Dataset Information

Recombinant Thrombomodulin Suppresses Histone-Induced Neutrophil Extracellular Trap Formation.

ABSTRACT: Histones, the major protein components of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and subsequently mediate organ failure. Extracellular histones can promote endothelial damage and platelet aggregation, which can be suppressed by administration of recombinant thrombomodulin (rTM). The present study aimed to clarify whether histones can activate neutrophils to induce NET formation and whether rTM can prevent histone-induced NET formation. NET formation was analyzed in vitro by stimulating human neutrophils with histones in the absence or presence of rTM. NET formation was further analyzed in vivo by intravenous infusion of histones into rats with or without rTM. Histones induced NET release in a dose-dependent manner in vitro and NET release was induced as early as 1 h after stimulation. Histone-induced NET release was independent of NADPH oxidase. rTM suppressed histone-induced NET release in vitro as well as in vivo. The suppression might be mediated by rTM binding to histones, as suggested by analysis using a quartz crystal microbalance system. The present findings suggest that histones can activate neutrophils to form NETs and that rTM can inhibit histone-induced NET formation.

SUBMITTER: Shrestha B

PROVIDER: S-EPMC6828967 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Recombinant Thrombomodulin Suppresses Histone-Induced Neutrophil Extracellular Trap Formation.

Shrestha Binita B Ito Takashi T Kakuuchi Midori M Totoki Takaaki T Nagasato Tomoka T Yamamoto Mika M Maruyama Ikuro I

Frontiers in immunology 20191029

Histones, the major protein components of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and subsequently mediate organ failure. Extracellular histones can promote endothelial damage and platelet aggregation, which can be suppressed by administration of recombinant thrombomodulin (rTM). The present study aimed to clarify whether histones can activate neutrophils to induce NET formation and whether rTM can prevent histone-induced NET f ...[more]

PMID: 31736962

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Project description:Neutrophils undergo a unique form of cell death to generate neutrophil extracellular traps (NETs). It is well established that citrullination of histones (e.g., CitH3) facilitates chromatin decondensation during NET formation (NETosis), particularly during calcium-induced NETosis that is independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activation. However, the importance of other forms of histone modifications in NETosis has not been established. We considered that acetylation of histones would also facilitate NETosis. To test this hypothesis, we induced NOX-dependent NETosis in human neutrophils with phorbol myristate acetate or lipopolysaccharide (from Escherichia coli 0128), and NOX-independent NETosis with calcium ionophores A23187 or ionomycin (from Streptomycesconglobatus) in the presence or absence of two pan histone deacetylase inhibitors (HDACis), belinostat and panobinostat (within their half maximal inhibitory concentration (IC50) range). The presence of these inhibitors increased histone acetylation (e.g., AcH4) in neutrophils. Histone acetylation was sufficient to cause a significant increase (~20%) in NETosis in resting neutrophils above baseline values. When acetylation was promoted during NOX-dependent or -independent NETosis, the degree of NETosis additively increased (~15⁻30%). Reactive oxygen species (ROS) production is essential for baseline NETosis (mediated either by NOX or mitochondria); however, HDACis did not promote ROS production. The chromatin decondensation step requires promoter melting and transcriptional firing in both types of NETosis; consistent with this point, suppression of transcription prevented the NETosis induced by the acetylation of histones. Collectively, this study establishes that histone acetylation (e.g., AcH4) promotes NETosis at baseline, and when induced by both NOX-dependent or -independent pathway agonists, in human neutrophils. Therefore, we propose that acetylation of histone is a key component of NETosis.

Project description:Myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA)-associated vasculitis is a systemic small-vessel vasculitis, wherein, MPO-ANCA plays a critical role in the pathogenesis. Neutrophil extracellular traps (NETs) released from activated neutrophils are composed of extracellular web-like DNA and antimicrobial proteins, including MPO. Diverse stimuli, such as phorbol myristate acetate (PMA) and ligands of toll-like receptors (TLR), induce NETs. Although TLR-mediated NET formation can occur with preservation of living neutrophilic functions (called vital NETosis), PMA-stimulated neutrophils undergo cell death with NET formation (called suicidal NETosis). In the process of suicidal NETosis, histones are citrullinated by peptidylarginine deiminase 4 (PAD4). Since this step is necessary for decondensation of DNA, PAD4 plays a pivotal role in suicidal NETosis. Although NETs are essential for elimination of microorganisms, excessive formation of NETs has been suggested to be implicated in MPO-ANCA production. This study aimed to determine if pan-PAD inhibitors could suppress MPO-ANCA production in vivo. At first, NETs were induced in peripheral blood neutrophils derived from healthy donors (1 × 10(6)/ml) by stimulation with 20 nM PMA with or without 20 μM propylthiouracil (PTU), an anti-thyroid drug. We then determined that the in vitro NET formation was inhibited completely by 200 μM Cl-amidine, a pan-PAD inhibitor. Next, we established mouse models with MPO-ANCA production. BALB/c mice were given intraperitoneal (i.p.) injection of PMA (50 ng at days 0 and 7) and oral PTU (2.5 mg/day) for 2 weeks. These mice were divided into two groups; the first group was given daily i.p. injection of PBS (200 μl/day) (n = 13) and the other group with daily i.p. injection of Cl-amidine (0.3 mg/200 μl PBS/day) (n = 7). Two weeks later, citrullination as an indicator of NET formation in the peritoneum and serum MPO-ANCA titer was compared between the two groups. Results demonstrated that citrullination in the peritoneum was significantly reduced in the Cl-amidine-treated mice compared with the vehicle-injected control mice (38% reduction). Additionally, the serum MPO-ANCA titer of the Cl-amidine-treated mice (32.3 ± 31.0 ng/ml) was significantly lower than that in the vehicle-injected mice (132.1 ± 41.6 ng/ml). The collective findings indicate that excessive formation of NETs may be implicated in MPO-ANCA production in vivo.

Project description:Tonicity of saline (NaCl) is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET) formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA), Gram-negative bacterial cell wall component lipopolysaccharide (LPS), calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus), and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus). We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2)-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12), and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol). This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic solutions do not suppress NOX2-independent NETosis. Although hypertonic saline partially suppresses ionomycin-induced NETosis, it enhances A23187-induced NETosis, and it does not alter S. aureus-induced NETosis. Overall, this study determined that hypertonic saline suppresses NOX2-dependent NETosis induced by several agonists; in contrast, it has variable effects on neutrophil death induced by NOX2-independent NETosis agonists. These findings are important in understanding the regulation of NETosis and apoptosis in neutrophils.

Project description:IntroductionRecent studies have shown that histones, the chief protein component of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and act as major mediators of the death of an organism. This study was designed to elucidate the cellular and molecular basis of histone-induced lethality and to assess the protective effects of recombinant thrombomodulin (rTM). rTM has been approved for the treatment of disseminated intravascular coagulation (DIC) in Japan, and is currently undergoing a phase III clinical trial in the United States.MethodsHistone H3 levels in plasma of healthy volunteers and patients with sepsis and DIC were measured using enzyme-linked immunosorbent assay. Male C57BL/6 mice were injected intravenously with purified histones, and pathological examinations were performed. The protective effects of rTM against histone toxicity were analyzed both in vitro and in mice.ResultsHistone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC. These levels were higher in non-survivors than in survivors. Extracellular histones triggered platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries and subsequent right-sided heart failure in mice. These mice displayed symptoms of DIC, including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding. Platelet depletion protected mice from histone-induced death in the first 30 minutes, suggesting that vessel occlusion by platelet-rich thrombi might be responsible for death during the early phase. Furthermore, rTM bound to extracellular histones, suppressed histone-induced platelet aggregation, thrombotic occlusion of pulmonary capillaries, and dilatation of the right ventricle, and rescued mice from lethal thromboembolism.ConclusionsExtracellular histones cause massive thromboembolism associated with consumptive coagulopathy, which is diagnostically indistinguishable from DIC. rTM binds to histones and neutralizes the prothrombotic action of histones. This may contribute to the effectiveness of rTM against DIC.

Dataset Information

Recombinant Thrombomodulin Suppresses Histone-Induced Neutrophil Extracellular Trap Formation.

Publications

Recombinant Thrombomodulin Suppresses Histone-Induced Neutrophil Extracellular Trap Formation.

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