Redox modifications of cysteine residues regulate the cytokine activity of HMGB1.

BACKGROUND: High mobility group box 1 (HMGB1) is a nuclear protein with extracellular inflammatory cytokine activity. It is passively released during cell death and secreted by activated cells of many lineages. HMGB1 contains three conserved redox-sensitive cysteine residues: cysteines in position 23 and 45 (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. METHODS: Primary human macrophages or murine macrophage-like RAW 264.7 cells were activated in cell cultures by redox-modified or point-mutated (C45A) recombinant HMGB1 preparations or by lipopolysaccharide (E. coli.0111: B4). Cellular phosphorylated NF-κB p65 subunit and subsequent TNF-α release were quantified by commercial enzyme-linked immunosorbent assays. RESULTS: Cell cultures with primary human macrophages and RAW 264.7 cells demonstrated that fully reduced HMGB1 with all three cysteines expressing thiol side chains failed to generate phosphorylated NF-КB p65 subunit or TNF-α. Mild oxidation forming a C23-C45 disulfide bond, while leaving C106 with a thiol group, was required for HMGB1 to induce phosphorylated NF-КB p65 subunit and TNF-α production. The importance of a C23-C45 disulfide bond was confirmed by mutation of C45 to C45A HMGB1, which abolished the ability for cytokine induction. Further oxidation of the disulfide isoform also inactivated HMGB1. CONCLUSIONS: These results reveal critical post-translational redox mechanisms that control the proinflammatory activity of HMGB1 and its inactivation during inflammation

HMGB1 promotes recruitment of inflammatory cells to damaged tissues by forming a complex with CXCL12 and signaling via CXCR4

After tissue damage, inflammatory cells infiltrate the tissue and release proinflammatory cytokines. HMGB1 (high mobility group box 1), a nuclear protein released by necrotic and severely stressed cells, promotes cytokine release via its interaction with the TLR4 (Toll-like receptor 4) receptor and cell migration via an unknown mechanism. We show that HMGB1- induced recruitment of inflammatory cells depends on CXCL12. HMGB1 and CXCL12 form a heterocomplex, which we characterized by nuclear magnetic resonance and surface plasmon resonance, that acts exclusively through CXCR4 and not through other HMGB1 receptors. Fluorescence resonance energy transfer data show that the HMGB1–CXCL12 heterocomplex promotes different conformational rearrangements of CXCR4 from that of CXCL12 alone. Mononuclear cell recruitment in vivo into air pouches and injured muscles depends on the heterocomplex and is inhibited by AMD3100 and glycyrrhizin. Thus, inflammatory cell recruitment and activation both depend on HMGB1 via different mechanisms

Mutually exclusive redox forms of HMGB1 promote cell recruitment or proinflammatory cytokine release

Tissue damage causes inflammation, by recruiting leukocytes and activating them to release proinflammatory mediators. We show that high-mobility group box 1 protein (HMGB1) orchestrates both processes by switching among mutually exclusive redox states. Reduced cysteines make HMGB1 a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine and further cysteine oxidation to sulfonates by reactive oxygen species abrogates both activities. We show that leukocyte recruitment and activation can be separated. A nonoxidizable HMGB1 mutant in which serines replace all cysteines (3S- HMGB1) does not promote cytokine production, but is more effective than wild-type HMGB1 in recruiting leukocytes in vivo. BoxA, a HMGB1 inhibitor, interferes with leukocyte recruitment but not with activation. We detected the different redox forms of HMGB1 ex vivo within injured muscle. HMGB1 is completely reduced at first and disulfide-bonded later. Thus, HMGB1 orchestrates both key events in sterile inflammation, leukocyte recruitment and their induction to secrete inflammatory cytokines, by adopting mutually exclusive redox states

Mutually exclusive redox forms of HMGB1 promote cell recruitment or proinflammatory cytokine release

Tissue damage causes inflammation, by recruiting leukocytes and activating them to release proinflammatory mediators. We show that high-mobility group box 1 protein (HMGB1) orchestrates both processes by switching among mutually exclusive redox states. Reduced cysteines make HMGB1 a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine and further cysteine oxidation to sulfonates by reactive oxygen species abrogates both activities. We show that leukocyte recruitment and activation can be separated. A nonoxidizable HMGB1 mutant in which serines replace all cysteines (3S- HMGB1) does not promote cytokine production, but is more effective than wild-type HMGB1 in recruiting leukocytes in vivo. BoxA, a HMGB1 inhibitor, interferes with leukocyte recruitment but not with activation. We detected the different redox forms of HMGB1 ex vivo within injured muscle. HMGB1 is completely reduced at first and disulfide-bonded later. Thus, HMGB1 orchestrates both key events in sterile inflammation, leukocyte recruitment and their induction to secrete inflammatory cytokines, by adopting mutually exclusive redox states

Génération et analyse fonctionnelle d'antagonistes de l'oncostatine m et de l'interleukine-31, cytokines impliquées dans l'inflammation cutanée et le développement tumoral

Oncostatin M (OSM) and interleukin-31 (IL-31), cytokines of the IL-6 family, are involved in inflammation associated with skin diseases such as atopic dermatitis or psoriasis. In a therapeutic perspective, two first studies were based on the generation of antagonists acting as soluble receptors and intended to trap these cytokines. In order to have a new model for the IL-31 in vivo study, we also identified and cloned this protein in rat. In a latest study, we highlighted IL-31 involvement in tumor development by demonstrating its cytostatic activity on tumor cell lines, in a similar way to OSM. This study has also led us to analyze the intracellular signaling pathways involved in these anti-tumor effects and we demonstrated the activation of a new transcription factor in response to these two cytokines.L'oncostatine M (OSM) et l'interleukine-31 (IL-31), cytokines de la famille de l'IL-6, sont impliquées dans l'inflammation cutanée associée à des pathologies telles que la dermatite atopique ou le psoriasis. Dans une perspective thérapeutique, deux premières études sont basées sur la génération d'antagonistes agissant comme des récepteurs solubles et destinés à piéger ces cytokines. Afin de disposer d'un nouveau modèle d'étude de l'IL-31 in vivo, nous avons également identifié et cloné cette protéine chez le rat. Dans une dernière étude, nous avons mis en évidence l'implication de l'IL-31 dans le développement tumoral en démontrant son activité cytostatique sur certaines lignées tumorales, de manière similaire à l'OSM. Cette étude nous a également amenés à analyser les voies de signalisation intracellulaires impliquées dans ces effets anti-tumoraux et nous avons ainsi démontré l'activation d'un nouveau facteur de transcription en réponse à ces deux cytokines

Génération et analyse fonctionnelle d'antagonistes de l'oncostatine M et de l'interleukine-31, cytokines impliquées dans l'inflammation cutanée et le développement tumoral

L'oncostatine M (OSM) et l'interleukine-31 (IL-31), cytokines de la famille de l'IL-6, sont impliquées dans l'inflammation cutanée associée à des pathologies telles que la dermatite atopique ou le psoriasis. Dans une perspective thérapeutique, deux premières études sont basées sur la génération d'antagonistes agissant comme des récepteurs solubles et destinés à piéger ces cytokines. Afin de disposer d'un nouveau modèle d'étude de l'IL-31 in vivo, nous avons également identifié et cloné cette protéine chez le rat. Dans une dernière étude, nous avons mis en évidence l'implication de l'IL-31 dans le développement tumoral en démontrant son activité cytostatique sur certaines lignées tumorales, de manière similaire à l'OSM. Cette étude nous a également amenés à analyser les voies de signalisation intracellulaires impliquées dans ces effets anti-tumoraux et nous avons ainsi démontré l'activation d'un nouveau facteur de transcription en réponse à ces deux cytokines..Oncostatin M (OSM) and interleukin-31 (IL-31), cytokines of the IL-6 family, are involved in inflammation associated with skin diseases such as atopic dermatitis or psoriasis. In a therapeutic perspective, two first studies were based on the generation of antagonists acting as soluble receptors and intended to trap these cytokines. In order to have a new model for the IL-31 in vivo study, we also identified and cloned this protein in rat. In a latest study, we highlighted IL-31 involvement in tumor development by demonstrating its cytostatic activity on tumor cell lines, in a similar way to OSM. This study has also led us to analyze the intracellular signaling pathways involved in these anti-tumor effects and we demonstrated the activation of a new transcription factor in response to these two cytokines.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

HMGB1 is upregulated in the airways in asthma and potentiates airway smooth muscle contraction via TLR4

[First paragraph] Asthma is characterized by variable airflow obstruction, airway hyperresponsiveness, and inflammation. Airway smooth muscle (ASM) contributes to asthma pathophysiology via hypercontractility, increased mass, and inflammatory mediator release.1 Clinical studies and animal models demonstrate a role for high-mobility group box 1 (HMGB1) and its receptors in airway inflammation and asthma.2 ; 3 HMGB1's activity and receptor interactions is determined by its redox state, with oxidation rendering HMGB1 inactive.4 We have investigated the redox state of airway HMGB1 and the role of HMGB1 in ASM function

Oncostatin M Secreted by Skin Infiltrating T Lymphocytes Is a Potent Keratinocyte Activator Involved in Skin Inflammation

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