Influences Of TFN Antagonist Therapies On Th Subsets And Related Mechanisms In RA Patients

Objecive :Rheumatoid arthritis （RA） is a chronic inflammatory disease characterized by intense immune activation within the synovial compartment of joints and a variety of systemic manifestations. The inflammatory process leads to cartilage and bone destruction. Although the pathophysiology of RA is not completely understood, the abundance of T cells within the mononuclear infiltrates of the hyperplastic synovial membrane in RA together with the local production of T cell-derived cytokines suggest that T cells are important in the autoimmune response in RA. According to the cytokine profiles after activation, CD4⁺ T cells are subdivided into different subclasses termed T helper lymphocyte type 1（Th1）, Th2, and others. The up-regulation of Th1 response has been shown within the synovial compartment, and it had been considered that Th1/Th2 imbalance plays an important role in the pathogenesis of RA. However, this dichotomy has been disrupted by the identification of other two CD4⁺ T cell lineages: CD4⁺CD25high regulatory T （Treg） cells and IL-17-producing Th17 cells, which have the opposite effects on the pathogenesis of RA.Treg cells are characterized by expression of the forkhead/winged helix transcription factor （Foxp3）. This T cell subset has an anti-inflammatory role and can maintain tolerance to self components by contact-dependent suppression or releasing anti-inflammatory cytokines （IL-10 and TGF-β1） . Th17 cells express retinoic acid-related orphan receptorγt （RORγt） as key transcription factor and play some critical roles in the development of autoimmunity and allergic reactions by producing IL-17 and, to a lesser extent, TNF-αand IL-6. So the balance between Th17 and Treg has long been hypothesized as one of the important factors in the development/prevention of autoimmune diseases, including RA.In recent years, TNF antagonist therapies with monoclonal antibodies or soluble receptor antagonists have developed in suppressing signs of inflammation and preventing progression of structural damage in virtually all RA patients. In both early and established RA, two-thirds of the patients achieve meaningful clinical response, yet one-third do not respond. Additionally, a number of patients who initially respond would develop acquired drug resistance or gradual drug failure, and some would have to discontinue the biologic treatment because of adverse events . So far, few studies have investigated the underlying basis of the drug resistance and a complete set of clinical and biological criteria capable of classifying RA patients and predicting their responses to different treatments are expected. So far there have been few reports of the effect of Etanercept（ETA）, a TNF-αinhibitor, on T cell subsets in RA patients. In view of the above problems, this study aims to: 1. explore the mechanism of ETA in the treatment of RA, we investigated whether the Th1/Th2 and Th17/Treg imbalance could be reversed by ETA and whether the reversal was related to the improvement of clinical indications; 2. investigate the frequencies of Th cell subsets and expression of related cytokines in different disease stages or in different disease activities, and especially investigate the expression of inflammatory phenotype and chemokine receptors（CCR） on Th17 in patients with different disease stages or different disease activities; 3. investigate the frequencies of Th cell subsets and expression of related cytokines in patients with different responses to anti-TNF-αtherapies and explore the likely mechanisms of resistance to anti-TNF-αor mechanisms of recurrence of disease.Methods （three parts） :1. Influences of etanercept（ETA） on Th1/Th2, Th17/Treg cell subsets and their related cytokines We studied 20 active RA patients. 10 patients were given a stable weekly dose of MTX alone as monotherapy and 10 patients received Etanercept plus MTX as combine-therapy, both for 12 weeks. Before and after treatment, frequencies of Th1、Th2、Th17 and Treg cells were quantified using flow cytometry, and plasma levels of IL-1β、IFN-γ、TNF-α、IL-4、IL-10、IL-6、IL-17、TGF-βand IL-23 were measured by ELISA. Control specimens were obtained from 10 healthy blood donors. Laboratory measurements, including erythrocyte sedimentation rate （ESR） and C-reactive protein（CRP） assessed at each visit. Disease activity was assessed by the composite 28-joint count Disease Activity Score （DAS28）, and was measured prior to the Etanercept injection. Meanwhile, Health Assessment Questionnaire （HAQ） was done by the patients as the functional and quality-of-life assement.2. Th1/Th2, Th17/Treg cell subsets and their related CCR and transcription factors in RA patients in different disease stages or with different disease activities Choose outpatient or hospitalized RA patients with disease druation<1year（41） or duration≥1 year（50）, according disease activity, all the patients were divided into low activity（DAS28≤2.6） and moderate to severe activity（DAS28≥3.2）. Intracellular cytokines such as IFN-γ、IL-17、RORc、Foxp3 and phenotype markers such as CCR4, CCR6 and IL-23R were detected using flow cytometry, and mRNA of transcription factors such as T-bet、RORc and Foxp3 were quantified by real-time rt-PCR.3. Changes of Th1/Th2, Th17/Treg cell subsets and their related CCR and transcription factors in RA patients with different responses to anti-TNF-αtherapy Peripheral blood lymphocytes were obtained from RA patients（15） who received ACR50 reponse criterion after anti-TNF-αtherapy and patients（10） who failed to receive ACR20 reponse criterion after anti-TNF-αtherapy. Intracellular cytokines such as IFN-γ、IL-17、RORc、Foxp3 and phenotype markers such as CCR4, CCR6 and IL-23R were detected using flow cytometry, and mRNA of transcription factors such as T-bet、RORc and Foxp3 were quantified by real-time RT-PCR before and after anti-TNF-αtherapy within 12 months.Results:1. Percentagies of IFN-γ⁺Th1 and IL-17⁺Th17 cells among CD4⁺ T cell subsets were significantly higher in RA patients than that in healthy controls, while percentages of CD4⁺CD25^highFoxp3⁺Treg cells were significantly lower in RA patients than that in healthy controls. After 12 weeks’therapy by MTX single or by MTX and Etanercept combination, the circulating Th17/Treg ratio were significantly decreased, while the circulating Th1/Th2 ratio were not significantly decreased in patients of both thrapy groups. The Th17/Treg ratio was positively correlated with the remittence of disease activity （ΔDAS28） in MTX+Etanercept combine-therapy group. Finally, in patients who received MTX+Etanercept combine-therapy, the serum levels of pro-inflammatory cytokines such as IL-1β, TNF-α, IL-6, IL-17 and IL-23 were significantly degraded after 12 weeks’therapy, while the mean concentration of TGF-βwas significantly elevated after the therapy. Furthermore, in patients treated with MTX+Etanercept combine-therapy, the Th17/Treg ratio was positively related to serum TGF-βbut negatively correlated with serum IL-6.2. Early RA patients regardless of disease activities, have a high level of IL-17 +Th17 cells （p < 0.05）, and patients whose duration≥1 year have higher expression level of the IFN-γ⁺Th1 cells （p < 0.05）. CCR4⁺CCR6^-T cells and CCR4⁺CCR6-T cells are significantly higher in early patients than in established patients （p < 0.05）, and frequencies of CCR4⁺CCR6⁺CD4⁺T cells are significantly related to percentages of IL-17+Th17 cells （r = 0.972, p < 0.001）, and only with CCR4 may related to IFN-γsecretion （r = 0.625, p < 0.05）, while only with CCR6 may related IFN-γor IL-17 expression（r = 0.619, p < 0.05）. The expression of transcription factor T-bet in each group did not differ significantly. mRNA expression of RORc and Foxp3 in early RA patients have no significant differences but differ significantly in established patients, indicating that RORc expression was higher in moderate and severe patients than in low activatied disease （p < 0.05）, with significantly diseased Foxp3 mRNA expression（p < 0.05）.3. After anti- TNF-αtherapy, frequencies of Th1 in CD4⁺T cells did not significantly reduced in neither groups, but frequencies of Th17 in CD4⁺T cells reduced significantly, especially in patients who reached ACR50 response criterion. Frequencies of Treg cells reduced significantly in patients with good response, with no changes in patients with poor response.RA patients with satisfied response have a higher reduction of IL-17⁺ Th17 cells （p < 0.05）. CCR4⁺CCR6^-T cells and CCR4⁺CCR6-T cells are significantly decreased in patients who catch ACR50（p < 0.05）. The expression of transcription factor T-bet in poor responsed patients increased significantly. mRNA expression of RORc in well responsed RA patients have significantly decreased with no significant diseased Foxp3 mRNA expression.Conclusions:Taken together, our data indicate that an alteration in the balance of Th1, Th2, Th17 and Treg exists in patients with RA and that Etanercept in combination with MTX can reverse the imbalance between Th17 and Treg and change the levels of their corresponding cytokines. Moreover, for the first time, our findings suggest that anti-TNF-αtherapy can ameliorate the activity of RA by influencing the balance between Th17 and Treg and their related cytokines, which is considered to be an important mechanism of anti-TNF-αtherapy in RA treatment. We need to further prove our conclusion in a large scale of the population and ongoing efforts should be made to identify the precise effect and mechanism by which TNF-αinhibitors influence Th17/Treg imbalance. Th17/Treg balance may provide a new target or indication for the treatment of RA and novel insights into new therapeutic interventions.