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Plasma and Synovial Fluid TrxR Levels are Correlated With Disease Risk and Severity in Patients With Rheumatoid Arthritis

Abstract This study was designed and performed to establish the relationship between plasma and synovial fluid (SF) levels of thioredoxin reductase (TrxR) and disease activity in Chinese patients with rheumatoid arthritis (RA). This study consisted of a total of 224 patients diagnosed with RA, 224 age and sex-matched healthy controls, and 156 patient controls. The disease activity of RA patients was calculated as diseases activity score that include 28-joint counts (DAS 28), which was divided into low-diseases activity (LDA) and high-diseases activity (HDA) groups. Increased plasma TrxR was detected in patients with RA than healthy controls (P < 0.0001). With an area under the curve (AUC) of 0.874, plasma TrxR showed a evidently greater discriminatory ability than C-reactive protein (CRP; AUC, 0.815), antistreptolysin-O (ASO; AUC, 0.631), rheumatoid factor (RF, AUC, 0.793), and erythrocyte sedimentation rate (ESR, AUC, 0.789) in diagnosing RA. RA patients with HDA had significantly elevated TrxR levels in plasma and SF than did those with LDA (P < 0.0001). With an AUC of 0.874, plasma TrxR levels as an indicator for screening of HDA showed a significantly greater discriminatory ability than CRP (AUC, 0.690), ASO (AUC, 0.597), RF (AUC, 0.657), and ESR (AUC, 0.603). Similarly, SF TrxR levels as an indicator for screening of HDA also showed a significantly greater discriminatory ability as compared with above biomarkers. TrxR levels in plasma and SF were positively correlated with the severity of RA. TrxR levels may therefore serve as a new biomarker in addition of the traditional biomarkers for assessing the risk and severity of RA. Further analysis of TrxR release machinery may give us a new understanding of pathogenesis of RA.


INTRODUCTION
R heumatoid arthritis (RA) is a common long-lasting autoimmune disorder that primarily affects joints resulted from the inflammation of the synovial membrane of multiple joints and further causing damage to the cartilage and bones. 1 The cause of RA is unknown, and the prognosis is guarded, where half of the risk for RA is believed to be genetic 2 . Furthermore, smoking is suggested to be the most significant nongenetic risk factor; besides, vitamin D deficiency is more common in people with RA than in the general population. Importantly, cytokines are directly implicated in the pathogenesis of RA as part of a complex regulatory network related to specific immunological processes that promote autoimmunity, chronic inflammation, and tissue destruction. 3 In addition, oxidative stress is a feature of much pathology of human diseases, which has been proposed to be closely correlated with the pathogenesis of RA. 4,5 The clear relationship between cellular redox homeostasis imbalance and increased oxidative damage has been proposed, possible reason might be that there was a concomitant association of increased expression of proinflammatory factors at sites of inflammation, including cytokines, prostaglandins, reactive nitrogen species (RNS), and reactive oxygen species (ROS). 6 These reactive species function significantly in the following 3 aspects: leading to the destructive, proliferative synovitis evident in RA; working as a key modulators of the immune responses (synovial T lymphocytes) that take place in the inflamed synovium of RA patients; and playing a critical role in regulating events of cell-signaling. 7 Coincidently, there was previous evidence proved this supposition, for example, in the study performed by Rall et al, 8 observably higher levels of oxidative stress was found in subjects with RA when compared to those in the healthy individuals. Besides, there was research mentioned that via the application of antioxidants, a suppressing of its serum levels was documented to predispose for the development of RA, 9 emphasizing the effect of elevated ROS production in the disease clinically. Furthermore, multiple products generated by ROS have been identified in clinical samples from the joints in patients with RA. 10 According to the previous evidence on the exploration of the relationship between oxidative stress and RA, we speculated that redox imbalance might play a role in the pathology of RA associated with previous studies. 11 Thioredoxin system included thioredoxin reductase (TrxR) and thioredoxin (TRX) is 1 of the important regulators of the redox state of proteins. 12 The mammalian TrxRs are consisting of a conserved-Cys-Val-Asn-Val-Gly-Cys-redox catalytic site, to glutathione reductases. 13 Importantly, TrxRs can contribute to the catalysis of the NADPHdependent reduction of the redox protein TRX. In addition, TRX gene was found to have a novel cis-regulatory element responsible for oxidative stress in its promoter region, and can be strongly induced by oxidative stress. 14 Increased production of TRX in synovial tissue and blood of RA patients were recently reported consistent with the hypothesis that TRX/TrxR activities may contribute to disease activity in RA. 15,16 For instance, Jikimoto et al 17 showed that plasma TRX could be regarded as a useful biomarker for RA disease activity and as an index of oxidative activity and may therefore reflect higher oxidative stress levels in RA, which was also proved by another paper indicated that increased expression of TRX presented a positive association with the disease activity in RA. 1 In sharp contrast, few studies investigate the relationship of the TrxR in plasma and synovial fluid (SF) with the screening of RA and the confirmation of disease activity of RA. Herein, this study was designed to establish the association between plasma and SF levels of TrxR and disease activity via the detection of TrxR in the plasma and SF in Chinese patients with RA.

Ethnic Statement
The ethics committee of the Zhejiang Chinese Medical University approved this study before the performance of the operation. All participants (or their relatives) were notified of the study protocol and signed informed consent approved by the institutional ethics committee.

Patients
In 2014, a total of 224 patients with RA who fulfilled the 2010 18 American College of Rheumatology criteria for RA were recruited from the Outpatient unit of Rheumatology Department at the Second Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou and enrolled in this study for the following experiment. The included eligible patients should have paired plasma and SF samples. Exclusion criteria: patients who have previous history of smoking history over 5 years; patients had alcohol intake during the past 1 years; patients with narcotic drugs intake; patients were diagnosed with hypertension, diabetes mellitus, hypothyroidism/hyperthyroidism, and any other form of arthritis except RA; and patients who received a previous treatment of steroids ([10 mg/d, including parenteral administration) and have a previous history of biologic therapy.
Two hundred eighteen age and sex-matched subjects who were enrolled for health physical examination during the same hospital with no clinical and radiological evidence of RA served as the control group. In addition, 156 age and sex-matched patients who diagnosed as ankylosing spondylitis, Reiter's syndrome, or infectious arthritis were designated as the patient control group. These patients came from our orthopedic clinic. The exclusion criteria for RA patients also apply to the control cases.

Clinical Variable
Standard demographic details and biomarkers were collected including age, sex, disease duration, white blood cells (WBC), C-reactive protein (CRP), rheumatoid factor (RF), antistreptolysin-O (ASO), anticitrullinated protein/peptide antibodies (ACPA), and erythrocyte sedimentation rate (ESR). Disease activity was assessed using a variety of variables including ESR and CRP, swollen joint count (SJC), tender joint count (TJC), and diseases activity score 28 (DAS 28) according to the method of Prevoo et al. 19 Consisting of 28 joint swelling and 28 joint tender, values DAS 28 3.

Laboratory Test
Morning fasting blood samples were collected in 5 mL BD Vacutainer Rapid Serum Tube, Mississauga, Ontario, Canada (NJ) from each participant, were quickly centrifuged to separate the plasma from the cells, and were then immediately frozen at À808C until analysis. Before any treatment, SF was obtained from RA patients. Control subjects gave only plasma samples. All samples and indicators examination were detected and analyzed in duplicates. Investigators who were recruited to measure the concentration of the included biomarkers were blinded to the clinical outcome. Levels of TrxR were measured by enzyme-linked immunosorbent assay (ELISA). The coefficients of variation (CVs) of inter-assay and intra-assay were 5.8% to 8.9% and 6.8% to 9.9%, respectively. ACPA levels in plasma and SF in RA patients were tested by ELISA method. Other biomarkers, such as WBC, Hs-CRP, ASO, RF, and ESR, were also tested. In addition, undetectable levels were preset to equal to the lower limit in the experiment.

Statistical Analysis
In this experiment, continuous variables were expressed as means (standard deviation, SD) and medians (interquartile ranges, IQR). The difference between groups was estimated via the application of the Mann-Whitney U test and chi-squared test. Spearman rank correlation test analyzed the possible correlations among laboratory parameters. The diagnostic accuracy of TrxR in RA and its disease severity were assessed by receiver operating characteristic (ROC) curve. Area under the curve (AUC) was used to assess cut-point choices to ensure the accuracy of the test. In addition, with multivariate adjustment for possible confounders (age, gender, body mass index [BMI], disease duration, levels of WBC, CRP, ASO, RF, and ESR), ordered logistic regression models were applied for the investigation of the association between the levels of TrxR and disease severity. The association between TrxR and disease severity of RA were expressed as adjusted odds ratios (ORs) and 95% confidence interval (CI). Statistical calculation was tested by SPSS (version 19.0, SPSS, Inc., Chicago, IL) and the version1.0-2ROCR package (http://cran.r-project.org/). Differences were considered significant at P < 0.05.

Patient Demographics
In our study, there was a sum of 224 patients recruited who were diagnosed with RA that had paired plasma and SF samples. In the study population, 80 (36.7%) were male and the median age was 51 years (IQR, 37-58). In those patients, 188 (83.9%) patients had ACPA-positive results. The TrxR levels in plasma and SF were no significantly difference in those RA patients who had ACPA-positive as compared with those who had ACPA-negative (All P > 0.05). Out of the 224 patients, 109 (48.7%) had HDA disease (DAS-28 > 3.2). Detailed information with respect to the baseline characteristics between groups were shown in Table 1.

DISCUSSION
There was previous data suggested that oxidative stress measurement could help to monitor disease severity in RA, 20 where DNA oxidation, lipid peroxidation, ROS, 6 protein carbonylation, 21 and genetic polymorphisms 22 were used as biomarkers evaluating oxidative stress in RA. In this study, we have proposed that there was a strong correlation established between TrxR both in peripheral blood or SF and DAS 28 score, emphasizing that measurement of TrxR, irrespective of the source contribute a lot to predict RA development as well as to elucidate the mechanisms of RA pathogenesis. Similarly, another study suggested that oxidative/nitrosative stress markers may be important for the prediction of the development of RA. 23 Our study also showed significant correlation between the HDA and higher levels TrxR, which was also proved by previous evidence. 4,6 However, there were other authors insisted that there might be no observably association of this topic in patients with RA. 24 Do the elevated TrxR levels in plasma of RA patients merely reflect the local TrxR production in inflamed joints? Interestingly, the positive correlation between plasma and SF level of TrxR and SF highlighted that the important role of the measurement of TrxR in peripheral blood might contribute to the indirect measure of the oxidant status at the sites of tissue damage. These findings suggest that plasma TrxR might be originated from the SF in RA patients. TrxR produced in synovial cavity can move to peripheral circulation due to the  cavity pressure is raised in inflamed joints and exceeded the capillary perfusion pressure. 25 What kind of cells produces TrxR in SF of RA patients? To be specific, the production of TRX might be stimulated by local environment in the rheumatic joint and there was a positive association between TRX levels and the leukocyte number in the SF, 1 this result was also examined in our study. Thus, leukocytes could be seen as a candidate of TRX producers in the RA synovial tissue. 26 Previous study reported that SF of RA patients contained large amount of cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-1. 17 Thus, the TrxR may be produced by synovial cells, because H 2 O 2 and TNF were reported to induce TRX/TrxR in synovial fibroblasts. 1 This study was also performed to confirm whether elevated TrxR levels in plasma and SF have physiological or pathological roles in RA patients. In this study, no link between ACPA and TrxR had been found. Some other possible mechanisms should be taken into account. One study provided strong evidence that oxidative stress is significantly involved in cartilage degradation in experimental arthritis. 27 TrxR might contribute to the disease activity in RA associated with its multiple effects in immunologic and inflammatory responses. First, local and systemic oxidative stress was indicated to be deeply involved in the development of chronic and destructive inflammation. 28 Redox status can be disturbed by excessive production of ROS, in which ROS production was suggested to modulate the expression of inflammatory chemokines and affecting tissue damage in RA. 29 However, in multivariate analysis, there was an increased risk of HDA associated with elevated plasma TrxR levels, showing a different mechanism of TRX production and consumption from the reactive protein.
Second, comparisons between mediators suggest a unique correlation of TRX with macrophage migration inhibitory factor (MIF). 30 Previous study suggested that MIF is an important member of the cytokine hierarchy in RA. 31 In addition, Radstake et al 32 found in their established mice model that MIF may be responsible for the development of collagen type IIinduced arthritis. Third, pivotal role of TNF has been mentioned in the pathogenesis of RA. 33 TNF could result in the induction of increased TRX expression by fibroblast-like synoviocytes. 1  In this regard, the positive feedback regulation of both mediators could be part of a self-perpetuating inflammatory response. Fourth, oxidative stress at site of chronic inflammation can cause permanent genetic change. 34 Genetic polymorphism of the p53 has been reported to be detected in synovial cells in RA and may contribute to the transformedappearing phenotype. 35 Lastly, by exerting the role of antiapoptosis and growth promoting capacities, TrxR may therefore contribute to both survival of inflammatory cells and hypertrophy of synoviocytes in RA. 36 Kabuyama et al 37 demonstrated that TrxR1 suppresses hydrogen peroxide and inhibits apoptosis of RA synovial cells. Meanwhile, TRX can trans activate activator protein 1 and nuclear factor kB, their activation may influence in the synovial macrophages and fibroblast-like synoviocytes. 1 This study has several potential limitations. First, the sample size was not large enough to reach definitive conclusions. Further studies with great numbers are warranted. Second, this cross-sectional design study must be confirmed by future longitudinal studies. Third, with respect to the adoption of a ''gold standard,'' ELISA, for the detection of TrxR, hemoglobin interference can frequently cause a false positive signal. Fourth, only TrxR was measured, and in this regard we failed to determine the relationship of other antioxidant defense parameters with TrxR levels and RA. Last, we did not assess the differences of TrxR levels in SF levels between RA patients and control subjects because of ethical concerns.

CONCLUSIONS
TrxR levels in plasma and SF were positively correlated with the severity of RA. TrxR levels in plasma may serve as a new biomarker in addition of the traditional methods for assessing the risk and severity of RA. Further analysis of TrxR release machinery may give us a new understanding of pathogenesis of RA, leading a chance of exogenous TrxR as a new tool for the treatment of RA.