| BackgroundRheumatoid arthritis (RA) is a systemic, chronic inflammatory disease that manifests itself predominantly as inflammation of joints, including small and large joints, which leads to pain and joint failure, eventually resulting in disability. The etiology and pathogenesis of RA is still not fully understood. At present, there is no specific remedy available in clinic for curing rheumatoid arthritis.T cell-mediated immune response has been considered to play an important role in the pathogenesis of RA. Historically, RA was considered a Thl disease, which implies that the immunological process is dominated by IFN-γ-secreting CD4+T cells. A large number of IFN-γ+Th1cells cloning were found in the synovium of patients with RA, but IL-4, the Th2cytokines, was too low to be detected. The percentage of CD4+IFN-γ+T cells in the synovial fluid was higher than peripheral blood; meanwhile Th1/Th2ratio in synovial fluid was highly correlated with the disease activity. However, in the recent years this dogma has been challenged by several lines of evidence:IFN-γ-deficient (KO) mice do not show resistance to autoimmunity. On the contrary, these mice are even more susceptible to autoimmunity; treatment by blocking of IFN-γ does not improve the CIA mice; which led us and others to hypothesize that there may be additional Th subsets that are distinct from Thl cells. IL-17-producing T cells (Th17), and CD4+CD25+regulatory T cells (Tregs) were firstly reported by independent groups. Since then much attention have been carried out on the characteristics and functions of Thl7and Tregs in autoimmune diseases.Tregs are characterized by immune suppression, it is essential for the maintenance of peripheral tolerance and to control the immune response by direct contact inhibition and secretion of inflammatory cytokines, such as IL-10and transforming growth factor-beta(TGF-β). Foxp3is the most specific marker of Tregs, and contol the development of Tregs; reverse transcript Foxp3can transformed the initial CD4+CD25-T cells into Tregs with regulatory activity; on the contrary, Foxp3KO mice can develop fatal inflammatory diseases. Studies have shown that mice with defection of Tregs suffered severe erosive arthritis early; the percentages of peripheral CD4+CD25+Tregs in patients with early RA was lower than healthy controls.Th17cells are characterized by the production of the eponymous cytokines, IL-17, and are an important component of the adaptive immune response to certain microbes, particularly extracellular bacteria and fungi, which involved in chronic inflammation and damaged in RA by a variety of ways. Transcription factor isolated nuclear receptor (RORyt) is the Th17cell-specific transcription factor, which have been confirmed that RORyt is the essential transcriptional regulation of Th17cell differentiation factor, regardless of in vitro cytokine-induced differentiation of Th17cells or in vivo Th17cell-mediated inflammatory response. Local over expression of IL-17can significantly increase the expression of RANKL and its receptor, resulting in destroys the balance of RANKL/OPG in the synovial fluid and aggravates the bone destruction in CIA. The preliminary results of animal experiments show that application anti-IL-17neutralize antibodies in the early stages can significantly abate arthritis severity. Another study using anti-IL-17antibody or vaccine for blocking treatment achieved relieves arthritis symptoms and joint damage.There is sophisticated relationship between Tregs and Th17cells, which antagonism in function and inhibition in differentiation. TGF-b plays an essential role in the differentiation of Thl7and Treg. TGF-β promote the generation of Treg through induced Foxp3expression, thus inhibiting the inflammation and prevent the autoimmune response; when infection occured, endogenous TGF-β combined with IL-6or IL-21, inflammatory mediators, to induced Th17cell differentiation by inhibiting the expression of Foxp3; when the inflammatory mediators such as IL-6decreased, the expression of Foxp3inhibited RORyt while promoting Treg differentiation, maintain the function of Treg, retain the immune homeostasis. TGF-β signal pathway is relatively simple. Smads family plays an important role. Smad2/3, a receptor-activated Smad (R-Smads), is the first signaling molecule in the TGF-P signaling, synergies with co-receptor Smad (Co-Smad) in cytoplasm and involved in signal transduction. Smad7inhibited Smads complex formation or phosphorylation of Smad2, Smad3and prevent the signal transduction process. Smads are important for the dynamic regulation of TGF-β signaling. Activation of Smad proteins complex translocation into the nucleus and regulate transcription of target genes together with other nuclear factors.A considerable portion of patients with RA using conventional treatment is lack of efficacy, and the lack of evidence to confirm that these treatments can inhibit the progress of the joints destruct and to improve the prognosis of the disease. In the past10years, biological agents have become a major breakthrough of RA treatment. TNF-α antagonist achieve successful in the treatment of active RA, clinical trials showed that anti-TNF-α therapy can not only improve the symptoms of RA patients, but also effectively control the progress of the disease, which is one of the clinical treatment of RA leap. Differen trandomized clinical trials have confirmed the effectiveness of a variety of TNF-α antagonist, but its mechanism needs further explore.A balance between Th17and Treg is crucial for immune homeostasis. However, the role of Treg/Th17imbalance in RA and the synovial lesion formation, as well as the signaling pathway which involved in Tregs and Th17cells differentiation has yet to be further explored. In order to clarify the above problems, our study established the CIA model in rats to observe the impact of TNF-α antagonist therapy on the synovial lesions and Treg/Th17balance; detected key transcription factors of Treg and Th17and related cytokines expression in patients with RA to investigate peripheral Treg/Th17imbalance and the relationship with RA disease activity; observed the impact on Treg/Th17balanced by TNFR-Fc treatment,TNF-α antagonist, and for the first time to study its mechanism by explored the impact on differentiation of Treg and Th17cells and TGF-(3/Smad pathway; to further clarify the RAimmunological pathogenesis and provide new ideas for clinical intervention of RA treatment.Objective1. To explore the impact of TNF-α antagonist treatment on the Treg/Th17balance and related cytokines in synovium in CIA.2. To explore Tregs and Thl7cells and cytokine imbalance in RA and the association with disease activity.3. To explore the impact of TNF-alpha antagonist, TNFR-Fc, on the differentiation of Treg/Th17and the pathway of TGF-β/Smad.Methods 1. The balance of Treg/Thl7in synovium of CIA and the impact of TNF-α antagonist30Lewis rats were randomly divided into normal control group, model group and treatment group. Bovine-derived collagen type Ⅱ and incomplete Freund’s adjuvant mixing, emulsifying, multi-point subcutaneous injected0.4ml per rat in model group and treatment group rats, the control group rats were injected normal saline. From day13to the end of the experiment, treatment group was administered intraperitoneally10mg/kgTNFR-Fc per injection every other day. The Model group of rats were received PBS as control. Grading and assessment of arthritis weekly, rats were killed at day35. Plasma TNF-alpha was detected by ELISA, pathological changes in synovium was observed by toluidine blue and HE staining, the expression of Treg/Th17in synovium was detected by double stain immunofluorescence, and the expression of TGF-betal, IL-10, IL-17protein were observed by immunohistochemistry assay.2. The associations of imbalance Treg/Th17cells in peripheral blood with disease activity in patients of RA40active RA patients (35females and5males) were enrolled in, all patients meeting the American College of Rheumatology (ACR) criteria for RA, who exhibited an active disease defined as≥4swollen joints,≥6tender joints and morning stiffness greater than45minutes, or erythrocyte sedimentation rate (Wilcoxon)≥28mm/h, or C-reactive protein (CRP)>10mg/L. Clinical assessments including tender and swollen joint counts, pain visual analogue score (VAS score), health assessment questionnaire (HAQ) and DAS28score, and laboratory parameters such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF).40healthy persons, including35females and5males were enrolled as the healthy control group, with an average of44.7 years (20-70years). Studies after informed consent, Blood samples were drawn.ELISA was used to measure serum cytokines levels (including TGF-β1,IL-10, and IL-17). The mRNA expression of Treg/Th17transcription factor Foxp3/RORyt was detected by realtime-PCR.3. The impact and mechanism of TNF-α antagonist on the balance of Treg/Th17in patients of RA40active RA patients enrolled in and clinical assessment identical the first part. all patients gave their written consent according to the Declaration of Helsinki. All patients were randomized to rhTNFR:Fc treatment group or placebo group on a background of a stable dosage of methotrexate by2:1, subcutaneously,25mg each time, twice a week, during12weeks. Patients were assessed at weeks0,6,12by the same physician. DAS28score improvement≥1.2or DAS28≤3.2is defined as responders at the end of the study.ELISA was used to measure serum cytokines levels (including TGF-β1, IL-10, and IL-17) before and after treatment. The expression of Treg/Thl7transcription factor Foxp3/RORyt and cytokines (including TGF-β1, IL-10, IL-17), as well as TGF-β/Smad pathway mRNA (including Smad2, Smad3, Smad7) were detected by realtime-PCR.4. Statistical analysisValues are expressed as mean±SD. Firstly, homogeneity of variance, the data were analyzed by independent samples t test or ANOVA when homogeneity of variance, non-parametric Mann-Whitney U test and Kruskal-Wallis test was used when heterogeneity of variance; If significance was found, then Student-Newman-Keuls or Mann-Whitney test was performed to detect the difference among groups; before and after treatment comparison using a paired t-test or the Wilcoxon’s test,depending on the type of data distribution; Spearman’s correlation was used as a test of correlation between two continuous variables. P Values less than0.05were considered significant.Results1. The balance of Treg/Th17in synovium of CIA and the impact of TNF-a antagonistObvious symptoms of arthritis emerged on an average of day18in model group, hind feet first appeared red and swollen, and then the front feet swelling, with time peaked at about day21, severity joint movement handicap, indicated CIA rat model established, emergence time of symptoms of arthritis in treatment group was similar to model group, but peak time delayed to day28, and arthritis grade was significantly mitigate than model group (P<0.001). The joint showed no abnormal changes in control group.The pathological observation showed the regular arrangement of normal rat synovial tissue cells, found no lymphocytes and plasma cells infiltration and cartilage staining missing; model rats synovial tissue showed severe hyperplasia, the synovial layer thicken and disorganized, a large number of lymphocytes, neutrophils, and mononuclear cell infiltration, cartilage deficiency severely; treatment group synovial hyperplasia and inflammatory cell infiltration and cartilage loss lessen than model group which stained by HE and toluidine blue.ELISA results of TNF-alpha concentrations analyzed by Non-parametric Kruskal-Wallis test between groups were significantly different(χ2=15.158, P=0.001). The level of Serum TNF-α of model group was significantly higher as compared with the control group (713.33±145.83pg/ml vs24.86±3.12pg/ml, P<0.05), whereas no significant difference was found between treatment group and control group (67.62±20.05pg/ml vs24.86±3.12pg/ml, P>0.05). There was significant difference between groups in the expression of CD4+Foxp3+Treg/CD4+in synovium (F=6.096, P=0.012), so does the expression of CD4+Foxp3+Treg/CD4+(F=40.791, P<0.001). There was no significant difference between model group and normal control group in the expression of CD4+Foxp3+Treg/CD4+in synovium (23.12±4.93%vs24.66±5.82%, P>0.05), whereas the level of treatment group(33.07±5.14%) was significantly higher than model group and normal control group (P<0.05). The expression of CD4+ROR γ t+Thl7/CD4+of model group was significantly higher as compared with the normal control group and treatment group (9.74±2.23%vs1.00±0.59%、5.63±1.76%, P<0.05), whereas treatment group higher than control group (P<0.05).Immunohistochemical analysis showed that TGF-β1was dispersed in the synovial lining cell layer and around blood vessels, in addition to the cartilage surface; IL-10and IL-17were mainly located in the synovial lining cell layer and around blood vessels. ANOVA between three groups of TGF-β1(F=19.841, P=0.000), IL-10(F=21.798, P=0.000), IL-17(F=8.958, P=0.003) expression were statistically difference. TGF-β1expression of model group was significantly higher as compared with the normal control group and treatment group (P<0.05), no significant difference between normal group and treatment group (P=0.12); the expression of IL-10in model group was significantly lower than control group and treatment group (P<0.05); whereas IL-17was significantly higher (P<0.05).2. The associations of imbalance Treg/Th17cells in peripheral blood with disease activity in patients of RAThe expression of Foxp3mRNA was significantly decrease in RA (0.6542±0.3037vs.1.4811±0.2958, t=-10.099, P=0.000), whereas the expression of RORγt mRNA was increase when compared with control group (1.0278±0.3038vs. 0.4066±0.1519, U=12.000, P=0.000); the level of IL-10was much diminish in RA(115.16±83.80vs.204.86±66.01, t=-5.238, P<0.001), while TGF-β1and IL-17significantly higher than the healthy control group (P=0.000and P<0.001, respectively).Spearman analysis showed there was no correlation between the expression of Foxp3mRNA and disease activity parameter (including tender joint counts, swollen joint counts, visual analog scale of patients, HAQ, disease activity score in28jioints, ESR, CRP, etc), and laboratory parameters (including ESR, CRP, RF, anti-CCP antibody). The expression of Foxp3mRNA correlated with no disease activity parameter and laboratory parameters except positive correlated with the tender joint counts (r=0.495, P<0.05) and RF (r=0.453, P=0.014).RA patients were sub-divided into three groups, ESR<28,28≤ESR<100and ESR≥100, and found no difference for the expression of Foxp3mRNA between groups by ANOVA analysis (F=2.36, P=0.114), though ESR≥100patients with high degree of inflammation which the expression of Foxp3mRNA were lower than normal ESR patients but not significant (0.3623±0.2233vs.0.7707±0.2848, P>0.05), and the expression of RORyt mRNA showed significant difference among three groups (F=6.334, P=0.006), the expression of RORyt mRNA of ESR≥100patients is higher than the ESR normal and28≤ESR<100patients (1.5297±0.1652vs.0.9844±0.2446,0.9608±0.2762, P<0.05).The level of IL-10was positive correlated with the expression of Foxp3mRNA (r=0.495, P=0.01); while the level of IL-17was positive correlated with the expression of RORyt mRNA (r=0.461, P=0.02). The level of TGF-β1was no correlated with any parameters. The level of IL-17was also positive correlated with tender joint counts, swollen joint counts (r=0.341, P=0.042and r=0.448, P=0.006; respectively). 3. The impact and mechanism of TNF-α antagonist on the imbalance of Treg/Th17in patients of RAAfter12weeks of anti-TNF treatment, the expression of Foxp3mRNA and the levels of IL-10mRNA and protein was significant increased (P<0.05), while RORyt mRNA expression and IL-17mRNA, protein level was depress than before treatment (P<0.05); but the control group with MTX alone treatment, the expression of Foxp3mRNA was significantly increased (P<0.01), while RORyt mRNA and IL-10, of IL-17mRNA, protein levels found no significant changes before and after treatment.When RA patients were divided into responders and non responders according to DAS28, the increase of Foxp3mRNA and IL-10mRNA and protein was still significant for both groups (P<0.05), and the decrease of ROR γ t mRNA was still significant for both groups (P<0.05); But the decrease of IL-17mRNA and protein only found in responders.Further analysis of the correlation of DAS28score after treatment and Treg and Th17cells and cytokines in all patients, we found that DAS28score was negatively correlated with the baseline Foxp3mRNA expression (r=-0.380, P=0.042), and positively correlated with the baseline RORyt mRNA expression (r=0.407, P=0.028) and12week IL-17protein level (r=0.362, P=0.030). In TNF-a antagonist treatment group, the positive correlation between DAS28score after treatment and RORyt mRNA expression was found (r=0.430, P=0.046).RA patients after anti-TNF treatment for12weeks, the expression of Smad2decreased (0.3361±0.0903vs.0.2936±0.0976, Z=-2.728, P=0.006) and Smad3ascent (0.3132±0.1216vs.0.3841±0.1288, t=-4.124, P=0.001) when compared with baseline, Smad7is slightly lower but not statistically significance (1.1218±0.2157vs.1.0387±0.2268, t=1.566, P=0.133);the expression of Smad2, Smad3, Smad7mRNA found no significant difference before and after treatment in MTX treatment group (P>0.05).Conclusion1. Treg/Th17imbalance and cytokines disturbance were found in RA peripheral blood and synovium, differentiation imbalance is more significant in patients with higher disease activity, indicated that the imbalance of Treg/Th17may play an important role in the pathogenesis of RA.2. The imbalance of Treg/Th17can be restored by anti-TNF therapy (TNFR:Fc), but not MTX.3. The expression of RORγt mRNA at baseline can be an useful predictor for the responders in TNF-α antagonist therapy, while IL-17levels may be more relevant to disease activity.4. TGF-β/Smad pathway was involved in the regulation the balance of Treg/Th17cells by TNF-α antagonists, by down-regulating the expression of Smad2, increase Smad3expression, promoting Foxp3expression and Treg differentiation and functional recovery, meanwhile inhibit Th17cell generation and IL-17expression. |
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