| Rheumatoid arthritis(RA)is an autoimmune disease targeting synovium,which is characterized by synovial hyperplasia,cartilage destruction,pannus formation and systemic inflammation.The pathogenesis of RA is complex,and a variety of immune cells are involved,such as B cells,T cells,macrophages and dendritic cells.As an important adaptive immune cell,B cells play an important role in the pathogenesis of RA.T/B cells interact to promote the differentiation and maturation of plasma cells,which are responsible for the production of autoantibodies and participate in disease development;Activated B cells induce differentiation of effector T cells,effector T cells produce a large number of pro-inflammatory cytokines such as interleukin-1(IL-1),IL-6,IL-17 and tumor necrosis factor-α(TNF-α);B cells also participate in RA by secreting cytokines(such as IL-10)on other immune or non-immune cells(such as macrophages,synovial cells and chondrocytes).Immunoglobulin D(IgD),a member of immunoglobulins family,was first found in multiple myeloma.Because of its special structure and low content,the function of IgD is still unclear.The previous studies of our group showed that the level of IgD increased significantly in autoimmune diseases such as RA,and IgDR was expressed on T cells,B cells,fibroblast-like synoviocytes and macrophages and other immune cells,In addition IgD could promote the activation of peripheral blood monocytes in patients with RA,suggesting that IgD may play an important role in the pathogenesis of RA by binding IgDR.B cell receptor(BCR)is a complex receptor formed by membrane immunoglobulin(mIg)non-covalently coupled Igα and Igβ.BCR is vital for maintaining the survival,differentiation,proliferation and function of B cells.When the antigen binds to BCR,the immune receptor tyrosine activating motif(ITAM)in the cytoplasmic tail of Igα and Igβ will recruit and phosphorylate tyrosine kinases such as spleen tyrosine kinase(Syk)and Bruton tyrosine kinase(BTK),so that antigen stimulation signals are amplified and transmitted downstream.Src family tyrosine kinase Lyn,which can binds to CD22 and other intracellular immune receptors tyrosine inhibition motif(ITIM))and to be phosphorylated,negatively regulate the activation of BCR signal,play a role in regulating the activation threshold of BCR.In the early stage,the recombinant plasmid pET28a(+)-DG was successfully constructed by overlapping PCR,and the recombinant fusion protein DG was successfully purified in Escherichia coli.Can it inhibit the binding of IgD to IgD receptor(IgD receptor,IgDR)on B cell surface? Does DG have therapeutic effect on CIA mice? Can DG regulate the differentiation and function of B cells by inhibiting the binding of IgD to IgDR? If it can regulate the differentiation and function of B cells,does it play a regulatory role through the BCR signal pathway? Those question still unclear.This project intends to use CIA mice,Daudi and Ramos cells as the research objects,using flow cytometry,laser confocal technology,quantitative real-time polymerase chain reaction(q RT-PCR),and immune protein Western blot(WB),protein chip technology,Hematoxylin and Eosin(HE)staining and immunohistochemistry methods to study whether DG has a therapeutic effect on CIA mice and whether it has a therapeutic effect on B cell subgroups and functions influences.Constructing Lyn overexpression plasmid in vitro and transfecting the overexpression plasmid into Daudi cells.BCR knockout Ramos cells were constructed by Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/ Cas9 to further clarify the mechanism of IgD activation of B cells through IgD-IgDR-Syk-Btk-NF-κB signal pathway.The purpose of this study is to study the mechanism of IgD promoting the pathological process of RA by activating B cells,to evaluate the therapeutic effect of DG on mouse CIA model,and to provide experimental basis for developing DG as a new drug for treating RA.Objective:1.To study the activation effect of IgD on B cells by regulating IgD-IgDR-Syk-Btk-NF-κB signal pathway.2.To determine the therapeutic effect of DG on mouse CIA and its effect on the differentiation,activation and proliferation of B cells.3.It is revealed that DG has a therapeutic effect on mouse CIA by inhibiting the binding of IgD to IgDR on the surface of B cells and inhibiting IgD-IgDR-Syk-Btk-NF-κB signal pathway.Methods:1.The heat shock method was used to transform the pET28a(+)-DG recombinant plasmid into BL21 competent bacteria,culture the positive transformant and induce the recombinant plasmid to express DG by IPTG,and separate the cell pellet by centrifugation.Ultrasonic disruption,protein purification instrument purification and molecular sieve filtration to remove impurities to obtain the fusion protein DG.2.Fluorescein labeling kit were used to label IgD and DG with FITC labels.In vitro,mouse CD19+ B cells were sorted by immunomagnetic beads.By setting the cell density to 1×106/ml and adjusting the concentration of IgD-FITC or DG-FITC to 0,0.03,0.1,0.3,1,3,10,30 and 100 μg/ml,flow cytometry was used to detect the FITC fluorescence intensity of mouse B cells incubated with different concentrations of IgD-FITC or DG-FITC,and the expression of IgD receptors was the MFI value.The same method was used to detect the equilibrium dissociation constants KD and Bmax of IgD and DG on Daudi and Ramos cells,and the IC50 of DG inhibiting the binding of IgD to mouse B cells,Ramos and Daudi cells.3.About 20 g male DBA1 mice were selected to establish CIA model,and the successful DBA1 mice were randomly divided into CIA group,DG low dose group(1.625 mg/kg),DG medium dose group(3.25 mg/kg),DG high dose group(6.5 mg/kg),Ig G1-Fc(6.5 mg/kg)negative control group,enalapril(5 mg/kg)and rituximab group(5 mg/kg).DBA1 mice without modeling were taken as normal control group.After intraperitoneal injection,the therapeutic effects were evaluated by body weight,arthritis index,paw swelling,HE staining of ankle joints and spleens.4.Blood was collected from the tail vein on days 19,31,42 and 66.Flow cytometry was used to detect the total CD19+ levels in the peripheral blood and spleen tissues of CIA mice after the first immunization,after the second immunization,during the peak period of inflammation,and when the inflammation subsided.Changes in the levels of B cells,CD19+CD27+ memory B cells,CD19-CD138+ plasma cells,CD19+Ig M+immature B cells and CD19+IgD+ mature B cells,to study the effects of DG on the B cell subsets in peripheral blood and spleen tissues of CIA mice.5.The level of immunoglobulin subtypes in serum of each group of mice was analyzed by protein chip technology,and the effect of DG on B cell function of CIA mice was evaluated.6.The levels of p-Lyn,p-Syk,p-Btk,p-P38 and p-P65 in spleen tissue were detected by immunofluorescence and WB.7.Immunohistochemistry was used to detect the expression of MMP-13,RANKL and type Ⅱ collagen in the ankle joint and synovial tissue of each mice.8.Daudi cell line was stimulated by IgD and intervention with different gradients of IgD-Fc-Ig in vitro.WB was used to detect the levels of p-Lyn,p-Syk,p-Btk,p-P38 and p-P65.Co-immunoprecipitation was used to detect the interaction between Lyn and Syk,Syk and Btk.9.Nuclear translocation of NF-κB p65 and expression of p-NF-κB p65 in Daudi cells stimulated by IgD were detected by immunofluorescence technique.10.The levels of Bach2,Bcl6,IRF4,E2 A,Pax5 and Blimp-1 in Daudi cells were detected by q RT-PCR and the intervention effect of DG was detected.11.The single vector Cas9-U6-sgRNA(Igα+Igβ)plasmid was used to construct Igα-/-Igβ-/-Ramos cells,which were stimulated by IgD.The levels of p-Lyn,p-Syk,p-BTK and p-NF-κB p65 in Igα-/-Igβ-/-Ramos cells were detected by WB.12.Igβ-/-Ramos was constructed with single-vector lentivirus to further detect the activation of BCR signaling pathway by IgD and to find the mechanism of activation of B cells by IgD;13.The expression of BACH2,Bcl6,IRF4,E2 a,PAX5 and Blimp-1 in Igα-/-Igβ-/-and Igβ-/-Ramos cells stimulated by IgD was detected by q RT-PCR.Results:1.IgDR exists on the surface of B cells,and DG can inhibit IgD binds to IgDR on the surface of mouse B cells,Daudi and Ramos cell lines.Immunofluorescence results showed that IgDR existed on the surface of mouse B cells and co-localized with IgD-BCR and Ig M-BCR.Flow cytometry results show that IgD and DG can bind to mouse CD19+ B cells,Daudi and Ramos cells in vitro,and DG can inhibit the binding of IgD to IgDR on mouse B cells,Daudi and Ramos cells.The half inhibitory concentration(half maximal The inhibitory concentration,IC50)were 7.58 μg/ml,8.86 μg/ml and 11.45 μg/ml,respectively.2.DG has a therapeutic effect on CIA miceDG was administrated by intraperitoneal injection every three days.At the same time,the body weight of mice in each group was weighed,and the number of paw swelling and arthritis index of mice in each group were scored.The results showed that DG had significant therapeutic effect on CIA mice,alleviated the symptoms of foot swelling in CIA mice,alleviated the weight loss of CIA mice,and inhibited arthritis index and paw swelling.3.DG can inhibit the activity of T/B cells in vitro,and decrease the thymus and spleen index of CIA mice in vivo.CCK-8 was used to detect the effect of DG on the viability of T/B cells under the stimulation of Con A or LPS.The results showed that DG could inhibit the viability of T/B cells in vitro.By calculating the thymus and spleen index of mice in each group,the results showed that DG could inhibit the thymus and spleen index of CIA mice.4.DG can regulate the levels of memory B cells,plasma cells and mature B cells in the peripheral blood of CIA miceFlow cytometry was used to detect the percentage of B cell subsets in the peripheral blood of mice in different stages of inflammation.The results showed that DG reduced the abnormally elevated levels of memory B cells(CD19+CD27+),plasma cells(CD19-CD138+)and mature B cells(CD19+IgD+),had no significantly affected on the immature B cells(CD19+Ig M+).5.DG can improve the pathological scores of spleen and ankle in CIA miceThe results of HE staining showed that DG could improve the lesions of spleens and ankle joints.DG could reduce the density of lymphatic sheath and the number of lymphatic follicles around the artery in spleen tissue of CIA mice,and improve the marginal zone and erythromyeloid hyperplasia.DG inhibited the infiltration of lymphocytes and synovial hyperplasia in the ankle tissues of CIA mice,and also reduced the formation of pannus and bone destruction.6.DG can regulate B cell subsets in spleen of CIA miceFlow cytometry was used to detect the changes of B cell subsets in the spleen of mice in each group.The results showed that DG could reduce the percentage of total B cells,memory B cells,plasma cells and mature B cells in the spleen of CIA mice,but had no significant effect on immature B cells.7.DG can regulate the level of immunoglobulin in CIA miceProtein chip was used to detect the changes of immunoglobulin subtypes in the serum of mice in each group.DG could inhibit the abnormally increased levels of Ig A,Ig M,IgD,Ig G1,Ig G2 b,Ig G3 and lambda in CIA mice,but had no significant change in the levels of Ig E,kappa and Ig G2 a,indicating that DG could regulate the function of B cells and reduce the secretion of immunoglobulin.8.DG can reduce the expression of MMP-13 and RANKL in ankle cartilage and synovium of CIA mice,and has a protective effect on type Ⅱ collagenThe results of immunohistochemistry showed that DG could increase the level of type Ⅱ collagen in the joints of CIA mice,while reduce the levels of RANKL and MMP-13 in synovium and cartilage tissue,indicating that DG has obvious protective effect on the joints of CIA mice.9.DG can increase the level of p-Lyn and decrease the expression of p-Syk,p-Btk,NF-κ B p65 and p-P38 in spleenThe results of immunofluorescence showed that DG could increase the phosphorylation of Lyn in B cells of spleen tissue of CIA mice and decrease the phosphorylation of Syk and Btk.The results of WB detection were consistent with those of immunofluorescence.DG could reduce the expression of p-Syk,p-Btk,NF-κB p65 and p-P38,indicating that DG could regulate the BCR signal pathway.10.IgD can activate the IgD-IgDR-Syk-Btk-NF-κB signaling pathway of Daudi cells,and DG can inhibit the activation of IgD on BCR signaling in vitroIgD can activate the BCR signal pathway of Daudi cells in vitro,increase the phosphorylation level of Syk and Btk,and inhibit the phosphorylation of Lyn.DG 10μg/ml can significantly inhibit the activation of BCR signal by IgD and reduce p-Syk,p-Btk,p-NF-κB p65 and p-P38 levels.11.DG can inhibit the nuclear translocation of NF-κB p65 in Daudi cells stimulated by IgD and decrease the level of NF-κB p65The results of immunofluorescence showed that IgD 5 μg/ml could significantly promote the translocation of NF-κB p65 into the nucleus of Daudi cells,and the expression of NF-κB p65 in the nucleus was significantly increased.DG 10 μg/ml could significantly reduce the entry of NF-κB p65 into the nucleus and the phosphorylation level of NF-κB p65.12.DG can reverse the effects of IgD on the levels of Bach2,Bcl6,IRF4,E2 A,Pax5 and Blimp-1 transcription factors in Daudi cellsq RT-PCR results showed that IgD 5 μg/ml could increase the transcription levels of Blimp-1,PAX5,E2 A and IRF4 in Daudi cells,while inhibit the transcription of Bach2 and Bcl6.DG 10 μg/ml could significantly inhibit the transcription of Blimp-1,PAX5,E2 A and IRF4 stimulated by IgD,and increase the transcription level of Bach2 and Bcl6.13.Deletion of IgαIgβ or Igβ can inhibit the activation of BCR signal by IgD in Ramos cellsWB results showed that Igβ knockout could inhibit the activation of BCR signal induced by IgD in Ramos cells.Compared with WT+IgD group,the phosphorylation levels of Syk,Btk,NF-κB p65 and p38 were significantly decreased in Ig α-/-Igβ-/-and Ig β-/-Ramos cells under the stimulation of IgD.14.Deletion of IgαIgβ or Igβ can reverse the effect of IgD on the level of transcription factors in Ramos cellsIn Ramos cells,IgD could inhibit the m RNA level of Bach2 and Bcl6 and promote the PAX5 transcription of E2 A and IRF4,but had no significant effect on BLIMP-1.The deletion of Igβ or Igα Igβ could reverse the increase of IRF4 under the stimulation of IgD.In addition,in Igβ-/-or Igα-/-Igβ-/-Ramos cell,the levels of Bcl6,E2 A,PAX5 and BLIMP-1 m RNA were significantly lower than those of WT.Conclusion:1.IgD can activate B cells in vitro by regulating IgD-IgDR-Syk-Btk signal pathway.2.DG fusion protein can inhibit the binding of IgD to mouse B cells,Daudi and Ramos cells in vitro.3.DG has significant therapeutic effect on CIA mice and regulates B cell subsets and functions in CIA mice.4.DG can inhibit the activation of IgD-IgDR-Syk-Btk signal pathway by inhibiting the binding of IgD and IgDR,and further inhibit the activation of B cells to play a therapeutic effect on CIA mice.5.DG may be a new biological agent for the treatment of RA,which has potential therapeutic effect in clinic. |
