Extracellular Histones Bind Vascular Glycosaminoglycans and Inhibit the Anti-Inflammatory Function of Antithrombin.

Binding of histones to molecular pattern recognition receptors on endothelial cells and leukocytes provokes proinflammatory responses and promotes activation of coagulation. Histones also bind therapeutic heparins, thereby neutralizing their anticoagulant functions. The aim of this study was to test the hypothesis that histones can interact with the antithrombin (AT)-binding vascular glycosaminoglycans (GAGs) to induce inflammation and inhibit the anti-inflammatory function of AT. METHODS: We evaluated the heparin-binding function of histones by an AT-dependent protease-inhibition assay. Furthermore, we treated endothelial cells with histones in the absence and presence of AT and monitored cellular phenotypes employing established signaling assays. RESULTS: Histones neutralized AT-dependent anticoagulant function of heparin in both purified protease-inhibition and plasma-based assays. Histones also disrupted endothelial cell barrier-permeability function by a GAG-dependent mechanism as evidenced by the GAG-antagonist, surfen, abrogating their disruptive effects. Further studies revealed histones and AT compete for overlapping binding-sites on GAGs, thus increasing concentrations of one protein abrogated effects of the other. Histones elicited proapoptotic effects by inducing nuclear localization of PKC-δ in endothelial cells and barrier-disruptive effects by destabilizing VE-cadherin, which were inhibited by AT, but not by a D-helix mutant of AT incapable of interacting with GAGs. Finally, histones induced release of Weibel-Palade body contents, VWF and angiopoietin-2, and promoted expression of cell adhesion molecules on endothelial cells, which were all downregulated by AT but not by D-helix mutant of AT. CONCLUSION: We conclude that histones and AT compete for overlapping binding sites on vascular GAGs to modulate coagulation and inflammation.

Histone H3 Cleavage in Severe COVID-19 ICU Patients

The severity of coronavirus disease 19 (COVID-19) is associated with neutrophil extracellular trap (NET) formation. During NET formation, cytotoxic extracellular histones are released, the presence of which is linked to the initiation and progression of several acute inflammatory diseases. Here we study the presence and evolution of extracellular histone H3 and several other neutrophil-related molecules and damage-associated molecular patterns (DAMPs) in the plasma of 117 COVID-19-positive ICU patients. We demonstrate that at ICU admission the levels of histone H3, MPO, and DNA-MPO complex were all significantly increased in COVID-19-positive patients compared to control samples. Furthermore, in a subset of 54 patients, the levels of each marker remained increased after 4+ days compared to admission. Histone H3 was found in 28% of the patients on admission to the ICU and in 50% of the patients during their stay at the ICU. Notably, in 47% of histone-positive patients, we observed proteolysis of histone in their plasma. The overall presence of histone H3 during ICU stay was associated with thromboembolic events and secondary infection, and non-cleaved histone H3 was associated with the need for vasoactive treatment, invasive ventilation, and the development of acute kidney injury. Our data support the validity of treatments that aim to reduce NET formation and additionally underscore that more targeted therapies focused on the neutralization of histones should be considered as treatment options for severe COVID-19 patients.

Prognostic role of levels of extracellular histones H3 for diagnosis of early dysfunction of transplant after hepatic transplantation

Objective. The aim of this study was to determine the prognostic role of extracellular histones in the diagnosis of early graft dysfunction after liver transplantation.. Materials and methods. The 93 recipients undergoing LDLT were enrolled in this prospective study. Blood samples of patients were collected on postoperative day 1 and histone levels in the plasma samples were measured with Total Histone H3 sandwich ELISA kits. 19 (20.4%) subjects had early graft dysfunction (EAD) which was diagnosed on postoperative day 7 according to Ol-thoff’s criteria, based on liver function tests and coagulation profiles. Other 74 (79.6%) recipients did not have EGD. Results. Levels of circulating histones were depressed in patients with EGD (0,808±0.026, 95% (CI) 0.752-0.864) than in patients without EGD (0.820±0.017, 95% (CI) 0.786-0.854) (P=0.727). These differences were not significant. The sources of histones in the circulation are not etiologies specific and levels of total histone H 3 after 24 h of operation had not stronger predictive value with AUC 0.477 (95 % CI 0.329 to 0.625) for liver dysfunction. The AUC value of the total bilirubin (AUC 0.685, 95 % CI 0.546 to 0.825) in predicting early graft dysfunction outperformed other LFTs and was less than CRP (AUC 0.705, CI 0.573 to 0.838). The optimal cutoff value of total bilirubin obtained from the analysis of ROC curves was 4,5 and surpassed all other parameters with a sensitivity of 94.4% and a specificity of 40.7% respectively for prognoses of EGD (P=0.012). The univariate analysis determined that postoperative neutrophils level and CRP were identified as independent risk factors for early graft dysfunction. Neutrophils had a higher predictive value for liver dysfunction than any other parameter within 24 h (Odds ratio (OR) 16.3; 95% CI: 1.7-156.3, P = 0.016). Conclusion. Collectively, extracellular histone H3 levels were depressed, total bilirubin and CRP levels were elevated in patients with EGD, which can be used as early predictors for liver tissue damage and early allograft dysfunction in patients after liver transplantation.

Intrabronchial application of extracellular histones shows no proinflammatory effects in swine in a translational pilot study

Objective Extracellular histones have been identified as one molecular factor that can cause and sustain alveolar damage and were linked to high mortality rates in critically ill patients. In this pilot study, we wanted to validate the proinflammatory in vivo effects of local histone application in a prospective translational porcine model. This was combined with the evaluation of an experimental acute lung injury model using intrabronchial lipopolysaccharides, which has been published previously. Results The targeted application of histones was successful in all animals. Animals showed decreased oxygenation after instillation, but no differences could be detected between the sham and histone treatments. The histologic analyses and inflammatory responses indicated that there were no differences in tissue damage between the groups.

Plasma Protein Layer Concealment Protects Streptococcus pyogenes From Innate Immune Attack

Early recognition and elimination of invading pathogens by the innate immune system, is one of the most efficient host defense mechanisms preventing the induction of systemic complications from infection. To this end the host can mobilize endogenous antimicrobials capable of killing the intruder by perforating the microbial cell wall. Here, we show that Streptococcus pyogenes can shield its outer surface with a layer of plasma proteins. This mechanism protects the bacteria from an otherwise lytic attack by LL-37 and extracellular histones, allowing the bacteria to adjust their gene regulation to an otherwise hostile environment.

Extracellular histones are a target in myocardial ischaemia reperfusion injury

Acute myocardial infarction causes lethal cardiomyocyte injury during ischaemia and reperfusion (I/R). Histones have been described as important Danger Associated Molecular Proteins (DAMPs) in sepsis. Aims The objective of this study was to establish whether extracellular histone release contributes to myocardial infarction. Methods and results Isolated, perfused rat hearts were subject to I/R. Nucleosomes and histone H4 release was detected early during reperfusion. Sodium-β-O-Methyl cellobioside sulfate (mCBS), a newly developed histone-neutralising compound, significantly reduced infarct size whilst also reducing the detectable levels of histones. Histones were directly toxic to primary adult rat cardiomyocytes in vitro. This was prevented by mCBS, or HIPe, a recently described, histone-H4 neutralizing peptide, but not by an inhibitor of TLR4, a receptor previously reported to be involved in DAMP-mediated cytotoxicity. Furthermore, TLR4-reporter HEK293 cells revealed that cytotoxicity of histone H4 was independent of TLR4 and NF-κB. In an in vivo rat model of I/R, HIPe significantly reduced infarct size. Conclusion Histones released from the myocardium are cytotoxic to cardiomyocytes, via a TLR4-independent mechanism. The targeting of extracellular histones provides a novel opportunity to limit cardiomyocyte death during I/R injury of the myocardium. Translational perspective Acute myocardial infarction causes lethal cardiomyocyte injury during ischaemia and reperfusion (I/R). New approaches are needed to prevent cardiomyocyte injury and limit final infarct size. We show that histones released from damaged cells, and histone-H4 in particular, causes rapid cardiomyocyte death during I/R. mCBS, a compounds targeting histones non-specifically, was cardioprotective in ex vivo rat hearts, while HIPe, a targeting histone H4 specifically, was cardioprotective in an in vivo rat model. HIPe may have potential as a therapeutic agent in the setting of acute myocardial infarction.