The Role And Molecular Mechanism Of E3 Ubiquitin Ligase Grail In B Lymphocytes Activation And Immune Tolerance

Background and Object B lymphocytes will produce autoantibodies against host cells during development,differentiation.And the long-standing reactive B lymphocytes in the body,will cause B lymphocytes immune interruption.While long-term activation of B lymphocytes are closely associated with the occurrence of autoimmune diseases.In order to prevent the activation of autoreactive B lymphocytes and the eventual immune response,the immune system can recognize autoantigens through BCR,elite autoreactive B lymphocytes by cloning clearance,edit autoreactive BCR receptors,or mediate B lymphocytes anergy during central and peripheral immune tolerance.However,the mechanism leading to autoreactive B lymphocytes is still unclear,which is an important problem to be solved urgently.Studies have shown the balance between ubiquitination and deubiquitination is an important mechanism to maintain T lymphocytes anergy and prevent autoimmunity.E3 ubiquitin ligase members have been identified as important molecules mediating T lymphocytes anergy.GRAIL（Gene Related Anergy In Lymphocyte,RNF128）,also known as RNF128,a novel ubiquitin protein ligase.Grail can inhibit the differentiation,proliferation and activation of naive CD4+ T lymphocytes,and inhibit the antigen presentation function that mediates CD4+ and CD8+T lymphocytes immune tolerance.Grail is closely related to the occurrence of many autoimmune diseases,such as autoimmune arthritis,ulcerative colitis,asthma,etc.However,the current understanding about the mechanism regulating B lymphocytes tolerance is limited,and no studies have been reported about Grail and B lymphocytes immune tolerance.Therefore,the purpose of this study is to determine whether Grail mediate B lymphocytes tolerance,to explore the specific mechanism of Grail-mediated B lymphocytes tolerance,and further to confirm the regulation of Grail on B lymphocytes activation and tolerance by establishing mice rheumatoid arthritis model.To create theoretical foundation for clarifying the pathogenesis of autoimmune diseases and exploring new therapeutic targets.Method 1.Analysis Grail expression in B lymphocytes.Western Blot was used to detect Grail protein expression in healthy donor and WT mice B lymphocytes.2.Analysis the effects of Grail on B lymphocytes development.Firstly,bone marrow chimeric mice μMT/TCRβ^-/-and μMT/TCRβ^-/- Grail^-/-were established,to compare B lymphocytes development in bone marrow and spleen.3.Analysis the effects of Grail on B lymphocytes activation.After activation of WT B lymphocytes by anti-IgM,anti-CD40 or IL4,Grail expression were analyzed.Flow cytometry was used to detect the expression of B lymphocytes surface activation markers.The bone marrow chimeric mice were immunized with DNP-Ficoll.Then collected B lymphocytes from spleen and lymph nodes on the 7th days after immunization to detect B lymphocytes number and expression of B lymphocytes surface activation markers.4.Analysis the effects of Grail on B lymphocytes antigen presentation function.Bone marrow chimeric mice were immunized with DNP-Ficoll and KLH/CFA to establish “T cell-independent” and “T cell-dependent” B lymphocytes activation models.The serum anti-DNP,antiKLH specific IgG and IgM,the formation of germinal centers and the proportion of B lymphocytes in the germinal centers were analyzed.T helper lymphocytes were differentiated in vitro,and the proportion of T helper lymphocytes subsets were detected by flow cytometry.5.Analysis the correlation between Grail and B lymphocytes anergy.Grail expression were analyzed by Western Blot and q RT-PCR in transgenic Ars/A1 and WT mice.Grail gene was knocked out in anergic IgHELs HEL mice,the proportion of immature B lymphocytes to follicular B lymphocytes was compared.The serum anti-HEL specific IgM was detected by ELISA to determine the changes of B lymphocytes effector function.6.Analysis the effects of Grail on B lymphocytes proliferation,activation and survival.WT and Grail KO mice were activated by antiIgM,IL-4 and anti-CD40.The proliferation of B lymphocytes was detected by 3H-Thymidine adulteration.Analysis the expression of B lymphocytes surface activation markers and B lymphocytes survival.7.Analysis the regulation of Grail on BCR signaling pathway.B lymphocytes of WT and Grail KO mice were activated,protein expression of major BCR signaling molecules and phosphorylated protein expression were detected by Western Blot,and anti-Ub（K48）ubiquitination analysis was performed to clarify the molecular mechanism of Grail regulating BCR signaling pathway.8.Analysis the molecular pathway regulating Grail expression.mTOR signaling pathway was inhibited by mTOR signaling inhibitors and PTEN inducers,the expression of Grail protein and downstream molecule S6 phosphorylated protein of mTOR pathway in B lymphocytes of WT mice and healthy donor were analyzed.B lymphocytes of WT and Grail KO mice were activated to detect the mTOR,Akt protein and phosphorylated protein.At the same time,PTEN expression and ubiquitinated protein were also be detected.9.A rheumatoid arthritis mice model was established to confirm Grail mediate B lymphocytes immune tolerance.CII/CFA was used to induce WT and Grail KO mice to develop rheumatoid arthritis.Digital arthritis score was performed,and analysis B lymphocyte subsets,serum antigen specific IgG,IgM and IgG2 c.Results 1.Grail expressed in WT mice and human B lymphocytes.And Grail protein expression increased after B lymphocytes activation.2.Grail didn’t affect B lymphocytes development.There was no difference in the proportion of B lymphocytes subsets,such as Immature,mature,Pro-B,pre-B,transitional B（T1,T2,T3）,marginal zone and follicular B lymphocytes in bone marrow and spleen between Grail KO and WT mice.3.Grail inhibited B lymphocytes activation.Flow cytometry analysis showed that the expression of surface activation markers,CD80,CD86 and CD40 in Grail KO B lymphocytes in resting and activated states were higher than those in WT mice（P<0.01）,suggesting that Grail could inhibit B lymphocytes activation.4.Grail inhibited B lymphocytes antigen presentation function.After DNP-Ficoll and KLH/CFA immunization,Grail KO mice produced more antigen specific IgG and IgM,with increased germinal center B lymphocytes than WT mice（P<0.01）.In vitro,Grail KO mice B lymphocytes promoted TH1,TH2,TH17,and TFH lymphocytes differentiation（P<0.01）.Both in vivo and in vitro studies confirmed Grail inhibited B lymphocytes antigen presentation.5.Grail was associated with B lymphocytes anergy.Western Blot showed that anergic T3 B lymphocytes increased in transgenic Ars/A1 mice,with increased Grail expression.Grail mRNA also increased in anergic T3 B lymphocytes.This results showed that Grail was related to B lymphocytes anergy.After Grail gene was knocked out in transginic IgHELs HEL mice,the proportion of FO B lymphocytes increased.In addition,Grail KO mice produced more antigen specific IgM（P<0.01）,suggesting that Grail expression break B lymphocytes anergy and restore B lymphocytes effector function.6.Grail inhibited B lymphocytes proliferation and activation through BCR signaling pathway.After anti-IgM activation,B lymphocytes proliferation of Grail KO mice was stronger than WT mice,while there was no difference in B lymphocytes proliferation activity between Grail KO mice and WT mice after IL-4 and anti-CD40 activation.B lymphocytes activation markers,CD80（P<0.01）,CD86（P<0.01）,ICOSL（P<0.05）expression increased in Grail KO mice.There was no difference in survival analysis,suggesting that the enhanced B lymphocytes proliferation in Grail KO mice was not related to survival.Grail regulates B lymphocytes proliferation and activation through BCR signaling pathway.7.Grail ubiquitinate major BCR signaling molecules,mediating B lymphocytes tolerance.Western Blot showed major BCR signaling molecules expression in Grail KO mice were increased compared with WT mice,including Igα,PLcγ2,NFATC1,PI3 k,AKt and mTOR protein,the expressions of Igα,AKt,PLcγ2 and PI3 k phosphorylated protein were also increased.Ubiquitination analysis showed that the dispersion bands of Igα,Akt,PI3 K and PLcγ2 in Grail KO mice were weaker than those in WT mice,suggesting that Grail inhibit B lymphocytes activation and effector function,mediate immune tolerance by ubiquitination major BCR signaling molecules Igα,Akt,PI3 K,and PLcγ2.8.PTEN,mTOR signaling pathways regulated Grail expression.After Rapamycin treatment,expression of Grail protein increased in B lymphocytes from WT mice and healthy human compared with control groups,and the downstream phosphorylated protein p S6 was decreased in healthy human but not detected in WT mice,suggesting that Grail protein expression was negatively correlated with mTOR signaling pathway.After Raji cells were knocked out of PTEN and treated with PTEN inducer Doxycycline,Grail protein expression increased after induced PTEN expression compared with the control group,the expression increased with the extension of incubation.However,the expression of downstream phosphorylated protein p S6 in mTOR signaling pathway was significantly decreased.In addition,Grail KO mice showed decreased expression of PTEN protein and increased expression of Akt and mTOR phosphorylated proteins in B lymphocytes.It was confirmed that PTEN inhibited mTOR signaling pathway to promote Grail expression,Grail and PTEN might have a synergistic effect.However,there was no significant difference between Grail KO and WT mice in the diffusion bands of PTEN ubiquitinated protein K48,the specific synergistic mechanism between them should be further explored.9.Grail mediated B lymphocytes tolerance in vivo and reduce the risk of autoimmune diseases.Compared with WT mice,Grail KO mice with more severe arthritis,increased percentages of germinal center B lymphocytes and TFH cells（P<0.01）,decreased percentages of Treg cells（P<0.01）,and increased serum IgM,IgG,and IgG2 c.It was confirmed that Grail reduce the occurrence of autoimmune diseases by mediating B lymphocytes immune tolerance.Conclusion 1.E3 ubiquitin ligase Grail express in mice and human B lympho cytes.Grail doesn’t affect B lymphocytes development,but inhibites B lymphocytes activation and antigen presentation.2.Grail is associated with B lymphocytes anergy.Grail expression deficiency will break B lymphocytes anergy and promote B lymphocytes function recover.3.Grail negatively regulates BCR signaling pathway,inhibiting B Lymphocytes activation and effector function,mediating B lymphocytes immune tolerance through ubiquitination major BCR signaling molecules,such as Igα,Akt,PI3 K,PLcγ2.In rheumatoid arthritis mice model,Grail has been shown to inhibit arthritis by mediating B lymphocytes tolerance.4.PTEN promotes Grail expression in B lymphocytes by negatively regulating AKT/mTOR signaling pathway.Grail may have a synergistic effect with PTEN,but the specific mechanism needs to be further explored.