Complex Cell Type-Specific Roles of Autophagy in Liver Fibrosis and Cirrhosis.

The lysosomal degradation pathway, or autophagy, plays a fundamental role in cellular, tissue, and organismal homeostasis. A correlation between dysregulated autophagy and liver fibrosis (including end-stage disease, cirrhosis) is well-established. However, both the up and downregulation of autophagy have been implicated in fibrogenesis. For example, the inhibition of autophagy in hepatocytes and macrophages can enhance liver fibrosis, whereas autophagic activity in hepatic stellate cells and reactive ductular cells is permissive towards fibrogenesis. In this review, the contributions of specific cell types to liver fibrosis as well as the mechanisms underlying the effects of autophagy are summarized. In view of the functional effects of multiple cell types on the complex process of hepatic fibrogenesis, integrated approaches that consider the role of autophagy in each liver cell type should be a focus of future research.

Nerve growth factor induced farnesoid X receptor upregulation modulates autophagy flux and protects hepatocytes in cholestatic livers.

Upregulation of nerve growth factor (NGF) in parenchymal hepatocytes has been shown to exert hepatoprotective function during cholestatic liver injury. However, the modulatory role of NGF in regulation of liver autophagy remains unclear. This study aimed to scrutinize the regulatory role of NGF in hepatic expression of farnesoid X receptor (FXR), a bile acid (BA)-activated nuclear receptor, and to determine its cytoprotective effect on BA-induced autophagy and cytotoxicity. Livers of human hepatolithiasis and bile duct ligation (BDL)-induced mouse cholestasis were used for histopathological and molecular detection. The regulatory roles of NGF in autophagy flux and FXR expression, as well as its hepatoprotection against BA cytotoxicity were examined in cultured hepatocytes. FXR downregulation in human hepatolithiasis livers showed positive correlation with hepatic NGF levels. NGF administration upregulated hepatic FXR levels, while neutralization of NGF decreased FXR expression in BDL-induced cholestatic mouse livers. In vitro studies demonstrated that NGF upregulated FXR expression, increased cellular LC3 levels, and exerted hepatoprotective effect in cultured primary rat hepatocytes. Conversely, autophagy inhibition abrogated NGF-driven cytoprotection under BA exposure, suggesting involvement of NGF-modulated auophagy flux. Although FXR agonistic GW4064 stimulation did not affect auophagic LC3 levels, FXR activity inhibition significantly potentiated BA-induced cytotoxicity and increased cellular p62/SQSTM1 and Rab7 protein in SK-Hep1 hepatocytes. Moreover, FXR gene silencing abolished the protective effect of NGF under BA exposure. These findings support that NGF modulates autophagy flux via FXR upregulation and protects hepatocytes against BA-induced cytotoxicity. NGF/FXR axis is a novel therapeutic target for treatment of cholestatic liver diseases.

CD8+PD-1-ILT2+ T Cells Are an Intratumoral Cytotoxic Population Selectively Inhibited by the Immune-Checkpoint HLA-G.

Only some cancer patients respond to the immune-checkpoint inhibitors being used in the clinic, and other therapeutic targets are sought. Here, we investigated the HLA-G/ILT2 checkpoint in clear-cell renal-cell carcinoma (ccRCC) patients and focused on tumor-infiltrating CD8+ T lymphocytes (TIL) expressing the HLA-G receptor ILT2. Using transcriptomics and flow cytometry, we characterized both peripheral blood and tumor-infiltrating CD8+ILT2+ T cells from cancer patients as late-differentiated CD27-CD28-CD57+ cytotoxic effectors. We observed a clear dichotomy between CD8+ILT2+ and CD8+PD-1+ TIL subsets. These subsets, which were sometimes present at comparable frequencies in TIL populations, barely overlapped phenotypically and were distinguished by expression of exclusive sets of surface molecules that included checkpoint molecules and activating and inhibitory receptors. CD8+ILT2+ TILs displayed a more mature phenotype and higher expression of cytotoxic molecules. In ex vivo functional experiments with both peripheral blood T cells and TILs, CD8+ILT2+ T cells displayed significantly higher cytotoxicity and IFNγ production than their ILT2- (peripheral blood mononuclear cells, PBMC) and PD-1+ (TILs) counterparts. HLA-G expression by target cells specifically inhibited CD8+ILT2+ T-cell cytotoxicity, but not that of their CD8+ILT2- (PBMC) or CD8+PD-1+ (TIL) counterparts, an effect counteracted by blocking the HLA-G/ILT2 interaction. CD8+ILT2+ TILs may therefore constitute an untapped reservoir of fully differentiated cytotoxic T cells within the tumor microenvironment, independent of the PD1+ TILs targeted by immune therapies, and specifically inhibited by HLA-G. These results emphasize the potential of therapeutically targeting the HLA-G/ILT2 checkpoint in HLA-G+ tumors, either concomitantly with anti-PD-1/PD-L1 or in cases of nonresponsiveness to anti-PD-1/PD-L1.

A critical role of autophagy in regulating the mesenchymal transition of ductular cells in liver cirrhosis.

Our previous studies have shown that autophagy mediates the link between ductular reaction (DR) and liver cirrhosis. Whether the subsequent fibrogenic response is regulated by increased autophagy in DR remains unclear. Here, using both human liver specimens and a rat model of liver cirrhosis induced by 2-acetylaminofluorene (AAF) and carbon tetrachloride (CCL4), we explored the involvement of autophagy in regulating mesenchymal transition of ductular cells. Ductular cells from AAF/CCL4 livers exhibited increased autophagy compared to those of normal livers. These cells showed morphological and functional characteristics of mesenchymal cells. Blocking autophagy using bafilomycin A1 or siRNA targeting ATG7 reduced the expression of mesenchymal markers in these ductular cells from AAF/CCL4 livers, indicating a role for autophagy in regulating the mesenchymal phenotype of ductular cells. Furthermore, we show that the mesenchymal transition in DR requires the activation of transforming growth factor-ß (TGF-ß) signaling in an autophagy-dependent manner. Importantly, in cirrhotic human livers, ductular cells that are positive for LC3B also showed increased expression of TGF-ß and fibroblast-specific protein-1. Our data suggest activation of autophagy in ductular cells, and also demonstrate that it is required for the mesenchymal transition during the DR, processes that are critically involved in the pathogenesis of cirrhosis.

Antifibrotic Activity of Human Placental Amnion Membrane-Derived CD34+ Mesenchymal Stem/Progenitor Cell Transplantation in Mice With Thioacetamide-Induced Liver Injury.

: Liver fibrosis represents the end stage of chronic liver inflammatory diseases and is defined by the abnormal accumulation of extracellular matrix in the liver. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension. Liver transplantation has been the most effective treatment for these diseases, but the procedure is limited by the shortage of suitable donors. Mesenchymal stromal cells (MSCs) have shown great potential in the treatment of chronic inflammatory diseases associated with fibrosis. This study aimed to evaluate the therapeutic effect of MSC-based cell transplantation as an alternative treatment for liver fibrosis. A CD34-positive subpopulation of human placental amnion membrane-derived stem/progenitor cells (CD34+ AMSPCs) was isolated through the depletion of CD34-negative stromal fibroblasts (CD34- AMSFCs) facilitated by CD34 fluorescence-activated cell sorting, enriched and expanded ex vivo. These cells express pluripotency markers and demonstrate multidirectional differentiation potentials. Comparative analysis was made between CD34+ AMSPCs and CD34- AMSFCs in terms of the expressions of stemness surface markers, embryonic surface antigens, and multilineage differentiation potentials. A mouse model of liver fibrosis was established by thioacetamide (TAA) administration. When injected into the spleen of TAA-injured mice, human placental amnion membrane-derived MSCs (hAM-MSCs) can engraft into the injury site, ameliorate liver fibrosis, and restore liver function, as shown by pathological and blood biochemical analysis and downregulated gene expressions associated with liver damage. CD34+ AMSPCs represent a more primitive subset of hAM-MSCs and could be a suitable candidate with a potentially better safety profile for cell-based therapy in treatment of liver diseases associated with fibrosis. SIGNIFICANCE: In this study, a CD34+ subpopulation of stem/progenitor cells derived from neonatal placental amnion membrane, denoted as CD34+ AMSPCs, were identified, enriched, and characterized. These cells are highly proliferative, express mesenchymal stromal cells and pluripotent stem cell markers, and demonstrate multidirectional differentiation potentials, indicating their promising application in clinical regenerative therapies. CD34+ AMSPC transplantation ameliorated liver fibrosis in mice with drug-induced liver injury. These cells represent a potential therapeutic agent for treating liver diseases associated with fibrosis.

Bai-Hu-Jia-Ren-Shen-Tang Decoction Reduces Fatty Liver by Activating AMP-Activated Protein Kinase In Vitro and In Vivo.

Obesity and associated conditions, such as type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD), are currently a worldwide health problem. In Asian traditional medicine, Bai-Hu-Jia-Ren-Shen-Tang (BHJRST) is widely used in diabetes patients to reduce thirst. However, whether it has a therapeutic effect on T2DM or NAFLD is not known. The aim of this study was to examine whether BHJRST had a lipid-lowering effect using a HuS-E/2 cell model of fatty liver induced by palmitate and in a db/db mouse model of dyslipidemia. Incubation of HuS-E/2 cells with palmitate markedly increased lipid accumulation and expression of adipose triglyceride lipase (ATGL), which is involved in lipolysis. BHJRST significantly decreased lipid accumulation and increased ATGL levels and phosphorylation of AMP-activated protein kinase (AMPK) and its primary downstream target, acetyl-CoA carboxylase (ACC), which are involved in fatty acid oxidation. Furthermore, after twice daily oral administration for six weeks, BHJRST significantly reduced hepatic fat accumulation in db/db mice, as demonstrated by increased hepatic AMPK and ACC phosphorylation, reduced serum triglyceride levels, and reduced hepatic total lipid content. The results show that BHJRST has a lipid-lowering effect in the liver that is mediated by activation of the AMPK signaling pathway.

Increased Autophagy Markers Are Associated with Ductular Reaction during the Development of Cirrhosis.

Autophagy is a regulatory pathway in liver fibrosis. We investigated the roles of autophagy in human cirrhotic livers. Cirrhotic and noncirrhotic liver tissues were obtained from patients with hepatocellular carcinoma, and liver tissues from live donors served as control. Patients with cirrhotic livers had significantly increased levels of various essential autophagy-related genes compared with noncirrhotic livers. In addition, colocalization of autophagy marker microtubule-associated protein 1 light chain 3B (LC3B) with lysosome-associated membrane protein-1, increased levels of lysosome-associated membrane protein-2, and increased maturation of lysosomal cathepsin D were observed in cirrhotic livers. By using dual-immunofluorescence staining, we demonstrated that increased LC3B was located mainly in the cytokeratin 19-labeled ductular reaction (DR) in human cirrhotic livers and in an experimental cirrhosis induced by 2-acetylaminofluorene (AAF) with carbon tetrachloride (CCl4), indicating a conserved response to chronic liver damage. Furthermore, an AAF/CCl4-mediated increase in DR and fibrosis were attenuated after chloroquine treatment, suggesting that the autophagy-lysosome pathway was essential for AAF/CCl4-induced DR-fibrosis. In conclusion, we demonstrated that increased autophagy marker positively correlated with DR during the development of cirrhosis. Therefore, targeting autophagy may hold therapeutic value for liver cirrhosis.

Hepatitis C virus NS5A protein enhances gluconeogenesis through upregulation of Akt-/JNK-PEPCK signalling pathways.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is highly associated with the type 2 diabetes mellitus, but the detailed mechanisms by which the viral proteins are involved in the clinical outcome remain unclear. METHODS: A cDNA microarray analysis was performed following introducing an NS5A-encoding plasmid or a control vector into a mouse system by hydrodynamics- based transfection. Differentially expressed genes that are associated with gluconeogenesis were selected and their expression levels in HCV patients, in NS5A-expressing systems, and in the viral subgenomic replicon system were further examined by real-time quantitative polymerase chain reaction and Western blot analysis. RESULTS: Differential gene expression including an upregulation of the gluconeogenic rate-limiting enzyme phosphoenolpyruvate carboxykinase (PEPCK) compared with controls was detected in mouse hepatocytes expressing HCV NS5A and in HCV patients with diabetes. In addition, an NS5A-dependent increase in glucose production was demonstrated in human primary hepatocytes. The upregulation of PEPCK and peroxisome proliferator-activated receptor-c coactivator-1a (PGC-1a) were also detected in NS5A-expressing cells and in the viral genotype 1b subgenomic replicon system. Further studies demonstrated that the NS5A-mediated upregulation of PEPCK and PGC-1a genes were resulted from the activation of PI3K-Akt and JNK signalling pathways. In addition, the expression levels of the forkhead transcription factor FoxO1 and the liver-enriched transcription factor HNF-4a were increased in HCV NS5A expressing cells. CONCLUSIONS: By upregulating the expression of PEPCK gene via its transactivators FoxO1 and HNF-4a, and the coactivator PGC-1a, the NS5A promotes the production of hepatic glucose which may contribute to the development of HCV-associated type 2 diabetes mellitus.

(-)-Epigallocatechin-3-gallate inhibits entry of hepatitis B virus into hepatocytes.

Hepatitis B virus (HBV) is a major cause of liver disease and hepatocellular carcinoma. Chronic HBV infection is currently managed with either nucleoside/nucleotide-based or interferon-based therapies, but fails to clear infection in a substantial proportion of cases, and antiviral strategies targeting the early stages of infection are therefore required for the prevention of HBV infection. In this study, we examined some common phytochemicals and identified epigallocatechin-3-gallate (EGCG) as a new inhibitor of HBV entry. EGCG, a flavonoid present in green tea extract, belongs to the subclass of catechins. We demonstrated that EGCG at a concentration of 50µM inhibited HBV entry into immortalized human primary hepatocytes by more than 80%, whereas the other four catechins tested had much weaker inhibitory effects. DMSO-differentiated HuS-E/2 cells expressed sodium taurocholate cotransporting polypeptide (NTCP), which is a receptor for HBV. Application of EGCG during HBV inoculation markedly inhibited infection in both DMSO-differentiated HuS-E/2 cells and HA-NTCP-expressing Huh7 cells. Interestingly, EGCG induced clathrin-dependent endocytosis of NTCP from the plasma membrane followed by protein degradation. In addition, EGCG inhibited the clathrin-mediated endocytosis of transferrin. Treatment of cells with EGCG had no effect on HBV genome replication or virion secretion. Moreover, the characteristic of HBV virion and the expression of known HBV entry factors were unaltered by EGCG. Finally, the antiviral activity of EGCG on HBV entry was observed using four different genotypes, A to D. These results show that the green tea-derived molecule EGCG potently inhibits HBV entry and could be used in prevention of HBV reinfection.

miRSeq: a user-friendly standalone toolkit for sequencing quality evaluation and miRNA profiling.

MicroRNAs (miRNAs) present diverse regulatory functions in a wide range of biological activities. Studies on miRNA functions generally depend on determining miRNA expression profiles between libraries by using a next-generation sequencing (NGS) platform. Currently, several online web services are developed to provide small RNA NGS data analysis. However, the submission of large amounts of NGS data, conversion of data format, and limited availability of species bring problems. In this study, we developed miRSeq to provide alternatives. To test the performance, we had small RNA NGS data from four species, including human, rat, fly, and nematode, analyzed with miRSeq. The alignments results indicate that miRSeq can precisely evaluate the sequencing quality of samples regarding percentage of self-ligation read, read length distribution, and read category. miRSeq is a user-friendly standalone toolkit featuring a graphical user interface (GUI). After a simple installation, users can easily operate miRSeq on a PC or laptop by using a mouse. Within minutes, miRSeq yields useful miRNA data, including miRNA expression profiles, 3' end modification patterns, and isomiR forms. Moreover, miRSeq supports the analysis of up to 105 animal species, providing higher flexibility.

Up-regulation of microRNA-190b plays a role for decreased IGF-1 that induces insulin resistance in human hepatocellular carcinoma.

BACKGROUND & AIMS: Insulin-like growth factor, (IGF)-1, is produced mainly by the liver and plays important roles in promoting growth and regulating metabolism. Previous study reported that development of hepatocellular carcinoma (HCC) was accompanied by a significant reduction in serum IGF-1 levels. Here, we hypothesized that dysregulation of microRNAs (miRNA) in HCC can modulate IGF-1 expression post-transcriptionally. METHODS: The miRNAs expression profiles in a dataset of 29 HCC patients were examined using illumina BeadArray. Specific miRNA (miR)-190b, which was significantly up-regulated in HCC tumor tissues when compared with paired non-tumor tissues, was among those predicted to interact with 3'-untranslated region (UTR) of IGF-1. In order to explore the regulatory effects of miR-190b on IGF-1 expression, luciferase reporter assay, quantitative real-time PCR, western blotting and immunofluorecence analysis were performed in HCC cells. RESULTS: Overexpression of miR-190b in Huh7 cells attenuated the expression of IGF-1, whereas inhibition of miR-190b resulted in up-regulation of IGF-1. Restoration of IGF-1 expression reversed miR-190b-mediated impaired insulin signaling in Huh7 cells, supporting that IGF-1 was a direct and functional target of miR-190b. Additionally, low serum IGF-1 level was associated with insulin resistance and poor overall survival in HCC patients. CONCLUSIONS: Increased expression of miR-190 may cause decreased IGF-1 in HCC development. Insulin resistance appears to be a part of the physiopathologic significance of decreased IGF-1 levels in HCC progression. This study provides a novel miRNA-mediated regulatory mechanism for controlling IGF-1 expression in HCC and elucidates the biological relevance of this interaction in HCC.

Downregulation of alpha-fetoprotein expression by LHX4: a critical role in hepatocarcinogenesis.

LHX4 is a member of the LIM-homeobox family and plays a critical role in pituitary development and differentiation. Several lines of evidences have reported their aberrant expression in cancers. However, the exact roles of LHX4 in carcinogenesis remain unclear. In this study, LHX4 expression was analyzed in tumor and paired non-tumor tissues obtained from patients with hepatocellular carcinoma (HCC) using western blotting and immunohistochemistry. LHX4 was found to be downregulated in tumor tissues and negatively correlated with differentiation grade and alpha-fetoprotein (AFP) levels in 66 HCC patients. To clarify the biological functions of LHX4, transient or stable transfectants overexpressing LHX4 were generated in human hepatoma cells (Huh7 and HepG2). LHX4 overexpression in Huh7 and HepG2 cells induced a more differentiated phenotype by reducing AFP expression. Using in silico analysis, the evolutionary conserved region within the AFP promoter containing LHX4-binding site was identified, implying that AFP is a putative target for LHX4. Moreover, ectopic LHX4 overexpression attenuated Huh7 and HepG2 proliferation. Importantly, the growth-inhibitory effect of LHX4 was reversed by replenishing AFP to the LHX4-overexpressing cells, providing a functional relevance between LHX4 and AFP. Finally, we analyzed expressions of LHX4 and AFP during normal liver development. Hepatic LHX4 expression increased in adult liver in a manner that parallel AFP repression. In conclusion, these data indicate that LHX4 may act as a potential tumor suppressor in hepatocarcinogenesis, suggesting that targeting LHX4 to downregulate AFP might have therapeutic implications.

In trans interaction of hepatitis C virus helicase domains mediates protease activity critical for internal NS3 cleavage and cell transformation.

Hepatitis C virus (HCV) internal non-structural protein 3 (NS3) cleavage can occur in trans in the presence of NS4A. In this study, we have further demonstrated a critical role of the helicase domain in the internal NS3 cleavage, different from HCV polyprotein processing which requires only the serine protease domain. The NTPase domain of NS3 helicase interacts with the RNA binding domain to facilitate internal NS3 cleavage. In addition, NS3 protease activity contributes to the transforming ability of the major internal cleavage product NS3(1-402). These findings imply important roles of the internal cleavage and protease activity of the NS3 protein in the pathogenesis of HCV.

A novel nonsynonymous variant of matrix metalloproteinase-7 confers risk of liver cirrhosis.

UNLABELLED: Liver cirrhosis is characterized by progressive accumulation of extracellular matrix following chronic liver injuries. In the extracellular space, the constant turnover of liver matrix is regulated by the matrix metalloproteinase (MMP) class of enzyme. To assess whether genetic variations in MMP would result in diversity of liver cirrhosis, a case-control study of 320 patients with hepatocellular carcinoma, with or without cirrhosis, was conducted. Ten single-nucleotide polymorphism markers from four potential fibrosis-associated genes were selected for genotyping. Among these genes, a nonsynonymous single-nucleotide polymorphism which generates the variation of Gly-137 and Asp-137 in the MMP-7 gene was found to be strongly associated with the development of liver cirrhosis. In contrast to MMP-7(Gly-137) that predominantly secretes out into the cell culture medium, the cirrhosis-associated MMP-7(Asp-137) variant is preferentially localized on the extracellular membranes where it exerts its proteolytic activity on pericellular substrates. Functional analysis demonstrated an increased ability of the MMP-7(Asp-137) variant to associate with the cell surface CD151 molecule. In wound-healing and Boyden chamber assays, cell motility was specifically enhanced with the expression of MMP-7(Asp-137) as compared to the cells expressing MMP-7(Gly-137). These results demonstrate that the MMP-7(Asp-137) variant confers a gain-of-function phenotype for MMP-7. CONCLUSION: We have identified a novel genetic association of MMP-7(Asp-137) variant with liver cirrhosis in patients with hepatocellular carcinoma. Whether the MMP-7 variant can be a new marker for liver cirrhosis will be further studied.

Differential requirements of NS4A for internal NS3 cleavage and polyprotein processing of hepatitis C virus.

The NS3 protein of hepatitis C virus (HCV) possesses protease activity responsible for the proteolytic cleavage of the viral polyprotein at the junctions of nonstructural proteins downstream of NS3. The NS3 protein was also found to be internally cleaved. In this study, we demonstrated that internal cleavages occurred on the NS3 protein of genotype 1b in the presence of NS4A, both in culture cells and with a mouse model system. No internal cleavage products were detected with the NS3 and NS4A proteins of genotype 2a. Three potential cleavage sites were detected in the NS3 protein (genotype 1b), with IPT(402)|S being the major one. The internal cleavage requires the polyprotein processing activity of NS3 protease, but when supplemented in trans, the internal cleavage efficiency is reduced. In addition, several mutations in NS4A disrupted the internal cleavage of NS3 but did not affect polyprotein processing, indicating that NS4A contributes differently to these two proteolytic activities. Furthermore, Ile-25, Val-26, and Ile-29 of the NS4A protein, important for the NS4A-dependent internal cleavages, were also shown to be critical for the transforming activity of NS3, but mutations at these critical residues resulted only in a slight increase of HCV replicating efficiency. The internal cleavage-associated enhancement of the transforming activity of NS3 was reduced when a T402A substitution at the major internal cleavage site was introduced. The multiple roles of NS4A in viral multiplication and pathogenesis make NS4A an ideal molecular target for HCV therapy.