Study On The Mechanism Of Hepatic Macrophage Infiltration In Fulminant Hepatitis

Background and objectiveFulminant hepatitis is one of the most important causes of death for patients with liver diseases,characterized by massive necrosis of hepatocytes and infiltration of inflammatory cells.The main causes of fulminant hepatitis vary in diverse regions,which is hepatitis virus in developing countries in Asia and drug poisoning in developed countries in the West.The incidence of fulminant hepatitis is low,while the clinical mortality rate is as high as 40～60%.The pathogenesis of fulminant hepatitis is still not clear,and current studies consider that regulation of the immune system is closely related to the occurrence and progression of it.MHV-3-induced acute liver failure is the most common model for fulminant hepatitis.Several researches have shown that parenchymal hepatocytes,cytokines and complements are all involved in the liver injury induced by MHV-3,and macrophages play an important role.The early depletion of liver macrophages can significantly reduce the virus titer and cytokine expression.In the progression of disease,peripheral monocytes are recruited to the liver via Ccr2 to promote the inflammatory response,while Kupffer cells are activated to secrete inflammatory cytokines and chemokines that recruit other immune cells into the liver so as to exacerbate the accumulation of inflammation.Lack of Ccr2 is associated with decreased monocyte infiltration and improved liver injury.Macrophage therapy has been recognized as a potential therapeutic option for liver diseases,for example,adoptive transfer of bone marrow-derived macrophages can effectively promote the regression of liver fibrosis in mouse models.Thus,liver macrophages are not only inflammatory cells,but also have the ability to regulate and limit the inflammatory response.The aim of this study was to investigate whether macrophage infiltration played an important role in the pathogenesis of fulminant hepatitis in mice model induced by MHV-3.Method1.The fulminant hepatitis group（MHV-3）and healthy control group（control）were established by intraperitoneal injection of MHV-3 and saline to WT C57BL/6 mice. After 56h,the liver nonparenchymal cells of mice were extracted for single-cell RNA sequencing,and bioinformatic analysis was conducted by using R language to determine the cell subsets,differential genes among cell subsets,biological functions of cells and ligand-receptor interactions between cells.2.The fulminant hepatitis mouse model was established by intraperitoneal injection of MHV-3 to WT C57BL/6 mice and Ccr2 KO C57BL/6 mice,followed by survival rate experiment.And after infection of MHV-3 0h,24h,48h and 72h,the liver tissues were collected for HE staining,serum ALT and AST levels were detected to evaluate the liver function,and liver nonparenchymal cells were extracted for flow cytometry to analyze the infiltration of liver immune cells.Result1.In the liver of mice with fulminant hepatitis,the account and proportion of T cell, ILC and Neutrophil were increased,while the account and proportion of B cell,DC and Macrophage were decreased.2.The top 10 genes significantly up-regulated in the liver macrophages of mice with fulminant hepatitis were Tnfrsf4/Tmem123/Bcl2a1d/Ly6a/Socs2/Il7r/Ppia/Rgs1/Ms4a6c/Ms4a4c,while the top 10 genes significantly down-regulated were Ngp/Gm5483/Retnlg/Mmp9/C1qa/C1qb/C1qc/Pglyrp1/Ifitm6/Wfdc17.3.The number and proportion of MHV-3.Mo were increased in the liver of mice with fulminant hepatitis,while the number and proportion of Monocyte（1）, Monocyte（2）,Monocyte（3）and Kupffer cell were decreased.4.The main functions were viral response and interferon-mediated signaling pathway for Monocyte（1）,cytokines-mediated signaling pathway for Monocyte（2）,cellular metabolism for Monocyte（3）,cytolysis for MHV3.Mo,and cytokine production for Kupffer cell.5.Compared with resting macrophages,viral response was weakened,cytokine production and phagocytosis were partially weakened or enhanced,as well as chemotaxis and cytolysis were enhanced for MHV3.Mo.6.In the liver of mice with fulminant hepatitis,ligand-receptor interactions between Macrophage and other type of nonchemical hepatocytes were enhanced,while that between Macrophage subsets were weakened.7.Compared with resting macrophages,the expression of Ccr2 was enhanced in MHV-3.Mo.In addition,the deletion of Ccr2 could alleviate the acute liver injury induced by MHV-3 though reducing the infiltration of inflammatory monocytes and increasing that of Kupffer cells.ConclusionIn the mouse model of fulminant hepatitis,hepatic macrophages had significant proinflammatory,chemotactic and cytolytic effects,while the viral response was weakened.The expression of Ccr2 was improved in MHV3.Mo,which might aggravate the injury of liver function through promoting the infiltration of inflammatory monocytes(Ly6C^hi).Thus,the study was of great significance in providing potential new therapeutic directions and targets for the immunotherapy of fulminant hepatitis.