The Role Of Prostaglandin E2 In Regulating Peripheral Blood Immune Cells In Acute-oh-Chronic Liver Failure And Acute Liver Failure

Background&Aims:Acute-on-chronic liver failure(ACLF)and acute liver failure(ALF)are a syndrome characterized by an acute deterioration of liver function accompanied by multiple organ failure and high short-term mortality.ACLF is developed on preexisting chronic liver disease and its primary pathogenesis is excessive systemic inflammation.Monocytes are one of the major innate immune members involved in the pathogenesis,progression and resolution of ALF.Prostaglandin E2(PGE2)acts on prostaglandin E receptors to exert a broad regulation of the body's immune response.Prostaglandin E Receptor 2(EP2)has been reported to be closely related to macrophage dysfunction in patients with decompensated cirrhosis.Peripheral blood monocytes and macrophages differentiated from monocytes in patients with alcoholic ACLF or acute decompensated cirrhosis showed immunosuppressive characteristics.However,the immune status of immune cells in patients with chronic hepatitis B related ACLF(HB-ACLF)is still unclear.The role of PGE2 in ACLF and D-GalN/LPS-induced ALF and the underlying mechanisms remain mystery.The aim of this study was to investigate the phenotypic and functional changes of various immune cells in patients with ACLF,and the regulation of PGE2 and its receptor EP2 on various immune cells,and to explore the alleviation effect of dmPGE2 on acute liver failure and the underlying mechanisms using mouse model of D-GalN/LPS-induced acute liver failure.These studies could help to further understand the immunological pathogenesis of ACLF,and provide a theoretical basis for the new treatment of ACLF and ALF.Part I:The role of PGE2 and its receptor EP2 in the regulation ofperipheral blood immune cells in patients with HB-ACLF.Objectives:To determine the immune status of peripheral blood immune cells in patients with HB-ACLF and the regulation of PGE2/EP2 on these immune cells.Methods:Peripheral blood samples from 135 patients with HB-ACLF,128 patients with chronic hepatitis B(CHB),and 180 healthy controls(HC)were collected.We measured the levels of plasma cytokines of ACLF using a multi-plex cytokine panel,and used flow cytometry to detect the expressions of EP2,Anti-infective immune related molecules like TLR4,spontaneous generation of reactive oxygen species(ROS)and ROS and cytokine production after Lipopolysaccharide(LPS)stimulation.We further detected the characteristics of EP2+immune cells,the correlation between the expression of EP2 and plasma cytokine levels or prognostic scores.MIP-1? and IP-10 were applied and changes in EP2 expression were observed.Finally,block or activate EP2 and detect changes in cytokine production,bacterial phagocytosis,and ROS production.Result:(1)Compared with HC,monocyte and neutrophil from HB-ACLF had up-regulated TLR-4;compared with HC and CHB,monocytes and neutrophils from HB-ACLF have increased spontaneous and E.coli-or PMA-stimulated ROS production,and PBMC from HB-ACLF had enhanced LPS-stimulated cytokine secretion.(2)Compared with HC and CHB,plasma PGE2 levels were increased in HB-ACLF patients,and EP2 expressions were abnormal on peripheral blood cells,especially on CD8+T cells.EP2+monocytes or neutrophils had a higher expression of TLR4,TLR2,CD11b and HLA-DR and produced more ROS under E.coli stimulation than EP2-monocytes or neutrophils.In addition,EP2+PBMC secreted more IL-10 and IFN-y in response to LPS than EP2-PBMC.(3)The plasma levels of PGE2 in patients with HB-ACLF in the 30-day non-transplanted survival group were higher than that in the transplanted or dead group(p<0.05).The expressions of EP2 on CD8+T cells in patients with multi-organ failure during the 90-day follow-up period were lower than that in patients without multi-organ failure(p<0.05).Down-regulation of EP2 expression on CD8+T cells negatively correlated with plasma cytokine MIP-1?(r=-0.66,p=0.024),IP-10(r=-0.67,p=0.028)levels and prognostic scores MELD(r=-0.41,p=0.017),CLIF-C(r=-0.48,p=0.004)(4)A combination of MIP-1? and IP-10 down-regulated EP2 expression on CD8+T cells(p<0.05).(5)Blocking EP2 in vitro enhanced the production of cytokines and ROS in immune cells from patients with HB-ACLF,but attenuated phagocytosis.Activation of EP2 reduced cytokine production of PBMC from HB-ACLF patients but increased G-CSF production.Conclusion:(1)Peripheral blood immune cells of HB-ACLF showed hypersensitivity to LPS or E.coli stimulation,providing theory for early use of antibiotics in HB-ACLF patients.(2)Down-regulated EP2 expression is associated with the patient's condition and this hypersensitivity reaction,and selective agonist of EP2 suppressed this hypersensitivity reaction and increased the production of the favorable cytokine G-CSF,providing a new potential target for treatment of HB-ACLF.Part ?:The alleviation effect of dmPGE2 on mice model of D-GalN/LPS-induced ALF and regulation of monocyte apoptosis and its mechanism.Objectives:To determine the alleviating role and underlying mechanism of PGE2 analogue 16,16-dimethyl PGE2(dmPGE2)on D-galactosamine/Lipopolysaccharide-induced(D-GalN/LPS)acute liver failure and monocyte apoptosis.Methods:mice were grouped into saline control group,D-GalN/LPS induction model group and dmPGE2 treatment group or PGE2 synthase antagonist group.Hepatic inflammation,levels of serum transaminase,serum cytokines and apoptosis and autophagy of peripheral blood monocytes were observed.Further combined with autophagy inhibitors,changes in liver and systemic inflammation and apoptosis of monocytes were observed.In vitro experiments were performed using PGE2 and EP2 antagonists to treat monocyte line U937 to detect autophagic levels.Results:(1)dmPGE2 significantly reduced serum transaminase in D-GalN/LPS-induced ALF mice,improved intrahepatic inflammation and reduced serum cytokines.(2)dmPGE2 induced monocyte apoptosis in ALF mice(3)The PGE2-EP2 pathway promoted monocyte autophagy both in vitro and in vivo.(4)After combined with autophagy inhibitor,serum cytokines and transaminases increased and intrahepatic inflammation were aggravated again in ALF mice.Monocyte apoptosis was also reduced.Conclusion:dmPGE2 attenuated D-GalN/LPS-induced ALF.The mechanism may be through promoting autophagy-dependent apoptosis of monocytes.PGE2 pathway might be a new potential target for ALF treatment.