Therapeutic Effect Of IgD-Fc-Ig Fusion Protein On Rat Collagen-induced Arthritis And Regulation On IgD-IgDR-Lck-NF-KB Signaling Pathway In T Cell

Rheumatoid arthritis(RA)is a chronic,systemic,autoimmune disease in which T and B lymphocytes are involved.Joint inflammation,vasospasm formation,synovial hyperplasia,cartilage and bone injury are the main features of RA.There is increasing evidence that T lymphocytes play an important role in the development and progression of RA.Unbalanced and abnormal activation of T cell subsets involved in RA synovitis and immune response.Helper T(Th)cells can promote the destruction process of RA joint tissue,and activated T cells can regulate this inflammatory process by secreting effector cytokines.At present,the therapeutic drugs for RA are mainly steroidal anti-inflammatorydrugs,Non-steroidalanti-inflammatorydrugs(NSAIDs),disease-modifying drugs and biological agents.However,these drugs have only partial relief effects on joint inflammation,and there are significant gastrointestinal adverse reactions,hepatotoxicity and myelosuppression.Therefore,it is of great clinical significance to explore new therapeutic targets and develop new therapeutic for RA.Immunoglobulin D(IgD)includes secreted IgD(sIgD)and membrane-bound IgD(mIgD).Although the content of IgD in the body is particularly low,IgD plays an important immunomodulatory role in autoimmune diseases.Clinically,patients with IgD-type myeloma have significantly elevated sIgD levels,and sIgD levels in patients with infectious diseases and autoimmune diseases are also high,such as RA,systemic lupus erythematosus(SLE).These findings have attracted domestic and foreign scholars'attention to the role of IgD in autoimmune diseases.High levels of sIgD lead to immune tissue damage and inflammation,and injection of anti-IgD antibodies in animal models of arthritis significantly relieves joint inflammation.In 1980,IgDR was found in human T cells,and IgDR was also found on mouse T cells.sIgD exerts immunoregulatory function by specifically binding to IgDR.Experimental studies have found that binding of IgD to IgDR on T cells leads to an increase in antigen-specific T cell clones.In vitro studies in mice have found that protein tyrosine kinase(PTK)inhibitors can inhibit IgD-induced IgDR expression.Therefore,we hypothesize that IgD may induce IgDR expression through the PTK signaling pathway,thereby activating downstream signaling pathways.,producing a series of biological effects.The proteins involved in the PTK signaling pathway include the lymphocyte-specific protein tyrosine kinase(Lck)of the Src family and the zeta-associated protein 70(ZAP70)of the T cell-specific Syk family member.Lck mainly exists in T cells and is involved in the signal transduction process of T cell development,differentiation and activation.As one of the earliest activated upstream signal molecules in the process of T cell activation signal transduction,Lck plays a key role in mediating T cell immune response.When stimulated by some external factors,the expression and activity of Lck are abnormal,and Lck is involved in autoimmune diseases such as RA and SLE.It suggests that IgD-IgDR and its downstream pathway may become new therapeutic targets for autoimmune diseases,and specific blocking of IgD-IgDR signal transduction may become a new therapeutic method for RA.The research team successfully developted IgD-Fc-Ig fusion protein through linking the human IgD-Fc segment to the human IgG1-Fc segment targeting IgD-IgDR,and has applied for a patent of IgD-Fc-Ig fusion protein.The fusion protein aims to specifically block the IgD-IgDR pathway and highly selectively inhibit abnormal proliferation,activation and differentiation of T cells.The previous study of the research team found that the level of IgD serum in patients with RA was significantly higher than that in healthy controls.IgD could significantly promote the proliferation of T cells in peripheral blood of RA patients.IgD-Fc-Ig fusion protein significantly inhibits T cell proliferation in peripheral blood of RA patients.So,does the IgD-Fc-Ig fusion protein have therapeutic effects on experimental arthritis?Does the IgD-Fc-Ig fusion protein have an effect on the proliferation,activation and differentiation of experimental arthritis T cells?Can IgD-Fc-Ig fusion protein regulate the IgD-IgDR-Lck signaling pathway on T cells?It has been unclear.In this study,collagen-induced arthritis(CIA)rats were used as the research object to observe the therapeutic effect of IgD-Fc-Ig fusion protein on rat CIA and its effect on T cell proliferation,activation and differentiation.To elucidate the mechanism by which IgD-Fc-Ig fusion proteins inhibit T cell proliferation,activation and differentiation by blocking the IgD-IgDR-Lck signaling pathway.This study provides an experimental basis for further revealing the pathogenesis of RA and elucidating the role and mechanism of IgD-Fc-Ig fusion protein in the treatment of RA.ObjectiveTo study the therapeutic effect of igd-fc-ig fusion protein on CIA in rats,and to reveal the regulatory effect of IgD-Fc-Ig fusion protein on the IgD-IgDR-Lck-NF-?B signaling pathway on T cells.Methods1.Wistar male rats were established for CIA model.The experiments were grouped as follows:normal group,CIA model group,IgD-Fc-Ig fusion protein(1,3and 9 mg/kg)groups and Etanercept(3 mg/kg)group.There were 10 rats in each group.Each dose was calculated according to the body weight of the rats,and administered once every 3 days,all of which were administered by tail vein.The administration time is 6 weeks.The normal group and the CIA group were given a parallel control according to the body weight of the rats by intravenous injection.2.Body weight,arthritis index(AI),swollen joint count(SJC),and paw volume were measured and evaluated every 3 days.Calculate the thymus and spleen index.The spleen and joint tissues were stained with HE,and pathological changes were observed and the relevant indicators were scored.CCK-8 was used to detect the proliferation of thymic T cells and spleen B cells in rats.3.The percentage of T cell subsets in peripheral blood at different inflammatory stages and spleen cells of rats was determined by flow cytometry,including CD3~+total T cells,CD3~+CD4~+Th cells,CD3~+CD4~+CD25~+activated Th cells,Th1(CD4~+IFN-?~+),Th2(CD4~+IL4~+),Th17(CD4~+IL-17~+)and Treg(CD4~+CD25~+Foxp3~+)cells.4.Enzyme-linked immunosorbent assay(ELISA)and protein chip technology were used to detect the changes of inflammatory cytokines(such as IL-1?,IL-4,IL-6,IL-17,IFN-?,TNF-?,MCP-1,Leptin and TIMP)in serum of rats.5.Western blotting was used to detect the expression of Lck,p-Lck,ZAP70,p-ZAP70,P38,p-P38,NF-?B and p-NF-?B in rat spleen.6.In vitro experiments:The optimal concentration of IgD on T cell proliferation and the effect of IgD-Fc-Ig fusion protein alone on normal T cells were detected by CCK-8.The effect of IgD-Fc-Ig fusion protein on T cell proliferation was detected by CFSE.7.Flow cytometry was used to detect the percentages of CD3~+total T cells,CD3~+CD4~+Th cells,CD3~+CD4~+CD25~+activated Th cells,Th1(CD4~+INF-?~+),Th2(CD4~+IL-4~+),Th17(CD4~+IL-17~+)and Tregs(CD4~+CD25~+Foxp3~+)in IgD-stimulated spleen cells.8.Western blotting was used to detect the expression of Lck,p-Lck,ZAP70p-ZAP70,P38,p-P38,NF-?B and p-NF-?B in IgD-stimulated spleen cells.Results1.IgD-Fc-Ig fusion protein has therapeutic effect on rat CIAThe indicators of paw inflammation in CIA rats at different inflammatory stages were analyzed and evaluated.It was found that IgD-Fc-Ig fusion protein significantly reduced paw swelling in CIA rats.The IgD-Fc-Ig fusion protein can significantly reduce the AI,SJC and the swelling volume of the paw in CIA rats,and restore the reduced body weight of CIA rats.2.IgD-Fc-Ig fusion protein can reduce thymus and spleen index,inhibit T/B cell proliferation,and improve pathological changes of spleen and jointsCompared with the normal group,the thymus and spleen index of the CIA group increased significantly;the IgD-Fc-Ig fusion protein significantly decreased the thymus and spleen index of CIA rats.Compared with the normal group,the proliferation of thymic T cells and spleen B cells in the CIA group was significantly stronger;the IgD-Fc-Ig fusion protein significantly inhibited the proliferation of T and B cells in CIA rats.The pathological findings of the spleen showed that the number of germinal centers,inflammatory cell infiltration and lymphoid follicles increased significantly in the CIA group compared with the normal group;the IgD-Fc-Ig fusion protein significantly reduced the number of germinal centers and lymphoid follicles,and improved red pulp congestion.Joint pathology showed that inflammatory cell infiltration,bone erosion and synovial hyperplasia were more severe in the joints of CIA rats than in the normal group.IgD-Fc-Ig fusion protein significantly inhibits inflammatory cell infiltration and synovial hyperplasia.3.IgD-Fc-Ig fusion protein regulates differentiation and activation of T cells in CIA ratsFlow cytometry was used to detect the percentage of T cell subsets in peripheral blood at different stages of inflammation and spleen cells.In the late stage of inflammation,the IgD-Fc-Ig fusion protein significantly reduced the percentages of CD3~+total T cells,CD3~+CD4~+Th cells and CD3~+CD4~+CD25~+activated Th cells in peripheral blood lymphocytes.IgD-Fc-Ig fusion protein significantly reduced the percentages of CD3~+total T cells,CD3~+CD4~+Th cells and CD3~+CD4~+CD25~+activated Th cells,Th1(CD4+INF-?~+)and Th17(CD4~+IL-17~+),increased the percentage of Treg(CD4~+CD25~+Foxp3~+)cells in spleen cells.The IgD-Fc-Ig fusion protein had no significant effect on the percentage of Th2(CD4~+IL-4~+)cells.4.Effect of IgD-Fc-Ig fusion protein on the levels of inflammatory cytokines in serum of CIA ratsThe levels of inflammatory cytokines(such as IL-1?,IL-4,IL-6,IL-17,IFN-?,TNF-?,MCP-1,Leptin and TIMP in serum of CIA rats were detected by ELISA and protein chip technique.The results showed that the levels of IL-1?,IL-6,IL-17,IFN-?,TNF-?,MCP-1 and Leptin in the serum of CIA group were significantly higher than those in the normal group,and the level of TIMP was significantly decreased.Compared with the CIA group,the IgD-Fc-Ig fusion protein significantly decreased the levels of IL-1?,IL-6,IL-17,IFN-?,TNF-?,MCP-1 and Leptin,and increased TIMP level.The IgD-Fc-Ig fusion protein had no significant effect on IL-4 level.5.IgD-Fc-Ig fusion protein down-regulates p-Lck,p-ZAP70,p-P38 and p-NF-?B65 protein expression in CIA rat T cellsWestern Blot results showed that the expression of p-Lck,p-ZAP70,p-P38 and p-NF-?B65 protein in spleen cells of CIA group was significantly up-regulated compared with normal group rats.Compared with CIA group rats,IgD-Fc-Ig fusion protein significantly down-regulated p-Lck,p-ZAP70,p-P38 and p-NF-?B65 protein expression.6.In vitro assay:IgD-Fc-Ig fusion protein inhibits IgD-stimulated T cell proliferationThe results showed that IgD(9?g/ml)could significantly stimulate the proliferation of T cells at 48h.IgD-Fc-Ig fusion protein could significantly inhibit the proliferation of IgD-stimulated T cell.The IgD-Fc-Ig fusion protein alone had no significant effect on normal T cells.7.IgD-Fc-Ig fusion protein regulates IgD-stimulated T cell differentiation and activationFlow cytometry was used to detect the percentage of T cell subsets in IgD-stimulated spleen cells.The results showed that the IgD-Fc-Ig fusion protein significantly reduced the percentages of CD3~+total T cells,CD3~+CD4~+Th cells and CD3~+CD4~+CD25~+activated Th cells,Th1(CD4~+INF-?~+)and Th17(CD4~+IL-17~+),increased the percentage of Treg(CD4~+CD25~+Foxp3~+)cells in IgD-stimulated spleen cells.The IgD-Fc-Ig fusion protein had no significant effect on the percentage of Th2(CD4~+IL-4~+)cells.8.IgD-Fc-Ig fusion protein down-regulates p-Lck,p-ZAP70,p-P38 and p-NF-?B65 protein expression in IgD-stimulated spleen cellsWestern Blot results showed that IgD significantly up-regulated the expressions of p-Lck,p-ZAP70,p-P38 and p-NF-?B65 in IgD-stimulated spleen cells compared with Control group.The IgD-Fc-Ig fusion protein significantly down-regulated the expressions of p-Lck,p-ZAP70,p-P38 and p-NF-?B65 compared to the IgD(9?g/ml)group.Conclusions1.IgD-Fc-Ig fusion protein has a significant therapeutic effect on rat CIA.2.The IgD-Fc-Ig fusion protein may regulate T cell function by inhibiting T cell activation and differentiation.3.The IgD-Fc-Ig fusion protein may play a therapeutic role in rat CIA by regulating the IgD-IgDR-Lck-NF-?B signaling pathway on T cells.