Delineating the heterogeneity of senescence-induced-functional alterations in hepatocytes.

BACKGROUND AND AIM: Cellular senescence of hepatocytes involves permanent cell cycle arrest, disrupted cellular bioenergetics, resistance to cell death, and the release of pro-inflammatory cytokines. This 'zombie-like' state perpetuates harmful effects on tissues and holds potential implications for liver disease progression. Remarkably, senescence exhibits heterogeneity, stemming from two crucial factors: the inducing stressor and the cell type. As such, our present study endeavors to characterize stressor-specific changes in senescence phenotype, its related molecular patterns, and cellular bioenergetics in primary mouse hepatocytes (PMH) and hepatocyte-derived liver organoids (HepOrgs). METHODS: PMH, isolated by collagenase-perfused mouse liver (C57B6/J; 18-23 weeks), were cultured overnight in William's E-medium supplemented with 2% FBS, L-glutamine, and hepatocyte growth supplements. HepOrgs were developed by culturing cells in a 3D matrix for two weeks. The senescence was induced by DNA damage (doxorubicin, cisplatin, and etoposide), oxidative stress (H2O2, and ethanol), and telomere inhibition (BIBR-1532), p53 activation (nutlin-3a), DNA methyl transferase inhibition (5-azacitidine), and metabolism inhibitors (galactosamine and hydroxyurea). SA-ß galactosidase activity, immunofluorescence, immunoblotting, and senescence-associated secretory phenotype (SASP), and cellular bioenergetics were used to assess the senescence phenotype. RESULTS: Each senescence inducer triggers a unique combination of senescence markers in hepatocytes. All senescence inducers, except hydroxyurea and ethanol, increased SA-ß galactosidase activity, the most commonly used marker for cellular senescence. Among the SASP factors, CCL2 and IL-10 were consistently upregulated, while Plasminogen activator inhibitor-1 exhibited global downregulation across all modes of senescence. Notably, DNA damage response was activated by DNA damage inducers. Cell cycle markers were most significantly reduced by doxorubicin, cisplatin, and galactosamine. Additionally, DNA damage-induced senescence shifted cellular bioenergetics capacity from glycolysis to oxidative phosphorylation. In HepOrgs exposed to senescence inducers, there was a notable increase in γH2A.X, p53, and p21 levels. Interestingly, while showing a similar trend, SASP gene expression in HepOrgs was significantly higher compared to PMH, demonstrating a several-fold increase. CONCLUSION: In our study, we demonstrated that each senescence inducer activates a unique combination of senescence markers in PMH. Doxorubicin demonstrated the highest efficacy in inducing senescence, followed by cisplatin and H2O2, with no impact on apoptosis. Each inducer prompted DNA damage response and mitochondrial dysfunction, independent of MAPK/AKT.

Single-cell landscape of immune cells during the progression from HBV infection to HBV cirrhosis and HBV-associated hepatocellular carcinoma.

Introduction: Immune cells play crucial roles in the development of chronic hepatitis B virus (HBV) infection, leading to cirrhosis and hepatocellular carcinoma (HCC). However, their functions at different disease stages are not fully understood. Methods: In this study, we used single-cell RNA sequencing (scRNA-seq) to characterize the human liver immune microenvironment at different disease stages. We analyzed scRNA-seq data from 118,455 immune cells obtained from livers of six healthy individuals, four patients with HBV infection, five patients with HBV cirrhosis, and three patients with HBV-associated HCC. Results: Our results showed an accumulation of scar-associated macrophages during disease progression, and we identified two relevant immune subsets, Macrophage-CD9/IL18 and macrophage-CD9/IFI6. Macrophage-CD9/IL18 expanded from HBV infection to cirrhosis, while macrophage-CD9/IFI6 expanded from cirrhosis to HCC. We verified the existence of Macrophage-CD9/IFI6 using multiplex immunofluorescence staining. We also found an increase in cytotoxic NK Cell-GNLY during progression from cirrhosis to HCC. Additionally, the proportion of CD4 T cell-TNFAIP3, CD8 T cell-TNF (effector CD8 T cells), and CD8 T cell-CD53 increased, while the proportion of Treg cells decreased from HBV infection to cirrhosis. The proportion of Treg and CD8 T cell-LAG3 (Exhausted CD8 T cell) enhanced, while the proportion of CD8 T cell-TNF (effector CD8 T cells) decreased from cirrhosis to HCC. Furthermore, GSEA enrichment analyses revealed that MAPK, ERBB, and P53 signaling pathways in myeloid cells were gradually inhibited from HBV infection to cirrhosis and HCC. Discussion: Our study provides important insights into changes in the hepatic immune environment during the progression of HBV-related liver disease, which may help improve the management of HBV-infected liver diseases.

Changes of the bacterial composition in duodenal fluid from patients with liver cirrhosis and molecular bacterascites.

Small intestinal bacterial overgrowth and compositional changes of intestinal microbiota are pathomechanistic factors in liver cirrhosis leading to bacterial translocation and infectious complications. We analyzed the quantity and composition of duodenal bacterial DNA (bactDNA) in relation to bactDNA in blood and ascites of patients with liver cirrhosis. Duodenal fluid and corresponding blood and ascites samples from 103 patients with liver cirrhosis were collected. Non-liver disease patients (n = 22) served as controls. BactDNA was quantified by 16S-rRNA gene-based PCR. T-RFLP and 16S-rRNA amplicon sequencing were used to analyze bacterial composition. Duodenal bacterial diversity in cirrhosis was distinct to controls showing significantly higher abundances of Streptococcus, Enterococcus and Veillonella. Patients with bactDNA positive ascites revealed reduced spectrum of core microbiota with Streptococcus as key player of duodenal community and higher prevalence of Granulicatella proving presence of cirrhosis related intestinal dysbiosis. Regarding duodenal fluid bactDNA quantification, no significant differences were found between patients with cirrhosis and controls. Additionally, percentage of subjects with detectable bactDNA in blood did not differ between patients and controls. This study evaluated the diversity of bacterial DNA in different body specimens with potential implications on understanding how intestinal bacterial translocation may affect infectious complications in cirrhosis.

Comprehensive insights into the multifaceted roles of the transient receptor potential vanilloid 1 channel in the digestive system.

The transient receptor potential vanilloid (TRPV) channel, a family of calcium transporters comprising six distinct members (TRPV1-6), takes on a paramount role in maintaining intracellular Ca2+ homeostasis in mammalian cells. Notably, TRPV1, among its counterparts, has emerged as the subject of extensive scrutiny, owing to its pervasive presence in diverse cellular, tissue, and organ settings. This ubiquitous distribution underscores its fundamental involvement in the genesis of pain, making it a central focus in pain-related research. However, recent investigations have unveiled that TRPV1's functional significance transcends the realm of pain modulation, extending its influence to encompass a wide spectrum of physiological and pathological processes. The ambit of TRPV1's influence encompasses not only pain responses but also embraces the intricate domains of nervous system disorders, cancer metastasis, as well as afflictions pertaining to the skin and heart. Moreover, compelling evidence now demonstrates that TRPV1 also wields substantial sway in the domain of digestive diseases, further highlighting its versatility and far-reaching impact on human health. Therefore, this comprehensive review endeavors to delve into the multifaceted roles played by TRPV1 in the various organs constituting the digestive system.

Single-cell landscape of immune cells in human livers affected by HBV-related cirrhosis.

Background & Aims: HBV infection is one of the leading causes of liver cirrhosis. However, the immune microenvironment in patients with HBV cirrhosis remains elusive. Methods: Single-cell RNA sequencing was used to analyse the transcriptomes of 76,210 immune cells in the livers of six healthy individuals and in five patients with HBV cirrhosis. Results: Patients with HBV cirrhosis have a unique immune ecosystem characterised by an accumulation of macrophage-CD9/IL18, macrophage-C1QA, NK Cell-JUNB, CD4+ T cell-IL7R, and a loss of B cell-IGLC1 clusters. Furthermore, our analysis predicted enhanced cell communication between myeloid cells and all immune cells in patients with HBV-related cirrhosis. Pseudo-time analysis of myeloid cells, natural killer (NK) cells, and B cells demonstrated a significant accumulation of mature cells and a depletion of naive cells in HBV cirrhosis. In addition, we observed an increase in antigen processing and presentation capacities in myeloid cells in patients with HBV cirrhosis, whereas NK cell-mediated cytotoxicity was substantially reduced. Conclusions: Our results provide valuable insight into the immune landscape of HBV cirrhosis, suggesting that HBV cirrhosis is associated with the accumulation of activated myeloid cells and impaired cytotoxicity in NK cells. Impact and implications: The absence of single-cell transcriptome profiling of immune cells in HBV cirrhosis hinders our understanding of the underlying mechanisms driving disease progression. To address this knowledge gap, our study unveils a distinctive immune ecosystem in HBV cirrhosis and represents a crucial advancement in elucidating the impact of the immune milieu on the development of this condition. These findings constitute significant strides towards the identification of more effective therapeutic approaches for HBV cirrhosis and are relevant for healthcare professionals, researchers, and pharmaceutical developers dedicated to combating this disease.

Granulocyte colony stimulating factor in decompensated cirrhosis, acute alcoholic hepatitis, and acute-on-chronic liver failure: A comprehensive meta-analysis of randomized controlled trials.

BACKGROUND: GCSF may improve the prognosis of severe liver disease by promoting liver regeneration and immune restoration. Our Aim was to investigate its controversial efficacy in decompensated cirrhosis, acute alcoholic hepatitis (AAH), or acute-on-chronic liver failure (ACLF) through meta-analysis. METHODS: Meta-analysis of proportions (random effect model) including 19 RCTs (1287 patients from 16 Asian and 3 European studies including 487 ACLF, 231 AAH and 569 cirrhotic patients) evaluating survival at day-28, day-90, 6 months, one year, and/or occurrence of sepsis as major outcomes. RESULTS: In patients with decompensated cirrhosis, G-CSF administration was associated with a reduction in the weight-adjusted risk of mortality of 9% at day-90 (OR=0.33; 95%CI: 0.18-0.58; p = 0.0002), 16% at 6 months (OR=0.31; 95%CI: 0.15-0.62; p = 0.0009), 26% at one year (OR=0.21; 95%CI:0.12-0.38, p<0.0001) and a weight-adjusted 28% risk reduction for sepsis (OR=0.28; 95%CI: 0.16-0.49; p<0.0001). Only Asian studies were positive. In AAH, G-CSF was associated with an 18% reduction in weight-adjusted mortality risk at day-28 (OR=0.31; 95%CI:0.11-0.83, p = 0.021), 32% at day-90 (OR=0.20; 95%CI:0.09-0.46, p<0.0001) and a weight-adjusted 42% risk reduction for sepsis (OR=0.17; 95%CI: 0.08-0.38; p<0.0001). Only Asian studies, in which corticosteroids were not given systematically in case of severe AAH, were positive. In patients with ACLF, the results on mortality at day-28 were heterogeneous, and GCSF had no beneficial effect on sepsis or survival at day-90. CONCLUSION: G-CSF may be effective in patients with decompensated cirrhosis or AAH by reducing the occurrence of sepsis and mortality. Further meta-analyses of individual data, or new, powerful and methodologically flawless therapeutic trials, are warranted to confirm these results, which harbor wide divergences between Asian and European RCTs.

Pathogenetic mechanisms and therapeutic approaches of acute-to-chronic liver failure.

Liver cirrhosis is the end stage of all chronic liver diseases and contributes significantly to overall mortality of 2% globally. The age-standardized mortality from liver cirrhosis in Europe is between 10 and 20% and can be explained by not only the development of liver cancer but also the acute deterioration in the patient's overall condition. The development of complications including accumulation of fluid in the abdomen (ascites), bleeding in the gastrointestinal tract (variceal bleeding), bacterial infections, or a decrease in brain function (hepatic encephalopathy) define an acute decompensation that requires therapy and often leads to acute-on-chronic liver failure (ACLF) by different precipitating events. However, due to its complexity and organ-spanning nature, the pathogenesis of ACLF is poorly understood, and the common underlying mechanisms leading to the development of organ dysfunction or failure in ACLF are still elusive. Apart from general intensive care interventions, there are no specific therapy options for ACLF. Liver transplantation is often not possible in these patients due to contraindications and a lack of prioritization. In this review, we describe the framework of the ACLF-I project consortium funded by the Hessian Ministry of Higher Education, Research and the Arts (HMWK) based on existing findings and will provide answers to these open questions.

Mechanisms of immunity in acutely decompensated cirrhosis and acute-on-chronic liver failure.

The identification of systemic inflammation (SI) as a central player in the orchestration of acute-on-chronic liver failure (ACLF) has opened new avenues for the understanding of the pathophysiological mechanisms underlying this disease condition. ACLF, which develops in patients with acute decompensation of cirrhosis, is characterized by single or multiple organ failure and high risk of short-term (28-day) mortality. Its poor outcome is closely associated with the severity of the systemic inflammatory response. In this review, we describe the key features of SI in patients with acutely decompensated cirrhosis and ACLF, including the presence of a high blood white cell count and increased levels of inflammatory mediators in systemic circulation. We also discuss the main triggers (i.e. pathogen- and damage-associated molecular patterns), the cell effectors (i.e. neutrophils, monocytes and lymphocytes), the humoral mediators (acute phase proteins, cytokines, chemokines, growth factors and bioactive lipid mediators) and the factors that influence the systemic inflammatory response that drive organ failure and mortality in ACLF. The role of immunological exhaustion and/or immunoparalysis in the context of exacerbated inflammatory responses that predispose ACLF patients to secondary infections and re-escalation of end-organ dysfunction and mortality are also reviewed. Finally, several new potential immunogenic therapeutic targets are debated.

Ex Vivo Liver Machine Perfusion: Comprehensive Review of Common Animal Models.

The most common preservation technique for liver grafts is static cold storage. Due to the organ shortage for liver transplantation (LT), extended criteria donor (ECD) allografts are increasingly used-despite the higher risk of inferior outcome after transplantation. Ex vivo liver machine perfusion (MP) has been developed to improve the outcome of transplantation, especially with ECD grafts, and is currently under evaluation in clinical trials. We performed a literature search on PubMed and ISI Web of Science to assemble an overview of rodent and porcine animal models of ex vivo liver MP for transplantation, which is essential for the present and future development of clinical liver MP. Hypothermic, subnormothermic, and normothermic MP systems have been successfully used for rat and pig LT. In comparison with hypothermic systems, normothermic perfusion often incorporates a dialysis unit. Moreover, it enables metabolic assessment of liver grafts. Allografts experiencing warm ischemic time have a superior survival rate after MP compared with cold storage alone, irrespective of the temperature used for perfusion. Furthermore, ex vivo MP improves the outcome of regular and ECD liver grafts in animal models. Small and large animal models of ex vivo liver MP are available to foster the further development of this new technology. Impact Statement Ex vivo machine perfusion is an important part of current research in the field of liver transplantation. While evidence for improve storage is constantly rising, the development of future applications such as quality assessment and therapeutic interventions necessitates robust animal models. This review is intended to provide an overview of this technology in common large and small animal models and to give an outlook on future applications. Moreover, we describe developmental steps that can be followed by others, and which can help to decrease the number of animals used for experiments based on the replace, reduce, refine concept.

Combination of G-CSF and a TLR4 inhibitor reduce inflammation and promote regeneration in a mouse model of ACLF.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterised by high short-term mortality, systemic inflammation, and failure of hepatic regeneration. Its treatment is a major unmet medical need. This study was conducted to explore whether combining TAK-242, a Toll-like receptor-4 (TLR4) antagonist, with granulocyte-colony stimulating factor (G-CSF), could reduce inflammation whilst enhancing liver regeneration. METHODS: Two mouse models of ACLF were investigated. Chronic liver injury was induced by carbon tetrachloride; lipopolysaccharide (LPS) or galactosamine (GalN) were then administered as extrahepatic or hepatic insults, respectively. G-CSF and/or TAK-242 were administered daily. Treatment durations were 24 hours and 5 days in the LPS model and 48 hours in the GalN model. RESULTS: In a mouse model of LPS-induced ACLF, treatment with G-CSF was associated with significant mortality (66% after 48 hours vs. 0% without G-CSF). Addition of TAK-242 to G-CSF abrogated mortality (0%) and significantly reduced liver cell death, macrophage infiltration and inflammation. In the GalN model, both G-CSF and TAK-242, when used individually, reduced liver injury but their combination was significantly more effective. G-CSF treatment, with or without TAK-242, was associated with activation of the pro-regenerative and anti-apoptotic STAT3 pathway. LPS-driven ACLF was characterised by p21 overexpression, which is indicative of hepatic senescence and inhibition of hepatocyte regeneration. While TAK-242 treatment mitigated the effect on senescence, G-CSF, when co-administered with TAK-242, resulted in a significant increase in markers of hepatocyte regeneration. CONCLUSION: The combination of TAK-242 and G-CSF inhibits inflammation, promotes hepatic regeneration and prevents mortality in models of ACLF; thus, this combination could be a potential treatment option for ACLF. LAY SUMMARY: Acute-on-chronic liver failure is associated with severe liver inflammation and poor short-term survival. Therefore, effective treatments are urgently needed. Herein, we have shown, using mouse models, that the combination of granulocyte-colony stimulating factor (which can promote liver regeneration) and TAK-242 (which inhibits a receptor that plays a key role in inflammation) could be effective for the treatment of acute-on-chronic liver failure.

Final safety and efficacy results from a 106 real-world patients registry with an ascites-mobilizing pump.

BACKGROUND AND AIMS: Patients with cirrhotic refractory ascites ineligible for transjugular intrahepatic shunt (TIPSS) have limited treatment options apart from repeated large volume paracentesis. The alfapump® is an implantable device mobilizing ascites from the peritoneal cavity to the bladder, from where it can be excreted. The aim of this observational cohort study was to prospectively investigate safety and efficacy of the device in a real-world cohort with cirrhotic refractory ascites and contraindications for TIPSS. METHODS: A total of 106 patients received an implant at 12 European centres and were followed up for up to 24 months. Complications, device deficiencies, frequency of paracentesis, clinical status and survival were recorded prospectively. RESULTS: Approximately half of the patients died on-study, about a quarter was withdrawn because of serious adverse events leading to explant, a sixth were withdrawn because of liver transplant or recovery, and nine completed follow-up. The most frequent causes of on-study death and complication-related explant were progression of liver disease and infection. The device reduced the requirement for large-volume paracentesis significantly, with more than half of patients not having required any post-implant. Survival benefits were not observed. Device-related reinterventions were predominantly caused by device deficiencies. A post-hoc comparison of the first 50 versus the last 50 patients enrolled revealed a decreased reintervention rate in the latter, mainly related to peritoneal catheter modifications. CONCLUSIONS: The device reduced paracentesis frequency in a real-world setting. Technical complications were successfully decreased by optimization of management and device modification (NCT01532427).

Body composition and the skeletal muscle compartment in liver transplantation: Turning challenges into opportunities.

Frailty, nutritional status, and body composition are increasingly under the spotlight of interest in various clinical scenarios including liver transplantation. To address the rapidly accumulating evidence in this field, recent European and North American practice guidelines have clearly underlined the clinical importance of nutritional status and body composition with adopting their assessment in patients with liver disease and in transplant candidates into their recommendations. While earlier reports, and therefore present guidelines, were focusing predominantly on quantitative alterations of the skeletal muscle mass (sarcopenia), recent studies have identified qualitative alterations such as intramuscular fat accumulation (myosteatosis) and sarcopenic obesity as emerging risk factors for poor clinical outcomes. In this review, the role of body composition in the context of liver transplantation will be discussed with a focus on the skeletal muscle compartment. A brief overview of current assessment modalities including their limitations, diagnostic challenges, prognostic significance, and pathophysiology are included. Possibilities to incorporate body composition parameters into clinical decision making are discussed. In addition, novel trends and remaining challenges in the therapeutic targeting of body composition and the skeletal muscle compartment are highlighted.

Body fat composition determines outcomes before and after liver transplantation in patients with cirrhosis.

Cachexia occurs in late stages of liver cirrhosis, and a low-fat mass is potentially associated with poor outcome. This study compared different computed tomography (CT)-derived fat parameters with respect to its prognostic impact on the development of complications and death before and after liver transplantation. Between 2001 and 2014, 612 patients with liver cirrhosis without hepatocellular carcinoma listed for liver transplantation met the inclusion criteria, including abdominal CT scan (±200 days to listing). A total of 109 patients without cirrhosis served as controls. The subcutaneous fat index (SCFI), the paraspinal muscle fat index, and the visceral fat index were assessed at L3/L4 level and normalized to the height (cm2 /m2 ). Data were collected and analyzed retrospectively. Low SCFI was associated with a higher rate of ascites and increased C-reactive protein levels (p < 0.001). In addition, multivariate Cox regression analysis adjusting for sex, age, body mass index (BMI), and Model for End-Stage Liver Disease showed that decreasing SCFI was also associated with an increased risk of cirrhosis-related complications (p = 0.003) and death on the transplant wait list (p = 0.013). Increased paraspinal and visceral fat were not only positively correlated with creatinine levels (p < 0.001), BMI, and metabolic comorbidities (all p < 0.001) before transplantation, but also predictive for 1-year mortality after transplantation. Conclusion: The distribution of body fat is a major determinant for complications and outcome in cirrhosis before and after liver transplantation.

Consensus care recommendations for alfapump® in cirrhotic patients with refractory or recurrent ascites.

BACKGROUND: The alfapump® is an implantable class III medical device that pumps ascitic fluid from the peritoneal space to the urinary bladder from where it is excreted. The pump reduces or abrogates the need for repeated paracentesis in patients with recurrent or refractory ascites. AIMS: To improve outcomes for alfapump® implantation and pre- and post-implant patient management in both clinical trial and real-world settings by development of consensus recommendations. METHODS: The alfapump® working group consisting of hepatologists and surgeons with extensive experience in implantation of the alfapump® and patient management met on two occasions: (1) to determine the key areas where recommendations should be made; and (2) to discuss the experiences of the working group within those areas and formulate draft statements. Developed statements were submitted to the group and consensus sought on relevance and wording through a collaborative iterative approach in order to consolidate the recommendations into consensus statements. Only recommendations agreed upon unanimously were included. RESULTS: Twenty-three consensus recommendations were developed in the areas of pre-implantation procedure, (three statements), surgical implant procedure (11 statements), immediate post-implant care (three statements) and long-term management (six statements). CONCLUSIONS: The consensus statements are a valuable reference resource for physicians managing patients with the alfapump® and for those considering management strategies for patients with refractory ascites.

Prognostic and Predictive Molecular Markers in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is the second most common primary liver cancer and subsumes a heterogeneous group of malignant tumors arising from the intra- or extrahepatic biliary tract epithelium. A rising mortality from CCA has been reported worldwide during the last decade, despite significant improvement of surgical and palliative treatment. Over 50% of CCAs originate from proximal extrahepatic bile ducts and constitute the most common CCA entity in the Western world. Clinicopathological characteristics such as lymph node status and poor differentiation remain the best-studied, but imperfect prognostic factors. The identification of prognostic molecular markers as an adjunct to traditional staging systems may not only facilitate the selection of patients who would benefit the most from surgical, adjuvant or palliative treatment strategies, but may also be helpful in defining the aggressiveness of the disease and identifying patients at high-risk for tumor recurrence. The purpose of this review is to provide an overview of currently known molecular prognostic and predictive markers and their role in CCA.

The Potential Role of Cellular Senescence in Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) represents an increasing global health burden. Cellular senescence develops in response to cellular injury, leading not only to cell cycle arrest but also to alterations of the cellular phenotype and metabolic functions. In this review, we critically discuss the currently existing evidence for the involvement of cellular senescence in NAFLD in order to identify areas requiring further exploration. Hepatocyte senescence can be a central pathomechanism as it may foster intracellular fat accumulation, fibrosis and inflammation, also due to secretion of senescence-associated inflammatory mediators. However, in some non-parenchymal liver cell types, such as hepatic stellate cells, senescence may be beneficial by reducing the extracellular matrix deposition and thereby reducing fibrosis. Deciphering the detailed interaction between NAFLD and cellular senescence will be essential to discover novel therapeutic targets halting disease progression.