scholarly journals Histone Acetylation Promotes Neutrophil Extracellular Trap Formation

Biomolecules ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 32 ◽  
Author(s):  
Hussein Hamam ◽  
Meraj Khan ◽  
Nades Palaniyar
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylase Inhibitors ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Neutrophil Extracellular Trap ◽  
Human Neutrophils ◽  
Ros Production ◽  
Unique Form ◽  
Chromatin Decondensation ◽  
Extracellular Traps ◽  
Baseline Values

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 Streptomyces conglobatus) 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.

scholarly journals Histone Deacetylase Inhibitors Dose-Dependently Switch Neutrophil Death from NETosis to Apoptosis

Biomolecules ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 184 ◽  
Author(s):  
Hussein J. Hamam ◽  
Nades Palaniyar
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Histone Deacetylase Inhibitors ◽  
Significant Inhibition ◽  
The Body ◽  
Neutrophil Extracellular Trap ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Post Translational Modification

Acetylation is an important post translational modification of histone that plays a role in regulation of physiological and pathological process in the body. We have recently shown that the inhibition of histone deacetylases (HDAC) by low concentrations of HDAC inhibitors (HDACis), belinostat (up to 0.25 µM) and panobinostat (up to 0.04 µM) promote histone acetylation (e.g., AcH4) and neutrophil extracellular trap formation (NETosis). Clinical use of belinostat and panobinostat often leads to neutropenia and the in vivo concentrations vary with time and tissue locations. However, the effects of different concentrations of these HDACis on neutrophil death are not fully understood. We considered that increasing concentrations of belinostat and panobinostat could alter the type of neutrophil death. To test this hypothesis, we treated human neutrophils with belinostat and panobinostat in the presence or absence of agonists that promote NOX-dependent NETosis (phorbol myristate acetate or lipopolysaccharide from Escherichia coli 0128) and NOX-independent NETosis (calcium ionophores A23187 or ionomycin from Streptomyces conglobatus). Increasing concentrations of HDACis induced histone acetylation in a dose-dependent manner. ROS analyses showed that increasing concentrations of HDACis, increased the degree of NOX-derived ROS production. Higher levels (>1 µM belinostat and >0.2 µM panobinostat) of AcH4 resulted in a significant inhibition of spontaneous as well as the NOX-dependent and -independent NETosis. By contrast, the degree of neutrophil apoptosis significantly increased, particularly in non-activated cells. Collectively, this study establishes that increasing concentrations of belinostat and panobinostat initially increases NETosis but subsequently reduces NETosis or switches the form of cell death to apoptosis. This new information indicates that belinostat and panobinostat can induce different types of neutrophil death and may induce neutropenia and regulate inflammation at different concentrations.

scholarly journals Mast Cell Tryptase Potentiates Neutrophil Extracellular Trap Formation

2021 ◽  
pp. 1-14
Author(s):  
Gunnar Pejler ◽  
Sultan Alanazi ◽  
Mirjana Grujic ◽  
Jeremy Adler ◽  
Anna-Karin Olsson ◽  
...  
Keyword(s):  
Neutrophil Extracellular Trap ◽  
Human Neutrophils ◽  
Functional Communication ◽  
Extracellular Milieu ◽  
Extracellular Traps ◽  
Inflammatory Conditions ◽  
Histone 3 ◽  
Activated Neutrophils ◽  
Core Histones

Previous research has indicated an intimate functional communication between mast cells (MCs) and neutrophils during inflammatory conditions, but the nature of such communication is not fully understood. Activated neutrophils are known to release DNA-containing extracellular traps (neutrophil extracellular traps [NETs]) and, based on the known ability of tryptase to interact with negatively charged polymers, we here hypothesized that tryptase might interact with NET-contained DNA and thereby regulate NET formation. In support of this, we showed that tryptase markedly enhances NET formation in phorbol myristate acetate-activated human neutrophils. Moreover, tryptase was found to bind vividly to the NETs, to cause proteolysis of core histones and to cause a reduction in the levels of citrullinated histone-3. Secretome analysis revealed that tryptase caused increased release of numerous neutrophil granule compounds, including gelatinase, lactoferrin, and myeloperoxidase. We also show that DNA can induce the tetrameric, active organization of tryptase, suggesting that NET-contained DNA can maintain tryptase activity in the extracellular milieu. In line with such a scenario, DNA-stabilized tryptase was shown to efficiently degrade numerous pro-inflammatory compounds. Finally, we showed that tryptase is associated with NET formation in vivo in a melanoma setting and that NET formation in vivo is attenuated in mice lacking tryptase expression. Altogether, these findings reveal that NET formation can be regulated by MC tryptase, thus introducing a novel mechanism of communication between MCs and neutrophils.

scholarly journals A Lipid Mediator Hepoxilin A3 Is a Natural Inducer of Neutrophil Extracellular Traps in Human Neutrophils

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
David N. Douda ◽  
Hartmut Grasemann ◽  
Cecil Pace-Asciak ◽  
Nades Palaniyar
Keyword(s):  
Cystic Fibrosis ◽  
Nadph Oxidase ◽  
Neutrophil Extracellular Trap ◽  
Lipid Mediator ◽  
Human Neutrophils ◽  
Synthetic Analog ◽  
Extracellular Traps ◽  
Cystic Fibrosis Lung Disease ◽  
Higher Doses ◽  
Hepoxilin A3

Pulmonary exacerbations in cystic fibrosis airways are accompanied by inflammation, neutrophilia, and mucous thickening. Cystic fibrosis sputum contains a large amount of uncleared DNA contributed by neutrophil extracellular trap (NET) formation from neutrophils. The exact mechanisms of the induction of NETosis in cystic fibrosis airways remain unclear, especially in uninfected lungs of patients with early cystic fibrosis lung disease. Here we show that Hepoxilin A3, a proinflammatory eicosanoid, and the synthetic analog of Hepoxilin B3, PBT-3, directly induce NETosis in human neutrophils. Furthermore, we show that Hepoxilin A3-mediated NETosis is NADPH-oxidase-dependent at lower doses of Hepoxilin A3, while it is NADPH-oxidase-independent at higher doses. Together, these results demonstrate that Hepoxilin A3 is a previously unrecognized inducer of NETosis in cystic fibrosis lungs and may represent a new therapeutic target for treating cystic fibrosis and other inflammatory lung diseases.

scholarly journals In Vivo Transmigrated Human Neutrophils Are Highly Primed for Intracellular Radical Production Induced by Monosodium Urate Crystals

2020 ◽  
Vol 21 (11) ◽  
pp. 3750 ◽  
Author(s):  
Lisa Davidsson ◽  
Agnes Dahlstrand Rudin ◽  
Felix Peter Sanchez Klose ◽  
Alicia Buck ◽  
Lena Björkman ◽  
...  
Keyword(s):  
Neutrophil Extracellular Trap ◽  
Human Neutrophils ◽  
Ros Production ◽  
Monosodium Urate ◽  
Intracellular Compartments ◽  
Blood Neutrophils ◽  
Msu Crystals ◽  
Urate Crystals

Gout is an inflammatory disease caused by monosodium urate (MSU) crystals. The role of neutrophils in gout is less clear, although several studies have shown neutrophil extracellular trap (NET) formation in acutely inflamed joints of gout patients. MSU crystals are known to induce the production of reactive oxygen species (ROS) and NET formation in neutrophils isolated from blood, but there is inconclusive knowledge on the localization of ROS production as well as whether the ROS are required for NET formation. In this report we demonstrate that MSU crystals activate human neutrophils to produce ROS exclusively in intracellular compartments. Additionally, in vivo transmigrated neutrophils derived from experimental skin chambers displayed markedly increased ROS production as compared to resting blood neutrophils. We also confirmed that MSU stimulation potently induced NET formation, but this response was not primed in in vivo transmigrated neutrophils. In line with this we found that MSU-triggered NET formation was independent of ROS production and proceeded normally in neutrophils from patients with dysfunctional respiratory burst (chronic granulomatous disease (CGD) and complete myeloperoxidase (MPO) deficiency). Our data indicate that in vivo transmigrated neutrophils are markedly primed for oxidative responses to MSU crystals and that MSU triggered NET formation is independent of ROS production.

scholarly journals Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation

2009 ◽  
Vol 184 (2) ◽  
pp. 205-213 ◽  
Author(s):  
Yanming Wang ◽  
Ming Li ◽  
Sonja Stadler ◽  
Sarah Correll ◽  
Pingxin Li ◽  
...  
Keyword(s):  
Innate Immune ◽  
Neutrophil Extracellular Trap ◽  
Peptidylarginine Deiminase ◽  
Chromatin Decondensation ◽  
Extracellular Traps ◽  
Blood Neutrophils ◽  
Decondensed Chromatin ◽  
Nuclear Structures ◽  
Histone Citrullination ◽  
Histone H5

Peripheral blood neutrophils form highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs), that have been implicated in innate immune response to bacterial infection. Neutrophils express high levels of peptidylarginine deiminase 4 (PAD4), which catalyzes histone citrullination. However, whether PAD4 or histone citrullination plays a role in chromatin structure in neutrophils is unclear. In this study, we show that the hypercitrullination of histones by PAD4 mediates chromatin decondensation. Histone hypercitrullination is detected on highly decondensed chromatin in HL-60 granulocytes and blood neutrophils. The inhibition of PAD4 decreases histone hypercitrullination and the formation of NET-like structures, whereas PAD4 treatment of HL-60 cells facilitates these processes. The loss of heterochromatin and multilobular nuclear structures is detected in HL-60 granulocytes after PAD4 activation. Importantly, citrullination of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures. Together, these results suggest that histone hypercitrullination has important functions in chromatin decondensation in granulocytes/neutrophils.

scholarly journals Early and Late Processes Driving NET Formation, and the Autocrine/Paracrine Role of Endogenous RAGE Ligands

2021 ◽  
Vol 12 ◽  
Author(s):  
Olga Tatsiy ◽  
Vanessa de Carvalho Oliveira ◽  
Hugo Tshivuadi Mosha ◽  
Patrick P. McDonald
Keyword(s):  
Signaling Pathways ◽  
Neutrophil Extracellular Trap ◽  
Net Generation ◽  
Human Neutrophils ◽  
Chromatin Decondensation ◽  
Cellular Processes ◽  
Activated Neutrophils ◽  
Arginine Residues ◽  
Neutrophil Response

Neutrophil extracellular trap (NET) formation has emerged as an important response against various pathogens; it also plays a role in chronic inflammation, autoimmunity, and cancer. Despite a growing understanding of the mechanisms underlying NET formation, much remains to be elucidated. We previously showed that in human neutrophils activated with different classes of physiological stimuli, NET formation features both early and late events that are controlled by discrete signaling pathways. However, the nature of these events has remained elusive. We now report that PAD4 inhibition only affects the early phase of NET generation, as do distinct signaling intermediates (TAK1, MEK, p38 MAPK). Accordingly, the inducible citrullination of residue R2 on histone H3 is an early neutrophil response that is regulated by these kinases; other arginine residues on histones H3 and H4 do not seem to be citrullinated. Conversely, elastase blockade did not affect NET formation by several physiological stimuli, though it did so in PMA-activated cells. Among belated events in NET formation, we found that chromatin decondensation is impaired by the inhibition of signaling pathways controlling both early and late stages of the phenomenon. In addition to chromatin decondensation, other late processes were uncovered. For instance, unstimulated neutrophils can condition themselves to be poised for rapid NET induction. Similarly, activated neutrophils release endogenous proteic factors that promote and largely mediate NET generation. Several such factors are known RAGE ligands and accordingly, RAGE inbibition largely prevents both NET formation and the conditioning of neutrophils to rapidly generate NETs upon stimulation. Our data shed new light on the cellular processes underlying NET formation, and unveil unsuspected facets of the phenomenon that could serve as therapeutic targets. In view of the involvement of NETs in both homeostasis and several pathologies, our findings are of broad relevance.

scholarly journals The total terpenoids of Celastrus orbiculatus (TTC) inhibit NOX-dependent formation of PMA-induced neutrophil extracellular traps (NETs)

2018 ◽  
Vol 16 ◽  
pp. 205873921880566
Author(s):  
Li Tao ◽  
Min Xu ◽  
Yanqing Liu
Keyword(s):  
Enzymatic Activity ◽  
Early Stage ◽  
Neutrophil Extracellular Traps ◽  
Human Neutrophils ◽  
Ros Generation ◽  
Ros Production ◽  
Celastrus Orbiculatus ◽  
Traditional Chinese Herbal Medicine ◽  
Extracellular Traps ◽  
Anti Inflammatory

Previously, we identified that Celastrus orbiculatus, a traditional Chinese herbal medicine, exhibited prominent anti-inflammatory and anti-tumor activities. More recently, the formation of neutrophil extracellular traps (NETs) or NETosis has been recognized as a critical pathological event in the development of inflammatory and autoimmune diseases. The present study is aimed to explore the pharmacological effect of the total terpenoids from the stems of C. orbiculatus (TTC) on NETosis and underlying mechanisms, which may provide fundamental knowledge for future utilization of the Chinese medicine. Human neutrophils were isolated by density gradient centrifugation; lactase dehydrogenase (LDH) assay was used to detect cytotoxic effect of TTC on neutrophils. Moreover, we established phorbol-12-myristate-13-acetate (PMA)-induced NETosis. Quantitative and qualitative study of PMA-induced NET release was labeled by SYTOX™ Green. ROS production was determined by flow cytometry. The neutrophil NADPH oxidase (NOX) activity was assessed by lucigenin chemiluminescence assay, and the phosphorylation of NOX subunit was analyzed by immunoblot assay. TTC (5–80 μg.mL−1) had no predominant neutrophil cytotoxicity after 4 h exposure. PMA (200 ng.mL−1) significantly induced the formation of NETs after 4 h stimulus, whereas TTC dose-dependently (5–80 μg.mL−1) inhibited the process. TTC (40 μg.mL−1) blocked neutrophil elastase (NE) and myeloperoxidase (MPO) translocation from cytoplasm to nucleus and disrupted the formation of NET-associated deoxyribonucleic acid (DNA)–MPO and DNA–NE complexes. Moreover, TTC dose-dependently blocked PMA-mediated ROS production, and inhibited the NOX enzymatic activity of neutrophils upon PMA stimulus for 1 h. Finally, TTC suppressed PMA-induced phosphorylation of NOX subunit p40phox on Thr154 residue. TTC inhibited PMA-induced NOX phosphorylation, thereby suppressing NOX enzymatic activity and ROS generation in neutrophils undergoing NETosis. Consequently, TTC disrupted NETosis in the early stage of NOX-dependent NETs formation, which might serve as a promising anti-inflammatory agent by targeting suicidal NETosis.

scholarly journals The prolyl isomerase Pin1 acts as a novel molecular switch for TNF-α–induced priming of the NADPH oxidase in human neutrophils

Blood ◽  
2010 ◽  
Vol 116 (26) ◽  
pp. 5795-5802 ◽  
Author(s):  
Tarek Boussetta ◽  
Marie-Anne Gougerot-Pocidalo ◽  
Gilles Hayem ◽  
Silvia Ciappelloni ◽  
Houssam Raad ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Inflammatory Diseases ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Human Neutrophils ◽  
Ros Production ◽  
Prolyl Isomerase ◽  
Tnf Α ◽  
Factor Α

Abstract Neutrophils play a key role in host defense by releasing reactive oxygen species (ROS). However, excessive ROS production by neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can damage bystander tissues, thereby contributing to inflammatory diseases. Tumor necrosis factor-α (TNF-α), a major mediator of inflammation, does not activate NADPH oxidase but induces a state of hyperresponsiveness to subsequent stimuli, an action known as priming. The molecular mechanisms by which TNF-α primes the NADPH oxidase are unknown. Here we show that Pin1, a unique cis-trans prolyl isomerase, is a previously unrecognized regulator of TNF-α–induced NADPH oxidase hyperactivation. We first showed that Pin1 is expressed in neutrophil cytosol and that its activity is markedly enhanced by TNF-α. Inhibition of Pin1 activity with juglone or with a specific peptide inhibitor abrogated TNF-α–induced priming of neutrophil ROS production induced by N-formyl-methionyl-leucyl-phenylalanine peptide (fMLF). TNF-α enhanced fMLF-induced Pin1 and p47phox translocation to the membranes and juglone inhibited this process. Pin1 binds to p47phox via phosphorylated Ser345, thereby inducing conformational changes that facilitate p47phox phosphorylation on other sites by protein kinase C. These findings indicate that Pin1 is critical for TNF-α–induced priming of NADPH oxidase and for excessive ROS production. Pin1 inhibition could potentially represent a novel anti-inflammatory strategy.

scholarly journals Flavonoids and 5-Aminosalicylic Acid Inhibit the Formation of Neutrophil Extracellular Traps

2013 ◽  
Vol 2013 ◽  
pp. 1-14 ◽  
Author(s):  
Tina Kirchner ◽  
Eva Hermann ◽  
Sonja Möller ◽  
Matthias Klinger ◽  
Werner Solbach ◽  
...  
Keyword(s):  
Neutrophil Extracellular Traps ◽  
Human Neutrophils ◽  
Therapeutic Effects ◽  
Ros Production ◽  
Aminosalicylic Acid ◽  
Extracellular Traps ◽  
Catechin Hydrate ◽  
Pathophysiological Role ◽  
Chemical Stimuli

Neutrophil extracellular traps (NETs) have been suggested to play a pathophysiological role in several autoimmune diseases. Since NET-formation in response to several biological and chemical stimuli is mostly ROS dependent, in theory any substance that inhibits or scavenges ROS could prevent ROS-dependent NET release. Therefore, in the present comprehensive study, several antioxidative substances were assessed for their capacity to inhibit NET formation of primary human neutrophilsin vitro. We could show that the flavonoids (−)-epicatechin, (+)-catechin hydrate, and rutin trihydrate as well as vitamin C and the pharmacological substancesN-acetyl-L-cysteine and 5-aminosalicylic acid inhibited PMA induced ROS production and NET formation. Therefore, a broad spectrum of antioxidative substances that reduce ROS production of primary human neutrophils also inhibits ROS-dependent NET formation. It is tempting to speculate that such antioxidants can have beneficial therapeutic effects in diseases associated with ROS-dependent NET formation.

scholarly journals AB0083 SIGNALING OF PSGL-1 IN HUMAN NEUTROPHILS MIGHT CONTROL NET GENERATION AND APOPTOSIS AND MIGHT BE ALTERED IN SLE PATIENTS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1071.2-1071
Author(s):  
A. Muñoz-Callejas ◽  
E. González Sánchez ◽  
E. F. Vicente-Rabaneda ◽  
J. Garcia Perez ◽  
S. Castañeda ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Lupus Erythematosus ◽  
Neutrophil Extracellular Traps ◽  
Neutrophil Extracellular Trap ◽  
Rank Test ◽  
Net Generation ◽  
Human Neutrophils ◽  
Extracellular Traps ◽  
Healthy Donors ◽  
Cutaneous Lupus

Background:Neutrophil extracellular traps (NETs) are extracellular chromatin fibers decorated with neutrophil antimicrobial proteins and histones, which are formed by neutrophils to trap invading pathogens and facilitate their removal. Excessive presence of NETs has been reported in autoimmune diseases like Systemic Lupus Erythematosus (SLE) and has been related with disease pathogenesis [1]. Our previous work showed that P-selectin KO mice develop an autoimmune syndrome similar to human lupus and that patients with cutaneous lupus have reduced expression of P-selectin in skin vessels [2]. Although it has been reported that P-selectin might induce NET generation in mouse neutrophils [3], there are no studies performed with human neutrophils.Objectives:1)To analyze the contribution of PSGL-1/P-Selectin interaction to control the generation of NETs by human neutrophils from healthy donors and patients with SLE.2)To study the implication of PSGL-1/P-Selectin interaction in the control of other types of neutrophil death.Methods:Human neutrophils were isolated from healthy donors and patients with SLE. After incubation in vitro with BSA or P-Selectin in rolling-like conditions (60 rpm shaking at 370C) for 10 min, neutrophils were left to adhere for 1 hour. Then, NETs were labeled with Sytox Green and quantified by fluorimetry and by flow cytometry (FACSCanto II, BD Biosciences). For quantification of necrosis and apoptosis, cells were labeled with propidium iodide and annexing V and analyzed by flow cytometry. Data were analyzed with two-sided Student, two-sided Mann-Whitney U or two-sided Wilcoxon signed rank test using GraphPad Prism software (Version 8.0.1, La Jolla, CA). Results were considered significant at P<.05.Results:Our results show that rolling on BSA, compared to adhesion, induces NET generation and apoptosis of neutrophils isolated from blood of healthy donors. Rolling on P-selectin reduces apoptosis of neutrophils and increase the percentage of NET events, although reducing NET intensity. Comparing to healthy donors, neutrophils from active SLE patients generate NETs with higher intensity in adhesion and do not respond to P-Selectin.Conclusion:The interaction of PSGL-1 with P-Selectin preserves neutrophil death from apoptosis and controls NET generation. Our results suggest that this control might be altered in patients with active SLE.References:[1]Fousert, E., Toes, R., & Desai, J. (2020). Neutrophil extracellular traps (NETs) take the central stage in driving autoimmune responses. Cells, 9(4), 915.[2]González-Tajuelo, R., Silván, J., Pérez-Frías, A., De La Fuente-fernández, M., Tejedor, R., Espartero-Santos, M. & Urzainqui, A. (2017). P-Selectin preserves immune tolerance in mice and is reduced in human cutaneous lupus. Scientific reports, 7(1), 1-12.[3]Etulain, J., Martinod, K., Wong, S. L., Cifuni, S. M., Schattner, M., & Wagner, D. D. (2015). P-selectin promotes neutrophil extracellular trap formation in mice. Blood, 126(2), 242-246.Disclosure of Interests:None declared

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