| Background and purposes:Systemic lupus erythematosus(SLE)is a highly heterogeneous autoimmune disease characterized by autoantibody production.The production of multiple autoantibodies is a central part of SLE pathogenesis.Due to defective peripheral immune tolerance,B cells in SLE patients become hyperactive and eventually differentiate into auto-reactive plasma cells that produce large amounts of autoantibodies.However,the exact mechanism of B cell hyperactivity in SLE patients is not well understood.The activation and terminal differentiation of B cells after antigenic stimulation is an irreversible process.During this process,naive B cells gradually activate and finally differentiate into antibody secreting cells(ASCs)with specific effects,while exiting the cell cycle and losing the ability to divide cells,accompanied by the shutdown of the B cell transcriptional program and the opening of the ASC transcriptional program.The transcription factors that have been shown to maintain B cell identity include Pax5,Bcl6,Bach2,etc.m6A is the most abundant reversible post-transcriptional modification within eukaryotic mRNAs,occurring primarily in the 3’-UTR and 5’-UTR regions,and regulates mRNA stability,editing and translation,and thus regulating gene expression.Numerous studies have shown that m6A is mainly regulated by dynamic modifications of methylesterases(writers)such as METTL3,METTL14,WTAP,etc.,demethylases(erasers)such as FTO,ALKBH5 and readers such as YTHDF1 and YTHDF2.The m6A modification is essential in the development of B cells in the bone marrow and in the response of the germinal center.METTL3 and METTL14 are key factors in the maintenance of the germinal center.However,the role of m6A modification in B cell activation and terminal differentiation and whether they are involved in the pathogenesis of SLE are still unknown and need to be further investigated.Recent studies have shown that the expression of the demethylase ALKBH5decreased in the peripheral blood of SLE patients and was negatively correlated with anti-ds DNA antibody titers.METTL3 expression was decreased in peripheral blood CD4~+T cells of SLE patients,and METTL3 inhibition exacerbated symptoms in chronic graft versus host disease(c GVHD)lupus mice.However,the mechanisms by which METTL3 mediates B cell activation and terminal differentiation and participates in the defective B cell immune tolerance in SLE are unclear.Based on the above research background,we propose the following research objectives for this study:(1)To clarify whether METTL3 and m6A modification are involved in B cell activation and terminal differentiation.(2)To investigate the effect of METTL3 on B cell/ASC transcription factors during B cell activation and terminal differentiation.(3)To elucidate the role of m6A modification and METTL3 in B cells of SLE patients and lupus mouse models.Methods:(1)The m6A modification is involved in B cell activation and terminal differentiation.Firstly,we screened the m6A modification-related genes for possible changes during B cell activation and terminal differentiation by bioinformatics analysis.Then B cell activation and terminal differentiation were induced in vivo and in vitro by tail vein injection(2.5 mg/kg)or stimulation(20 ug/ml)with LPS.The m6A levels and changes of m6A modification related enzymes were detected by m6A dot blot,real-time quantitative polymerase chain reaction(RT-qPCR)and western blotting(WB).(2)Effect of overexpression or inhibition of METTL3 on B cell maturation and differentiation in mice.C57BL/6J mice were given tail vein injection of overexpressed AAV-METTL3 adeno-associated virus or intraperitoneal injection of METTL3 inhibitor STM2457(50 mg/kg),and the proportions of pre-B cells in bone marrow and B cell subsets in spleen of mice were analyzed by flow analysis.Then the activation and terminal differentiation of B cells in mice were induced in vivo by using LPS tail vein(2.5 mg/kg)injection,and the ratio of activated B cells and ASCs in spleen was analyzed by flow cytometry.The level of Ig M in mouse serum was detected by ELISA.(3)METTL3 interacts with the transcription factor PAX5 to maintain B cell identity.First,a bioinformatics analysis was performed on genes with altered expression and related pathways after B cell specific knockout of Mettl3.Then si RNA was used to knock down METTL3 or PAX5 in Raji cells,and changes in PAX5 or METTL3were detected by RT-qPCR,WB and flow cytometry.The possible sites of METTL3 promoter binding to PAX5 were predicted in the JARSPAR database,and whether PAX5 binds to the promoter of METTL3 in vitro was clarified using gel electrophoretic mobility shift assay(EMSA).A dual luciferase reporter gene assay was further used to explore whether PAX5 binds to the promoter-specific locus of the METTL3 gene and affects its promoter transcriptional activity.The results of m6A binding protein immunoprecipitation(RNA immunoprecipitation,RIP)sequencing of wild-type and knockdown METTL3 in GSE223728database HG3 cells(B lymphoma cell line)were analyzed by bioinformatics to determine whether the m6A site on PAX5 mRNA was changed.The METTL3-RIP assay was used to clarify whether METTL3 could bind to PAX5 mRNA,and then actinomycin D assay was used to explore whether METTL3 had an effect on the stability of PAX5 mRNA.(4)Changes of m6A levels and METTL3 in B cells of lupus mice and SLE patients.The lupus mouse model was established by intraperitoneal injection of pristane,and METTL3 levels in the spleen,bone marrow and lymph nodes of the lupus mice were detected by flow cytometry.Magnetic beads were used to sort mouse spleen B cells.m6A levels and changes of m6A modification related enzymes in spleen B cells of lupus mice were detected by m6A dot blot,RT-qPCR and WB.By collecting peripheral blood from patients with naive SLE patients and healthy controls,CD19~+B cells were sorted by magnetic beads.m6A dot blot was performed to detect the changes in the overall level of m6A in peripheral blood B cells of SLE patients,and METTL3expression changes in B cells and plasma cells of SLE patients were detected by flow cytometry.Finally,the clinical data of SLE patients were collected,and the correlation between the expression level of METTL3 in B cells of SLE patients and SLEDAI score,complement C3,anti-C1q levels,inflammatory factors and other clinical information was analyzed.Results:(1)By analyzing the differential expression of m6A related enzyme genes in GSE71698,a transcriptome dataset of B cell subsets,Mettl3 was found to be increased in activated B cells and decreased in pre-plasmablast cells.The results of m6A dot blot showed that the overall level of m6A exhibited a dynamic change,first increasing and then decreasing during the activation and terminal differentiation of B cells in vivo and in vitro.RT-qPCR and WB results showed that METTL3 expression was increased during early activation of B cells,and decreased in late activation and the final differentiation process.(2)After overexpression of METTL3 in mice,flow cytometry results showed that the proportion of pre-B cells in the bone marrow of mice increased,and the proportion of marginal zone B cells(MZBs)in the spleen decreased.After the activation of B cells in mice induced by LPS,the proportion of ASCs in the spleen decreased,and ELISA results showed a decrease in serum Ig M titers.After the mice were treated with the METTL3 inhibitor,flow cytometry results showed that the proportion of pre-B cells in the bone marrow decreased,and the proportion of MZBs in the spleen increased.After the activation of B cells in the mice induced by LPS,the proportion of ASCs in the spleen increased,and ELISA results showed an increase in serum Ig M titers.(3)By performing differentially expressed gene analysis on the dataset GSE180359 with B cell specific knockout of Mettl3,it was found that Mettl3 knockout in B cells of the germinal center leads to decreased expression of B cell transcription factor genes such as PAX5and affects a series of pathways such as B cell proliferation,homeostasis and activation.After knocking down METTL3 in Raji cells,RT-qPCR,WB and flow cytometry results showed decreased PAX5expression.By analyzing the m6A RIP sequencing results of wild-type HG3 cells(B lymphoma cell line)and METTL3 knockout in GSE223728 database,the results showed that the m6A peak on PAX5mRNA was significantly decreased after METTL3 knockout.METTL3RIP assay showed that METTL3 was able to bind to PAX5 mRNA,and actinomycin D assay showed that METTL3 regulated PAX5 mRNA expression by promoting its stability.After knocking down PAX5 in Raji cells,RT-qPCR,WB and flow cytometry results showed decreased METTL3 expression.There were three sites predicted in the JARSPAR database where the METTL3 promoter might bind to PAX5,and the highest scoring binding site(-347～-365 bp)was selected for EMSA and dual luciferase reporter gene experiments,which exhibited that PAX5 directly binds to the promoter of METTL3 and initiates its transcriptional activity.(4)The lupus mouse model was successfully established by intraperitoneal injection of pristane.Results from m6A dot blot showed increased m6A levels in spleen B cells in mice with lupus compared to control mice.RT-qPCR,WB and flow cytometry showed that METTL3was up-regulated in B cells of spleen,bone marrow and lymph node of lupus mice.(5)Peripheral blood samples were collected from 90 newly diagnosed SLE patients and 59 healthy controls.CD19~+B cells were sorted by magnetic beads.m6A dot blot analysis showed an increase in the overall level of m6A in peripheral B cells of SLE patients.Flow cytometry showed that the expression of METTL3 in B cells of SLE patients was increased compared with healthy controls,and was positively correlated with SLEDAI score,anti-C1q antibody and TNFαlevels,while the expression of METTL3 in plasma cells of SLE patients was unchanged.Conclusions:(1)During the activation and terminal differentiation of B cells induced by LPS,the overall levels of m6A and METLL3 expression first increase and then decrease.(2)METTL3 promotes the development of bone marrow pre-B cells and maintains the homeostasis of splenic B cell subsets in mice.(3)Overexpression of METTL3 preserves B cell identity and reduces the production of ASCs.Inhibition of METTL3 promotes terminal differentiation of B cells into ASCs and antibody secretion.(4)METTL3 binds to PAX5 mRNA and promotes the stability of PAX5 mRNA to up-regulate its expression.PAX5 directly binds to the promoter of METTL3 and up-regulates its expression,thereby maintaining B cell identity.(5)The expression of METTL3 is elevated in the B cells of lupus mice and patients,and positively correlates with disease activity in SLE patients.Figures 41,tables 25,references 125... |
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