Recent advances in the prevention and treatment of decompensated cirrhosis and acute-on-chronic liver failure (ACLF) and the role of biomarkers.

The progression of cirrhosis with clinically significant portal hypertension towards decompensated cirrhosis remains clinically challenging and the evolution towards acute-on-chronic liver failure (ACLF), with one or more extrahepatic organ failures, is associated with very high mortality. In the last decade, significant progress has been made in the understanding of the mechanisms leading to decompensation and ACLF. As portal hypertension advances, bacterial translocation across an impaired gut barrier culminates in endotoxaemia, systemic inflammation and cirrhosis-associated immune dysfunction (CAID). Gut-derived systemic inflammation and CAID have become the logical targets for innovative therapies that prevent hepatic decompensation episodes and the progression to ACLF.Furthermore, classification of disease and biomarker discovery to personalise care have advanced in the field. This review discusses progress in biomarker discovery and personalisation of treatment in decompensated cirrhosis and ACLF.

Clinical, histological and molecular profiling of different stages of alcohol-related liver disease.

OBJECTIVE: Alcohol-related liver disease (ALD) ranges from never-decompensated ALD (ndALD) to the life-threatening decompensated phenotype, known as alcohol-related hepatitis (AH). A multidimensional study of the clinical, histological and molecular features of these subtypes is lacking. DESIGN: Two large cohorts of patients were recruited in an international, observational multicentre study: a retrospective cohort of patients with ndALD (n=110) and a prospective cohort of patients with AH (n=225). Clinical, analytical, immunohistochemistry and hepatic RNA microarray analysis of both disease phenotypes were performed. RESULTS: Age and mean alcohol intake were similar in both groups. AH patients had greater aspartate amino transferase/alanine amino transferase ratio and lower gamma-glutamyl transferase levels than in ndALD patients. Patients with AH demonstrated profound liver failure and increased mortality. One-year mortality was 10% in ndALD and 50% in AH. Histologically, steatosis grade, ballooning and pericellular fibrosis were similar in both groups, while advanced fibrosis, Mallory-Denk bodies, bilirubinostasis, severe neutrophil infiltration and ductular reaction were more frequent among AH patients. Transcriptome analysis revealed a profound gene dysregulation within both phenotypes when compare to controls. While ndALD was characterised by deregulated expression of genes involved in matrisome and immune response, the development of AH resulted in a marked deregulation of genes involved in hepatocyte reprogramming and bile acid metabolism. CONCLUSIONS: Despite comparable alcohol intake, AH patients presented with worse liver function compared with ndALD patients. Bilirubinostasis, severe fibrosis and ductular reaction were prominent features of AH. AH patients exhibited a more profound deregulation of gene expression compared with ndALD patients.

Rifaximin-α reduces gut-derived inflammation and mucin degradation in cirrhosis and encephalopathy: RIFSYS randomised controlled trial.

BACKGROUND & AIMS: Rifaximin-α is efficacious for the prevention of recurrent hepatic encephalopathy (HE), but its mechanism of action remains unclear. We postulated that rifaximin-α reduces gut microbiota-derived endotoxemia and systemic inflammation, a known driver of HE. METHODS: In a placebo-controlled, double-blind, mechanistic study, 38 patients with cirrhosis and HE were randomised 1:1 to receive either rifaximin-α (550 mg BID) or placebo for 90 days. PRIMARY OUTCOME: 50% reduction in neutrophil oxidative burst (OB) at 30 days. SECONDARY OUTCOMES: changes in psychometric hepatic encephalopathy score (PHES) and neurocognitive functioning, shotgun metagenomic sequencing of saliva and faeces, plasma and faecal metabolic profiling, whole blood bacterial DNA quantification, neutrophil toll-like receptor (TLR)-2/4/9 expression and plasma/faecal cytokine analysis. RESULTS: Patients were well-matched: median MELD (11 rifaximin-α vs. 10 placebo). Rifaximin-α did not lead to a 50% reduction in spontaneous neutrophil OB at 30 days compared to baseline (p = 0.48). However, HE grade normalised (p = 0.014) and PHES improved (p = 0.009) after 30 days on rifaximin-α. Rifaximin-α reduced circulating neutrophil TLR-4 expression on day 30 (p = 0.021) and plasma tumour necrosis factor-α (TNF-α) (p <0.001). Rifaximin-α suppressed oralisation of the gut, reducing levels of mucin-degrading sialidase-rich species, Streptococcus spp, Veillonella atypica and parvula, Akkermansia and Hungatella. Rifaximin-α promoted a TNF-α- and interleukin-17E-enriched intestinal microenvironment, augmenting antibacterial responses to invading pathobionts and promoting gut barrier repair. Those on rifaximin-α were less likely to develop infection (odds ratio 0.21; 95% CI 0.05-0.96). CONCLUSION: Rifaximin-α led to resolution of overt and covert HE, reduced the likelihood of infection, reduced oralisation of the gut and attenuated systemic inflammation. Rifaximin-α plays a role in gut barrier repair, which could be the mechanism by which it ameliorates bacterial translocation and systemic endotoxemia in cirrhosis. CLINICAL TRIAL NUMBER: ClinicalTrials.gov NCT02019784. LAY SUMMARY: In this clinical trial, we examined the underlying mechanism of action of an antibiotic called rifaximin-α which has been shown to be an effective treatment for a complication of chronic liver disease which effects the brain (termed encephalopathy). We show that rifaximin-α suppresses gut bacteria that translocate from the mouth to the intestine and cause the intestinal wall to become leaky by breaking down the protective mucus barrier. This suppression resolves encephalopathy and reduces inflammation in the blood, preventing the development of infection.

Hepatic encephalopathy and depression in chronic liver disease: is the common link systemic inflammation?

Hepatic encephalopathy and depression share a number of clinical features, such as cognitive impairment and psychomotor retardation, and are highly prevalent in patients with chronic liver disease. Both conditions signify a poor prognosis, carry an increased mortality and are major determinants of reduced health related quality of life. The pathophysiology of hepatic encephalopathy is complex. Whilst cerebral accumulation of ammonia is well-recognised as being central to the development of hepatic encephalopathy, systemic inflammation, which acts in synergy with hyperammonaemia, is emerging as a key driver in its development. The pro-inflammatory state is also widely documented in depression, and peripheral to brain communication occurs resulting in central inflammation, behavioural changes and depressive symptoms. Gut dysbiosis, with a similar reduction in beneficial bacteria, increase in pathogens and decreased bacterial diversity, has been observed in both hepatic encephalopathy and depression, and it may be that the resultant increased bacterial translocation causes their shared inflammatory pathophysiology. Whilst the literature on a positive association between hepatic encephalopathy and depression in cirrhosis remains to be substantiated, there is evolving evidence that treatment with psychobiotics may be of dual benefit, improving cognition and mood in cirrhosis.

Activation and Functional Priming of Blood Neutrophils in Non-Alcoholic Fatty Liver Disease Increases in Non-Alcoholic Steatohepatitis.

INTRODUCTION: In non-alcoholic fatty liver disease (NAFLD), neutrophils in liver infiltrates are activated, which may contribute to disease progression towards non-alcoholic steatohepatitis (NASH). However, the functional status of the blood neutrophils remains unknown and their role in the disease mechanisms is thus uncertain. We therefore characterized activation and function of blood neutrophils in patients with NAFLD in relation to clinical disease markers and the NAFLD plasma milieu. METHODS: We studied 20 patients with NAFLD, among these 6 patients with NASH, and 14 healthy persons. Neutrophil activation, interleukin (IL)-8 production and oxidative burst were measured by flow cytometry on participants´ neutrophils and on healthy neutrophils exposed in vitro to plasma from the study participants. RESULTS: Blood neutrophils from the NASH patients showed a doubling in their expression of the activation marker CD62L. Also, all NAFLD patients had 50-100% increased expression of CD11b. Functionally, NASH neutrophils had 30% elevated IL-8 production and more than doubled spontaneous oxidative burst. In all NAFLD patients, higher spontaneous oxidative burst was associated with worse liver function. Incubation of healthy neutrophils with NAFLD plasma paradoxically slightly reduced CD62L and CD11b expression, and NASH plasma also reduced the frequency of IL-8-producing neutrophils. CONCLUSION: In NAFLD, blood neutrophils are activated, and in NASH also functionally primed. This suggests a progressive neutrophil aggressiveness already present with liver fat infiltration. However, NAFLD plasma in vitro, if anything, had the opposite effect on the healthy neutrophils so the NAFLD-related neutrophil activation cannot be attributed to humoral factors and remains unexplained.

Targeting the gut-liver-immune axis to treat cirrhosis.

Cirrhotic portal hypertension is characterised by development of the decompensating events of ascites, encephalopathy, portal hypertensive bleeding and hepatorenal syndrome, which arise in a setting of cirrhosis-associated immune dysfunction (CAID) and define morbidity and prognosis. CAID describes the dichotomous observations that systemic immune cells are primed and display an inflammatory phenotype, while failing to mount robust responses to pathogen challenge. Bacterial infections including spontaneous bacterial peritonitis are common complications of advanced chronic liver disease and can precipitate variceal haemorrhage, hepatorenal syndrome and acute-on-chronic liver failure; they frequently arise from gut-derived organisms and are closely linked with dysbiosis of the commensal intestinal microbiota in advanced chronic liver disease.Here, we review the links between cirrhotic dysbiosis, intestinal barrier dysfunction and deficits of host-microbiome compartmentalisation and mucosal immune homoeostasis that occur in settings of advanced chronic liver disease. We discuss established and emerging therapeutic strategies targeted at restoring intestinal eubiosis, augmenting gut barrier function and ameliorating the mucosal and systemic immune deficits that characterise and define the course of decompensated cirrhosis.

Ammonia-Induced Brain Edema Requires Macrophage and T Cell Expression of Toll-Like Receptor 9.

BACKGROUND & AIM: Ammonia is central in the pathogenesis of brain edema in acute liver failure (ALF) with infection and systemic inflammation expediting development of intracranial hypertension (ICH). Patients with acetaminophen-induced ALF have increased neutrophil TLR9 expression which can be induced by ammonia. We determined whether ammonia-induced brain edema and immune dysfunction are mediated by TLR9 and if this could be prevented in a TLR9-deficient mouse model. METHODS: Ammonium acetate (NH4-Ac; 4mmol/kg) was injected intraperitoneally in wild type (WT), Tlr9-/- and Lysm-Cre Tlr9fl/fl mice (TLR9 absent in neutrophils and macrophages including Kupffer cells) and compared to controls. Six hours after NH4-Ac injection, intracellular cytokine production was determined in splenic macrophages, CD4+ and CD8+ T cells. Brain water (BW) and total plasma DNA (tDNA) were also measured. The impact of the TLR9 antagonist ODN2088 (50µg/mouse) was evaluated. RESULTS: Following NH4-Ac injection, BW, macrophage and T cell cytokine production increased (P < .0001) in WT but not Tlr9-/- mice (P < .001). ODN2088 inhibited macrophage and T cell cytokine production (P < .05) and prevented an increase in BW (P < .0001). Following NH4-Ac injection, macrophage cytokine production and BW were ameliorated in Lysm-Cre Tlr9fl/fl mice compared to WT mice (P < .05) but there was no difference compared to Tlr9-/- mice. Following NH4-Ac injection, plasma tDNA levels increased in WT and Tlr9-/- mice (P < .05) suggesting that TLR9 may be activated by DNA released from ammonia-stimulated cells. CONCLUSION: Ammonia-induced brain edema requires macrophage and T cell expression of TLR9. Amelioration of brain edema and lymphocyte cytokine production by ODN2088 supports exploration of TLR9 antagonism in early ALF to prevent progression to ICH.