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. 2015 Jun 16:13:193.
doi: 10.1186/s12967-015-0552-7.

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

Michal Ganz  1 , Terence N Bukong  2 , Timea Csak  3 , Banishree Saha  4 , Jin-Kyu Park  5 , Aditya Ambade  6 , Karen Kodys  7 , Gyongyi Szabo  8
Affiliations

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

Michal Ganz et al. J Transl Med. .

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.

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Figures

Figure 1
Figure 1
HF–HC–HSD leads to an increase in liver to body weight ratio, insulin resistance and steatohepatitis. Male C57Bl/6 mice were fed with either a high fat diet supplemented with 10% cholesterol and sucrose and fructose in the drinking water, or with a control chow diet for 8, 27, or 49 weeks. Body weight over time (a) and liver to body weight ratio (b) is shown at each time point. Blood glucose (c) over time is shown for each time point. Insulin levels are shown in the chow fed and HFD fed mice both before and 30 min post glucose injection (d). Liver tissue was subjected to (e) Oil-Red-O staining. One representative slide from n = 6/group is shown. Liver triglyceride levels (f) and serum alanine aminotransferase (ALT) levels (g) were measured at each time point. *p < 0.01. n = 6–12/group. #p < 0.01—compared to HFD prior to glucose injection, +p < 0.01, ++p = 0.02—compared to mice fed chow diet prior to glucose injection. n = 6–12/group.
Figure 2
Figure 2
Long-term HF–HC–HSD results in hepatic inflammation. Representative H&E (a) slides of chow and HFD fed mice are shown at each time point. Histology scoring was performed on tissue sections at 8, 27 and 49 weeks (b). MCP-1 levels in the liver were evaluated at both the c mRNA and d protein level. Liver TNFα mRNA was evaluated (e). We also looked at IL-1β mRNA in the liver (f) and IL-1β protein levels in the serum (g). IL-1β was undetectable in the serum at 8 and 27 weeks. Electrophoretic mobility assay was performed from nuclear extracts for NFκB at 8,27 and 49 weeks compared to control chow diet (h). *p < 0.01, **p = 0.02, %p = 0.05 versus chow fed controls, n = 6–12/group.
Figure 3
Figure 3
Long-term HF–HC–HSD induces inflammasome upregulation and activation as well as upregulation of danger signals. Hepatic mRNA expression of the inflammasome components NLRP3 (a), Pannexin-1 (b), P2X7 (c), ASC (d), and caspase-1 (e), were measured using qPCR at each of the different HF–HC–HSD time points. Activation of inflammatory caspases was evaluated by measurement of caspase-1 activity (f). Cell death was assessed by immunohistochemistry of cleaved PARP (g) on paraffin embedded liver sections. Level of danger signals in the serum, uric acid (h), ATP (i) were determined. Western blot was performed to determine levels of total HMGB1 (j) and immunoprecipitation and western blot for the active form of HMGB1, acetyl-HMGB1 (k). Hepatic mRNA expression of receptors for danger signals, TLR2 (l), TLR4 (m), TLR9 (n) and RAGE (o) were measured using qPCR. Serum endotoxin was measured at 8, 27 and 49 weeks of HF–HC–HSD (p). *p < 0.01, **p = 0.02, ^p = 0.03, #p = 0.04 versus chow fed controls, n = 6–12/group.
Figure 4
Figure 4
M1 macrophage markers are increased, whereas M2 macrophage markers are decreased with long-term HFD feeding. F4/80 (a), CD11b (b), and CD68 (c) mRNA levels were evaluated using qPCR to look for the presence of M1 macrophages. qPCR was also used to look for the presence of M2 markers CD163 (d) and Arg1 (e) mRNA levels. mRNA expression of CD3 in liver samples was determined by qPCR (f). FACS was performed to determine levels of CD3 (g, h), inflammatory monocytes (g, i), NK cells (g, j) NKT cells (g, k). *p < 0.01, **p = 0.02, versus chow fed controls, n = 6–12/group.
Figure 5
Figure 5
Long-term HF–HC–HSD induces hepatic fibrosis. Hepatic mRNA expression of αSMA (a), pro-collagen-1 (b), TGFβ (c), TIMP-1 (d), TIMP-3 (e), MMP2 (f), and MMP-9 (g) were measured using qPCR in male mice at each time point. Representative images of Sirius red staining to evaluate fibrosis at 27 and 49 weeks are shown (h).
Figure 6
Figure 6
Long-term HF–HC–HSD induces hepatic tumor development. Liver mRNA was determined for CD133 (a), Nanog (b) and vimentin (c) by qPCR. Gross images of tumors that were detected in two of the mice and tumor histopathology (d). Detectable liver p53 protein expression was observed by western blotting after 27 and 49 weeks of HF–HC–HSD (e). Low but detectable AFP was observed in liver tumor samples after 49 weeks of HF–HC–HSD on immunohistochemistry (f). *p < 0.01, #p = 0.03, %p = 0.05 versus chow fed controls, n = 6–12/group.
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References

    1. Ng M, Fleming T, Robinson M, Thomson B, Graetz N, Margono C, et al. Global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2014;384:766–781. doi: 10.1016/S0140-6736(14)60460-8. - DOI - PMC - PubMed
    1. Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, et al. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology. Gastroenterology. 2012;142:1592–1609. doi: 10.1053/j.gastro.2012.04.001. - DOI - PubMed
    1. Tiniakos DG, Vos MB, Brunt EM. Nonalcoholic fatty liver disease: pathology and pathogenesis. Annu Rev Pathol. 2010;5:145–171. doi: 10.1146/annurev-pathol-121808-102132. - DOI - PubMed
    1. Tarantino G, Savastano S, Colao A. Hepatic steatosis, low-grade chronic inflammation and hormone/growth factor/adipokine imbalance. World J Gastroenterol. 2010;16:4773–4783. doi: 10.3748/wjg.v16.i38.4773. - DOI - PMC - PubMed
    1. Sheth SG, Gordon FD, Chopra S. Nonalcoholic steatohepatitis. Ann Intern Med. 1997;126:137–145. doi: 10.7326/0003-4819-126-2-199701150-00008. - DOI - PubMed

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