Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome
Avlin B. Imaeda, … , Richard A. Flavell, Wajahat Z. Mehal
Avlin B. Imaeda, … , Richard A. Flavell, Wajahat Z. Mehal
Published January 26, 2009
Citation Information: J Clin Invest. 2009;119(2):305-314. https://doi.org/10.1172/JCI35958.
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Research Article

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-1β and pro–IL-18 in sinusoidal endothelial cells. By activating caspase-1, the enzyme responsible for generating mature IL-1β and IL-18 from pro–IL-1β 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.

Authors

Avlin B. Imaeda, Azuma Watanabe, Muhammad A. Sohail, Shamail Mahmood, Mehdi Mohamadnejad, Fayyaz S. Sutterwala, Richard A. Flavell, Wajahat Z. Mehal

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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.

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DNA from apoptotic hepatocytes increases pro–IL-1β and pro–IL-18 transcr...
(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.