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Imaeda AB, Watanabe A, Sohail MA, Mahmood S, Mohamadnejad M, Sutterwala FS, et al. Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome. J Clin Invest 2009;119:305–314. (Reprinted with permission.)

Abstract

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  2. Abstract
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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 acetaminopheninduced 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.

Comment

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  2. Abstract
  3. Comment
  4. References

The molecular mechanisms of sterile inflammation in response to toxic liver injury are incompletely understood. Acetaminophen (AAP)-induced liver necrosis is a model for sterile inflammation: AAP causes an initial toxic insult leading to a mixture of both necrotic and apoptotic hepatocytes (Fig. 1). Although it is generally known that components released from necrotic cells can trigger immune cell activation, only a few molecular pathways have been characterized. One well-established mechanism by which necrotic cell death induces inflammation is the release of the chromatin protein High-Mobility-Group-Box-1 (HMGB-1). Indeed, blockade of HMGB-1 has been reported to rescue AAP-treated mice from mortality, and this indicates that the immune response to HMGB-1 is the culprit in this model.1 HMGB-1 has been reported to activate the receptor for advanced glycation end products (RAGE) on innate immune cells.2 Another report has shown that when infected cells undergo cell death, microbial DNA and released HMGB-1 engage toll-like receptor 9 (TLR9) and RAGE, respectively, to cooperatively induce the production of type I interferon (IFN).3 HMGB-1 released from necrotic tumor cells has been reported to interact with TLR4 on dendritic cells (DCs) to induce cross-priming of cytotoxic T cells.4 It has been shown only recently that CD8α+ DCs express a C-type lectin (C-type lectin domain family 9 member A) that recognizes a ligand exposed by necrotic cells, leading to DC cross-presentation of necrotic cell–derived antigens.5 However, despite the role of HMGB-1 in inducing inflammation and activation of the adaptive immune system, molecules such as CD24 and sialic acid binding immunoglobulin-like lectin G have recently been shown to down-modulate the host response to HMGB-1 released by damaged cells.1 Furthermore, the mode of cell death is a critical determinant of the resulting immune response. Although necrotic cell death or pyroptosis of infected cells results in inflammation, apoptosis during normal tissue turnover is a silent process that induces regulatory and tissue repair genes. Indeed, we have recently shown that in contrast to the innate immune recognition of apoptotic cells, which induces the development of regulatory T cells, the recognition of infected apoptotic cells is a potent trigger for T helper 17 immune responses.6

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Figure 1. AAP or other hepatotoxic compounds damage the liver, causing hepatocyte AO as well as (secondary) NE. Nonparenchymal cells sense components released from dead hepatocytes. DNA fragments stimulate TLR9 in LSECs and possibly Kupffer cells, leading to activation of MyD88 and subsequent transcription of NF-κB–dependent cytokines, among which is pro-IL-1β. A hitherto unidentified trigger activates the NALP3 inflammasome, leading to Casp1 activation and IL-1β processing and release. Possible candidates for NALP3 inflammasome triggers are ATP and uric acid from necrotic hepatocytes. Abbreviations: AAP, acetaminophen; AO, apoptosis; Asc, apoptosis-associated speck-like protein containing a caspase recruitment domain; ATP, adenosine triphosphate; Casp1, caspase-1; IL, interleukin; MyD88, myeloid differentiation protein 88; NALP3, NACHT domain-, leucine-rich repeat-, and PYD-containing protein 3; NE, necrosis; NF-κB, nuclear factor kappa B; LSEC, liver sinusoidal endothelial cell; TLR9, toll-like receptor 9.

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A new study by Imaeda and colleagues7 took a closer look at the mechanisms responsible for AAP-induced inflammation. On the basis of previous observations [i.e., the inflammatory cytokine interleukin-1β (IL-1β) is strongly up-regulated during AAP-induced hepatotoxicity, and IL-1R signaling is required for sterile inflammation caused by AAP8], they investigated the pathways responsible for IL-1β production. Proforms of IL-1β and IL-18 (and IL-33) are produced in response to various signals involving nuclear factor kappa B activation. Processing of the zymogens into their active form requires activation of a cytosolic protein complex termed the inflammasome, which generally consists of oligomers of NOD-like receptors (NLRs), a large group of intracellular pattern recognition receptors, and the adaptor molecule apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which recruits the effector caspase-1.9 Caspase-1 proteolytically cleaves pro-IL-1β into its active form. The present study demonstrates that cooperative signaling between TLR9 and the NACHT domain-, leucine-rich repeat-, and PYD-containing protein 3 (NALP3) inflammasome mediates hepatotoxicity in mice.7 TLR9-deficient mice were protected from AAP-mediated hepatotoxicity, showing strongly reduced liver damage and improved survival. Furthermore, direct administration into the portal vein of DNA isolated from primary hepatocytes made apoptotic in vitro caused liver damage, as evidenced by elevated alanine aminotransferase levels, and induced pro-IL-1β and pro-IL-18 transcripts in wild-type mice but not in TLR9-deficient mice. This effect was much weaker upon the administration of DNA extracted from viable hepatocytes. The authors further investigated the cell type responding to TLR9 stimulation. They proposed liver sinusoidal endothelial cells (LSECs) as the major cell population activated by DNA released from dead hepatocytes; they based this on observations made in vitro with isolated LSECs and in vivo in mice deficient for the genes encoding recombination activation gene 1 (RAG1) and the common gamma chain (Rag1−/−γ−/−) and thus lacking all T, B, natural killer T, and natural killer cell lineages. However, the role of Kupffer cells, known as potent producers of IL-1β and recently shown to strongly aggravate concanavalin A–induced hepatitis in response to TLR9 ligands,10 was not investigated here. The authors next demonstrated that inflammasome activation was required for AAP-induced hepatotoxicity because mice lacking caspase-1, ASC, or the NLR NALP3 were at least partially protected from liver damage and showed significantly reduced mortality. Interestingly, the authors found no difference among these strains in total IL-1β levels in the liver, whereas serum IL-1β was significantly decreased in Nalp3−/− mice following AAP administration; this indicated that extrahepatic cells likely contribute to hepatotoxicity and sterile inflammation in this model. Notably, both inhibition of IL-1R(8)/IL-1β and IL-18 and administration of TLR9 inhibitors were protective in the murine model of AAP-induced liver damage, and this indicated cooperation among these pathways. This could suggest novel liver-protective strategies for AAP intoxication in humans. The authors further presented evidence that a low-dose aspirin treatment could have protective effects during AAP-induced liver damage and that this is mediated through the inhibition of pro-IL-1β and pro-IL-18 transcription. However, the underlying mechanisms are not clear, and previous results from clinical trials are less promising.

These results show a nonredundant role for TLR9 and NALP3 in mediating the sterile inflammation following AAP toxicity–induced hepatocyte death. Several questions still remain regarding the molecular mechanisms leading to TLR9 and NALP3 activation. It is unclear how genomic DNA specifically from apoptotic cells activates TLR9 because classical apoptosis does not involve the release of DNA. Alternatively, given that both necrosis and apoptosis are induced in AAP-induced liver injury, stimulatory DNA may be derived from necrotic hepatocytes, although this DNA still needs to gain access to intracellular endosomal compartments in which TLR9 resides. However, there is published evidence for surface expression of TLR9 on epithelial cells,11 and a previous report indicated CpG DNA uptake and TLR9 activation in LSECs.12 The exact mechanisms of TLR9 activation in nonphagocytic cells require further characterization.

Another unresolved question is which signals activate the NALP3 inflammasome during AAP-induced liver injury. Inflammasome activation is a tightly controlled process that is only beginning to be understood in greater detail.9 One well-established activator of the NALP3 inflammasome is adenosine triphosphate (ATP), which in combination with LPS leads to prominent IL-1β release from macrophages.13 It is conceivable that ATP released from necrotic hepatocytes could activate the NALP3 inflammasome. This hypothesis could be tested in mice lacking the purinergic receptor P2X7 because ATP-mediated NALP3 activation is dependent on the interaction of P2X7 with the hemichannel protein pannexin 1.14 Another possibility for inflammasome activation could be uric acid released from necrotic hepatocytes, which was previously shown to be responsible for sterile inflammation in gout.15

The authors have shown that direct delivery of TLR9 ligands to the liver induces tissue damage and up-regulation of pro-IL-1β and pro-IL-18. One major effect of TLR9 stimulation is the induction of type I IFN, which is important for antiviral and antitumor immune responses. To what extent type I IFN production contributes to the detrimental effects of TLR9 activation during AAP-induced liver injury remains to be investigated. Interestingly, IFN-β has been shown to enhance inflammasome activation and IL-1β production in macrophages.16 One further point that requires consideration is that although AAP-induced liver injury serves as an important model for studying sterile inflammation, the liver is exposed to a considerable amount of endotoxins derived from the intestine through the portal vein. Therefore, it remains to be investigated whether the mechanism described by Imaeda et al.7 can be transferred to other models of sterile inflammation.

Finally, it will be of special interest to further characterize the role of TLR-NLR cooperation during chronic inflammation and carcinogenesis. Notably, it has previously been shown that the TLR/IL-1R signaling adaptor myeloid differentiation protein 88 (MyD88) is required for the development of hepatocellular carcinoma in a sterile liver injury model in mice, whereas the ligands activating this pathway are still unknown.17 Perpetuation of chronic hepatitis due to either viral infection or alcoholic hepatotoxicity might also involve mechanisms similar to those depicted in the present study. Although inhibitors of IL-1β are now available for the treatment of chronic inflammatory conditions such as arthritis, these treatments are not suitable for the therapy of liver disorders, despite the demonstration of a pivotal role for IL-1β in several murine models of hepatitis and fibrosis. Moreover, intervening before the release of this important cytokine may be preferred; this could possibly yield a more targeted molecular therapy for inflammatory disorders of the liver. Therefore, it will be important to further investigate the mechanisms of immune responses to dying hepatocytes in order to gain deeper insight into the pathophysiology of acute and chronic liver injury and also open new horizons for therapeutic strategies.

References

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