HMGB1 Controls Peripheral B Cell Differentiation Via Regulating BCR Signaling And Actin Recombination

High-mobility group box-1(HMGB1)protein is a kind of non-histone nucleoprotein,which is widely express in eukaryotic cells and plays an important role in all kinds of life activities.The biological activity of HMGB1 varies with its location,environment and post-translational modification.When it is located in the nucleus,HMGB1 binds with the small groove on DNA,changes the spatial conformation,regulates the cells structure of chromatin,plays a part in DNA recombination,replication,transcriptional regulation and repair,and maintains the stability of chromatin.When the body is stimulated,the spatial position of HMGB1 changes and released from necrotic cells or activated immune cells into chemokines and pro-inflammatory cytokines.HMGB1,as a pro-inflammatory cytokine,is released extracellular and combined with the corresponding receptors to promote the inflammatory response,thus triggering a series of biological effects.After mature in the bone marrow,B lymphocytes migrate to the peripheral immune organs via blood,produce antibodies to destory pathogens,participate in immune regulation,and mediate humoral immune response.B cells bind antigen through B cell receptor(BCR),Iga and Igβ proteins strands transmit the first signal that recognizes the antigen,initiate the activation of downstream cascade signal molecules,and lead to the proliferation and differentiation of B cells.BCR mediated signal transduction is essential for B cell development and adaptive immunity.Research background:HMGB1 plays an important regulatory role in various cells such as neutrophils,dendritic cells,macrophages,lymphocytes and participate in the development of autoimmune diseases.At present,a lot of research advances on the role of HMGB 1 in lymphocytes have already been reported,but there are few related studies on the role of HMGB 1 in B cells,especially the regulation of BCR signal and its mechanism have not been reported.Objective:This study focused on the deletion of Hmgb1 gene in mice,aiming to study the effect of Hmgb1 deficiency on the development and differentiation of B lymphocytes,and the regulation of HMGB1 on BCR signaling and its mechanism.Finally,a model was established to detect the effect of Hmgb1 deficiency on t-cell-dependent immune response and the functional role which plays in Schistosoma Japonicum infection.Methods:A model was constructed in which Hmgb1 gene was conditionally knocked out in B cells.All mice were genetically identified to confirm the genotype.The age of 6-8w were selected,and the mice were divided into the control group(WT)and knockout group(KO)for the following experimental study according to the genotype results.Using flow cytometry to detect the effects of Hmgbl deficiency in bone marrow,peripheral B cells subsets and B1 cells,and compare the expression in two groups in total B cells and other subsets by calculating the mean fluorescence intensity(MFI).After statistics analysis,differential molecules were found for correlation study.Determine whether HMGB1 participates in BCR activation,explore the effects of Hmgbl deficiency on the activation of BCR proximal signal molecules and the expression of molecules after activation,studies were investigated by confocal analysis and western blot.According to the previous experimental results,explore the effect of Hmgb1 deletion on BCR metabolic signaling molecules was detected.To study the effect of Hmgb1 deficiency on actin cytoskeleton.To study the effect of Hmgb1 deficiency on the expansion of B cells and BCR cluster.To study the changes of partial nuclear transcription factors in B cells of Hmgb1 deficiency.To study the role of HMGB1 in humoral immunity.Results:(1)HMGB1 is not necessary for the development of bone marrow B cells,but it plays an important role in the differentiation of peripheral B cells and innate immunity.Compared with WT mice,there was no significant difference in the proportion,cell number,and expression of CD127 and CD22 in six subsets of bone marrow B cells in Hmgb1 KO mice.Compared with WT mice,there was no significant difference in the proportion and number of FO,T1 and GC B subsets in spleen B cells of KO mice.However,the proportion of T2 B cells in KO mice was significantly higher than that in WT mice,and the proportion and number of MZ B cells were significantly higher than that in WT mice.The proportion and number of B1a cells in KO mice were significantly higher than that in WT mice.IgG deposition in glomeruli of KO mice was significantly higher than that of WT.(2)HMGB1 participates in BCR activation,down-regulates BCR signaling and negatively regulates molecules related to BCR metabolic signaling.It was observed under confocal microscope that HMGB1 was redistributed on the plasma membrane and co-located with the BCR cluster with the activation of BCR.After antigen stimulation,the co-localization of pCD19,pY,pBTK,pSHIP and BCR in Hmgb1 KO B cells was higher than those in WT B cells,and the expression of pCD19/CD19,pY/actin,pBTK/BTK and pSHIP/SHIP in KO B cells was higher than that in WT B cells.After antigen stimulation,the expressions of pPI3K/PI3K,pAKT/AKT,pFOXO1/FOXO1,pS6/S6 and pmTOR/mTOR were also higher in KO B cells than in WT B cells.(3)Hmgb1 deficiency affects actin polymerization increases during B cell activation,which enhances B cell expansion and BCR clustering,positive BCR signaling.The aggregation of F-actin in Hmgb1 KO B cells were increased after antigen stimulation than in WT B cells,the colocalization between pWASP and BCR was increased.Phosphorylation flow detection showed that the expression of F-actin and pWASP in KO B cells was higher than that in WT B cells after antigen stimulation.The expression levels of proteins in KO B cells were higher than that in WT B cells after antigen stimulation by WB detection of pWASP,WASP,pEZRIN,WIP and DOCK8.It was observed under total reflection fluorescence microscope that KO B cells had higher degree of expansion and BCR aggregation than WT B cells,and the signals of f-actin,pWASP,pBTK,pY and pSHIP increased.(4)Hmgb1 deficiency affects the activation of transcription factor NF-kappaB,STAT1 and STAT5.Confocal microscopy showed that after antigen stimulation of HMGB1 KO B cells,the colocalization between pSTAT1 and BCR was significantly reduced at 5min,while the colocalization between pSTAT1,pSTAT5 and HMGB1 was still highly maintained after activation.The co-localization between pSTAT1,pSTAT5 and HMGB1 was also significantly increased at 5min and decreased at 30min.The expression of pSTAT1 was decreased and the expression of pSTAT5 was increased by WB.(5)The humoral immune response of Hmgb1 KO mice was decreased.After immunization,the proportion and number of FO in B cells of HMGB1 KO mice were significantly lower than those of WT,and the percentage of T1 was also reduced,and the number of FO,T1,T2 and MZ cells were all significantly increased.The percentage and number of GC B cells in KO mice were significantly lower than that in WT.The ratio of MBC in KO mice significantly decreased after immunization,and the number of Switched cells also significantly decreased.No significant difference was found between PC and PBC.Conclusion:HMGB1 participates in the activation of BCR and affects the humoral immune response.HMGB1 influences the actin recombination in the activation process of B cells,then influences the amplification of B cells and the aggregation of BCR,which regulates the signal transmission of BCR and negatively regulates the signal molecules of BCR.Finally,affecting the differentiation of peripheral B cells.