Lena Klevenvall

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‐1β, 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.

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.

High mobility group box protein 1 (HMGB1)-partner molecule complexes enhance cytokine production by signaling through the partner molecule receptor.

The nuclear protein high mobility group box protein 1 (HMGB1) promotes inflammation upon extracellular release. HMGB1 induces proinflammatory cytokine production in macrophages via Toll-like receptor (TLR)-4 signaling in a redox-dependent fashion. Independent of its redox state and endogenous cytokine-inducing ability, HMGB1 can form highly immunostimulatory complexes by interaction with certain proinflammatory mediators. Such complexes have the ability to enhance the induced immune response up to 100-fold, compared with induction by the ligand alone. To clarify the mechanisms for these strong synergistic effects, we studied receptor requirements. Interleukin (IL)-6 production was assessed in supernatants from cultured peritoneal macrophages from mice each deficient in one of the HMGB1 receptors (receptor for advanced glycation end products [RAGE], TLR2 or TLR4) or from wild-type controls. The cultures were stimulated with the TLR4 ligand lipopolysaccaride (LPS), the TLR2 ligand Pam3CysSerLys4 (Pam3CSK4), noninflammatory HMGB1 or each TLR ligand in complex with noninflammatory HMGB1. The activity of the HMGB1-TLR ligand complexes relied on engagement of the same receptor as for the noncomplexed TLR ligand, since HMGB1-LPS complexes used TLR4 and HMGB1-Pam3CSK4 complexes used TLR2. Deletion of any of the intracellular adaptor molecules used by TLR2 (myeloid differentiation factor-88 (MyD88), TIR domain-containing adaptor protein (TIRAP)) or TLR4 (MyD88, TIRAP, TIR domain-containing adaptor-inducing interferon-β (TRIF), TRIF-related adaptor molecule (TRAM)) had similar effects on HMGB1 complex activation compared with noncomplexed LPS or Pam3CSK4. This result implies that the enhancing effects of HMGB1-partner molecule complexes are not regulated by the induction of additional signaling cascades. Elucidating HMGB1 receptor usage in processes where HMGB1 acts alone or in complex with other molecules is essential for the understanding of basic HMGB1 biology and for designing HMGB1-targeted therapies.

Protective targeting of high mobility group box chromosomal protein 1 in a spontaneous arthritis model

OBJECTIVE High mobility group box chromosomal protein 1 (HMGB-1) is a DNA binding nuclear protein that can be released from dying cells and activated myeloid cells. Extracellularly, HMGB-1 promotes inflammation. Clinical and experimental studies demonstrate that HMGB-1 is a pathogenic factor in chronic arthritis. Mice with combined gene deficiency for DNase II and IFNRI spontaneously develop chronic, destructive polyarthritis with many features shared with rheumatoid arthritis. DNase II is needed for macrophage degradation of engulfed DNA. The aim of this study was to evaluate a potential pathogenic role of HMGB-1 in this novel murine model. METHODS The course of arthritis, assessed by clinical scoring and histology, was studied in DNase II(-/-) × IFNRI(-/-) mice, in comparison with heterozygous and wild-type mice. Synovial HMGB-1 expression was analyzed by immunohistochemistry. Serum levels of HMGB-1 were determined by Western immunoblotting and enzyme-linked immunosorbent assay (ELISA), and anti-HMGB-1 autoantibodies were detected by ELISA. Macrophage activation was studied by immunostaining for intracellular interleukin-1β and HMGB-1. HMGB-1 was targeted with truncated HMGB-1-derived BoxA protein, acting as a competitive antagonist, with intraperitoneal injections every second day for 5 weeks. RESULTS DNase II(-/-) × IFNRI(-/-) mice developed symmetric polyarthritis with strong aberrant cytosolic and extracellular HMGB-1 expression in synovial tissue, in contrast to that observed in control animals. Increased serum levels of HMGB-1 and HMGB-1 autoantibodies were recorded in DNase II(-/-) × IFNRI(-/-) mice, both prior to and during the establishment of disease. Systemic HMGB-1-specific blockade significantly ameliorated the clinical disease course, and a protective effect on joint destruction was demonstrated by histologic evaluation. CONCLUSION HMGB-1 is involved in the pathogenesis of this spontaneous polyarthritis, and intervention with an HMGB-1 antagonist can mediate beneficial effects.

A novel high mobility group box 1 neutralizing chimeric antibody attenuates drug-induced liver injury and postinjury inflammation in mice

Acetaminophen (APAP) overdoses are of major clinical concern. Growing evidence underlines a pathogenic contribution of sterile postinjury inflammation in APAP‐induced acute liver injury (APAP‐ALI) and justifies development of anti‐inflammatory therapies with therapeutic efficacy beyond the therapeutic window of the only current treatment option, N‐acetylcysteine (NAC). The inflammatory mediator, high mobility group box 1 (HMGB1), is a key regulator of a range of liver injury conditions and is elevated in clinical and preclinical APAP‐ALI. The anti‐HMGB1 antibody (m2G7) is therapeutically beneficial in multiple inflammatory conditions, and anti‐HMGB1 polyclonal antibody treatment improves survival in a model of APAP‐ALI. Herein, we developed and investigated the therapeutic efficacy of a partly humanized anti‐HMGB1 monoclonal antibody (mAb; h2G7) and identified its mechanism of action in preclinical APAP‐ALI. The mouse anti‐HMGB1 mAb (m2G7) was partly humanized (h2G7) by merging variable domains of m2G7 with human antibody‐Fc backbones. Effector function‐deficient variants of h2G7 were assessed in comparison with h2G7 in vitro and in preclinical APAP‐ALI. h2G7 retained identical antigen specificity and comparable affinity as m2G7. 2G7 treatments significantly attenuated APAP‐induced serum elevations of alanine aminotransferase and microRNA‐122 and completely abrogated markers of APAP‐induced inflammation (tumor necrosis factor, monocyte chemoattractant protein 1, and chemokine [C‐X‐C motif] ligand 1) with prolonged therapeutic efficacy as compared to NAC. Removal of complement and/or Fc receptor binding did not affect h2G7 efficacy. Conclusion: This is the first report describing the generation of a partly humanized HMGB1‐neutralizing antibody with validated therapeutic efficacy and with a prolonged therapeutic window, as compared to NAC, in APAP‐ALI. The therapeutic effect was mediated by HMGB1 neutralization and attenuation of postinjury inflammation. These results represent important progress toward clinical implementation of HMGB1‐specific therapy as a means to treat APAP‐ALI and other inflammatory conditions. (Hepatology 2016;64:1699‐1710).

The synthetic retinoid RO 13-6307 induces neuroblastoma differentiation in vitro and inhibits neuroblastoma tumour growth in vivo

Retinoids modulate cell proliferation, differentiation and apoptosis in a variety of tumour cells including leukaemia and neuroblastoma, a childhood tumour of the sympathetic nervous system. 13‐cis retinoic acid is in clinical use against minimal residual disease in neuroblastoma, where the effect seems to depend on dose, scheduling and tumour mass. Novel retinoids are searched for, to improve potency and lower toxicity. We investigated the effect of the synthetic retinoid Ro 13‐6307 on neuroblastoma growth in vitro on SK‐N‐BE(2) and SH‐SY5Y cells. Furthermore, effects on tumour growth and the toxicity profile were investigated in a rat xenograft model. Effects of Ro 13‐6307 were compared to 13‐cis RA (retinoic acid) in vitro and in vivo. Neuroblastoma cells treated with 1 μM Ro 13‐6307 exhibited neuronal differentiation, decreased proliferation and accumulation of cells in G1 phase in at least the same magnitude as 5 μM 13‐cis RA. No apoptosis was detected in vitro. Treatment of nude rats with neuroblastoma using Ro 13‐6307, 0.12 mg p.o. daily, decreased neuroblastoma growth in vivo, in terms of tumour volume during treatment and tumour weight at sacrifice (p < 0.05). In contrast, Ro 13‐6307, 0.08 mg p.o. daily, resulted in no significant reduction in tumour growth. All rats treated with Ro 13‐6307 gained less weight than control rats, but they exhibited no other signs of toxicity. The toxicity profile of Ro 13‐6307 was similar to what we found with 13‐cis RA. Our preclinical results suggest that Ro 13‐6307 may be a candidate retinoid for clinical oral therapy of neuroblastoma in children.

Bioavailability and dose-dependent anti-tumour effects of 9-cis retinoic acid on human neuroblastoma xenografts in rat.

Neuroblastoma, the most common extracranial solid tumour in children, may undergo spontaneous differentiation or regression, but the majority of metastatic neuroblastomas have poor prognosis despite intensive treatment. Retinoic acid regulates growth and differentiation of neuroblastoma cells in vitro, and has shown activity against human neuroblastomas in vivo. The retinoid 9-cis RA has been reported to induce apoptosis in vitro, and to inhibit the growth of human neuroblastoma xenografts in vivo. However, at given dosage, the treatment with 9-cis RA caused significant toxic side effects. In the present study we investigated the bioavailability of 9-cis RA in rat. In addition, we compared two different dose schedules using 9-cis RA. We found that a lower dose of 9-cis RA (2 mg day−1) was non-toxic, but showed no significant effect on tumour growth. The bioavailability of 9-cis RA in rat was 11% and the elimination half-life (t1/2) was 35 min. Considering the short t1/2, we divided the toxic, but tumour growth effective dose 5 mg day−1 into 2.5 mg p.o. twice daily. This treatment regimen showed no toxicity but only limited effect on tumour growth. Our results suggest that 9-cis RA may only have limited clinical significance for treatment of children with poor prognosis neuroblastoma.

HMGB1-partner molecule complexes enhance cytokine production by signaling through the partner molecule receptor

Background and objectives The pro-inflammatory protein HMGB1 is a pathogenic mediator in several inflammatory diseases as demonstrated by beneficial effects of HMGB1-blocking therapy in experimental models including arthritis. High levels of extracellular HMGB1 are found both in experimental models of arthritis and in synovial biopsies from RA patients. Furthermore, intraarticular injection of HMGB1 causes destructive synovitis in mice. The authors and other groups have recently demonstrated that besides its endogenous pro-inflammatory properties, HMGB1 can induce cytokine release by complex formation with various proinflammatory molecules resulting in strong synergy. Direct HMGB1-mediated cytokine induction is mediated via TLR4. Receptor usage by HMGB1-complex formations is however an unresolved issue. The authors set out to test a hypothesis that HMGB1-complexes stimulate cytokine induction via dual receptor interaction. The authors postulated that HMGB1 signals via RAGE and the partner molecule via its reciprocal receptor and that the complexes bring these receptors in close proximity. Methods Recombinant HMGB1 was complexed with low amounts of either the TLR4 ligand LPS or the TLR2 ligand Pam3CSK4. The complexes were used to stimulate intraperitoneal macrophages obtained either from mice deficient in RAGE, TLR2, TLR4, MyD88, TIRAP, TRIF or TRAM or from wild type mice on a C57BL/6 background. Supernatant IL-6 levels assessed by ELISA (R&D Systems) were used as the read-out method. Results HMGB1-LPS and HMGB1-Pam3CSK4 complexes both stimulated IL-6 release in a synergistic mode compared to the individual components. Lack of TLR4 eliminated HMGB1-LPS complex-induced IL-6 production whereas absence of TLR2 eliminated that activated by HMGB1-Pam3CSK4 complexes. Deleting RAGE had however no effect on stimulation by either complex. To study whether the complex stimulation caused changes in the intracellular signaling the authors stimulated cells deficient in the adaptor molecules used by TLR2 (MyD88, TIRAP) or TLR4 (MyD88, TIRAP, TRIF, TRAM) with HMGB1-LPS and HMGB1-Pam3CSK4 complexes. Deletion of any of the adaptor molecules had similar effects on HMGB1 complex activation as compared with non-complexed LPS or Pam3CSK4. Conclusions Cytokine production induced by LPS or Pam3CSK4 in complex with HMGB1 occurred via their reciprocal receptors. The authors could not demonstrate that HMGB1-RAGE interaction or changes in the signalling pathways was responsible for the synergistic cytokine release. Elucidating HMGB1 receptor usage in processes where HMGB1 acts alone or in complex with other molecules is essential for the understanding of basic HMGB1 biology and for designing HMGB1-targeted therapies.

Neuroinflammation in Response to Intracerebral Injections of Different HMGB1 Redox Isoforms

Background: Neuroinflammation triggered by infection or trauma is the cause of central nervous system dysfunction. High-mobility group box 1 protein (HMGB1), released from stressed and dying brain cells, is a potent neuroinflammatory mediator. The proinflammatory functions of HMGB1 are tightly regulated by post-translational redox modifications, and we here investigated detailed neuroinflammatory responses induced by the individual redox isoforms. Methods: Male Dark Agouti rats received a stereotactic injection of saline, lipopolysaccharide, disulfide HMGB1, or fully reduced HMGB1, and were accessed for blood-brain barrier modifications using magnetic resonance imaging (MRI) and inflammatory responses by immunohistochemistry. Results and Conclusions: Significant blood-brain barrier disruption appeared 24 h after injection of lipopolysaccharide, disulfide HMGB1, or fully reduced HMGB1 compared to controls, as assessed in post-gadolinium T1-weighted MRI images and confirmed by increased uptake of FITC-conjugated dextran. Immunohistochemistry revealed that both HMGB1 isoforms also induced a local production of IL-1β. Additionally, disulfide HMGB1 increased major histocompatibility complex class II expression and apoptosis. Together, the results demonstrate that extracellular, cerebral HMGB1 causes significant blood-brain barrier disruption in a redox-independent manner and activates several components of neuroinflammation. Blocking HMGB1 might potentially improve clinical outcome in conditions such as stroke and traumatic brain injury.

A2.28 Characterisation and potential function of HMGB1 in juvenile idiopathic arthritis

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.