Therapeutic Effects Of IgD-Fc-Ig Fusion Protein,inhibitor Targeting IgD-IgDG,on Collagen Induced Arthritis Mice And The Regulation Of T Cell Activity

IntroductionRheumatoid Arthritis?RA?is the most familiar systemic,chronic autoimmune disease,characterized by pain,swelling,persistent synovitis and progressive destruction of small joints like hands and feet,finally resulting in functional disability.There are about 20-30 percentages of patients with RA who have not been treated have the risk of permanent function loss in one year,which badly impact on their healthy and life quality.Plenty of routine drugs like disease-modifying antirheumatic drugs?DMARDs?and nonsteroidal anti-inflammatory drugs?NSAIDs?,were widely used in the treatment of RA.However,long-term use of these drugs could cause serious adverse reactions.Along with the better understanding of the inflammatory and immune pathways,new treatments that target inflammatory cytokines like B cells,T-cell co-stimulation and TNF-?for RA treatment have been cast into a new light,they have better efficacy,but can also cause more serious adverse reactions,extensive immunosuppression,and induce malignant infections and tumors,which are difficult to long-term use.These treatments are also likely ineffective or have low response to some RA patients,or have therapeutic effect at the initial stage,and drug resistance appears after a period of times.These problems can be caused by a variety of reasons,and elevated levels of immunoglobulin D may be one of them.Immunoglobulin D?IgD?includes the membrane-bound form?mIgD?and secreted form?sIgD?.Many lymphoid and nonlymphoid cells express surface membrane receptors,whose specific structures are located in the constant region of immunoglobulin molecules?FcR?,the presence of IgDR on T cells was been proved in1985,IgD binding to T cells and basophilic cell lines has been demonstrated through quantitative immunofluorescence techniques,suggesting that IgD may be involved in regulating T cell function through IgDR.Our previous research found that the average concentration of sIgD in 54 RA patients was 91.93?g/ml,which was much higher than that in healthy controls?19.8?g/ml?.It has been reported that the concentration of anti-nuclear IgD antibody is increased in about 35%of RA patients,while for adult RA patients,the concentration of anti-IgD antibody is significantly increased in about 55%of these patients,suggesting that IgD-IgDR could be considered as an ideal target for the treatment of IgD overexpressed RA.Selective blocking of IgD-IgDR signal transduction may be a novel immunotherapy for RA which accompanied with high IgD expression.Our group considered IgD-IgDR as a novel target and successfully synthesised IgD-Fc-Ig fusion protein through connecting IgD-Fc and IgG₁-Fc domains,aim to block IgD-IgDR pathway specifically and highly selectively inhibit the abnormal T cell proliferation and activation,we already applied china patent.In this study,PBMCs of RA patients were used to detect the effect of IgD on the function of PBMCs of RA patients,and the influence of IgD-Fc-Ig on the role of IgD mentioned above.The collagen induced arthritis?CIA?model was established,the overall therapeutic effect of IgD-Fc-Ig on CIA mice and partial mechanism were observed.We take IgD-IgDR as a novel target and provide experimental basis for innovative drug research on IgD-Fc-Ig in the treatment of IgD overexpressed RA.ObjectiveClarify the effect of IgD on PBMCs function in RA patients and the role of IgD-Fc-Ig,and to reveal the therapeutic effect and part of the mechanism of IgD-Fc-Ig on CIA mice.MethodsPeripheral blood was collected from RA patients,PBMCs of RA patients were purified using ficoll gradient centrifugation.Effects of IgD-Fc-Ig on the proliferation of IgD-induced PBMCs in RA patients were detected by CCK-8.Effects of IgD-Fc-Ig on the percentage of IgD-induced T cell surfactant molecules?CD4⁺/CD69⁺,CD4⁺/CD154⁺?and T cell subsets?CD4⁺/INF?⁺,CD4⁺/IL-4⁺,CD4⁺/IL-17⁺,CD4⁺/CD25⁺/FoxP3⁺?of PBMC in RA patients were detected by flow cytometry.The effects of IgD-Fc-Ig on IgD-induced mRNA expression of T cell specific nuclear transcription factors?Tbet,GATA,ROR?t,FoxP3?in RA patients were detected by QPCR.Level of plasma IgD on CIA mice was detected by ELISA.The correlation of plasma IgD levels with swollen joint count and arthritis index in mice treated with IgD-Fc-Ig was analyzed;To study the effect of IgD-Fc-Ig on overall index?body weight,swollen joint count,arthritis index?and pathological changes of spleen and ankle in CIA mice.To discuss the effects of IgD-Fc-Ig on thymus index and spleen index of CIA mice.Effects of IgD-Fc-Ig on the proliferation of thymus T cells and spleen B cells in CIA mice were measured by CCK-8.Effects of IgD-Fc-Ig on the percentage of T cell surfactant molecules?CD4⁺/CD154⁺?andcellsubsets?CD4⁺/INF?⁺,CD4⁺/IL-4⁺,CD4⁺/IL-17⁺,CD4⁺/CD25⁺/FoxP3⁺,B220⁺/IgM⁺/IgD⁺?were analyzed by flow cytometry.Level of IL-1??IL-15?MCP-5?MCSF in CIA mice plasma was detected by antibody microarrays.The correlation of plasma IgD levels with IL-1??IL-15?MCP-5?MCSF levels in mice treated with IgD-Fc-Ig was analyzed;Protein expression of Lck,p-Lck,ZAP70,p-ZAP70 in CIA mice spleen was detected by western blot.Results1.Demographic characteristics and clinical indicators of RA patients33 RA patients were collected,most of the patients were females?female/male ratio=27:6?.Means±standard error of age?years?,RF?IU/ml?,CRP?mg/L?,ESR?mm/h?and Anti-CCP?RU/ml?were as follows:?57.24±2.07?,?171.1±23.8?,?36.71±5.903?,?64.7±4.872?,?683.9±55.04?.2.Effects of IgD and IgD-Fc-Ig on T cell function of RA patientsAfter stimulation for 48h,IgD?3?g/ml?could obviously promote the proliferation of PBMCs from RA patients,IgD-Fc-Ig?10?g/ml?significantly inhibited the proliferation of PBMCs from RA patients induced by IgD.IgD?3?g/ml?also clearly increased the percentage of T cell surfactant molecules?CD4⁺/CD154⁺,CD4⁺/CD69⁺?,IgD-Fc-Ig?10?g/ml?significantly reduced the percentage of IgD-induced CD4⁺/CD69⁺T cells in RA patients,10?g/ml of IgD-Fc-Ig could downregulate the proportion of IgD-induced CD4⁺/CD154⁺T cells in RA patients.IgD?3?g/ml?stimulation for 48h in RA patients could upregulate the percentage of Th17?CD4⁺/IL-17⁺?cells and mRNA expression of Th17-specific nuclear transcription factor ROR-?t,downregulate the percentage of Treg?CD4⁺/CD25⁺/FoxP3⁺?cells and mRNA expression of Treg-specific nuclear transcription factor FoxP3,treatment of IgD-Fc-Ig?10?g/ml?could decrease the IgD-inudced percentage of Th17 cells,IgD-Fc-Ig?3,10?g/ml?treatment clearly downregulated ROR-?t mRNA expression in RA patients induced by IgD,IgD-Fc-Ig?3,10?g/ml?administration significantly increased the IgD-induced percentage of Treg cells,IgD-Fc-Ig?1,3,10?g/ml?significantly upregulated FoxP3 mRNA expression in RA patients induced by IgD.While IgD?3?g/ml?stimulation for 48h and IgD-Fc-Ig administration had no obvious effects on the number of Th1 and Th2 cells and mRNA expression of Th1 and Th2 cell-specific nuclear transcription factors Tbet and GATA in RA patients.3.Effects of IgD-Fc-Ig on overall indexes of CIA miceThe weight of mice in the CIA group began to drop from d21,and the change had significance compared with normal group from d33 to d44.After d44,There was no significant difference between CIA group and normal group,and no obvious changes in the weight were found among the mediated groups.Administration of anti-mouse IgD antibody clearly decreased the arthritis index and swollen joint counts from d33 to d54,rhTNFR:Fc treatment had significance from d37,IgD-Fc-Ig?3.25,6.5 mg/kg?treatment decreased the arthritis index and swollen joint counts from d47,13 mg/kg of IgD-Fc-Ig had significance from d50,while IgD-Fc-Ig?1.625mg/kg?and negative control IgG₁-Fc?13mg/kg?groups had no obvious effects on the arthritis index and swollen joint counts of CIA mice.4.Changes of plasma IgD levels in CIA miceCompared with normal group,the levels of plasma IgD in CIA mice were obviously elevated.5.The correlation of the plasma IgD levels with swollen joint count and arthritis index in mice treated with IgD-Fc-IgThe plasma IgD levels were negatively correlated with swollen joint count and arthritis index in mice treated with IgD-Fc-Ig.In the IgD-Fc-Ig groups with high plasma IgD levels,swollen joint count and arthritis index of mice were lower,and joints swelling was obviously ameliorated.6.Effect of IgD-Fc-Ig on histopathological changes of ankle joints and spleens in CIA miceCompared with the normal group,synovial tissue hyperplasia,inflammatory cell infiltration,vascular congestion,pannus,bone and cartilage destruction were observed in the joint of CIA mice,IgD-Fc-Ig?1.625mg/kg?and negative control IgG₁-Fc?13mg/kg?groups had no obvious ameliorative effects on the histopathological changes of ankle joints,administration of IgD-Fc-Ig?3.25,6.5mg/kg?,rhTNFR:Fc and anti-mouse IgD antibody could clearly ameliorate the situation of synovial hyperplasia,inflammatory cell infiltration,vascular congestion and pannus information.Compared with the normal group,CIA mice spleen showed white pulp hyperplasia,germinal center emergence,red pulp hyperplasia,lymphoid follicular hyperplasia and inflammatory cell infiltration.IgD-Fc-Ig?1.625mg/kg?and negative control IgG₁-Fc?13mg/kg?groups had no obvious ameliorative effects on the histopathological changes of spleen.Treatment of IgD-Fc-Ig?3.25,6.5,13mg/kg?,rhTNFR:Fc and anti-mouse IgD antibody could significantly alleviate lymphoid follicular hyperplasia and inflammatory cell infiltration,as well as germinal centers formation.7.Effects of IgD-Fc-Ig on thymus index and spleen index in CIA miceCompared with the normal group,the thymus and spleen index of CIA mice were significantly increased.Treatment of IgD-Fc-Ig?3.25,6.5,13mg/kg?,rhTNFR:Fc group and anti-mouse IgD antibody could clearly downregulate thymus index of CIA mice,anti-mouse IgD antibody could decrease the spleen index of CIA mice,while IgD-Fc-Ig,rhTNFR:Fc and IgG₁-Fc administration had no significant effect on the spleen index.8.Effects of IgD-Fc-Ig on the proliferation of thymus T cells and spleen B cells in CIA miceThe proliferation of thymus T cells and spleen B cells in CIA mice was significantly increased.Administration of IgD-Fc-Ig?3.25,6.5mg/kg?could apparently prevent proliferation of thymus T cells in CIA mice,and the proliferation of spleen B cells in CIA mice was significantly downregulated after IgD-Fc-Ig?3.25,6.5,13mg/kg?treatment.IgD-Fc-Ig?1.625mg/kg?had no significant effect on the proliferation of thymus T cells and spleen B cells in CIA mice.9.Effects of IgD-Fc-Ig on the percentage of T cell subsets and B cell subsets in CIA miceCompared with the normal group,percentage of total helper T cells?Th cells,CD3⁺/CD4⁺?,Th cells expressed CD40L?CD4⁺/CD154⁺?,Th1 cells?CD4⁺/IFN-?⁺?,Th17cells?CD4⁺/IL-17⁺?and mature B cells?B220⁺/IgM⁺/IgD⁺?was obviously increased in CIAmicespleen,proportionofTh2cells?CD4⁺/IL-4⁺?andTreg cells?CD4⁺/CD25⁺/Foxp3⁺?wasdecreased,IgD-Fc-Ig?3.25,6.5,13mg/kg?administration could reduce Th cells,Th cells that expressed CD40L,Th1 and Th17cells proportions,IgD-Fc-Ig?3.25,6.5mg/kg?could increase percentage of Th2 cells,and?6.5,13mg/kg?administration of IgD-Fc-Ig could increase Treg cells proportion,treatment of IgD-Fc-Ig?3.25,6.5,13mg/kg?could also decrease the number and percentage of mature B cells in CIA mice spleen.10.Effects of IgD-Fc-Ig on the levels of IL-1?,IL-15,MCP5 and MCSF in CIA mice plasmaCompared with normal group,the expression of IL-1?,IL-15,MCP5 and MCSF in the plasma of CIA mice was significantly increased,expression of IL-1?was obviously downregulated after IgD-Fc-Ig?1.625,3.25,6.5,13mg/kg?treatment,administration of IgD-Fc-Ig?1.625,3.25,6.5mg/kg?decreased the level of IL-15,and?3.25,6.5,13mg/kg?of IgD-Fc-Ig could also reduce the expression of MCP5 and MCSF.11.The correlation of plasma IgD levels with IL-1?,IL-15,MCP-5,MCSF levels in mice treated with IgD-Fc-IgPlasma IgD levels were negatively correlated with IL-1?,IL-15 levels in mice treated with IgD-Fc-Ig,plasma IL-1?and IL-15 levels in IgD-Fc-Ig treated mice with high plasma IgD level decreased more significantly,but plasma IgD levels in IgD-Fc-Ig groups were not obviously correlated with MCP-5 and MCSF levels.12.Effects of IgD-Fc-Ig on the protein expression of Lck,p-Lck,ZAP70 and p-ZAP70 in CIA mice spleenThe expression of total Lck and ZAP70 had no significant difference among normal,model or the medicated groups,but p-Lck and p-ZAP70 expression was obviously increased in CIA mice,and IgD-Fc-Ig?3.25,6.5 and 13mg/kg?could significantly downregulate the expression of p-Lck and p-ZAP70 in mice with CIA.Conclusions1.IgD could promote the proliferation and activation of PBMCs in RA patients,resulting in the imbalance of Th17/Treg cell subsets,while IgD-Fc-Ig could downregulate the abnormal cell proliferation and activation induced by IgD and restore the IgD induced-imbalance of Th17/Treg cell subsets.2.Firstly revealed that IgD-Fc-Ig had therapeutic effect on experimental arthritis,IgD-Fc-Ig could attenuate the paw swelling and histopathological changes of CIA mice,the plasma IgD levels of CIA mice were significantly increased,plasma IgD levels in the IgD-Fc-Ig groups were negatively correlated with swollen joint count and arthritis index,IgD-Fc-Ig inhibited the abnormal proliferation and activation of T cells,and reversed the imbalance of Th1/Th2,Th17/Treg cell subsets,as well as downregulating the levels of inflammatory cytokines and chemokines in CIA mice,and these procedure might be related to the downregulation of Lck and ZAP-70 phosphorylation.3.This study suggests that IgD-IgDR may be a novel target for RA,IgD-Fc-Ig may have an important prospect for the treatment of RA accompanied with increased IgD levels.