NASH is an inflammatory disorder: Pathogenic, prognostic and therapeutic implications

While non-alcoholic fatty liver disease (NAFLD) is highly prevalent (15% to 45%) in modern societies, only 10% to 25% of cases develop hepatic fibrosis leading to cirrhosis, end-stage liver disease or hepatocellular carcinoma. Apart from pre-existing fibrosis, the strongest predictor of fibrotic progression in NAFLD is steatohepatitis or non-alcoholic steatohepatitis (NASH). The critical features other than steatosis are hepatocellular degeneration (ballooning, Mallory hyaline) and mixed infl ammatory cell infi ltration. While much is understood about the relationship of steatosis to metabolic factors (over-nutrition, insulin resistance, hyperglycemia, metabolic syndrome, hypoadiponectinemia), less is known about infl ammatory recruitment, despite its importance for the perpetuation of liver injury and fi brogenesis. In this review, we present evidence that liver infl ammation has prognostic signifi cance in NAFLD. We then consider the origins and components of liver infl ammation in NASH. Hepatocytes injured by toxic lipid molecules (lipotoxicity) play a central role in the recruitment of innate immunity involving Toll-like receptors (TLRs), Kupffer cells (KCs), lymphocytes and neutrophils and possibly infl ammasome. The key pro-infl ammatory signaling pathways in NASH are nuclear factor-kappa B (NF-κB) and c-Jun N-terminal kinase (JNK). The downstream effectors include adhesion molecules, chemokines, cytokines and the activation of cell death pathways leading to apoptosis. The upstream activators of NF-κB and JNK are more contentious and may depend on the experimental model used. TLRs are strong contenders. It remains possible that infl ammation in NASH originates outside the liver and in the gut microbiota that prime KC/TLR responses, infl amed adipose tissue and circulating infl ammatory cells. We briefl y review these mechanistic considerations and project their implications for the effective treatment of NASH

Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH

Cholesterol crystals form within hepatocyte lipid droplets in human and experimental nonalcoholic steatohepatitis (NASH) and are the focus of crown-like structures (CLSs) of activated Kupffer cells (KCs). Obese, diabetic Alms1 mutant ( foz/foz ) mice were a fed high-fat (23%) diet containing 0.2% cholesterol for 16 weeks and then assigned to four intervention groups for 8 weeks: a ) vehicle control, b ) ezetimibe (5 mg/kg/day), c ) atorvastatin (20 mg/kg/day), or d ) ezetimibe and atorvastatin. Livers of vehicle-treated mice developed fibrosing NASH with abundant cholesterol crystallization within lipid droplets calculated to extend over 3.3% (SD, 2.2%) of liver surface area. Hepatocyte lipid droplets with prominent cholesterol crystallization were surrounded by TNFα -positive (activated) KCs forming CLSs ( ≥ 3 per high-power field). KCs that formed CLSs stained positive for NLRP3, implicating activation of the NLRP3 inflammasome in response to cholesterol crystals. In contrast, foz/foz mice treated with ezetimibe and atorvastatin showed near-complete resolution of cholesterol crystals [0.01% (SD, 0.02%) of surface area] and CLSs (0 per high-power field), with amelioration of fibrotic NASH. Ezetimibe or atorvastatin alone had intermediate effects on cholesterol crystallization, CLSs, and NASH. These findings are consistent with a causative link between exposure of hepatocytes and KCs to cholesterol crystals and with the development of NASH possibly mediated by NLRP3 activation

Endoplasmic reticulum stress does not contribute to steatohepatitis in obese and insulin resistant high-fat diet fed foz/foz mice

Non-alcoholic fatty liver (steatosis) and steatohepatitis [non-alcoholic steatohepatitis (NASH)] are hepatic complications of the metabolic syndrome. Endoplasmic reticulum (ER) stress is proposed as a crucial disease mechanism in obese and insulin-resistant animals (such as ob/ob mice) with simple steatosis, but its role in NASH remains controversial. We therefore evaluated the role of ER stress as a disease mechanism in foz/foz mice, which develop both the metabolic and histological features that mimic human NASH. We explored ER stress markers in the liver of foz/foz mice in response to a high-fat diet (HFD) at several time points. We then evaluated the effect of treatment with an ER stress inducer tunicamycin, or conversely with the ER protectant tauroursodeoxycholic acid (TUDCA), on the metabolic and hepatic features. foz/foz mice are obese, glucose intolerant and develop NASH characterized by steatosis, inflammation, ballooned hepatocytes and apoptosis from 6 weeks of HFD feeding. This was not associated with activation of the upstream unfolded protein response [phospho-eukaryotic initiation factor 2α (eIF2α), inositol-requiring enzyme 1α (IRE1α) activity and spliced X-box-binding protein 1 (Xbp1)]. Activation of c-Jun N-terminal kinase (JNK) and up-regulation of activating transcription factor-4 (Atf4) and CCAAT/enhancer-binding protein-homologous protein (Chop) transcripts were however compatible with a ‘pathological’ response to ER stress. We tested this by using intervention experiments. Induction of chronic ER stress failed to worsen obesity, glucose intolerance and NASH pathology in HFD-fed foz/foz mice. In addition, the ER protectant TUDCA, although reducing steatosis, failed to improve glucose intolerance, hepatic inflammation and apoptosis in HFD-fed foz/foz mice. These results show that signals driving hepatic inflammation, apoptosis and insulin resistance are independent of ER stress in obese diabetic mice with steatohepatitis

The damage-associated molecular pattern HMGB1 is released early after clinical hepatic ischemia/reperfusion.

OBJECTIVE AND BACKGROUND: Activation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice. METHODS: Plasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury. RESULTS: In patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects. CONCLUSION: HMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage

NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice

Background & Aims: NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. Methods: We fed foz/foz and wild-type mice an atherogenic diet for 16 weeks, gavaged MCC950 or vehicle until 24 weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6 weeks and determined the effects on liver fibrosis. Results: In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1β, active caspase-1 and IL-1β increased at 24 weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1β, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1β expression, plasma IL-1β, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1β; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1β, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. Conclusion: MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH.Supported by NIH Project grants R2 AA023574 and U01 AA022489 (to AEF), Australian NHMRC project grants 1084136 and 1044288 (to GCF), and 1086786 (to MAC and AABR), and Deutsche Forschungsgemeinschaft WR 173/3-1 (to AW). Matthew Cooper is an NHMRC Principle Research Fellow (1059354

NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice

Background & Aims NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. Methods We fed foz/foz and wild-type mice an atherogenic diet for 16\ua0weeks, gavaged MCC950 or vehicle until 24\ua0weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6\ua0weeks and determined the effects on liver fibrosis. Results In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1β, active caspase-1 and IL-1β increased at 24\ua0weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1β, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1β expression, plasma IL-1β, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1β; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1β, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. Conclusion MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH. Lay summary Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis

Cholesterol as a mediator of hepatic injury in non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is highly prevalent. 20-30% of patients with NAFLD progress to non-alcoholic steatohepatitis (NASH), characterised by steatosis, ballooning degeneration of hepatocytes, inflammation and fibrosis, but the cause(s) of this transition remains unclear. Foz/foz (Alms1 mutant) mice develop hyperphagia and obesity-related NAFLD. High fat (HF)-feeding induces NASH transition by 12 weeks, and fibrosis by 24 weeks. The aims of this research were to investigate the cause and effect of hepatic cholesterol dysregulation in foz/foz mice, its pathogenic significance in NASH, and the potential of dietary and pharmacological approaches to treat NASH. After 24 weeks HF-feeding, hepatic free cholesterol (FC) and cholesteryl ester (CE) content were markedly increased in foz/foz mice. Hepatic cholesterol accumulation was associated with increased expression of low-density lipoprotein receptor (LDLR), but not cholesterol synthesis. Bile acid (BA) biosynthesis genes were suppressed, as was expression of cholesterol and BA export proteins. Transcriptional regulators of cholesterol metabolism were differentially expressed in foz/foz mice with NASH: SREBP-2, a nuclear regulator of LDLR increased significantly, while LXR-a and HNF-4a decreased. Importantly, expression of liver receptor homolog-1 (LRH-1), while significantly elevated in HF-fed WT mice, was unaltered in HF-fed foz/foz mice. To test whether hyperinsulinaemia was responsible for some or all of these changes, experiments were conducted in primary murine hepatocyte cultures. Insulin activated both SREBP-2 and LDLR, while simultaneously decreasing BA export proteins and LRH-1 expression. Removing dietary cholesterol in HF-fed foz/foz mice ameliorated liver injury, whereas increasing dietary cholesterol accentuated injury. Steatohepatitis severity correlated with hepatic FC and CE, but not hepatic triglyceride or free fatty acids. Hepatic cholesterol loading activated nuclear factor (NF)-kB and c-Jun N-terminal kinase (JNK), and increased serum monocyte chemotactic protein (MCP)-1. Finally, quantitative analysis of sirius red staining demonstrated a positive relationship between hepatic cholesterol content and fibrosis severity. To establish whether pharmacological modulation of cholesterol turnover could modulate NASH outcome, HF-fed foz/foz and WT mice were administered atorvastatin and/or ezetimibe after NASH onset. Both agents significantly lowered hepatic CE and FC, serum ALT, total cholesterol and HDL and normalised liver size in foz/foz mice. Hepatocellular apoptosis and expression of inflammatory mediators was suppressed as was hepatic macrophage infiltration. While histological steatosis and ballooning scores were unaltered, lobular inflammation and liver fibrosis were significantly reduced in drug-treated foz/foz mice. In foz/foz mice, NASH is associated with profoundly disordered hepatic cholesterol turnover. Insulin-induced activation of SREBP-2 and suppression of LRH-1 likely result in increased LDLR expression and suppression of genes involved in cholesterol biotransformation and export. Consequently, cholesterol accumulates within the livers of foz/foz mice. The evidence from both dietary and pharmacological intervention experiments is that hepatic cholesterol mediates hepatocellular apoptosis, inflammatory cell recruitment and liver fibrosis in this model. Conversely, inhibition of cholesterol uptake/redistribution and biosynthesis, and dietary cholesterol restriction, reduces liver injury, inflammation, and fibrosis in foz/foz mice with NASH. These findings strongly support the hypothesis that hepatic cholesterol, secondary to insulin resistance, acts as a lipotoxic mediator of pro-fibrotic liver injury and inflammation in experimental NASH. -- provided by Candidate

Hepatic endoplasmic reticulum stress in obesity : Deeper insights into processes, but are they relevant to nonalcoholic steatohepatitis?

The endoplasmic reticulum (ER) is the main site of protein and lipid synthesis, membrane biogenesis, xenobiotic detoxification and cellular calcium storage, and perturbation of ER homeostasis leads to stress and the activation of the unfolded protein response. Chronic activation of ER stress has been shown to have an important role in the development of insulin resistance and diabetes in obesity. However, the mechanisms that lead to chronic ER stress in a metabolic context in general, and in obesity in particular, are not understood. Here we comparatively examined the proteomic and lipidomic landscape of hepatic ER purified from lean and obese mice to explore the mechanisms of chronic ER stress in obesity. We found suppression of protein but stimulation of lipid synthesis in the obese ER without significant alterations in chaperone content. Alterations in ER fatty acid and lipid composition result in the inhibition of sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and ER stress. Correcting the obesity-induced alteration of ER phospholipid composition or hepatic Serca over-expression in vivo both reduced chronic ER stress and improved glucose homeostasis. Hence, we established that abnormal lipid and calcium metabolism are important contributors to hepatic ER stress in obesity. (HEPATOLOGY 2011