Drug Induced Liver Injury (DILI) is associated with high morbidity and mortality rates. It is the leading cause of acute liver failure, accounting for 50% of all cases. Moreover, DILI is the most frequent cause of post marketing drug withdrawal and is often cited as a cause of compound attrition during the drug-development process. The High Mobility Group Box 1 (HMGB1) protein is an important inflammatory mediator which alters the immune system to tissue stress and injury. HMGB1 has been implicated in the pathogenesis of multiple inflammatory diseases including immune-mediated DILI. It has been identified as a potential biomarker of hepatic injury and a target for therapeutic intervention. Research is required to elucidate the pro-inflammatory role of HMGB1. HMGB1 has been reported to interact with a diverse range of endogenous (IL-1, DNA, nucleosomes, CXCL12) and exogenous (LPS) molecules to promote inflammation. However, the mechanisms responsible for these synergistic interactions remain poorly defined and therefore, the overall aim of this work was to characterise the interactions of HMGB1. Specifically, this work has focused on the interaction with IL-1β, which is of particular interest since both molecules often co-exist at the site of inflammation. The interaction between HMGB1 and IL-1β was investigated using combined cellular and Nuclear Magnetic Resonance (NMR) methodologies. LPS-free, isotopically labelled recombinant HMGB1 (full length protein, amino acids 1-215) and IL-1β proteins were expressed and purified from BL21 (DE3) cells. To facilitate these studies, three additional HMGB1 mutants were sub-cloned from the HMGB1 plasmid: Δ30 (1-185), A box (1-85) and B box (89-163). The recombinant proteins were characterised using Mass Spectrometry (MS) and NMR spectroscopy. Synovial fibroblasts were isolated from synovial tissue obtained from rheumatoid arthritis patients undergoing joint replacement surgery. Cells were treated with HMGB1 (full length, Δ30, A box or B box) alone or combination with IL-1β. Cell supernatants were collected after 24hr and IL-6 levels were quantified by ELISA. Untreated fibroblasts or cells treated with any HMGB1 construct, or IL-1β alone had no detectable IL-6 release (<9.375pg/mL). In contrast, full length HMGB1, the Δ30 and the B box domain (but not the A box domain) all acted in synergy with IL-1β to substantially enhance IL-6 production. In one patient, HMGB1, Δ30 and the B box in combination with IL-1β induced IL-6 levels of 28,473 ± 127pg/mL, 18,491 ± 2388pg/mL and 18,710 ± 2792pg/mL, respectively. The synergistic interaction was mediated via the Interleukin-1 receptor (IL-1R) and could be inhibited when the cells were pre-treated with 5µg/mL anakinra, a selective IL-1R antagonist but not detoxified LPS, a TLR4 receptor antagonist. To investigate if there is a direct interaction between HMGB1 and IL-1β, a comprehensive biophysical analysis was performed using NMR methodologies. However, despite performing the experiments in various ways, no evidence of a direct interaction between IL-1β and either full length HMGB1, Δ30 or the B box was detected. This suggests that the synergistic interaction between HMGB1 and IL-1β is mediated via an alternative cellular mechanism in which HMGB1 is required. In conclusion, the work presented in this thesis has identified that the B box domain of HMGB1 is critical for the synergistic effect observed with IL-1β. However, this is not due to the formation of a binary complex between HMGB1 and IL-1β. Instead, it would appear that the synergistic effect is mediated via an alternative cellular mechanism in which HMGB1 is required and additional proteins are involved. Future work could focus on discovering what these other proteins might be. These findings help to elucidate the pro-inflammatory role of HMGB1 and provide a novel an insight into HMGB1 biology.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:617514 |
| Date | January 2014 |
| Creators | Aucott, Hannah |
| Contributors | Williams, Dominic; Park, Kevin |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://livrepository.liverpool.ac.uk/16877/ |
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