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. 2011 Jul;54(1):133-44.
doi: 10.1002/hep.24341.

Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells

Timea Csak  1 , Michal Ganz, Justin Pespisa, Karen Kodys, Angela Dolganiuc, Gyongyi Szabo
Affiliations

Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells

Timea Csak et al. Hepatology. 2011 Jul.

Abstract

The pathogenesis of nonalcoholic steatohepatitis (NASH) and inflammasome activation involves sequential hits. The inflammasome, which cleaves pro-interleukin-1β (pro-IL-1β) into secreted IL-1β, is induced by endogenous and exogenous danger signals. Lipopolysaccharide (LPS), a toll-like receptor 4 ligand, plays a role in NASH and also activates the inflammasome. In this study, we hypothesized that the inflammasome is activated in NASH by multiple hits involving endogenous and exogenous danger signals. Using mouse models of methionine choline-deficient (MCD) diet-induced NASH and high-fat diet-induced NASH, we found up-regulation of the inflammasome [including NACHT, LRR, and PYD domains-containing protein 3 (NALP3; cryopyrin), apoptosis-associated speck-like CARD-domain containing protein, pannexin-1, and pro-caspase-1] at the messenger RNA (mRNA) level increased caspase-1 activity, and mature IL-1β protein levels in mice with steatohepatitis in comparison with control livers. There was no inflammasome activation in mice with only steatosis. The MCD diet sensitized mice to LPS-induced increases in NALP3, pannexin-1, IL-1β mRNA, and mature IL-1β protein levels in the liver. We demonstrate for the first time that inflammasome activation occurs in isolated hepatocytes in steatohepatitis. Our novel data show that the saturated fatty acid (FA) palmitic acid (PA) activates the inflammasome and induces sensitization to LPS-induced IL-1β release in hepatocytes. Furthermore, PA triggers the release of danger signals from hepatocytes in a caspase-dependent manner. These hepatocyte-derived danger signals, in turn, activate inflammasome, IL-1β, and tumor necrosis factor α release in liver mononuclear cells.

Conclusion: Our novel findings indicate that saturated FAs represent an endogenous danger in the form of a first hit, up-regulate the inflammasome in NASH, and induce sensitization to a second hit with LPS for IL-β release in hepatocytes. Furthermore, hepatocytes exposed to saturated FAs release danger signals that trigger inflammasome activation in immune cells. Thus, hepatocytes play a key role in orchestrating tissue responses to danger signals in NASH.

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Conflict of interest statement

Potential conflict of interest: Nothing to report.

Figures

Fig. 1
Fig. 1
MCD diet–induced steatohepatitis is associated with increased IL-1β production and NALP3 inflammasome activation in the liver. C57Bl/6 mice were fed an MCD diet or an MCS diet for 5 weeks. (A) The serum IL-1β levels and (B) the liver mRNA fold changes for IL-1β and the NALP3 inflammasome complex (including NALP3, ASC, caspase-1, and pannexin-1) were determined. The functional activity of the inflammasome was evaluated by measurements of (C) the caspase-1 activity and (D) the mature IL-1β protein levels in the liver. (E) Liver triglycerides were measured (six mice per group). *P < 0.05 versus the MCS diet–fed mice.
Fig. 2
Fig. 2
Long-term HFD diet–induced steatohepatitis is associated with increased IL-1β production and NALP3 inflammasome activation in the liver. C57Bl/6 mice were fed an HFD or a control diet for 4 weeks or 9 months. The liver mRNA fold changes for IL-1β and the NALP3 inflammasome complex (including NALP3, ASC, caspase-1, and pannexin-1) were determined after (A) 4 weeks or (B) 9 months of HFD feeding. The functional activity of the inflammasome was evaluated by measurements of (C) the caspase-1 activity and (D) the mature IL-1β protein levels in the liver. (E) Liver triglycerides were measured (six mice per group). *P < 0.05 versus the corresponding control group.
Fig. 3
Fig. 3
Increased inflammasome expression in human NASH. The mRNA expression of NALP3, ASC, caspase-1, and pannexin-1 was measured with qPCR in the livers of NASH patients, in commercially available, normal human livers (n = 4), and in liver samples from HCV-infected patients. *P < 0.05 versus the control.
Fig. 4
Fig. 4
LPS induces the up-regulation of the inflammasome in the liver. C57Bl/6 mice were fed an MCD diet or an MCS diet for 5 weeks and were injected with LPS (0.5 mg/kg of body weight) intraperitone-ally for 2 hours. Hepatic mRNA expression for (A) NALP3 and (B) IL-1β was analyzed with qPCR, and (C) mature IL-1β protein levels were measured with an enzyme-linked immunosorbent assay (six mice per group). *P < 0.05 versus the MCS diet–fed mice at the baseline. #P < 0.05 versus the MCS diet–fed mice after LPS stimulation.
Fig. 5
Fig. 5
The inflammasome is up-regulated in hepatocytes in NASH. Saturated FAs (but not unsaturated FAs) induce NALP3 mRNA expression in hepatocyte and macrophage cell lines. C57Bl/6 mice were fed an MCD diet or an MCS diet for 5 weeks, and primary hepatocytes were isolated as described in the Materials and Methods section. (A) The hepatocyte mRNA fold changes of IL-1β and the NALP3 inflammasome complex (including NALP3, ASC, caspase-1, and pannexin-1) were determined. (B) Serum endotoxin data are presented as means and standard errors of the mean (four to six mice per group). (C) Hepa1–6 cells, which were used as prototypes for hepatocytes, and (D) RAW macrophages, which were used as prototypes for macrophages, were exposed to the saturated FA PA (0.165 or 0.33 mM) or unsaturated FAs such as oleic acid (0.33 or 0.66 mM) and linoleic acid (0.33 or 0.66 mM). NALP3 mRNA expression was analyzed with qPCR. *P < 0.05 versus the control.
Fig. 6
Fig. 6
FAs induce inflammasome activation and sensitization to LPS-induced IL-1β secretions in hepatocytes in vitro that involve inflammasome-dependent (caspase-1) and inflammasome-independent (caspase-8–dependent) pathways. (A) Isolated hepatocytes from C57Bl/6 mice on a normal rodent diet were treated with PA (0.33 mM), LPS (1000 ng/mL), or both for 6 hours, and the NALP3 mRNA levels were determined with qPCR. (B) The IL-1β protein levels in the supernatants of hepatocytes were measured with an enzyme-linked immunosorbent assay after their exposure to PA (0.33 mM), LPS (1000 µg/mL), or both for 2, 6, or 18 hours. The activation of (C) inflammatory caspase-1 and (D) apoptotic caspase-8 was determined with an enzyme activity assay. *P < 0.05 versus the control.
Fig. 7
Fig. 7
PA-treated hepatocytes transmit danger signals and induce inflammasome activation in LMNCs. (A) Isolated hepatocytes from C57Bl/6 mice on a normal rodent diet were treated with PA (0.33 mM) or LPS (1000 ng/mL) in the presence or absence of the pancaspase inhibitor ZVAD (40 µM). LDH release as a marker of cell death was determined. The hepatocyte mRNA fold changes of (B) NALP3 and (C) IL-1β were analyzed with qPCR. Supernatants of hepatocytes that were treated with PA for 6 hours and then cultured in fresh media without PA were transferred to LMNCs. The mRNA expression of (D) NALP3 and (E) IL-1β was analyzed. *P < 0.05 versus the control.
Fig. 8
Fig. 8
Summary of inflammasome activation in NASH.
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