Regulation Of CD4~+CD25~+ Regulatory T Cells On Peritoneal Macrophages In Collagen-induced Arthritis Rats And The Effect Of Leflunomide On The Regulation

Rheumatoid arthritis is a chronic, systemic and self-immunological disease, whose origin and pathogenesis is still unknown. In recent years, CD4+CD25+ regulatory T cells (CD4+CD25+Tregs) that possess unique functions on regulating immunity have been founnd. Its role in RA have been emphasized a lot which show that it could be a new target point of anti-arthritic drugs.Many studies demonstrated that CD4+CD25+Tregs could inhibit the activity and function of autoreactive T cells, which maintain immunologic tolerance and homoiostasis in a positive way. The abnormality of CD4+CD25+Tregs in quantity and function related to the development of autoimmune disease. More and more evidence indicate that CD4+CD25+Tregs are abnormal in RA patients and its disability is related to the occurrence and development of RA. The expression of CD4+CD25+Tregs in autoimmune disease has been focused on, but the expression and mechanism of CD4+CD25+Tregs in RA is still not well-known and need further studying. In this study, collagen-induced arthritis (CIA) in rats which well represents RA is chosen, based on abstracting and purification of collagen type II. The kinetics of CD4+CD25+Tregs population was determined to analyze its effect in CIA course. The main cytokine TGF-β1 was chose to investigate the mechanism of regulation of CD4+CD25+Tregs on peritoneal macrophages in CIA rats and the effects of anti-arthritic drug leflunomide on the regulation, to clarify the mechanism of RA and to find new pathways in cure RA. The study contains three sections: 1. Extract and purify collagen type II, and induce collagen-induced arthritis in rats.Soluble collagen typeⅡ(CⅡ) was released from the chicken sternums with pepsin digestion and passed over a DE-52 column. Soluble collagen typeⅡ(CⅡ) was released from the chicken sternums with pepsin digestion and passed over a DE-52 column. The yield of CⅡis 0.17%. SDS-PAGE and Ultra-violet scanning results of the sample and standard CⅡfrom Sigma Company were the same. Collagen-induced arthritis in rats was induced with this product, and the animal model was successful by determing body weight, paw swelling, polyarthritis index, anti-CⅡantibody in serum and histopathology examination. The achievement ratio of 4 experiments is 42%.2. Study the mechanism of regulation of CD4+CD25+ regulatory T cells on peritoneal macrophages in CIA rats.Detect the kinetics of CD4+CD25+Foxp3+ T cell ratio during the course of CIA in peripheral blood of rats, and analyze it with paw swelling. The results indicated that the paw swelling is negatively related to the percentage of CD4+CD25+Tregs in CIA rats, and the coefficient correlation is -0.786. The results in immunofluorescence and RT-PCR showed that CD4+CD25+Tregs and peritoneal macrophages (PMΦ) in CIA rats express TGF-βand Tβ-RII, respectively. This indicated that CD4+CD25+Tregs could effect on macrophages through TGF-β, which is in accordance with the literature.TGF-βwas incubated with PMΦfrom CIA rats in vitro to study the effects and mechanism of it on the immune function of CIA rats. The results showed that TGF-β(5 ng/ml, 36h) could suppress the secretion and mRNA expression of TNF-αand IL-6, improve the secretion and mRNA expression of anti-inflammatory IL-10, and inhibit the secretion of NO and mRNA expression of iNOS in PMΦfrom CIA rats. The results of RT-PCR, flow cytometry and Western blot indicated that TGF-β(5 ng/ml, 36h) inhibits mRNA and protein expression of TLR 4 in PMΦof CIA rats, which suggests that TGF-βcould regulate the immune function of PMΦfrom CIA rats through LPS-TLR4 pathway.3. Investigate the effects of leflunomide on the regulation of CD4+CD25+Tregs on PMΦof CIA rats.Firstly, leflunomide has effects on CD4+CD25+Tregs of CIA rats. The results in vivo experiment showed that leflunomide (2 mg/kg, d12-d33) has therapeutic effect on CIA rats, could increase the percentage of CD4+CD25+T cell and the expression of Foxp 3 mRNA. In in vitro Con A-induced splenic lymphocytes proliferation system, A771726 (0.1, 1, 10μM) promoted lymphocytes differentiation to CD4+CD25+Tregs and increased the secretion of TGF-β, which results in inhibiting the proliferation in Con A-stimulated splenic lymphocytes system.Secondly, leflunomide directly inhibited the immune function of PMΦfrom CIA rats. In vitro, A771726 (0.1, 1, 10μM) suppressed the secretion function of PMΦfrom CIA rats by inhibiting LPS-TLR4 pathway.Thirdly, leflunomide could enhance the regulation of CD4+CD25+Tregs on PMΦof CIA rats. In vitro, A771726 (0.1, 1, 10μM) reinforced the suppression of TGF-βon secretion of PMΦfrom CIA rats, and enhanced the anti-inflammatory cytokine secretion. The results in RT-PCR and Western-blot indicated that A771726 strengthened the regulation of TGF-βon PMΦof CIA rats by inhibiting TLR4 expression in PMΦof CIA rats.Conclusion:①C IA rat model induced with our production of CⅡis successful as indicated the similarities in clinical manifestation, immunology and histopathology with human RA.②T he mechanisms of regulation of CD4+CD25+Tregs on PMΦof CIA rats: CD4+CD25+Tregs combined with PMΦthrough TGF-β/Tβ-RII, inhibiting TLR 4 expression of PMΦwhich leads to suppression of the immune function of LPS-stimulated PMΦof CIA rats.③Leflunomide has therapeutic effects on CIA rats, and the mechanisms contains three aspects: improving the percentage of suppressive CD4+CD25+Tregs, directly inhibiting the immune function of PMΦ, and enhancing the regulation of CD4+CD25+Tregs on PMΦby LPS-TLR4 pathway.