High mobility group box protein 1 in complex with lipopolysaccharide or IL-1 promotes an increased inflammatory phenotype in synovial fibroblasts

Inflammation can be infectious and/or sterile depending on the initiating event. The proinflammatory mediator High mobility group box protein 1 (HMGB1) is a nuclear protein released from cells during both sterile and infectious inflammation and once extracellular, initates and potentiates inflammation by inducing cytokine production and by recruiting inflammatory cells. Autoimmune diseases are characterised by chronic sterile inflammation leading to tissue destruction. HMGB1 has been implicated in the pathogenesis of several autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosis, multiple sclerosis and myositis. The involvement of HMGB1 in arthritis has been shown by overexpression of HMGB1 in RA synovial tissue and synovial fluid, by beneficial outcome of therapeutic HMGB1-blockade in several experimental arthritis models and by the induction of arthritis by intra-articular injection of recombinant HMGB1 into mice. In this thesis work I set out to investigate the potential role of HMGB1 in juvenile idiopathic arthritis (JIA), to further delineate mechanisms by which HMGB1 can contribute to arthritis pathogenesis and to study the means by which HMGB1 activity can be suppressed. I could report for the first time that HMGB1 levels were increased in synovial fluid as compared to plasma during JIA. HMGB1 levels in synovial fluid did not correlate to disease duration. In contrast, the recorded levels of IL-8 and S100 proteins were higher in synovial fluid during early phases of disease. This indicates a change in the inflammatory phenotype during the progression of JIA. High HMGB1 levels in synovial fluid correlated with early JIA onset, suggesting differences in immunopathogenesis between patient groups. I have also demonstrated that HMGB1 may form complexes with the exogenous TLR ligand LPS or the endogenous inflammatory mediators IL-1α and IL-1β, respectively. Compared to each mediator alone such complexes stimulated synovial fibroblasts from arthritis patients to enhanced production of cytokines and tissue degrading enzymes. This enhancement is mediated via the reciprocal receptor for each HMGB1-partner molecule. Since all the studied mediators are present in arthritic joint during inflammation, this is a potential mechanism through which HMGB1 enhances ongoing inflammation and destruction during rheumatic diseases. Finally, I have demonstrated that the proinflammatory activity of HMGB1 can be therapeutically targeted, either by inhibiting its active release by clinically approved anti-rheumatic drugs or by neutralization with a HMGB1-specific monoclonal antibody. Extracellular secretion of HMGB1 from LPS+IFN-γ stimulated human primary monocytes was inhibited by dexamethasone, chloroquine and gold sodium thiomalate in vitro as recorded using an ELISPOT assay. Therapeutic administration of an HMGB1-specific HMGB1 monoclonal antibody ameliorated arthritis in two separate experimental models. In conclusion, my thesis work has added to the growing evidence that HMGB1 is involved in the pathogenesis of arthritis, has revealed a potential mechanism for its proinflammatory function and has demonstrated a means by which HMGB1-mediated activities can be counteracted

Antibodies against High Mobility Group Box protein-1 (HMGB1) versus other anti-nuclear antibody fine-specificities and disease activity in systemic lupus erythematosus

Introduction: The non-histone nuclear protein high mobility group box protein-1 (HMGB1) is typically associated with nucleosomes, but may shuttle between the nucleus and the cytoplasm, and under some conditions also be released extracellularly and participate in systemic inflammation. Monoclonal HMGB1-targeting antibodies can ameliorate murine polyarthritis and lupus-like disease. Interestingly, autoantibodies against HMGB1 have also been described in patients with systemic lupus erythematosus (SLE), but their clinical implications remain elusive. The main aims of this study were to detect serum anti-HMGB1 antibodies in patients with SLE and relate them to other types of antinuclear antibodies (ANA), and to disease activity. Methods: 188 Swedish SLE patients meeting the 1982 American College of Rheumatology classification criteria and/or the 2012 Systemic Lupus International Collaborating Clinics classification criteria participated in the study. Anti-HMGB1 antibody levels were analysed in patient and control (n = 112) sera by an in-house ELISA using recombinant histidine-tagged HMGB1. SLE sera were also analysed for ANA by immunofluorescence (IF) microscopy (IF-ANA) using fixed HEp-2 cells, and by a line-blot assay for antigen fine-specificities. To quantify antibodies to double-stranded DNA, a fluoroenzyme-immunoassay was employed. Results: At inclusion, 23 % of the SLE patients were anti-HMGB1 antibody positive compared to 5 % of the controls. Anti-HMGB1 antibodies occurred in 49 % of the IF-ANA positive SLE patients, and in 34 % of IF-ANA negative cases (p = 0.004). Levels of anti-HMGB1 antibodies correlated with anti-dsDNA antibody levels (r = 0.49; p less than 0.001). Significant, but less pronounced correlations were found regarding anti-HMGB1 and SLE disease activity index (SLEDAI-2K: r = 0.15; p = 0.04), classical complement function (r = -0.24; p = 0.002) and complement protein C4 (r = -0.23; p = 0.002). Average anti-HMGB1 antibody levels were significantly higher among patients with homogenous +/- other IF-ANA staining patterns (median 180 AU) compared to IF-ANA negative cases (median 83 AU) (p = 0.004). Rabbit anti-HMGB1 antibodies gave rise to cytoplasmic, but not nuclear, staining of HEp-2 cells. Conclusions: We confirm that anti-HMGB1 antibodies are common in SLE and correlate with disease activity variables. Although anti-HMGB1 antibodies measured by ELISA often coincide with nuclear IF-ANA staining, our results indicate that anti-HMGB1 antibodies do not give rise to nuclear staining of the predominantly used commercial HEp-2 cell slides.Funding Agencies|Swedish Society for Medical Research, Region Ostergotland; Swedish Research Council; Swedish Rheumatism Association; Swedish Society of Medicine; Professor Nanna Svartz foundation; King Gustaf Vs 80-year foundation</p

Digestibility index and factors affecting rate of starch digestion in vitro in conventional food preparation

The rate of starch hydrolysis in ten cereal-based food preparations was studied using an in vitro dialysis system. The foods were incubated with human saliva and porcine pancreatin, The sugars released after 3 h digestion were expressed as digestibility index (DI), the percentage starch digested was determined and correlated with the degree of gelatinization (DG). Granule morphology was also investigated and related with starch availability for hydrolysis. Significant differences were observed in the in vitro starch digestibility of the 10 foods (P < 0.05). The DI ranged from 53 for chapathi to 78 for rice flakes. DI was inversely related to the protein (r = -0.79, P < 0.01) fat (r = -0.63, P < 0.05) and energy (r = -0.61, P < 0.01), Percent starch digested was inversely related to the insoluble (r = -0.49, P < 0.05) and total dietary fiber (r = -0.63, P < 0.01) content of the foods. The SEM results provided a better understanding of granular morphology on cooking and the effect of protein on limiting DG. The results suggest that carbohydrate: foods of potential use in the therapeutic diets may be identified by their in vitro digestion characteristics

Additional file 1: of Antibodies against High Mobility Group Box protein-1 (HMGB1) versus other anti-nuclear antibody fine-specificities and disease activity in systemic lupus erythematosus

Figure shows anti-high mobility group box protein-1 ( HMGB1 ) antibody levels at highest and lowest disease activity. Highest and lowest disease activity was defined by peak systemic lupus erythematosus disease activity index (SLEDAI)-2K score and remission in the 18 patients selected for consecutive analysis. Dashed line indicates cut-off level for positive test. (TIF 150 kb

Monoclonal Anti-HMGB1 (High Mobility Group Box Chromosomal Protein 1) Antibody Protection in Two Experimental Arthritis Models

High mobility group box chromosomal protein 1 (HMGB1) is a DNA-binding nuclear protein that can be released from dying cells and activated myeloid cells. Extracellularly, HMGB1 promotes inflammation. Experimental studies demonstrate HMGB1 to be a pathogenic factor in many inflammatory conditions including arthritis. HMGB1-blocking therapies in arthritis models alleviate disease and confer significant protection against cartilage and bone destruction. So far, the most successful HMGB1-targeted therapies have been demonstrated with HMGB1-specific polyclonal antibodies and with recombinant A box protein, a fragment of HMGB1. The present study is the first to evaluate the potential of a monoclonal anti-HMGB1 antibody (2G7, mouse IgG2b) to ameliorate arthritis. Effects of repeated injections of this antibody have now been studied in two conceptually different models of arthritis: collagen type II–induced arthritis (CIA) in DBA/1 mice and in a spontaneous arthritis disease in mice with combined deficiencies for genes encoding for the enzyme DNase type II and interferon type I receptors. These mice are unable to degrade phagocytozed DNA in macrophages and develop chronic, destructive polyarthritis. Therapeutic intervention in CIA and prophylactic administration of anti-HMGB1 monoclonal antibody (mAb) in the spontaneous arthritis model significantly ameliorated the clinical courses. Anti-HMGB1 mAb therapy also partially prevented joint destruction, as demonstrated by histological examination. The beneficial antiarthritic effects by the anti-HMGB1 mAb in two diverse models of arthritis represent additional proof-of-concept, indicating that HMGB1 may be a valid target molecule to consider for development of future clinical therapy

Immunomodulatory Drugs Regulate HMGB1 Release from Activated Human Monocytes

Several HMGB1-specific antagonists have provided beneficial results in multiple models of inflammatory disease–preclinical trials including arthritis. Since no HMGB1-specific targeted therapy has yet reached the clinic, we have performed in vitro studies to investigate whether any of a selection of well-established antirheumatic drugs inhibit HMGB1 release as part of its mode of action. Freshly purified peripheral blood monocytes from healthy donors were stimulated in cultures with LPS and IFNγ to cause HMGB1 and TNF release detected in ELISPOT assays. Effects on the secretion were assessed in cultures supplemented with dexamethasone, cortisone, chloroquine, gold sodium thiomalate, methotrexate, colchicine, etanercept or anakinra. Pharmacologically relevant doses of dexamethasone, gold sodium thiomalate and chloroquine inhibited the extracellular release of HMGB1 in a dose-dependent mode. Immunostaining demonstrated that dexamethasone caused intracellular HMGB1 retention. No effects on HMGB1 secretion were observed in cultures with activated monocytes by any of the other studied agents. TNF production in LPS/IFNγ-activated monocytes was readily downregulated by dexamethasone and, to some extent, by chloroquine and etanercept. We conclude that dexamethasone, gold sodium thiomalate and chloroquine share a capacity to inhibit HMGB1 release from activated monocytes