Neutrophil Extracellular Traps Induce Platelet Adhesion and Thrombus Formation.

Abstract Abstract 1345 Introduction Thrombus stability is provided by very large polymers adhering to platelets and anchoring the thrombus to the vessel wall. The best described polymers are fibrin and von Willebrand Factor (VWF). Activated neutrophils and other leukocytes can form an extracellular fibrous network which is composed of DNA, histones, and granular proteins. These neutrophil extracellular traps (NETs) are present in various inflammatory diseases. In deep vein thrombosis (DVT) inflammation closely cooperates with thrombosis. Here we examine whether NETs provide a new means to support the adhesion and recruitment of platelets and whether NETs are present in DVT. Methods and Results: To study the interaction of platelets with NETs, we isolated human neutrophils, induced NET formation and perfused over the NETs human platelets in plasma or whole blood anticoagulated with the thrombin inhibitor PPACK. Microscopic analysis revealed that under flow platelets adhere avidly to NETs. Perfusion of whole blood at physiological shear resulted in formation of thrombi on NETs in a time dependent manner. Addition of DNase1 degraded NETs and removed all platelets and thrombi demonstrating their adhesion to NETs. Thrombus formation on NETs was absent if blood was supplemented with EDTA indicating the requirement for divalent cations. Perfusion of NETs with heparinized blood dismantled NETs and prevented thrombus formation. Incubation of NETs with heparin alone released histones from NETs, indicating that heparin destroys the chromatin backbone of NETs. Furthermore, immunocytochemistry revealed that NETs were able to bind platelet adhesion molecules VWF and fibronectin from human plasma. Immunohistochemical analysis of a baboon deep vein thrombus showed abundant extracellular chromatin which co-localized with fibronectin and VWF. Conclusions: We show that extracellular traps are able to promote thrombosis in vitro and are abundant in vivo in DVT. We propose that extracellular chromatin provides a new type of scaffold that promotes platelet adhesion, activation, and aggregation and may be important for thrombus initiation or stability. Disclosures No relevant conflicts of interest to declare.

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Microfluidic and Flow Cytometric Studies Support a Role for Monocytes and Coated Platelets in the Prothrombotic State in Heparin-Induced Thrombocytopenia (HIT)

Blood ◽

10.1182/blood.v118.21.539.539 ◽

2011 ◽

Vol 118 (21) ◽

pp. 539-539

Author(s):

Valerie Tutwiler ◽

Hyun Sook Ahn ◽

Douglas B. Cines ◽

Rodney M. Camire ◽

Mortimer Poncz ◽

...

Keyword(s):

Whole Blood ◽

Platelet Adhesion ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Passive Immunization ◽

Annexin V ◽

Thrombin Inhibitor ◽

Prothrombotic State ◽

Flow Cytometric ◽

Activated State

Abstract Abstract 539 HIT is an immune thrombocytopenia associated with a high risk of developing thrombosis. A passive immunization murine model of this disorder has provided important insights into the underlying pathogenesis of this disease, but is limited by its inability to study human cells and limited ability to define the contribution of various hematopoeitic and vascular cells to the prothrombotic state. We used a microfluidic system in conjunction with flow cytometry to further our understanding of the prothrombotic nature of HIT. Platelet adhesion and aggregation was studied in whole blood labeled with Calcein AM, perfused through a microfluidic channel (BioFlux 200 system, Fluxion) coated with von Willebrand factor (vWf) at shear stress of 20 dyne/cm2 at 37°C. A 40–60% increase in platelet adhesion (relative area covered by platelets) with up to a 4 fold increase in average aggregate size was seen in the presence of the pathogenic HIT-like monoclonal antibody (moAb) KKO (50 μg/ml) in conjunction with PF4 (10 μg/ml) when compared to control samples with PF4 only or with PF4 plus a non-pathogenic anti-PF4 moAb RTO (p <0.01). Monocyte-depletion decreased platelet aggregation by 20 – 40% relative to whole blood or after monocyte-repletion (P<0.0001). In HIT, thrombin plays a key role in the formation of platelet aggregates. Addition of thrombin inhibitor PPACK to the whole blood stimulated by KKO and PF4 decreased thrombus formation in the microfluidic chamber by 40% (p<0.001). Coated platelets are prothrombotic and characterized by phosphatidylserine (PS) exposure and binding of FVa and FXa. This activated state requires dual stimulation via thrombin and ITAM receptors. Flow cytometric studies of annexin V and FXa binding showed extensive induction of coated platelets in whole blood by KKO plus PF4 in contrast to PF4 or PF4 plus RTO (annexin V: p<0.0001; Factor Xa p<0.01). These new studies, focused on human blood, support our finding in the passive murine HIT model as to the importance of monocytes to thrombus formation and suggest that the prothrombotic nature of HIT may also be promoted by the generation of coated platelets. Identification of coated platelets may also lead to new diagnostic tests and new therapeutic interventions in HIT. Disclosures: No relevant conflicts of interest to declare.

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Abstract 56: Coagulation Factor XI Promotes Distal Platelet Activation and Single Platelet Consumption in the Bloodstream Under Shear Flow

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.56 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Jenya Zilberman-Rudenko ◽

Chantal Wiesenekker ◽

Asako Itakura ◽

Owen J McCarty

Keyword(s):

Shear Flow ◽

Platelet Activation ◽

Platelet Adhesion ◽

Ex Vivo ◽

Thrombus Formation ◽

Coagulation Factor ◽

Thrombin Inhibitor ◽

Dependent Manner ◽

Factor Xi ◽

Primate Model

Objective: Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor (TF)-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was designed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. Approach and Results: Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization and fibrin formation on immobilized collagen and TF under shear flow, ex vivo . Downstream of the thrombus formed on immobilized collagen or collagen and 10 pM TF, platelet CD62P expression and microaggregate formation and progressive platelet consumption were significantly reduced in the presence of FXI-function blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. Conclusions: This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlights FXI as a novel therapeutic target for inhibiting distal platelet activation without affecting proximal platelet adhesion.

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Neutrophil Extracellular Traps in Arterial and Venous Thrombosis

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0038-1677040 ◽

2019 ◽

Vol 45 (01) ◽

pp. 086-093 ◽

Cited By ~ 41

Author(s):

Elodie Laridan ◽

Kimberly Martinod ◽

Simon De Meyer

Keyword(s):

Myocardial Infarction ◽

Thrombus Formation ◽

Neutrophil Extracellular Traps ◽

Major Health ◽

Vein Thrombosis ◽

Extracellular Traps ◽

Thrombotic Complications ◽

Decondensed Chromatin ◽

Deep Vein ◽

Novel Therapeutic

AbstractThrombotic complications are still a major health risk worldwide. Our view on the pathophysiology of thrombosis has significantly changed since the discovery of neutrophil extracellular traps (NETs) and their prothrombotic characteristics. Generated by neutrophils that release their decondensed chromatin as a network of extracellular fibers, NETs promote thrombus formation by serving as a scaffold that activates platelets and coagulation. The thrombogenic involvement of NETs has been described in various settings of thrombosis, including stroke, myocardial infarction, and deep vein thrombosis. The aim of this review is to summarize existing evidence showing the presence of NETs in human thrombus material. Following an introduction on NETs and their role in thrombus formation, the authors address studies showing the presence of NETs in arterial or venous thrombi. In addition, they focus on potential novel therapeutic opportunities to resolve or prevent thrombosis by targeting NETs.

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3′-Nucleotidase/Nuclease Activity Allows Leishmania Parasites To Escape Killing by Neutrophil Extracellular Traps

Infection and Immunity ◽

10.1128/iai.01232-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1732-1740 ◽

Cited By ~ 66

Author(s):

Anderson B. Guimarães-Costa ◽

Thiago S. DeSouza-Vieira ◽

Rafael Paletta-Silva ◽

Anita Leocádio Freitas-Mesquita ◽

José Roberto Meyer-Fernandes ◽

...

Keyword(s):

Leishmania Infantum ◽

Nuclease Activity ◽

Neutrophil Extracellular Traps ◽

Human Neutrophils ◽

Dependent Manner ◽

Content Type ◽

Extracellular Traps ◽

Leishmania Parasites ◽

High Phosphate

ABSTRACTLeishmaniasis is a widespread neglected tropical disease caused by parasites of theLeishmaniagenus. These parasites express the enzyme 3′-nucleotidase/nuclease (3′NT/NU), which has been described to be involved in parasite nutrition and infection. Bacteria that express nucleases escape the toxic effects of neutrophil extracellular traps (NETs). Hence, we investigated the role of 3′NT/NU inLeishmaniasurvival of NET-mediated killing. Promastigotes ofLeishmania infantumwere cultured in high-phosphate (HP) or low-phosphate (LP) medium to modulate nuclease activity. We compared the survival of the two different groups ofLeishmaniaduring interaction with human neutrophils, assessing the role of neutrophil extracellular traps. As previously reported, we detected higher nuclease activity in parasites cultured in LP medium. Both LP and HP promastigotes were capable of inducing the release of neutrophil extracellular traps from human neutrophils in a dose- and time-dependent manner. LP parasites had 2.4 times more survival than HP promastigotes. NET disruption was prevented by the treatment of the parasites with ammonium tetrathiomolybdate (TTM), a 3′NT/NU inhibitor. Inhibition of 3′NT/NU by 3′-AMP, 5′-GMP, or TTM decreased promastigote survival upon interaction with neutrophils. Our results show thatLeishmania infantuminduces NET release and that promastigotes can escape NET-mediated killing by 3′-nucleotidase/nuclease activity, thus ascribing a new function to this enzyme.

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Platelet-Delivered Recombinant ADAMTS13 Inhibits Platelet Adhesion and Aggregation on Collagen Surface Under Flow in the Presence of Autoantibodies from Acquired Thrombotic Thrombocytopenic Purpura

Blood ◽

10.1182/blood.v128.22.1374.1374 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1374-1374

Author(s):

Mohammad S. Abdelgawwad ◽

Jenny K. McDaniel ◽

Wenjing Cao ◽

Huy P. Pham ◽

Lance A. Williams ◽

...

Keyword(s):

Flow Cytometry ◽

Thrombotic Thrombocytopenic Purpura ◽

Whole Blood ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Effective Therapy ◽

Confocal Imaging ◽

Dependent Manner ◽

Thrombocytopenic Purpura ◽

Platelet Adhesion And Aggregation

Abstract Background: Acquired thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal syndrome. It is primarily caused by severe deficiency of plasma ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity. ADAMTS13 is a plasma metalloprotease that cleaves von Willebrand factor (VWF), thereby reducing the platelet adhesion and aggregation under flow. Plasma exchange is the only effective initial therapy for acquired autoimmune TTP. However, in hospital mortality rate remains as high as 10-20% in addition to the concern about the complications associated with the placement of central catheter and the use of plasma products. Therefore, a better and more effective therapy is urgently needed. Objectives: To develop a more effective therapy, we determined the uptake and package of recombinant ADAMTS13 (rA13) in platelets and assessed the efficacy of platelets-delivered rA13 in inhibiting platelet adhesion and aggregation and thrombus formation under arterial shear. Methods: Human platelets were isolated from whole blood, washed, and incubated with various concentrations of rA13 (1-100 microgram/ml) at 25 and 37 degree Celsius for various times. The amount of rA13 uptake by platelets was determined in cell lysate by Western blotting and FRETS-VWF73 assays and in fixed cells by immunofluoresent microscopy and flow cytometry. The function of platelet delivered rA13 was also determined by shear-induced platelet adhesion and aggregation on collagen surface using a microfluidic system. Results: Freshly isolated and blood bank stored platelets were able to update rA13 in a temperature, concentration, and time-dependent manner. When rA13 (5 microgram/ml) was incubated with platelets, nearly all platelets were positive for rA13, assessed by immunofluoresent microscopy and flow cytometry. The rA13 inside platelets remained intact and was proteolytically active in cleaving FRETS-VWF73. Confocal imaging revealed that rA13 was partially co-localized with VWF in alpha granules of platelets. Microfluidic assay demonstrated that platelet-delivered rA13 was able to dramatically inhibit platelet adhesion and aggregation on collagen-coated surface under arterial shear (100 dyne/cm2) in the absence and presence of a human monoclonal antibody against ADAMTS13 (scFv4-20) that was isolated from a patient with acquired autoimmune TTP. These results were consistent with the inhibitory effects observed with mouse transgenic platelets expressing rA13 under the same conditions. When 1/3 of whole blood Adamts13-/- platelets were replaced with transgenic platelets (containing rA13), the coverage of all fluorescent platelets onto a VWF-collagen surface was dramatically reduced (data not shown). Conclusion: Our results demonstrate that platelets uptake and deliver rA13 to the site of thrombus formation under flow and the platelet-delivered rA13 may be efficacious for treating acquired TTP with inhibitors. Disclosures Zheng: Ablynx: Consultancy; Alexion: Research Funding.

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Neutrophil Extracellular Traps Activate Proinflammatory Functions of Human Neutrophils

Frontiers in Immunology ◽

10.3389/fimmu.2021.636954 ◽

2021 ◽

Vol 12 ◽

Author(s):

Daniel Dömer ◽

Tabea Walther ◽

Sonja Möller ◽

Martina Behnen ◽

Tamás Laskay

Keyword(s):

B Cell ◽

Adaptive Immune System ◽

Neutrophil Extracellular Traps ◽

Microbial Pathogens ◽

Human Neutrophils ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Extracellular Traps ◽

Proinflammatory Role

Neutrophil extracellular traps (NETs) consist of decondensed nuclear chromatin that is associated with proteins and are released by neutrophils during an inflammatory response. Released NETs are able to capture pathogens, prevent their dissemination and potentially kill them via antimicrobial peptides and proteins that are associated with the decondensed chromatin. In addition to their antimicrobial functions, NETs have also been shown to exert immunomodulatory effects by activation and differentiation of macrophages, dendritic cells and T cells. However, the effect of NETs on neutrophil functions is poorly understood. Here we report the first comprehensive study regarding the effects of NETs on human primary neutrophils in vitro. NETs were isolated from cultures of PMA-exposed neutrophils. Exposure of neutrophils to isolated NETs resulted in the activation of several neutrophil functions in a concentration-dependent manner. NETs induced exocytosis of granules, the production of reactive oxygen species (ROS) by the NADPH oxidase NOX2, NOX2-dependent NET formation, increased the phagocytosis and killing of microbial pathogens. Furthermore, NETs induced the secretion of the proinflammatory chemokine IL-8 and the B-cell-activating cytokine BAFF. We could show that the NET-induced activation of neutrophils occurs by pathways that involve the phosphorylation of Akt, ERK1/2 and p38. Taken together our results provide further insights into the proinflammatory role of NETs by activating neutrophil effector function and further supports the view that NETs can amplify inflammatory events. On the one hand the amplified functions enhance the antimicrobial defense. On the other hand, NET-amplified neutrophil functions can be involved in the pathophysiology of NET-associated diseases. In addition, NETs can connect the innate and adaptive immune system by inducing the secretion of the B-cell-activating cytokine BAFF.

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Carboxyl-Terminal Thrombospondin-Type 1 (TSP-1) Repeats of ADAMTS13 Inhibits Platelet Adhesion to Collagen-Coated Surface Under Shear Stress, Independent of Proteolytic Activity

Blood ◽

10.1182/blood.v118.21.195.195 ◽

2011 ◽

Vol 118 (21) ◽

pp. 195-195 ◽

Cited By ~ 1

Author(s):

Juan (Jenny) Xiao ◽

X. Long Zheng

Keyword(s):

Shear Stress ◽

Whole Blood ◽

Platelet Adhesion ◽

Surface Coverage ◽

Thrombus Formation ◽

Microfluidic System ◽

Dependent Manner ◽

C Terminus ◽

Coated Surface

Abstract Abstract 195 ADAMTS13 contains multiple free thiols on its surface, which may form disulfide bonds with surface-exposed free thiols on plasma-derived von Willebrand factor (VWF). This interaction may prevent lateral association of apposed VWF under arterial shear stress. However, the functional consequence of ADAMTS13-VWF interaction without proteolysis is not known. We hypothesize that the interaction between the C-terminus of ADAMTS13 and the C-terminus of VWF inhibits thrombus formation under shear stress. Using a BioFlux microfluidic system, we showed that under arterial shear stress, 10 dyn/cm2, fluorescein-labeled platelets from PPACK (thrombin inhibitor) anti-coagulated human whole blood adhered to collagen (type I)-coated surface in a time-dependent manner. Addition of human recombinant full-length ADAMTS13 (10 nM) into whole blood dramatically reduced the surface coverage of fluorescein-labeled platelets. Conversely, addition of an inhibitory polyclonal anti-ADAMTS13 IgGs (150 ug/ml) to whole blood dramatically accelerated the accumulation of fluorescein-labeled platelets. These results suggest that this microfluidic system is highly sensitive for the assessment of anti-thrombotic function of ADAMTS13. Under the same conditions, we were able to further show that addition of recombinant C-terminal fragment of ADAMTS13 comprising of the 5th to 8th thrombospondin type 1 (TSP1) repeats and two CUB domains (T5C) or the 2nd to 8th TSP1 repeats and two CUB domains (T2C) into whole blood also inhibited the surface coverage of fluorescein-labeled platelets on collagen-coated surface in a concentration-dependent manner. In the presence of 0.1 μM and 0.5 μM of recombinant T2C or T5C, the surface coverage of fluorescein-labeled platelets was reduced by ∼40% and ∼60%, respectively. The inhibitory activity of these recombinant C-terminal fragments was nearly abolished if pre-treated with 40 mM of N-ethylmaleimide which blocked surface-exposed free thiols. Moreover, recombinant CUB domains at the highest concentration tested (1.0 μM) did not appear to alter the surface coverage of fluorecein-labeled platelets under the same conditions. These results suggest that the C-terminal TSP1 repeats of ADAMTS13 inhibit platelet adhesion and aggretion or thrombus formation through thiol-thiol interactions between ADAMTS13 and VWF (or other proteins). We conclude that the C-terminal TSP1 repeats may modulate thrombus formation independent of proteolytic activity. Disclosures: No relevant conflicts of interest to declare.

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Human Neutrophil Peptides (HNPs) Enhance Thrombus Formation Under Flow By Inhibiting Proteolytic Activity of Plasma ADAMTS13 Metalloprotease

Blood ◽

10.1182/blood.v126.23.105.105 ◽

2015 ◽

Vol 126 (23) ◽

pp. 105-105

Author(s):

Vikram G. Pillai ◽

Jialing Bao ◽

Jenny McDaniel ◽

Catherine B. Zander1 ◽

Wenjing Cao ◽

...

Keyword(s):

Amino Acid ◽

Inhibitory Activity ◽

Human Neutrophil ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Human Neutrophils ◽

Microfluidic Channels ◽

Dependent Manner ◽

Amino Acid Residues ◽

Adamts13 Activity

Abstract Human neutrophil peptides (HNPs) or alpha (α)-defensins are a family of small antimicrobial peptides which play important roles in innate immunity against invading bacteria, fungi, and viruses. HNPs contain 29-33 amino acid residues which share a distinct pattern of disulfide bonding. HNPs can be further subdivided into myeloid (HNPs 1-4) and enteric (HD5-6) forms. HNPs-1, -2, and -3 are structurally identical except for the first amino acid residue and are predominantly expressed in human neutrophils from which they are released at sites of infection or inflammation. Previous studies have demonstrated that HNP1 promotes thrombus formation. However, the mechanisms underlying its prothrombotic effects are not fully understood. In the present study, we demonstrate that HNP-1, -2, and -3 all inhibit plasma-derived and recombinant ADAMTS13 activity in a concentration-dependent manner as determined by the cleavage of FRETS-VWF73 (Fig. 1A & 1B) and multimeric VWF under denaturing conditions. At final concentrations of ~10-15 µM, purified and synthetic HNP-1, -2, and -3 completely abolish ADAMTS13Õs ability to cleave FRETS-VWF73 (Fig. 1A & 1B). The concentrations required for complete inhibition of proteolytic cleavage of pre-denatured VWF by ADAMTS13 using the urea dialysis method is higher, likely resulting from the removal of peptides from the reaction. HNP-1 binds to ultra large VWF released from endothelial cells, soluble multimeric VWF, GST-VWF73 peptide, and ADAMTS13 as determined by cultured endoethelial cells in a microfluidic channels and by surface plasmon resonance. The affinity (the dissociation constant, KD) for HNP-1 to bind VWF, GST-VWF73, and ADAMTS13 is 8.0 micro mol/L, 1.0 micro mol/L, and 3.2 micro mol/L, respectively. Sequence analysis reveals that the amino acid residues of HNP-1, -2, and -3 all contain a RRY motif that is also found in the spacer domain (i.e. 659RRYGEEY665) of ADAMTS13. We hypothesize that competition by HNPs with ADAMTS13 for binding to VWF-A2 domain mediates their inhibition. As shown, a deletion or alanine substitution of RRY within HNP1 nearly abolishes its ability to inhibit ADAMTS13 activity determined by the cleavage of FRETS-VWF73 (in Fig. 1C) and multimeric VWF under denaturing conditions. Similarly, HNP-beta and aliphatic (with no aromatic rings directly on the nitrogen atom) HNP-1 exhibit no inhibitory activity on ADAMTS13 (Fig. 1C). To further demonstrate the inhibitory activity of HNP1 towards ADAMTS13 under more physiological conditions, a BioFlux microfluidic system is employed. Addition of purified (native) HNP-1 (6-15 micro mol/L) to D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK) anticoagulated whole blood dramatically augments the rate of platelet adhesion and aggregation to the fibrillar collagen-coated surface under arterial shear stress (approximately 100 dyne per square centimeter). These results indicate that HNP-1 plays a role in the inhibition of VWF proteolysis by ADAMTS13 under flow. We conclude that HNP-1, -2, and -3 released from activated neutrophils at sites of infection or inflammation could significantly augment thrombus formation by inhibiting the local or residual plasma ADAMTS13 activity, when the circulating ADAMTS13 activity is already at critically low levels as in cases of hereditary or acquired autoimmune TTP and HUS, thereby triggering the onset of thrombotic complications. (*authors contribute equally to this work). Figure 1. Figure 1. HNPs are potent inhibitors of ADAMTS13 metalloprotease. A. Purified HNPs inhibit ADAMTS13-mediated cleavage of FRETS-VWF73; B. HNP-1, -2, and -3 all inhibit the cleavage of FRETS-VWF73 by ADAMTS13; C. No inhibition of the ADAMTS13 dependent cleavage of FRETS-VWF73 by HNP1 mutants, HNP-beta, and aliphatic HNP1 compared with WT control. Disclosures No relevant conflicts of interest to declare.

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Silver Nanoparticles Induce Neutrophil Extracellular Traps Via Activation of PAD and Neutrophil Elastase

Biomolecules ◽

10.3390/biom11020317 ◽

2021 ◽

Vol 11 (2) ◽

pp. 317

Author(s):

HanGoo Kang ◽

Jinwon Seo ◽

Eun-Jeong Yang ◽

In-Hong Choi

Keyword(s):

Silver Nanoparticles ◽

Neutrophil Elastase ◽

Mammalian Cells ◽

Antimicrobial Properties ◽

Neutrophil Extracellular Traps ◽

Arginine Deiminase ◽

Human Neutrophils ◽

Extracellular Traps ◽

Peptidyl Arginine Deiminase ◽

Key Factor

Silver nanoparticles (AgNPs) are widely used in various fields because of their antimicrobial properties. However, many studies have reported that AgNPs can be harmful to both microorganisms and humans. Reactive oxygen species (ROS) are a key factor of cytotoxicity of AgNPs in mammalian cells and an important factor in the immune reaction of neutrophils. The immune reactions of neutrophils include the expulsion of webs of DNA surrounded by histones and granular proteins. These webs of DNA are termed neutrophil extracellular traps (NETs). NETs allow neutrophils to catch and destroy pathogens in extracellular spaces. In this study, we investigated how AgNPs stimulate neutrophils, specifically focusing on NETs. Freshly isolated human neutrophils were treated with 5 or 100 nm AgNPs. The 5 nm AgNPs induced NET formation, but the 100 nm AgNPs did not. Subsequently, we investigated the mechanism of AgNP-induced NETs using known inhibitors related to NET formation. AgNP-induced NETs were dependent on ROS, peptidyl arginine deiminase, and neutrophil elastase. The result in this study indicates that treatment of 5 nm AgNPs induce NET formation through histone citrullination by peptidyl arginine deiminase and histone cleavage by neutrophil elastase.

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Neutrophil Extracellular Traps Exhibit Antibacterial Activity against Burkholderia pseudomallei and Are Influenced by Bacterial and Host Factors

Infection and Immunity ◽

10.1128/iai.00806-12 ◽

2012 ◽

Vol 80 (11) ◽

pp. 3921-3929 ◽

Cited By ~ 51

Author(s):

Donporn Riyapa ◽

Surachat Buddhisa ◽

Sunee Korbsrisate ◽

Jon Cuccui ◽

Brendan W. Wren ◽

...

Keyword(s):

Burkholderia Pseudomallei ◽

Capsular Polysaccharide ◽

Neutrophil Extracellular Traps ◽

Polymorphonuclear Neutrophils ◽

Dependent Manner ◽

Predisposing Factor ◽

Bacterial Killing ◽

Content Type ◽

Extracellular Traps ◽

Type Iii Protein Secretion

ABSTRACTBurkholderia pseudomalleiis the causative pathogen of melioidosis, of which a major predisposing factor is diabetes mellitus. Polymorphonuclear neutrophils (PMNs) kill microbes extracellularly by the release of neutrophil extracellular traps (NETs). PMNs play a key role in the control of melioidosis, but the involvement of NETs in killing ofB. pseudomalleiremains obscure. Here, we showed that bactericidal NETs were released from human PMNs in response toB. pseudomalleiin a dose- and time-dependent manner.B. pseudomallei-induced NET formation required NADPH oxidase activation but not phosphatidylinositol-3 kinase, mitogen-activated protein kinases, or Src family kinase signaling pathways.B. pseudomalleimutants defective in the virulence-associated Bsa type III protein secretion system (T3SS) or capsular polysaccharide I (CPS-I) induced elevated levels of NETs. NET induction by such mutants was associated with increased bacterial killing, phagocytosis, and oxidative burst by PMNs. Taken together the data imply that T3SS and the capsule may play a role in evading the induction of NETs. Importantly, PMNs from diabetic subjects released NETs at a lower level than PMNs from healthy subjects. Modulation of NET formation may therefore be associated with the pathogenesis and control of melioidosis.

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