The Effect Of Casitas B-lineage Lymphoma B On Regulating T Follicular Helper In Lupus Nephritis

ObjectiveThe purpose of this study is to investigate the regulatory mechanism of T follicular helper（Tfh）cells by Casitas B-lineage Lymphoma b（CBLB）and its potential role in the occurrence and development of lupus nephritis.Methods and Method1.Mice were divided into 4 groups（20/group）,WT,Cblb^-/-,B6-lpr and B6-lpr.Cblb^-/-mice.24h-proteinuria,serum creatinine,urea nitrogen,anti-ds DNA and ANA titer of mice were measured.PAS staining of kidney from mice were performed to observe the pathological variation.The Tfh（CD4⁺CXCR5⁺PD-1⁺）cells in peripheral blood and renal were analyzed by flow cytometry.Immunofluorescence staining was used to detect Ig G expression in kidney.Ig G levels in peripheral blood in mice were detected with ELISA.2.Tfh cells were isolated from peripheral blood of B6-lpr mice and B cells were isolated from peripheral blood of WT mice.The expression of CBLB in Tfh cells were regulated by transfecting si RNA and overexpression plasmid,and the relationship between the expression of CBLB and Ig G was observed.3.The sorted CD4⁺T cells from WT mice were induced to differentiate into Tfh cells in vitro,and the expression of Bcl-6 in Tfh cells was detected at diverse time points.The interaction between Bcl-6 and CBLB was detected with co-immunoprecipitation.The CD4⁺T cells were isolated from WT and Cblb^-/-mice,The cells were induced to differentiate into Tfh cells.The changes of ubiquitination and degradation of Bcl-6 protein were observed.4.The WT,Cblb^-/-,B6-lpr and B6-lpr.Cblb^-/-mice were treated with Bcl-6inhibitor FX1 by intraperitoneal injection for 4 weeks.The clinical symptoms and pathological variation of LN and the expression of Tfh cells in mice were observed.Results1.Deletion of CBLB aggravated kidney aamage in lpr miceThe results showed that the 24h-proteinuria levels of B6-lpr mice and B6-lpr.Cblb^-/-mice were higher than those of WT and Cblb^-/-mice,and the levels of24h-proteinuria in B6-lpr.Cblb^-/-mice were higher than those of B6-lpr mice（p<0.05）.The mice BUN levels were no significant difference in each groups before 20 weeks of age.Compared with the control groups,the BUN level of B6-lpr.Cblb^-/-mice was significantly increased at 20 weeks of age（p<0.05）.Compared with the control groups,the serum creatinine levels of B6-lpr mice and B6-lpr.Cblb^-/-mice increased significantly at 20 weeks of age（p<0.05）,and the serum creatinine levels in B6-lpr.Cblb^-/-mice were higher than those of B6-lpr mice（p<0.05）.The anti-ds DNA and ANA titer of B6-lpr mice and B6-lpr.Cblb^-/-mice were higher than those of WT and Cblb^-/-mice,and the anti-ds DNA and ANA titer in B6-lpr.Cblb^-/-mice were higher than those of B6-lpr mice（p<0.05）.The B6-lpr mice and B6-lpr.Cblb^-/-mice renal PAS staining showed hyperplasia of glomerular mesangial cells,and the segmental fibrinoid necrosis was observed in glomeruli of B6-lpr.Cblb^-/-mice.2.Deletion of CBLB increased Tfh cells in peripheral blood in lpr miceThe results of flow cytometry showed that the number of Tfh cells in peripheral blood and kidney tissue were significantly increased in B6-lpr mice and B6-lpr.Cblb^-/-mice compared with in WT and Cblb^-/-mice.The number of Tfh cells of peripheral blood and kidney tissue in B6-lpr.Cblb^-/-mice were significantly increased more than in B6-lpr mice（p<0.05）.The results of ELISA showed that the levels of Ig G in peripheral blood of B6-lpr mice and B6-lpr.Cblb^-/-mice were higher than those of WT and Cblb^-/-mice,and the levels of Ig G in B6-lpr.Cblb^-/-mice were higher than those of B6-lpr mice（p<0.05）.Immunofluorescence staining showed that the expression of Ig G was negative in WT and Cblb^-/-mice,and the expression of Ig G was strongly positive in B6-lpr mice and B6-lpr.Cblb^-/-mice.Compared with B6-lpr mice,the expression of Ig G in B6-lpr.Cblb^-/-mice was stronger.3.The changes of CBLB expression in Tfh cells affect the function of Tfh cellsAfter Tfh cells from lpr mice were transfected with CBLB si RNA and co-cultured with normal B cells,the prodution of Ig G increased significantly（p<0.05）;After Tfh cells from lpr mice were transfected with CBLB plasmid and co-cultured with normal B cells,the prodution of Ig G was significantly decreased（p<0.05）.4.The regulation of CBLB on Tfh proliferation realized by ubiquitination of Bcl-6.The expression of Bcl-6 was lower in CD4⁺T cells of WT mice.Under the condition of inducing Tfh differentiation,the expression of Bcl-6 increased and binding of CBLB to Bcl-6 enhanced in Tfh cells.Further study showed that the level of Bcl-6 ubiquitination and the degradation of Bcl-6 were reduced in Cblb^-/-Tfh cells.5.Bcl-6 inhibitor inhibited the proliferation of Tfh cells and alleviated renal injury in lpr miceCompared with the control groups,the 24-hour urinary protein and autoantibody titer were significantly decreased,when B6-lpr mice and B6-lpr.Cblb^-/-mice were treated with FX1,（Bcl-6 inhibitor）（p<0.05）,At the same time,it also alleviated the pathological changes of kidney in B6-lpr mice and B6-lpr.Cblb^-/-mice,and the proliferation of like-Tfh cells in peripheral blood and kidney were inhibited（p<0.05）.ConclusionsThe studies have proved that CBLB is a negative regulator of Tfh cells.Down regulation of CBLB expression induces the increased of Tfh cells in LN and aggravates the symptoms of LN.Simultaneously,we found CBLB regulates Tfh through targeting Bcl-6 ubiquitination.Therefore,Bcl-6 may be a new potential therapeutic target for LN.