| ?BACKGROUND & AIMS? Severe hepatitis is a liver failure syndrome that involves a variety of pathogenic factors which leads to a large or sub-block necrosis of the liver in a short term.Currently,severe hepatitis in foreign countries are mainly acute liver failure caused by poisoning such as drugs and alcohol.Severe hepatitis in our country is mostly caused by hepatitis virus,especially hepatitis B virus(about 70%).Although reasonable antiretroviral therapy is effective in reducing liver failure caused by HBV,severe hepatitis caused by HBV is still a common disease in many remote areas due to inappropriate antiretroviral therapy or even blind withdrawal.The disease is serious,rapid progressed,lacking of clinical sensitive early warning indicators and effective means of specific interventions and multiple complications,mortality is still as high as 40-60%.Therefore,it is very necessary to study the pathogenesis in depth and provide a scientific basis and target of intervention for clinical treatment.Abnormal host immune response caused by HBV under certain conditions is the central part of the pathogenesis of severe hepatitis.Pathomorphology and immunological studies have shown that early microcirculation and apoptosis are critical for the progression of severe hepatitis and also demonstrate that short-term progressive necrosis of hepatocytes in severe hepatitis is closely related to host immune damage,but the specific mechanism still remains to be elucidated.With the development of modern life sciences,immunologists have developed a new consensus on the liver,the composition and functional characteristics of liver-specific hematopoietic immune cells revealed that liver is not only a substantial organ,but also an unique innate immunity predominanced immune Organ which can directly affect the occurrence and development of liver disease and prognosis.In the unique double blood support,a large number of innate immune cells in the liver immune microenvironment constitutes an absolute dominant group,maintaining the liver immune tolerance.Kupffer cells(KCs)occupy the highest proportion,accounting for 80%-90% of the total amount of human macrophages,far beyond the composition of similar cells in the peripheral and other tissues and organs.It is reported that every 100 hepatocytes are accompanied by 20-40 macrophages in a healthy rodent liver.Numerous studies of mononuclear macrophages in recent years have enriched our understanding of liver monocyte-macrophages,it became apparent that hepatic macrophages represent heterogeneous populations in health and disease.The tremendous progress in understanding macrophage heterogeneity in health and disease not only has great implications for our basic knowledge of liver immunology but also opens the possibility to develop new strategies to therapeutically modulate liver macrophages for the treatment of liver diseases.CD11b-F4/80 hi KCs originate from the embryonic yolk sac and are capable of self-replication during homeostasis and after liver injury.In addition,peripherally infiltrating monocytes(IMs)can give rise to monocytes Cell-derived macrophages(Mo MFs)to replenish macrophage pools.During liver injury,monocytes are strongly recruited to the liver,and the phenotype is gradually matured from CD11b+F4/80-into CD11bhiF4/80 int Mo MFs which can acquire a KC-like state.Previous studies have shown that macrophages exist in a series of continuous functional states,which can be divided into two types of polydispersion according to their phenotypes and the secreted cytokines,ie Classically activated M1 macrophage and Alternatively activated M2 macrophage,.M1 macrophages mainly mediate proinflammatory effects,while M2 macrophages can inhibit inflammation,immune regulation and tissue repair by expressing anti-inflammatory cytokines.IMs can also be divided into Ly6 Chi Mo and Ly6 Clo Mo according to the expression level of Ly6 C.The Ly6 Chi monocytes expressed exclusively proinflammatory genes associated with classical activation M1 phenotype including triggering receptor expressed on myeloid cells-1(Trem1),NO synthase 2(Nos2),COX2(Ptgs2),and the chemokines Ccl2 and Ccl7.Ly6 Chi monocytes were short-lived and soon converted to Ly6 Clo monocytes which expressed a wider panel of genes associated with both alternatively activated regulatory M2 and prorestorative wound-healing MF phenotypes,including arginase I(Arg1),Lcn2,chitinase-3-like protein 3(Chil3l3,alsoknownas Ym1),IL-4Rsubunit a(Il4ra),TNF ligand superfamily member 14(Tnfsf14),TNF,TNFAIP3-interacting protein 3(Tnip3,also named Abin3),and the a-induced protein3(Tnfaip3,also named A20).In different histopathological conditions,monocyte-macrophages can affect the progression and prognosis of the disease through the M1 / M2 polarization balance shift and Ly6 Chi / Ly6 Clo conversion.Therefore,in-depth study of subtypes conversion,function and regulation of monocyte-macrophage in liver inflammation injury is of great significance to understand the mechanism of liver injury.The mechanism of polarization of monocyte-macrophage cells is extremely complicated.Our previous study confirmed that FGL2 was highly expressed in the liver of severe hepatitis B patients and fulminant hepatitis mouse model.In vitro differentiation of THP-1 cells into M2 macrophages or M1 macrophages,M1 macrophages successfully secreted large amounts of proinflammatory cytokines such as TNF? and highly expressed fgl2 compare to M2 macrophages.CCR7 and CD80 were highly expressed on Fgl2 + M1 macrophages.On the basis of previous study,we intend to use liver monocyte-macrophage cells as the core to study the effect of fgl2 on macrophage polarization and their relationship with the progression of severe viral hepatitis based on the animal model,clinical specimens and in vitro experiments,and further explore the relevant signaling pathways and mechanisms to provide a theoretical basis for further elucidating the immunological pathogenesis of severe hepatitis and provide new ideas for the development of a new type of treatment for severe hepatitis.?METHODS? 1.The liver tissue serial sections were immunohistochemically examined for the distribution of M1(CD68 + CD80 +)and M2 macrophages and the expression of fgl2 in the liver specimens of healthy controls and patients with severe hepatitis B.Evaluate the co-expression of fgl2,CD68 and CD80;2.Establish fulminant Hepatitis(FH)mice model by intraperitoneal injection of mouse strain hepatitis virus 3(MHV-3).HE staining of liver slices and measurement of plasma alkaline phoshatase(ALT)and alanine aminotransterase(AST)level were used to assess the degree of liver inflammation.Liver macrophages were isolated by hepatic portal vein perfusion / digestion and density gradient centrifugation.The number of intrahepatic KCs(CD11b-F4/80hi)and Mo MFs(CD11bhiF4/80int),the cytokine profile of KCs,the ratio / proportion of M1(i NOS+)and M2(CD206+)in KCs,the ratio / proportion of Ly6 ChiMo MFs and Ly6 CloMo MFs and the expression of fgl2 on the above mentioned cells were dynamically detected by Flow Cytometry;3.Wild-type(WT)and fgl2 knock-out(fgl2-/-)BALB/c mice were separately injected with chlodronate liposomes or PBS liposomes through tail vein to deplete liver macrophages 24 hours prior FH establishment,HE staining,MPO immunohistochemistry staining of liver slices and measurement of plasma ALT and AST level were used to assess the degree of liver inflammation.4.FH model was established in WT and fgl2-/-mice.Flow cytometry was used to dynamically evaluate the changes of M1 and M2 subtypes of KCs and their cytokines profiles,the number of IMs and the ratio of Ly6 Chi and Ly6 Clo monocytes between these two groups.5.WT and fgl2-/-bone marrow-derived macrophages(BMDM)were differentiated and induced by culture bone marrow cells with medium containing macrophage colony-stimulating factor(M-CSF)in vitro.Different stimulators were used to differentiate BMDM into M1 or M2(LPS,MHV-3,IL-4).Cytokine and metabolite levels in cell culture medium were measured by flow cytometry bead array(CBA),Griess reagent system and ELISA,M1 or M2 associated m RNA expression levels were measured by Real-time PCR between these two groups;6.Peritoneal macrophages(PEM)were recruited by intraperitoneal injection of starch broth and extract by peritoneal lavage,and then stimulated in vitro,the levels of cytokines,metabolites and M1 Or M2 m RNA expression were detected by the same methods mentioned in method 5(same as BMDM).7.The WT and fgl2-/-PEMs were extracted by pre-stimulation of starch broth or MHV-3 injection.Flow cytometry was used to compare the surface expression of co-stimulatory receptors(CD80,CD86,MHCI And MHCII)and a Phagotest kit was used to test the PEMs phagocytosis ability of two groups;8.WT and fgl2-/-mice BMDM cells were stimulated in vitro with LPS and MHV-3 for 15 min and 30 min,protein of the stimulated cells and unstimulated control cells were extracted,protein phosphorylation of pro-inflammation signal pathways were detected by Western Blot;9.293 T cells were transfected by lentivirus(produced by lipofectamine3000-plasmid transfected 293T)and further screened by puromycin to established a flag-labeled fgl2 over-expressed and flag control 293 T cells,Western Blot verify flag and fgl2 protein overexpression;(Plasmids used in this study were kindly provided by Dr.Xiaoyang Wan)10.Flag-labeled fgl2 over-expressed cells(or control)and THP-1 cells were lysed and amplified,proteins which binding fgl2 were screened by co-immunoprecipitation(CO-IP)and then were sent to the company for proteomics analysis;?RESULTS? 1.The number of CD68 + macrophages in the liver of patients with severe hepatitis B increased significantly compared with healthy controls,and CD68+ macrophage were mainly concentrated in the area of inflammation and necrosis.The number of CD80 + M1 macrophages was also significantly increased compare to healthy control;The expression of fgl2(mianly co-expressed with CD80 on CD68+macrophages)in the liver of patients with severe hepatitis B were significantly higher than healthy controls.2.FH model was successfully established in BALB/c mice.Post 48 hours of MHV-3 injection,the liver showed multiple flake or massive necrosis,the plasma level of aminotransferase were extremely high.Mice died around 72 hours post MHV-3 injection with a final mortality rate of 100%.We observed a significant depletion of KCs in the liver during FH progression but also an extensive periphery monocytes infiltration into the liver.KCs and monocytes in healthy mice were in a relatively anti-inflammatory state(M2 and LY6 Clo dominant).During the early phase of FH,KCs experienced a M1 polarization(M1 KCs increased significantly)and fgl2 expression on M1 macrophages was significantly increased;The proportion of Ly6 Chi monocytes in Mo MFs increased significantly and the expression of fgl2 on Ly6 Chi IMs was also increased markedly along with the progress of liver inflammation,while the expression of fgl2 in Ly6 Clo did not change during the whole course of FH;3.The severity of liver inflammation in WT-CL and fgl2-/--PBS groups were significantly reduced compare to WT-PBS group and there was no significant difference in the severity degree between WT-CL and fgl2-/--PBS group.The mice in fgl2-/--CL group showed less severe histological appearance and lower transaminase level than those in WT-CL and fgl2-/--PBS groups,but there was no significantly difference in MPO infiltration between these three groups.4.Compare to WT FH mice,the severity of liver inflammation was significantly reduced in fgl2-/-FH mice,the early M1 polarization of KCs were significantly weakened,number of IMs was decreased and Ly6 Chi Mo MFs ratio was also much lower in fgl2-/-FH mice;5.After stimulated by LPS and MHV-3,the release of pro-inflammatory cytokines of BMDM was significantly higher than that of unstimulated cells.The supernatant concentration of M1 cytokines and NO metabolites(IL-6,TNF,IL-1?,MCP-1 and NO2-)of fgl2-/-BMDM were markedly decreased compare to WT BMDM and so was the expressions of M1-related m RNAs(NOS2,IL-6,TNF-?,IL-1?,IL-12 and marco);similar results were obtained by stimulating PEM in vitro;IL-10 concentration were significantly increased in IL-4 stimulated fgl2-/-BMDM than that of WT BMDM and so was M2-related m RNAs(YM-1,Alox15,ARG1 and fizz1)expression;6.After stimulation by starch broth or MHV-3,the expression of costimulatory molecule MHC II on fgl2-/-PEMs surface decreased obviously and the phagocytic function was weakened compare to WT PEMs.7.The expression of fgl2 in BMDM of WT mice was significantly increased after stimulation with LPS and MHV-3.Compare to WT BMDM,the phosphorylation levels of multiple pro-inflammatory pathways including MAPK pathway(P38,ERK1 / 2 and JNK),NF-k B pathway(Ik Ba),by-pass pathway(BTK y223)and IRF3 were significantly reduced in fgl2-/-BMDM cells no matter LPS or MHV-3 stimulation;8.successfully established flag labeled fgl2 over-expression cell lines;9.Complete co-immuno precipitation pre-experiment,to be repeated and verified.?CONCLUSIONS? 1.In this study,we found that with the aggravation of liver inflammation,Kupffer cellsand experienced a persistent depletion with a strong infiltration of peripheral monocytes into the liver for replenishment,and both Kupffer cells and monocyte-derived macrophages exhibited a pro-inflammatory polarization state;2.This study confirmed that fgl2 could regulate the pro-inflammatory polarization of hepatic macrophages and promote the development of severe hepatitis through the study of liver tissue of severe hepatitis B patients,WT and fgl2-/-fulminant hepatitis mice and in vitro experiments.3.This study further revealed that fgl2 promotes the pro-inflammatory process of macrophages by regulating the phosphorylation of NF-k B,MAPK,IRF and BTK signal pathways;... |
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