Study On The Mechanism Of Innate Immune Signal In Ischemia-reperfusion Liver Injury Disease

PTEN-β-Catenin Signaling Modulates Regulatory T Cells and Inflammatory Responses in Mouse Liver Ischemia and Reperfusion InjuryObjective:In order to deeply study the pathogenesis of liver injury accompanied by hepatic resection and liver transplantation,we used 70%liver ischemia-reperfusion injury model to simulate the IRI-environment and explore the immune mechanism in the process of liver injury.The study is the first time to explore the relationship between PTEN/beta-Catenin signal axis in macrophages and the differentiation of T cells with a stable mouse ischemia reperfusion liver injury model.To further clarify the mechanism of innate and acquired immunity response in the process of ischemia reperfusion liver injuryMethods:Using the conventional IRI model,the experimental animals were FL/FL divided into 4 groups.Group A was sham operation group(sham+PTENFL/FL group),group B was sham operation group(sham+PTENM-KO group),group C:(IR+PTENEL/FL group),group D:(IR+PTENM-KO group)Liver sections were stained with hematoxylin and eosin(H&E).The IRI severity was graded using Suzuki’scriteria.Liver CD11b+macrophages were detected by immunohistochemistry staining with a rat anti-mouse CD11b mAb.Immunofluorescence staining was used to identify CD68+macrophages with a goat anti-mouse CD68 mAb.First in vivo,we separate liver macrophages and determine the protein expression of beta-Catenin and AKT、test the protein level of inflammatory cytokines and mRNA levels of Foxp3+,IL-17A,RORyt.Second in vitro,we transfected a small interfering RNA(si β-cat)to BMMs,followed by determine the protein expression of beta-Catenin,PPARy,NICD and test the protein level of inflammatory cytokines and mRNA levels of Foxp3+,IL-17A,RORyt from co-cultured T cells Third,after transfecting a small interfering RNA(si PPARy)to BMMs,we determine the protein expression of PPARy,NICD and test the protein level of inflammatory cytokines and mRNA levels of Foxp3+,IL-17A,RORyt from co-cultured T cells Finally,we determine the protein expression of NICD and test the protein level of inflammatory cytokines and mRNA levels of Foxp3+,IL-17A,RORyt from co-cultured T cells followed by transfecting a small interfering RNA(si Jagged-1)to BMMs.The important step,in vivo after intravenous injecting Notch inhibitor DAPT,we collect liver sections for hematoxylin and eosin(H&E),evaluate the IRI severity by using Suzuki’scriteria,determine the necrosis of liver tissue injury by using tunel assay and Hepatocellular function assay,test the protein expression of Hesl and the percentage of Treg/TH 17Results:The hepatocellular damage was evaluated in mouse livers subjected to 90 min of warm ischemia followed by 6h of reperfusion.Livers in PTENFL/FL mice showed severe edema,sinusoidal congestion,and necrosis.However,livers in PTENM KO mice showed mild to moderate edema andsinusoidal congestion.Consistent with the histopathological data,the serum ALT levels(IU/L)in PTENM KO mice were significantly decreased compared to the PTENFL/FL controls.Moreover,PTENM-KO reduced frequency of TUNEL+cells in ischemic livers compared to the PTENFL/FL controls.PTEN deficiency decreased CD68+macrophage infiltration compared to the PTENFL/FL controls and reduced CD11b+macrophages but not PTENFL/FL mice.After 90 min of ischemia,myeloid PTEN Knockout up-regulated the protein expression of(3-catenin and phosphorylated Akt compared to PTENFL/FL livers.The mRNA levels of proinflammatory genes coding for TNF-α,IL-1β,and IL-6 was decreased,whereas TGF-βexpression was increased in PTENM KO livers compared to the PTENFL/FL controls.Moreover,PTENM KO promoted M2 macrophage differentiation as evidenced by increased arginasel(Argl)while reduced M1 macrophage inducible nitric oxide synthase(iNOS)expression in ischemic livers compared to the PTENFL/FL controls.Myeloid PTEN Knockout up-regulated the mRNA expression of Foxp3 while inhibited the expression of RoRyt and IL-17A.In vitro,PTEN KO increased the expression ofβ-catenin,PPARy and Jagged-1 after LPS-stimulated BMMs.Meantime,knockdown ofβ-catenin with si β-cat pre-treatment in PTENM-KO-BMMs resulted in reduced PPARγ and Jagged-1 expression after LPS stimulation.Pre-treament with si β-cat augmented the mRNA levels of TNF-α,IL-1β,and IL-6 while reduced TGF-β.Meantime,the decreased Argl and increased iNOS expression was observed in siβ-cat-treated PTENM KO-BMMs.Knockdown ofβ-catenin in PTENM KO BMMs before co-culture decreased cleaved Notch1 protein expression and mRNA levels coding for Notchl,Hes1,and RBP-J,which was accompanied by reduced Foxp3 but augmented RORyt and IL-17A in spleen T cells.In vitro,knockdown of PPARy with si PPARy pre-treatment in PTENM-KO-BMMs resulted in reduced PPARy and Jagged-1 expression after LPS stimulation.Pre-treament with si PPARy augmented the mRNA levels of TNF-α,IL-1β,and IL-6 while reduced TGF-β.Si PPARy treatment decreased Argl but increased iNOS expression in PTEN-deficient macrophages compared to the NS siRNA-treated cells.Moreover,PPARy knockdown in macrophages reduced cleaved Notch1 protein expression and Notch1 and RBP-J mRNA levels with reduced Foxp3 but increased RORyt and IL-17A in spleen T cells after co-culture.In vitro,si Jagged-1 pre-treatment with BMMs inhibited cleaved Notchl and Hesl protein expression in spleen T cells after co-culture.The mRNA levels coding for Notch1 and RBP-J were reduced in si Jagged-1 but not NS siRNA-treated cells.Consistent with these data,knockdown of Jagged-1 resulted in reduced Foxp3 while increased RORyt and IL-17A expression in T cells.Intravenous injection of DAPT reversed phenoment produced by PTENM KO in IR-indced liver injury,increased the degree of liver injury and ALT index,promoted liver cell apoptosis,inhibited the expression of Hesl,inhibit the mRNA expression of Foxp3+,while promoted IL-17A expression in T cells.Conclusion:In the model of ischemia-reperfusion liver injury,myeloid-specific PTEN gene knockout protects liver injury.The mechanism may be that PTEN knockout promotes beta-catenin expression,then increases the activity of Jagged-1/Notch signaling cascasde-mediated by PPAR-gamma,leading to augmented Foxp3+Treg induction while inhibited RORyt/IL-17A in IR-triggered liver inflammation.Loss of ATF3 Exacerbates Liver Damage through Activation of mTOR/p70S6K/HIF-1α Signaling Pathway in Liver inflammatory InjuryActivating transcription factor 3(ATF3),a stress-induced transcription factor,plays important roles in regulating immune and metabolic homeostasis.Activation of the mechanistic target of rapamycin(mTOR)and hypoxia-inducible factor(HIF)transcription factors are crucial for the regulation immune cell function during inflammatory response.This study investigates the functional roles and molecular mechanisms by which ATF3-mediated mTOR-HIF-1 axis regulates innate immunity and adaptive T cell differentiation in a mouse model of liver inflammatory injury induced by ischemia and reperfusion.We found that mice with ATF3 knockout(ATF3 KO)exacerbated liver damage as evidenced by increased levels of serum ALT,intrahepatic macrophage/neutrophil trafficking,hepatocellular apoptosis,and pro-inflammatory mediators compared to the wild type(WT)controls.ATF3 deficiency promoted mTOR and Ribosomal protein S6 kinase(p70S6K)phosphorylation,activated HMGB1 and TLR4,with reduced prolyl hydroxylase domain protein(PHD)1 and increased HIF-1α activity,leading to reduced Foxp3 while increasing RORyt and IL-17A expression in ischemic livers.Blocking mTOR or its downstream target gene p70S6K in ATF3 KO mice reduced HMGB1,TLR4,and HIF-1α while increasing PHD 1,with augmented Foxp3 and reduced IL-17A in macrophage/T cell co-cultures.Moreover,disruption of HIF-1α in ATF3 KO mice ameliorated liver damage after liver IRI,which was accompanied by decreased RORyt-mediated IL-17A levels in ATF3-deficient mice.Our findings demonstrate that ablation of ATF3 exacerbates IR-induced liver damage.ATF3 deficiency activates mTOR/p70S6K/HIF-1α signaling,which is crucial for the modulation of innate TLR4-driven inflammatory response and adaptive T cell development.Our study provides potential therapeutic targets in liver IRI followed by liver transplantation.