated drug that has a modest anti-inflammatory effect in patients with moderate to highly active RA. Statins have been shown to reduce the level of C-reactive protein, endothelial adhesion molecule (ICAM-1), monocyte chemotaxis, and to induce apoptosis of inflammatory cells. In consequence, statins had antiinflammatory effects independent of their cholesterollowering effects in patients with RA [20][21][22].
In our study, AV therapy produced a significant reduction in TC, LDL-C, TG, and atherogenic ratios with a significant increase in HDL-C. Moreover, the percentage of FMD was significantly increased after the treatment in group two under AV therapy. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase, involved in cholesterol biosynthesis. LDL-C concentration is lowered by reducing its production in the liver and increasing removal from the circulation. Lowering blood levels of LDL-C was a major risk factor for cardiovascular disease. This reduced the chance of having cardiovascular disease.
De Vera et al. [23] reported that an increased risk for adverse outcomes was associated with poor statin adherence. Moreover, in our previous study [24] we found that AV therapy in patients with RA significantly reduced dyslipidemia, disease activity variables, serum MDA, tumor necrosis factor-α, resistin, and adiponectin. On the other hand, there was an improvement in FMD. Statins reduce disease activity and conventional and novel vascular risk factors that promote the atheromatous lesion [25].
Our study is the first one to examine the effect of AV therapy when combined with biologicals in patients with active RA who have been treated unsuccessfully with MTX. Moreover; the study examined its effect on CD39 + Tregs.
In this study, the levels of CD39 + Tregs were increased in group two, which received AV therapy combined with ETA when compared with group one receiving ETA only. In addition, the increased levels of CD39 + Tregs after treatment was found to correlate with the improvement of FMD and reduction of US7 sum score. This is in acceptance with findings by Blache et al. [26] who reported that neither ETA nor adalimumab modified the percentages or absolute numbers of circulating CD4+ CD25 high Tregs and their phenotypes after being administered for 6 and 12 weeks to RA patients.
Tang et al. [27] concluded that AT significantly upregulates the frequency and impaired function of Treg and decreases clinical activity in patients with RA. They proposed that AT induced Treg by reduction of the synthesis of isoprenoid intermediates, such as farnesyl pyrophosphate or geranylgeranyl pyrophosphate, thus affecting the activation of the small GTPases, which regulate FoxP3 expression in activated naive T cells. Moreover, Zhang et al. [28] demonstrated that AT induced an increase in the number of Tregs in atherosclerotic plaques from patients with acute coronary syndrome, which correlates with a better prognosis and in elevated ST-segment. Moreover, Kaneider et al. [29] suggested that the mechanism of action of statins to produce anti-inflammatory and antithrombotic effects could be through an enhancement in CD39 activity. However, Jalkanen et al. [30] observed that the use of statins was linked with lower ADP levels but not associated with CD39 activity. Statins could inhibit Rho-GTPase-dependent endocytosis of ectonucleotidases.

Conclusion
AV is a safe drug that has a potent immunomodulatory effect through enhancement of the immune suppression mediated by Treg cells. Statins can be used in combination with antitumor necrosis factor drugs to control disease activity and atherosclerotic changes in patients with RA, who are treated unsuccessfully with MTX.

Strengths and limitations
This study is the first to examine the effect of AV therapy when combined with biologicals in active RA patients who have been treated unsuccessfully with MTX. Moreover, the study examined its effect on CD39 + Tregs.
The chief limitation of our study was the small sample size. Therefore, various results yielded by the present study would have been more significant if a large sample had been included.

Financial support and sponsorship
Nil.

Conflicts of interest
There are no conflicts of interest.