Effects Of Kinsenoside,A Potential Immunosuppressive Drug For Autoimmune Hepatitis,on Dendritic Cells/CD8~+T Cells Communication

BACKGROUNDAutoimmune hepatitis(AIH)is defined as a chronic liver disease with loss of tolerance against liver tissue eventually leading to cirrhosis if left untreated.80%-90%of patients can be treated with a life-long immunosuppression.Unfortunately,there are strong drug-related side effects and steroid-refractory patients.Therefore,there is a need for investigating the complex immunopathogenesis of this chronic disease and subsequently developing new therapeutic interventions.CD8+T-cell responses to liver-expressed antigens range from deletional tolerance to full effector differentiation resulting in overt hepatotoxicity.Dendritic cells(DCs)are antigen-presenting cells(APCs)that regulate innate and adaptive immune responses.In autoimmunity,DCs have two apparently conflicting effects.On the one hand they initiate adaptive self-reactive responses,but on the other they promote and maintain tolerance.DCs may also have a specialized capacity to process exogenous antigens via the major histocompatibility complex(MHC)class pathway,becoming capable of activating CD8+T cells.Activation of immune cells is associated with a dramatic increase in metabolism,associated with enhanced production of energy required to stimulate protein synthesis,growth,and proliferation of activated immune cells.The development of DCs from progenitor cells is associated with mitochondrial biogenesis,which is driven by peroxisome proliferator-activated receptor-y(PPARy),AKT,mammalian target of rapamycin(mTOR),and so on.Anoectochilus roxburghii(A.roxburghii)is a traditional herb in China,and is widespread in tropical regions from India through the Himalayas and Southeast Asia to Hawaii.This herb is also referred to as "the Medicine of Kings" because of its diverse pharmacological effects,including anti-hyperglycemia,anti-osteoporosis,anti-adiposity,anti-fatigue,and,notably,hepatoprotection.Kinsenoside(KD)is a biologically active compound isolated from A.roxburghii.Our previous studies show that KD has normoglycemic effects in diabetic mice.Other reports indicate that KD reduces lipopolysaccharide(LPS)-stimulated inflammatory reactions and thioacetamide-induced hepatic fibrosis in mice,but the mechanism is not clear.AIMSThis study aims to identify the responsible immune cells and key molecule target for induction of AIH and KD treatment.METHODST cell-deficient murine was used to investigate the important effect of T cells in AIH.The effect of KD for AIH and immune cells was studied by histological,immunohistological,flow cytometry(FCM)analysis,enzyme-linked immunosorbent assay(ELISA),NO release analyses,and so on in concanavalin A(ConA)induced AIH murine models.The communication of DCs and CD8+T cells was illuminated using a novel murine AIH model by injections of DCs loaded with hepatocellular carcinoma cells(DC/Hepal-6)plus IL-12.And by means of adoptive transfer models,we next investigated DCs and CD8+T cells targeted therapy by KD.In vitro,the DCs-CD8+T cells communication was tested by mixed lymphocyte reaction(MLR),cytotoxicity assay,magnetic cell sorting(MACS),DC cross-presentation assay,and so on.The immune cells metabolic inhibition of KD was investigated by glucose uptake and non-esterified fatty acid(NEFA)release tests,and assessment of mitochondrial membrane potential(MMP).The glycolysis and fat metabolism related genes expression in DCs,T cells and DC-treated CD8+T cells were detected by quantitative real time PCR(qPCR)analysis.The target and the cross-talk of signal pathways,which affected by KD were performed by 2D fingerprint screen and molecular docking,qPCR analysis,western blotting analysis,electrophoretic mobility shift assay(EMSA),and so on.In DCs,the direct interaction between forkhead box protein 01(FoxO1)and the CC chemokine receptor 7(CCR7)promoter or programmed death ligand-1(PD-L1)promoter were demonstrated by the chromatin immunoprecipitation assay(ChIP assay).RESULTSTreatment with KD substantially reduced hepatic histopathological damage,induced by lymphocyte infiltration and pro-inflammatory cytokines,in ConA-induced murine AIH,and in DC/Hepal-6 induced murine AIH.Interactions between immune cells after KD treatment in AIH were detected by anti-CD8 antibody blocking,CD8+T cells sorting,adoptively transferred to naive mice by direct splenic inoculation,and vaccinated mice with KD-pretreated DCs in a DC/Hepal-6 model.The results showed that KD inhibited the elevated expressions of CD86 and MHC-II,densities of chemokine receptor CCR7,and extensive migration to lymph nodes of DCs.KD increased the PD-L1 level of DCs,followed by suppressing CD8+T cells characterized as low differentiation and cytotoxicity,and eliciting cytokines balance.Furthermore,biochemical analysis,2D fingerprint screen and 3D molecular docking results showed that KD bound to the VEGFR2 kinase domain,which inhibiting the metabolism-related PI3K-AKT pathway in DCs and DC-modulated CD8+T cells to lower the MMP and glucose/lipid utilization ratio in both cells.KD also inhibited glycolysis and fat metabolism related genes expression in DCs and DC-treated CD8+T cells.KD reversed the activation of the PI3K-AKT pathway by 740 Y-P(PI3K agonist),thereby impeding the translocation and dimerization of STAT3 and synergistically blocking the inflammation-related JAK2-STAT3 pathway in DCs and DC-modulated T cells.When DCs were stimulated with LPS the detection of FoxO1 binding to the CCR7 promoter was increased and FoxO]binding to the PD-L1 promoter was decreased with anti-FoxO1 antibody compared to matched IgG control.On the contrary,KD could reverse the responses.CONCLUSIONSKD treatment elicits immunosuppression against autoimmune liver injury by targeting VEGFR2,followed by diminishing the cross-talk of metabolism-related PI3K-AKT and inflammation-related JAK2-STAT3 pathways,thereby disrupts DC-induced cross-priming of CD8+T cell responses.