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Liver fibrosis is the most common feature of liver disease, and activated hepatic stellate cells (HSCs) are the main contributors to liver fibrosis. Thus, finding key targets that modulate HSC activation is important to prevent liver fibrosis. Previously, we showed that thymosin β4 (Tβ4) influenced HSC activation by interacting with the Hedgehog pathway in vitro. Herein, we generated Tβ4 conditional knockout (Tβ4-flox) mice to investigate in vivo functions of Tβ4 in liver fibrosis. To selectively delete Tβ4 in activated HSCs, double-transgenic (DTG) mice were generated by mating Tβ4-flox mice with α-smooth muscle actin (α-Sma)-Cre-ERT2 mice, and these mice were administered carbon tetrachloride (CCl4) or underwent bile duct ligation to induce liver fibrosis. Tβ4 was selectively suppressed in the activated HSCs of DTG mouse liver, and this reduction attenuated liver injury, including fibrosis, in both fibrotic models by repressing Hedgehog (Hh) signaling. In addition, the re-expression of Tβ4 by an adeno-associated virus reversed the effect of HSC-specific Tβ4 deletion and led to liver fibrosis with Hh activation in CCl4-exposed mice treated with tamoxifen. In conclusion, our results demonstrate that Tβ4 is a crucial regulator of HSC activation, suggesting it as a novel therapeutic target for curing liver fibrosis. [ABSTRACT FROM AUTHOR]

Drug-induced liver injury (DILI) is a major contributor to high attrition rates among candidate and market drugs and a key regulatory, industry, and global health concern. While acute and dose-dependent DILI, namely, intrinsic DILI, is predictable and often reproducible in preclinical models, the nature of idiosyncratic DILI (iDILI) limits its mechanistic understanding due to the complex disease pathogenesis, and recapitulation using in vitro and in vivo models is extremely challenging. However, hepatic inflammation is a key feature of iDILI primarily orchestrated by the innate and adaptive immune system. This review summarizes the in vitro co-culture models that exploit the role of the immune system to investigate iDILI. Particularly, this review focuses on advancements in human-based 3D multicellular models attempting to supplement in vivo models that often lack predictability and display interspecies variations. Exploiting the immune-mediated mechanisms of iDILI, the inclusion of non-parenchymal cells in these hepatoxicity models, namely, Kupffer cells, stellate cells, dendritic cells, and liver sinusoidal endothelial cells, introduces heterotypic cell–cell interactions and mimics the hepatic microenvironment. Additionally, drugs recalled from the market in the US between 1996–2010 that were studies in these various models highlight the necessity for further harmonization and comparison of model characteristics. Challenges regarding disease-related endpoints, mimicking 3D architecture with different cell–cell contact, cell source, and the underlying multi-cellular and multi-stage mechanisms are described. It is our belief that progressing our understanding of the underlying pathogenesis of iDILI will provide mechanistic clues and a method for drug safety screening to better predict liver injury in clinical trials and post-marketing. [ABSTRACT FROM AUTHOR]

Hepatic stellate cell (HSC) activation via the autophagy pathway is a critical factor in liver fibrogenesis. This study tests the hypothesis that chloroquine (CQ) treatment can prevent autophagy and HSC activation in vitro and in vivo in bile-duct-ligated (BDL) mice. Sham-operated and BDL mice were treated with either PBS or CQ in two 60 mg/kg doses the day (D) before and after surgery. On day 2 (2D), HSCs were isolated, and their biological activities were evaluated by measuring intracellular lipid content, α-sma/collagen, and expression of autophagy lc3, sqstm1/p62 markers. The treatment efficacy on liver function was evaluated with serum albumin, transaminases (AST/ALT), and hepatic histology. Primary HSCs were treated in vitro for 24 h with CQ at 0, 2.5, 5, 10, 30, and 50 µM. Autophagy and HSC activation were assessed after 2D of treatment. CQ treatment improved serum AST/ALT, albumin, and bile duct proliferation in 2D BDL mice. This is associated with a suppression of HSC activation, shown by higher HSC lipid content and collagen I staining, along with the blockage of HSC autophagy indicated by an increase in p62 level and reduction in lc3 staining. CQ 5 µM inhibited autophagy in primary HSCs in vitro by increasing p62 and lc3 accumulation, thereby suppressing their in vitro activation. The autophagy inhibitor CQ reduced HSC activation in vitro and in vivo. CQ improved liver function and reduced liver injury in BDL mice. [ABSTRACT FROM AUTHOR]

Objective: To study the effect and mechanism of the Clostridium metabolite p-Cresol sulfate (PCS) in primary biliary cholangitis (PBC). Methods: Gas chromatography-mass spectrometry (GC-MS) was used to detect differences in tyrosine, phenylalanine, tryptophan, PCS, and p-Cresyl glucuronide (PCG) between the serum of PBC patients and healthy controls. In vivo experiments, mice were divided into the normal control, PBC group, and PBC tyrosine group. GC-MS was used to detect PCS and PCG. Serum and liver inflammatory factors were compared between groups along with the polarization of liver Kupffer cells. Additionally, PCS was cultured with normal bile duct epithelial cells and Kupffer cells, respectively. PCS-stimulated Kupffer cells were co-cultured with lipopolysaccharide-injured bile duct epithelial cells to detect changes in inflammatory factors. Results: Levels of tyrosine and phenylalanine were increased, but PCS level was reduced in PBC patients, with PCG showing a lower concentration distribution in both groups. PCS in PBC mice was also lower than those in normal control mice. After oral administration of tyrosine feed to PBC mice, PCS increased, liver inflammatory factors were decreased, and anti-inflammatory factors were increased. Furthermore, Kupffer cells in the liver polarized form M1 transitioned to M2. PCS can damage normal bile duct epithelial cells and suppress the immune response of Kupffer cells. But PCS protects bile duct epithelial cells damaged by LPS through Kupffer cells. Conclusions: PCS produced by Clostridium-metabolized tyrosine reduced PBC inflammation, suggesting that intervention by food, or supplementation with PCS might represent an effective clinical strategy for treating PBC. [ABSTRACT FROM AUTHOR]

Chronic liver diseases (CLDs) are complex diseases that cause long-term inflammation and infection, which in turn accelerate their development. The usage of albumin in patients with CLDs has been debated for years. Human serum albumin (HSA) plays a key role in immunomodulation during the process of CLDs. The correlation between albumin and C-reactive protein (CRP) in CLD patients was analyzed by linear regression with the Pearson statistic. The damage of THP-1 and primary cells was evaluated by measuring the lactate dehydrogenase (LDH) in the supernatant. Immunofluorescence staining was performed to determine underlying pathways in Kupffer cells (KCs). Albumin negatively correlated with infection in patients with CLDs. In vitro experiments with THP-1 cells and KCs showed that albumin reduced LDH release after stimulation with bacterial products, while no differences in hepatic stellate cells (HSCs) and sinusoidal endothelial cells (SECs) were detected. Moreover, immunofluorescence staining revealed an increase of p-ERK and p-NF-kB p65 density after albumin treatment of KCs stimulated by bacterial products. In conclusion, albumin could assist CLD patients in alleviating inflammation caused by bacterial products and might be beneficial to patients with CLDs by securing KCs from bacteria-induced damage, providing a compelling rationale for albumin therapy in patients with CLDs. [ABSTRACT FROM AUTHOR]

Considering that the heterogenic population of a hepatic progenitor cell line (HPCL) can play a vital role in autoimmune hepatitis (AIH), we decided to conduct pioneering retrospective evaluation of these cells in pediatric AIH by means of transmission electron microscopy (TEM). The aim of the study was to assess the ultrastructure of the HPCL in children with untreated AIH. Ultrastructural analysis of the HPCL population, preceded by immunohistochemical staining for cytokeratin 7 (CK7), was performed using pretreatment liver biopsies from 23 children with clinicopathologically diagnosed AIH. Immunohistochemical assessment for CK7 allowed detection of proliferating immature epithelial cells differentiating towards periportal and intralobular intermediate hepatocytes without marked formation of ductular reactions in AIH children. Using TEM, we distinguished three morphological types of HPCs: I—the most undifferentiated progenitor cells; III—intermediate hepatocyte-like cells; II—intermediate bile duct cells. Most frequent were the cells differentiating towards hepatocytes, most rare—those differentiating towards cholangiocytes. The results indicate that an HPCL may be an important source of hepatocyte regeneration. Ultrastructural analyses of the HPCL population, combined with immunohistochemistry for CK7, might be a useful tool to evaluate liver cell regeneration, including fibrogenesis, and may help better understand the morphological pattern of the disease, in pediatric AIH. Frequent appearance of an HPCL in the vicinity of fibrotic foci, often accompanied by hyperactive Kupffer cells and transitional hepatic stellate cells, may indicate their significant involvement in liver fibrogenesis. [ABSTRACT FROM AUTHOR]

A high fat Western-style diet leads to hepatic steatosis that can progress to steatohepatitis and ultimately cirrhosis or liver cancer. The mechanism that leads to the development of steatosis upon nutritional overload is complex and only partially understood. Using click chemistry-based metabolic tracing and microscopy, we study the interaction between Kupffer cells and hepatocytes ex vivo. In the early phase of steatosis, hepatocytes alone do not display significant deviations in fatty acid metabolism. However, in co-cultures or supernatant transfer experiments, we show that tumor necrosis factor (TNF) secretion by Kupffer cells is necessary and sufficient to induce steatosis in hepatocytes, independent of the challenge of hepatocytes with elevated fatty acid levels. We further show that free fatty acid (FFA) or lipopolysaccharide are both able to trigger release of TNF from Kupffer cells. We conclude that Kupffer cells act as the primary sensor for both FFA overload and bacterial lipopolysaccharide, integrate these signals and transmit the information to the hepatocyte via TNF secretion. Hepatocytes react by alteration in lipid metabolism prominently leading to the accumulation of triacylglycerols (TAGs) in lipid droplets, a hallmark of steatosis. [ABSTRACT FROM AUTHOR]

Liver fibrosis is a common feature of sustained liver injury and represents a major public health problem worldwide. Fibrosis is an active research field and discoveries in the last years have contributed to the development of new antifibrotic drugs, although none of them have been approved yet. Liver sinusoidal endothelial cells (LSEC) are highly specialized endothelial cells localized at the interface between the blood and other liver cell types. They lack a basement membrane and display open channels (fenestrae), making them exceptionally permeable. LSEC are the first cells affected by any kind of liver injury orchestrating the liver response to damage. LSEC govern the regenerative process initiation, but aberrant LSEC activation in chronic liver injury induces fibrosis. LSEC are also main players in fibrosis resolution. They maintain liver homeostasis and keep hepatic stellate cell and Kupffer cell quiescence. After sustained hepatic injury, they lose their phenotype and protective properties, promoting angiogenesis and vasoconstriction and contributing to inflammation and fibrosis. Therefore, improving LSEC phenotype is a promising strategy to prevent liver injury progression and complications. This review focuses on changes occurring in LSEC after liver injury and their consequences on fibrosis progression, liver regeneration, and resolution. Finally, a synopsis of the available strategies for LSEC-specific targeting is provided. [ABSTRACT FROM AUTHOR]