Role of the inflammasome in acetaminophen-induced liver injury and acute liver failure

Abstract

Drug-induced acute liver failure carries a high morbidity and mortality rate. Acetaminophen overdose is the number one cause of acute liver failure and remains a major problem in Western medicine. Administration of N-acetyl cysteine is an effective antidote when given before the initial rise in toxicity; however, many patients present to the hospital after this stage occurs. As such, treatments which can alleviate late-stage acetaminophen-induced acute liver failure are imperative. While the initial mechanisms of toxicity are well described, a debate has recently occurred in the literature over whether there is a second phase of injury, mediated by inflammatory processes. Critical to this potential inflammatory process is the activation of caspase-1 and interleukin-1β by a molecular complex known as the inflammasome. Several different stimuli for the formation of multiple different inflammasome complexes have been identified. Formation of the NACHT, leucine-rich repeat (LRR) and pyrin (PYD) domains-containing protein 3 (Nalp3) inflammasome in particular, has directly been attributed to late-stage acetaminophen toxicity. In this review, we will discuss the mechanisms of acetaminophen-induced liver injury in mice and man with a particular focus on the role of inflammation and the inflammasome.

Keywords: Acetaminophen; Hepatotoxicity; Inflammasome, neutrophil; Monocyte; Sterile inflammation; Toll-like receptor.

Figure 1

Proposed mechanism of inflammasome activation by ATP:P2XR7 interaction in macrophages. Elevated ATP levels in serum released from dying cells activates P2XR7 causing pannexin-1 pore opening and potassium release. In addition, activation of P2XR7 causes activation of the protein Nek-7, which has a currently undefined function, but leads to formation of the Nalp3 inflammasome with activation of pro-caspase-1. Stimulation of toll like receptors, e.g. TLR4, by substrates such as high mobility group box 1 (HMGB1) protein causes NF-κB activation and transcriptional induction of pro-IL-1β formation. The active caspase-1 cleaves pro-IL-1β and the mature cytokine is being released. Activation of pro-inflammatory caspases, either directly by LPS in the case of caspase-4/5 or caspase-11, or through the inflammasome in the case of caspase-1, results in cleavage of Gasdermin D into the N-Terminal cleaved form of Gasdermin D. The N-terminal form mediates cell death via perforation of the plasma membrane after binding plasma membrane components such as phosphatidyl inositol or cardiolipin. Pore formation results in cellular collapse in cells undergoing pyroptosis and passive release of constituents such as IL-1ß. P2XR7 – purinergic receptor P2X 7, Nek-7 – NIMA-related kinase 7, Nalp3 – NACHT, LRR and PYD domains-containing protein 3, ASC - Apoptosis-associated speck-like protein containing a CARD, IL-1ß – interleukin-1ß, Gas D – Gasdermin D, Gas D-NT – Gasdermin D N-Terminal cleavage product. PIP – phosphatidylinositol phosphate

Figure 2

Central role of mitochondrial dysfunction in the intracellular signaling mechanisms of acetaminophen (APAP)-induced cell death. APAP is converted to NAQPI mainly by cytochrome P450 2E1. NAPQI binds to proteins, including mitochondrial proteins, causing a mitochondrial oxidant stress, which leads to activation of a mitogen-activated protein kinase cascade ultimately causing JNK phosphorylation. The translocation of p-JNK to mitochondria triggers an amplification of the oxidant stress resulting in the opening of the mitochondrial permeability transition (MPT) pores with loss of the membrane potential and declining ATP production. The MPT triggers matrix swelling with rupture of the outer member and release of intermembrane proteins including apoptosis-inducing factor (AIF) and endonuclease G, which translocate to the nucleus and cause nuclear DNA fragmentation. The injury process can be attenuated by treatment with N-acetylcysteine (NAC), which stimulates GSH synthesis. GSH can scavenge NAPQI, and reactive oxygen species and peroxynitrite in mitochondria. Figure adapted from [48]. ASK-1, apoptosis signal-regulating kinase 1; Mkp-1, mitogen-activated protein kinase phosphatase; MLK3, Mixed-lineage protein kinase 3; NAPQI, N-acetyl-p-benzoquinone imine; JNK, c-Jun N-terminal kinase; ROS, reactive oxygen species.

Figure 3

Proposed schematic of APAP-induced inflammatory liver injury. DAMPs released from hepatocytes during the first phase of necrosis, including ATP, HMGB1, and mtDNA, cause transcriptional activation of cytokine formation and activation of the inflammasome in resident macrophages such as Kupffer cells. This causes formation and release of cytokines including IL-1ß, which then amplify the immune signal by activating and recruiting of neutrophils thereby causing more liver damage through release of cytotoxic reactive oxygen species or proteases. This is the second (inflammatory) phase of necrotic cell death mediated by macrophages and neutrophils. DAMP, damage associated molecular pattern; mtDNA, mitochondrial DNA; IL-1ß, interleukin-1ß; Casp 1, caspase-1; IL-1R, interleukin-1 receptor; P2XR7, purinergic receptor P2X 7; TLR, toll-like receptor.

Figure 4

Plasma IL-1ß levels and alanine aminotransferase (ALT) activities in patients with acetaminophen-induced acute liver injury. IL-1ß levels were measured as part of a multiplex cytokine array and ALT activities were determined with a kinetic enzyme assay in healthy volunteers (HV), patients with acetaminophen overdose but no major increase in serum transaminases (NLT), and in patients with severe APAP-induced liver injury. The data show the very low levels of IL-1β in APAP overdose patients with severe liver injury suggesting limited inflammasome activation in patients. Data represent means ± SE of n = 8-10 patients per group. *P<0.05 versus HV. Data adapted from [74].