Giant mitochondria in human liver disease.

This thematic review aims to provide an overview of the current state of knowledge about the occurrence of giant mitochondria or megamitochondria in liver parenchymal cells. Their presence and accumulation are considered to be a major pathological hallmark of the health and fate of liver parenchymal cells that leads to overall tissue deterioration and eventually results in organ failure. The first description on giant mitochondria dates back to the 1960s, coinciding with the availability of the first generation of electron microscopes in clinical diagnostic laboratories. Detailed accounts on their ultrastructure have mostly been described in patients suffering from alcoholic liver disease, chronic hepatitis, hepatocellular carcinoma and non-alcoholic fatty liver disease. Interestingly, from this extensive literature survey, it became apparent that giant mitochondria or megamitochondria present themselves with or without highly organised crystal-like intramitochondrial inclusions. The origin, formation and potential role of giant mitochondria remain to-date largely unanswered. Likewise, the biochemical composition of the well-organised crystal-like inclusions and their possible impact on mitochondrial function is unclear. Herein, concepts about the possible mechanism of their formation and three-dimensional architecture will be approached. We will furthermore discuss their importance in diagnostics, including future research outlooks and potential therapeutic interventions to cure liver disease where giant mitochondria are implemented.

Human Precision-Cut Liver Slices: A Potential Platform to Study Alcohol-Related Liver Disease.

Alcohol-related liver disease (ALD) encompasses a range of pathological conditions that are complex to study at the clinical and preclinical levels. Despite the global burden of ALD, there is a lack of effective treatments, and mortality is high. One of the reasons for the unsuccessful development of novel therapies is that experimental studies are hindered by the challenge of recapitulating this multifactorial disorder in vitro, including the contributions of hepatotoxicity, impaired lipid metabolism, fibrosis and inflammatory cytokine storm, which are critical drivers in the pathogenesis of ALD in patients and primary targets for drug development. Here, we present the unique characteristics of the culture of human precision-cut liver slices (PCLS) to replicate key disease processes in ALD. PCLS were prepared from human liver specimens and treated with ethanol alone or in combination with fatty acids and lipopolysaccharide (FA + LPS) for up to 5 days to induce hepatotoxic, inflammatory and fibrotic events associated with ALD. Alcohol insult induced hepatocyte death which was more pronounced with the addition of FA + LPS. This mixture showed a significant increase in the cytokines conventionally associated with the prototypical inflammatory response observed in severe ALD, and interestingly, alcohol alone exhibited a different effect. Profibrogenic activation was also observed in the slices and investigated in the context of slice preparation. These results support the versatility of this organotypic model to study different pathways involved in alcohol-induced liver damage and ALD progression and highlight the applicability of the PCLS for drug discovery, confirming their relevance as a bridge between preclinical and clinical studies.

A review of alcohol-pathogen interactions: New insights into combined disease pathomechanisms.

Progression of chronic infections to end-stage diseases and poor treatment results are frequently associated with alcohol abuse. Alcohol metabolism suppresses innate and adaptive immunity leading to increased viral load and its spread. In case of hepatotropic infections, viruses accelerate alcohol-induced hepatitis and liver fibrosis, thereby promoting end-stage outcomes, including cirrhosis and hepatocellular carcinoma (HCC). In this review, we concentrate on several unexplored aspects of these phenomena, which illustrate the combined effects of viral/bacterial infections and alcohol in disease development. We review alcohol-induced alterations implicated in immunometabolism as a central mechanism impacting metabolic homeostasis and viral pathogenesis in Simian immunodeficiency virus/human immunodeficiency virus infection. Furthermore, in hepatocytes, both HIV infection and alcohol activate oxidative stress to cause lysosomal dysfunction and leakage and apoptotic cell death, thereby increasing hepatotoxicity. In addition, we discuss the mechanisms of hepatocellular carcinoma and tumor signaling in hepatitis C virus infection. Finally, we analyze studies that review and describe the immune derangements in hepatotropic viral infections focusing on the development of novel targets and strategies to restore effective immunocompetency in alcohol-associated liver disease. In conclusion, alcohol exacerbates the pathogenesis of viral infections, contributing to a chronic course and poor outcomes, but the mechanisms behind these events are virus specific and depend on virus-alcohol interactions, which differ among the various infections.

Inflammation in alcohol-associated liver disease progression.

Chronic alcohol consumption induces stress and damage in alcohol metabolising hepatocytes, which leads to inflammatory and fibrogenic responses. Besides these direct effects, alcohol disrupts intestinal barrier functions and induces gut microbial dysbiosis, causing translocation of bacteria or microbial products through the gut mucosa to the liver and, which induce inflammation indirectly. Inflammation is one of the key drivers of alcohol-associated liver disease progression from steatosis to severe alcoholic hepatitis. The current standard of care for the treatment of severe alcoholic hepatitis is prednisolone, aiming to reduce inflammation. Prednisolone, however improves only short-term but not long-term survival rates in those patients, and even increases the risk for bacterial infections. Thus, recent studies focus on the exploration of more specific inflammatory targets for the treatment of severe alcoholic hepatitis. These comprise, among others interference with inflammatory cytokines, modulation of macrophage phenotypes or targeting of immune cell communication, as summarized in the present overview. Although several approaches give promising results in preclinical studies, data robustness and ability to transfer experimental results to human disease is still not sufficient for effective clinical translation.

The multifactorial mechanisms of bacterial infection in decompensated cirrhosis.

Infections, due to a dysfunctional immune response, pose a great risk to patients with decompensated cirrhosis and herald the beginning of the terminal phase of this disease. Infections typically result from breaches in innate immune barriers and inadequate clearance by immune cells. This leads to bacterial and bacterial product translocation to the systemic circulation, which is already primed by ongoing hepatic inflammation in patients with cirrhosis, who are particularly prone to developing organ failure in the presence of an infection. Early identification of bacterial infection, along with the prompt use of appropriate antibiotics, have reduced the mortality associated with certain infections in patients with decompensated cirrhosis. Judicious use of antibiotic therapy remains imperative given the emergence of multidrug-resistant infections in the cirrhotic population. Important research over the last few years has identified molecular targets on immune cells that may enhance their function, and theoretically prevent infections. Clinical trials are ongoing to delineate the beneficial effects of targeted molecules from their off-target effects. Herein, we review the mechanisms that predispose patients with cirrhosis to bacterial infections, the clinical implications of infections and potential targets for the prevention or treatment of infections in this vulnerable population.

Dynamin-1-Like Protein Inhibition Drives Megamitochondria Formation as an Adaptive Response in Alcohol-Induced Hepatotoxicity.

Despite the growing global burden of alcoholic liver diseases, therapeutic options are limited, and novel targets are urgently needed. Accumulating evidence suggests that mitochondria adapt in response to ethanol and formation of megamitochondria in the livers of patients is recognized as a hallmark of alcoholic liver diseases. The processes involved in ethanol-induced hepatic mitochondrial changes, the impact on mitochondria-shaping proteins, and the significance of megamitochondria formation remain unknown. In this study, we investigated the mitochondrial and cellular response to alcohol in hepatoma cell line VL-17A. The mitochondrial architecture rapidly changed after 3 or 14 days of ethanol exposure with double-pronged presentation of hyperfragmentation and megamitochondria, and cell growth was inhibited. Dynamin-1-like protein (Drp1) was identified as the main mediator driving these mitochondrial alterations, and its genetic inactivation was determined to foster megamitochondria development, preserving the capacity of the cells to grow despite alcohol toxicity. The role of Drp1 in mediating megamitochondria formation in mice with liver-specific inactivation of Drp1 was further confirmed. Finally, when these mice were fed with ethanol, the presentation of hepatic megamitochondria was exacerbated compared with wild type fed with the same diet. Ethanol-induced toxicity was also reduced. Our study demonstrates that megamitochondria formation is mediated by Drp1, and this phenomenon is a beneficial adaptive response during alcohol-induced hepatotoxicity.

Perturbations in Mitochondrial Dynamics Are Closely Involved in the Progression of Alcoholic Liver Disease.

BACKGROUND: Mitochondria play a fundamental role in the pathogenesis of alcoholic liver disease (ALD). The preservation of functional mitochondria during toxic alcohol insults is essential for cell survival and is maintained by key processes known as mitochondrial dynamics, including fragmentation and fusion, which are regulated by mitochondria-shaping proteins (MSP). We have shown mitochondrial dynamics to be distorted by alcohol in cellular and animal models, but the effect in humans remains unknown. METHODS: Hepatic gene expression of the main MSP involved in the mitochondrial fusion and fragmentation pathways was evaluated in patients with alcoholic hepatitis (AH) by DNA microarray (n = 15) and Reverse Transcription Polymerase Chain Reaction (n = 32). The activation of dynamin-1-like protein (Drp1) was also investigated in mitochondria isolated from liver biopsies of ALD patients (n = 8). The effects of alcohol on mitochondrial dynamics and on MSP protein expression were studied in human precision-cut liver slices (PCLS) exposed for 24 hours to increasing doses of ethanol (EtOH; 50 to 250 mM). RESULTS: A profound hyperactivation of the fragmentation pathway was observed in AH patients, with a significant increase in the expression of Drp1 and its adapters/receptors. The translocation of Drp1 to the mitochondria was also induced in patients with severe ALD and was affected in the PCLS with short-term exposure to EtOH but only mildly. The fusion pathway was not altered in ALD, and this was confirmed in the PCLS model. CONCLUSIONS: The present study reveals the role of mitochondrial dynamics in human ALD, confirming our previous observations in animal and cell culture models of ALD. Taken together, we show that alcohol has a significant impact on the fragmentation pathway, and we confirm Drp1 as a potential therapeutic target in severe ALD.

Mucosa-associated invariant T cells link intestinal immunity with antibacterial immune defects in alcoholic liver disease.

BACKGROUND/AIMS: Intestinal permeability with systemic distribution of bacterial products are central in the immunopathogenesis of alcoholic liver disease (ALD), yet links with intestinal immunity remain elusive. Mucosa-associated invariant T cells (MAIT) are found in liver, blood and intestinal mucosa and are a key component of antibacterial host defences. Their role in ALD is unknown. METHODS/DESIGN: We analysed frequency, phenotype, transcriptional regulation and function of blood MAIT cells in severe alcoholic hepatitis (SAH), alcohol-related cirrhosis (ARC) and healthy controls (HC). We also examined direct impact of ethanol, bacterial products from faecal extracts and antigenic hyperstimulation on MAIT cell functionality. Presence of MAIT cells in colon and liver was assessed by quantitative PCR and immunohistochemistry/gene expression respectively. RESULTS: In ARC and SAH, blood MAIT cells were dramatically depleted, hyperactivated and displayed defective antibacterial cytokine/cytotoxic responses. These correlated with suppression of lineage-specific transcription factors and hyperexpression of homing receptors in the liver with intrahepatic preservation of MAIT cells in ALD. These alterations were stronger in SAH, where surrogate markers of bacterial infection and microbial translocation were higher than ARC. Ethanol exposure in vitro, in vivo alcohol withdrawal and treatment with Escherichia coli had no effect on MAIT cell frequencies, whereas exposure to faecal bacteria/antigens induced functional impairments comparable with blood MAIT cells from ALD and significant MAIT cell depletion, which was not observed in other T cell compartments. CONCLUSIONS: In ALD, the antibacterial potency of MAIT cells is compromised as a consequence of contact with microbial products and microbiota, suggesting that the 'leaky' gut observed in ALD drives MAIT cell dysfunction and susceptibility to infection in these patients.

Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis (NASH) fibrosis.

INTRODUCTION: Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH. METHODS: Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis. RESULTS: MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFß mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice. CONCLUSION: OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH.

Alisporivir inhibition of hepatocyte cyclophilins reduces HBV replication and hepatitis B surface antigen production.

BACKGROUND & AIMS: Cyclophilins are host factors required for hepatitis C virus replication. Cyclophilin inhibitors such as alisporivir have shown strong anti-hepatitis C virus activity in vitro and in clinical studies. However, little is known about whether hepatocyte cyclophilins are involved in the hepatitis B virus (HBV) life cycle. We investigated the effects of 2 cyclophilin inhibitors (alisporivir and NIM811) on HBV replication and hepatitis B surface antigen (HBsAg) production in cell lines. METHODS: Liver-derived cell lines producing full-length HBV and HBsAg particles, owing to stable (HepG2215) or transient (HuH-7) transfection, or infected with HBV (HepaRG cells; Invitrogen [Carlsbad, CA]), were incubated with alisporivir or NIM811 alone, or alisporivir in combination with a direct antiviral (telbivudine). The roles of individual cyclophilins in drug response was evaluated by small interfering RNA knockdown of cyclophilin (CYP)A, CYPC, or CYPD in HepG2215 cells, or CYPA knockdown in HuH-7 cells. The kinetics of antiviral activity were assessed based on levels of HBV DNA and HBsAg and Southern blot analysis. RESULTS: In HepG2215, HuH-7, and HepaRG cells, alisporivir reduced intracellular and secreted HBV DNA, in a dose-dependent manner. Knockdown of CYPA, CYPC, or CYPD (reduced by 80%) significantly reduced levels of HBV DNA and secreted HBsAg. Knockdown of CYPA significantly reduced secretion of HBsAg, leading to accumulation of intracellular HBsAg; the addition of alisporivir greatly reduced levels of HBsAg in these cells. The combination of alisporivir and telbivudine had greater antiviral effects than those of telbivudine or alisporivir alone. CONCLUSIONS: Alisporivir inhibition of cyclophilins in hepatocyte cell lines reduces replication of HBV DNA and HBsAg production and secretion. These effects are potentiated in combination with direct antiviral agents that target HBV-DNA polymerase.

Blockade of PD1 and TIM3 restores innate and adaptive immunity in patients with acute alcoholic hepatitis.

BACKGROUND & AIMS: Susceptibility to bacterial infection is a feature of alcohol-related liver disease. Programmed cell death 1 (PD1), the T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3, also known as hepatitis A virus cellular receptor 2), and their respective ligands-CD274 (also known as PD ligand 1 [PDL1]) and galectin-9-are inhibitory receptors that regulate the balance between protective immunity and host immune-mediated damage. However, their sustained hyperexpression promotes immune exhaustion and paralysis. We investigated the role of these immune inhibitory receptors in driving immune impairments in patients with alcoholic liver disease. METHODS: In a prospective study, we collected blood samples from 20 patients with acute alcoholic hepatitis (AAH), 16 patients with stable advanced alcohol-related cirrhosis, and 12 healthy individuals (controls). Whole blood or peripheral blood mononuclear cells were assessed for expression of PD1, PDL1, TIM3, galectin-9, and Toll-like receptors on subsets of innate and adaptive immune effector cells. We measured antibacterial immune responses to lipopolysaccharide (endotoxin) using ELISpot assays, and used flow cytometry to quantify cytokine production, phagocytosis, and oxidative burst in the presence or absence of blocking antibodies against PD1 or TIM3. RESULTS: Antibacterial innate and adaptive immune responses were greatly reduced in patients with AAH, compared with controls, and patients with alcohol-related cirrhosis had less severe dysfunctions in innate immune effector cells and preserved functional T-cell responses. Fewer T cells from patients with AAH produced interferon gamma in response to lipopolysaccharide, compared with controls. In addition, patients with AAH had greater numbers of interleukin 10-producing T cells, and reduced levels of neutrophil phagocytosis and oxidative burst in response to Escherichia coli stimulation, compared with controls. T cells from patients with AAH, but not alcohol-related cirrhosis, expressed higher levels of PD1 and PDL1, or TIM3 and galectin-9, than T cells from controls. Antibodies against PD1 and TIM3 restored T-cell production of interferon gamma, reduced the numbers of interleukin 10-producing T cells, and increased neutrophil antimicrobial activities. Circulating levels of endotoxin in plasma from patients with AAH caused over expression of immune inhibitory receptors on T cells via Toll-like receptor 4 binding to CD14(+) monocytes. CONCLUSIONS: Antibacterial immune responses are impaired in patients with AAH. Lymphocytes from these patients express high levels of immune inhibitory receptors, produce lower levels of interferon gamma, and have increased IL10 production due to chronic endotoxin exposure. These effects can be reversed by blocking PD1 and TIM3, which increase the antimicrobial activities of T cells and neutrophils.

Neutrophil Toll-Like Receptor 9 Expression and the Systemic Inflammatory Response in Acetaminophen-Induced Acute Liver Failure.

OBJECTIVES: There is a marked propensity for patients with acetaminophen-induced acute liver failure to develop sepsis, which may culminate in multiple organ failure and death. Toll-like receptors sense pathogens and induce inflammatory responses, but whether this is protective or detrimental in acetaminophen-induced acute liver failure remains unknown. DESIGN, SETTING, AND PATIENTS: We assessed Toll-like receptor expression on circulating neutrophils and their function in 24 patients with acetaminophen-induced acute liver failure and compared with 10 healthy controls. INTERVENTIONS: Neutrophil Toll-like receptor 2, -4, and -9 expression and cytokine production and function were studied ex vivo at baseline and following stimulation with lipopolysaccharide, oligodeoxynucleotides, ammonium chloride, and interleukin-8. To examine the influence of acetaminophen-induced acute liver failure plasma and endogenous DNA on Toll-like receptors-9 expression, healthy neutrophils were incubated with acetaminophen-induced acute liver failure plasma with and without deoxyribonuclease-I. MEASUREMENTS AND MAIN RESULTS: Circulating neutrophil Toll-like receptor 9 expression was increased in acetaminophen-induced acute liver failure on day 1 compared with healthy controls (p = 0.0002), whereas Toll-like receptor 4 expression was decreased compared with healthy controls (p < 0.0001). Toll-like receptor 2 expression was unchanged. Neutrophil phagocytic activity was decreased, and spontaneous oxidative burst increased in all patients with acetaminophen-induced acute liver failure compared with healthy controls (p < 0.0001). Neutrophil Toll-like receptor 9 expression correlated with plasma interleukin-8 and peak ammonia concentration (r = 0.6; p < 0.05) and increased with severity of hepatic encephalopathy (grade 0-2 vs 3/4) and systemic inflammatory response syndrome score (0-1 vs 2-4) (p < 0.05). Those patients with advanced hepatic encephalopathy (grade 3/4) or high systemic inflammatory response syndrome score (2-4) on day 1 had higher neutrophil Toll-like receptor 9 expression, arterial ammonia concentration, and plasma interleukin-8 associated with neutrophil exhaustion. Healthy neutrophil Toll-like receptor 9 expression increased upon stimulation with acetaminophen-induced acute liver failure plasma, which was abrogated by preincubation with deoxyribonuclease-I. Intracellular Toll-like receptor 9 was induced by costimulation with interleukin-8 and ammonia. CONCLUSION: These data point to neutrophil Toll-like receptor 9 expression in acetaminophen-induced acute liver failure being mediated both by circulating endogenous DNA as well as ammonia and interleukin-8 in a synergistic manner inducing systemic inflammation, neutrophil exhaustion, and exacerbating hepatic encephalopathy.

MacroH2A1 isoforms are associated with epigenetic markers for activation of lipogenic genes in fat-induced steatosis.

The importance of epigenetic changes in the development of hepatic steatosis is largely unknown. The histone variant macroH2A1 under alternative splicing gives rise to macroH2A1.1 and macroH2A1.2. In this study, we show that the macroH2A1 isoforms play an important role in the regulation of lipid accumulation in hepatocytes. Hepatoma cell line and immortalized human hepatocytes transiently transfected or knocked down with macroH2A1 isoforms were used as in vitro model of fat-induced steatosis. Gene expressions were analyzed by quantitative PCR array and Western blot. Chromatin immunoprecipitation analysis was performed to check the association of histone H3 lysine 27 trimethylation (H3K27me3) and histone H3 lysine 4 trimethylation (H3K4me3) with the promoter of lipogenic genes. Livers from knockout mice that are resistant to lipid deposition despite a high-fat diet were used for histopathology. We found that macroH2A1.2 is regulated by fat uptake and that its overexpression caused an increase in lipid uptake, triglycerides, and lipogenic genes compared with macroH2A1.1. This suggests that macroH2A1.2 is important for lipid uptake, whereas macroH2A1.1 was found to be protective. The result was supported by a high positivity for macroH2A1.1 in knockout mice for genes targeted by macroH2A1 (Atp5a1 and Fam73b), that under a high-fat diet presented minimal lipidosis. Moreover, macroH2A1 isoforms differentially regulate the expression of lipogenic genes by modulating the association of the active (H3K4me3) and repressive (H3K27me3) histone marks on their promoters. This study underlines the importance of the replacement of noncanonical histones in the regulation of genes involved in lipid metabolism in the progression of steatosis.

Truncated CXCL10 is associated with failure to achieve spontaneous clearance of acute hepatitis C infection.

UNLABELLED: The pathogenesis of hepatitis C virus (HCV) infection is strongly influenced by the nature of the host's antiviral immunity. Counterintuitively, elevated serum concentrations of C-X-C chemokine 10 (CXCL10), a potent chemoattractant for antiviral T-cells and NK-cells, are associated with poor treatment outcomes in patients with chronic HCV. It has been reported that an N-terminal truncated form of CXCL10, generated by the protease dipeptidylpeptidase 4 (DPP4), can act as chemokine antagonist. We sought to investigate CXCL10 antagonism in the clinical outcome and evolution of acute HCV infection. We collected serial blood samples from 16 patients, at the clinical onset of acute HCV infection and at 12 standardized follow-up timepoints over the first year. Intact and truncated CXCL10 and DPP4 activity were quantified in all longitudinal samples. In addition, NK-cell frequency/phenotype, and HCV-specific T-cell responses were assessed. Subjects developing chronicity (n = 11) had higher concentrations of CXCL10 (P < 0.001), which was predominantly in a truncated form (P = 0.036) compared to patients who spontaneously resolved infection (n = 5). Truncated CXCL10 correlated with HCV-RNA (r = 0.40, P < 0.001) and DPP4 activity (r = 0.53, P < 0.001). Subjects who resolved infection had a higher frequency of HCV-specific interferon-gamma (IFNγ)-producing T-cells (P = 0.017) and predominance of cytotoxic NK-cells (P = 0.005) compared to patients who became chronic. Patients who became persistently infected had higher proportions of cytokine-producing NK-cells, which were correlated with concentrations of truncated CXCL10 (r = 0.92, P < 0.001). CONCLUSION: This study provides the first evidence of chemokine antagonism during acute HCV infection. We suggest that the DPP4-CXCL10 axis inhibits antiviral innate and adaptive host immunity and favors establishment of viral persistence.

Alcohol-Associated Liver Disease Outcomes: Critical Mechanisms of Liver Injury Progression.

Alcohol-associated liver disease (ALD) is a substantial cause of morbidity and mortality worldwide and represents a spectrum of liver injury beginning with hepatic steatosis (fatty liver) progressing to inflammation and culminating in cirrhosis. Multiple factors contribute to ALD progression and disease severity. Here, we overview several crucial mechanisms related to ALD end-stage outcome development, such as epigenetic changes, cell death, hemolysis, hepatic stellate cells activation, and hepatic fatty acid binding protein 4. Additionally, in this review, we also present two clinically relevant models using human precision-cut liver slices and hepatic organoids to examine ALD pathogenesis and progression.

The diversity and management of chronic hepatitis B virus infections in the United Kingdom: a wake-up call.

BACKGROUND: Through migration, diversity of chronic hepatitis B virus (HBV) infection has changed, affecting disease burden and control. We describe clinical and viral characteristics of chronic HBV in the United Kingdom. METHODS: A total of 698 individuals with chronic HBV infection were recruited from referral liver centers. Demographic, clinical, and laboratory data were collected. RESULTS: Sixty-one percent of patients were male, 80% were not born in the United Kingdom, and the largest ethnicity was East/Southeast Asian (36%). Twenty-two percent were hepatitis B e antigen (HBeAg) seropositive; 20.4% (59/289) had cirrhosis and 10 (1.7%) had hepatocellular carcinoma. Genotype D was most common (31%) followed by A, C, B, and E (20%, 20%, 19%, and 9%, respectively). Genotype was significantly associated with country of birth, length of time in the United Kingdom, HBeAg status, and precore and basal core promoter mutations. One-third were on treatment, with men independently more likely to be treated. Only 18% of those on treatment were on recommended first-line therapies, and 30% were on lamivudine monotherapy. Among treated individuals, 27% had antiviral drug resistance. Testing rates for human immunodeficiency virus, hepatitis C virus, and delta coinfections were low. CONCLUSIONS: We demonstrated diversity of chronic HBV infections in UK patients, suggesting that optimal management requires awareness of the variable patterns of chronic HBV in countries of origin. We also found less-than-optimal clinical management practices, possible gender-based treatment bias, and the need to improve testing for coinfections.

A Pathogenic Role of Non-Parenchymal Liver Cells in Alcohol-Associated Liver Disease of Infectious and Non-Infectious Origin.

Now, much is known regarding the impact of chronic and heavy alcohol consumption on the disruption of physiological liver functions and the induction of structural distortions in the hepatic tissues in alcohol-associated liver disease (ALD). This review deliberates the effects of alcohol on the activity and properties of liver non-parenchymal cells (NPCs), which are either residential or infiltrated into the liver from the general circulation. NPCs play a pivotal role in the regulation of organ inflammation and fibrosis, both in the context of hepatotropic infections and in non-infectious settings. Here, we overview how NPC functions in ALD are regulated by second hits, such as gender and the exposure to bacterial or viral infections. As an example of the virus-mediated trigger of liver injury, we focused on HIV infections potentiated by alcohol exposure, since this combination was only limitedly studied in relation to the role of hepatic stellate cells (HSCs) in the development of liver fibrosis. The review specifically focusses on liver macrophages, HSC, and T-lymphocytes and their regulation of ALD pathogenesis and outcomes. It also illustrates the activation of NPCs by the engulfment of apoptotic bodies, a frequent event observed when hepatocytes are exposed to ethanol metabolites and infections. As an example of such a double-hit-induced apoptotic hepatocyte death, we deliberate on the hepatotoxic accumulation of HIV proteins, which in combination with ethanol metabolites, causes intensive hepatic cell death and pro-fibrotic activation of HSCs engulfing these HIV- and malondialdehyde-expressing apoptotic hepatocytes.

Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing.

BACKGROUND: The exploitation of anti-tumour immunity, harnessed through immunomodulatory therapies, has fundamentally changed the treatment of primary liver cancer (PLC). However, this has posed significant challenges in preclinical research. Novel immunologically relevant models for PLC are urgently required to improve the translation from bench to bedside and back, explore and predict effective combinatorial therapies, aid novel drug discovery and develop personalised treatment modalities. METHODS: We used human precision-cut tissue slices (PCTS) derived from resected tumours to create a patient-specific immunocompetent disease model that captures the multifaceted and intricate heterogeneity of the tumour and the tumour microenvironment. Tissue architecture, tumour viability and treatment response to single agent and combination therapies were assessed longitudinally over 8 days of ex vivo culture by histological analysis, detection of proliferation/cell death markers, ATP content via HPLC. Immune cell infiltrate was assessed using PCR and immunofluorescence. Checkpoint receptor expression was quantified via Quantigene RNA assay. FINDINGS: After optimising the culture conditions, PCTS maintained the original tissue architecture, including tumour morphology, stroma and tumour-infiltrated leukocytes. Moreover, PCTS retained the tumour-specific immunophenotype over time, suggesting the utility of PCTS to investigate immunotherapeutic drug efficacy and identify non-responsiveness. INTERPRETATION: Here we have characterised the PCTS model and demonstrated its effectiveness as a robust preclinical tool that will significantly support the development of successful (immuno)therapeutic strategies for PLC. FUNDING: Foundation for Liver Research, London.

Interaction between estrogen receptor-α and PNPLA3 p.I148M variant drives fatty liver disease susceptibility in women.

Fatty liver disease (FLD) caused by metabolic dysfunction is the leading cause of liver disease and the prevalence is rising, especially in women. Although during reproductive age women are protected against FLD, for still unknown and understudied reasons some develop rapidly progressive disease at the menopause. The patatin-like phospholipase domain-containing 3 (PNPLA3) p.I148M variant accounts for the largest fraction of inherited FLD variability. In the present study, we show that there is a specific multiplicative interaction between female sex and PNPLA3 p.I148M in determining FLD in at-risk individuals (steatosis and fibrosis, P < 10-10; advanced fibrosis/hepatocellular carcinoma, P = 0.034) and in the general population (P < 10-7 for alanine transaminase levels). In individuals with obesity, hepatic PNPLA3 expression was higher in women than in men (P = 0.007) and in mice correlated with estrogen levels. In human hepatocytes and liver organoids, PNPLA3 was induced by estrogen receptor-α (ER-α) agonists. By chromatin immunoprecipitation and luciferase assays, we identified and characterized an ER-α-binding site within a PNPLA3 enhancer and demonstrated via CRISPR-Cas9 genome editing that this sequence drives PNPLA3 p.I148M upregulation, leading to lipid droplet accumulation and fibrogenesis in three-dimensional multilineage spheroids with stellate cells. These data suggest that a functional interaction between ER-α and PNPLA3 p.I148M variant contributes to FLD in women.

CD8(+) T cell control of hepatitis B virus replication: direct comparison between cytolytic and noncytolytic functions.

Resolution of hepatitis B virus (HBV) infection was believed to be attributed to the cytotoxic T cell-mediated killing of infected hepatocytes. However, studies in HBV transgenic mice and HBV-infected chimpanzees revealed that T cell control of HBV replication also involves cytokine-mediated noncytolytic mechanisms. The relative role of cytolytic and noncytolytic functions of virus-specific CD8(+) T cells during interaction with HBV-producing hepatocytes is not well understood. By using HLA-A2 matched effector cells (CD8(+) T cell line or clone) and target cells supporting full HBV replication, we demonstrate that virus-specific CD8(+) T cells can inhibit HBV replication in HBV-producing hepatocytes with minimal cell lysis. Although CD8(+) T cells kill a fraction of infected cells, this effect is minimal, and most of the viral inhibition is mediated by noncytolytic mechanisms. CD8(+) T cells produce an array of cytokines, among which IFN-gamma and TNF-alpha are responsible for HBV inactivation in the target cells. Blockade of IFN-gamma and TNF-alpha abrogated the noncytolytic inhibition of HBV, indicating that these two cytokines mediate the control of HBV by noncytolytic mechanisms. Furthermore, treatment of the HBV-producing hepatocytes with rIFN-gamma and rTNF-alpha resulted in an efficient suppression of viral replication without cytotoxicity. In contrast, coculture of the same target cells with activated HLA-mismatched mitogen-activated lymphomononuclear cells caused a marked cytolytic effect and was less effective in HBV control. These results provide direct evidence that virus-specific CD8(+) T cells efficiently control HBV replication by noncytolytic mechanisms, and this effect is mediated by IFN-gamma and TNF-alpha.