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1.
Nat Commun ; 15(1): 1201, 2024 Feb 08.
Article in English | MEDLINE | ID: mdl-38331917

ABSTRACT

Chemokine heterodimers activate or dampen their cognate receptors during inflammation. The CXCL12 chemokine forms with the fully reduced (fr) alarmin HMGB1 a physiologically relevant heterocomplex (frHMGB1•CXCL12) that synergically promotes the inflammatory response elicited by the G-protein coupled receptor CXCR4. The molecular details of complex formation were still elusive. Here we show by an integrated structural approach that frHMGB1•CXCL12 is a fuzzy heterocomplex. Unlike previous assumptions, frHMGB1 and CXCL12 form a dynamic equimolar assembly, with structured and unstructured frHMGB1 regions recognizing the CXCL12 dimerization surface. We uncover an unexpected role of the acidic intrinsically disordered region (IDR) of HMGB1 in heterocomplex formation and its binding to CXCR4 on the cell surface. Our work shows that the interaction of frHMGB1 with CXCL12 diverges from the classical rigid heterophilic chemokines dimerization. Simultaneous interference with multiple interactions within frHMGB1•CXCL12 might offer pharmacological strategies against inflammatory conditions.


Subject(s)
Chemokine CXCL12 , HMGB1 Protein , Humans , Chemokine CXCL12/metabolism , HMGB1 Protein/metabolism , Receptors, CXCR4/metabolism , Inflammation , Signal Transduction
2.
Mol Med ; 28(1): 108, 2022 09 07.
Article in English | MEDLINE | ID: mdl-36071400

ABSTRACT

BACKGROUND: High-mobility group box 1 protein (HMGB1) is an ubiquitous nuclear protein that once released in the extracellular space acts as a Damage Associated Molecular Pattern and promotes inflammation. HMGB1 is significantly elevated during Pseudomonas aeruginosa infections and has a clinical relevance in respiratory diseases such as Cystic Fibrosis (CF). Salicylates are HMGB1 inhibitors. To address pharmacological inhibition of HMGB1 with small molecules, we explored the therapeutic potential of pamoic acid (PAM), a salicylate with limited ability to cross epithelial barriers. METHODS: PAM binding to HMGB1 and CXCL12 was tested by Nuclear Magnetic Resonance Spectroscopy using chemical shift perturbation methods, and inhibition of HMGB1·CXCL12-dependent chemotaxis was investigated by cell migration experiments. Aerosol delivery of PAM, with single or repeated administrations, was tested in murine models of acute and chronic P. aeruginosa pulmonary infection in C57Bl/6NCrlBR mice. PAM efficacy was evaluated by read-outs including weight loss, bacterial load and inflammatory response in lung and bronco-alveolar lavage fluid. RESULTS: Our data and three-dimensional models show that PAM is a direct ligand of both HMGB1 and CXCL12. We also showed that PAM is able to interfere with heterocomplex formation and the related chemotaxis in vitro. Importantly, PAM treatment by aerosol was effective in reducing acute and chronic airway murine inflammation and damage induced by P. aeruginosa. The results indicated that PAM reduces leukocyte recruitment in the airways, in particular neutrophils, suggesting an impaired in vivo chemotaxis. This was associated with decreased myeloperoxidase and neutrophil elastase levels. Modestly increased bacterial burdens were recorded with single administration of PAM in acute infection; however, repeated administration in chronic infection did not affect bacterial burdens, indicating that the interference of PAM with the immune system has a limited risk of pulmonary exacerbation. CONCLUSIONS: This work established the efficacy of treating inflammation in chronic respiratory diseases, including bacterial infections, by topical delivery in the lung of PAM, an inhibitor of HMGB1.


Subject(s)
Chemokine CXCL12 , HMGB1 Protein , Naphthols , Pneumonia, Bacterial , Animals , Chemokine CXCL12/antagonists & inhibitors , Chemotaxis/drug effects , Disease Models, Animal , HMGB1 Protein/antagonists & inhibitors , Inflammation/drug therapy , Inflammation/pathology , Mice , Mice, Inbred C57BL , Naphthols/pharmacology , Pneumonia, Bacterial/drug therapy , Pseudomonas aeruginosa/metabolism
3.
J Virol ; 96(19): e0112222, 2022 10 12.
Article in English | MEDLINE | ID: mdl-36121298

ABSTRACT

Zika virus (ZIKV) is an arbovirus member of the Flaviviridae family that causes severe congenital brain anomalies in infected fetuses. The key target cells of ZIKV infection, human neural progenitor cells (hNPCs), are highly permissive to infection that causes the inhibition of cell proliferation and induces cell death. We have previously shown that pharmaceutical-grade heparin inhibits virus-induced cell death with negligible effects on in vitro virus replication in ZIKV-infected hNPCs at the "high" multiplicity of infection (MOI) of 1. Here, we show that heparin inhibits formation of ZIKV-induced intracellular vacuoles, a signature of paraptosis, and inhibits necrosis and apoptosis of hNPCs grown as neurospheres (NS). To test whether heparin preserved the differentiation of ZIKV-infected hNPCs into neuroglial cells, hNPCs were infected at the MOI of 0.001. In this experimental condition, heparin inhibited ZIKV replication by ca. 2 log10, mostly interfering with virion attachment, while maintaining its protective effect against ZIKV-induced cytopathicity. Heparin preserved differentiation into neuroglial cells of hNPCs that were obtained from either human-induced pluripotent stem cells (hiPSC) or by fetal tissue. Quite surprisingly, multiple additions of heparin to hNPCs enabled prolonged virus replication while preventing virus-induced cytopathicity. Collectively, these results highlight the potential neuroprotective effect of heparin that could serve as a lead compound to develop novel agents for preventing the damage of ZIKV infection on the developing brain. IMPORTANCE ZIKV is a neurotropic virus that invades neural progenitor cells (NPCs), causing inhibition of their proliferation and maturation into neurons and glial cells. We have shown previously that heparin, an anticoagulant also used widely during pregnancy, prevents ZIKV-induced cell death with negligible inhibition of virus replication. Here, we demonstrate that heparin also exerts antiviral activity against ZIKV replication using a much lower infectious inoculum. Moreover, heparin interferes with different modalities of virus-induced cell death. Finally, heparin-induced prevention of virus-induced NPC death allows their differentiation into neuroglial cells despite the intracellular accumulation of virions. These results highlight the potential use of heparin, or pharmacological agents derived from it, in pregnant women to prevent the devastating effects of ZIKV infection on the developing brain of their fetuses.


Subject(s)
Heparin , Neural Stem Cells , Neuroprotective Agents , Zika Virus , Anticoagulants/pharmacology , Antiviral Agents/pharmacology , Cell Death/drug effects , Cell Differentiation , Heparin/pharmacology , Humans , Neural Stem Cells/cytology , Neural Stem Cells/virology , Neuroglia/cytology , Neuroglia/virology , Neuroprotective Agents/pharmacology , Virus Replication , Zika Virus/drug effects , Zika Virus/physiology , Zika Virus Infection/drug therapy
4.
EMBO Mol Med ; 13(7): e13591, 2021 07 07.
Article in English | MEDLINE | ID: mdl-34096686

ABSTRACT

Cachexia syndrome develops in patients with diseases such as cancer and sepsis and is characterized by progressive muscle wasting. While iNOS is one of the main effectors of cachexia, its mechanism of action and whether it could be targeted for therapy remains unexplored. Here, we show that iNOS knockout mice and mice treated with the clinically tested iNOS inhibitor GW274150 are protected against muscle wasting in models of both septic and cancer cachexia. We demonstrate that iNOS triggers muscle wasting by disrupting mitochondrial content, morphology, and energy production processes such as the TCA cycle and acylcarnitine transport. Notably, iNOS inhibits oxidative phosphorylation through impairment of complexes II and IV of the electron transport chain and reduces ATP production, leading to energetic stress, activation of AMPK, suppression of mTOR, and, ultimately, muscle atrophy. Importantly, all these effects were reversed by GW274150. Therefore, our data establish how iNOS induces muscle wasting under cachectic conditions and provide a proof of principle for the repurposing of iNOS inhibitors, such as GW274150 for the treatment of cachexia.


Subject(s)
Cachexia , Neoplasms , Animals , Humans , Mice , Mitochondria , Muscles , Muscular Atrophy
5.
Sci Transl Med ; 13(596)2021 06 02.
Article in English | MEDLINE | ID: mdl-34078746

ABSTRACT

Muscular dystrophies (MDs) are a group of genetic diseases characterized by progressive muscle wasting associated to oxidative stress and persistent inflammation. It is essential to deepen our knowledge on the mechanism connecting these two processes because current treatments for MDs have limited efficacy and/or are associated with side effects. Here, we identified the alarmin high-mobility group box 1 (HMGB1) as a functional link between oxidative stress and inflammation in MDs. The oxidation of HMGB1 cysteines switches its extracellular activities from the orchestration of tissue regeneration to the exacerbation of inflammation. Extracellular HMGB1 is present at high amount and undergoes oxidation in patients with MDs and in mouse models of Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy 3 (LGMDR3) compared to controls. Genetic ablation of HMGB1 in muscles of DMD mice leads to an amelioration of the dystrophic phenotype as evidenced by the reduced inflammation and muscle degeneration, indicating that HMGB1 oxidation is a detrimental process in MDs. Pharmacological treatment with an engineered nonoxidizable variant of HMGB1, called 3S, improves functional performance, muscle regeneration, and satellite cell engraftment in dystrophic mice while reducing inflammation and fibrosis. Overall, our data demonstrate that the balance between HMGB1 redox isoforms dictates whether skeletal muscle is in an inflamed or regenerating state, and that the nonoxidizable form of HMGB1 is a possible therapeutic approach to counteract the progression of the dystrophic phenotype. Rebalancing the HMGB1 redox isoforms may also be a therapeutic strategy for other disorders characterized by chronic oxidative stress and inflammation.


Subject(s)
HMGB1 Protein , Muscular Dystrophy, Duchenne , Animals , HMGB1 Protein/metabolism , Humans , Mice , Mice, Inbred mdx , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/metabolism , Oxidation-Reduction , Protein Isoforms/metabolism
6.
Front Chem ; 8: 598710, 2020.
Article in English | MEDLINE | ID: mdl-33324614

ABSTRACT

HMGB1 is a key molecule that both triggers and sustains inflammation following infection or injury, and is involved in a large number of pathologies, including cancer. HMGB1 participates in the recruitment of inflammatory cells, forming a heterocomplex with the chemokine CXCL12 (HMGB1·CXCL12), thereby activating the G-protein coupled receptor CXCR4. Thus, identification of molecules that disrupt this heterocomplex can offer novel pharmacological opportunities to treat inflammation-related diseases. To identify new HMGB1·CXCL12 inhibitors we have performed a study on the ligandability of the single HMG boxes of HMGB1 followed by a virtual screening campaign on both HMG boxes using Zbc Drugs and three different docking programs (Glide, AutoDock Vina, and AutoDock 4.2.6). The best poses in terms of scoring functions, visual inspection, and predicted ADME properties were further filtered according to a pharmacophore model based on known HMGB1 binders and clustered according to their structures. Eight compounds representative of the clusters were tested for HMGB1 binding by NMR. We identified 5,5'-methylenedi-2,3-cresotic acid (2a) as a binder of both HMGB1 and CXCL12; 2a also targets the HMGB1·CXCL12 heterocomplex. In cell migration assays 2a inhibited the chemotactic activity of HMGB1·CXCL12 with IC50 in the subnanomolar range, the best documented up to now. These results pave the way for future structure activity relationship studies to optimize the pharmacological targeting of HMGB1·CXCL12 for anti-inflammatory purposes.

7.
Front Immunol ; 11: 1122, 2020.
Article in English | MEDLINE | ID: mdl-32670275

ABSTRACT

Acute inflammation is a complex biological response of tissues to harmful stimuli, such as pathogens or cell damage, and is essential for immune defense and proper healing. However, unresolved inflammation can lead to chronic disorders, including cancer and fibrosis. The High Mobility Group Box 1 (HMGB1) protein is a Damage-Associated Molecular Pattern (DAMP) molecule that orchestrates key events in inflammation by switching among mutually exclusive redox states. Fully reduced HMGB1 (frHMGB1) supports immune cell recruitment and tissue regeneration, while the isoform containing a disulphide bond (dsHMGB1) promotes secretion of inflammatory mediators by immune cells. Although it has been suggested that the tissue itself determines the redox state of the extracellular space and of released HMGB1, the dynamics of HMGB1 oxidation in health and disease are unknown. In the present work, we analyzed the expression of HMGB1 redox isoforms in different inflammatory conditions in skeletal muscle, from acute injury to muscle wasting, in tumor microenvironment, in spleen, and in liver after drug intoxication. Our results reveal that the redox modulation of HMGB1 is tissue-specific, with high expression of dsHMGB1 in normal spleen and liver and very low in muscle, where it appears after acute damage. Similarly, dsHMGB1 is highly expressed in the tumor microenvironment while it is absent in cachectic muscles from the same tumor-bearing mice. These findings emphasize the accurate and dynamic regulation of HMGB1 redox state, with the presence of dsHMGB1 tightly associated with leukocyte infiltration. Accordingly, we identified circulating, infiltrating, and resident leukocytes as reservoirs and transporters of dsHMGB1 in tissue and tumor microenvironment, demonstrating that the redox state of HMGB1 is controlled at both tissue and cell levels. Overall, our data point out that HMGB1 oxidation is a timely and spatially regulated process in physiological and pathological conditions. This precise modulation might play key roles to finetune inflammatory and regenerative processes.


Subject(s)
HMGB1 Protein/metabolism , Animals , Cachexia/immunology , Cachexia/metabolism , Chemical and Drug Induced Liver Injury/immunology , Chemical and Drug Induced Liver Injury/metabolism , Disease Models, Animal , HMGB1 Protein/deficiency , HMGB1 Protein/immunology , Inflammation/immunology , Inflammation/metabolism , Leukocytes/immunology , Leukocytes/metabolism , Liver/immunology , Liver/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Muscle, Skeletal/immunology , Muscle, Skeletal/injuries , Muscle, Skeletal/metabolism , Oxidation-Reduction , Spleen/immunology , Spleen/metabolism , Tumor Microenvironment/immunology , Tumor Microenvironment/physiology
8.
J Exp Med ; 215(1): 303-318, 2018 01 02.
Article in English | MEDLINE | ID: mdl-29203538

ABSTRACT

Inflammation and tissue regeneration follow tissue damage, but little is known about how these processes are coordinated. High Mobility Group Box 1 (HMGB1) is a nuclear protein that, when released on injury, triggers inflammation. We previously showed that HMGB1 with reduced cysteines is a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine. Here we report that fully reduced HMGB1 orchestrates muscle and liver regeneration via CXCR4, whereas disulfide HMGB1 and its receptors TLR4/MD-2 and RAGE (receptor for advanced glycation end products) are not involved. Injection of HMGB1 accelerates tissue repair by acting on resident muscle stem cells, hepatocytes, and infiltrating cells. The nonoxidizable HMGB1 mutant 3S, in which serines replace cysteines, promotes muscle and liver regeneration more efficiently than the wild-type protein and without exacerbating inflammation by selectively interacting with CXCR4. Overall, our results show that the reduced form of HMGB1 coordinates tissue regeneration and suggest that 3S may be used to safely accelerate healing after injury in diverse clinical contexts.


Subject(s)
HMGB1 Protein/metabolism , Liver Regeneration/physiology , Muscles/metabolism , Muscles/physiology , Receptors, CXCR4/metabolism , Animals , Cell Line , Chemotactic Factors/metabolism , Cytokines/metabolism , HEK293 Cells , Hepatocytes/metabolism , Hepatocytes/physiology , Humans , Inflammation/metabolism , Male , Mice , Mice, Inbred C57BL , Wound Healing/physiology
9.
Front Immunol ; 6: 422, 2015.
Article in English | MEDLINE | ID: mdl-26347745

ABSTRACT

Our body handles tissue damage by activating the immune system in response to intracellular molecules released by injured tissues [damage-associated molecular patterns (DAMPs)], in a similar way as it detects molecular motifs conserved in pathogens (pathogen-associated molecular patterns). DAMPs are molecules that have a physiological role inside the cell, but acquire additional functions when they are exposed to the extracellular environment: they alert the body about danger, stimulate an inflammatory response, and finally promote the regeneration process. Beside their passive release by dead cells, some DAMPs can be secreted or exposed by living cells undergoing a life-threatening stress. DAMPs have been linked to inflammation and related disorders: hence, inhibition of DAMP-mediated inflammatory responses is a promising strategy to improve the clinical management of infection- and injury-elicited inflammatory diseases. However, it is important to consider that DAMPs are not only danger signals but also central players in tissue repair. Indeed, some DAMPs have been studied for their role in tissue healing after sterile or infection-associated inflammation. This review is focused on two exemplary DAMPs, HMGB1 and adenosine triphosphate, and their contribution to both inflammation and tissue repair.

10.
J Exp Med ; 209(3): 551-63, 2012 Mar 12.
Article in English | MEDLINE | ID: mdl-22370717

ABSTRACT

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.


Subject(s)
Chemokine CXCL12/physiology , HMGB1 Protein/physiology , Inflammation/etiology , Receptors, CXCR4/physiology , Animals , Base Sequence , Calcium Signaling , Cell Movement/physiology , Chemokine CXCL12/chemistry , DNA, Complementary/genetics , Fibroblasts/physiology , Fluorescence Resonance Energy Transfer , HEK293 Cells , HMGB1 Protein/chemistry , Humans , Inflammation/pathology , Inflammation/physiopathology , MAP Kinase Signaling System , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Monocytes/physiology , Multiprotein Complexes/chemistry , NIH 3T3 Cells , Nuclear Magnetic Resonance, Biomolecular , Receptor for Advanced Glycation End Products , Receptors, CXCR4/chemistry , Receptors, CXCR4/genetics , Receptors, Immunologic/physiology , Signal Transduction , Surface Plasmon Resonance , Toll-Like Receptors/physiology , Transfection
11.
PLoS One ; 4(7): e6234, 2009 Jul 29.
Article in English | MEDLINE | ID: mdl-19641621

ABSTRACT

A number of nuclear events occur during apoptosis, including DNA laddering, nuclear lamina breakdown, phosphorylation of histones H2B and histone H2AX, and the tight binding to chromatin of HMGB1 and CAD, the nuclease responsible for DNA laddering. We have performed an epistasis analysis to investigate whether these events cluster together in pathways. We find that all depend directly or indirectly on caspase-3 activation. CAD activation, H2AX phosphorylation and DNA laddering cluster together into a pathway, but all other events appear to be independent of each other downstream of caspase-3, and likely evolved subject to different functional pressures.


Subject(s)
Apoptosis , Caspase 3/metabolism , Cell Nucleus/enzymology , Base Sequence , Caspase 6/metabolism , Cell Line , Chromatin/metabolism , DNA Primers , Enzyme Activation , Fluorescence , HMGB1 Protein/metabolism , Histones/metabolism , Humans , Phosphorylation
12.
AIDS ; 23(5): 567-77, 2009 Mar 13.
Article in English | MEDLINE | ID: mdl-19194273

ABSTRACT

OBJECTIVE: High mobility group box-1 (HMGB1) is a nuclear chromatin protein. Furthermore, it induces chemotaxis and inflammation once released in the extracellular milieu, and it has been reported to upregulate, but also to inhibit HIV-1 replication in different cell types. We here investigated the potential role of extracellular HMGB1 in both R5 and X4 HIV-1 replication in primary human monocyte-derived macrophages (MDM) and U937 promonocytic cells, respectively. DESIGN: MDM or U937 cells were infected with R5 and X4 HIV-1 strains, respectively, in the presence or absence of endotoxin-free recombinant (r) HMGB1 or necrotic cell supernatants either containing or depleted of endogenous HMGB1. METHODS: HIV replication was measured by means of virion-associated reverse transcriptase activity in culture supernatants and cell-associated viral protein expression. Cytokine and chemokine production were measured by enzyme-linked immunosorbent assay; cell surface expression of CD4, CC chemokine receptor 5, receptor for advanced glycation end-products, Toll-like receptor-2 and Toll-like receptor-4 were analyzed by flow cytometry. RESULTS: Both rHMGB1 and necrotic cell supernatant-associated HMGB1 inhibited replication of R5 HIV-1 in MDM. Surprisingly enough, no upregulation of CC chemokine receptor 5-binding chemokines or of other chemokines and cytokines was observed in rHMGB1-stimulated MDM. HMGB1 also induced chemotaxis and strongly inhibited the replication of X4 HIV-1 in the 'Minus' subset of U937 cell clones expressing high levels of putative HMGB1 receptors (receptor for advanced glycation end-products, Toll-like receptors 2 and 4). CONCLUSION: Extracellular HMGB1 is a potent inhibitor of both R5 and X4 HIV-1 replication in mononuclear phagocytic cells without inducing the release of HIV-Modulatory chemokines or cytokines.


Subject(s)
Anti-HIV Agents/pharmacology , HIV Infections/virology , HIV-1/drug effects , HMGB1 Protein/pharmacology , Macrophages/virology , Animals , Cells, Cultured , Chemokines/biosynthesis , Chemotaxis/drug effects , Cytokines/biosynthesis , Drug Evaluation, Preclinical/methods , HIV Infections/immunology , HIV-1/physiology , HMGB1 Protein/physiology , Humans , Macrophages/drug effects , Macrophages/immunology , Mice , Monocyte-Macrophage Precursor Cells/drug effects , Monocyte-Macrophage Precursor Cells/immunology , Monocyte-Macrophage Precursor Cells/virology , Receptor for Advanced Glycation End Products , Receptors, Immunologic/metabolism , Recombinant Proteins/pharmacology , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism , U937 Cells , Virus Replication/drug effects
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