The Mechanism Of TLRs-induced CD1d Expression And Sphingolipid Metabolism Of B Cell In SLE Progression

Systemic lupus erythematosus(SLE)is a typical human autoimmune disease,which may induce body system and the multiple organ damage,and leads to significant morbidity and mortality.It is generally recognized that both genetic factor and environmental factor contributed to the development of SLE,although the exact mechanism is still unknown.Numerous studies have been demonstrated that B cell could not only contribute to the pathogenesis of SLE by secreting auto-antibodies directly,but also participate in SLE by cytokines secretion or severed as a antigen presentation cell,which indicated a key role of B cell disorder in the onset and development of SLE.However,B cell targeted therapy for SLE still has not achieved the desired effect.Notably,recently it has been found that B cell not play a pathogenic role in the pathogenesis of SLE,but also have a protective effect by its immunoregulatory function.But whether the unsatisfactory effect of B cell targeted therapy attributes to the different role of B cells in specific pathological condition of SLE still have no question.Thus further study of B cell in the pathogenesis of SLE still need to do,and the research will provide effective theoretical basis for clinical treatment.The recent studies shown that CD1d mediated the immunoregulatory function of B cell.B cell could recognize lipid antigen by CD1d and crosstalk with iNKT cell,which was responsible for the autoimmune diseases.Although the defect of CD1d expression on B cell was related to the pathogenesis of SLE,but which factor induced the defective expression of CD1d on B cell under SLE condition has not reported.Moreover,the lipid antigen is essential for the immunoregulatory function of B cell mediated by CD ld,and it has been reported a direct correlation of lipid and SLE progression.But whether the lipid metabolism is changed and what is the associated mechnicam in B cell under SLE condition are still under question.To analyze these questions,the paper conducted the following study.1.TLR9-induced miR-155 and Etsl expression contributed to the decreased level of CD Id on B cells under SLE condition.Firstly,we analyzed the expression of CD1d on B cells from B6.MRL-lpr SLE model mice.And the result showed decreased level of CD1d on B cells in SLE mice,which was consistent with the results from SLE patients.The activation of TLR signaling pathway was responsible for SLE,and our previous results demonstrated that TLR signaling pathway could regulate SLE through B cells.So,we stimulated mouse B cells with TLR9 agonist in vitro,and the results showed the activation of TLR9 signaling pathway could decrease CD1d expression significantly.To analyze the mechanism underling this phenomenon,we detected the miRNA expression and CD1d level in TLR9-induced B cells and in human B cell lines with different EBV background according to the the reported disorder of microRNA(miRNA)in SLE,and we found that miR-155 correlated with the level of CD1d.The subsequent bioinformatics technology and associated assays confirmed that miR-155 could inhibit CD1d expression directly.More importantly,the inhibitory effects of miR-155 on CD1d expression impaired its antigen-presenting capacity to iNKT cells,we found decreased activation of iNKT cells and less IFN-y secretion.Moreover,we found Etsl,one of the susceptibility genes of SLE,also regulated the level of CD1d after TLR9 activation.Above all,theses resulsts demonstrated the defect of CD1d on B cells in SLE was responsible for TLR9-induced miR-155 and Ets-1 expression.2.The defect of CDld on B cells was responsible for the excessive activation of B cells in SLE.To clarify the effect of CD1d disorder on B cells,we analyzed the level of CD1d and the activation state of B cells from B6.MRL-lpr mice of four staged(6 weeks,16 weeks,21 weeks and 26 weeks),and the results showed decreased expression of CD1d at early stage of SLE mice,but the up-regulation of CD86 was at a relative late stage.To confirm this phenomenon further,we also treated the B6 mice with imiquimod(IMQ),a TLR7 agonist,for 7 weeks,10 weeks and 13 weeks separately,and the results in PBMC and splenic B cells showed early decreased CD1d level and subsequent increased CD86 expression,too.And we found a significant negative correlation of CD1d and CD86 on B cells,but not on DCs.Moreover,to confirm this correlation,we analyzed B cells from healthy human beings and the negative correlation was also found,but not found on B cells form SLE patients.Lastly,our in vitro assay demonstrated that the ligation of CD1d could inhibit TLR7-induced CD86 expression.So,our results demonstrated that the defect of CD1d on B cells was responsible for the excessive activation of B cells in SLE.3.TLR singnaling pathway induced sphingolipid metabolism defect,which enhances inflammation response and severe SLE condition.It has been confirmed that the lipid antigen is essential for the immunoregulatory function of B cell mediated by CD1d,and it has been reported a direct correlation of lipid and SLE progression.To find whether the lipid metabolism was changed in B cells under SLE condition,we re-analyzed our B cell gene microarray of SLE patients.The result showed that the genes,such as sphingomyelin phosphodiesterase 3(SMPD3),associated with sphingolipid metabolism expressed differently in SLE B cells,which suggested the disorder of sphingolipid metabolism in B cells under SLE condition.Then,we confirmed the gene expression in B cells from SLE mice.What's more,we found that TLR signaling pathway could regulate these gene expression in B cells and macrophage cell line,and the defect of SMPD3 could enhance TLR-induced inflammation response in turn,which induced more pro-inflammation cytokines secretion,but supressed IL-10 expression.What's more,we also found TLR signaling pathway could regulate the transportation of SMPD3 and the ceramide level,all this could promote TLR-induced inflammation response.The above results demonstrated the disorder of sphingolipid metabolism in SLE patients and in SLE model mice,and we also found TLR signaling pathway contributed to the defect,which enhanced TLR-induced inflammation response in turn.Finally,all this was responsible for the development of SLE.In conclusion,(1)we found the defect expression of CD1d on B cells in SLE model mice,and TLR9-induced miR-155 and Etsl expression was responsible for this defect;(2)The decreased expression of CD1d on B cells was found at a early stage of SLE mice,and the down-regulation could contribute to over expression of CD86 on B cells;(3)The sphingolipid metabolism was defect in B cells under SLE condition,and TLR signaling pathway was responsible for this disorder,which enhanced TLR-induced inflammation response in turn.