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. 2015 Nov;63(5):1147-55.
doi: 10.1016/j.jhep.2015.06.013. Epub 2015 Jun 20.

Inhibition of sterile danger signals, uric acid and ATP, prevents inflammasome activation and protects from alcoholic steatohepatitis in mice

Arvin Iracheta-Vellve  1 , Jan Petrasek  1 , Abhishek Satishchandran  1 , Benedek Gyongyosi  1 , Banishree Saha  1 , Karen Kodys  1 , Katherine A Fitzgerald  1 , Evelyn A Kurt-Jones  1 , Gyongyi Szabo  2
Affiliations

Inhibition of sterile danger signals, uric acid and ATP, prevents inflammasome activation and protects from alcoholic steatohepatitis in mice

Arvin Iracheta-Vellve et al. J Hepatol. 2015 Nov.

Abstract

Background & aims: The inflammasome is a well-characterized inducer of inflammation in alcoholic steatohepatitis (ASH). Inflammasome activation requires two signals for mature interleukin (IL)-1β production. Here we asked whether metabolic danger signals trigger inflammasome activation in ASH.

Methods: Wild-type mice, ATP receptor 2x7 (P2rx7)-KO mice, or mice overexpressing uricase were fed Lieber-DeCarli ethanol or control diet. We also implemented a pharmacological approach in which mice were treated with probenecid or allopurinol.

Results: The sterile danger signals, ATP and uric acid, were increased in the serum and liver of alcohol-fed mice. Depletion of uric acid or ATP, or lack of ATP signaling attenuated ASH and prevented inflammasome activation and its major downstream cytokine, IL-1β. Pharmacological depletion of uric acid with allopurinol provided significant protection from alcohol-induced inflammatory response, steatosis and liver damage, and additional protection was achieved in mice treated with probenecid, which depletes uric acid and blocks ATP-induced P2rx7 signaling. We found that alcohol-damaged hepatocytes released uric acid and ATP in vivo and in vitro and that these sterile danger signals activated the inflammasome in LPS-exposed liver mononuclear cells.

Conclusions: Our data indicate that the second signal in inflammasome activation and IL-1β production in ASH results from the endogenous danger signals, uric acid and ATP. Inhibition of signaling triggered by uric acid and ATP may have therapeutic implications in ASH.

Keywords: Alcoholic steatohepatitis; Damage-associated molecular patterns; Determinants of liver inflammation; Inflammasome; Pathogen-associated molecular patterns; Sterile inflammatory response.

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Conflict of interest statement

Conflict of interest: none to declare

Figures

Fig. 1
Fig. 1. Endogenous inflammasome activators, ATP and uric acid, are increased in ASH
Wild-type mice were fed control (pair-fed) or alcohol Lieber-DeCarli diet. After 4 weeks, liver damage and uric acid and ATP in the serum (A) and in the liver were evaluated (B). N= 15 (ethanol-fed); 5 (pair-fed). *P < 0.05 vs. baseline.
Fig. 2
Fig. 2. ATP signaling is required for inflammasome activation and increased TNF-α in ASH
WT or P2rx7-KO mice were fed control (pair-fed) or Lieber-DeCarli alcohol diet. After 4 weeks, liver damage was assessed by liver histology (H&E) and serum ALT (A). Oil-red-O staining was performed and liver triglycerides were measured to evaluate steatosis (B). We evaluated levels of TNF-α in the serum and in the liver (C) and IL-1β in the serum (D). We evaluated cleavage of Casp-1 in the liver (E). N= 7–15 (ethanol-fed, per genotype); 5 (pair-fed, per genotype). Numbers in the graphs indicate P values. *P < 0.05 vs. baseline. Original magnification, ×200.
Fig. 3
Fig. 3. Uric acid is required for inflammasome activation and increased TNF-α in ASH
WT, ssUOX-Tg or intUOX-Tg mice were fed control (pair-fed) or alcohol Lieber-DeCarli diet. After 4 weeks, we evaluated levels of uric acid in serum and in the liver (A). Liver damage was assessed by liver histology (H&E) and serum ALT. Oil-red-O staining and liver triglycerides were measured to evaluate steatosis (B). Levels of TNF-α in the serum and in the liver were evaluated (C). Levels of IL-1β in were evaluated in the serum (D). We evaluated cleavage of Casp-1 in the liver (E). N= 5–7 (ethanol-fed, per genotype); 3–5 (pair-fed, per genotype). Numbers in the graphs indicate P values. *P < 0.05 vs. baseline. Original magnification, ×200.
Fig. 4
Fig. 4. Pharmacologic depletion of uric acid and inhibition of ATP signaling prevents ASH
WT mice treated with daily intragastric doses of vehicle (PBS), allopurinol or probenecid (100 mg/kg body weight) were fed control (pair-fed) or alcohol Lieber-DeCarli diet. After 4 weeks, we evaluated the levels of uric acid in the serum (A). Liver damage was assessed by histology (H&E) and serum ALT, and Oil-red-O staining was performed and liver triglycerides were measured to evaluate steatosis (B). We evaluated levels of TNF-α in the serum and in the liver (C), levels of IL-1β in the serum (D), and cleavage of Casp-1 in the liver (E). Murine splenocytes were treated with 100 ng/mL of LPS. After 2 hours, allopurinol or probenecid was added at indicated doses, and ATP (5 mM) was added 30 minutes later. One hour afterwards, inflammasome activation was assessed by measuring IL-1β protein in cell-free supernatants. Stimulations were performed in triplicates (F). N= 9–10 mice (ethanol-fed, per treatment); 3–5 mice (pair-fed, per treatment). Numbers in the graphs indicate P values. *P < 0.05 vs. baseline. Original magnification, ×200.
Fig. 5
Fig. 5. Damaged hepatocytes, but not liver mononuclear cells, release endogenous metabolic activators of the inflammasome, ATP and soluble uric acid
Primary murine or human hepatocytes were treated with 800 μM ethanol, and levels of LDH, indicating hepatocyte death, ATP and uric acid were evaluated in supernatants at indicated time points (A). Primary murine or human hepatocytes were cultured at 45°C (heat-shock) or 37°C (control) and levels of LDH, ATP and uric acid were evaluated in supernatants at indicated time points (B). Murine liver mononuclear cells (LMNCs) were treated with 800 mM ethanol, and levels of LDH, indicating cell death, ATP and uric acid were evaluated in supernatants at indicated time points. Murine LMNCs were cultured at 45°C (heat-shock) or 37°C (control) and levels of LDH, ATP and uric acid were evaluated in supernatants at indicated time points (C). LMNCs isolated from wild-type mice were pretreated with indicated doses of LPS. In some groups, ATP (5 mM) was added after 2 hours of LPS stimulation and supernatant was collected 1 hour afterward. In other groups, soluble uric acid (100 μg/mL) or monosodium urate crystals (MSU, 250 μg/mL) were added after 2 hours of LPS stimulation and supernatant was collected at 24 hours (D). Stimulations were performed in triplicates. *P < 0.05 vs. baseline.
Fig. 6
Fig. 6. DAMPs released from primary hepatocytes damaged from in vivo ethanol gavage result in inflammasome activation
Primary murine hepatocytes and LMNCs were isolated four hours after EtOH (in vivo EtOH Gavage) or isocaloric sucrose gavage (in vivo Sugar Gavage) or from untreated mice. Hepatocytes were plated and non-adherent cells were washed off with PBS four hours later. Hepatocyte supernatant was collected 3 hours later. LMNCs were primed for 2 hours with LPS (10 ng/mL) or water (unstimulated). LMNCs were then stimulated with hepatocyte supernatant collected as described. LMNCs were stimulated with supernatant from primary murine hepatocytes treated with 800 mM EtOH for 1 hour in vitro (in vitro EtOH). LMNCs were stimulated with ATP (5 mM) for 6 hours (in vitro ATP). Some LMNCs were analyzed via immunofluorescence microscopy to assess inflammasome activation (A). Supernatant was collected from other LMNCs and analyzed for IL-1β secretion at 2 and 6 hours (B).
Fig. 7
Fig. 7. Uric acid and ATP represent a second signal involved in LPS-driven inflammatory response in ASH
(A–C) WT or P2rx7-KO mice were fed control (pair-fed) or Lieber-DeCarli alcohol diet. After 4 weeks, levels of ethanol were measured in the liver (A), levels of endotoxin were measured in the liver (B) and expression of pro-IL-1B gene (mRNA) were measured in the liver (C). N= 6–9 (ethanol-fed, per genotype); 4–5 (pair-fed, per genotype). (D–F) WT, ssUOX-Tg or intUOX-Tg mice were fed control (pair-fed) or alcohol Lieber-DeCarli diet. After 4 weeks, levels of ethanol were measured in the liver (D), levels of endotoxin were measured in the liver (E) and expression of pro-IL-1B gene (mRNA) were measured in the liver (F). N= 6–9 (ethanol-fed, per genotype); 4–5 (pair-fed, per genotype). Numbers in the graphs indicate P values. (G) Proposed role of uric acid and ATP control inflammation in ASH. Exposure to alcohol induces hepatocyte death. In the same time, alcohol increases exposure of liver immune cells to gut-derived bacterial LPS, which provides the signal for induction of the immature pro-IL-1β in liver immune cells (first signal). The signal for IL-1β activation in the liver is probably derived from damaged hepatocytes which release uric acid and ATP (second signal). These molecules activate the inflammasome in liver immune cells. As a result, Caspase-1, the effector molecule of the inflammasome, cleaves pro-IL-1β into the bioactive IL-1β. Active IL-1β plays a crucial role in alcoholic steatohepatitis [2].
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