Study On The Function Of Metabotropic Glutamate Receptor 3 In Systemic Lupus Erythematosus And Myeloma B Cells

B lymphocytes(referred to as B cells)are derived from bird bursa and mammalian bone marrow.Its development and functional activity are affected by various factors in the body such as anatomical site,microenvironment,cell and humoral immune system,etc.The immune response of B lymphocytes to pathogens under the physiological condition is that the body is resistant to the invasion of pathogen on important members of the immune system.However,B lymphocyte differentiation,development or dysfunction will lead to a variety of diseases such as systemic lupus erythematosus(SLE),thyrotoxicosis(Graves),myasthenia gravis(Sjogren Syndrome),Multiple Myeloma(MM),B lymphoma leukemia and so on.Therefore,the study of the influence of B lymphocyte differentiation,development and function will help understand the etiology of such diseases and provide a meaningful reference for clinical diagnosis and treatment.The etiology and course of disease of SLE are closely related to B lymphocyte dysfunction.In the clinic,targeted B-cell therapy is also carried out in addition to conventional treatments,such as belimumab which targets B cell activating factor(BAFF),effectively alleviates the symptoms of SLE and obtains a positive effect,but it has no significant effect on the recurrence of SLE.MM is that B lymphocytes abnormally differentiated into monoclonal malignant plasma cells and lead to a large number of monoclonal globulin secretion,causing a series of clinical diseases such as extensive bone destruction,recurrent infection,anemia,hypercalcemia,hyperviscosity,renal insufficiency and so on.Traditional chemotherapy drugs can alleviate the symptoms of MM and prolong survival time,but they also have high side effects.The targeted therapy in recent years is mainly for MM cell growth and survival in the bone marrow microenvironment,it still cannot fundamentally cure the disease.Many studies have been carried out on BAFF as B cell activation positive regulatory factor,but the study on the negative regulatory factor of B cell immune response is less.Our previous studies have shown that TACI-Ig G,a fused protein formed by a receptor molecule TACI of BAFF(interacting molecule of transmembrane activators and calmodulin ligands)and Ig G Fc,can effectively block BAFF effect;TACI-Ig G in the body can reduce the generation of plasma cells that secretes autoantibodies and reduce SLE mice autoimmune symptoms by effectively reducing the activation of B cells.In order to screen the negative regulatory factors of B cell activation,we used gene chip technology to analyze the changes of B cell gene expression in TACI-Ig G treated SLE mice.It was found that after blocking BAFF,the gene level of the metabolic glutamate receptor 3(Grm3)increased abnormally.Grm3 is an inhibitory molecule on the surface of mammalian cells and is a subtype of the metabotropic glutamate receptor group II,which is a G protein coupled receptor that inhibits adenylate cyclase system and reduces c AMP formation after being activated.We suggest that Grm3 be an inhibitory molecule on the surface of B cells and its abnormal level be related to the etiology and course of B lymphocyte dysfunction and B-cell abnormalities-associated diseases.We have carried out related exploratory studies intended to provide a new perspective and a new target in clinical treatment for SLE,MM and other diseases.Objective: To explore the expression of Grm3 in B cells of SLE and MM and its possible mechanism,so as to provide a new theoretical and experimental basis for Grm3 intervention in SLE and MM,and to provide a new potential target for its treatment.Methods and results: The correlation between expression level of Grm3 in B cells and SLE 1.Gene chip technique was used to screen B cell activation negative regulatory factor in TA cells treated with TACI-Ig G in SLE mice.It was found that the Grm3 gene level in B cells of SLE was abnormally increased after blocking BAFF.2.Further,at animal level,SLE model mice were treated with TACI-Ig G and the expression of Grm3 in B cells was detected by Real-time-PCR and WB method.The results showed that expression of Grm3 in B cells of SLE model mice was significantly decreased comparing with that of normal mice.The expression of Grm3 m RNA and protein was up-regulated in B cells after SCI model mice were treated with TACI-Ig G.3.Blood samples were collected from patients with clinical SLE and MS,the expression of Grm3 in peripheral blood B cells of patients with SLE and MS was detected by Real-time-PCR and WB.The results showed that the expression of Grm3 in SLE and MS patients was decreased.4.To determine whether Grm3 is specifically expressed in B cells,the Grm3 m RNA and protein expression in different cells such as macrophages,neutrophils,natural killer cells,T cells,dendritic cells and B cells were further detected by Realtime-PCR and WB,the results showed: Grm3 was mainly expressed in B cells,with a low expression in other immune cells.5.To further clarify the expression of Grm3 in B cells at different stages of developmental differentiation,the expression of Grm3 in naive B cells(CD19+ B220+ CD138-GL7-),activated B cells(GL7+ B220+)and plasma cells(CD138+ B220+)were detected by Real-time-PCR and WB,The results showed that: Grm3 was mainly expressed in the na?ve B cells,with a low expression in plasma cells and activated B cells.6.To further verify the relationship between Grm3 and B cell activation and differentiation into plasma cell,we used Blimp-1(the key transcription factors that determines plasma cell production)and Bcl6(the key transcription factor that determines the germ center B cell production)as lentivirus expression vector to infect the B cells of the mice,the results showed that: the overexpression of Blimp-1 and Bcl6 was able to inhibit the expression of Grm3.7.To study the effect of Grm3 on B cell proliferation,Grm3-sh RNA lentivirus transfection method was used to knock down Grm3 in B cells.The results showed that Grm3 knockdown B cells increased the response to LPS.8.To further investigate the effect of Grm3 on B cell proliferation,we detected the effect of Grm3 agonists on B cell proliferation.Cell proliferation analysis suggested that Grm3 agonist LY379268 was able to inhibit LPS-induced B cell proliferation,whereas Grm3 agonist LY379268 did not inhibit the increase of the response of Grm3 knockdown B cells to LPS.9.In order to verify the above conclusion again,the same method was used to detect the effect of Grm3 agonist LY 341495 and antagonist on B cells,and it was found that Grm3 agonist LY354740 could reduce the number of B cells while Grm3 antagonists increased the number of B cells.10.In order to detect the effect of Grm3 in SLE disease,after Grm3 agonists wereinjected into SLE mice,the concentration of c AMP and the titers of anti-ds-DNA antibody in the mice were detected by ELISA,the ratio of total B cells,GL7+ and Ig G+ B cells was detected by FACS and the urine protein was quantified in accordance with the BCA protein quantitative kit instructions,the results showed that: Grm3 agonists reduced the c AMP concentration,anti-ds-DNA antibody titers,the numbers of the total B cells,GL7+ and Ig G+ B cells,and the level of urine protein.In addition,the kidneys were sliced and stained with HE,it was found that SLE mice treated with agonists had no inflammatory cell infiltration in renal interstitium and their glomerular space were returned to normal.The above results suggest that Grm3 can effectively relieve SLE mice autoimmune symptoms.The correlation between the expression level of Grm3 and the apoptosis of myeloma cell line SP2/0 In our previous study,it has been demonstrated that Grm3 can be expressed on the surface of B cells and that targeting Grm3 is effective in the treatment of SLE mice.So what is the role of Grm3 in B cell-associated tumors such as multiple myeloma or Bcell lymphoma? To this end,we have taken two representative cell lines: Nalm-6(human pre-B lymphoblastic leukemia cell line)and SP2/0(mouse myeloma cell line),the former can be used as a cell model of B cells differentiation into plasma cells,the latter can be used as a cell model of abnormal plasma cells.Relevant research on these two cell lines was carried out,methods and results are given as follows: 1.The expression of Grm3 and CD138 in SP2/0 and Nalm-6 cell lines were detected by FACS.It was found that SP2/0 expressed CD138 and Nalm-6 did not express CD138,Grm3 was expressed in SP2/0 and Nalm-6,respectively,but Grm3 was not expressed in CD138+ SP2/0.Furthermore,Annexin V and PI were used to label apoptotic cells.The results showed that Grm3 was expressed on the surface of Annexin V+/PI+ Nalm-6 and SP2/0 cells,while the living cells did not express Grm3,Grm3 was mainly expressed in the medium phase of apoptosis.2.To investigate the effect of apoptosis on Grm3 expression,FK506 was first used to induce apoptosis.It was found that FK506 can effectively induce the apoptosis of SP2/0 cells by FACS,and the conclusion was further confirmed by Real-time-PCR and Western Blot.FACS also found that FK506 induced SP2/0 cell apoptosis in a dosedependent manner.In human embryonic kidney cell 293 T cells,FK506 did not induceGrm3 expression,which again confirmed our previous conclusion that Grm3 is mainly expressed in B cells.In addition,FACS found that anti-tumor drugs such as nocodazole and monastrol can effectively induce cell apoptosis and Grm3 expression.3.In order to verify the role of Grm3 in apoptosis,the effect of knockdown of Grm3 on apoptosis was observed in vitro.The expression of Grm3 in SP2/0 cells was knocked down by Grm3 sh RNA,and the knockdown of Grm3 could inhibit the apoptosis of SP2/0 cells by FACS,and could also reduce the apoptosis of SP2/0 cells induced by FK506,rapamycin,monastrol,nocodazole and other drugs.4.In vivo experiments to observe the effects of knockdown of Grm3 on the apoptosis,subcutaneous transplanted tumor method was used.Balb/c mice and nude mice were selected and injected with SP2/0 cells transfected with Grm3-sh RNA or control sh RNA,respectively.The results showed that Grm3 deletion could accelerate the development of tumor in Balb/c mice and nude mice.The volume of the tumor is larger than that of the control group.5.In order to further verify the role of Grm3 in apoptosis,Grm3 overexpression was used to observe the effect of Grm3 on apoptosis.The Grm3-expressing lentivirus and control lentivirus with EGFP were transferred into SP2/0 cells.The apoptosis of EGFP-labeled cells was analyzed by FACS.The results showed that overexpression of Grm3 could increase the apoptosis of cells.6.Previous studies have shown that Grm3 could inhibit c AMP signaling,and c AMP-PKA could phosphorylate Foxo1.In order to explore the effect of Foxo1 on cell apoptosis,liposome transfection method was used to overexpress Foxo1,FACS suggested that Foxo1 could lead to increase of SP2/0 apoptosis.To observe the effect of knockdown of Foxo1 on the apoptosis,Foxo1 sh RNA lentivirus transfection method was used to knock down the expression of Foxo1.FACS suggested that FK506 induced SP2/0 cell apoptosis was inhibited.Then we observed the effect of FK506-or Bortezomib-induced apoptosis in Foxol knockdown mice,and FACS suggested that FK506 or Bortezomib-induced apoptosis was inhibited after Foxo1 was knocked down.Conclusion: Based on the previous work,we first found that Grm3 is related to B cell differentiation and development.The expression of Grm3 in B cells of SLE and other autoimmune diseases was reduced,the symptoms of SLE in mice can be alleviated byactivating Grm3,the symptoms of SLE can be aggravated by increasing the number of B cells after antagonizing Grm3.Grm3 is also involved in the process of apoptosis of mouse myeloma cell line SP2/0 induced by anti-tumor drugs,it is mainly expressed in the middle stage of apoptosis,knockdown of Grm3 can inhibit cell apoptosis,Grm3 overexpression can aggravate cell apoptosis.Our results suggest that Grm3 as an inhibitory molecule suppresses the symptoms of autoimmune diseases by inhibiting the expansion of B cells in autoimmune diseases,as a regulatory molecule for B cell apoptosis it inhibits the formation of tumor of myeloma cell line SP2/0 in the body.This study provides a basis for the clinical validation of the effect of Grm3 in the abnormal differentiation of B cells of SLE and MM patients into plasma cells and provides a theoretical and experimental basis for Grm3 acting as the intervention target of SLE,MM and other related diseases.