USP25 Attenuates Anti-GBM Nephritis In Mice By Negative Feedback Regulation Of Th17 Cell Differentiation

PurposeAnti-glomerular basement membrane（GBM）nephritis is a serious acute glomerular disease with various types of lesions.It is characterized by crescent glomerulonephritis,including crescent formation,vascular loop necrosis,mesangial dilation,etc.,with rapid onset and poor prognosis.In crescent glomerulonephritis,CD4⁺helper T cells differentiate into different effector T cell subsets and resulting in inflammatory cell infiltration,inflammatory mediators release,and immune damage.The activation and proliferation of Th17 cells are considered to be key cells in the early stage of crescent glomerulonephritis.Therefore,inhibition of Th17 cell-mediated immune response will directly affect the pathological process of crescent glomerulonephritis.Reversible ubiquitin modification of cell signaling molecules is considered to be a key mechanism for cell response to extracellular stimuli,and ubiquitin-specific proteinase 25（USP25）has been reported to play an important role in regulating immunity.Therefore,in this study,the anti-GBM nephritis mice model was established to explore the influence of USP25 on the immune process of anti-GBM nephritis.It will provide theoretical basis for early intervention of immune activation and reduction of immune damage.MethodsFirst,the anti-GBM nephritis model was established by injecting nephrotoxic serum from the tail vein of mice,and then dynamically monitor its kidney function and renal pathological changes.Flow cytometry,ELISA,RT-PCR and other methods were used to detect the changes of immune cells,inflammatory factors or inflammatory cell transcription factors in peripheral lymphoid organs,blood and kidney over time to understand the immune process of nephritis in mice.Then,the USP25 expression level of C57BL/6 mice with anti-GBM nephritis and spontaneous systemic lupus erythematosus mice(MRL/Mp J-Fas ^lpr/J mice)was detected by RT-PCR to study its relationship with various types of crescent nephritis.And,USP25 gene knockout mice were used to establish anti-GBM nephritis.Under the condition that USP25 gene deletion did not affect the growth,immune organs and immune cell development of the mice,its renal function（serum creatinine and urea nitrogen）and pathological changes at different time were detected.Also,the levels of spleen immune cells（detected by flow cytometry）,peripheral inflammatory cytokines IFN-g、IL4、IL17A（detected by ELISA）,and characteristic transcription factors of renal helper lymphocytes（T-bet、GATA3、FOXP3、RORgt）（detected by RT-PCR）were compared with those of wild mice with anti-GBM nephritis,in order to explore the specific role of USP25 in anti-GBM nephritis immune process.In the last section,mice spleen lymphocytes were extracted and naive na?ve CD4⁺T cells were sorted by magnetic beads.Then,the isolated CD4⁺T cells were stimulated to differentiate into Th1,Th2 and Th17 cells respectively in a 96-well cell culture plate.Flow cytometry was used to determine whether the induction was successful.After stable induction conditions were established,the directional differentiation ability of CD4⁺T lymphocytes in wild mice and USP25^-/-mice was compared to explore the effect of USP25 on CD4⁺T lymphocyte differentiation.Then,CD4⁺T cells selected from the wild mice and USP25^-/-mice were activated to proliferate,and the m RNA levels of characteristic transcription factors（T-bet、GATA-3和RORγt）of Th1,Th2 and Th17cells were detected by RT-PCR,in order to investigate whether USP25 affects its directional differentiation by changing the level of transcription factors.Finally,CD4⁺T lymphocytes of wild mice were induced to differentiate into Th17 cells,and then the expression level of USP25 protein was detected to clarify the relationship between Th17cell differentiation and USP25.ResultsCompared with the normal mice,the levels of serum creatinine（Scr）and urea nitrogen（BUN）in the anti-GBM nephritis mice were increased at the 14th day of the anti-GBM nephritis model,and were significantly higher than normal mice.The expression ratio of Th1 and Th2 lymphocytes increased gradually within 21 days after the anti-GBM nephritis model,while Th17 cells increased first and then decreased,and the peak value was about 14 days after nephritis.There was no significant difference in serum IFN-γ,IL-4 and IL-17A levels between the nephritis model group and the control group on day7,but the levels in the model group were significantly higher than those in the control group on day 14 and 21.In general,the levels of IFN-g、IL-4 and IL-17A showed an increasing trend after the establishment of the anti-GBM nephritis model.With the progression of nephritis,transcription factors T-bet、GATA3 and FOXP3 of Th1 cells,Th2 cells and Treg cells were higher than those in the control group,showing a trend of first increasing and then decreasing,and reaching a peak at about day 14.The level of RORgt,the characteristic transcription factor of Th17 cells,reached a peak at about day7 and then continuously decreasing.RT-PCR results showed that the m RNA level of USP25 in anti-GBM nephritis mice was higher than those in control mice after the establishment of nephritis model,and showed a trend of first increasing and then decreasing.The m RNA levels of USP25 in MRL/Mp J mice and Fas^lpr mice increased with time,and the m RNA levels of USP25 in Fas^lpr mice were higher than those in MRL/Mp J mice of the same weeks of age,and the difference was significant.Scr level in USP25^-/-mice was significantly higher than that in wild mice on day 14 of nephritis model,and BUN level was significantly higher than that of wild nephritis mice on day 14 and 21.The infiltration of Th1 and Th17 cells in spleen of USP25^-/-nephritis mice was increased compared with that of wild nephritis mice,but there was no significant difference in Th2lymphocyte infiltration.The levels of IFN-g,IL-4 and IL-17A in peripheral blood of USP25^-/-nephritis mice were not statistically different from those of wild nephritis mice.The level of RORgt,the characteristic transcription factor of Th17 cells,in kidney of USP25^-/-nephritis mice was higher than that of wild nephritis mice,while the level of transcription factors FOXP3 of kidney Treg cells in USP25^-/-nephritis mice was lower than that of wild nephritis mice.The characteristic transcription factors T-bet and GATA3of Th1 cells and Th2 cells showed no significant difference between the two groups.CD4⁺T lymphocytes were successfully isolated from spleen lymphocytes.Then they were stimulated and induced to differentiate into Th1,Th2 and Th17 cells in vitro.The results showed that there was no difference in the proportion of CD4⁺T lymphocytes directed to Th1 or Th2 cells between wild mice and USP25^-/-mice,while the proportion of Th17positive cells was significantly increased in the USP25^-/-mice group.After activated proliferation of spleen CD4⁺T cells from wild mice and USP25^-/-mice,RORγt m RNA level was higher in USP25^-/-mice group,while T-bet and GATA-3 m RNA levels showed no significant difference between the two groups.After inducing differentiation of CD4⁺T lymphocytes into Th17 cells,the USP25 protein level was higher than that in control group.ConclusionIn the early stage of anti-GBM nephritis,Th1 and Th17 cells were the main immune cells in CD4⁺T cell immune response.With the progression of the disease,the level of renal Th17 cells decreased before Th1 cells,while the concentration of peripheral inflammatory cytokines continued to increase.In vivo,USP25 can reduce renal histopathological and functional damage in the process of anti-GBM nephritis.The mechanism is mainly through changing the balance between Th17 cells and Treg cells to promote immune tolerance.Meanwhile,USP25 can negatively regulate the proliferation of proinflammatory cells Th1 and Th17 in peripheral immune organs and reduce systemic inflammatory response.USP25 inhibits the differentiation of na?ve CD4⁺T cells into Th17 cells,and the mechanism may be related to the down-regulation of RORγt.When the inflammatory response worsens or Th17 cell differentiation increases,USP25 can be activated to form a negative feedback regulatory pathway and attenuate immune activation.