Cutting Edge: Hepatic Stellate Cells Drive the Phenotype of Monocyte-derived Macrophages to Regulate Liver Fibrosis in Metabolic Dysfunction-associated Steatohepatitis.

Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by infiltration of monocyte-derived macrophages (MdMs) into the liver; however, the function of these macrophages is largely unknown. We previously demonstrated that a population of MdMs, referred to as hepatic lipid-associated macrophages (LAMs), assemble into aggregates termed hepatic crown-like structures in areas of liver fibrosis. Intriguingly, decreasing MdM recruitment resulted in increased liver fibrosis, suggesting that LAMs contribute to antifibrotic pathways in MASH. In this study, we determined that hepatic crown-like structures are characterized by intimate interactions between activated hepatic stellate cells (HSCs) and macrophages in a collagen matrix in a mouse model of MASH. MASH macrophages displayed collagen-degrading capacities, and HSCs derived from MASH livers promoted expression of LAM marker genes and acquisition of a collagen-degrading phenotype in naive macrophages. These data suggest that crosstalk between HSCs and macrophages may contribute to collagen degradation MASH.

Nimodipine ameliorates liver fibrosis via reshaping liver immune microenvironment in TAA-induced in mice.

Nimodipine, a calcium antagonist, exert beneficial neurovascular protective effects in clinic. Recently, Calcium channel blockers (CCBs) was reported to protect against liver fibrosis in mice, while the exact effects of Nimodipine on liver injury and hepatic fibrosis remain unclear. In this study, we assessed the effect of nimodipine in Thioacetamide (TAA)-induced liver fibrosis mouse model. Then, the collagen deposition and liver inflammation were assessed by HE straining. Also, the frequency and phenotype of NK cells, CD4+T and CD8+T cells and MDSC in liver and spleen were analyzed using flow cytometry. Furthermore, activation and apoptosis of primary Hepatic stellate cells (HSCs) and HSC line LX2 were detected using α-SMA staining and TUNEL assay, respectively. We found that nimodipine administration significantly attenuated liver inflammation and fibrosis. And the increase of the numbers of hepatic NK and NKT cells, a reversed CD4+/CD8+T ratio, and reduced the numbers of MDSC were observed after nimodipine treatment. Furthermore, nimodipine administration significantly decreased α-SMA expression in liver tissues, and increased TUNEL staining adjacent to hepatic stellate cells. Nimodipine also reduced the proliferation of LX2, and significantly promoted high level of apoptosis in vitro. Moreover, nimodipine downregulated Bcl-2 and Bcl-xl, simultaneously increased expression of JNK, p-JNK, and Caspase-3. Together, nimodipine mediated suppression of growth and fibrogenesis of HSCs may warrant its potential use in the treatment of liver fibrosis.

Human placental mesenchymal stromal cells promote the formation of CD8+CD122+PD-1+Tregs via CD73/Foxo1 to alleviate liver injury in graft-versus-host disease mice.

BACKGROUND: Human placental mesenchymal stromal cells (hPMSCs) are known to limit graft-versus-host disease (GVHD). CD8+CD122+PD-1+Tregs have been shown to improve the survival of GVHD mice. However, the regulatory roles of hPMSCs in this subgroup remain unclear. Here, the regulatory mechanism of hPMSCs in reducing liver fibrosis in GVHD mice by promoting CD8+CD122+PD-1+Tregs formation and controlling the balance of IL-6 and IL-10 were explored. METHODS: A GVHD mouse model was constructed using C57BL/6J and BALB/c mice and treated with hPMSCs. LX-2 cells were explored to study the effects of IL-6 and IL-10 on the activation of hepatic stellate cells (HSCs). The percentage of CD8+CD122+PD-1+Tregs and IL-10 secretion were determined using FCM. Changes in hepatic tissue were analysed by HE, Masson, multiple immunohistochemical staining and ELISA, and the effects of IL-6 and IL-10 on LX-2 cells were detected using western blotting. RESULTS: hPMSCs enhanced CD8+CD122+PD-1+Treg formation via the CD73/Foxo1 and promoted IL-10, p53, and MMP-8 levels, but inhibited IL-6, HLF, α-SMA, Col1α1, and Fn levels in the liver of GVHD mice through CD73. Positive and negative correlations of IL-6 and IL-10 between HLF were found in liver tissue, respectively. IL-6 upregulated HLF, α-SMA, and Col1α1 expression via JAK2/STAT3 pathway, whereas IL-10 upregulated p53 and inhibited α-SMA and Col1α1 expression in LX-2 cells by activating STAT3. CONCLUSIONS: hPMSCs promoted CD8+CD122+PD-1+Treg formation and IL-10 secretion but inhibited HSCs activation and α-SMA and Col1α1 expression by CD73, thus controlling the balance of IL-6 and IL-10, and alleviating liver injury in GVHD mice.

Fluorescence imaging sheds light on the immune evasion mechanisms of hepatic stellate cells mediated by superoxide anion.

Whether and how the reactive oxygen species generated by hepatic stellate cells (HSCs) promote immune evasion of hepatocellular carcinoma (HCC) remains mysterious. Therefore, investigating the function of superoxide anion (O2•-), the firstly generated reactive oxygen species, during the immune evasion become necessary. In this work, we establish a novel in situ imaging method for visualization of O2•- changes in HSCs based on a new two-photon fluorescence probe TPH. TPH comprises recognition group for O2•- and HSCs targeting peptides. We observe that O2•- in HSCs gradually rose, impairing the infiltration of CD8+ T cells in HCC mice. Further studies reveal that the cyclin-dependent kinase 4 is deactivated by O2•-, and then cause the up-regulation of PD-L1. Our work provides molecular insights into HSC-mediated immune evasion of HCC, which may represent potential targets for HCC immunotherapy.

Chimeric antigen receptor-modified macrophages ameliorate liver fibrosis in preclinical models.

BACKGROUND & AIMS: Treatments directly targeting fibrosis remain limited. Given the unique intrinsic features of macrophages and their capacity to engraft in the liver, we genetically engineered bone marrow-derived macrophages with a chimeric antigen receptor (CAR) to direct their phagocytic activity against hepatic stellate cells (HSCs) in multiple mouse models. This study aimed to demonstrate the therapeutic efficacy of CAR macrophages (CAR-Ms) in mouse models of fibrosis and cirrhosis and to elucidate the underlying mechanisms. METHODS: uPAR expression was studied in patients with fibrosis/cirrhosis and in murine models of liver fibrosis, including mice treated with carbon tetrachloride, a 5-diethoxycarbonyl-1, 4-dihydrocollidine diet, or a high-fat/cholesterol/fructose diet. The safety and efficacy of CAR-Ms were evaluated in vitro and in vivo. RESULTS: Adoptive transfer of CAR-Ms resulted in a significant reduction in liver fibrosis and the restoration of function in murine models of liver fibrosis. CAR-Ms modulated the hepatic immune microenvironment to recruit and modify the activation of endogenous immune cells to drive fibrosis regression. These CAR-Ms were able to recruit and present antigens to T cells and mount specific antifibrotic T-cell responses to reduce fibroblasts and liver fibrosis in mice. CONCLUSION: Collectively, our findings demonstrate the potential of using macrophages as a platform for CAR technology to provide an effective treatment option for liver fibrosis. CAR-Ms might be developed for treatment of patients with liver fibrosis. IMPACT AND IMPLICATIONS: Liver fibrosis is an incurable condition that afflicts millions of people globally. Despite the clear clinical need, therapies for liver fibrosis are limited. Our findings provide the first preclinical evidence that chimeric antigen receptor (CAR)-macrophages (CAR-Ms) targeting uPAR can attenuate liver fibrosis and cirrhosis. We show that macrophages expressing this uPAR CAR exert a direct antifibrotic effect and elicit a specific T-cell response that augments the immune response against liver fibrosis. These findings demonstrate the potential of using CAR-Ms as an effective cell-based therapy for the treatment of liver fibrosis.

Antiviral Toll-like Receptor Signaling in Non-Parenchymal Liver Cells Is Restricted to TLR3.

The role of non-parenchymal liver cells as part of the hepatic, innate immune system in the defense against hepatotropic viruses is not well understood. Here, primary human Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells were isolated from liver tissue obtained after tumor resections or liver transplantations. Cells were stimulated with Toll-like receptor 1-9 ligands for 6-24 h. Non-parenchymal liver cells expressed and secreted inflammatory cytokines (IL6, TNF and IL10). Toll-like receptor- and cell type-specific downstream signals included the phosphorylation of NF-κB, AKT, JNK, p38 and ERK1/2. However, only supernatants of TLR3-activated Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells contained type I and type III interferons and mediated an antiviral activity in the interferon-sensitive subgenomic hepatitis C virus replicon system. The antiviral effect could not be neutralized by antibodies against IFNA, IFNB nor IFNL, but could be abrogated using an interferon alpha receptor 2-specific neutralization. Interestingly, TLR3 responsiveness was enhanced in liver sinusoidal endothelial cells isolated from hepatitis C virus-positive donors, compared to uninfected controls. In conclusion, non-parenchymal liver cells are potent activators of the hepatic immune system by mediating inflammatory responses. Furthermore, liver sinusoidal endothelial cells were identified to be hyperresponsive to viral stimuli in chronic hepatitis C virus infection.

Crosstalk between hepatic stellate cells and surrounding cells in hepatic fibrosis.

Hepatic fibrosis represents as a dynamic pathological process characterized by the net accumulation of extracellular matrix in the progression of various chronic liver diseases, including viral hepatitis, alcoholic liver disease, and metabolic associated fatty liver disease (MAFLD). Activation of hepatic stellate cells (HSCs) is well-defined to play a central role in the initiation and progression of hepatic fibrosis. However, the activation of HSCs is affected by the complicated microenvironments in liver, which largely attributes to the communication between hepatocytes and multiple tissue-resident cells, including sinusoidal endothelial cells, bile duct epithelial cells, platelets, T cells, B cells, macrophages, natural killer cells, neutrophils, dendritic cells, in the direct or indirect mechanisms. Cellular crosstalk between HSCs and surrounding cells contributes to the activation of HSCs and the progression of hepatic fibrosis. Currently, accumulating evidence have proven the complexity and plasticity of HSCs activation, and further clarification of cellular communication between HSCs and surrounding cells will provide sufficient clue to the development of novel diagnostic methods and therapeutic strategies for hepatic fibrosis.

MiR-130a-3p Alleviates Liver Fibrosis by Suppressing HSCs Activation and Skewing Macrophage to Ly6Clo Phenotype.

Emerging evidences have highlighted the crucial role of microRNAs (miRNAs) in the liver cirrhosis, but the relationship between miR-130a-3p and liver cirrhosis is not entirely clear. As we all know, schistosomiasis, as one of the zoonoses, can lead to liver cirrhosis when it advances. In this study, we investigated the biological functions of miR-130a-3p on the liver fibrosis of schistosomiasis in vivo and in vitro. The mice infected with Schistosoma japonicum (S. japonicum) were treated with lentivirus vector (LV)-miR-130a-3p by hydrodynamic injection through the tail vein. Our findings showed significantly decreased expression of miR-130a-3p both in the serum of patients with cirrhosis and in the liver of mice infected with S. japonicum. The results showed that LV-miR-130a-3p could effectively enter into the liver and alleviate liver granulomatous inflammation and collagen deposition. Simultaneously, LV-miR-130a-3p-promoted macrophages presented the Ly6Clo phenotype, concomitant with the decreased expression of the tissue inhibitor of metalloproteinases (TIMP) 1, and increased the expression of matrix metalloproteinase (MMP) 2, which contributed to the dissolution of collagen. Furthermore, overexpression of miR-130a-3p not only inhibited the activation and proliferation of hepatic stellate cells (HSCs) but also induced the apoptosis of HSCs. In addition, we also confirmed that miR-130a-3p enables to bind with mitogen-activated protein kinase (MAPK) 1 and transforming growth factor-beta receptors (TGFBR) 1 and TGFBR2 genes and inhibit the expressions of these genes. Our findings suggested that miR-130a-3p might represent as the potential candidate biomarker and therapeutic target for the prognosis identification and treatment of schistosomiasis liver fibrosis.

CD8+ tissue-resident memory T cells promote liver fibrosis resolution by inducing apoptosis of hepatic stellate cells.

Non-alcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease that can progress to liver fibrosis. Recent clinical advance suggests a reversibility of liver fibrosis, but the cellular and molecular mechanisms underlying NASH resolution remain unclarified. Here, using a murine diet-induced NASH and the subsequent resolution model, we demonstrate direct roles of CD8+ tissue-resident memory CD8+ T (CD8+ Trm) cells in resolving liver fibrosis. Single-cell transcriptome analysis and FACS analysis revealed CD69+CD103-CD8+ Trm cell enrichment in NASH resolution livers. The reduction of liver CD8+ Trm cells, maintained by tissue IL-15, significantly delayed fibrosis resolution, while adoptive transfer of these cells protected mice from fibrosis progression. During resolution, CD8+ Trm cells attracted hepatic stellate cells (HSCs) in a CCR5-dependent manner, and predisposed activated HSCs to FasL-Fas-mediated apoptosis. Histological assessment of patients with NASH revealed CD69+CD8+ Trm abundance in fibrotic areas, further supporting their roles in humans. These results highlight the undefined role of liver CD8+ Trm in fibrosis resolution.

Single-Cell Transcriptomic Analysis Reveals a Hepatic Stellate Cell-Activation Roadmap and Myofibroblast Origin During Liver Fibrosis in Mice.

BACKGROUND AND AIMS: HSCs and portal fibroblasts (PFs) are the major sources of collagen-producing myofibroblasts during liver fibrosis, depending on different etiologies. However, the mechanisms by which their dynamic gene expression directs the transition from the quiescent to the activated state-as well as their contributions to fibrotic myofibroblasts-remain unclear. Here, we analyze the activation of HSCs and PFs in CCL4 -induced and bile duct ligation-induced fibrosis mouse models, using single-cell RNA sequencing and lineage tracing. APPROACH AND RESULTS: We demonstrate that HSCs, rather than PFs, undergo dramatic transcriptomic changes, with the sequential activation of inflammatory, migrative, and extracellular matrix-producing programs. The data also reveal that HSCs are the exclusive source of myofibroblasts in CCL4 -treated liver, while PFs are the major source of myofibroblasts in early cholestatic liver fibrosis. Single-cell and lineage-tracing analysis also uncovers differential gene-expression features between HSCs and PFs; for example, nitric oxide receptor soluble guanylate cyclase is exclusively expressed in HSCs, but not in PFs. The soluble guanylate cyclase stimulator Riociguat potently reduced liver fibrosis in CCL4 -treated livers but showed no therapeutic efficacy in bile duct ligation livers. CONCLUSIONS: This study provides a transcriptional roadmap for the activation of HSCs during liver fibrosis and yields comprehensive evidence that the differential transcriptomic features of HSCs and PFs, along with their relative contributions to liver fibrosis of different etiologies, should be considered in developing effective antifibrotic therapeutic strategies.

Hepatic Dendritic Cells in the Development and Progression of Metabolic Steatohepatitis.

Metabolic Associated Fatty liver disease (MAFLD) is a global health problem and represents the most common cause of chronic liver disease in the world. MAFLD spectrum goes from simple steatosis to cirrhosis, in between metabolic steatohepatitis with progressive fibrosis, which pathogenesis is not completely understood. Hence, the role of the immune system has become an important fact in the trigger of inflammatory cascades in metabolic steatohepatitis and in the activation of hepatic stellate cells (HSCs). Among, the more studied immune cells in the pathogenesis of MAFLD are macrophages, T cells, natural killer and dendritic cells. In particular, hepatic dendritic cells had recently attracted a special attention, with a dual role in the pathogenesis of MAFLD. These cells have the capacity to switch from a tolerant state to active state inducing an inflammatory cascade. Furthermore, these cells play a role in the lipid storage within the liver, having, thus providing a crucial nexus between inflammation and lipid metabolism. In this review, we will discuss the current knowledge on the dual role of dendritic cells in lipid accumulation, as wells as in the triggering of hepatic inflammation and hepatocytes cell death in metabolic steatohepatitis.

MicroRNA-124 inhibits hepatic stellate cells inflammatory cytokines secretion by targeting IQGAP1 through NF-κB pathway.

Liver fibrosis is a health concern that leads to organ failure mediated via production of inflammatory cytokines and fibrotic biomarkers. To date, there was no direct approved antifibrotic therapy, and current treatment was mainly the removal of the causative factor. Recent studies demonstrated that aberrant expression of miR-124 was involved in the progression of various liver diseases including hepatocellular carcinoma (HCC). However, whether miR-124 could function as a transcriptional regulator in the inflammatory cytokines secretion of liver fibrosis remains unclear. In this study, we demonstrated that the expression of miR-124 was downregulated in liver fibrosis tissues and TNF-α-induced LX-2 cells, concomitant with the upregulated expression of IQGAP1, suggesting that miR-124 and IQGAP1 might be associated with the development of inflammation in liver fibrosis. Therefore, we demonstrated that the overexpression of miR-124 and knockdown of IQGAP1 could lead to the downregulation of TNF-α, IL-1ß and IL-6. While knockdown of miR-124 or overexpression of IQGAP1 showed reversed results. Moreover, dual luciferase reporter assays demonstrated that miR-124 specifically targeted the 3'-UTR of IQGAP1, and thus inhibited the expression of IQGAP1. Mechanistically, we found that the expression changes of miR-124 and IQGAP1 could be involved in inhibition or activation of NF-κB signaling pathway in response to TNF-α. In conclusion, these results indicated that miR-124 plays a crucial role in TNF-α-induced LX-2 cells via regulating NF-κB signaling pathway.

Resveratrol increases the activation markers and changes the release of inflammatory cytokines of hepatic stellate cells.

The phytoalexin Resveratrol (3,5,4'-trihydroxystilbene; RSV) has been related to numerous beneficial effects on health by its cytoprotection and chemoprevention activities. Liver fibrosis is characterized by the extracellular matrix accumulation after hepatic injury and can lead to cirrhosis. Hepatic stellate cells (HSC) play a crucial role during fibrogenesis and liver wound healing by changing their quiescent phenotype to an activated phenotype for protecting healthy areas from damaged areas. Strategies on promoting the activated HSC death, the quiescence return or the cellular activation stimuli decrease play an important role on reducing liver fibrosis. Here, we evaluated the RSV effects on some markers of activation in GRX, an HSC model. We further evaluated the RSV influence in the ability of GRX on releasing inflammatory mediators. RSV at 1 and 10 µM did not alter the protein content of α-SMA, collagen I and GFAP; but 50 µM increased the content of these activation-related proteins. Also, RSV did not change the myofibroblast-like morphology of GRX. Interestingly, RSV at 10 and 50 µM decreased the GRX migration and collagen-I gel contraction. Finally, we showed that RSV triggered the increase in the TNF-α and IL-10 content in culture media of GRX while the opposite occurred for the IL-6 content. Altogether, these results suggested that RSV did not decrease the activation state of GRX and oppositely, triggered a pro-activation effect at the 50 µM concentration. However, despite the increase of TNF- α in culture media, these results on IL-6 and IL-10 secretion were in accordance with the anti-inflammatory role of RSV in our model.

Phenotypic Changes in Macrophage Activation in a Model of Nonalcoholic Fatty Liver Disease using Microminipigs.

AIM: Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver disorders associated with metabolic syndrome, and its prevalence has been on the rise. The pathogenesis of NAFLD has not yet been sufficiently elucidated due to the multifactorial nature of the disease, although the activation of macrophages/Kupffer cells is considered to be involved. We previously reported an animal model of NAFLD using MicrominipigsTM (µMPs) fed high-fat diets containing cholesterol with or without cholic acid. The aim of this study was to investigate the phenotypic changes of macrophages that occur during the development of NAFLD. METHODS: Immunohistochemistry of macrophages, lymphocytes, and stellate cells was performed using liver samples, and the density of positive cells was analyzed. RESULTS: The number of Iba-1-positive macrophages increased with increasing cholesterol content in the diet. The numbers of CD163-positive macrophages and CD204-positive macrophages also increased with increasing cholesterol content in the diet; however, the proportion of CD204-positive macrophages among Iba-1-positive macrophages was significantly reduced by cholic acid supplementation. CONCLUSION: The results suggest that lipid accumulation induced macrophage recruitment in swine livers, and that the number of M2-like macrophages increased at the early stage of NAFLD, while the number of M1-like macrophages increased at the late stage of NAFLD, resulting in a liver condition like non-alcoholic steatohepatitis. We provide evidence of the phenotypic changes that occur in macrophages during the development of NAFLD that has never been reported before using µMPs.

Inflammation response after the cessation of chronic arsenic exposure and post-treatment of natural astaxanthin in liver: potential role of cytokine-mediated cell-cell interactions.

Ongoing groundwater arsenic contamination throughout China was first recognized in the 1960s. Groundwater arsenic contamination is a high risk for human and animal health worldwide. Apart from drinking water, diet is the second pathway for arsenic to enter the human body and eventually cause liver injury. Natural astaxanthin extracted from the green algae Haematococcus pluvialis has dominated the nutraceutical market for potential health benefits. Nevertheless, the molecular mechanism underlying the protective effect post astaxanthin against arsenic-induced hepatotoxicity remains largely obscure. In this study, we investigate the effect of natural astaxanthin (derived from Haemotococcus pluvialis) on oxidative stress and liver inflammatory response in rats after the cessation of chronic arsenic exposure. Wistar rats were given astaxanthin (250 mg kg-1) daily for 2 weeks after the cessation of exposure to sodium arsenite (300 µg L-1, drinking water, 24 weeks) by intragastric administration. The results showed that post treatment with astaxanthin attenuated liver injury induced by long-term exposure to arsenic in rats. Most importantly, post treatment with astaxanthin decreased the increasing of inflammatory cytokine NF-κB, tumor necrosis factor-α, interleukin-1ß, oxidative stress level, and total arsenic content in livers of rats exposed to arsenic. In addition, post treatment with astaxanthin reversed the increasing of protein levels of alpha-smooth muscle actin and collagen Iα1, which are the activation markers of hepatic stellate cells (HSCs). Collectively, these data demonstrate that post astaxanthin treatment attenuates inflammation response in the liver after the cessation of chronic arsenic exposure via inhibition of cytokine-mediated cell-cell interactions. Daily ingestion of natural astaxanthin might be a potential and beneficial candidate for the treatment of liver damage after the cessation of chronic exposure to sodium arsenite.

Role of Kupffer Cells in Driving Hepatic Inflammation and Fibrosis in HIV Infection.

While the interactions between HIV and various liver cell populations have been explored, the relevance of these interactions when patients are well-controlled on ART is less clear. Therefore, we focus this perspective on HIV-related alterations that may drive hepatic inflammation and fibrosis in aviremic patients, with a focus on Kupffer cells and Hepatic Stellate Cells. Persistent CD4+ T cell depletion in the gut resulting in increased gut permeability has been postulated to play a role in systemic immune activation in HIV patients. The liver, with its unique location, remains the gatekeeper between the gut and the systemic circulation. The resident liver macrophage, Kupffer cell, is responsible for clearing and responding to these products. We propose that changes in Kupffer cell biology, in the context of HIV infection, creates a mileu that drives hepatic inflammation and fibrosis in response to microbial translocation. Targeting these pathways may be helpful in improving liver-related outcomes in HIV patients.

Gan Shen Fu Fang ameliorates liver fibrosis in vitro and in vivo by inhibiting the inflammatory response and extracellular signal-regulated kinase phosphorylation.

BACKGROUND: Liver fibrosis is a common health problem worldwide and there is still a lack of effective medicines. The Chinese herbal medicine, Gan Shen Fu Fang (GSFF) is composed of salvianolic acid B and diammonium glycyrrhizinate. In this study, we observed the effects of GSFF on liver fibrosis in vivo and in vitro in an attempt to provide some hope for the treatment. AIM: To observe the effects of GSFF on liver fibrosis in vivo and in vitro and investigate the mechanism from the perspective of the inflammatory response and extracellular signal-regulated kinase (ERK) phosphorylation. METHODS: Common bile duct-ligated rats were used for in vivo experiments. Hepatic stellate cells-T6 (HSC-T6) cells were used for in vitro experiments. Hematoxylin and eosin staining and Masson staining, biochemical assays, hydroxyproline (Hyp) assays, enzyme-linked immunoasorbent assay and western blotting were performed to evaluate the degree of liver fibrosis, liver function, the inflammatory response and ERK phosphorylation. The CCK8 assay, immunofluorescence and western blotting were applied to test the effect of GSFF on HSC-T6 cell activation and determine whether GSFF had an effect on ERK phosphorylation in HSC-T6 cells. RESULTS: GSFF improved liver function and inhibited liver fibrosis in common bile duct-ligated rats after 3 wk of treatment, as demonstrated by histological changes, hydroxyproline assays and collagen I concentrations. GSFF alleviated inflammatory cell infiltration and reduced the synthesis of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α) and interlukin-1ß] and NF-κB. In addition, GSFF decreased ERK phosphorylation. In vitro, GSFF inhibited the viability of HSC-T6 cells with and without transforming growth factor ß1 (TGF-ß1) stimulation and decreased the synthesis of collagen I. GSFF had the greatest effect at a concentration of 0.5 µmol/L. GSFF inhibited the expression of α-smooth muscle actin (α-SMA), a marker of HSC activation, in HSC-T6 cells. Consistent with the in vivo results, GSFF also inhibited the phosphorylation of ERK and downregulated the expression of NF-κB. CONCLUSION: GSFF inhibited liver fibrosis progression in vivo and HSC-T6 cell activation in vitro. These effects may be related to an alleviated inflammatory response and downregulated ERK phosphorylation.

Metabolic inflammation as an instigator of fibrosis during non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive storage of fatty acids in the form of triglycerides in hepatocytes. It is most prevalent in western countries and includes a wide range of clinical and histopathological findings, namely from simple steatosis to steatohepatitis and fibrosis, which may lead to cirrhosis and hepatocellular cancer. The key event for the transition from steatosis to fibrosis is the activation of quiescent hepatic stellate cells (qHSC) and their differentiation to myofibroblasts. Pattern recognition receptors (PRRs), expressed by a plethora of immune cells, serve as essential components of the innate immune system whose function is to stimulate phagocytosis and mediate inflammation upon binding to them of various molecules released from damaged, apoptotic and necrotic cells. The activation of PRRs on hepatocytes, Kupffer cells, the resident macrophages of the liver, and other immune cells results in the production of proinflammatory cytokines and chemokines, as well as profibrotic factors in the liver microenvironment leading to qHSC activation and subsequent fibrogenesis. Thus, elucidation of the inflammatory pathways associated with the pathogenesis and progression of NAFLD may lead to a better understanding of its pathophysiology and new therapeutic approaches.

Regulation of macrophage activation in the liver after acute injury: Role of the fibrinolytic system.

The liver functions, in part, to prevent exposure of the body to potentially harmful substances ingested in the diet. While it is highly efficient at accomplishing this, it is frequently prone to liver injury due to the biotransformation of xenobiotics into toxic metabolites. To counter this injury, the liver has evolved a unique capacity to rapidly and efficiently repair itself. Successful resolution of acute liver injury relies on hepatic macrophage populations that orchestrate the reparative response. After injury, Kupffer cells, the resident macrophages of the liver, become activated and secrete proinflammatory cytokines. These cytokines recruit other immune cells, including monocyte-derived macrophages, to the liver where they contribute to the repair process. Monocyte-derived macrophages traffic into the necrotic foci where they rapidly phagocytose dead cell debris. Simultaneous with this process, these cells change phenotype from a proinflammatory macrophage to a pro-restorative macrophage that produce pro-mitogenic growth factors and anti-inflammatory cytokines. Ultimately this process triggers resolution of inflammation, and along with proliferation of other hepatic cells, restores the liver architecture and function. While the mechanisms regulating specific macrophage functions during repair remain to be elucidated, recent studies indicate a key role for the fibrinolytic system in coordinating macrophage function during repair. In this review, we will highlight the function and role of hepatic macrophages in repair after acute liver injury, and will discuss the role of the fibrinolytic enzyme, plasmin, in regulation of these various processes.

Macrophages in metabolic associated fatty liver disease.

Metabolic associated fatty liver disease (MAFLD), formerly named non-alcoholic fatty liver disease is the most common liver disorder in many countries. The inflammatory subtype termed steatohepatitis is a driver of disease progression to cirrhosis, hepatocellular carcinoma, liver transplantation, and death, but also to extrahepatic complications including cardiovascular disease, diabetes and chronic kidney disease. The plasticity of macrophages in response to various environmental cues and the fact that they can orchestrate cross talk between different cellular players during disease development and progression render them an ideal target for drug development. This report reviews recent advances in our understanding of macrophage biology during the entire spectrum of MAFLD including steatosis, inflammation, fibrosis, and hepatocellular carcinoma, as well as for the extra-hepatic manifestations of MAFLD. We discuss the underlying molecular mechanisms of macrophage activation and polarization as well as cross talk with other cell types such as hepatocytes, hepatic stellate cells, and adipose tissue. We conclude with a discussion on the potential translational implications and challenges for macrophage based therapeutics for MAFLD.