β-Conglycinin induces the formation of neutrophil extracellular traps dependent on NADPH oxidase-derived ROS, PAD4, ERK1/2 and p38 signaling pathways in mice

2021 ◽  
Author(s):  
Xiao Liu ◽  
Yunhe Fu ◽  
Jingjing Wang ◽  
Di Wu ◽  
Shuangqiu Li ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Signaling Pathways ◽  
Neutrophil Extracellular Traps ◽  
Schematic Representation ◽  
Extracellular Traps

Schematic representation of β-conglycinin induced NETosis.

scholarly journals The FcγRIII Engagement Augments PMA-Stimulated Neutrophil Extracellular Traps (NETs) Formation by Granulocytes Partially via Cross-Talk between Syk-ERK-NF-κB and PKC-ROS Signaling Pathways

Biomedicines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1127
Author(s):  
Cheng-Hsun Lu ◽  
Ko-Jen Li ◽  
Cheng-Han Wu ◽  
Chieh-Yu Shen ◽  
Yu-Min Kuo ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Inflammatory Cytokines ◽  
Cross Talk ◽  
Neutrophil Extracellular Traps ◽  
Ros Generation ◽  
Dependent Manner ◽  
Extracellular Traps ◽  
Tnf Α ◽  
The Impact ◽  
Pro Inflammatory Cytokines

Polymorphonuclear neutrophils (PMNs) are the most abundant white blood cell in the circulation capable of neutrophil extracellular traps (NETs) formation after stimulation. Both NADPH oxidase-dependent and -independent pathways are involved in NET formation. The IgG is the most abundant immunoglobulin in human serum. However, the impact of the circulating IgG on NET formation is totally unexplored. In this study, the all-trans retinoic acid (ATRA)-induced mature granulocytes (dHL-60) were pre-treated with monomeric human IgG, papain-digested Fab fragment, crystallizable IgG Fc portion, rituximab (a human IgG1), or IgG2. The NET formation of the dHL-60 in the presence/absence of phorbol 12-myristate 13-acetate (PMA) stimulation was then measured by the fluorescent area after SYTOX green nucleic acid stain. The intracellular reactive oxygen species (ROS) generation was measured by flow cytometry. Total and phosphorylated Syk, SHP-1, and ERK were detected by immunoblot. We found that human monomeric IgG and its subclasses IgG1 and IgG2 per se induced negligible NET formation of dHL-60, but the FcγRIII engagement by these IgG subclasses and Fc portion augment PMA-stimulated dHL-60 NET formation in a dose-dependent manner. Furthermore, we found that increased Syk and ERK phosphorylation, intracellular ROS generation, and pro-inflammatory cytokines, IL-8 and TNF-α, production could be induced after FcγRIII engagement. Blocking FcγRIII engagement by a specific antibody diminished the augmented NET formation. In conclusion, we discovered that cross-talk between FcγRIII engagement-induced Syk-ERK and PMA-induced PKC signaling pathways augment NET formation of dHL-60 via increased ROS generation and pro-inflammatory cytokines, IL-8 and TNF-α, production.

The Role of Rac and Pak in Neutrophil Histone Hypercitrullination and Neutrophil Extracellular Traps Formation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 462-462 ◽  
Author(s):  
Mathilde Gavillet ◽  
Kimberly Martinod ◽  
Denisa D. Wagner ◽  
David A. Williams
Keyword(s):  
Flow Cytometry ◽  
Nadph Oxidase ◽  
Autoimmune Diseases ◽  
Actin Polymerization ◽  
Genetic Model ◽  
Neutrophil Extracellular Traps ◽  
Extracellular Traps ◽  
Downstream Effector ◽  
Group A

Abstract Under specific activating conditions, polymorphonuclear neutrophils (PMNs) release neutrophil extracellular traps (NETs) composed of decondensed chromatin lined with microbicidal protein such as neutrophil elastase and myeloperoxidase. NETs contribute to innate immunity but can also foster autoimmune diseases and thrombus formation. NET formation (NETosis) requires reactive oxygen species (ROS) production by NADPH oxidase and histone hypercitrullination by peptidylarginine deiminase 4 (PAD4), allowing for chromatin decondensation. Rac GTPases are expressed in three isoforms: Rac1 is ubiquitously expressed and plays a role in PMN migration and oxidase function; Rac2 is hematopoietic-specific and the major isoform in PMNs and Rac3 is mostly neuronal. Rac1 and Rac2 regulate the cytoskeleton in PMNs, controlling actin polymerization, cell shape, adhesion and migration and are essential components of the NADPH oxidase complex. The present study aimed to explore the role of the Rac pathway on NETosis in PMNs, including the upstream guanosine exchange factor (GEF) activator, Vav, and a downstream effector of Rac, p21 activated kinase, Pak. We developed a flow cytometry-based quantification of H3 hypercitrullination (H3Cit). In response to phorbol myristate ester (PMA) stimulation, H3Cit is increased to 136% of basal in WT cells, compared with 103% in Rac2-/- (P<0.01) (Table). H3Cit levels observed by flow were confirmed in a NET formation assay. Rac2-/- PMNs formed significantly fewer NETs both spontaneously and after PMA stimulation (WT unstimulated 2.79%, Rac2-/- unstimulated 0.72%, WT+PMA 10.84%, Rac2-/-+PMA 1.39%, P< 0.05 for all pair comparisons). Furthermore, Rac2-/- mice demonstrated a trend towards reduced frequency of provoked thrombosis in an in vivo vena cava stenosis model (WT 78% and Rac2-/- 56% of mice with thrombus). Deletion of floxed Rac1 sequences in a Rac2-/- background in vivoallows generation and purification of PMNs lacking both Rac isoforms. Rac1Δ/Δ,Rac2-/- PMNs, which are defective in actin polymerization, had reduced basal H3Cit and a nearly complete lack of PMA-induced increase in H3Cit (136% vs 69%, WT vs Rac1Δ/Δ,Rac2-/-, P<0.01) (Table). Null knockouts of the GEFs Vav1 (hematopoietic-specific), Vav2, Vav3 or both Vav1 and 3 did not impair H3Cit response to PMA (Table). We next studied downstream effectors of Rac. Group A Paks include Pak1, 2 and 3 isoforms. Pretreatment of wild-type PMNs with either PF3758309 or IPA-3, two group A Pak inhibitors with distinct mechanisms of action, led to reduced H3Cit after PMA stimulation (induction reduced from 36% to 11% for both PF3758309 and IPA-3 treated (Table). To validate this in a genetic model, we studied Pak2Δ/Δ PMNs, since we have recently demonstrated the dependence of hematopoietic stem cell migration on Pak2. Pak2Δ/Δ demonstrated a reduced basal level of H3Cit and a significantly reduced PMA-induced increase in H3Cit (136% vs 94%, WT vs Pak2Δ/Δ, P<0.05, Table). In summary, we describe a flow-based assay that quantitates the early processes of NET formation and validated that this assay reliably predicts agonist-induced NET formation in a genetic model. The results establish that both Rac1 and Rac2, and the downstream effector Pak2, regulate histone H3 hypercitrullination and NET formation in PMNs, while suggesting that Vav does not activate the Rac pathway in PMA-induced NET formation. These data further delineate the role of the Rac pathway in NETosis, linking cytoskeleton and oxidase functions. Furthermore, these data indicate Pak could represent a therapeutic target for a wide array of pathological processes related to NETosis such as thrombosis and numerous autoimmune diseases. Table Intensity of H3Cit staining as determined by flow cytometry-based assay. Basal H3Cit level PMA-induced H3Cit PMA-induced change WT PMN 100±2% 136±5% 36% Rac2-/- 82±9% ns 103±15.3% ** 21% Rac1Δ/Δ, Rac2-/- 64±10% *** 69±10% ** 5% Vav1-/- 86±9% ns 145±15% ns 59% Vav2-/- 91±4% ns 134±18% ns 43% Vav3-/- 153±30% * 171±28% ns 18% Vav1,3-/- 125±20% ns 144±24% ns 19% WT+ PF 5nM 76±17% ns 87±8% * 11% WT+ IPA 5µM 100±4% ns 111±10% ns 11% Pak2Δ/Δ 75±7% * 94±11% * 19% Results are expressed as mean±SEM % of the untreated WT control of each experiment. Results are from ≥3 independent experiments. * P<0.05, **P<0.01. P<0.001, ns non-significant, by two-tailed t-test. Disclosures No relevant conflicts of interest to declare.

scholarly journals Novel cell death program leads to neutrophil extracellular traps

2007 ◽  
Vol 176 (2) ◽  
pp. 231-241 ◽  
Author(s):  
Tobias A. Fuchs ◽  
Ulrike Abed ◽  
Christian Goosmann ◽  
Robert Hurwitz ◽  
Ilka Schulze ◽  
...  
Keyword(s):  
Cell Death ◽  
Nadph Oxidase ◽  
Neutrophil Extracellular Traps ◽  
Death Process ◽  
Extracellular Traps ◽  
Cell Death Pathway ◽  
Antimicrobial Function ◽  
Cell Death Program ◽  
Cell Death Process ◽  
Extracellular Structures

Neutrophil extracellular traps (NETs) are extracellular structures composed of chromatin and granule proteins that bind and kill microorganisms. We show that upon stimulation, the nuclei of neutrophils lose their shape, and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate, allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death process is distinct from apoptosis and necrosis and depends on the generation of reactive oxygen species (ROS) by NADPH oxidase. Patients with chronic granulomatous disease carry mutations in NADPH oxidase and cannot activate this cell-death pathway or make NETs. This novel ROS-dependent death allows neutrophils to fulfill their antimicrobial function, even beyond their lifespan.

scholarly journals Restoration of NET formation by gene therapy in CGD controls aspergillosis

Blood ◽  
2009 ◽  
Vol 114 (13) ◽  
pp. 2619-2622 ◽  
Author(s):  
Matteo Bianchi ◽  
Abdul Hakkim ◽  
Volker Brinkmann ◽  
Ulrich Siler ◽  
Reinhard A. Seger ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Antimicrobial Activity ◽  
Nadph Oxidase ◽  
Invasive Pulmonary Aspergillosis ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Neutrophil Extracellular Traps ◽  
Pulmonary Aspergillosis ◽  
Extracellular Traps ◽  
Healthy Persons

AbstractChronic granulomatous disease (CGD) patients have impaired nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function, resulting in poor antimicrobial activity of neutrophils, including the inability to generate neutrophil extracellular traps (NETs). Invasive aspergillosis is the leading cause of death in patients with CGD; it is unclear how neutrophils control Aspergillus species in healthy persons. The aim of this study was to determine whether gene therapy restores NET formation in CGD by complementation of NADPH oxidase function, and whether NETs have antimicrobial activity against Aspergillus nidulans. Here we show that reconstitution of NET formation by gene therapy in a patient with CGD restores neutrophil elimination of A nidulans conidia and hyphae and is associated with rapid cure of preexisting therapy refractory invasive pulmonary aspergillosis, underlining the role of functional NADPH oxidase in NET formation and antifungal activity.

scholarly journals Characterization of Bovine Neutrophil Extracellular Traps Induced by Histamine

2020 ◽  
Author(s):  
Ershun Zhou ◽  
Zhikai Wu ◽  
Xingyi Zhu ◽  
Peixuan Li ◽  
Jingjing Wang ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Nadph Oxidase ◽  
Molecular Mechanisms ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Allergic Diseases ◽  
Neutrophil Extracellular Traps ◽  
Cell Counting ◽  
Dependent Manner ◽  
Extracellular Traps ◽  
Main Components

Abstract BackgroundHistamine plays an central role in many allergic diseases including allergic asthma and allergic rhinitis, and is also involved in bovine laminitis through regulating immune responses. Neutrophil extracellular traps (NETs) formation is a novel effector mechanism of neutrophils to defend against various stimuli. In the present study, we aimed to investigate the role of histamine on bovine NET formation and examined its fundamental molecular mechanisms. ResultsFirstly, the effects of histamine on neutrophil viability was measured by Cell Counting Kit-8 (CCK-8) and Lactate dehydrogenase (LDH) assays. The results showed that histamine had no significant influence on neutrophil viability. Then we characterized histamine-triggered NET formation by confocal microscopy and PicoGreen-derived NETs quantification. Confocal microscopy analyses illustrated NET structures by co-localizing the main components of NETs, and NET quantification revealed that histamine-triggered NETs were released in a dose-dependent manner. In addition, we found reactive oxygen species (ROS) production, phosphorylated extracellular signal-regulated kinase (ERK) and p38 proteins were significantly elevated in histamine-challenged neutrophils. By applying functional inhibitors of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), ERK and p38, histamine-triggered NETs were markedly reduced, indicating their importance in histamine-triggered NET formation. ConclusionsOur findings described histamine-triggered NET formation, and revealed its potential molecular mechanisms via NADPH oxidase, ERK and p38 pathways. This is the first study to depict NET formation induced by histamine, which could provide a new insight into histamine-related diseases.

scholarly journals Candida albicans triggers NADPH oxidase-independent neutrophil extracellular traps through dectin-2

2019 ◽  
Vol 15 (11) ◽  
pp. e1008096 ◽  
Author(s):  
Sheng-Yang Wu ◽  
Chia-Lin Weng ◽  
Min-Jhen Jheng ◽  
Hung-Wei Kan ◽  
Sung-Tsang Hsieh ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Nadph Oxidase ◽  
Neutrophil Extracellular Traps ◽  
Extracellular Traps
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