Therese Östberg

RAGE is the Major Receptor for the Proinflammatory Activity of HMGB1 in Rodent Macrophages

High‐mobility group box chromosomal protein 1 (HMGB1) is a protein with both intranuclear functions and extracellular cytokine‐like effects. In this report, we study possible candidate receptors for HMGB1 on macrophages (Mφ) and define pathways activated by HMGB1 binding. Bone marrow Mφ were prepared from Dark Agouti (DA) rats and stimulated in vitro with HMGB1. The kinetics of tumour necrosis factor (TNF) production, NO production, activation of p38 mitogen‐activated protein kinase (MAPK), p44/42 MAPK‐ and SAPK/JNK‐signalling pathways, nuclear translocation of nuclear factor kappa B (NF‐κB) and HMGB1‐induced upregulation of major histocompatibility complex (MHC) class II and CD86 were analysed. Mφ from interleukin (IL)‐1 receptor type I–/–, Toll‐like receptor 2 (TLR2–/–) and RAGE–/– mice were used to investigate the role of these receptors in HMGB1 signalling. HMGB1 induced TNF and NO production by Mφ, phosphorylation of all investigated MAP kinase pathways and NF‐κB translocation, and expression of MHC class II was increased. Mφ from RAGE–/– mice produced significantly lower amounts of TNF, IL‐1β and IL‐6, while IL‐1RI–/– and TLR2–/– Mφ produced cytokine levels comparable with wildtype controls in response to HMGB1 stimulation. We conclude that HMGB1 has the potential to induce a proinflammatory phenotype in Mφ, with RAGE as the major activation‐inducing receptor.

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.

Effects of HMGB1 on in vitro responses of isolated muscle fibers and functional aspects in skeletal muscles of idiopathic inflammatory myopathies

Idiopathic inflammatory myopathies (IIMs) are heterogeneous rheumatic disorders of unknown cause characterized by muscle weakness, inflammatory cell infiltrates, and major histocompatibility complex (MHC) class I expression on muscle fibers. The nonhistone nuclear protein alarmin high‐mobility group box 1 protein (HMGB1) has been detected extranuclearly in muscle biopsies from patients with IIMs. We hypothesize that HMGB1 has a central role in the cause of muscle weakness, particularly in the early phases of IIMs. Experiments were performed on skeletal muscle fibers isolated from adult mice, which were exposed to recombinant interferon (IFN)‐γ or HMGB1. The myoplasmic free [Ca2+] was measured. Stimulation with IFN‐γ resulted in increased HMGB1 expression in muscle nuclei and the myoplasm. Exposure to HMGB1 induced a reversible up‐regulation of MHC class I in the muscle fibers. However, HMGB1 exposure caused an irreversible decrease in Ca2+ release from the sarcoplasmic reticulum during fatigue, induced by repeated tetanic contractions. HMGB1 and MHC class I were frequently colocalized in the myoplasm of muscle fibers in muscle biopsies from patients with early IIMs. However, HMGB1‐expressing fibers outnumbered fibers expressing MHC class I. Our data indicate that HMGB1 could be an early inducer of skeletal muscle dysfunction in IIMs.—Grundtman, C., Bruton, J., Yamada, T., Östberg, T., Pisetsky, D. S., Harris, H. E., Andersson, U., Lundberg, I. E., Westerblad, H. Effects of HMGB1 on in vitro responses of isolated muscle fibers and functional aspects in skeletal muscles of idiopathic inflammatory myopathies. FASEB J. 24, 570–578 (2010). www.fasebj.org

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‐γ, polyinosinic:polycytidylic acid, IFN‐β, or NO in the presence of GST, ranging from 0 μM to 250 μM. Secretion and intracellular location of HMGB1 were assessed by Western blotting, HMGB1‐specific ELISPOT assay, and immunofluorescent staining. In parallel, TNF and IFN‐β 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‐β 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.

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.

Oxaliplatin retains HMGB1 intranuclearly and ameliorates collagen type II-induced arthritis

IntroductionHigh mobility group box chromosomal protein 1 (HMGB1) is a nuclear protein that acts as a pro-inflammatory mediator following extracellular release. The protein is aberrantly expressed extracellularly in the settings of clinical and experimental synovitis. Therapy based on HMGB1 antagonists has shown encouraging results in experimental arthritis and warrants further scientific exploration using independent methods. In the present study we asked whether nuclear sequestration of HMGB1 preventing HMGB1 release would be beneficial for synovitis treatment.MethodsOxaliplatin-based therapy was evaluated in collagen type II-induced arthritis in DBA/1 mice by clinical scoring and immunostaining of articular tissue. Oxaliplatin is an antineoplastic platinum-based compound that generates DNA adducts which tightly bind HMGB1. Secretion and intracellular location of HMGB1 were assessed by a novel HMGB1-specific ELISPOT assay and immunofluorescent staining.ResultsIntraperitoneal injections of oxaliplatin in early collagen type II-induced arthritis trapped HMGB1 with a distinct biphasic response pattern. Oxaliplatin therapy showed beneficial results for approximately 1 week. Microscopic evaluation of synovitis during this period showed strong nuclear HMGB1 staining in the oxaliplatin treated animals with much lower quantities of extracellular HMGB1 when compared to control treated animals. Furthermore, cellular infiltration, as well as cartilage and bone damage, were all reduced in the oxaliplatin treated group. A dramatic and as yet unexplained clinical relapse occurred later in the oxaliplatin exposed animals, which coincided with a massive synovial tissue expression of extracellular HMGB1 in all treated animals. This rebound-like reaction was also accompanied by a significantly increased incidence of arthritis in the oxaliplatin treated group. These results indicate a distinct temporal and spatial relationship between the clinical course of disease and the cellular localization of HMGB1. Beneficial effects were noted when extracellular HMGB1 expression was low, while severe inflammation coincided with substantial extracellular synovial HMGB1 expression.ConclusionTherapeutic compounds like oxaliplatin and gold salts share a capacity to inhibit nuclear HMGB1 release and to ameliorate the course of synovial inflammation. These observations support the hypothesis that HMGB1 plays an important functional role in the pathogenesis of arthritis and may represent a novel target molecule for therapy.

Impaired myofibrillar function in the soleus muscle of mice with collagen-induced arthritis.

OBJECTIVE Progressive muscle weakness is a common feature in patients with rheumatoid arthritis (RA). However, little is known about whether the intrinsic contractile properties of muscle fibers are affected in RA. This study was undertaken to investigate muscle contractility and the myoplasmic free Ca2+ concentration ([Ca2+](i)) in the soleus, a major postural muscle, in mice with collagen-induced arthritis (CIA). METHODS Muscle contractility and [Ca2+](i) were assessed in whole muscle and intact single-fiber preparations, respectively. The underlying mechanisms of contractile dysfunction were assessed by investigating redox modifications using Western blotting and antibodies against nitric oxide synthase (NOS), superoxide dismutase (SOD), 3-nitrotyrosine (3-NT), carbonyl, malondialdehyde (MDA), and S-nitrosocysteine (SNO-Cys). RESULTS The tetanic force per cross-sectional area was markedly decreased in the soleus muscle of mice with CIA, and the change was not due to a decrease in the amplitude of [Ca2+](i) transients. The reduction in force production was accompanied by slowing of the twitch contraction and relaxation and a decrease in the maximum shortening velocity. Immunoblot analyses showed a marked increase in neuronal NOS expression but not in inducible or endothelial NOS expression, which, together with the observed decrease in SOD2 expression, favors peroxynitrite formation. These changes were accompanied by increased 3-NT, carbonyl, and MDA adducts content in myofibrillar proteins from the muscles of mice with CIA. Moreover, there was a significant increase in SNO-Cys content in myosin heavy-chain and troponin I myofibrillar proteins from the soleus muscle of mice with CIA. CONCLUSION These findings show impaired contractile function in the soleus muscle of mice with CIA and suggest that this abnormality is due to peroxynitrite-induced modifications in myofibrillar proteins.

The HLA locus contains novel foetal susceptibility alleles for congenital heart block with significant paternal influence

The main aim of this study was to identify foetal susceptibility genes on chromosome six for Ro/SSA autoantibody‐mediated congenital heart block.

Augmented Th17 differentiation in Trim21 deficiency promotes a stable phenotype of atherosclerotic plaques with high collagen content

Aims Patients with hyperlipidemia are at risk of atherosclerosis, but not all develop cardiovascular disease, highlighting the importance of other risk factors such as inflammation. Both the innate and adaptive arms of the immune system have been suggested in the initiation and propagation of plaque formation. Tri-partite motif (TRIM) 21 is a regulator of tissue inflammation and pro-inflammatory cytokine production, and has been implicated in chronic inflammatory disease. Here, we investigate a potential role for TRIM21 in coronary artery disease. Methods and results Trim21-deficient or wild-type bone marrow was transplanted into Ldlr-/- mice fed a hypercholesterolemic diet. The Trim21-/-->Ldlr-/- mice developed larger atherosclerotic plaques, with significantly higher collagen content compared to mice transplanted with wild-type cells. High collagen content of the atheroma is stabilizing, and has recently been linked to IL-17. Interestingly, Trim21-/-->Ldlr-/- mice had elevated CD4 and IL-17 mRNA expression in plaques, and increased numbers of activated CD4+ T cells in the periphery. An increased differentiation of naïve T cells lacking Trim21 into Th17 cells was confirmed in vitro, with transcriptomic analysis revealing upregulation of genes of a non-pathogenic Th17 phenotype. Also, decreased expression of matrix metalloproteinases (MMPs) was noted in aortic plaques. Analysis of human carotid plaques confirmed that TRIM21 expression negatively correlates with the expression of key Th17 genes and collagen, but positively to MMPs also in patients, linking our findings to a clinical setting. Conclusion In this study, we demonstrate that TRIM21 influences atherosclerosis via regulation of Th17 responses, with TRIM21 deficiency promoting IL-17 expression and a more fibrous, stable, phenotype of the plaques.

A7.23 The HLA Locus Contains Novel Foetal Susceptibility Alleles for Congenital Heart Block with Significant Paternal Influence

Objective To identify foetal susceptibility genes in Ro/SSA autoantibody-mediated congenital heart block on chromosome six. Methods Single nucleotide polymorphism (SNP) genotyping of individuals included in the Swedish Congenital Heart Block (CHB) study population was performed. Low resolution HLA-A, -Cw and -DRB1 allele typing was carried out in 86 families of the study population comprising 339 individuals (86 Ro/SSA autoantibody positive mothers, 71 fathers, 87 CHB index cases and 95 unaffected siblings). Results A case-control comparison between index cases and population-based out of study controls (n = 1710) revealed an association of CHB with fifteen SNPs in the 6p21.3 MHC locus at a chromosome-wide significance of p < 2.59 × 10–6 (OR 2.21–3.12). In a family-based analysis between SNP markers as well as distinct MHC class I and II alleles with CHB we observed associations to HLA-DRB1*04 and HLA-Cw*05 variants that were significantly more often transmitted to affected individuals (p < 0.03 and p < 0.05, respectively), and HLA-DRB1*13 and HLA-Cw*06 variants which were significantly less often transmitted to affected children (p < 0.05 and p < 0.04). We further observed a significant association of increased paternal, but not maternal, HLA-DRB1*04 transmissions to the affected offspring (p < 0.02). Conclusions Our study identifies HLA-DRB1*04 and HLA-Cw*05 as novel foetal HLA-allele variants that confer susceptibility to develop CHB in response to exposure to Ro/SSA autoantibodies, while DRB1*13 and Cw*06 emerged as protective alleles. For the first time, we also demonstrate paternal contribution to foetal susceptibility to CHB.