BackgroundA therapeutic revolution in the past decade is still a considerable unmet need in the treatment of rheumatoid arthritis (RA). On the other hand, dysfunction of regulatory T cells (Tregs) has been considered to be a pivotal cause of RA and correction of this dysfunction to be a potential RA therapy1. However, abnormalities of Tregs in patients with RA were reported controversially in previous studies2, in which only proportion was measured and Tregs were defined using different protein markers.ObjectivesIn this study, we measured both absolute numbers and proportions of CD4+CD25+Foxp3+ Tregs in peripheral blood of RA patients and investigated the effects of low-dose recombinant human IL-2 (rhIL-2) on Tregs and CD4+ effector T cell subsets in patients with RA.MethodsBoth absolute numbers and proportions of Treg and Th17 cells in peripheral blood, defined as the CD4+CD25+FOXp3+T or CD4+IL-17 + T cell population, were examined by flow cytometry in 342 patients with RA with different 28-joint Disease Activities (DAS28), including 75 who had never received disease-modifying antirheumatic drugs (DMARD) and 151 who were receiving or had received DMARD. Among these patients, 112 consented at enrollment to receive rhIL-2 treatment. Before and after treatment, the Th17 and Treg cells in peripheral blood were analyzed by flow cytometry.ResultsThe absolute count of Treg cells in patients with RA was significantly low compared with that of healthy controls (P<0.05), but the absolute count of Th17 cell was no different between RA and healthy controls. After the course of rhIL-2 treatment, there was a significant increase in the absolute count of Th17 and Treg cells in the CD4+ T cell population (P<0.01), but Th17/Treg was significantly low after the treatment.ConclusionsThe data suggest that, besides propotion, the decrease of Treg cell number, defined as the CD4+CD25+FOXp3+population, may contribute to the pathogenesis of RA. Over the treatment of rhIL-2, there was a more significant increase in the absolute count of Treg cells than that of Th17, and consequently restore the balance of Th17/Treg.References Miyara M, Ito Y, Sakaguchi S. TREG-cell therapies for autoimmune rheumatic diseases. Nature reviews Rheumatology 2014;10:543–51.Morita T, Shima Y, Wing JB, Sakaguchi S, Ogata A, Kumanogoh A. The Proportion of Regulatory T Cells in Patients with Rheumatoid Arthritis: A Meta-Analysis. PloS one 2016;11:e0162306. Disclosure of InterestNone declared

BackgroundThe molecular target rapamycin (mTOR) signaling can regulate between effector and regulatory T cell lineage commitment [1]. Rapamycin, the inhibitor of mTOR, has appeared to be a new therapy for several autoimmune diseases, such as systemic lupus erythematosus [2].ObjectivesTo evaluate whether rapamycin is beneficial in patients with Rheumatoid Arthritis (RA), and compared with Methotrexate in efficiency and safety.MethodsFifty-eight DMARDs-naive RA patients were enrolled, thirty-eight were treated with Rapamycin (0.5 mg every 2 days, combined with IL-2 50WIU per day for 5 days), the others with Methotrexate (10mg per week) taken as control. Clinical improvement and immunological assessments were performed at baseline, 1 and 12 weeks. Treatment group assessed CD4+ T cell subsets by flow cytometry at baseline, 1 and 12 weeks.ResultsWe enrolled 58 patients. At baseline, patients had a mean DAS28 of 3.34 (0.81). Rapamycin group and Methotrexate group included 38 and 20 patients, respectively, with no significant differences in baseline characteristics. At 1 week, the mean DAS28 after Rapamycin treatment (2.43 [0.77]) and Methotrexate (2.25 [0.86]) was not significantly different (P=0.43). Same as ESR (24.74 [24.53], 21.76 [24.27], P=0.66). The dose of glucocorticoid during hospitalization of rapamycin treatment group (720.8 [554.3]) was lower than Methotrexate (1202.3 [943.1], P=0.042). The length of hospital stay of Rapamycin (14.5 [3.9]) was lower than Methotrexate (21.0 [3.8], P<0.001). Rapamycin administration resulted in an increase in the absolute counts of Treg cells from a median of 36.82 cell/ul (at week 0) to 99.80 cell/ul (at week 1) (P<0.001). The ratios of Th17/Treg cells showed a reduction from a median of 0.16 to 0.09, and the difference was significant (P=0.047). At 12 week, 5 patients treated with Rapamycin dropped out because of non-compliance. the mean DAS28 was not significantly different (2.36 [0.97], 2.16 [0.86], P=0.51). The same as the daily dose of glucocorticoid (10.21 [32.3], 9.16 [40.1], P=0.804). The absolute counts of Treg cells increased from a median of 36.82 cell/ul (at baseline) to 43.26 cell/ul after Rapamycin administration (P=0.028). The ratios of Th17/Treg had no significant difference from a median of 0.16 at baseline to 0.12 at week 12 (P=0.937). Liver enzyme elevations occurred on 2 patients after Methotrexate therapy for 1 week. However, there were no serious adverse events observed during the 12-week period of rapamycin treatment.ConclusionsRapamycin combined with the low-dose IL-2 appears to be a safe and effective therapy for RA, by a rapid increase of circulating Treg cells and a correction of the ratio of Th17/Treg cells, which has gotten a same response compared with Methotrexate.References Zheng Y. The mTOR kinase differentially regulates effector and regulatory T cell lineage commitment.[J]. Immunity, 2009, 30(6):832–844.Fernandez D, Bonilla E, Mirza N, et al. Rapamycin Reduces Disease Activity and Normalizes T Cell Activation–Induced Calcium Fluxing in Pati...

BackgroundRheumatoid arthritis (RA) is a systemic autoimmune disease characterized by irreversible joint destruction and disability. At present, the biggest challenge in the field of RA treatment is the emergence of multidrug resistance (MDR) in the application of disease-modifying anti-rheumatic drugs (DMARDs).The Multidrug resistance Related Protein and Multi Drug Resistance protein 1, also called P-glycoprotein can decreases the intracellular concentration of different drugs. Moreover, cytokines play an increasingly important role in the expression regulation mechanisms of P-gp, especially inflammatory cytokines IL-6. Previously, We have indicated that Interleukin-6 up-regulates P-gp in peripheral blood lymphocytes via the JAK2-STAT3 pathway in RA patients. Through more than ten-years' clinical research, we observed that the therapeutic cycle alliance of methotrexate (MTX) and cyclophosphamide (CTX) has had better curative effect and lower rate of adverse reaction in treating RA. And we didn't found the emergence of MDR. How can the therapeutic alliance of MTX and CTX overcome MDR in RA patients? This study will give the answer.ObjectivesTo Clarify whether the therapeutic alliance of MTX and 4-hydroperoxycyclophosphamide (4-HC) suppress expression and mRNA of P-gp in peripheral blood lymphocytes of RA through JAK2-STAT3 pathway.MethodsRA patients without any DMARDs and biologic therapy (n=15) were enrolled. P-gp expression level was detected by Flow Cytometry. P-gp mRNA of peripheral blood lymphocytes and the intracellular signaling pathway mediating the effects of MTX and 4-HC on IL-6-stimulated JAK2-STAT3 activation was assessed by RT-PCR.ResultsCompared with blank control group, IL-6 induced P-gp, JAK2 and STAT3 expression levels increased significantly (P<0.05). Compared with IL-6 group, P-gp, JAK2 and STAT3 expression levels of 4-HC group and low MTX+4-HC group both decreased (P<0.05). the expression levels of P-gp, JAK2 and STAT3 in low MTX group, middle MTX group and high MTX group were lower than IL-6 group, but there were no statistically significant differences (P>0.05).ConclusionsOur data indicated that MTX combined with CTX significantly inhibited IL-6 induced JAK2-STAT3 activation, as well as the induction of P-gp. Inhibition of IL-6-mediated multidrug resistance signaling pathways by the alliance of MTX and CTX may represents a new reversing drug-resistance therapeutic strategy for RA.References Li HX, Zhao W, Shi Y, et al. Retinoic acid amide inhibits JAK/STAT pathway in lung cancer which leads to apoptosis. Tumour Biol. 2015; 36(11): 8671–8678.Oh HM, Lee SW, Yun BR, et al. Vigna angularis inhibits IL-6-induced cellular signalling and ameliorates collagen-induced arthritis. Rheumatology (Oxford). 2014; 53(1): 56–64.Li R, Cai L, Ren DY, Xie XF, Hu CM, Li J. Therapeutic effect of 7, 3'-dimethoxy hesperetin on adjuvant arthritis in rats through inhibiting JAK2-STAT3 signal pathway. Int Immunopharmacol. 2012; 14(2): 157–163.Yun M, Lee D, Park MN, et al. Cinnamaldehyde derivative (CB-PIC) sensitizes c...

BackgroundCD4+CD25+Foxp3+ T regulatory (Treg) cells play a key role in peripheral tolerance. Rapamycin was approved by the FDA to preserve renal allografts and to be efficacious in patients with several autoimmune diseases [1].ObjectivesTo investigate the status of Treg cells in active refractory rheumatoid arthritis (RA) and the effects of rapamycin on patients with RA.MethodsForty-five active refractory RA patients were enrolled. Rapamycin was used at a dose of 0.5 mg every 2 days [the preliminary, open-label clinical trial of rapamycin (Clinical Trials.gov number: ChiCTR-IPR-17010307)]. Clinical improvement and immunological assessments were performed before 1st rapamycin dose and 12 weeks post treatment. Blood samples were obtained from RA patients and 75 healthy volunteers for estimation of CD4+ T cell subsets.ResultsTwo patients dropped out due to non-compliance. As compared to healthy controls (median of Treg cells: 33.32 cell/ul), the absolute counts of circulating Treg cells were significantly decreased in patients with active refractory RA (median: 27.17 cell/ul; P=0.046). While the median ratios of Th17/Treg cells in patients with active refractory RA (median: 0.26) were significantly higher than those of healthy volunteers (median: 0.19; P=0.029). No difference in the absolute counts of circulating Th17 cells and Th1 cells was observed between patients with active refractory RA and healthy subjects. Rapamycin treatment led to clinical improvement with the median post-treatment DAS28-ESR decreasing when compared to baseline (from 4.19 to 3.78) in active refractory RA patients. Sixteen patients (16/43 patients, 37.21%) achieved an EULAR moderate response and 6 patients (6/43 patients, 13.95%) reached good response at week 12. Rapamycin administration resulted in an increase in the absolute counts of Treg cells in active refractory RA patients, from a median of 27.17 cell/ul (at week 0) to 37.57 cell/ul (at week 12) (P=0.041). The ratios of Th17/Treg cells shows a reduction from a median of 0.26 at baseline to 0.20 at week 12, but the difference is not significant (P =0.376). No significant difference was observed in the absolute counts of circulating Th17 and Th1 cells after rapamycin treatment. Interestingly, we observed that Treg cells increased before the complete remission of the disease (DAS28 score <2.6) in patients with active refractory RA. At week 12, the mean dose of prednisone which refractory RA patients were receiving decreased from 11.98 mg/d to 8.31 mg/d, with a dose reduced by ≥30% than that at baseline. The categories of DMARDs use were also reduced (P<0.05). No serious adverse events was observed during the 12-week period of rapamycin treatment.ConclusionsReduced absolute number of Treg cells was found in the patients with active refractory RA, indicating an imbalance between Th17 and Treg cells. Rapamycin elicits rapid improvement of disease activity via restoring circulating Treg cells numbers in patients with active refractory RA.References Perl A. Activation of mTOR (mechanistic tar...