Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome

Abstract

Hepatocyte death results in a sterile inflammatory response that amplifies the initial insult and increases overall tissue injury. One important example of this type of injury is acetaminophen-induced liver injury, in which the initial toxic injury is followed by innate immune activation. Using mice deficient in Tlr9 and the inflammasome components Nalp3 (NACHT, LRR, and pyrin domain-containing protein 3), ASC (apoptosis-associated speck-like protein containing a CARD), and caspase-1, we have identified a nonredundant role for Tlr9 and the Nalp3 inflammasome in acetaminophen-induced liver injury. We have shown that acetaminophen treatment results in hepatocyte death and that free DNA released from apoptotic hepatocytes activates Tlr9. This triggers a signaling cascade that increases transcription of the genes encoding pro-IL-1beta and pro-IL-18 in sinusoidal endothelial cells. By activating caspase-1, the enzyme responsible for generating mature IL-1beta and IL-18 from pro-IL-1beta and pro-IL-18, respectively, the Nalp3 inflammasome plays a crucial role in the second step of proinflammatory cytokine activation following acetaminophen-induced liver injury. Tlr9 antagonists and aspirin reduced mortality from acetaminophen hepatotoxicity. The protective effect of aspirin on acetaminophen-induced liver injury was due to downregulation of proinflammatory cytokines, rather than inhibition of platelet degranulation or COX-1 inhibition. In summary, we have identified a 2-signal requirement (Tlr9 and the Nalp3 inflammasome) for acetaminophen-induced hepatotoxicity and some potential therapeutic approaches.

Figure 1. APAP-mediated hepatotoxicity is dependent on Tlr9.

(A) Increase in total liver pro–IL-1β transcript in Tlr9^+/+ mice 12 hours after APAP (500 mg/kg), which is significantly smaller in Tlr9^–/– compared with Tlr9^+/+ mice (*P < 0.01). (B) Significantly lower serum transaminase levels in Tlr9^–/– compared with Tlr9^+/+ mice 12 hours after a single toxic dose of APAP (*P < 0.01). (C) Less liver hemorrhage and necroinflammation in Tlr9^–/– compared with Tlr9^+/+ mice 12 hours after APAP (H&E staining; original magnification, ×20). (D) Kaplan-Meier survival curves for Tlr9^+/+ and Tlr9^–/– mice over 72 hours after a single toxic dose of APAP (Tlr9^+/+: n = 15, Tlr9^–/–: n = 17, P < 0.04). Error bars indicate 1 SD. Ctrl, control.

Figure 2. Reduction in liver injury by Tlr9 antagonist and induction of liver injury with apoptotic DNA.

(A and B) Treatment of Tlr9^+/+ mice with the Tlr9 antagonist ODN2088 significantly reversed the APAP-induced rise in liver pro–IL-1β transcript and serum transaminase levels (*P < 0.01). (C and D) The Tlr7 and Tlr9 antagonist IRS 954 significantly decreased mortality from APAP over 72 hours and also reduced elevations in serum ALT at 12 hours after APAP (control: n = 14, IRS 954: n = 14, P < 0.006; *P < 0.01). (E–G) Direct administration of DNA from apoptotic hepatocytes into the blood supplying the liver resulted in an increase in hepatic transcripts of pro–IL-1β and pro–IL-18 and serum transaminases in Tlr9^+/+ mice. Both serum transaminase and pro–IL-1β and pro–IL-18 transcript levels were lower after injection of DNA from healthy hepatocytes (*P < 0.01). (H–J) In control Tlr9^–/– mice, there were no significant changes in serum transaminases and hepatic transcripts of IL-1β and IL-18 after direct administration of DNA from apoptotic or healthy hepatocytes. Error bars indicate 1 SD.

Figure 3. DNA from apoptotic hepatocytes increases pro–IL-1β and pro–IL-18 transcript levels in primary liver endothelial cells, and this is inhibited by Tlr9 antagonist.

(A and B) To determine whether APAP-induced upregulation of pro–IL-1β and pro–IL-18 was dependent on immune cells, we examined the livers of Rag1^–/–γ^–/– mice, which lack most immune cell populations. There was significant upregulation of the transcripts of both cytokines in the livers of Rag1^–/–γ^–/– mice (*P < 0.001 and **P < 0.01). (C and D) Culture of primary mouse endothelial cells from Tlr9^+/+ mice with DNA from apoptotic (APOP) but not healthy hepatocytes results in upregulation of pro–IL-1β and pro–IL-18, and this is downregulated by Tlr9 antagonist ODN2088 (*P < 0.001). (E and F) Culture of mouse endothelial cells from Tlr9^–/– mice with DNA from apoptotic and healthy hepatocytes does not result in upregulation of pro–IL-1β and pro–IL-18. (G) To establish the importance of IL-1β in APAP hepatotoxicity, an anti–IL-1β antibody (0.2 mg per mouse) was used for in vivo neutralization. This demonstrates a significant increase in survival of wild-type mice in the presence of IL-1β neutralization compared with control antibody after APAP (control antibody: n = 10, anti–IL-1β: n = 10, P < 0.02). (H) To establish the importance of IL-18 in APAP hepatotoxicity, we treated Il18^–/– and Il18^+/+ mice with APAP. There was significantly better survival in Il18^–/– compared with Il18^+/+ mice (Il18^+/+: n = 10, Il18^–/–: n = 7, P < 0.036). Error bars indicate 1 SD.

Figure 4. APAP-mediated hepatotoxicity is dependent on the Nalp3, but not the Ipaf, inflammasome.

(A) Survival of Casp1^–/– and control mice after i.p. injection of 500 mg/kg APAP (Casp1^+/+: n = 12, Casp1^–/–: n = 12, P < 0.04) (B) Survival of ASC^–/– and control mice after APAP (ASC^+/+: n = 15, ASC^–/–: n = 15, P < 0.03). (C) Survival of Nalp3^–/– and control mice after APAP (Nalp3^+/+: n = 15, Nalp3^–/–: n = 15, P < 0.006). (D) Survival of Ipaf^–/– and control mice after APAP (Ipaf^+/+: n = 12, Ipaf^–/–: n = 8, P = NS). (E) H&E staining of livers (original magnification, ×20) from wild-type, Casp1^–/–, ASC^–/–, and Nalp3^–/– mice 12 hours after i.p. injection of PBS or APAP showing reduced necroinflammation and hemorrhage in all the mice lacking components of the Nalp3 inflammasome. (F) Serum ALT from wild-type, Casp1^–/–, Nalp3^–/–, and Ipaf^–/– mice 12 hours after APAP. In Casp1^–/– and Nalp3^–/– serum, ALT levels were significantly lower than those in wild-type serum after APAP (P < 0.03) (G) To confirm in vivo Casp1 activation in endothelial cells, 24 hours after administration of APAP or control PBS, liver sinusoidal cells were isolated and cleavage of caspase-1 detected by Western blotting. Error bars indicate 1 SD.

Figure 6. Aspirin downregulates pro–IL-1β and pro–IL-18 transcripts.

(A) Survival after i.p. injection of APAP with and without clopidogrel by gavage (30 mg/kg every 24 hours) (PBS gavage: n = 15, clopidogrel gavage: n = 15, P = 0.31). Clopidogrel or PBS was gavaged every 24 hours beginning 48 hours prior and ending 24 hours after APAP injection. (B) Survival after i.p. injection of APAP with and without the COX-1 inhibitor SC-560. SC-560 (5 mg/kg) or control PBS was gavaged twice daily beginning 60 hours prior to and ending 48 hours after APAP injection (PBS gavage: n = 10, SC-560 gavage: n = 10, P = 0.97). (C–F) Q-PCR for pro–IL-1β, pro–IL-18, TNF-α, and IFN-γ from whole livers of mice treated as describe above. Shown are results from 1 representative experiment of 4; each group represents 3 mice (*P < 0.03, **P < 0.005). (G) ELISA for IL-1β from serum of mice given APAP with and without aspirin in drinking water (^†P < 0.02). (H) ELISA for IL-1β from THP1 cells that were incubated overnight with control vehicle or various doses of aspirin and then for 8 hours with or without LPS. Data shown are from 1 representative experiment of 3 in which each treatment was performed in triplicate (^‡P < 0.05, ^#P < 0.05). Error bars indicate 1 SD.

Figure 7. Production of mature IL-1β in APAP hepatotoxicity.

Release of mature IL-1β requires transcription of pro–IL-1β and subsequent cleavage and secretion by activated caspase-1. In APAP hepatotoxicity, apoptotic mammalian DNA has been shown to increase transcription of pro–IL-1β via a Tlr9-dependent pathway, and caspase-1 has been shown to be activated via a Nalp3/ASC pathway. The identity of the presumed danger-associated molecules responsible for activating the Nalp3 inflammasome in APAP hepatotoxicity remains to be determined.

Figure 5. Aspirin inhibits the Nalp3 pathway and reduces APAP-induced liver injury.

(A) Nalp3^+/+ or Nalp3^–/– mice were injected with MSU crystals i.p. (3 mg/mouse). After 3 hours, peritoneal lavage was performed and the number of GR-1–positive neutrophils quantified (*P < 0.0001). (B) Wild-type mice were treated with or without aspirin (60 mg/l) in the drinking water for 3 days and then injected i.p. with MSU crystals or PBS. After 3 hours, peritoneal lavage was performed and the number of Gr-1–positive neutrophils quantified (*P < 0.0001). (C) Survival analysis of mice treated with and without aspirin (60 mg/l) in the drinking water for 3 days and then injected i.p. with APAP (500 mg/kg) (control drinking water: n = 13, aspirin drinking water: n = 17, P < 0.02). (D) H&E-stained liver tissue sections (original magnification, ×20) from wild-type mice 12 hours after i.p. injection with APAP or PBS. Mice were on aspirin or regular drinking water for 3 days prior to APAP injection. There is substantial reduction in APAP-induced liver injury and hemorrhage in mice receiving aspirin. (E) ALT levels in serum from wild-type mice 12 hours after APAP injection with and without pretreatment with aspirin. The aspirin-treated group had significantly lower serum ALT levels (P < 0.04). Error bars indicate 1 SD.