title: 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.
abstract: BACKGROUND
  Non-alcoholic fatty liver disease (NAFLD) is becoming a pandemic. While multiple 'hits' 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, TNFα, IL-1β 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.
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, 364 Plantation St, LRB-208, Worcester, MA, 01605, USA. mganz1@gmail.com.
authors.forename: Michal
authors.initials: M
authors.lastname: Ganz
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, 364 Plantation St, LRB-208, Worcester, MA, 01605, USA. terence.bukong@umassmed.edu.
authors.forename: Terence N
authors.initials: TN
authors.lastname: Bukong
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, 364 Plantation St, LRB-208, Worcester, MA, 01605, USA. timea.csak@umassmed.edu.
authors.forename: Timea
authors.initials: T
authors.lastname: Csak
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, 364 Plantation St, LRB-208, Worcester, MA, 01605, USA. banishree.saha@umassmed.edu.
authors.forename: Banishree
authors.initials: B
authors.lastname: Saha
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, 364 Plantation St, LRB-208, Worcester, MA, 01605, USA. jin-kyu.park@yale.edu.
authors.forename: Jin-Kyu
authors.initials: JK
authors.lastname: Park
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, 364 Plantation St, LRB-208, Worcester, MA, 01605, USA. aditya.ambade@umassmed.edu.
authors.forename: Aditya
authors.initials: A
authors.lastname: Ambade
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, 364 Plantation St, LRB-208, Worcester, MA, 01605, USA. karen.kodys@umassmed.edu.
authors.forename: Karen
authors.initials: K
authors.lastname: Kodys
authors.affiliation: Department of Medicine, University of Massachusetts Medical School, 364 Plantation St, LRB-208, Worcester, MA, 01605, USA. gyongyi.szabo@umassmed.edu.
authors.forename: Gyongyi
authors.initials: G
authors.lastname: Szabo
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001835:Body Weight; D002784:Cholesterol / Q000009:adverse effects; D016207:Cytokines / Q000378:metabolism; D059305:Diet, High-Fat / Q000009:adverse effects; D004040:Dietary Carbohydrates / Q000009:adverse effects; D004195:Disease Models, Animal; D018450:Disease Progression; D005247:Feeding Behavior; D005260:Female; D058847:Inflammasomes / Q000378:metabolism; D007249:Inflammation / Q000473:pathology*; D018836:Inflammation Mediators / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D007728:Kupffer Cells / Q000473:pathology; D008099:Liver / Q000473:pathology*; D008103:Liver Cirrhosis / Q000150:complications / Q000473:pathology*; D008113:Liver Neoplasms / Q000150:complications / Q000473:pathology*; D008262:Macrophage Activation; D008297:Male; D008810:Mice, Inbred C57BL; D065626:Non-alcoholic Fatty Liver Disease / Q000150:complications / Q000473:pathology*; D009929:Organ Size; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D015415:Biomarkers; D016207:Cytokines; D004040:Dietary Carbohydrates; D058847:Inflammasomes; D018836:Inflammation Mediators; D007328:Insulin; D002784:Cholesterol