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

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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Neisseria gonorrhoeae Evades Calprotectin-Mediated Nutritional Immunity and Survives Neutrophil Extracellular Traps by Production of TdfH

Infection and Immunity ◽

10.1128/iai.00319-16 ◽

2016 ◽

Vol 84 (10) ◽

pp. 2982-2994 ◽

Cited By ~ 26

Author(s):

Sophonie Jean ◽

Richard A. Juneau ◽

Alison K. Criss ◽

Cynthia N. Cornelissen

Keyword(s):

Neisseria Gonorrhoeae ◽

Defense Mechanism ◽

Outer Membrane Proteins ◽

Neutrophil Extracellular Traps ◽

Host Protein ◽

Dependent Manner ◽

Bacterial Survival ◽

Content Type ◽

Extracellular Traps ◽

Nutritional Immunity

Neisseria gonorrhoeaesuccessfully overcomes host strategies to limit essential nutrients, termed nutritional immunity, by production of TonB-dependent transporters (TdTs)—outer membrane proteins that facilitate nutrient transport in an energy-dependent manner. Four gonococcal TdTs facilitate utilization of iron or iron chelates from host-derived proteins, including transferrin (TbpA), lactoferrin (LbpA), and hemoglobin (HpuB), in addition to xenosiderophores from other bacteria (FetA). The roles of the remaining four uncharacterized TdTs (TdfF, TdfG, TdfH, and TdfJ) remain elusive. Regulatory data demonstrating that production of gonococcal TdfH and TdfJ are unresponsive to or upregulated under iron-replete conditions led us to evaluate the role of these TdTs in the acquisition of nutrients other than iron. In this study, we found that production of gonococcal TdfH is both Zn and Zur repressed. We also found that TdfH confers resistance to calprotectin, an immune effector protein highly produced in neutrophils that has antimicrobial activity due to its ability to sequester Zn and Mn. We found that TdfH directly binds calprotectin, which enables gonococcal Zn accumulation in a TdfH-dependent manner and enhances bacterial survival after exposure to neutrophil extracellular traps (NETs). These studies highlight Zn sequestration by calprotectin as a key functional arm of NET-mediated killing of gonococci. We demonstrate for the first time thatN. gonorrhoeaeexploits this host strategy in a novel defense mechanism, in which TdfH production hijacks and directly utilizes the host protein calprotectin as a zinc source and thereby evades nutritional immunity.

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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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Immunomodulatory Role of Clarithromycin in Acinetobacter baumannii Infection via Formation of Neutrophil Extracellular Traps

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02063-15 ◽

2015 ◽

Vol 60 (2) ◽

pp. 1040-1048 ◽

Cited By ~ 26

Author(s):

Theocharis Konstantinidis ◽

Konstantinos Kambas ◽

Alexandros Mitsios ◽

Maria Panopoulou ◽

Victoria Tsironidou ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Ex Vivo ◽

Neutrophil Extracellular Traps ◽

Macrolide Antibiotics ◽

Dependent Manner ◽

Biofilm Inhibition ◽

Content Type ◽

Extracellular Traps

ABSTRACTMacrolide antibiotics have been shown to act as immunomodulatory molecules in various immune cells. However, their effect on neutrophils has not been extensively investigated. In this study, we investigated the role of macrolide antibiotics in the generation of neutrophil extracellular traps (NETs). By assessingex vivoandin vivoNET formation, we demonstrated that clarithromycin is able to induce NET generation bothin vitroandin vivo. Clarithromycin utilizes autophagy in order to form NETs, and these NETs are decorated with antimicrobial peptide LL-37. Clarithromycin-induced NETs are able to inhibitAcinetobacter baumanniigrowth and biofilm formation in an LL-37-dependent manner. Additionally, LL-37 antimicrobial function depends on NET scaffold integrity. Collectively, these data expand the knowledge on the immunomodulatory role of macrolide antibiotics via the generation of LL-37-bearing NETs, which demonstrate LL-37-dependent antimicrobial activity and biofilm inhibition againstA. baumannii.

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Role of neutrophil extracellular traps in radiation resistance of invasive bladder cancer

Nature Communications ◽

10.1038/s41467-021-23086-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Surashri Shinde-Jadhav ◽

Jose Joao Mansure ◽

Roni F. Rayes ◽

Gautier Marcq ◽

Mina Ayoub ◽

...

Keyword(s):

Bladder Cancer ◽

Neutrophil Extracellular Traps ◽

Polymorphonuclear Neutrophils ◽

Dependent Manner ◽

Potential Therapeutic Target ◽

Extracellular Traps ◽

Tumor Immune Microenvironment

AbstractRadiation therapy (RT) is used in the management of several cancers; however, tumor radioresistance remains a challenge. Polymorphonuclear neutrophils (PMNs) are recruited to the tumor immune microenvironment (TIME) post-RT and can facilitate tumor progression by forming neutrophil extracellular traps (NETs). Here, we demonstrate a role for NETs as players in tumor radioresistance. Using a syngeneic bladder cancer model, increased NET deposition is observed in the TIME of mice treated with RT and inhibition of NETs improves overall radiation response. In vitro, the protein HMGB1 promotes NET formation through a TLR4-dependent manner and in vivo, inhibition of both HMGB1 and NETs significantly delays tumor growth. Finally, NETs are observed in bladder tumors of patients who did not respond to RT and had persistent disease post-RT, wherein a high tumoral PMN-to-CD8 ratio is associated with worse overall survival. Together, these findings identify NETs as a potential therapeutic target to increase radiation efficacy.

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Burkholderia pseudomallei Capsule Exacerbates Respiratory Melioidosis but Does Not Afford Protection against Antimicrobial Signaling or Bacterial Killing in Human Olfactory Ensheathing Cells

Infection and Immunity ◽

10.1128/iai.01546-15 ◽

2016 ◽

Vol 84 (7) ◽

pp. 1941-1956 ◽

Cited By ~ 8

Author(s):

Samantha J. Dando ◽

Deepak S. Ipe ◽

Michael Batzloff ◽

Matthew J. Sullivan ◽

David K. Crossman ◽

...

Keyword(s):

Burkholderia Pseudomallei ◽

Capsular Polysaccharide ◽

Molecular Transport ◽

Olfactory Ensheathing Cells ◽

Bacterial Invasion ◽

Bacterial Killing ◽

Content Type ◽

Cytokine Responses ◽

Ensheathing Cells

Melioidosis, caused by the bacteriumBurkholderia pseudomallei, is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery ofB. pseudomalleito establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improvedB. pseudomalleisurvivalin vivoand triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response toB. pseudomalleiinfection, we infected human olfactory ensheathing cells (OECs)in vitroand measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of theB. pseudomalleiin a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I inB. pseudomalleisurvivalin vivofollowing inhalation infection and the antibacterial signaling network that exists in human OM and OECs.

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Neutrophil Extracellular Traps May Contribute to the Pathogenesis in Adult-onset Still Disease

The Journal of Rheumatology ◽

10.3899/jrheum.181058 ◽

2019 ◽

Vol 46 (12) ◽

pp. 1560-1569 ◽

Cited By ~ 4

Author(s):

Mi-Hyun Ahn ◽

Jae Ho Han ◽

Young-Jun Chwae ◽

Ju-Yang Jung ◽

Chang-Hee Suh ◽

...

Keyword(s):

Immunohistochemical Analysis ◽

Serum Levels ◽

Neutrophil Extracellular Traps ◽

Polymorphonuclear Neutrophils ◽

Adult Onset ◽

Cell Free Dna ◽

Extracellular Traps ◽

Free Dna ◽

Still Disease

Objective.Release of neutrophil extracellular traps (NET) has been described as an effector mechanism of polymorphonuclear neutrophils in several inflammatory diseases. Thus, this study was performed to evaluate the role of NET in the pathogenesis of adult-onset Still disease (AOSD).Methods.We determined the serum levels of NET molecules and investigated their associations with clinical disease activities in patients with AOSD. Further, we analyzed the differences in the NETosis response in AOSD patients compared to healthy controls (HC). To explore the in vivo involvement of NET in AOSD, we performed immunohistochemical analysis of skin and lymph node (LN) biopsies for proteins related to NET in patients with active AOSD.Results.Serum levels of cell-free DNA, myeloperoxidase (MPO)-DNA complex, and α-defensin were significantly increased in patients with AOSD compared to HC. Serum levels of the NET molecules, cell-free DNA, MPO-DNA, and α-defensin were correlated with several disease activity markers for AOSD. In followup of patients with AOSD after treatment with corticosteroid, the levels of cell-free DNA and α-defensin decreased significantly. On immunohistochemistry, neutrophil elastase–positive and MPO-positive inflammatory cells were detected in skin and LN of patients with AOSD, and were expressed in fiber form in the lesions. The serum from patients with active AOSD induced NETosis in neutrophils from HC. NET molecules induced interleukin 1β production in monocytes, representing a novel mechanism in the pathogenesis of AOSD.Conclusion.The findings presented here suggest that NET may contribute to the inflammatory response and pathogenesis in AOSD.

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Ovine vital neutrophil extracellular traps bind and impairHaemonchus contortusL3 in a breed-dependent manner

Parasite Immunology ◽

10.1111/pim.12572 ◽

2018 ◽

Vol 40 (9) ◽

pp. e12572 ◽

Cited By ~ 3

Author(s):

Javier J. Garza ◽

Scott P. Greiner ◽

Scott A. Bowdridge

Keyword(s):

Neutrophil Extracellular Traps ◽

Dependent Manner ◽

Extracellular Traps

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Neutrophil Extracellular Traps Induce Platelet Adhesion and Thrombus Formation.

Blood ◽

10.1182/blood.v114.22.1345.1345 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1345-1345 ◽

Cited By ~ 4

Author(s):

Tobias Fuchs ◽

Alexander Brill ◽

Daniel Dürschmied ◽

Daphne Schatzberg ◽

John H. Hartwig ◽

...

Keyword(s):

Whole Blood ◽

Platelet Adhesion ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Neutrophil Extracellular Traps ◽

Thrombin Inhibitor ◽

Human Neutrophils ◽

Dependent Manner ◽

Extracellular Traps ◽

Deep Vein

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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In VivoAssessment of Drug Efficacy against Mycobacterium abscessus Using the Embryonic Zebrafish Test System

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00142-14 ◽

2014 ◽

Vol 58 (7) ◽

pp. 4054-4063 ◽

Cited By ~ 41

Author(s):

Audrey Bernut ◽

Vincent Le Moigne ◽

Tiffany Lesne ◽

Georges Lutfalla ◽

Jean-Louis Herrmann ◽

...

Keyword(s):

Drug Efficacy ◽

Test System ◽

Mycobacterium Abscessus ◽

Zebrafish Embryos ◽

Dependent Manner ◽

Bacterial Killing ◽

Content Type ◽

Fluorescence Measurements ◽

Drug Concentrations

ABSTRACTMycobacterium abscessusis responsible for a wide spectrum of clinical syndromes and is one of the most intrinsically drug-resistant mycobacterial species. Recent evaluation of thein vivotherapeutic efficacy of the few potentially active antibiotics againstM. abscessuswas essentially performed using immunocompromised mice. Herein, we assessed the feasibility and sensitivity of fluorescence imaging for monitoring thein vivoactivity of drugs against acuteM. abscessusinfection using zebrafish embryos. A protocol was developed where clarithromycin and imipenem were directly added to water containing fluorescentM. abscessus-infected embryos in a 96-well plate format. The status of the infection with increasing drug concentrations was visualized on a spatiotemporal level. Drug efficacy was assessed quantitatively by measuring the index of protection, the bacterial burden (CFU), and the number of abscesses through fluorescence measurements. Both drugs were active in infected embryos and were capable of significantly increasing embryo survival in a dose-dependent manner. Protection from bacterial killing correlated with restricted mycobacterial growth in the drug-treated larvae and with reduced pathophysiological symptoms, such as the number of abscesses within the brain. In conclusion, we present here a new and efficient method for testing and compare thein vivoactivity of two clinically relevant drugs based on a fluorescent reporter strain in zebrafish embryos. This approach could be used for rapid determination of thein vivodrug susceptibility profile of clinical isolates and to assess the preclinical efficacy of new compounds againstM. abscessus.

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