The Underlying Biological Mechanism Of Itaconate Inducing DC Immune Tolerance In Alleviating AIH

AIMSAutoimmune hepatitis（AIH）is a chronic autoimmune-mediated inflammatory response against liver cells.Under the condition of dysregulation of immune homeostasis,a loss of tolerance against the own liver antigens is regarded as the main underlying pathogenetic mechanism.Dendritic cells are pivotal master antigen-presenting cells that play an important role in the pathological progress of AIH.Activation and effector function of DCs are dependent on reprogramming of intracellular metabolic pathway.Inflammatory activation of DCs causes impaired mitochondrial respiration and tricarboxylic acid cycle disruption,resulting in the accumulation of endogenous metabolites capable of adopting immunomodulatory roles.Itaconate,a product of the decarboxylation of the TCA cycle intermediate cis-aconitate,has recently attracted the attention of the scientific community due to its broad immunomodulatory properties linked to immunological tolerance.Itaconate and its derivates have been successfully applied as an immunoregulatory effector in the treatment of inflammation diseases,including ulcerative colitis,liver ischemia reperfusion,systemic lupus erythematosus and systemic sclerosis.The production of itaconate is encoded by immune-response gene 1（IRG-1）,which can be elevated in most activated immune cells,the extent and peak time of the increase in IRG-1 will vary under different circumstances inflammation and autoimmune disease.In the present study,we investigated itaconate possesses an immunomodulatory effect on regulating DC activation and liver inflammation in AIH to detect immunology-related regulatory mechanism.This study aims to determine explore the therapeutic effect of exogenous itaconate on AIH to elucidate the regulatory mechanism of itaconate on DCs immune environment and immune function.METHODS1.In order to verify the role of itaconate in AIH,we successfully constructed a mouse model capable of mimicking human AIH using isolated S100 protein.The levels of endogenous Itaconate secretion in liver tissues were targeted by liquid chromatography mass spectrometry and the expression levels of IRG-1 in liver tissues were measured by WB technique.The mice were divided into three groups:control group,model group and treatment group.The model group was injected intraperitoneally with saline and the treatment group was injected intraperitoneally with exogenous itaconate to evaluate the anti-inflammatory effects of exogenous itaconate on S100 induced AIH model.2.To further explore the changes of immune cells in AIH mice induced by S100,the cross-talk of DCs and Tregs impacted by itaconate was illuminated.The proportion of DCs and Tregs immune cells in liver group was analyzed by tissue multiple immune fluorescence.The mature indexes of DCs and the balance of Tregs/Th17 cells in mouse liver and spleen were analyzed by immunohistochemistry and flow cytometry.Since the immune status of DCs is closely related to energy metabolism,energy metabolomics analysis of DCs in spleen tissue were performed after DCs sorted by MACS.We successfully cultured bone marrow-derived DCs and initial CD4⁺T cells.In vitro,the DCs-Tregs cells communication was tested by mixed lymphocyte reaction T cell proliferation,and so on.In vivo,BMDCs were adopted transfer into AIH mice through caudal vein to explore the effect of anti-inflammation of BMDCS induced by itaconate.3.To verify that itaconate may participate in the development of AIH by regulating apoptosis and autophagy levels of DCs.In vitro,BMDC apoptosis-related tests,mitochondrial membrane potential（MMP）,intracellular reactive oxygen species（ROS）,the activity of apoptosis pathway protein Bcl-2/Bax and Caspase3 were analyzed to investigate the regulation of Itaconate on DCs apoptosis.To confirm that the autophagy in BMDC were regulated by itaconate,the late stage autophagic vacuoles were observed under a fluorescence microscope,the autophagy-related protein expression was evaluated by western blot.Autophagy involves multiple functions of DCs,and m TOR is closely related to the regulation of DCs autophagy function.Furthermore,in vivo and in vitro,the phosphorylation levels of PI3K/AKT,the key signaling molecules upstream of m TOR pathway and the main molecules downstream of m TOR pathway were examed by western blotting.In order to further verify the effect of m TOR gene deletion in DCs cells on AIH,conditioned gene knockout mice(m TOR^DC-/-)were re-modeled with S100,liver inflammation indicators were analyzed after HE staining,autophagy pathway protein LC-Ⅱ\LC-Ⅰ,Beclin-1 and P62 was analyzed by WB.m TOR agonist MHY1485 was used to further reverse validate the effect of m TOR pathway activation on DCs autophagy,liver inflammation indicators and autophagy pathway proteins were analyzed to clarify the specific mechanism of itaconate improving the regulation of liver inflammation.RESULTS1.IRG-1-Itaconate pathway is altered during AIH,exogenous itaconate alleviates liver injury in mice with S100-induced AIH.A significant increase in intracellular endogenous itaconate levels in S100-induced AIH mice,consistent with upregulation of IRG-1 in liver tissues,which suggest that the IRG-1-Itaconate pathway is altered in AIH,which is relation to liver inflammation.The findings suggested that the production of itaconate is stimulus-dependent.Exogenous itaconate attenuated the pathological changes of the liver and spleen,including the diminished infiltrated lymphocytes in the portal area,the intact structure of hepatic lobule and the normal arrangement of hepatic cord.It can reduce ALT level released after hepatocyte destruction caused by liver antigen,increase ALB,reduce the release of inflammatory factor IL-6,demonstrating the protective effect of itaconate in AIH.2.Itaconate induced DC immune tolerance function by regulated the balance of Th17/Tregs to prevent autoimmune hepatitis.In AIH model,the proportion of Treg cells（CD25⁺Foxp3⁺）increased significantly with the number of DCs（CD11⁺）increased in liver tissue.The frequency of immature DCs and Tregs in liver tissues in the itaconate treated group increased compared with those in the AIH group.We found that there was a positive correlation of DCs frequency with ALT,suggesting that the mitigation effect of Itaconate on AIH may be related to the regulation of DCs and Treg cells.Itaconate induces DCs immunotolerance phenotype,and DCs with low expression of MHC-Ⅱand CD86 in the portal vein area of liver and spleen,suggesting that Itaconate inhibits the antigen delivery of DCs to T cells.Targeted metabolic analysis also showed that Itaconate ameliorate the AIH by changing DCs metabolism,by switch from glycolysis to aerobic phosphorylation and down-regulates the main glycolysis enzymes,such as HK2,PKM2 and LDHA.It is suggested that itaconate enhances the immune tolerance of DCs by inhibiting glycolysis and is beneficial for the expression of genes related to immune tolerance.T cells subgroup analysis shows that the percentage of CD4⁺CD25⁺Foxp3⁺（Tregs）expression were decreased,the CD4⁺IL-17A⁺IFN-γ⁺（Th17）expression were increased in AIH;itaconate significantly reverse this change.Mixed lymphocyte reaction ex vivo showed that:S100-induced BMDCs were exhibited a pro-inflammatory ability as CD4⁺T cells proliferation.On the contrary,itaconate treatment BMDCs displayed inhibition of CD4⁺T proliferation,CD44^highCD62L^lowT cells decreased.In itaconate induced-BMDC transferred group,the inflammation was alleviated.Taken together,these findings suggest that itaconate has a direct regulatory effect on T cell differentiation and promotes the generation of Tregs cells by inhibiting DCs maturation and antigen-presenting ability.3.Itaconate promotes DCs apoptosis and inhibition of autophagy,and reduces liver inflammation of AIH mice through inhibition of DC autophagy mediated by m TOR signaling pathwayThe results of apoptosis assay showed that the levels of early and late apoptotic cells decreased,mitochondrial membrane potential increased,and intracellular reactive oxygen species decreased in S100-treated BMDCs.However,itaconate-treated BMDCs,the apoptosis rate were increased,MMP was decreased and ROS was increased.Furthermore,quantitative analysis of protein levels showed that the Bcl-2 was deregulated,the Bax was upregulated,the activity of caspase-3 was upregulated in S00+ITA group compared with S100 group.These results suggest that itaconate promoted apoptosis of BMDC,immature DCs can rapidly phagocytose apoptotic DCs,restrict antigen presentation to T cells and induce immune tolerance.To confirm the effect of itaconate on autophagy and autophagy pathway of BMDC,the WB results showed that LC3-Ⅱ,beclin-1 were upregulated after treatment with S100,more MDC-labelled vacuoles accumulated in the cytoplasm,itaconate reversed the results.Chloroquine,which is a useful inhibitor of autophagy tool,was used to assessment of autophagy flux.As shown in results,the ratio LC3-Ⅱ/LC3-Ⅰdid not increase further.These results demonstrated that itaconate depressed autophagy by blocking the fusion between autophagosomes and lysosomes,rather autophagy activation.In vitro,itaconate enhanced the phosphorylation level of m TOR and inhibited autophagy.To prove that Itaconate may inhibit autophagy of BMDC through m TOR signal,signal molecules in the upstream and downstream of the m TOR pathway were analysed in vivo.The results showed that P-AKT and P-m TOR expressions were down-regulated in AIH mice;P-m TOR,P-P70-S6,P-S6 and P-4EPP-1 are increased after Itaconate treatment,suggesting that Itaconate may inhibit the autophagy level of AIH through PI3K-AKT-m TOR pathway and protect S100-induced AIH.We bred mice bearing floxed m TOR with a CD11c-Cre delete strain,resulting in specific genetic ablation,confirmed that m TOR deletion can aggravate liver injury and improve autophagy level.Itaconate could reverse this results.MHY1485 is an m TOR agonist that can activate downstream targets of m TOR,improve liver inflammation,significantly decrease IL-6,and inhibit the expression level of autophagy protein.Itaconate is consistent with the treatment of MHY1485,suggesting that itaconate may inhibit autophagy by activating PI3K/AKT/m TOR pathway in DCs and improve liver inflammation caused by m TOR knock out.CONCLUSIONS1.Itaconate ameliorates AIH inflammation by inhibiting the maturation of DCs.2.Itaconate suppresses CD4+T cell immune response by inhibiting glycolytic metabolism to induce immune tolerance in DCs and regulating the balance of Th17/Tregs.3.Itaconate inhibits autophagy in DCs by phosphorylating the PI3K/AKT/m TOR pathway and attenuates liver inflammation and connects the metabolism and function of DCs,providing new ideas and approaches for AIH treatment.