Distinct responses between healthy and cirrhotic human livers upon lipopolysaccharide challenge: possible implications for acute-on-chronic liver failure.

Patients with acute-on-chronic liver failure (ACLF) are at risk of developing acute hepatic decompensation and organ failures with an unraveled complex mechanism. An altered immune response toward insults in cirrhotic compared with healthy livers may contribute to the ACLF development. Therefore, we aim to investigate the differences in inflammatory responses between cirrhotic and healthy livers using human precision-cut liver slices (PCLSs) upon the lipopolysaccharide (LPS) challenge. PCLSs prepared from livers of patients with cirrhosis or healthy donors of liver transplantation were incubated ex vivo with or without LPS for up to 48 h. Viability test, qRT-PCR, and multiplex cytokine assay were performed. Regulation of the LPS receptors during incubation or with LPS challenge differed between healthy versus cirrhotic PCLSs. LPS upregulated TLR-2 in healthy PCLSs solely (P < 0.01). Culturing for 48 h induced a stronger inflammatory response in the cirrhotic than healthy PCLS. Upon LPS stimulation, cirrhotic PCLSs secreted more proinflammatory cytokines (IL-8, IL-6, TNF-α, eotaxin, and VEGF) significantly and less anti-inflammatory cytokine (IL-1ra) than those of healthy. In summary, cirrhotic PCLSs released more proinflammatory and less anti-inflammatory cytokines after LPS stimuli than healthy, leading to dysregulated inflammatory response. These events could possibly resemble the liver immune response in ACLF.NEW & NOTEWORTHY Precision-cut liver slices (PCLSs) model provides a unique platform to investigate the different immune responses of healthy versus cirrhotic livers in humans. Our data show that cirrhotic PCLSs exhibit excessive inflammatory response accompanied by a lower anti-inflammatory cytokine release in response to LPS; a better understanding of this alteration may guide the novel therapeutic approaches to mitigate the excessive inflammation during the onset of acute-on-chronic liver failure.

Host microbiota dictates the proinflammatory impact of LPS in the murine liver.

Gut microbiota can impact liver disease development via the gut-liver axis. Liver inflammation is a shared pathological event in various liver diseases and gut microbiota might influence this pathological process. In this study, we studied the influence of gut microbiota on the inflammatory response of the liver to lipopolysaccharide (LPS). The inflammatory response to LPS (1-10 µg/ml) of livers of specific-pathogen-free (SPF) or germ-free (GF) mice was evaluated ex vivo, using precision-cut liver slices (PCLS). LPS induced a more pronounced inflammatory response in GF PCLS than in SPF PCLS. Baseline TNF-α gene expression was significantly higher in GF slices as compared to SPF slices. LPS treatment induced TNF-α, IL-1ß, IL-6 and iNOS expression in both SPF and GF PCLS, but the increase was more intense in GF slices. The anti-inflammatory markers SOCS3 and IRAK-M gene expression was significantly higher in GF PCLS than SPF PCLS at 24h with 1 µg/ml LPS treatment, and IL-10 was not differently expressed in GF PCLS than SPF PCLS. In addition, TLR-4 mRNA, but not protein, at basal level was higher in GF slices than in SPF slices. Taken together, this study shows that, in mice, the host microbiota attenuates the pro-inflammatory impact of LPS in the liver, indicating a positive role of the gut microbiota on the immune homeostasis of the liver.

Murine Precision-cut Intestinal Slices as a Potential Screening Tool for Antifibrotic Drugs.

BACKGROUND: Intestinal fibrosis is a hallmark of Crohn's disease. Here, we investigated the impact of several putative antifibrotic compounds on the expression of fibrosis markers using murine precision-cut intestinal slices. METHODS: Murine precision-cut intestinal slices were cultured for 48 hours in the presence of profibrotic and/or antifibrotic compounds. The fibrotic process was studied on gene and protein level using procollagen 1a1 (Col1α1), heat shock protein 47 (Hsp47), fibronectin (Fn2), and plasminogen activator inhibitor-1 (Pai-1). The effects of potential antifibrotic drugs mainly inhibiting the transforming growth factor ß (TGF-ß) pathway (eg, valproic acid, tetrandrine, pirfenidone, SB203580, and LY2109761) and compounds mainly acting on the platelet-derived growth factor (PDGF) pathway (eg, imatinib, sorafenib, and sunitinib) were assessed in the model at nontoxic concentrations. RESULTS: Murine precision-cut intestinal slices remained viable for 48 hours, and an increased expression of fibrosis markers was observed during culture, including Hsp47, Fn2, and Pai-1. Furthermore, TGF-ß1 stimulated fibrogenesis, whereas PDGF did not have an effect. Regarding the tested antifibrotics, pirfenidone, LY2109761, and sunitinib had the most pronounced impact on the expression of fibrosis markers, both in the absence and presence of profibrotic factors, as illustrated by reduced levels of Col1α1, Hsp47, Fn2, and Pai-1 after treatment. Moreover, sunitinib significantly reduced Hsp47 and Fn2 protein expression and the excretion of procollagen 1. CONCLUSIONS: Precision-cut intestinal slices can successfully be used as a potential preclinical screening tool for antifibrotic drugs. We demonstrated that sunitinib reduced the expression of several fibrosis markers, warranting further evaluation of this compound for the treatment of intestinal fibrosis.

Peribiliary Glands Are Key in Regeneration of the Human Biliary Epithelium After Severe Bile Duct Injury.

Peribiliary glands (PBG) are a source of stem/progenitor cells organized in a cellular network encircling large bile ducts. Severe cholangiopathy with loss of luminal biliary epithelium has been proposed to activate PBG, resulting in cell proliferation and differentiation to restore biliary epithelial integrity. However, formal evidence for this concept in human livers is lacking. We therefore developed an ex vivo model using precision-cut slices of extrahepatic human bile ducts obtained from discarded donor livers, providing an intact anatomical organization of cell structures, to study spatiotemporal differentiation and migration of PBG cells after severe biliary injury. Postischemic bile duct slices were incubated in oxygenated culture medium for up to a week. At baseline, severe tissue injury was evident with loss of luminal epithelial lining and mural stroma necrosis. In contrast, PBG remained relatively well preserved and different reactions of PBG were noted, including PBG dilatation, cell proliferation, and maturation. Proliferation of PBG cells increased after 24 hours of oxygenated incubation, reaching a peak after 72 hours. Proliferation of PBG cells was paralleled by a reduction in PBG apoptosis and differentiation from a primitive and pluripotent (homeobox protein Nanog+/ sex-determining region Y-box 9+) to a mature (cystic fibrosis transmembrane conductance regulator+/secretin receptor+) and activated phenotype (increased expression of hypoxia-inducible factor 1 alpha, glucose transporter 1, and vascular endothelial growth factor A). Migration of proliferating PBG cells in our ex vivo model was unorganized, but resulted in generation of epithelial monolayers at stromal surfaces. Conclusion: Human PBG contain biliary progenitor cells and are able to respond to bile duct epithelial loss with proliferation, differentiation, and maturation to restore epithelial integrity. The ex vivo spatiotemporal behavior of human PBG cells provides evidence for a pivotal role of PBG in biliary regeneration after severe injury.

In vitro and ex vivo anti-fibrotic effects of LY2109761, a small molecule inhibitor against TGF-ß.

Fibrosis is a pathophysiological state characterized by the excessive formation/deposition of fibrous extracellular matrix. Transforming growth factor-beta (TGF-ß) is a central profibrotic mediator, and targeting TGF-ß is a promising strategy in the development of drugs for the treatment of fibrosis. Therefore, the effect of LY2109761, a small molecule inhibitor against TGF-ß with targets beyond TGF-ß signaling, on fibrogenesis was elucidated in vitro (HepG2 cells and LX-2 cells) and ex vivo (human and rat precision-cut liver slices). Our results displayed an anti-fibrotic effect of LY2109761, as it markedly down-regulated gene and protein expression of collagen type 1, as well as gene expression of the inhibitor of metalloproteinases 1. This effect on fibrosis markers was partially mediated by targeting TGF-ß signaling, seeing that LY2109761 inhibited TGF-ß1 gene expression and SMAD2 protein phosphorylation. Interestingly, particularly at a high concentration, LY2109761 decreased SMAD1 protein phosphorylation and gene expression of the inhibitor of DNA binding 1, which appeared to be TGF-ß-independent effects. In conclusion, LY2109761 exhibited preclinical anti-fibrotic effects via both TGF-ß-dependent and -independent pathways. These results illustrate that small molecule inhibitors directed against TGF-ß could possibly influence numerous signaling pathways and thereby mitigate fibrogenesis.

Targeting Oxidative Stress for the Treatment of Liver Fibrosis.

Oxidative stress is a reflection of the imbalance between the production of reactive oxygen species (ROS) and the scavenging capacity of the antioxidant system. Excessive ROS, generated from various endogenous oxidative biochemical enzymes, interferes with the normal function of liver-specific cells and presumably plays a role in the pathogenesis of liver fibrosis. Once exposed to harmful stimuli, Kupffer cells (KC) are the main effectors responsible for the generation of ROS, which consequently affect hepatic stellate cells (HSC) and hepatocytes. ROS-activated HSC undergo a phenotypic switch and deposit an excessive amount of extracellular matrix that alters the normal liver architecture and negatively affects liver function. Additionally, ROS stimulate necrosis and apoptosis of hepatocytes, which causes liver injury and leads to the progression of end-stage liver disease. In this review, we overview the role of ROS in liver fibrosis and discuss the promising therapeutic interventions related to oxidative stress. Most importantly, novel drugs that directly target the molecular pathways responsible for ROS generation, namely, mitochondrial dysfunction inhibitors, endoplasmic reticulum stress inhibitors, NADPH oxidase (NOX) inhibitors, and Toll-like receptor (TLR)-affecting agents, are reviewed in detail. In addition, challenges for targeting oxidative stress in the management of liver fibrosis are discussed.

A nonalcoholic fatty liver disease cirrhosis model in gerbil: the dynamic relationship between hepatic lipid metabolism and cirrhosis.

Nonalcoholic fatty liver disease (NAFLD) usually takes decades to develop into cirrhosis, which limits the longitudinal study of NAFLD. This work aims at developing a NAFLD-caused cirrhosis model in gerbil and examining the dynamic relationship between hepatic lipid metabolism and cirrhosis. We fed gerbil a high-fat and high-cholesterol diet (HFHCD) for 24 weeks, and recorded the gerbil's phenotype at 3, 6, 9, 12, 15, 18, 21, 24 weeks. The model's pathological process, lipid metabolism, oxidative stress, liver collagen deposition and presence of relevant cytokines were tested and evaluated during the full-time frame of disease onset. The gerbil model can induce non-alcoholic steatohepatitis (NASH) within 9 weeks, and can develop cirrhosis after 21 weeks induction. The model's lipids metabolism disorder is accompanied with the liver damage development. During the NAFLD progression, triglycerides (TG) and free fatty acids (FFA) have presented distinct rise and fall tendency, and the turning points are at the fibrosis stage. Besides that, the ratios of total cholesterol (CHO) to high-density lipoprotein cholesterol (HDL-C) exhibited constant growth tendency, and have a good linear relationship with hepatic stellate cells (HSC) (R2 = 0.802, P < 0.001). The gerbil NAFLD cirrhosis model has been developed and possesses positive correlation between lipids metabolism and cirrhosis. The compelling rise and fall tendency of TG and FFA indicated that the fibrosis progression can lead to impairment in lipoprotein synthesis and engender decreased TG level. CHO/HDL-C ratios can imply the fibrosis progress and be used as a blood indicator for disease prediction and prevention.

Evaluating the antifibrotic potency of galunisertib in a human ex vivo model of liver fibrosis.

BACKGROUND AND PURPOSE: Liver fibrosis is a major cause of liver-related mortality and, so far, no effective antifibrotic drug is available. Galunisertib, a TGF-ß receptor type I kinase inhibitor, is a potential candidate for the treatment of liver fibrosis. Here, we evaluated the potency of galunisertib in a human ex vivo model of liver fibrosis. EXPERIMENTAL APPROACH: Antifibrotic potency and associated mechanisms were studied ex vivo, using both healthy and cirrhotic human precision-cut liver slices. Fibrosis-related parameters, both transcriptional and translational level, were assessed after treatment with galunisertib. KEY RESULTS: Galunisertib showed a prominent antifibrotic potency. Phosphorylation of SMAD2 was inhibited, while that of SMAD1 remained unchanged. In healthy and cirrhotic human livers, spontaneous transcription of numerous genes encoding collagens, including collagen type I, α 1, collagen maturation, non-collageneous extracellular matrix (ECM) components, ECM remodelling and selected ECM receptors was significantly decreased. The reduction of fibrosis-related transcription was paralleled by a significant inhibition of procollagen I C-peptide released by both healthy and cirrhotic human liver slices. Moreover, galunisertib showed similar antifibrotic potency in human and rat lives. CONCLUSIONS AND IMPLICATIONS: Galunisertib is a drug that deserves to be further investigated for the treatment of liver fibrosis. Inhibition of SMAD2 phosphorylation is probably a central mechanism of action. In addition, blocking the production and maturation of collagens and promoting their degradation are related to the antifibrotic action of galunisertib.