Acetaminophen hepatotoxicity and repair: the role of sterile inflammation and innate immunity

Abstract

Acetaminophen (APAP) hepatotoxicity because of overdose is the most frequent cause of acute liver failure in the western world. Metabolic activation of APAP and protein adduct formation, mitochondrial dysfunction, oxidant stress, peroxynitrite formation and nuclear DNA fragmentation are critical intracellular events in hepatocytes. However, the early cell necrosis causes the release of a number of mediators such as high-mobility group box 1 protein, DNA fragments, heat shock proteins (HSPs) and others (collectively named damage-associated molecular patterns), which can be recognized by toll-like receptors on macrophages, and leads to their activation with cytokine and chemokine formation. Although pro-inflammatory mediators recruit inflammatory cells (neutrophils, monocytes) into the liver, neither the infiltrating cells nor the activated resident macrophages cause any direct cytotoxicity. In contrast, pro- and anti-inflammatory cytokines and chemokines can directly promote intracellular injury mechanisms by inducing nitric oxide synthase or inhibit cell death mechanisms by the expression of acute-phase proteins (HSPs, heme oxygenase-1) and promote hepatocyte proliferation. In addition, the newly recruited macrophages (M2) and potentially neutrophils are involved in the removal of necrotic cell debris in preparation for tissue repair and resolution of the inflammatory response. Thus, as discussed in detail in this review, the preponderance of experimental evidence suggests that the extensive sterile inflammatory response during APAP hepatotoxicity is predominantly beneficial by limiting the formation and the impact of pro-inflammatory mediators and by promoting tissue repair.

Fig. 1

Phases of acetaminophen (APAP)-induced hepatotoxicity. APAP overdose results in metabolic activation of the drug to the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which depletes glutathione and forms APAP protein adducts triggering the initiation of the injury process with mitochondrial oxidative and nitrosative stress and compromised respiratory function. In the subsequent amplification phase, these mitochondrial events result in activation of mediators such as c-jun-N-terminal kinase (JNK), followed by initiation of the mitochondrial permeability transition with resultant translocation of mitochondrial proteins such as apoptosis-inducing factor and endonuclease G to the nucleus, producing DNA fragmentation and necrotic cell death. A number of cellular components released during necrosis including nuclear DNA fragments, formyl peptides and HMGB1 can act as damage-associated molecular patterns (DAMPs) to activate resident liver macrophages (Kupffer cells). In the regeneration and repair phase, chemokine and cytokine secretion because of Kupffer cell activation results in homing and transmigration of neutrophils and macrophages into the damaged tissue to facilitate removal of dead cells and activate regenerative pathways.

Fig. 2

Nuclear DNA damage and release of DNA fragments into plasma. DNA damage was assessed after 300 mg/kg acetaminophen (APAP) in mice using the TUNEL assay (indicates DNA strand breaks), DNA fragments in an agarose gel (DNA Ladder) (represents mono- and polyoligonucleosomes generated by endonucleases) and the DNA fragmentation assay (measures oligonucleosomes in cytosol or plasma based on detection of histone proteins). The data indicate early DNA strandbreaks (TUNEL assay at 3 h) and nuclear DNA degradation at 6 h (DNA ladder, DNA fragmentation assay). DNA ladder caused by APAP is indistinguishable from apoptosis-mediated DNA fragmentation after galactosamine/LPS. At 6 h after APAP treatment, a substantial amount of DNA fragments is released into the plasma. Data adapted from Cover et al. (30) (reproduced with permission).

Fig. 3

Priming of liver accumulated neutrophils for reactive oxygen formation. Mice were treated with saline, 100 µg endotoxin/kg for 90 min or 300 mg APAP/kg for 6 h. Hepatic nonparenchymal cells were isolated and then stimulated ex vivo with phorbol ester (PMA). Upon PMA-induced ROS production DHR-123 is converted to R-123 and quantified in neutrophils by flow cytometry. Representative ROS histograms or mean fluorescence intensities for saline, the positive control endotoxin and APAP are shown. *P < 0.05 (compared with saline control). (Figure reproduced from Williams et al. (86) with permission.)