Effects And Underlying Mechanisms Of Regulation Of Macrophage-mediated Immune Inflammatory Response On Acute Liver Injury

Acute liver injury is a common liver disease in daily clinical practice.The major causes of acute liver injury include drug overdose,toxin exposure,hepatitis virus infection and ischemia/reperfusion（IR）.Serious acute liver injury may lead to acute liver failure and threaten patients’life.Although the pathogenesis of acute liver injury remains unclear,the activation of hepatic immune inflammatory response has been proposed to play critical roles in the progression of acute liver injury.Macrophage which is the most population of innate immune cells in the liver is responsible for maintenance of hepatic immune homeostasis.Upon different stimulations,macrophages are activated to initiate hepatic immune inflammatory response resulting in exacerbated liver injury.Thus,regulation of macrophage-mediated proinflammatory response have been currently the focus and difficulty in studies of acute liver injury.This present study aimed to investigate whether and how regulation of macrophage function engaged in acute liver injury using two different types of models through genetic and pharmacological methods,respectively.Our findings might provide the rationale and new ideas for the application of regulating macrophage-mediated proinflammatory response in treating acute liver injury.The subject includes the following two parts:Part 1:TGR5/Cat E signaling regulates macrophage migration and polarization in liver ischemia/reperfusion injuryBackground:Hepatic immune inflammatory response represents one of the major causes of liver ischemia/reperfusion（IR）injury,macrophages-mediated proinflammatory response plays critical roles in IR-induced hepatic inflammation.Studies have suggested bile acid-responsive G-protein-coupled receptor（TGR5）as a key regulator of innate immune activation.Our previous study has demonstrated that TGR5 protects against liver IR injury by suppressing hepatic inflammation,however,the underlying mechanisms remains unclear.Aim:To investigate the function and the mechanism of TGR5 in macrophages in a murine partial warm hepatic ischemia model.Methods:To study the function of TGR5 in bone marrow derived macrophages（BMDMs）in a murine partial warm hepatic ischemia model,we generated chimeric mice through bone marrow transplantation using wild-type（WT）and TGR5 knockout(TGR5^-/-)mice as follows:WT→WT（expressing TGR5 in both myeloid cells and nonmyeloid cells）,WT→TGR5^-/-（expressing TGR5 only in myeloid cells）and TGR5^-/-→WT（expressing TGR5 only in nonmyeloid cells）.BMDMs isolated from WT and TGR5^-/-mice were used to investigate the effect of TGR5 on migration and polarization of BMDMs in vitro.A microarray was performed to compare the gene profiles between WT and TGR5^-/-BMDMs primed with LPS.Ten successive patients who were preparing for partial hepatectomy with portal triad clamping were randomly divided into two groups according to the presence or absence of ursodesoxycholic acid（UDCA）administration.Peripheral blood mononuclear cells（PBMCs）were collected from these patients 1h before the operation and 24 h after the operation and were subjected to flow cytometry.Results:The TGR5^-/-→WT chimeric mice developed more severe liver IR injury with an enhanced proinflammatory immune response in the livers compared with the WT→WT and WT→TGR5^-/-mice.Upon LPS stimulation,TGR5-deficient bone marrow-derived macrophages（BMDMs）displayed increased mobility toward MCP-1and were more likely to be polarized to the proinflammatory type（M1）compared to WT BMDMs.INT-777,a TGR5 agonist,enhanced the anti-inflammatory effect of TGR5 both in vivo and in vitro.Microarray profiling of WT and TGR5^-/-BMDMs primed with LPS revealed that TGR5^-/-BMDMs exhibited increased cytokine secretion and enhanced chemotactic characteristics.Cathepsin E（Cat E）was the most upregulated gene in TGR5^-/-BMDMs,and knockdown of Cat E with Cat E-siRNA significantly abolished the enhanced mobility and shift of macrophage phenotypes induced by TGR5 deficiency.Moreover,Cat E knockdown attenuated liver IR injury and hepatic inflammation in TGR5^-/-→WT chimeric mice.Correspondingly,IR stress significantly activated TGR5 in human peripheral macrophages,correlating with the shift in macrophage M2 polarization.UDCA administration enhanced TGR5activation and the trend of macrophage M2 polarization.Conclusion:TGR5 inhibits macrophage migration and M1 polarization by suppressing Cat E expression,leading to a reduced proinflammatory response in IR-stressed livers.Thus,targeting TGR5/Cat E axis could provide therapeutic benefit for liver IR injury.Part 2: Spermine regulates liver-resident macrophages proinflammatory response in acute liver injuryBackground: Liver-resident macrophages,Kupffer cells（KCs）play critical roles in toxin-induced acute liver injury.Recent evidence indicates that autophagy can regulate macrophage M1/M2 polarization under different inflammatory conditions.Spermine is one of the polyamines which possess anti-oxidative and anti-aging properties.Emerging evidence has indicated that spermine plays protective effect on several organ or cell injury by inducing autophagy,however,the function and mechanism of spermine in toxin-induced acute liver injury remains ambiguous.Aim: To explore the function and mechanism of spermine in modulating KCs proinflammtory response in thioacetamide（TAA）-induced acute liver injury.Methods: TAA was peritoneally injected to generate murine TAA-induced acute liver injury model.The severity of TAA-induced liver injury and hepatic inflammation were examined by HE staining and immunohistochemical staining respectively.KCs were isolated from TAA-stressed livers and were cultured in vitro.The effect of TAA and spermine on KCs inflammatory activation and autophagy induction were assessed.si RNA was used to investigate the relationship between spermine-mediated autophagy and TAA-induced KCs proinflammatory activation both in vivo and in vitro.Results: Spermine pretreatment significantly alleviated TAA-induced acute liver injury and restricted hepatic inflammatory response.KCs in TAA-stressed livers polarized towards M1 phenotype and enhanced hepatic inflammation.Spermine inhibited TAA-induced KCs M1 polarization but shifted KCs towards M2 phenotype,as evidenced by decreased IL-1β and i NOS gene induction but increased Arg-1 and Mrc-1 gene induction accompanied by decreased proinflammatory cytokines（TNF-α,IL-6）secretion and increased anti-inflammatory cytokine（IL-10）secretion.Furthermore,spermine enhanced autophagy by activating ATG5,as revealed by increased LC3B-II protein levels,decreased p62 protein levels and increased ATG5 protein levels in TAA-stressed KCs.Knockdown of ATG5 in spermine-pretreated KCs by si RNA resulted in a significant increase in proinflammatroy TNF-α and IL-6 secretion and decreased anti-inflammatory IL-10 secretion after TAA treatment,while no significant changes were observed in cytokine production in the TAA treatment alone.Conclusion: Spermine-mediated autophagy inhibits M1 polarization,while promoting M2 polarization of KCs in TAA-treated livers via upregulation of ATG5 expression,leading to attenuated liver injury.Our results provides a novel target for the prevention of acute liver injury.