Alexander Wree

From NAFLD to NASH to cirrhosis—new insights into disease mechanisms

NAFLD has evolved as a serious public health problem in the USA and around the world. In fact, NASH—the most serious form of NAFLD—is predicted to become the leading cause of liver transplantation in the USA by the year 2020. The pathogenesis of NAFLD and NASH, in particular the mechanisms responsible for liver injury and fibrosis, is the result of a complex interplay between host and environmental factors, and is at the centre of intense investigation. In this Review, we focus on recently uncovered aspects of the genetic, biochemical, immunological and molecular events that are responsible for the development and progression of this highly prevalent and potentially serious disease. These studies bring new insight into this complex disorder and have led to the development of novel therapeutic and diagnostic strategies that might enable a personalized approach in the management of this disease.

NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice

Inflammasome activation plays a central role in the development of drug‐induced and obesity‐associated liver disease. However, the sources and mechanisms of inflammasome‐mediated liver damage remain poorly understood. Our aim was to investigate the effect of NLRP3 inflammasome activation on the liver using novel mouse models. We generated global and myeloid cell‐specific conditional mutant Nlrp3 knock‐in mice expressing the D301N Nlrp3 mutation (ortholog of D303N in human NLRP3), resulting in a hyperactive NLRP3. To study the presence and significance of NLRP3‐initiated pyroptotic cell death, we separated hepatocytes from nonparenchymal cells and developed a novel flow‐cytometry–based (fluorescence‐activated cell sorting; FACS) strategy to detect and quantify pyroptosis in vivo based on detection of active caspase 1 (Casp1)‐ and propidium iodide (PI)‐positive cells. Liver inflammation was quantified histologically by FACS and gene expression analysis. Liver fibrosis was assessed by Sirius Red staining and quantitative polymerase chain reaction for markers of hepatic stellate cell (HSC) activation. NLRP3 activation resulted in shortened survival, poor growth, and severe liver inflammation; characterized by neutrophilic infiltration and HSC activation with collagen deposition in the liver. These changes were partially attenuated by treatment with anakinra, an interleukin‐1 receptor antagonist. Notably, hepatocytes from global Nlrp3‐mutant mice showed marked hepatocyte pyroptotic cell death, with more than a 5‐fold increase in active Casp1/PI double‐positive cells. Myeloid cell‐restricted mutant NLRP3 activation resulted in a less‐severe liver phenotype in the absence of detectable pyroptotic hepatocyte cell death. Conclusions: Our data demonstrate that global and, to a lesser extent, myeloid‐specific NLRP3 inflammasome activation results in severe liver inflammation and fibrosis while identifying hepatocyte pyroptotic cell death as a novel mechanism of NLRP3‐mediated liver damage. (Hepatology 2014;59:898–910)

Circulating Extracellular Vesicles with Specific Proteome and Liver MicroRNAs Are Potential Biomarkers for Liver Injury in Experimental Fatty Liver Disease

Background & Aim Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy. Design Choline Deficient L-Amino Acid (CDAA) and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens. Results We observed statistically significant differences in the level of extracellular vesicles (EVs) in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2 = 0.64, p<0.05), fibrosis (r2 = 0.66, p<0.05) and pathological angiogenesis (r2 = 0.71, p<0.05). Extensive characterization of blood EVs identified both microparticles (MPs) and exosomes (EXO) present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192 - two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver. Conclusions These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD.

Biomarkers of liver cell death

Hepatocyte cell death during liver injury was classically viewed to occur by either programmed (apoptosis), or accidental, uncontrolled cell death (necrosis). Growing evidence from our increasing understanding of the biochemical and molecular mechanisms involved in cell demise has provided an expanding view of various modes of cell death that can be triggered during both acute and chronic liver damage such as necroptosis, pyroptosis, and autophagic cell death. The complexity of non-invasively assessing the predominant mode of cell death during a specific liver insult in either experimental in vivo models or in humans is highlighted by the fact that in many instances there is significant crosstalk and overlap between the different cell death pathways. Nevertheless, the realization that during cell demise triggered by a specific mode of cell death certain intracellular molecules such as proteins, newly generated protein fragments, or MicroRNAs are released from hepatocytes into the extracellular space and may appear in circulation have spurred a significant interest in the development of non-invasive markers to monitor liver cell death. This review focuses on some of the most promising markers, and their potential role in assessing the presence and severity of liver damage in humans.

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 16weeks, gavaged MCC950 or vehicle until 24weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6weeks 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 24weeks, 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.

Adipocyte cell size, free fatty acids and apolipoproteins are associated with non-alcoholic liver injury progression in severely obese patients

PURPOSE Obesity is a modern pandemic with continuous expansion and represents an independent risk factor for non-alcoholic fatty liver disease (NAFLD), the most common liver disease in westernized countries. The crosstalk between adipose tissue and the liver is key to the development of NAFLD. PROCEDURES Therefore, in an observational study blood, visceral adipose tissue and liver tissue were obtained from 93 severely obese patients with a mean age of 43 years and mean BMI of 52 kg/m2 at the time of weight loss surgery. In a subset of patients a follow-up blood sample was obtained 6 weeks after surgery to assess acute effects of weight loss. In addition to routine parameters of liver injury, serum samples were analyzed for leptin, adiponectin, free fatty acids (FFAs), and several apolipoproteins. MAIN FINDINGS The diameter of visceral adipocytes correlated to liver injury, serum markers of inflammation and serum adipokine levels. Liver injury assessed by serology (ALT, AST) and histology (NAFLD activity score, NAS) was independent of the BMI. However, serum levels of triglycerides and Apolipoprotein CIII (ApoCIII) were associated with NAS. Serum levels and composition of FFAs, especially long chain FFAs, also correlated with NAS. Analysis of serum samples six weeks after surgery revealed beneficial changes in serum triglycerides, levels of ApoCIII and several FFAs. CONCLUSIONS In severely obese patients beneficial effects on liver injury can been observed as early as six weeks after bariatric surgery. These effects may be explained by the observed changes in adipose tissue and lipid metabolism. Collectively, these findings underline the importance of the link between adipose tissue and the liver.

Fetuin-A mRNA expression is elevated in NASH compared with NAFL patients

Fetuin-A is a pro-inflammatory protein expressed by hepatocytes. Its course in morbidly obese patients with NAFLD (non-alcoholic fatty liver disease) following weight loss by BAS (bariatric surgery) has not been fully elucidated yet. In the present study, we prospectively examined the effects of weight loss on various metabolic factors at 4 weeks and 6 months after surgery. Blood and liver tissues were retrieved from 108 morbidly obese NAFLD patients before/during BAS, and 50 of these individuals met the criteria for NASH (non-alcoholic steatohepatitis). Fetuin-A expression was measured by qPCR (quantitative real-time PCR), Western blotting and immunohistochemistry. Hepatocyte apoptosis was quantified via M30 (caspase-cleaved cytokeratin-18 fragments). Plasma concentrations of adiponectin and fetuin-A were determined by ELISA. Serum-derived parameters were additionally taken at 4 weeks and 6 months post-operatively. In addition, primary human hepatocytes were treated with NEFA (non-esterified fatty acid) to investigate changes in fetuin-A. BMI (body mass index) decreased significantly from 53.0±1.1 to 36.4±1.9 kg/m2 in the NAFL group and from 53.3±1.1 to 37.6±1.2 kg/m2 in the NASH group (P<0.0001) at 6 months post-surgery. This was associated with diminishing M30 and M65 (total cytokeratin-18) levels over 6 months after surgery. Adiponectin levels increased continuously in NASH patients, whereas NAFL patients plateaued at 4 weeks post-operatively. Hepatic fetuin-A mRNA and protein expression was elevated before surgery-induced weight loss. However, plasma concentrations of fetuin-A increased signficantly in NASH patients 4 weeks post-operatively. Treatment of hepatocytes with NEFA led to up-regulation of fetuin-A expression. BAS probably has a beneficial effect on NAFLD, as indicated by reduced hepatocyte apoptosis and improved adipokine profiles. In addition, fetuin-A expression is more prominent in NASH.

Arginase 2 deficiency results in spontaneous steatohepatitis: A novel link between innate immune activation and hepatic de novo lipogenesis

BACKGROUND & AIMS Innate immune activation has been postulated as a central mechanism for disease progression from hepatic steatosis to steatohepatitis in obesity-related fatty liver disease. Arginase 2 competes with inducible nitric oxide synthase (iNOS) for its substrate and the balance between these two enzymes plays a crucial role in regulating immune responses and macrophage activation. Our aim was to test the hypothesis that arginase 2 deficiency in mice favours progression from isolated hepatic steatosis, induced by high fat feeding, to steatohepatitis. METHODS Arginase 2-knockout (Arg2(-/-)) mice were studied for changes in liver histology and metabolic phenotype at baseline and after a short term course (7 week) feeding with a high fat (HFAT) diet. In additional experiments, Arg2(-/-) mice received tail vein injections of liposome-encapsulated clodronate (CLOD) over a three-week period to selectively deplete liver macrophages. RESULTS Unexpectedly, Arg2(-/-) mice showed profound changes in their livers at baseline, characterized by significant steatosis as demonstrated with histological and biochemical analysis. These changes were independent of systemic metabolic parameters and associated with marked mRNA level increases of genes involved in hepatic de novo lipogenesis. Liver injury and inflammation were present with elevated serum ALT, marked infiltration of F4/80 positive cells, and increased mRNA levels of inflammatory genes. HFAT feeding exacerbated these changes. Macrophage depletion after CLOD injection significantly attenuated lipid deposition and normalized lipogenic mRNA profile of livers from Arg2(-/-) mice. CONCLUSIONS This study identifies arginase 2 as a novel link between innate immune responses, hepatic lipid deposition, and liver injury.

Steroid and Ursodesoxycholic Acid Combination Therapy in Severe Drug-Induced Liver Injury

Background: Drug-induced liver injury (DILI) is the leading cause of acute severe liver disease in Western countries. Treatment strategies for DILI are still not well defined. Aim: We studied the safety and outcomes of steroid/ursodesoxycholic acid (UDCA) combination therapy in DILI patients. Patients, Materials and Methods: 15 consecutive patients with severe DILI were analyzed for clinical, biochemical and histological data. Nine patients were treated with a steroid step-down therapy with reduction of the daily dose over several weeks; 6 patients received a steroid pulse therapy for 3 days. UDCA was administered for several weeks in both groups. Results: Patients without histological signs of preexistent liver damage (n = 10) showed the most favorable clinical course. Bilirubin and serum transaminases dropped to <50% of peak values within 2 weeks, and normalized within 4–8 weeks. In contrast, patients with positive autoimmune antibodies (anti-nuclear antibodies and/or soluble liver antigen) and/or histological features of chronic hepatitis (n = 3) exhibited a slower reduction in bilirubin and serum transaminase levels. These patients were given immunosuppressants (steroids, azathioprine) for a further 6 months. Conclusion: Treatment of severe DILI with corticosteroids (both pulse and step-down therapy) and UDCA appears to be safe, and leads to a more rapid reduction in bilirubin and transaminases after DILI.

Bioelectrical impedance analysis in clinical practice: implications for hepatitis C therapy BIA and hepatitis C.

BackgroundBody composition analysis using phase angle (PA), determined by bioelectrical impedance analysis (BIA), reflects tissue electrical properties and has prognostic value in liver cirrhosis. Objective of this prospective study was to investigate clinical use and prognostic value of BIA-derived phase angle and alterations in body composition for hepatitis C infection (HCV) following antiviral therapy.Methods37 consecutive patients with HCV infection were enrolled, BIA was performed, and PA was calculated from each pair of measurements. 22 HCV genotype 3 patients treated for 24 weeks and 15 genotype 1 patients treated for 48 weeks, were examined before and after antiviral treatment and compared to 10 untreated HCV patients at 0, 24, and 48 weeks. Basic laboratory data were correlated to body composition alterations.ResultsSignificant reduction in body fat (BF: 24.2 ± 6.7 kg vs. 19.9 ± 6.6 kg, genotype1; 15.4 ± 10.9 kg vs. 13.2 ± 12.1 kg, genotype 3) and body cell mass (BCM: 27.3 ± 6.8 kg vs. 24.3 ± 7.2 kg, genotype1; 27.7 ± 8.8 kg vs. 24.6 ± 7.6 kg, genotype 3) was found following treatment. PA in genotype 3 patients was significantly lowered after antiviral treatment compared to initial measurements (5.9 ± 0.7° vs. 5.4 ± 0.8°). Total body water (TBW) was significantly decreased in treated patients with genotype 1 (41.4 ± 7.9 l vs. 40.8 ± 9.5 l). PA reduction was accompanied by flu-like syndromes, whereas TBW decline was more frequently associated with fatigue and cephalgia.DiscussionBIA offers a sophisticated analysis of body composition including BF, BCM, and TBW for HCV patients following antiviral regimens. PA reduction was associated with increased adverse effects of the antiviral therapy allowing a more dynamic therapy application.