The Ubiquitin-conjugating Enzyme E2O(UBE2O) Ameliorates Autoimmune Hepatitis In A Novel Mouse Model

BACKGROUND&AIMS:Autoimmune hepatitis(AIH)is a type of immune-mediated liver injury with progressive chronic liver damage.AIH is more common in western countries.However,the reported cases of AIH have increased these years in China.The pathogenesis of AIH has not been clarified yet and the related basic research in this field is lacking.This situation is largely because of a lack of a suitable animal model of AIH.The diagnosis of AIH is difficult and highly depends on the liver biopsy.The early diagnosis and treatment of AIH are very important for a good clinical prognosis.Corticosteroid therapy is regarded as the standard treatment for AIH clinically.However,the side effects of the corticosteroids remain a tough issue.Therefore,exploring the potential pathogenesis,the diagnostic biomarker,and potential treatment target for AIH are urgent issues to be solved.In 1992,Tiegs et al.established a T cell-mediated acute hepatitis mouse model using concanavalin A(ConA),which is a typical and well-recognized mouse model used to investigate T cell-mediated liver injury.However,hepatitis in mice livers induced by ConA is acute and often disappears after 48h;furthermore,there is no autoantibody production or liver fibrosis in this mouse model.Thus,the ConA-induced hepatitis model may not fully mimic AIH in the human body.There is no well-recognized chronic AIH mouse model so far worldwide.Therefore,establishing a true sense of the AIH mouse model to mimic the disease process of AIH in the human body remains an urgent issue.The impaired imbalance of regulatory T cells(Tregs)and T helper 17(Th17) cells have been demonstrated to play a very important role in the development of many kinds of autoimmune diseases.Improving the ratio of Treg/Th17 cells has been proven to be an important treatment target for many autoimmune diseases.However,its role remains unclear in AIH.Th17 cells mainly secrete IL-17,which has been reported to increase in the sera of AIH patients and contributing to liver injury.Treg can secrete anti-inflammatory cytokines,which can inhibit the proliferation and function of Th17 cells effectively.However,whether the imbalance of Treg and Th17 cells and the function of Treg remain unclear in AIH,which also largely because of the lack of a suitable AIH mouse model.Protein ubiquitination is a specific modification of target proteins,which involves ubiquitin-activating enzyme E1,ubiquitin-conjugating enzymes E2 and ubiquitin-ligase enzymes E3.More evidence has shown that ubiquitination play important role in autoimmune diseases.Recent the important role of ubiquitin-conjugating enzymes E2 has been proven in regulating immune cell function and differentiation.Recent studies proved that ubiquitin-ligase enzyme E3.is important in regulating the activity of immune cells such as the development,activation,and differentiation of T cells,thereby maintaining the immune tolerance in the human body.The ubiquitin-conjugating enzyme E2O(UBE2O)is a novel ubiquitin-conjugating enzyme with both E2 and E3 function.A recent study also suggests it can inhibit the polyubiquitination of K63 by combination with tumor necrosis factor receptor(TNFR)-associated factor 6,thereby inhibiting the activation of NF-κstimulated by LPS and IL-1βand the following secretion of inflammatory cytokines.However,the study of UBE2O in AIH is lacking.This prompted us to consider that if UBE2O can regulate the differentiation of Treg and Th17 in AIH,promoting the Treg ratio to ameliorate the liver inflammation in AIH.To confirm this hypothesis,we overexpressed UBE2O in the AIH mouse liver and detected liver inflammation and fibrosis over time.Besides,we assessed the Treg/Th17 ratio in the AIH liver and explored the potential mechanism.In this study,we established a true sense AIH mouse model,providing a stable tool for the AIH basic research field.Besides,we screened the potential biomarkers based on this AIH model to provide insights for clinical diagnosis.In addition,we also explore the protective effect of UBE2O for AIH,which may provide a novel viewpoint for AIH pathogenesis and clinical treatment target.METHODS:(1)Mice were first infected with adenovirus through tail vein injection on day 0 to promote the inductionAIH using human CYP2D6.Next,we injected the plasmid expressing human CYP2D6(pCYP2D6)on day 1,4,9,13 via a rapid tail vein injection using the hydrodynamic-based liver targeted gene delivery technique to establish the AIH model.(2)Evaluate the liver inflammation with the H&E staining.(3)Asses the liver fibrosis using Sirius red staining andα-SMA.(4)Detect the production of antibodies and the infiltration of CD4~+T and CD8~+T cells using immunofluorescence.(5)Comparison of characteristics between ConA acute hepatitis model and chronic AIH mouse model using H&E staining,cytometric bead array,and flow cytometry.(6)Perform plasma proteomic analysis using AIH mouse model then do KEGG and GO enrichment based on the differentially expressed proteins(DEPs).(7)Detect the expression of UBE2O in the liver of AIH using WB,RT-PCR,and IHC.(8)Evaluate the liver inflammation,fibrosis,and related inflammatory cytokines in AIH mouse model after overexpression of UBE2O.(9)Use flow cytometry to detect the role of UBE2O in regulating the Treg/Th17 ratio.(10)Overexpress UBE2O in human hepatocytes L02 using lentivirus and detect the cytokines(IL-6)in the supernatant to find a potential mechanism for UBE2O-related-Treg/Th17 regulation.RESULTS:Part I:A true sense of the AIH mouse model was established,which can mimic the disease process of AIH in the human body.The characteristics were compared between the acute ConA mouse model and chronic AIH mouse model.(1)CYP2D6 was successfully overexpressed in the mouse liver via a rapid tail vein injection using the hydrodynamic-based liver targeted gene delivery technique.(2)The autoantibodies were observed in AIH mice sera at 4 weeks from the first injection.(3)The specific pathologic features such as interfacial hepatitis,rosettes,and lymphocytes invasion were observed in the liver of AIH mouse 4 weeks after the first injection.(4)Liver fibrosis was detected in AIH mice at 5 weeks.(5)The hepatitis was relieved in 48h after ConA injection.The pro-inflammatory and anti-inflammatory cytokines increased in 24h and reduced to normal in 48h in the ConA model.The pro-inflammatory cytokines increased in the sera of AIH mice with no obvious change in the anti-inflammatory cytokines.(6)Hepatic Tregs and Th17 cells increased after ConA injection and reduced gradually in 48h.Th17 cells increased in the liver of the AIH mouse but Treg showed a downregulated trend.Part II:To find a potential biomarker for AIH diagnosis clinically,plasma proteomic analysis was performed based on isobaric tag(IBT)technology.(1)A total of 176 DEPs was found in this experiment,of which 148 DEPs were up-regulated and 28 DEPs were down-regulated.(2)Thirty significant KEGG pathways(P<0.05)were detected.Arginine biosynthesis was found to be the most significant pathway involved in the AIH process.(3)During the GO analysis,most DEPs were found to be involved in the binding,cellular,and metabolic processes.(4)The most overexpressed DEP is SAA1 based on the i TRAQ results.(5)Using ELISA,SAA1 was confirmed to be increased specifically in the plasma of patients with AIH compared to other chronic hepatitis,which may be a potential biomarker for AIH diagnosis.Part III:UBE2O ameliorate liver injury in ConA mouse model and AIH mouse model and regulating the Treg/Th17 ratio.(1)WB,RT-PCR,and IHC results suggest UBE2O was downregulated in the liver of the ConA mouse model and chronic AIH mouse model.(2)Overexpression of UBE2O in mouse liver can relieve hepatitis in ConA-mediated liver injury and AIH mouse.(3)Overexpression of UBE2O can ameliorate liver fibrosis in AIH mice.(4)UBE2O can reduce the pro-inflammatory cytokines in AIH mouse liver and ConA mouse liver while the anti-inflammatory cytokines were upregulated.(5)UBE2O can upregulate the proportion of hepatic Treg and decrease the proportion of Th17 cells,thereby reconstructing immune tolerance in AIH mice.Part IV:UBE2O regulates Treg/Th17 balance via affecting the IL-6 secretion by hepatocytes.(1)The cell culture supernatant of hepatocytes from AIH mice and control mice was used for the culture of na(?)ve CD4~+T cells.72h later,Treg and Th17 differentiation was detected.The proportion of Th17 is much higher in the AIH group compared with supernatant from control mice.In contrast,the Treg proportion is less in the AIH group.(2)UBE2O was overexpressed in normal hepatocytes cell line L02,and the cell supernatant was used to culture CD4~+T cells from peripheral blood.Compared with the control group,the UBE20-overexpressed supernatant increase the proportion of Treg and decrease Th17 cells.(3)The cell supernatant from L02 cell line transfected with UBE2O-overexpressed lentivirus or vector was detected the human XL Cytokine Array Coordinates.The hepatocytes can secret abundant IL-6,which is down-regulated after UBE2O overexpression.(4)The murine hepatocytes were isolated and then cultured with the anti-IL-6 antibody for 24h.The culture supernatant was used to culture na(?)ve CD4~+T cells,and the Treg differentiation was detected unchanged.CONCLUSIONS:Collectively,this study established a novel AIH mouse model,which can mimic the chronic disease prose of AIH in the human body and provide a research tool for the AIH research field.The plasma proteomic analysis based on this novel AIH mouse model revealed the importance of the metabolic process in the pathogenesis in AIH.The most differentially expressed protein SAA1 was also confirmed specifically increased in AIH patients,which may work as a biomarker for AIH diagnosis clinically.This study also elucidates the protective role of UBE2O in AIH.UBE2O downregulate IL-6 secretion by hepatocytes,thereby promoting Treg to differentiate into Tregs rather than Th17.This study provides novel insights for the basic research,clinical diagnosis,and therapy for AIH.