Study On The Expression Of Blimp1and Its Regulation On BCMA In Systemic Lupus Erythematosus

Systemic lupus erythematosus(SLE) is a chronic autoimmune disease with a wide spectrum of clinical and immunological abnormalities.It is characterized by overactivation of autoreactive B lymphocytes,the presence of various autoantibodies and circulating immune complexes deposit in tissues and organs.Both the accurate etiology and the pathogenesis of SLE are still unexplained. Autoantibodies are thought to have a primary role in the pathogenesis of SLE.Drugs that have demonstrated efficacy in lupus include hormone and non-specific immune inhibitors,such as glucocorticoid and cyclophosphamide and so on.However, although these pharmacological agents have a role to play in some patients with lupus, broad based effects have led to problems with side effects and adverse reactions.Because of autoreactive lymphocytes permanent after the medication,it is difficult to cure the SLE.This finding implies that self-reactive B cells are playing important pathogenic roles in autoimmune disorders beyond the production of autoantibodies.Therefore, elimination of B cells may represent a new therapy for SLE.In recent years the B lymphocytes targeted therapy SLE made a lot of progress,however, because of plasma cells is the direct source of the pathogenic autoantibodies, only split B cells can’t remove the fundamental source of autoantibodies, resulting in patipatient’s condition recurrent.Depletion of B cells with rituximab or other agents will have no effect on autoantibodies produced by long-lived plasma cells,while40%secretory autoantibodies plasma cells is the long-life plasma cells.It is produce antibodies canbe activated again potential pathological immune response.Antibodies specific for self-antigens mediate lifethreatening pathology in several autoimmune diseases.Clearly the ability to target the plasma cells (PCs)producing the autoantibodies would be of great clinical benefit. Visible,clearly the ability to target the plasma cells producing the autoantibodies would be of great clinical benefit of SLE.Blimpl(B-lymphocyte-induced maturation proteinl), a98-Kd transcriptional factor that contains five zinc finger motifs, has long been considered a master regulator of the terminal differentiation of B cells into antibody-secreting plasma cells.Blimpl is required for the formation of plasma cells and for the maintenance of nondividing, long-lived plasma cells in the bone marrow. Blimpl is also required for long-term maintenance of antigen-specific immunoglobulin in serum.At present,Blimpl maintain plasma cells alive and antibody secretion of the mechanism is not clear. Blimpl, conversely, is upregulated during plasma cell development and is implicated in playing a role in plasma cell survival.Immunofluorescent staining and flow cytometry analysis showed that inhibition of Blimpl expression in J558L cells is accompanied with reduced expression of CD138(Syndecan-1) and reappearance of CD19.The XBP-1,J-chain,BCMA gene expression was decreased and c-myc gene expression was elevated following knockdown of Blimpl expression in J558L cells as shown by semi-quantitative RT-PCR.The results indicated that knockdown of Blimpl expression could induce reprogramming and dedifferentiation of plasma cells and reactivation of the B cell transcriptional program.B-cell maturation antigen (BCMA),a type I transmembrane receptor and belongs to the tumor necrosis factor receptor family (TNFR),binds to BAFF and APRIL.BCMA is expressed on the mature B and plasma cell surfaces. Recent studies have underscored the importance of BLyS and APRIL as factors that can support the survival of B lineage lymphocytes.The BCMA gene is only expreesed preferentially in mature B lymphocytes,which encodes a184amino acid preptide, containing a80 amino acid intracellular domain. However, little is known about its function and how the expression of BCMA is regulated. The function of the third receptor,BCMA, which is primarily restricted to the B cell lineage, remains unresolved.A comprehensive analysis of BCMA mice have shown that loss of the BCMA receptor does not impact on the generation of mature peripheral B cells, alter the quality or magnitude of TI and T cell-dependent (TD) humoral immune responses, nor alter germinal center (GC) formation or the generation of short-lived PCs. Thus, previous analyses have not yet identified a function for the BCMA receptor.BCMA is a critical factor of the survival of plasma cells as significant short life-span of plasma cells are observed in BCMA-/-mice. BAFF can bind to BCMA and upregulated the expression of apoptin gene such as Bcl-2, Bclw,Mcl-1, supporting the survival of plasma cells.Our previous study has shown that the BCMA and Blimpl genes are expressed in mature B cells and plasma cells and Blimpl is an essential regulator of the development and survival of plasma cells in the mouse bone marrow. Apparently, both BCMA and Blimpl are involved in the regulation of activated B cell differentiation and plasma cell development and survival. However, whether Blimpl can regulate the expression of BCMA is unknown.Analyse B cell growth process of gene expression patterns,expression increase gradually along with the development of mature B cells, and only present in the mature B lymphocytes and plasma cells stage in order to maintain plasma cells survival.Previous studies have demonstrated that Blimpl can directly target to several genes, such as c-myc, CIITA, Pax-5, Spi-B, Id3and others and Blimpl can bind to the consensus sequence of "NNNNGAAAGNN" in these genes. Analysis of the DNA sequences in the BCMA promoter region revealed that two potential Blimpl binding sequences were located at "-46to-36" and"-31to-21" from the potential TSS of the BCMA gene, respectively. These suggest that these sequences may be important for Blimpl to regulate the expression of BCMA in mice.the transcriptional activity of the BCMA promoter was activated by Blimpl in adose-dependent manner.The results indicated that Blimpl maybe a trans-activated factor of BCMA expression.The"CTTAGAAACGG"located in-36to-46upstream of BCMA gene was probably the binding site of Blimp1.The regulation of BCMA by Blimpl may play an important role in maintain plasma survival.so we suggest that BCMA expression was directly regulated by transcription factor Blimpl.If this happens in human,treatment with the blockage of Blimpl expression may inhibit the development and survival of pathogenic plasma cells, reducing autoantibody production and inhibiting antibody-mediated autoimmune diseases。Objective1、 To indentify the role of Blimpl in the regulation of BCMA gene and to indentify the transcription and relationship between Blimpl and BCMA.2、 To indentify the expression of Blimpl gene in SLE and provides a new of thinking for the development of targeted plasma cell therapy.Methods1、 Blimpl regulation on BCMA1) EMSAThe binding feature of Blimpl to the specific sequence of the BCMA promoter was determined by the competitive EMSA.Nuclear proteins were extracted from unmanipulated J558L cells using the specific reagents, according to the manufacturer’s instructions.Combining product for the degeneration PAGE electrophoresis.2) CHIPCollected cell and treated it with1%formaldehyde.Cross-linked chromatin was prepared from cells and sonieated to an average length of200-1000nucleotides, precleaned with salmon sperm DNA Protein G Agarose beads,and immunoprecipitated with normalantibody.Then Collected antibody-protein complex.PCR amplifieated precipitated chromosome.2、 RT-PCR:Extraction of spleen and lymph nodes from MRL/lpr mouse group and normal group, then extraction of total RNA, reverse transcription into cDNA, doing real-time fluorescence quantitative PCR finally.To Determine thespecificity of PCR product through the melting curve。Take an average of two Ct values2-ΔΔCT,as the specimen relative mRNA expression levels.compration the Blimpl gene expression differences of MRL/lpr mouse groupand normal group.3、The statistical data was analyzed by SPSS13.0. Datas were reported as Medians. Because the data do not meet the normal distribution, thus we used the non-parametric test (Mann-Whitney Test and Wilcoxon test). The significance level setted at P<0.05.Result1、 EMSA showed that Blimpl recognized the "gaaag" motiflocated within"-38--42"bp of the BCMA promoter. ChIP revealed in vivo Blimpl binded to the BCMA promoter in the J558L.2、 The Blimpl mRNA expression level in spleen of MRL/lpr mouse group significantly higher than the normal control group (Z=-2.609,P=0.009).The Blimpl mRNA expression in Lymph node of MRL/lpr mouse group were obviously increased compared with the control groups (Z=-3.402,P=0.001).ConclusionThe expression of Blimpl mRNA in higher than the control group. So the Blimpl play a very important role in the development of SLE.Treatment with the blockage of Blimpl expression may inhibit the development and survival of pathogenic plasma cells, reducing autoantibody production and inhibiting antibody-mediated autoimmune diseases,which has establish the foundation for the development of new drug.Blimpl might be new target for targeted plasma cell therapy of SLE.