Neutralizing the pathological effects of extracellular histones with small polyanions.
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ABSTRACT:
SUBMITTER: Meara CHO
PROVIDER: S-EPMC7744542 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The uptake and clearance of apoptotic cells by macrophages and other phagocytic cells, a process called efferocytosis, is a major component in the resolution of inflammation. Increased concentrations of extracellular histones are found during acute inflammatory states and appear to contribute to organ system dysfunction and mortality. In these studies, we examined the potential role of histones in modulating efferocytosis. We found that phagocytosis of apoptotic neutrophils or thymocytes by macrophages was significantly diminished in the presence of histones H3 or H4, but not histone H1. Histone H3 demonstrated direct binding to macrophages, an effect that was diminished by preincubation of macrophages with the opsonins growth arrest-specific gene 6 (Gas6) and milk fat globule-epidermal growth factor (EGF) 8 (MFG-E8). Incubation of histone H3 with soluble ?(v)?? integrin and Mer, but not with ?(v)??, diminished its binding to macrophages. Phagocytosis of apoptotic cells by alveolar macrophages in vivo was diminished in the presence of histone H3. Incubation of histone H3 with activated protein C, a treatment that degrades histones, abrogated its inhibitory effects on efferocytosis under both in vitro and in vivo conditions. The present studies demonstrate that histones have inhibitory effects on efferocytosis, suggesting a new mechanism by which extracellular histones contribute to acute inflammatory processes and tissue injury.
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Project description:Heterozygous mutations in the GBA gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), are the strongest known genetic risk factor for Parkinson's disease (PD). The molecular mechanisms underlying the increased PD risk and the variable phenotypes observed in carriers of different GBA mutations are not yet fully elucidated. Extracellular vesicles (EVs) have gained increasing importance in neurodegenerative diseases since they can vehiculate pathological molecules potentially promoting disease propagation. Accumulating evidence showed that perturbations of the endosomal-lysosomal pathway can affect EV release and composition. Here, we investigate the impact of GCase deficiency on EV release and their effect in recipient cells. EVs were purified by ultracentrifugation from the supernatant of fibroblast cell lines derived from PD patients with or without GBA mutations and quantified by nanoparticle tracking analysis. SH-SY5Y cells over-expressing alpha-synuclein (α-syn) were used to assess the ability of patient-derived small EVs to affect α-syn expression. We observed that defective GCase activity promotes the release of EVs, independently of mutation severity. Moreover, small EVs released from PD fibroblasts carrying severe mutations increased the intra-cellular levels of phosphorylated α-syn. In summary, our work shows that the dysregulation of small EV trafficking and alpha-synuclein mishandling may play a role in GBA-associated PD.
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Project description:In response to various infectious and sterile stimuli neutrophils release chromatin decorated with bactericidal proteins, referred to as NETs. Their scaffolds are formed from chromatin fibers which display an apparent diameter of 15-17 nm and mainly consist from DNA (2 nm) and DNA-associated histones (11 nm). The NET-forming strands are thus not naked DNA but higher ordered chromatin structures. The histones may be released from the NET, especially if their tail arginines have been citrullinated. Several studies indicate that extracellular histones are toxic for mammalian epithelia and endothelia and contribute to the microvascular dysfunction observed e.g., in patients suffering from autoimmune diseases or sepsis. NETs formed at sites of very high neutrophil densities tend to clump and form fairly stable enzymatically active aggregates, referred to as aggNETs. The latter are endowed with a bunch of enzymes that cleave, bind, and/or modify autologous as well as foreign macromolecules. The tight binding of the serine proteases to the matrix precludes the spread of these toxic enzymes into the tissue but still allows the access of soluble inflammatory mediators to the enzymatic active internal surfaces of the NETs where they are degraded. Here, we describe that externally added histones are removed from culture supernatants of aggNETs. We will address the fate of the histones and discuss the feature on the background of neutrophil-driven diseases and the resolution of inflammation.
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Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had spread from China and, within 2 months, became a global pandemic. The infection from this disease can cause a diversity of symptoms ranging from asymptomatic to severe acute respiratory distress syndrome with an increased risk of vascular hyperpermeability, pulmonary inflammation, extensive lung damage, and thrombosis. One of the host defense systems against coronavirus disease 2019 (COVID-19) is the formation of neutrophil extracellular traps (NETs). Numerous studies on this disease have revealed the presence of elevated levels of NET components, such as cell-free DNA, extracellular histones, neutrophil elastase, and myeloperoxidase, in plasma, serum, and tracheal aspirates of severe COVID-19 patients. Extracellular histones, a major component of NETs, are clinically very relevant as they represent promising biomarkers and drug targets, given that several studies have identified histones as key mediators in the onset and progression of various diseases, including COVID-19. However, the role of extracellular histones in COVID-19 per se remains relatively underexplored. Histones are nuclear proteins that can be released into the extracellular space via apoptosis, necrosis, or NET formation and are then regarded as cytotoxic damage-associated molecular patterns that have the potential to damage tissues and impair organ function. This review will highlight the mechanisms of extracellular histone-mediated cytotoxicity and focus on the role that histones play in COVID-19. Thereby, this paper facilitates a bench-to-bedside view of extracellular histone-mediated cytotoxicity, its role in COVID-19, and histones as potential drug targets and biomarkers for future theranostics in the clinical treatment of COVID-19 patients.
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Project description:ObjectiveExtracellular 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.ResultsThe 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.
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Project description:ObjectiveExtracellular histone (EH) is involved in the development of septic myocardial injury (SMI). In this study, we explored whether EH could induce left ventricular diastolic dysfunction (LVDD) in sepsis, and investigated the potential mechanisms through in vivo and in vitro experiments using animal models.MethodsThe ratio between E-wave and A-wave (E/A ratio), left ventricular end diastolic volume, and isovolumic relaxation time (IVRT) were measured in cecal ligation and perforation (CLP)- and EH-treated male C57BL/6J mice using echocardiography. The protein and mRNA levels of apoptosis-related proteins (cleaved caspase-3, Bcl-2, and Bax) and cardiac troponin T (cTnT) in the left ventricular tissue/cardiomyocytes were measured using enzyme-linked immunosorbent assay, qRT-PCR, and western blotting. Cardiomyocyte apoptosis was detected by flow cytometry.ResultsCLP mice presented with LVDD, which was accompanied by increased circulating histones, cTnT and Bax protein levels. Circulating histones were correlated with cTnT, Bax, IVRT, and E/A ratio in CLP mice. Intraperitoneal injection of EH resulted in LVDD in mice. EH induced cardiomyocyte apoptosis, and histone neutralizing agents improved SMI and protected mice against CLP- and EH-induced death.ConclusionEH is involved in septic LVDD, and this alteration might be associated with EH-induced apoptosis. EH may serve as a potential therapeutic target for SMI.
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Project description:Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti-glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NET-related histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN.
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Project description:Neutrophil infiltration is a hallmark of periodontitis, a prevalent oral inflammatory condition in which Th17-driven mucosal inflammation leads to destruction of tooth-supporting bone. Herein, we document that neutrophil extracellular traps (NETs) are early triggers of pathogenic inflammation in periodontitis. In an established animal model, we demonstrate that neutrophils infiltrate the gingival oral mucosa at early time points after disease induction and expel NETs to trigger mucosal inflammation and bone destruction in vivo. Investigating mechanisms by which NETs drive inflammatory bone loss, we find that extracellular histones, a major component of NETs, trigger upregulation of IL-17/Th17 responses, and bone destruction. Importantly, human findings corroborate our experimental work. We document significantly increased levels of NET complexes and extracellular histones bearing classic NET-associated posttranslational modifications, in blood and local lesions of severe periodontitis patients, in the absence of confounding disease. Our findings suggest a feed-forward loop in which NETs trigger IL-17 immunity to promote immunopathology in a prevalent human inflammatory disease.
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Project description:Extracellular vesicles are released by all cell types and contain proteins, microRNAs, mRNAs, and other bioactive molecules. Extracellular vesicles play an important role in intercellular communication and in the modulation of the immune system and neuroinflammation. The cargo of extracellular vesicles (e.g., proteins and microRNAs) is altered in pathological situations. Extracellular vesicles contribute to the pathogenesis of many pathologies associated with sustained inflammation and neuroinflammation, including cancer, diabetes, hyperammonemia and hepatic encephalopathy, and other neurological and neurodegenerative diseases. Extracellular vesicles may cross the blood-brain barrier and transfer pathological signals from the periphery to the brain. This contributes to inducing neuroinflammation and cognitive and motor impairment in hyperammonemia and hepatic encephalopathy and in neurodegenerative diseases. The mechanisms involved are beginning to be understood. For example, increased tumor necrosis factor α in extracellular vesicles from plasma of hyperammonemic rats induces neuroinflammation and motor impairment when injected into normal rats. Identifying the mechanisms by which extracellular vesicles contribute to the pathogenesis of these diseases will help to develop new treatments and diagnostic tools for their easy and early detection. In contrast, extracellular vesicles from mesenchymal stem cells have therapeutic utility in many of the above pathologies, by reducing inflammation and neuroinflammation and improving cognitive and motor function. These extracellular vesicles recapitulate the beneficial effects of mesenchymal stem cells and have advantages as therapeutic tools: they are less immunogenic, may not differentiate to malignant cells, cross the blood-brain barrier, and may reach more easily target organs. Extracellular vesicles from mesenchymal stem cells have beneficial effects in models of ischemic brain injury, Alzheimer's and Parkinson's diseases, hyperammonemia, and hepatic encephalopathy. Extracellular vesicles from mesenchymal stem cells modulate the immune system, promoting the shift from a pro-inflammatory to an anti-inflammatory state. For example, extracellular vesicles from mesenchymal stem cells modulate the Th17/Treg balance, promoting the anti-inflammatory Treg. Extracellular vesicles from mesenchymal stem cells may also act directly in the brain to modulate microglia activation, promoting a shift from a pro-inflammatory to an anti-inflammatory state. This reduces neuroinflammation and improves cognitive and motor function. Two main components of extracellular vesicles from mesenchymal stem cells which contribute to these beneficial effects are transforming growth factor-β and miR-124. Identifying the mechanisms by which extracellular vesicles from mesenchymal stem cells induce the beneficial effects and the main molecules (e.g., proteins and mRNAs) involved may help to improve their therapeutic utility. The aims of this review are to summarize the knowledge of the pathological effects of extracellular vesicles in different pathologies, the therapeutic potential of extracellular vesicles from mesenchymal stem cells to recover cognitive and motor function and the molecular mechanisms for these beneficial effects on neurological function.
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Project description:Hyperinflammatory responses can lead to a variety of diseases, including sepsis. We now report that extracellular histones released in response to inflammatory challenge contribute to endothelial dysfunction, organ failure and death during sepsis. They can be targeted pharmacologically by antibody to histone or by activated protein C (APC). Antibody to histone reduced the mortality of mice in lipopolysaccharide (LPS), tumor necrosis factor (TNF) or cecal ligation and puncture models of sepsis. Extracellular histones are cytotoxic toward endothelium in vitro and are lethal in mice. In vivo, histone administration resulted in neutrophil margination, vacuolated endothelium, intra-alveolar hemorrhage and macro- and microvascular thrombosis. We detected histone in the circulation of baboons challenged with Escherichia coli, and the increase in histone levels was accompanied by the onset of renal dysfunction. APC cleaves histones and reduces their cytotoxicity. Co-infusion of APC with E. coli in baboons or histones in mice prevented lethality. Blockade of protein C activation exacerbated sublethal LPS challenge into lethality, which was reversed by treatment with antibody to histone. We conclude that extracellular histones are potential molecular targets for therapeutics for sepsis and other inflammatory diseases.
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