Therapeutic administration of etoposide coincides with reduced systemic HMGB1 levels in macrophage activation syndrome.

BACKGROUND: Macrophage activation syndrome (MAS) is a potentially fatal complication of systemic inflammation. HMGB1 is a nuclear protein released extracellularly during proinflammatory lytic cell death or secreted by activated macrophages, NK cells, and additional cell types during infection or sterile injury. Extracellular HMGB1 orchestrates central events in inflammation as a prototype alarmin. TLR4 and the receptor for advanced glycation end products operate as key HMGB1 receptors to mediate inflammation. METHODS: Standard ELISA and cytometric bead array-based methods were used to examine the kinetic pattern for systemic release of HMGB1, ferritin, IL-18, IFN-γ, and MCP-1 before and during treatment of four children with critical MAS. Three of the patients with severe underlying systemic rheumatic diseases were treated with biologics including tocilizumab or anakinra when MAS developed. All patients required intensive care therapy due to life-threatening illness. Add-on etoposide therapy was administered due to insufficient clinical response with standard treatment. Etoposide promotes apoptotic rather than proinflammatory lytic cell death, conceivably ameliorating subsequent systemic inflammation. RESULTS: This therapeutic intervention brought disease control coinciding with a decline of the increased systemic HMGB1, IFN-γ, IL-18, and ferritin levels whereas MCP-1 levels evolved independently. CONCLUSION: Systemic HMGB1 levels in MAS have not been reported before. Our results suggest that the molecule is not merely a biomarker of inflammation, but most likely also contributes to the pathogenesis of MAS. These observations encourage further studies of HMGB1 antagonists. They also advocate therapeutic etoposide administration in severe MAS and provide a possible biological explanation for its mode of action.

High mobility group box protein 1-A prognostic marker for structural joint damage in 10-year follow-up of patients with juvenile idiopathic arthritis.

OBJECTIVE: High mobility group box protein 1 (HMGB1) is an important pro-inflammatory mediator in adult rheumatoid arthritis. The diagnostic utility of HMGB1 in Juvenile Idiopathic Arthritis (JIA) is still unclear. The aim was to examine whether serum HMGB1 levels are associated with inflammation, radiological disease progression, and long-term prognosis in JIA. METHODS: We included 131 children with JIA from a population-based prevalence study; 38 of them were prospectively followed up for 10 years. Clinical and laboratory disease characteristics at study entry and after 10 years as well as radiological progression over 10 years were recorded. HMGB1 levels were analyzed by an ELISA. RESULTS: The HMGB1 levels were similar in children with different JIA subgroups and in children with established (53%) or newly diagnosed (47%) disease. HMGB1 levels did not differ between groups at entry into the study or at 10 years, by sex, or by the presence or absence of RF or ANA antibodies. HMGB1 levels at the study entry correlated with HMGB1 levels at 10 years and with blood neutrophil count. Most importantly, children with destructive arthritis at 10 years had a tendency toward higher HMGB1 levels at study entry (median 1.2 vs 0.6ng/ml, ns) and displayed 4-fold higher circulating HMGB1 levels (median 3.4 vs 0.8ng/ml, p = 0.0014) than children without radiological destructions. CONCLUSIONS: Our results suggest that HMGB1 is a marker of inflammatory activity in children with JIA. Higher serum HMGB1 levels are related to more destructive JIA and could be used as a negative prognostic marker at the disease start. TRIAL REGISTRATION: Clinicaltrials.gov NCT01905319. Registered July 16, 2013.

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 < 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.

High systemic levels of the cytokine-inducing HMGB1 isoform secreted in severe macrophage activation syndrome.

Macrophage activation syndrome (MAS) is a potentially fatal complication of systemic inflammation. High mobility group box 1 (HMGB1) is a nuclear protein extensively leaked extracellularly during necrotic cell death or actively secreted by natural killer (NK) cells, macrophages and additional cells during infection or sterile injury. Extracellular HMGB1 orchestrates key events in inflammation as a prototypic alarmin. The redox states of its three cysteines render the molecule mutually exclusive functions: fully reduced "all-thiol HMGB1" exerts chemotactic activity; "disulfide HMGB1" has cytokine-inducing, toll-like receptor 4 (TLR4)-mediated effects­while terminally oxidized "sulfonyl HMGB1" lacks inflammatory activity. This study examines the kinetic pattern of systemic HMGB1 isoform expression during therapy in four children with severe MAS. Three of the four patients with underlying systemic rheumatic diseases were treated with biologics and two suffered from triggering herpes virus infections at the onset of MAS. All patients required intensive care unit therapy due to life-threatening illness. Tandem mass-spectrometric analysis revealed dramatically increased systemic levels of the cytokine-inducing HMGB1 isoform during early MAS. Disease control coincided with supplementary etoposide therapy initiated to boost apoptotic cell death, when systemic HMGB1 levels drastically declined and the molecule emerged mainly in its oxidized, noninflammatory isoform. Systemic interferon (IFN)-γ and ferritin peaked concomitantly with HMGB1, whereas interleukin (IL)-18 and monocyte chemotactic protein (MCP)-1 levels developed differently. In conclusion, this work provides new insights in HMGB1 biology, suggesting that the molecule is not merely a biomarker of inflammation, but most likely also contributes to the pathogenesis of MAS. These observations encourage further studies of disulfide HMGB1 antagonists to improve outcome of MAS.

JAK/STAT1 signaling promotes HMGB1 hyperacetylation and nuclear translocation.

Extracellular high-mobility group box (HMGB)1 mediates inflammation during sterile and infectious injury and contributes importantly to disease pathogenesis. The first critical step in the release of HMGB1 from activated immune cells is mobilization from the nucleus to the cytoplasm, a process dependent upon hyperacetylation within two HMGB1 nuclear localization sequence (NLS) sites. The inflammasomes mediate the release of cytoplasmic HMGB1 in activated immune cells, but the mechanism of HMGB1 translocation from nucleus to cytoplasm was previously unknown. Here, we show that pharmacological inhibition of JAK/STAT1 inhibits LPS-induced HMGB1 nuclear translocation. Conversely, activation of JAK/STAT1 by type 1 interferon (IFN) stimulation induces HMGB1 translocation from nucleus to cytoplasm. Mass spectrometric analysis unequivocally revealed that pharmacological inhibition of the JAK/STAT1 pathway or genetic deletion of STAT1 abrogated LPS- or type 1 IFN-induced HMGB1 acetylation within the NLS sites. Together, these results identify a critical role of the JAK/STAT1 pathway in mediating HMGB1 cytoplasmic accumulation for subsequent release, suggesting that the JAK/STAT1 pathway is a potential drug target for inhibiting HMGB1 release.

HMGB1 levels are increased in patients with juvenile idiopathic arthritis, correlate with early onset of disease, and are independent of disease duration.

OBJECTIVE: High mobility group box chromosomal protein 1 (HMGB1) has been implicated as a mediator of inflammation in rheumatoid arthritis (RA), while its role in juvenile idiopathic arthritis (JIA) has not been described. To evaluate the role of HMGB1 in the inflammatory process in JIA and its potential as a therapeutic target, we investigated whether extracellular HMGB1 is detectable in JIA and if so, to correlate the levels with established inflammatory markers and clinical measures. METHODS: Matching samples of blood and synovial fluid (SF) were collected from 23 patients with JIA. Levels of HMGB1, soluble receptor for advanced glycation endproducts, S100A12, myeloid-related protein 8/14, and other inflammatory mediators were analyzed. RESULTS: Significantly increased HMGB1 levels were recorded in SF compared to blood samples from patients with JIA. The amount of HMGB1 was highest in patients with early disease onset irrespective of disease duration. In contrast, the proinflammatory S100 protein and interleukin 8 were highest in patients in early phases of disease. Matrix metalloproteinase-3, a marker of cartilage destruction, was higher in patients with late disease onset, indicating similarities with RA in that patient subgroup. CONCLUSION: Levels of extracellular HMGB1 are increased in the inflamed joints of patients with JIA. This warrants further studies of HMGB1 as a mediator of JIA pathogenesis as well as a biomarker for inflammatory activity and as a target for therapy. The variation in levels of HMGB1 and S100 proteins in relation to disease onset indicates a difference in inflammatory phenotype during disease progression.

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

INTRODUCTION: In addition to its direct proinflammatory activity, extracellular high mobility group box protein 1 (HMGB1) can strongly enhance the cytokine response evoked by other proinflammatory molecules, such as lipopolysaccharide (LPS), CpG-DNA and IL-1ß, through the formation of complexes. Extracellular HMGB1 is abundant in arthritic joint tissue where it is suggested to promote inflammation as intra-articular injections of HMGB1 induce synovitis in mice and HMGB1 neutralizing therapy suppresses development of experimental arthritis. The aim of this study was to determine whether HMGB1 in complex with LPS, interleukin (IL)-1α or IL-1ß has enhancing effects on the production of proinflammatory mediators by rheumatoid arthritis synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF). Furthermore, we examined the toll-like receptor (TLR) 4 and IL-1RI requirement for the cytokine-enhancing effects of the investigated HMGB1-ligand complexes. METHODS: Synovial fibroblasts obtained from rheumatoid arthritis (RA) and osteoarthritis (OA) patients were stimulated with HMGB1 alone or in complex with LPS, IL-1α or IL-1ß. Tumour necrosis factor (TNF) production was determined by enzyme-linked immunospot assay (ELISPOT) assessment. Levels of IL-10, IL-1-ß, IL-6 and IL-8 were measured using Cytokine Bead Array and matrix metalloproteinase (MMP) 3 production was determined by ELISA. RESULTS: Stimulation with HMGB1 in complex with LPS, IL-1α or IL-1ß enhanced production of TNF, IL-6 and IL-8. HMGB1 in complex with IL-1ß increased MMP production from both RASF and OASF. The cytokine production was inhibited by specific receptor blockade using detoxified LPS or IL-1 receptor antagonist, indicating that the synergistic effects were mediated through the partner ligand-reciprocal receptors TLR4 and IL-1RI, respectively. CONCLUSIONS: HMGB1 in complex with LPS, IL-1α or IL-1ß boosted proinflammatory cytokine- and MMP production in synovial fibroblasts from RA and OA patients. A mechanism for the pathogenic role of HMGB1 in arthritis could thus be through enhancement of inflammatory and destructive mechanisms induced by other proinflammatory mediators present in the arthritic joint.

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.

The alarmin HMGB1 acts in synergy with endogenous and exogenous danger signals to promote inflammation.

The nuclear protein HMGB1 has previously been demonstrated to act as an alarmin and to promote inflammation upon extracellular release, yet its mode of action is still not well defined. Access to highly purified HMGB1 preparations from prokaryotic and eukaryotic sources enabled studies of activation of human PBMC or synovial fibroblast cultures in response to HMGB1 alone or after binding to cofactors. HMGB1 on its own could not induce detectable IL-6 production. However, strong enhancing effects on induction of proinflammatory cytokine production occurred when the protein associated with each of the separate proinflammatory molecules, rhIL-1beta, the TLR4 ligand LPS, the TLR9 ligand CpG-ODN, or the TLR1-TLR2 ligand Pam3CSK4. The bioactivities were recorded in cocultures with preformed HMGB1 complexes but not after sequential or simultaneous addition of HMGB1 and the individual ligands. Individual A-box and B-box domains of HMGB1 had the ability to bind LPS and enhance IL-6 production. Heat denaturation of HMGB1 eliminated this enhancement. Cocultures with HMGB1 and other proinflammatory molecules such as TNF, RANKL, or IL-18 did not induce enhancement. HMGB1 thus acts broadly with many but not all immunostimulatory molecules to amplify their activity in a synergistic manner.

Pivotal advance: inhibition of HMGB1 nuclear translocation as a mechanism for the anti-rheumatic effects of gold sodium thiomalate.

Gold compounds such as gold sodium thiomalate (GST) can reduce the symptoms of rheumatoid arthritis (RA), although their mechanism of action is not well defined. As the proinflammatory mediator high mobility group box chromosomal protein 1 (HMGB1) may play a role in the pathogenesis of RA, we have performed in vitro studies to investigate whether GST inhibits HMGB1 release as the basis of its mode of action. Murine RAW 264.7 or human THP-1 macrophage cells were stimulated in culture with agents causing extracellular HMGB1 release, including LPS, IFN-gamma, polyinosinic:polycytidylic acid, IFN-beta, or NO in the presence of GST, ranging from 0 microM to 250 microM. Secretion and intracellular location of HMGB1 were assessed by Western blotting, HMGB1-specific ELISPOT assay, and immunofluorescent staining. In parallel, TNF and IFN-beta levels were analyzed by ELISPOT and/or ELISA. Supernatant NO production was analyzed by the Griess method. At pharmacologically relevant doses, GST inhibited the extracellular release of HMGB1 from activated macrophages and caused the nuclear retention of this protein; in contrast, no effects were observed on the secretion or production of TNF. Release of the key endogenous mediators of HMGB1 translocation, IFN-beta and NO, was inhibited by GST. This inhibition required gold, as sodium thiomalate did not affect the responses measured. Furthermore, gold chloride also inhibited release of HMGB1. Together, these results suggest a new mechanism for the anti-rheumatic effects of gold salts in RA and the potential of drugs, which interfere with intracellular HMGB1 transport mechanisms, as novel agents to treat RA.