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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.

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.

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.

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.

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.

Our body handles tissue damage by activating the immune system in response to intracellularmolecules 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, PAMPs). DAMPs are molecules that have a physiological role inside the cell, but acquire additional functions when they are released outside the cell: 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 ATP, and their contribution to both inflammation and tissue repair.

Mouse embryonic stem cells (ESCs) are pluripotent mammalian cells derived from the inner cell mass (ICM) of mouse blastocysts, which give rise to all three embryonic germ layers both in vivo and in vitro. Mouse ESCs share a distinct epigenetic landscape and a more decondensed chromatin compared to differentiated cells. Numerous studies have shown that distinct histone modifications in ESCs serve as landmarks of pluripotency. However, so far it is still unknown whether the total histone amount (as opposed to histone modifications) in cells of different developmental stage and differentiation capacity remains the same. In this work we show that the total histone content differs between pluripotent and differentiated cells. In vitro spontaneous differentiation from ESCs to Embryoid Bodies (EBs) and directed differentiation towards neuronal and endodermal cells entails an increase in histone content. Primary MEFs also contain more histones than ESCs. We suggest that the difference in histone content is an additional landmark of pluripotency, in addition to and besides histone modifications.

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.