P2X7 receptor-mediated purinergic signaling promotes liver injury in acetaminophen hepatotoxicity in mice

Abstract

Inflammation contributes to liver injury in acetaminophen (APAP) hepatotoxicity in mice and is triggered by stimulation of immune cells. The purinergic receptor P2X7 is upstream of the nod-like receptor family, pryin domain containing-3 (NLRP3) inflammasome in immune cells and is activated by ATP and NAD that serve as damage-associated molecular patterns. APAP hepatotoxicity was assessed in mice genetically deficient in P2X7, the key inflammatory receptor for nucleotides (P2X7-/-), and in wild-type mice. P2X7-/- mice had significantly decreased APAP-induced liver necrosis. In addition, APAP-poisoned mice were treated with the specific P2X7 antagonist A438079 or etheno-NAD, a competitive antagonist of NAD. Pre- or posttreatment with A438079 significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury in wild-type but not P2X7-/- mice. Pretreatment with etheno-NAD also significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury. In addition, APAP toxicity in mice lacking the plasma membrane ecto-NTPDase CD39 (CD39-/-) that metabolizes ATP was examined in parallel with the use of soluble apyrase to deplete extracellular ATP in wild-type mice. CD39-/- mice had increased APAP-induced hemorrhage and mortality, whereas apyrase also decreased APAP-induced mortality. Kupffer cells were treated with extracellular ATP to assess P2X7-dependent inflammasome activation. P2X7 was required for ATP-stimulated IL-1β release. In conclusion, P2X7 and exposure to the ligands ATP and NAD are required for manifestations of APAP-induced hepatotoxicity.

Fig. 1.

Genetic deficiency of P2X7 decreases hepatic necrosis and inflammation in acetaminophen (APAP)-induced acute liver injury. Wild-type (WT; n = 6) and P2X7−/− mice (n = 7) were administered APAP at 500 mg/kg ip for 6 and 12 h. WT (n = 2) and P2X7−/− mice (n = 2) were also given PBS as control groups. Liver histology was assessed 12 h at posttreatment (A and B), serum ALTs at 6 h posttreatment (C), and neutrophils in the liver at 12 h posttreatment (D and E) . NS, not significant; *P < 0.05. Arrows point to neutrophils.

Fig. 2.

Pre- and posttreatment with the antagonist A438079 prevents liver injury and inflammation in APAP-induced acute liver injury in a P2X7-dependent manner. WT mice were administered saline vehicle (n = 8) or A438079 at 300 mM/kg ip (n = 4) 1 h before or 2 h after (n = 5) administration of APAP at 500 mg/kg ip. P2X7−/− mice were administered saline vehicle (n = 5) or A438079 at 300 mM/kg ip (n = 5) 1 h before administration of APAP at 500 mg/kg ip. In control experiments, WT mice and P2X7−/− mice were also administered saline (n = 2 per group) or A438079 (n = 2 per group) 1 h prior to PBS vehicle. At 12 h post-APAP treatment, liver histology was assessed in WT mice (A and B) and P2X7−/− mice (C and D). Liver caspase-1 activity (E), liver CYP2E1 expression (F), and liver APAP adducts (F) were measured at 6 h post-APAP in WT mice (n = 5), P2X7−/− mice (n = 7), and WT pretreated with A438079 as above (n = 4). *P < 0.05.

Fig. 3.

Treatment with apyrase or etheno-NAD decreases liver injury and inflammation in APAP-induced acute liver injury and improves survival. Saline vehicle (n = 5) or etheno-NAD at 2 mg per mouse (n = 6) was administered at 1 h before and 6 h after administration of APAP at 500 mg/kg ip. Apyrase at 4 units ip per mouse (n = 6) was administered 30 min before and 1 h after APAP. A438079 at 300 mM/kg ip (n = 4) was administered 1 h before APAP. At 12 h post-APAP treatment, liver histology (A and C), neutrophils in the liver (B and D), and serum ALT (E) were assessed. Survival was assessed to 72 h in WT mice administered apyrase (n = 10) or saline (n = 10) as above with APAP (F). H&E, hematoxylin and eosin; HPF, ×40 magnification high-powered field. *P < 0.05 between bracketed groups. #P < 0.05 compared with WT mice given APAP. Arrows point to neutrophils.

Fig. 4.

Genetic deficiency of CD39 accelerates hemorrhage and increases mortality in APAP hepatotoxicity. WT (n = 6) and CD39−/− mice (n = 6) were administered APAP at 200 mg/kg ip. Liver histology was assessed at 12 h post-APAP treatment (A and B). Survival was assessed to 72 h in WT (n = 8) and CD39−/− mice (n = 8) administered APAP at 500 mg/kg ip (C). *P < 0.05.

Fig. 5.

Kupffer cells require P2X7 to activate the nod-like receptor family, pryin domain containing-3 (NLRP3) inflammasome in response to extracellular ATP. Kupffer cells and thioglycollate-elicited peritoneal macrophages were isolated from WT and P2X7−/− mice and treated with LPS (100 ng/ml) and ATP (5 mM). Supernatant was assessed for IL-1β release (A). Adherent cells from the mouse liver nonparenchymal cell isolation were assessed for CD45.2 and F4/80 or control IgG positivity with representative results shown (n = 4) (B). *P < 0.05.

Fig. 6.

Injured hepatocytes release ATP and NAD, which signal NLRP3 inflammasome activation through P2X7. DAMP, damage-associated molecular patterns; TLR, TOLL-like receptor.