Neutralization Of Histones Research Articles

Synthetic hydrogel nanoparticles for sepsis therapy

Sepsis is a life-threatening condition caused by the extreme release of inflammatory mediators into the blood in response to infection (e.g., bacterial infection, COVID-19), resulting in the dysfunction of multiple organs. Currently, there is no direct treatment for sepsis. Here we report an abiotic hydrogel nanoparticle (HNP) as a potential therapeutic agent for late-stage sepsis. The HNP captures and neutralizes all variants of histones, a major inflammatory mediator released during sepsis. The highly optimized HNP has high capacity and long-term circulation capability for the selective sequestration and neutralization of histones. Intravenous injection of the HNP protects mice against a lethal dose of histones through the inhibition of platelet aggregation and migration into the lungs. In vivo administration in murine sepsis model mice results in near complete survival. These results establish the potential for synthetic, nonbiological polymer hydrogel sequestrants as a new intervention strategy for sepsis therapy and adds to our understanding of the importance of histones to this condition.

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.

Heparin Attenuates Histone-Mediated Cytotoxicity in Septic Acute Kidney Injury.

Histones are considered potential risk factors that contribute to the development of septic acute kidney injury (SAKI) by inducing apoptosis and inflammation. This study aimed to explore the protective effects of heparin on septic acute kidney injury through the neutralization of extracellular histones (EH) and to uncover the underlying mechanism. C57BL mice (16 each) were randomly divided into the sham group, the sepsis group (established by cecal ligation and puncture operation, CLP), and the heparin intervention group. Mice in the heparin intervention group received a subcutaneous injection of unfractionated heparin (0.03 IU/g) 4 h after CLP. At 6 h after the operation, nine mice from each group were sacrificed by the removal of the eyeballs to harvest blood samples; the upper half of the right kidney was used as the study sample. Mice renal tubular epithelial cells cultivated in six-well plates were equally divided into five groups. We cultured cells treated with either histone (40 U), histone (40 U) + heparin (25 IU/ml), histone(40U) + lipopolysaccharides (LPS; 10 μg/ml), or histone (40 U) + LPS (10 μg/ml) + heparin (25 IU/ml) for 6 h. For the histone + heparin group and the histone + LPS + heparin group, histone (and LPS) were treated with heparin simultaneously. Mice in the heparin intervention group showed decreased levels of EH4, neutrophil gelatinase-associated lipocalin (NAGL), kidney injury molecule-1 (KIM-1), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6 in the blood serum, longer average 72-h survival rate, significantly decreased kidney tissue edema, and a clearer glomerular structure coupled with decreased protein and mRNA expression levels of kidney apoptosis-related proteins (cleaved Caspase-3/Caspase-3 and Bax/Bcl-2) compared with those in the sepsis group at 6 h after CLP (P < 0.05). Meanwhile, cells in the heparin intervention group exhibited lower expression levels of serum EH4 and inflammatory cytokines, a lower apoptosis rate, and decreased expression of apoptosis-related proteins, both at protein and mRNA levels, than those in the histone-stimulated group at 6 h after stimulation (P < 0.05). Heparin may alleviate apoptosis and inflammation through the neutralization of histones, thus playing a protective role against septic acute kidney injury.

Extracellular histones in lung dysfunction: a new biomarker and therapeutic target?

Extracellular histones released from injured or dying cells following trauma and other severe insults can act as potent damage-associated molecular patterns. In fact, elevated levels of histones are present in human circulation in hyperinflammatory states such as acute respiratory distress syndrome and sepsis. The molecular mechanisms owing to histone-induced pathologies are at the very beginning of elucidating. However, neutralization of histones with antibodies, histone-binding or histone-degrading proteins, and heparan sulfates have shown promising therapeutic effects in pre-clinical acute respiratory distress syndrome and sepsis models. Various cell types undergoing necrosis and apoptosis or activated neutrophils forming neutrophil extracellular traps have been implicated in excessive release of histones which further augments tissue injury and may culminate in multiple organ failure. At the molecular level, an uncontrolled inflammatory cascade has been considered as the major event; however, histone-activated coagulation and thrombosis represent additional pathologic events reflecting coagulopathy. Furthermore, epigenetic regulation and chemical modifications of circulating histones appear to be critically important in their biological functions as evidenced by increased cytotoxicity associated with citrullinated histone. Herein, we will briefly review the current knowledge on the role of histones in acute respiratory distress syndrome and sepsis, and discuss the future potential of anti-histone therapy for treatment of these life-threatening disorders.

RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome

The development of multiple organ dysfunction syndrome (MODS) following infection or tissue injury is associated with increased patient morbidity and mortality. Extensive cellular injury results in the release of nuclear proteins, of which histones are the most abundant, into the circulation. Circulating histones are implicated as essential mediators of MODS. Available anti-histone therapies have failed in clinical trials due to off-target effects such as bleeding and toxicity. Here, we describe a therapeutic strategy for MODS based on the neutralization of histones by chemically stabilized nucleic acid bio-drugs (aptamers). Systematic evolution of ligands by exponential enrichment technology identified aptamers that selectively bind those histones responsible for MODS and do not bind to serum proteins. We demonstrate the efficacy of histone-specific aptamers in human cells and in a murine model of MODS. These aptamers could have a significant therapeutic benefit in the treatment of multiple diverse clinical conditions associated with MODS.

HMGB1 and Extracellular Histones Significantly Contribute to Systemic Inflammation and Multiple Organ Failure in Acute Liver Failure.

Acute liver failure (ALF) is the culmination of severe liver cell injury from a variety of causes. ALF occurs when the extent of hepatocyte death exceeds the hepatic regenerative capacity. ALF has a high mortality that is associated with multiple organ failure (MOF) and sepsis; however, the underlying mechanisms are still not clear. Emerging evidence shows that ALF patients/animals have high concentrations of circulating HMGB1, which can contribute to multiple organ injuries and mediate gut bacterial translocation (BT). BT triggers/induces systemic inflammatory responses syndrome (SIRS), which can lead to MOF in ALF. Blockade of HMGB1 significantly decreases BT and improves hepatocyte regeneration in experimental acute fatal liver injury. Therefore, HMGB1 seems to be an important factor that links BT and systemic inflammation in ALF. ALF patients/animals also have high levels of circulating histones, which might be the major mediators of systemic inflammation in patients with ALF. Extracellular histones kill endothelial cells and elicit immunostimulatory effect to induce multiple organ injuries. Neutralization of histones can attenuate acute liver, lung, and brain injuries. In conclusion, HMGB1 and histones play a significant role in inducing systemic inflammation and MOF in ALF.

Factor VII-Activating Protease Is a Regulator of Histone Cytotoxicity

Background: During sepsis, ischemia-reperfusion injury and sterile inflammation, levels of circulating histones and nucleosomes have been found to correlate with disease severity. Although the pathophysiological effects of circulating nucleosomes are not fully understood, free histones have been described to mediate cytotoxicity and contribute to disease severity. Several plasma proteins have been identified that modulate these cytotoxic effects by neutralizing or degrading histones. We have previously described that the plasma serine protease Factor VII-activating protease (FSAP) is activated upon contact with late apoptotic or necrotic cells and induces release of nucleosomes from these cells. During this release, histone H1 is cleaved. In addition, it has been described that FSAP is (auto)activated upon contact with histones and subsequently degrades histone H3.Aims: To investigate the regulation of histone-induced cytotoxicity by FSAP. Moreover, we aimed to compare differences in the cytotoxicity of histones and nucleosomes (histone+DNA) and potential differences in their proteolysis by FSAP.Methods: To determine FSAP activation and proteolysis of histones in serum we incubated normal donor or FSAP-deficient serum with 100ug/ml calf thymus histones. Upon its activation FSAP is rapidly bound by plasma inhibitors and we measured complexes of FSAP and its plasma inhibitor alpha 2-antiplasmin as an indirect readout for FSAP activation. To detect FSAP mediated proteolysis of histones we performed immunoblotting for histone H3. Results were corroborated using plasma-purified or recombinant FSAP. To determine histone-mediated cytotoxicity, we measured LDH levels after overnight incubation of HEK293 or ECRF cells with 100ug/ml calf thymus histone that had undergone a pre-incubation step in the presence or absence of healthy donor serum, FSAP-deficient serum or FSAP-depleted serum. Moreover, we tested the cytotoxicity of purified nucleosomes that were pre-incubated in the presence or absence of benzonase to degrade DNA.Results: EndogenousFSAP in serum was activated upon contact with histones, and histone proteolysis was observed. Similarly, histones were proteolyzed upon incubation using active plasma-purified or pre-activated recombinant FSAP, whereas no histone proteolysis was observed using recombinant inactive FSAP. These results indicate that FSAP is responsible for the observed histone degradation. FSAP-mediated histone degradation was able to protect HEK293 and ECRF cells against histone-induced cytotoxicity, showing that the resultant histone breakdown products had lost their cytotoxic effects. Recombinant inactive FSAP did not affect histone-induced cytotoxicity, indicating that the catalytic activity of FSAP is required for the observed protective effect of FSAP on cytotoxicity. When FSAP-deficient or FSAP-depleted serum was used, this led to greatly diminished histone proteolysis and reduced histone-induced cytotoxicity. Using our assay, we found that nucleosomes did not show cytotoxic effects unless they had been pre-incubated with benzonase to release histones. Notably, whereas intact nucleosomes were resistant against degradation by FSAP, FSAP protected against the cytotoxicity induced by histones released from these nucleosomes.Conclusion: FSAP in serum is activated by histones and subsequently proteolyses histones ,resulting in protection of cells from histone-induced cytotoxicity. Histones in the form of nucleosomes did not induce cytotoxicity, however after DNA removal the histones regained cytotoxicity. FSAP efficiently protected against the cytotoxicity of the unbound histones. We predict that the form in which histones are released into the blood is highly relevant in the context of human health and disease. We speculate that when endogenous DNases in serum degrade the DNA in nucleosomes, or alternatively, when highly cytotoxic histones are inadvertently released from dead cells in their free form rather than in the form of nucleosomes, FSAP mediates their efficient degradation to protect against the damaging effects of free histones. Neutralization of histones by FSAP may be an effective therapeutic approach to treat systemic inflammation. DisclosuresZeerleder:ViroPharma: Other: Receives an unrestricted grant from Viropharma.

Extracellular Chromatin Is an Important Mediator of Ischemic Stroke in Mice

Recently, a growing number of studies have revealed a prothrombotic and cytotoxic role for extracellular chromatin. Cerebral ischemia/reperfusion injury is characterized by a significant amount of cell death and neutrophil activation, both of which may result in the release of chromatin. The goal of this study was to assess the effect of extracellular chromatin in ischemic stroke using a mouse model of transient middle cerebral artery occlusion. Similar to reports in stroke patients, we observed increased levels of circulating nucleosomes and DNA after ischemic stroke in mice. In addition, we observed that general hypoxia also augmented extracellular chromatin. We hypothesized that targeting extracellular chromatin components would be protective in ischemic stroke. Indeed, treatment with recombinant human DNase 1 significantly improved stroke outcome. Neutralization of histones using an antihistone antibody was also protective as evidenced by smaller infarct volumes, whereas increasing levels of extracellular histones via histone infusion exacerbated stroke outcome by increasing infarct size and worsening functional outcome. Our results indicate that extracellular chromatin is generated and is detrimental during cerebral ischemia/reperfusion in mice. Targeting DNA and histones may be a new therapeutic strategy to limit injury resulting from ischemic stroke.