HMGB1 : regulation of inflammatory functions and therapeutic blockade

Abstract

The intracellular protein High Mobility Group Box Protein 1 (HMGB1) has been identified as a pivotal mediator of inflammation. HMGB1 can be released by various mechanisms and as an inflammatory mediator it induces both migration of inflammatory cells and cytokine production. Consequently, HMGB1 has been demonstrated to contribute to pathology in several inflammatory conditions. Increasing evidence indicate that HMGB1 post-translational modifications (PTMs) regulate both secretion and function of HMGB1.

The focus of this thesis work has been to investigate how selected PTMs regulate HMGB1 function and release and to define the presence of such modifications on HMGB1 in synovial fluid from patients with juvenile idiopathic arthritis (JIA). Furthermore, I have set the basis for HMGB1-blockade as a clinical treatment option by generating and characterizing the first known chimeric, humanized anti-HMGB1 antibody.

To examine the impact of redox-dependent PTMs on HMGB1 function, we first generated several cysteine redox isoforms of HMGB1. We found that all cysteines residues (C23, C45 and C106) required a defined redox state. A disulfide bridge between C23 and C45 with a concomitant C106 thiol was necessary for HMGB1 mediated cytokine-induction. In this disulfide redox isoform, HMGB1 activates TLR4. Furthermore, I have studied PTMs and their impact on HMGB1 secretion. We demonstrated that NLRC4 inflammasome activation induces hyperacetylation of key lysine stretches known to be associated with HMGB1 secretion, independently of priming signals. Addition of a priming signal induced reactive oxygen species (ROS) that stimulated a structural transition of HMGB1 to its cytokine-inducing, disulfide form. Hyperacetylated HMGB1 correlated significantly with inflammatory HMGB1 redox isoforms in joint fluid from JIA patients, indicating that HMGB1 is actively secreted during JIA and possesses inflammatory properties.

In addition, I recorded beneficial effects of mouse monoclonal anti-HMGB1 antibody (m2G7) treatment in experimental arthritis and in acetaminopheninduced liver injury. Importantly, I could demonstrate that a partly humanized version of the antibody (h2G7) retained its in vitro properties and in vivo therapeutic effects.

In conclusion, this thesis has significantly increased the understanding of the regulation of HMGB1 secretion and function during inflammation. The generation of an anti-HMGB1 chimeric antibody is an important step in development of a clinical anti-HMGB1 treatment.