Lars Ottosson

Redox modification of cysteine residues regulates the cytokine activity of high mobility group box-1 (HMGB1).

High mobility group box 1 (HMGB1) is a nuclear protein with extracellular inflammatory cytokine activity. It is released passively during cell injury and necrosis, and secreted actively by immune cells. HMGB1 contains three conserved redox-sensitive cysteine residues: C23 and C45 can form an intramolecular disulfide bond, whereas C106 is unpaired and is essential for the interaction with Toll-Like Receptor (TLR) 4. However, a comprehensive characterization of the dynamic redox states of each cysteine residue and of their impacts on innate immune responses is lacking. Using tandem mass spectrometric analysis, we now have established that the C106 thiol and the C23-C45 disulfide bond are required for HMGB1 to induce nuclear NF-κB translocation and tumor necrosis factor (TNF) production in macrophages. Both irreversible oxidation to sulphonates and complete reduction to thiols of these cysteines inhibited TNF production markedly. In a proof of concept murine model of hepatic necrosis induced by acetaminophen, during inflammation, the predominant form of serum HMGB1 is the active one, containing a C106 thiol group and a disulfide bond between C23 and C45, whereas the inactive form of HMGB1, containing terminally oxidized cysteines, accumulates during inflammation resolution and hepatic regeneration. These results reveal critical posttranslational redox mechanisms that control the proinflammatory activity of HMGB1 and its inactivation during pathogenesis.

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.

Ro/SSA autoantibodies directly bind cardiomyocytes, disturb calcium homeostasis, and mediate congenital heart block

Congenital heart block develops in fetuses after placental transfer of Ro/SSA autoantibodies from rheumatic mothers. The condition is often fatal and the majority of live-born children require a pacemaker at an early age. The specific antibody that induces the heart block and the mechanism by which it mediates the pathogenic effect have not been elucidated. In this study, we define the cellular mechanism leading to the disease and show that maternal autoantibodies directed to a specific epitope within the leucine zipper amino acid sequence 200–239 (p200) of the Ro52 protein correlate with prolongation of fetal atrioventricular (AV) time and heart block. This finding was further confirmed experimentally in that pups born to rats immunized with p200 peptide developed AV block. p200-specific autoantibodies cloned from patients bound cultured cardiomyocytes and severely affected Ca2+ oscillations, leading to accumulating levels and overload of intracellular Ca2+ levels with subsequent loss of contractility and ultimately apoptosis. These findings suggest that passive transfer of maternal p200 autoantibodies causes congenital heart block by dysregulating Ca2+ homeostasis and inducing death in affected cells.

Interferon-α Induces Up-regulation and Nuclear Translocation of the Ro52 Autoantigen as Detected by a Panel of Novel Ro52-specific Monoclonal Antibodies

Interferon-α (IFN-α) has been implicated in the pathogenesis of Sjögren’s syndrome and systemic lupus erythematosus. Ro52, which was recently identified as an E3 ligase with anti-proliferative and pro-apoptotic properties, is a major autoantigen targeted in both these conditions. Microarray analyses have indicated up-regulation of Ro52 by INF-α, and the objective of the present study was to address the potential link between IFN-α and Ro52. To investigate the influence of IFN-α on Ro52 protein levels and cellular localization, we generated a panel of monoclonal antibodies to different domains of Ro52. These novel monoclonal antibodies were characterized by immunoprecipitation, Western blot, and enzyme-linked immunosorbent assay using cell lysates, recombinant Ro52 protein, and synthetic peptides. Ro52 was up-regulated in HeLa cells and human B cells at the messenger RNA and protein levels in response to IFN-α stimulation as detected by reverse transcriptase polymerase chain reaction and Western blot. After up-regulation, Ro52 translocated from the cytoplasm to the nucleus. The nuclear translocation of Ro52 was observed after staining with generated monoclonal antibodies specific for both the RING, coiled-coil, and B30.2 domains of Ro52 and the nuclear translocation of Ro52 preceded IFN-α-induced apoptotic cell death detected by caspase-3 and TUNEL staining in the treated cultures. In conclusion, our data show that IFN-α first induces up-regulation of Ro52 protein and then prompts translocation of the up-regulated Ro52 protein in to the nucleus. The translocation precedes apoptosis of the IFN-α exposed cells, suggesting a role for Ro52 in mediating the anti-proliferative or pro-apoptotic effects of the autoimmune-related cytokine IFN-α.

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.

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).

Structurally Derived Mutations Define Congenital Heart Block‐Related Epitopes Within the 200–239 Amino Acid Stretch of the Ro52 Protein

Congenital heart block is a passively transferred autoimmune condition, which affects the children of mothers with Ro/SSA autoantibodies. During pregnancy, the antibodies are transported across the placenta and affect the fetus. We have previously demonstrated that antibodies directed to the 200–239 amino acid (aa) stretch of the Ro52 component of the Ro/SSA antigen correlate with the development of congenital heart block. In this report, we investigated the antibody–antigen interaction of this target epitope in detail at a molecular and structural level. Peptides representing aa 200–239 (p200) with structurally derived mutations were synthesized to define the epitopes recognized by two Ro52 human monoclonal antibodies, S3A8 and M4H1, isolated from patient‐derived phage display libraries. Analyses by ELISA, circular dichroism and MALDI‐TOF‐MS demonstrate that the antibody recognition is dependent on a partly α‐helical fold within the putative leucine zipper of the 200–239 aa stretch and that the two human anti‐p200 monoclonal antibodies, M4H1 and S3A8, recognize different epitopic structures within the p200 peptide. In addition, we investigated the representation of each fine specificity within the sera of mothers with children born with congenital heart block, and in such sera, antibodies of the S3A8 idiotype were more commonly detected and at higher levels than M4H1‐like antibodies.

The Ile128Thr polymorphism influences stability and ligand binding properties of the microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTTP) is essential for the assembly of VLDLs. We recently observed that a polymorphism in the MTTP promoter (−493G>T), which is in allelic association with an isoleucine-to-theronine substitution at position 128 (Ile128Thr) in the expressed protein, confers an increased risk of coronary heart disease. Two variant proteins comprising amino acids 16–297 of intact MTTP, MTTPN-Ile128 and MTTPN-Thr128, had similar native secondary structure content, as judged by circular dichroism. However, the thermal stability of MTTPN-Thr128 was greatly reduced, and this protein was also more extensively cleaved in limited proteolysis experiments compared with MTTPN-Ile128; both of these findings support a less compact fold. On adding LDL, which includes natively folded apolipoprotein B (apoB), decreased stability of the MTTPN-Thr128-LDL complex was observed compared with that of the MTTPN-Ile128-LDL complex. In a refined model of the N-terminal domain of MTTP, residue 128 is located in a surface-exposed position, in the same region as an identified MTTP binding site in the homologous apoB protein. Thus, the Ile128Thr polymorphism confers reduced structural stability, leading to decreased binding of MTTP to LDL particles. Because the major MTTP binding target on LDL is apoB, the Ile128Thr polymorphism could target the MTTP-apoB interaction.

Maternal MHC Regulates Generation of Pathogenic Antibodies and Fetal MHC-Encoded Genes Determine Susceptibility in Congenital Heart Block

Congenital heart block develops in fetuses of anti-Ro52 Ab-positive women. A recurrence rate of 20%, despite the persistence of maternal autoantibodies, indicates that there are additional, yet unidentified, factors critical for development of congenital heart block. In this study, we demonstrate that besides the maternal MHC controlling Ab specificity, fetal MHC-encoded genes influence fetal susceptibility to congenital heart block. Using MHC congenic rat strains, we show that heart block develops in rat pups of three strains carrying MHC haplotype RT1av1 (DA, PVG.AV1, and LEW.AV1) after maternal Ro52 immunization, but not in LEW rats (RT1l). Different anti-Ro52 Ab fine specificities were generated in RT1av1 versus RT1l animals. Maternal and fetal influence was determined in an F2 cross between LEW.AV1 and LEW strains, which revealed higher susceptibility in RT1l than RT1av1 pups once pathogenic Ro52 Abs were present. This was further confirmed in that RT1l pups more frequently developed heart block than RT1av1 pups after passive transfer of RT1av1 anti-Ro52 sera. Our findings show that generation of pathogenic Ro52 Abs is restricted by maternal MHC, whereas the fetal MHC locus regulates susceptibility and determines the fetal disease outcome in anti-Ro52–positive pregnancies.

Autoantibodies to the functionally active RING-domain of Ro52/SSA are associated with disease activity in patients with lupus

The Ro52 protein of the Ro/SSA antigen was recently defined as an E3 ligase controlling cytokine production. Autoantibodies from systemic lupus erythematosus (SLE) patients targeting the Ro52-RING domain, containing the E3 ligase activity, have been shown to inhibit the E3 ligase activity of Ro52. The objective of the present study was to investigate correlations between clinical parameters in patients with SLE and levels of Ro/SSA (Ro52 and Ro60) and La/SSB autoantibodies, including autoantibodies directed towards the functional RING and B-box domains of the Ro52 protein. SLE patients (n = 232) were clinically examined and disease activity indices collected concurrently to blood sampling. The samples were analyzed for immunological parameters including autoantibodies. Ro52 autoantibody levels were associated with more variables than the other analyzed antibodies and were significantly associated with several individual items related to sSS and the diagnosis of sSS itself (p = 0.004). Other associated variables were high sedimentation rate (p = 0.0003), levels of immunoglobulins (p = 0.0003), and an inverse correlation with levels of lymphocytes (p = 0.003) and leukocytes (p = 0.01). Antibodies to the RING domain of Ro52, which is the functionally active domain with E3 ligase activity, were significantly correlated with disease activity as measured by the SLAM score. We conclude that autoantibodies against Ro52 and in particular its functional RING domain are important in lupus patients and associated with several clinical and laboratory features of the disease. The impact on disease activity of Ro52-RING specific antibodies was especially noted, and could imply a functional role for these autoantibodies in inhibiting Ro52 activity, which is important for the control of proinflammatory cytokine production, including type 1 interferons.