Regulatory Effect Of Combination Of MTX And CTX On The Balance Of Th17and Treg In Collagen-induced Arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammatory arthritis that frequently results in destruction of articular cartilage and bone. There is a broad consensus about the therapeutic alliance of disease modifying anti-rheumatic drugs (DMARDs) should be used in the initial stage of RA, such as methotrexate (MTX), leflunomide(LEF) and sulfasalazine(SSZ).Through more than ten years’clinical research, combination of MTX and CTX dependent cell cycle had been suggested to relieve inflammatory symptoms, postpone joint damage and decrease toxic side effects in treating RA patients. The rational explanation behind this strategy was that the two agents worked through different mechanisms:MTX specifically delayed the transition from G0/G1to S phase, whereas CTX non-specifically damaged cells in any phase. Clinical data showed that compared with the single drug, combination with MTX and CTX were associated with a higher percentage of remissions, less radiographic progression, less dosage of the drugs and less adverse reactions. In our previous studies, MTX combined with CTX synergistically downregulated the expression of p53and cyclin D1mRNA in collagen-induced arthritis (CIA).Auto immunity and inflammation are regulated by the balance between Th17and Treg cells, and inflammation and joint damage are two closely linked processes. More recently, studies have found that the breaking of balance between Th17and Treg and the changing of RANKL/RANK/OPG-related cytokines microenvironment are the main pathogenesis of RA and CIA. However, to our knowledge, the effect of combined MTX and CTX on Th17/Treg profiles and related cytokines of RA has not yet been determined. In this study, we aimed to investigate the regulation of MTX and CTX on Th subsets, relative cytokines and trans-singalling proteins, used alone or in combination, in a murine model collagen-induced arthritis (CIA) which is representative animal model used in studies of RA. CIA was induced in DBA/1J mice by means of immunization with collage type II (CII) emulsified in complete Freund’s adjuvant. All mice were divided into normal control group (injected only with vehicle, n=15); model control group with CIA that received NS (n=13); MTX group with CIA that received MTX1.5mg/kg, twice a week (n=15); CTX group with CIA that received CTX30mg/kg/10day (n=15) and MTX+CTX group with CIA that was received MTX1.5mg/kg, twice a week and CTX30mg/kg/10day (n=15). All treatments started from four weeks after the first collagen immunization, and sustained for3,7or11weeks through intraperitoneal administration. The severity of arthritis was evaluated by clinical visual scoring, histological assessment and sera IgG anti-collagen (CII) antibody level. We evaluated the effect of these treatments on the effect of Thl7, CD4+CD25+FoxP3+Tregs, Thl and Th2subsets using flow cytometry. Then we investigated the effect of these treatments on Thl/Th2/Th17/Treg cytokines and gene expression. Finally, the cytokine profiles involved in bone resorption were also measured.The results showed as follows:①Taken together, these clinical results confirm that combination of MTX and CTX was superior to mono-therapy in CIA mice, as verified by clinical visual scoring, histological assessment and sera IgG anti-collagen (CII) antibody level. MTX and CTX combination additively reduced the inflammatory symptoms and joint damage in CIA.②Combination therapy significantly increased IκB-a inhibitor, and effectively modulated the NF-κB pathway mediates proliferation, apoptosis and development of Th cells. Moreover, Combination therapy inhibited production proinflammatory cytokines by CⅡ-stimulated spleen MNCs, such as IFN-y, TNF-a, IL-17, IL-6and IL-2, and additively restored IL-17/IL-10ratio balance.③Combination of MTX and CTX decreased Th17cells and reciprocally increased Treg cells and restored the Th17/Treg balance by regulating STAT proteins in CIA. ROR-yT and Foxp3are two critical transcription factors involved in the development of Th17cells and Treg cells respectively. The mRNA expression of ROR-yT and Foxp3and number of CD4+ROR-yT+and CD4+Foxp3+from spleen MNCs provide novel evidence that the therapeutic effect of combination therapy were associated with the balance of Th17and Treg. Moreover, restore the Thl7/Treg balance was associated with significant inhibition of their key transcriptional factors, pSTAT3.④Combination of MTX and CTX effectively reduced Th17-type response and Th17cells related pro inflammatory cytokines expression, such as IL-17, IL-6, IL-1β and TNF-a, blocking IL-6/IL-17/STAT3positive feedback loop, influenced on the RANK/RANKL/OPG system, significantly retarded the cartilage and bone destruction.In conclusion, MTX and CTX combination have synergistic or additive effects, decreasing inflammation and joint damage in CIA mice by modulating the balance of Th17and Treg.