Fgl2 Regulates Liver Fibrosis Progression By Promoting The Macrophage Phenotype

?BACKGROUND&AIMS?China is one of the countries with a high incidence of chronic hepatitis.In the past decades,with the popularization of hepatitis B vaccine,advancement of antiviral therapy and the improvement of medical and health conditions,the infection rate of viral hepatitis has decreased,but the number of deaths caused by hepatitis is increasing.The main cause of death from viral hepatitis is chronic liver disease,especially cirrhosis.Currently,our country is still facing the great challenge of chronic liver disease,which is a public health issue.Therefore,it is very important to study the pathogenesis of liver fibrosis and cirrhosis and provide new ideas for its basic research and clinical treatment.The essence of hepatic fibrosis is the repair response of the liver to chronic injury.The mechanism underlying fibrogenic activation depends on a complex interplay of damaged hepatocytes,non-parenchymal cells,recruited immune cells,and their secreted factors and signaling pathways.If liver fibrosis is not intervened in the early stage of the disease,the repair reaction after persistent liver injury can lead to excessive deposition of extracellular matrix?ECM?,scar formation,and eventually progress to cirrhosis and even hepatocellular carcinoma.Studies have confirmed that the activation of hepatic stellate cells?HSCs?plays a central role in the occurrence and development of hepatic fibrosis.Activated HSCs recruit more inflammatory cells to the injury through proliferation,chemotaxis,contraction and collagen synthesis,amplify the inflammatory response,and further cause liver injury,liver dysfunction,intrahepatic structural reconstruction,and produce fibroid lesions.The activation of HSCs is directly regulated by hepatic macrophages.As one of the important immune organs of the body,the liver is the organ where cytokines and complement components are produced.It contains a large number of macrophages,lymphocytes,and antigen presenting cells.The innate immunity of the liver plays a key role in host defense against microbial invasion,hepatic injury,and repair by regulating the balance between anti-inflammatory and pro-inflammatory cytokines.Among them,liver macrophages play an important role in maintaining liver homeostasis in the complex liver immune microenvironment and are also"fuel"or"brake"for the progression of liver fibrosis.Liver macrophages play a pivotal role in liver fibrosis,which is determined by their highly heterogeneous biological characteristics.According to its source,liver macrophages can be divided into resident macrophages,namely Kupffer cells?KCs?,and monocyte-derived macrophages recruited from peripheral monocytes.In different liver microenvironments,macrophages of different phenotypes play different roles in the occurrence,development,and reversal of liver fibrosis.According to its phenotype and function,hepatic resident macrophages can be divided into proinflammatory M1 and regulatory M2 types.The former mainly plays the immune defense function of host anti-microorganism,though it can cause inflammation and aggravate injury of tissues.M2 macrophages play an anti-inflammatory role in the later stage of inflammation,promote tissue repair and remodeling,and have anti-fibrotic functions.Another important source of liver macrophages is derived from circulating monocytes.Studies on mouse models revealed two major phenotypes:Ly6C^hii and Ly6C^loo monocytes.Monocyte-derived macrophages recruited to the liver can differentiate into Ly6C^loo phenotype macrophages under the action of CX3CL1 in the specific microenvironment of the liver.The macrophages of this subset secretes matrix metalloproteinases?MMPs?which can degrade the extracellular matrix,thus promoting the reversion of liver fibrosis.Further study on the function of monocyte-derived macrophages and related molecular mechanisms will help us understand the pathogenesis of chronic liver injury and fibrosis more comprehensively,thus providing a new direction for the treatment of liver fibrosis.Fibrinogen-like protein 2?Fgl2?is a member of the fibrinogen-related protein superfamily.It was observed that Fgl2 was significantly up-regulated in inflammatory-related diseases such as fulminant hepatitis,severe hepatitis B and allograft rejection while promoting disease progression.Fgl2 is expressed in macrophages,endothelial cells,dendritic cells,and T cells,with the highest level found in macrophages.Previous studies have found that in patients with chronic obstructive pulmonary disease?COPD?,up-regulation of Fgl2 is associated with the activation of alveolar macrophages,while down-regulation of Fgl2 reduces the activation of macrophages,suggesting that Fgl2 may play a potential role in promoting macrophage polarization.However,there is no report on the role and mechanism of Fgl2 in the course of liver fibrosis.This study was designed to observe the expression of Fgl2 in liver tissues of clinically confirmed hepatitis B patients with hepatic fibrosis and mouse models,to determine the relationship between Fgl2 expression and the progression of liver fibrosis,and to further identify the main cell sources of Fgl2 in the liver.On this basis,combined with in vivo and in vitro experiments,the effects of Fgl2 on the process of hepatic fibrosis,the regulation of polarization function of hepatic macrophages and the activation of HSCs were discussed in order to elucidate the molecular mechanism of hepatic fibrosis and provide new ideas for the treatment of liver fibrosis.?METHODS?1.Transcription levels of FGL2 and COL1A1 in patients with clinically proven HBV-induced fibrosis were detected by Realtime-PCR.The correlation of hepatic COL1A1 mRNA with FGL2 was analyzed.2.Carbon tetrachloride?CCl₄?was used to induce hepatic fibrosis model in mice.Hepatic mRNA of Fgl2 and Col1a1 were measured by Realtime-PCR.Protein levels of Fgl2 and Col-I in fibrotic livers were analyzed by Western Blotting.3.Paraffin-embedded sections of liver tissues from mice injected with vehicle or CCl₄were stained with H&E,Sirius Red,and immunohistochemical staining to detect the expression and localization of Fgl2 in the liver.4.The primary hepatic macrophages and sinusoidal endothelial cells were isolated and purified by in situ perfusion digestion,density gradient centrifugation,and magnetic bead sorting.The expression of Fgl2 in different types of primary liver cells was further detected by Realtime-PCR.5.Carbon tetrachloride?CCl₄?and thioacetamide?TAA?were used to induce hepatic fibrosis in wild type?WT?mice and Fgl2 knockout(Fgl2^-/-)mice.In regards to the resolution of hepatic fibrosis,mice were sacrificed after a 14-day recovery period.Liver sections from WT and Fgl2^-/-mice were stained with H&E and Sirius Red.Liver function was assessed by serum levels of ALT and AST.And hepatic hydroxyproline was detected.6.Transcription levels of fibrogenic genes Col1a1,?-Sma,Tgfb1,and matrix metalloproteinases?Mmp-2,-9-13?were measured in WT and Fgl2^-/-mice.7.Immunohistochemistry was used to detect the expression of F4/80 in the liver in WT and Fgl2^-/-mice.8.Whole-liver protein levels of Tnf?,Il-1?,and Il-6 and Ccl2 were measured by ELISA to analyze the expression of macrophage activation-related cytokines.9.Flow cytometry was used to compare the proportion and the absolute number of hepatic resident macrophages(F4/80^hii CD11b^int)and monocyte-derived macrophages(CD11b^hii F4/80^int)in WT and Fgl2^-/-mice at fibrosis progression and rregression period,respectively.The proportion and absolute number of M1 and M2 resident macrophages and Ly6C^hii and LyC6^loo monocyte-derived macrophages were further analyzed.10.WT and Fgl2^-/-mice were subjected to Muramyl dipeptide?MDP?.Flow cytometry was used to detect the monocytes in peripheral blood.11.CCl₄-induced hepatic fibrosis model was established.MDP was administrated by intravenous injectionat at the 3rd week of mouse model.Flow cytometry was used to detect peripheral blood monocyte subsets and hepatic macrophage subsets in WT and Fgl2^-/-mice.Sirius Red staining was used to evaluate the intervention on hepatic fibrosis.12.Bone marrow-derived macrophages were isolated.M1 and M2 macrophages were stimulated and polarized by cytokines.Inflammatory cytokines in M1 and M2macrophages were detected by Realtime-PCR.13.Primary HSCs were isolated and purified by in situ perfusion digestion,density gradient centrifugation,and magnetic bead sorting.The quiescent and activated hepatic stellate cells were identified after 3 and 7 days of culture in vitro.14.M1 macrophages were co-cultured with primary HSCs in vitro.The morphology of HSCs was detected by phalloidin fluorescence staining,and the expression of?-Sma and Col1a1 in HSCs was detected by Realtime-PCR.?RESULTS?1.Increased expression of Fgl2 in liver tissues of patients with chronic hepatitis B and mice with hepatic fibrosis.Levels of FGL2 were significantly higher in patients with advanced fibrosis?stage 4-6?compared to those with lower stages of fibrosis?stage 2-3?.FGL2 was positively correlated with the level of COL1A1 in the fibrotic liver.Hepatic levels of FGL2 in patients were significantly decreased after 60 weeks of antiviral and antifibrotic treatment along with the histological regression of fibrosis??1 unit decrease by Ishak scoring?.Consistently,Fgl2 mRNA and protein levels were elevated in fibrotic liver from CCl₄-induced hepatic fibrosis model.Collectively,these data suggest that Fgl2 is predominantly induced in response to chronic liver injury.2.Fgl2 is mainly produced by hepatic macrophages in liver fibrosis.During liver fibrosis,Fgl2 was profoundly induced in F4/80⁺KCs and,to a lesser extent,in CD31⁺liver sinusoidal endothelial cells?LSECs?.Quantitative results showed that Fgl2 mRNA level in KCs was significantly higher than that in LSECs.3.Fgl2 deficiency retards murine liver fibrosis and promotes reversal.CCl₄ and TAA-induced liver fibrosis models were established in WT mice and Fgl2^-/-mice.During the progression period of liver fibrosis,we found that liver damage was significantly ameliorated in Fgl2^-/-mice.The levels of serum ALT and AST,collagen deposition and hydroxyproline in Fgl2^-/-mice were significantly lower than those in WT mice.The hepatic expression of the fibrotic gene Col1a1,and genes associated with HSC activation such as?-Sma and Tgfb1,were significantly decreased in Fgl2^-/-mice.Similarly,profibrotic and fibrolytic matrix metalloproteinases?Mmp-2,-9,and-13?were also coordinately altered in Fgl2^-/-mice.Taken together,these data show that Fgl2promotes liver fibrosis during chronic liver injury.4.Fgl2 deficiency successfully attenuated the liver inflammatory response to CCl₄treatment.In liver sections of Fgl2^-/-mice,the number of F4/80⁺macrophages per field was significantly decreased.In line with this alleviated macrophage accumulation,chemokines and inflammatory cytokines such as Tnf?,Il-1?,and Il-6 and Ccl2 were significantly decreased in Fgl2^-/-mice when compared with WT littermates,suggesting an overall change in macrophage phenotype.5.Loss of Fgl2 facilitates infiltrating macrophage transition from profibrotic into restorative function.The proportion and the absolute number of macrophage subsets,including resident macrophage(F4/80^hii CD11b^int)and monocyte-derived macrophage(CD11b^hii F4/80^int)in Fgl2^-/-mice were significantly decreased during fibrosis progression and regression.Further analysis showed that in Fgl2^-/-mice,the number of fibrogenic macrophages including M1 polarized KCs and Ly6C^hii monocytic macrophages decreased significantly,while restorative macrophages,including M2 KCs and Ly6C^lo,were remarkably increased in Fgl2^-/-mice compared to the same levels in WT mice during fibrosis progression.6.Muramyl dipeptide?MDP?slows liver fibrosis subsequent to the transition of profibrotic macrophages into restorative macrophages.MDP administration increased the monocyte pool and promoted monocytes transition from Ly6C^hii into Ly6C^loo subset in WT and Fgl2^-/-mice.Moreover,such a transition was more enhanced in Fgl2^-/-mice,compared to that in WT mice.MDP treatment inhibited ongoing fibrosis following a CCl₄ challenge in both WT and Fgl2^-/-mice.Furthermore,MDP administration promoted a faster transition of hepatic monocytes from Ly6C^hii into Ly6Cⁱntnt and terminal Ly6C^loo macrophages,as revealed by a higher level of the Ly6Cⁱntnt macrophage subset in Fgl2^-/-mice than in WT mice during fibrosis.7.Fgl2 regulates polarization of macrophages The expression of specific inflammatory factors?Tnfa,Nos2,Il-6,Il-1?,and Ccl2?were significantly down-regulated in Fgl2^-/-M1 macrophages,while the expression of inflammatory factors?Il-10,Tgfb1,Arg-1,Ym-1,and Fizz 1?in Fgl2^-/-M2 macrophages were significantly increased.8.An in vitro Fgl2 deficiency impaired activation of polarized macrophages to HSCs.In vitro co-culture system of Fgl2^-/-M1 macrophages with HSCs,the morphology and activation markers of HSCs,?-Sma and Col1a1 synthesis were significantly downregulated than those of WT group.?CONCLUSIONS?1.In this study,we found that increased expression of Fgl2 in liver tissues of patients with chronic hepatitis B and mice with hepatic fibrosis.Fgl2 is mainly produced by hepatic macrophages in liver fibrosis.2.Fgl2 regulates polarization of macrophages,and promotes liver fibrosis progression by maintaining the profibrotic macrophage phenotype.3.Fgl2 promotes activation of hepatic stellate cells by directly regulating polarization of M1 macrophages.