title: Mechanistic regulation of HMGB1 function in drug-induced liver injury
Creators: name: Lea, Jonathan
Publisher: name: University of Liverpool Repository
Year: 2015

description: Drug-induced liver injury (DILI) is a major cause of attrition during drug development, and also a leading cause of hospital admissions. In order to identify novel biomarkers that may improve the diagnosis and treatment of DILI, well characterised hepatotoxins such as acetaminophen (APAP) are used. High mobility group box 1 (HMGB1) protein is a ubiquitously expressed protein involved in the facilitation of gene transcription. However, extracellular HMGB1 acts as a key mediator of inflammation. During APAP-induced liver injury (AILI), HMGB1 is passively released from necrotic hepatocytes in a fully reduced, hypoacetylated isoform, or actively released from immune cells following acetylation of HMGB1 at key lysine residues within nuclear localisation sequences. Extracellular HMGB1 can bind to a range of receptors including TLR2, TLR4, TLR9, CXCR4 and RAGE to mediate cytokine-inducing or chemotactic effects and has been shown to be a more sensitive and predictive serum biomarker of AILI outcome than ALT. Additionally, anti-HMGB1 antibodies have been used to successfully attenuate injury in animal models of inflammatory conditions, including DILI. Mechanistic understanding of HMGB1 regulation and function is essential for this emerging biomarker of AILI. A murine monoclonal anti-HMGB1 antibody (2G7) has previously been used to attenuate AILI. A chimeric anti-HMGB1 monoclonal antibody (h2G7) was investigated, alongside 2G7 and a human isotype control antibody (E2) in an experimental model of AILI. C57BL/6 mice dosed with 530mg/kg APAP by intraperitoneal (i.p.) injection, followed by antibody or PBS treatment i.p. at 2hr post-APAP and euthanisation at 10hr post-APAP showed that 300µg h2G7 treatment afforded protection from AILI by significantly attenuating serum ALT, miR-122 and chemo/cytokine increases seen in the isotype control group. Liver histology and MPO revealed reduced neutrophil infiltration and activity in anti-HMGB1 treated animals. No significant differences were seen between 2G7 and h2G7 animals, and there was no effect on hepatic GSH in antibody-treated animals. Mechanism of action studies using Fc region modified forms of h2G7 ruled out complement-mediated and FcγR-mediated effects of h2G7 in the AILI model, as these did not significantly affect the h2G7 effect on AILI. Redox modulation of HMGB1 function had been proposed as a mechanism of regulation of proinflammatory activity of HMGB1. Investigations into the cytokine-inducing capability of different redox isoforms of HMGB1 in THP-1 and RAW264.7 cells demonstrated that HMGB1 needs to be in a Cys23-Cys45 disulphide conformation with a reduced thiol at Cys106 in order to induce cytokine release via NF-κB activation. During apoptosis, HMGB1 can be oxidised by activated caspases. Oxidation has previously been shown to regulate the structure of proteins and facilitate other post-translational modifications (PTMs). LC-MS/MS characterisation of HMGB1 from APAP overdose patients with high levels of apoptosis revealed ubiquitination at Lys30 (HMGB1-Ub). Ubiquitination was shown in vitro not to direct HMGB1 for proteasomal degradation, but HMGB1-Ub was detectable in apoptotic microparticles (MP), membrane vesicles that are shed from a cell during late apoptosis. In summary, HMGB1 was shown to be critical for the coordination of AILI, as antibody-mediated inhibition lead to a reduction in hepatic injury. Chimeric anti-HMGB1 antibody was effective at attenuating liver injury and advances anti-HMGB1 antibody treatment towards clinical utility. HMGB1 redox regulation of inflammatory signalling and the identification of HMGB1-Ub demonstrate the importance of PTMs in governing HMGB1 function. HMGB1 shows promise as an acute, sensitive and predictive biomarker of AILI in man. Through the use of in vitro and in vivo systems, the work presented in this thesis expands our current understanding of the mechanistic and functional roles of HMGB1 in DILI and has further defined mechanisms that can be used to inform the clinical situation.