scholarly journals Patients with recurrent PVL+-Staphylococcus aureus infections show enhanced sensitivity to PVL-mediated formation of atypical NETs

2021 ◽  
Author(s):  
Hina Jhelum ◽  
Dora Čerina ◽  
Christopher J Harbort ◽  
Andreas Lindner ◽  
Leif Gunnar Hanitsch ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Proteome Analysis ◽  
Neutrophil Extracellular Traps ◽  
Human Neutrophils ◽  
Proteinase 3 ◽  
Oxygen Species ◽  
Extracellular Traps ◽  
Enhanced Sensitivity ◽  
Microbial Toxin ◽  
Panton Valentine Leukocidin

Panton-Valentine leukocidin (PVL) is a Staphylococcus aureus (S. aureus) toxin that binds to and kills human neutrophils, resulting in the formation of neutrophil extracellular traps (NETs). Some individuals colonized with PVL-positive S. aureus (PVL-SA) suffer from recurring infections whereas others are asymptomatically colonized. We found that neutrophils from affected patients express higher levels of CD45, one of the PVL receptors, and are more susceptible to killing at a low concentration of recombinant PVL than control neutrophils. We verified that PVL induces the formation of NETs and provide genetic and pharmacological evidence that PVL-induced NET formation is independent of NADPH-oxidase and reactive oxygen species (ROS) production. Through NET proteome analysis we identified that the protein content of PVL-induced NETs is different from NETs induced by mitogen or the microbial toxin nigericin. The abundance of the proteins cathelicidin (CAMP), elastase (NE), and proteinase 3 (PRTN3) was lower on PVL-induced NETs, which were inefficient in killing S. aureus. Neutrophils from patients that suffer from recurring PVL-positive infections may be more sensitive to PVL-induced NET formation, which may impair their ability to combat the infection.

Cisplatin Fails To Stimulate Production Of Reactive Oxygen Species and Release Of Neutrophil Extracellular Traps By Human Neutrophils In Vitro

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4714-4714
Author(s):  
Leonardo Pasalic ◽  
Campbell Heather ◽  
Shane Thomas ◽  
Vivien M Chen
Keyword(s):  
Reactive Oxygen Species ◽  
Fluorescent Microscopy ◽  
Neutrophil Extracellular Traps ◽  
Vehicle Control ◽  
Human Neutrophils ◽  
Oxygen Species ◽  
Extracellular Traps ◽  
Spontaneous Production ◽  
Confocal Fluorescent Microscopy

Background Cisplatin is a commonly used antineoplastic agent for treatment of a broad range of cancers. Cisplatin-based treatment has been associated with a significant risk of venous thromboembolism. The mechanisms through which cisplatin contributes to a prothrombotic state remain unclear. Neutrophil extracellular traps (NETs) consist of web-like DNA–histone core decorated with granule proteins and are released from activated neutrophils in a process dependent on reactive oxygen species (ROS), in particular hypochlorous acid (HOCl). Recently, NETs have been shown to play an important role in initiation and propagation of venous thrombus in a number of animal models of deep vein thrombosis. The aim of this study was to investigate whether NETs may provide a potential link between cisplatin and venous thromboembolism. Methods and Results To assess the effect of cisplatin on release of NETs by ex vivo human neutrophils isolated by positive immunomagnetic selection we visualised NETs release by confocal fluorescent microscopy and performed fluorimetric quantification of cell-free DNA (CFDNA) using either SYTOX Green nucleic acid stain (10 µM) or an ultrasensitive fluorescent assay Picogreen Quant IT (Invitrogen). In contrast to stimulation with phorbol 12-myristate 13-acetate (PMA) (25 nM),which resulted in 22 ng/104neutrophils of detectable CFDNA, neither of these two assays could detect any significant release of CFDNA by human neutrophils exposed to cisplatin (15 µM) for 2 or 4 hours above baseline similar with vehicle control. Furthermore, confocal fluorescent microscopy imaging of neutrophils stained with non-cell permeable DNA dye SYTOX Red (Invitrogen) demonstrated no difference in NET formation between control and cisplatin treated human neutrophils. Thus we could not demonstrate that NETS are produced in response to cisplatin treatment. In view of consistent reports that NET formation is ROS dependent we decided to investigate whether cisplatin exposure leads to production of ROS by human neutrophils. Few published studies into the effects of cisplatin on the production of ROS by human neutrophils in vitro offer conflicting results. We used flow cytometry and fluorescent probe hydroethidine (HE) for detection of intercellular superoxide anion radical in HL60 granulocytic cells in the presence of cisplatin (up to 50 µM). Differentiation down the granulocytic lineage after stimulation with ATRA was confirmed by light microscopy and by flow cytometry. Capacity of differentiated HL60 cells to generate NET formation after PMA stimulation was confirmed by fluorescence microscopy. Cisplatin failed to augment the spontaneous production of ROS by ATRA differentiated HL60 cells. The number of viable ethidium-high cells in cisplatin treated group did not differ from the vehicle control indicating no detectable production of ROS in response to cisplatin. In contrast, positive control treatment with PMA (25 nM) and menadione (40 µM) resulted in 4- and 20-fold increase in viable ethidium-high population respectively. ROS generation by human neutrophils was measured by a colorimetric assay for chlorination of extracellular taurine to determine if exposure to cisplatin results in the production of HOCl by human neutrophils in vitro. Treatment of resting neutrophils with cisplatin (15 µM) for 30 min or 120 min was not associated with an increase in the spontaneous production of HOCl above the baseline. Furthermore, the PMA (25 nM)-activated generation of HOCl production was not increased by pre-treating neutrophils with cisplatin indicating that there was no potentiation of ROS by pre-treatment with cisplatin. Discussion and Conclusion Our results suggest that cisplatin fails to induce release of NETs or HOCl from human neutrophils in vitro. These negative findings seem to be at odds with the well described pro-oxidative actions of cisplatin. One possible explanation centres on reported findings that the pro-oxidative effects of cisplatin are dependent on the mitochondrial generation of ROS whilst the mitochondria-generated ROS appear not to be instrumental to NET formation. Therefore, we postulate that cisplatin may not be able to induce NET formation by human neutrophils, which are known to contain few mitochondria, due to a sub-threshold ROS signal. Therefore it appears that cisplatin-associated increased risk of venous thrombosis is unlikely to be mediated through NETs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Small GTPase Cdc42 Negatively Regulates the Formation of Neutrophil Extracellular Traps by Engaging Mitochondria

2021 ◽  
Vol 12 ◽  
Author(s):  
Heidi Tackenberg ◽  
Sonja Möller ◽  
Marie-Dominique Filippi ◽  
Tamás Laskay
Keyword(s):  
Neutrophil Extracellular Traps ◽  
Small Gtpase ◽  
Negative Regulator ◽  
Human Neutrophils ◽  
Neutrophil Granulocytes ◽  
Oxygen Species ◽  
Sk Channels ◽  
First Line ◽  
Extracellular Traps ◽  
Kinase C

Neutrophil granulocytes represent the first line of defense against invading pathogens. In addition to the production of Reactive Oxygen Species, degranulation, and phagocytosis, these specialized cells are able to extrude Neutrophil Extracellular Traps. Extensive work was done to elucidate the mechanism of this special form of cell death. However, the exact mechanisms are still not fully uncovered. Here we demonstrate that the small GTPase Cdc42 is a negative regulator of NET formation in primary human and murine neutrophils. We present a functional role for Cdc42 activity in NET formation that differs from the already described NETosis pathways. We show that Cdc42 deficiency induces NETs independent of the NADPH-oxidase but dependent on protein kinase C. Furthermore, we demonstrate that Cdc42 deficiency induces NETosis through activation of SK-channels and that mitochondria play a crucial role in this process. Our data therefore suggests a mechanistic role for Cdc42 activity in primary human neutrophils, and identify Cdc42 activity as a target to modulate the formation of Neutrophil Extracellular Traps.

scholarly journals Natural Variation in Clinical Isolates of Candida albicans Modulates Neutrophil Responses

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Madhu Shankar ◽  
Tricia L. Lo ◽  
Ana Traven
Keyword(s):  
Reactive Oxygen Species ◽  
Candida Albicans ◽  
Reactive Oxygen ◽  
Neutrophil Extracellular Traps ◽  
Prototype Strain ◽  
Human Neutrophils ◽  
Oxygen Species ◽  
Content Type ◽  
Extracellular Traps ◽  
Strain Sc5314

ABSTRACT Neutropenia predisposes patients to life-threatening infection with Candida albicans, a commensal and opportunistic fungal pathogen. How phenotypic variation in C. albicans isolates dictates neutrophil responses is poorly understood. By using a panel of clinical C. albicans strains, here we report that the prototype strain SC5314 induces the most potent accumulation of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) by human neutrophils of all tested isolates. ROS and NET accumulation positively correlated with the degree of hyphal formation by the isolates, the hypha being the fungal morphotype that promotes pathogenesis. However, there was no correlation of ROS and NET accumulation with fungal killing by neutrophils. Fungal killing was also not correlated with phagocytosis levels or oxidative stress susceptibility of the isolates. The bloodstream isolate P94015 cannot make hyphae and was previously shown to be hyperfit in the murine gut commensalism model. Our results show that P94015 displays poor phagocytosis by neutrophils, the least ROS and NET accumulation of all tested isolates, and resistance to neutrophil-mediated killing. Our data suggest that reduced susceptibility to neutrophils is likely to be independent from a previously described genetic mutation in P94015 that promotes commensalism. Reduced clearance by neutrophils could benefit commensal fitness of C. albicans and could also have promoted the virulence of P94015 in the human patient in the absence of hyphal morphogenesis. Collectively, our study provides new insights into neutrophil interactions with C. albicans and suggests that studying diverse isolates informs knowledge of the relevant aspects of this key immune interaction. IMPORTANCE Neutrophils are the key immune cell type for host defenses against infections with Candida albicans. C. albicans strains isolated from patients display large phenotypic diversity, but how this diversity impacts host-pathogen interactions with neutrophils is incompletely defined. Here, we show that important neutrophil responses, such as accumulation of reactive oxygen species and neutrophil extracellular traps, as well as the levels of phagocytosis and killing of the pathogen, differ when comparing diverse C. albicans isolates. A bloodstream patient isolate previously described as more suited to commensalism than pathogenesis in animal models is relatively “silent” to neutrophils and resistant to killing. Our findings illuminate the relationships between fungal morphogenesis, neutrophil responses, and C. albicans survival. Our findings suggest that host phenotypes of a commensally adapted strain could be driven by resistance to immune clearance and indicate that we should extend our studies beyond the “prototype” strain SC5314 for deeper understanding of Candida-neutrophil interactions.

scholarly journals Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps

2010 ◽  
Vol 191 (3) ◽  
pp. 677-691 ◽  
Author(s):  
Venizelos Papayannopoulos ◽  
Kathleen D. Metzler ◽  
Abdul Hakkim ◽  
Arturo Zychlinsky
Keyword(s):  
Immune Deficiency ◽  
Neutrophil Elastase ◽  
Serine Proteases ◽  
Neutrophil Extracellular Traps ◽  
Oxygen Species ◽  
Chromatin Decondensation ◽  
Unknown Mechanism ◽  
Extracellular Traps ◽  
Decondensed Chromatin ◽  
Chromatin Density

Neutrophils release decondensed chromatin termed neutrophil extracellular traps (NETs) to trap and kill pathogens extracellularly. Reactive oxygen species are required to initiate NET formation but the downstream molecular mechanism is unknown. We show that upon activation, neutrophil elastase (NE) escapes from azurophilic granules and translocates to the nucleus, where it partially degrades specific histones, promoting chromatin decondensation. Subsequently, myeloperoxidase synergizes with NE in driving chromatin decondensation independent of its enzymatic activity. Accordingly, NE knockout mice do not form NETs in a pulmonary model of Klebsiella pneumoniae infection, which suggests that this defect may contribute to the immune deficiency of these mice. This mechanism provides for a novel function for serine proteases and highly charged granular proteins in the regulation of chromatin density, and reveals that the oxidative burst induces a selective release of granular proteins into the cytoplasm through an unknown mechanism.

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

Biomolecules ◽  
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.

scholarly journals Capsular polysaccharides from Cryptococcus neoformans modulate production of neutrophil extracellular traps (NETs) by human neutrophils

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Juliana D. B. Rocha ◽  
Michelle T. C. Nascimento ◽  
Debora Decote-Ricardo ◽  
Suzana Côrte-Real ◽  
Alexandre Morrot ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Neutrophil Extracellular Traps ◽  
Human Neutrophils ◽  
Capsular Polysaccharides ◽  
Extracellular Traps

scholarly journals Staphylococcus aureus Degrades Neutrophil Extracellular Traps to Promote Immune Cell Death

Science ◽  
2013 ◽  
Vol 342 (6160) ◽  
pp. 863-866 ◽  
Author(s):  
V. Thammavongsa ◽  
D. M. Missiakas ◽  
O. Schneewind
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Death ◽  
Immune Cell ◽  
Neutrophil Extracellular Traps ◽  
Extracellular Traps

Exosomal MALAT1 derived from ox-LDL-treated endothelial cells induce neutrophil extracellular traps to aggravate atherosclerosis

2020 ◽  
Vol 401 (3) ◽  
pp. 367-376 ◽  
Author(s):  
Hailai Gao ◽  
XiaoLi Wang ◽  
Chaolan Lin ◽  
Zhujun An ◽  
Jiangbo Yu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Low Density Lipoprotein ◽  
Atherosclerotic Lesion ◽  
Density Lipoprotein ◽  
Neutrophil Extracellular Traps ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Extracellular Traps ◽  
Umbilical Vein Endothelial Cells

AbstractThe objective of this study was to reveal a novel mechanism underlying the progression of atherosclerosis (AS) associated with endothelial cells (ECs) and neutrophils. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to observe the morphology and particle size of isolated exosomes. Western blotting was applied to examine exosomal markers, while the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The production of inflammatory cytokines and reactive oxygen species (ROS) was determined by an enzyme-linked immunosorbent assay (ELISA) and a dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Circulating neutrophil extracellular traps (NETs) were represented by myeloperoxidase (MPO)-DNA complexes. NETs formation was assessed using immunofluorescence microscopy. Atherosclerotic lesion development was measured by Oil Red O (ORO) staining. In the results, MALAT1 expression was increased in exosomes extracted from oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs). When co-cultured with human neutrophils, exosomes derived from ox-LDL-treated HUVECs were revealed to promote NETs formation, which was mediated by exosomal MALAT1. Furthermore, ox-LDL-treated HUVECs-derived exosomes were demonstrated to trigger hyperlipidemia, inflammatory response and NETs release in a mouse model of AS. In conclusion, exosomal MALAT1 derived from ox-LDL-treated ECs initiated NETs formation, which in turn deteriorated AS.

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