Background and objectives High mobility group box protein 1 (HMGB1) is a prototypic alarmin being released from activated, stressed or dying cells. Extracellular HMGB1 has the ability to induce cell migration as well as cytokine production. Pathogenic effects of HMGB1 have been described in several inflammatory diseases, including arthritis, and HMGB1-specific blockade is beneficial in multiple experimental disease models. Recent reports have underlined the importance of post-translational modifications (PTMs) in determination of HMGB1 function and release mechanisms. We investigated the occurrence of PTMs of HMGB1 obtained from synovial fluid (SF) of juvenile idiopathic arthritis patients (JIA).
Materials and methods Synovial fluid was obtained from 17 JIA patients. Total Levels of HMGB1 were determined by ELISA. PTMs of HMGB1 were determined HMGB1-specific immunoprecipitation followed by Liquid chromatography tandem mass-spectrometry (LC-MS/MS).
Results Analyses of 17 JIA patients confirmed high HMGB1 levels in SF. Liquid chromatography tandem mass-spectrometry (LC-MS/MS) analyses of PTMs revealed that total HMGB1 levels did not associate with increased LDH activity but strongly correlated with nuclear location sequence 2 (NLS2) hyperacetylation, indicating active release of HMGB1. The correlation between total HMGB1 levels and NLS2 hypoacetylation suggests additional, acetylation-independent release mechanisms. Monomethylation of lysine 43 (K43), a proposed neutrophil-specific PTM, strongly associated with high HMGB1 levels, implying that neutrophils are a source of released HMGB1. Analysis of cysteine redox isoforms: fully reduced HMGB1, disulfide HMGB1 and oxidised HMGB1 revealed that HMGB1 acts as both a chemotactic and a cytokine-inducing mediator. These properties were associated with actively released HMGB1.
Conclusions This is the first report that characterises HMGB1-specific PTMs during a chronic inflammatory condition. HMGB1 in SF from JIA patients is actively released through both acetylation- and non-acetylation-dependent manners. The presence of various functional HMGB1 redox isoforms confirms the complexity of its pathogenic role during chronic inflammation. Defining HMGB1 release pathways and redox isoforms is critical for the understanding of the contribution of HMGB1 during inflammatory processes.