High fat diet feeding results in gender specific steatohepatitis and inflammasome activation

AIM: To develop an animal model that encompasses the different facets of non-alcoholic steatohepatitis (NASH), which has been a challenge. METHODS: In this study, we used a high fat diet (HFD) feeding supplemented with fructose and sucrose in the water mimicking the high-fructose corn syrup that is abundant in the diet in the United States. We used C57Bl/6 wild-type mice for short and long-term feedings of 6 and 16 wk respectively, and evaluated the extent of liver damage, steatosis, and inflammasome activation. Our methods included histopathological analysis to assess liver damage and steatosis, which involved H and E and oil-red-o staining; biochemical studies to look at ALT and triglyceride levels; RNA analysis using quantitative polymerase chain reaction; and cytokine analysis, which included the enzyme-linked immunosorbent assay method to look at interleukin (IL)-1beta and tumor necrosis factor-alpha (TNFalpha) levels. Furthermore, at each length of feeding we also looked at insulin resistance and glucose tolerance using insulin tolerance tests (ITT) and glucose tolerance tests. RESULTS: There was no insulin resistance, steatosis, or inflammasome activation at 6 wk. In contrast, at 16 wk we found significant insulin resistance demonstrated by impaired glucose and ITT in male, but not female mice. In males, elevated alanine aminotransferase and triglyceride levels, indicated liver damage and steatosis, respectively. Increased liver TNFalpha and monocyte chemoattractant protein-1 mRNA and protein, correlated with steatohepatitis. The inflammasome components, adaptor molecule, Aim2, and NOD-like receptor 4, increased at the mRNA level, and functional inflammasome activation was indicated by increased caspase-1 activity and IL-1beta protein levels in male mice fed a long-term HFD. Male mice on HFD had increased alpha-smooth muscle actin and pro-collagen-1 mRNA indicating evolving fibrosis. In contrast, female mice displayed only elevated triglyceride levels, steatosis, and no fibrosis. CONCLUSION: Our data indicate gender differences in NASH. Male mice fed a long-term HFD display steatohepatitis and inflammasome activation, whereas female mice have steatosis without inflammation

Increased oxidative capacity of circulating polymorphonuclear neutrophils (PMNs) in non-diabetic NASH patients

Background: Inflammation and oxidative stress are key factors in the pathogenesis of non-alcoholic steatohepatitis (NASH). Polymorphonuclear neutrophils are capable to produce significant amounts of reactive oxygen species (ROS) via the NADPH oxidase complex. Increased hepatic neutrophil infiltration has been described in steatohepatitis. We aimed to investigate the in vitro ROS generation by neutrophils of NASH patients and the hepatic NADPH oxidase activity in murine steatohepatitis. Material and methods: PMNs were isolated from peripheral blood of NASH patients (n=16) and healthy controls (n=16). In vitro ROS production was measured by luminol chemiluminescence after phorbol myristate acetate (PMA) or opsonized zymosan stimulation. Hepatic lipid peroxidation and NADPH oxidase activation were measured in mice fed with methionine-choline-deficient (MCD) or -supplemented (MCS) diets. Results: PMA activated oxidative burst both in patients and controls. However, ROS production was significantly increased in non-diabetic NASH patients (n=9) compared to controls 30 min after the PMA stimulation. PMNs from NASH patients with diabetes mellitus (n=7) did not have higher ROS production after PMA-stimulation compared to controls. The PMA-induced peak chemiluminescence was significantly higher in the non-diabetic NASH patients compared to controls and diabetic NASH patients. No significant difference was observed without any stimulation and in opsonized zymosan induced chemiluminescence. Consistent with the increased oxidative capacity of PMNs in NASH patients, we found increased hepatic lipid peroxidation, higher expression and activation of the NADPH oxidase complex in MCD-steatohepatitis. Conclusion: Our finding supports the role of neutrophil oxidative stress in NASH. Our novel data suggests that the increased oxidative capacity of the PMNs it is not only localized to the liver but can have systemic effects and serve as a potential biomarker of NASH

Progression of non-alcoholic steatosis to steatohepatitis and fibrosis parallels cumulative accumulation of danger signals that promote inflammation and liver tumors in a high fat-cholesterol-sugar diet model in mice

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is becoming a pandemic. While multiple \u27hits\u27 have been reported to contribute to NAFLD progression to non-alcoholic steatohepatitis (NASH), fibrosis and liver cancer, understanding the natural history of the specific molecular signals leading to hepatocyte damage, inflammation and fibrosis, is hampered by the lack of suitable animal models that reproduce disease progression in humans. The purpose of this study was first, to develop a mouse model that closely mimics progressive NAFLD covering the spectrum of immune, metabolic and histopathologic abnormalities present in human disease; and second, to characterize the temporal relationship between sterile/exogenous danger signals, inflammation, inflammasome activation and NAFLD progression. METHODS: Male C57Bl/6 mice were fed a high fat diet with high cholesterol and a high sugar supplement (HF-HC-HSD) for 8, 27, and 49 weeks and the extent of steatosis, liver inflammation, fibrosis and tumor development were evaluated at each time point. RESULTS: The HF-HC-HSD resulted in liver steatosis at 8 weeks, progressing to steatohepatitis and early fibrosis at 27 weeks, and steatohepatitis, fibrosis, and tumor development at 49 weeks compared to chow diet. Steatohepatitis was characterized by increased levels of MCP-1, TNFalpha, IL-1beta and increased liver NASH histological score. We found increased serum levels of sterile danger signals, uric acid and HMGB1, as early as 8 weeks, while endotoxin and ATP levels increased only after 49 weeks. Increased levels of these sterile and microbial danger signals paralleled upregulation and activation of the multiprotein complex inflammasome. At 27, 49 weeks of HF-HC-HSD, activation of M1 macrophages and loss of M2 macrophages as well as liver fibrosis were present. Finally, similar to human NASH, liver tumors occurred in 41% of mice in the absence of cirrhosis and livers expressed increased p53 and detectable AFP. CONCLUSIONS: HF-HC-HSD over 49 weeks induces the full spectrum of liver pathophysiologic changes that characterizes the progression of NAFLD in humans. NAFLD progression to NASH, fibrosis and liver tumor follows progressive accumulation of sterile and microbial danger signals, inflammasome activation, altered M1/M2 cell ratios that likely contribute to NASH progression and hepatic tumor formation

STING-IRF3 pathway links endoplasmic reticulum stress with hepatocyte apoptosis in early alcoholic liver disease

Emerging evidence suggests that innate immunity drives alcoholic liver disease (ALD) and that the interferon regulatory factor 3 (IRF3),a transcription factor regulating innate immune responses, is indispensable for the development of ALD. Here we report that IRF3 mediates ALD via linking endoplasmic reticulum (ER) stress with apoptotic signaling in hepatocytes. We found that ethanol induced ER stress and triggered the association of IRF3 with the ER adaptor, stimulator of interferon genes (STING), as well as subsequent phosphorylation of IRF3. Activated IRF3 associated with the proapoptotic molecule Bax [B-cell lymphoma 2 (Bcl2)-associated X protein] and contributed to hepatocyte apoptosis. Deficiency of STING prevented IRF3 phosphorylation by ethanol or ER stress, and absence of IRF3 prevented hepatocyte apoptosis. The pathogenic role of IRF3 in ALD was independent of inflammation or Type-I interferons. Thus, STING and IRF3 are key determinants of ALD, linking ER stress signaling with the mitochondrial pathway of hepatocyte apoptosis

IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice

Alcoholic liver disease (ALD) is characterized by steatosis and upregulation of proinflammatory cytokines, including IL-1beta. IL-1beta, type I IL-1 receptor (IL-1R1), and IL-1 receptor antagonist (IL-1Ra) are all important regulators of the IL-1 signaling complex, which plays a role in inflammation. Furthermore, IL-1beta maturation is dependent on caspase-1 (Casp-1). Using IL-1Ra-treated mice as well as 3 mouse models deficient in regulators of IL-1beta activation (Casp-1 and ASC) or signaling (IL-1R1), we found that IL-1beta signaling is required for the development of alcohol-induced liver steatosis, inflammation, and injury. Increased IL-1beta was due to upregulation of Casp-1 activity and inflammasome activation. The pathogenic role of IL-1 signaling in ALD was attributable to the activation of the inflammasome in BM-derived Kupffer cells. Importantly, in vivo intervention with a recombinant IL-1Ra blocked IL-1 signaling and markedly attenuated alcohol-induced liver inflammation, steatosis, and damage. Furthermore, physiological doses of IL-1beta induced steatosis, increased the inflammatory and prosteatotic chemokine MCP-1 in hepatocytes, and augmented TLR4-dependent upregulation of inflammatory signaling in macrophages. In conclusion, we demonstrated that Casp-1-dependent upregulation of IL-1beta and signaling mediated by IL-1R1 are crucial in ALD pathogenesis. Our findings suggest a potential role of IL-1R1 inhibition in the treatment of ALD

Presence of Anti-Microbial Antibodies in Liver Cirrhosis – A Tell-Tale Sign of Compromised Immunity?

Bacterial translocation plays important role in the complications of liver cirrhosis. Antibody formation against various microbial antigens is common in Crohn's disease and considered to be caused by sustained exposure to gut microflora constituents. We hypothesized that anti-microbial antibodies are present in patients with liver cirrhosis and may be associated with the development of bacterial infections.<0.001, OR:2.02) by Cox-regression analysis.The present study suggests that systemic reactivity to microbial components reflects compromised mucosal immunity in patients with liver cirrhosis, further supporting the possible role of bacterial translocation in the formation of anti-microbial antibodies

MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

BACKGROUND & AIM: MicroRNAs (miRs) regulate hepatic steatosis, inflammation and fibrosis. Fibrosis is the consequence of chronic tissue damage and inflammation. We hypothesized that deficiency of miR-155, a master regulator of inflammation, attenuates steatohepatitis and fibrosis. METHODS: Wild type (WT) and miR-155-deficient (KO) mice were fed methionine-choline-deficient (MCD) or -supplemented (MCS) control diet for 5 weeks. Liver injury, inflammation, steatosis and fibrosis were assessed. RESULTS: MCD diet resulted in steatohepatitis and increased miR-155 expression in total liver, hepatocytes and Kupffer cells. Steatosis and expression of genes involved in fatty acid metabolism were attenuated in miR-155 KO mice after MCD feeding. In contrast, miR-155 deficiency failed to attenuate inflammatory cell infiltration, nuclear factor kappa beta (NF-kappaB) activation and enhanced the expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFalpha) and monocyte chemoattractant protein-1 (MCP1) in MCD diet-fed mice. We found a significant attenuation of apoptosis (cleaved caspase-3) and reduction in collagen and alpha smooth muscle actin (alphaSMA) levels in miR-155 KO mice compared to WTs on MCD diet. In addition, we found attenuation of platelet derived growth factor (PDGF), a pro-fibrotic cytokine; SMAD family member 3 (Smad3), a protein involved in transforming growth factor-beta (TGFbeta) signal transduction and vimentin, a mesenchymal marker and indirect indicator of epithelial-to-mesenchymal transition (EMT) in miR-155 KO mice. Nuclear binding of CCAAT enhancer binding protein beta (C/EBPbeta) a miR-155 target involved in EMT was significantly increased in miR-155 KO compared to WT mice. CONCLUSIONS: Our novel data demonstrate that miR-155 deficiency can reduce steatosis and fibrosis without decreasing inflammation in steatohepatitis

Role of MicroRNAs in NAFLD/NASH

MicroRNAs (miRNAs) are highly conserved, small, 18-25 nucleotide, non-coding RNAs that regulate gene expression at the post-transcriptional level. Each miRNA can regulate hundreds of target genes, and vice versa each target gene can be regulated by numerous miRNAs, suggesting a very complex network and explaining how miRNAs play pivotal roles in fine-tuning essentially all biological processes in all cell types in the liver. Here, we summarize the current knowledge on the role of miRNAs in the pathogenesis and diagnosis of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) with an outlook to the broader aspects of metabolic syndrome. Furthermore, we discuss the role of miRNAs as potential biomarkers and therapeutic targets in NAFLD/NASH

Inflammasomes in liver diseases

Inflammation is a common element in the pathogenesis of most chronic liver diseases that lead to fibrosis and cirrhosis. Inflammation is characterized by activation of innate immune cells and production of pro-inflammatory cytokines IL-1alpha, IL-1beta, and TNFalpha. Inflammasomes are intracellular multiprotein complexes expressed in both parenchymal and non-parenchymal cells of the liver that in response to cellular danger signals activate caspase-1, and release IL-1beta and IL-18. The importance of inflammasome activation in various forms of liver diseases in relation to liver damage, steatosis, inflammation and fibrosis is discussed in this review. Elsevier B.V. All rights reserved