Adoptive Transfer Of Regulatory T Cells Stimulated By Allogeneic Hepatic Stellate Cells Mitigates Liver Injury In Mice With Concanavalin A-induced Autoimmune Hepatitis

BackgroundThe etiology and pathogenesis of autoimmune hepatitis(AIH)have not been fully elucidated.It is effective in the treatment of glucocorticoids or azathioprine and eventually develops into cirrhosis or liver failure.Regulatory T cells(Treg)in AIH patients have deficiencies in number and function.The actual role of Treg damage in AIH is still controversial.However,AIH treatment based on Treg may still be effective.Adoptive transfer of Treg cells may be a promising method for treating AIH.How to amplify Treg and exert its immunoregulatory effect is a hot research topic at present.Hepatic stellate cells(HSC)may play an important role in immune regulation which can selectively expand allogeneic Treg early after liver transplantation.In order to observe the induction and amplification effect of HSC on Treg cells,and to explore the therapeutic effect of adoptive transfer of Treg cells on AIH induced by Concanavalin A(ConA)in mice,we designed the experiment.Objective:In order to find an effective method for isolation and culture of primary hepatic stellate cells(HSC),observe the induction and amplification effect of HSC on Treg cells,and to explore the therapeutic effect and mechanism of adoptive transfer of Treg cells on AIH induced by Concanavalin A(ConA)in miceMethods:We used OptiPrep density gradient separation fluid to separate and culture primary HSC of BALB/c male mice by hand in situ perfusion.Spontaneous fluorescence was observed under inverted fluorescence microscope,and cells were identified by oil red O staining,alpha-SMA and Desmin immunocytochemical staining.The splenic CD4~+CD25~+Treg cells and CD4~+CD25~-Teff cells of C57BL/6 mice were isolated by immunomagnetic beads,and primary(0-HSC),2 generations of(2-HSC)were respectively co-cultured with Treg for 72h.The proliferation effect of HSC cells on Treg was observed,then the proliferation inhibition effect of expanded Treg on Teff were observed by flow cytometry.The CFSE-labeled CD4~+CD25~-effector T cells(CFSE-Teffs)were co-cultured with Teffs or Tregs in the presence or absence of HSC to determine the suppressive capacity of Tregs.Similarly,the CFSE-labeled Tregs were co-cultured with s in transwell plates to determine the potential cell-cell contact dependent.The effects of Tregs or-stimulated Tregs on AIH severity and the frequency of splenic Tregs and Th17cells were examined in mice.AIH mice induced by ConA,coded histological sections were scored independently by using Ishak modified Histology Activity Index(IMHAI).The number of Treg and Th17 cells and Treg/Th17 ratio in the spleen cells were observed.The Ex Vivo expanded Tregs were transferred to AIH mice.The therapeutic effect on hepatitis were observed after 24 hours of adoptive transfer including the pathological changes of liver HE staining,the Treg/Th17 ratio of the spleen and serum level of ALT and AST were observed,the serum levels of IL-2,IL-4,IL-6,TNF-alpha,IFN-gamma,IL-17A and IL-10 were detected by CBA(Cytometric Bead Array)with flow cytometry.Results:We isolated a high yield and high activity primary(0-HSC),which was fully activated after 2 generations of(2-HSC).The spontaneous blue-green fluorescence was observed at 328 nm wavelength under inverted microscope.The lipid droplets in the cytoplasm stained with oil red were red and the nucleus was blue.The cytoplasm of HSC(2-HSC)alpha-SMA and Desmin stained positive cells were brown granules,100%positive,and all activated.The proliferation rate of Treg induced by 2-HSC was significantly higher than that of Treg induced by 0-HSC.HSC induced the proliferation of Treg in vitro as a dose dependence.Transwell experiment showed that the proliferation of Treg induced by HSC was caused by the cell-cell contact,and the Treg cocultured with HSC inhibiting the proliferation of Teff was more effective than Treg alone in inhibiting Teff proliferation.Mouse AIH model was successfully made,the liver inflammation and hepatocyte necrosis was obvious,IMHAI and serum ALT and AST increased,the serum level of IL-2,IL-4,IL-6,TNF-alpha,IFN-gamma,IL-17A and IL-10 increased,the number of Treg and Th17 increased and Treg/Th17 ratio in the spleen was lower in the model mice than that of the control group.So,it proved imbalance of Treg/Th17 in AIH mice.After the adoptive transfer of Treg or expanded Treg induced by to AIH mice for24h,the treatment group compared with the model group,the liver inflammation and hepatocyte necrosis was relieved,IMHAI and serum ALT and AST level decreased,the levels of IL-6,TNF-alpha,IFN-gamma dencreased and the spleen Treg/Th17 ratio increased.The effect of expanded Treg induced by HSC was most obvious.Conclusion:High yield and high activity primary hepatic stellate cells can be isolated by OptiPrep density gradient separation fluid.The activated allogeneic HSC can induce the amplification of Treg cells in vitro.Treg cells amplified in vitro can play an immunosuppressive role on effector CD4~+CD25~-T cells.Treg/Th17 imbalance participates in the pathogenesis of AIH induced by ConA.Adoptive transfer the expanded Treg induced by HSC in vitro can effectively improve the imbalance of Treg/Th17,reduce the serum levels of IL-6,TNF-alpha,IFN-gamma and treat the AIH induced by ConA in mice.