Dataset Information

Classical ROS-dependent and early/rapid ROS-independent release of Neutrophil Extracellular Traps triggered by Leishmania parasites.

ABSTRACT: Neutrophil extracellular traps (NETs) extruded from neutrophils upon activation are composed of chromatin associated with cytosolic and granular proteins, which ensnare and kill microorganisms. This microbicidal mechanism named classical netosis has been shown to dependent on reactive oxygen species (ROS) generation by NADPH oxidase and also chromatin decondensation dependent upon the enzymes (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO). NET release also occurs through an early/rapid ROS-independent mechanism, named early/rapid vital netosis. Here we analyze the role of ROS, NE, MPO and PAD4 in the netosis stimulated by Leishmania amazonensis promastigotes in human neutrophils. We demonstrate that promastigotes induce a classical netosis, dependent on the cellular redox imbalance, as well as by a chloroamidine sensitive and elastase activity mechanism. Additionally, Leishmania also induces the early/rapid NET release occurring only 10?minutes after neutrophil-parasite interaction. We demonstrate here, that this early/rapid mechanism is dependent on elastase activity, but independent of ROS generation and chloroamidine. A better understanding of both mechanisms of NET release, and the NETs effects on the host immune system modulation, could support the development of new potential therapeutic strategies for leishmaniasis.

SUBMITTER: Rochael NC

PROVIDER: S-EPMC4682131 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Classical ROS-dependent and early/rapid ROS-independent release of Neutrophil Extracellular Traps triggered by Leishmania parasites.

Rochael Natalia C NC Guimarães-Costa Anderson B AB Nascimento Michelle T C MT DeSouza-Vieira Thiago S TS Oliveira Matheus P MP Garcia e Souza Luiz F LF Oliveira Marcus F MF Saraiva Elvira M EM

Scientific reports 20151217

Neutrophil extracellular traps (NETs) extruded from neutrophils upon activation are composed of chromatin associated with cytosolic and granular proteins, which ensnare and kill microorganisms. This microbicidal mechanism named classical netosis has been shown to dependent on reactive oxygen species (ROS) generation by NADPH oxidase and also chromatin decondensation dependent upon the enzymes (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO). NET release also occurs through an early/rap ...[more]

PMID: 26673780

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Project description:The inflammatory microenvironment is commonly characterized by extracellular acidosis (pH < 7.35). Sensitivity to pH, CO2 or bicarbonate concentrations allows neutrophils to react to changes in their environment and to detect inflamed areas in the tissue. One important antimicrobial effector mechanism is the production of neutrophil extracellular traps (NETs), which are released during a programmed reactive oxygen species (ROS)-dependent cell death, the so-called NETosis. Although several functions of neutrophils have been analyzed under acidic conditions, the effect of extracellular acidosis on NETosis remains mainly unexplored and the available experimental results are contradictory. We performed a comprehensive study with the aim to elucidate the effect of extracellular acidosis on ROS-dependent NETosis of primary human neutrophils and to identify the underlying mechanisms. The study was performed in parallel in a CO2-bicabonate-buffered culture medium, which mimics in vivo conditions, and under HEPES-buffered conditions to verify the effect of pH independent of CO2 or bicarbonate. We could clearly show that extracellular acidosis (pH 6.5, 6.0, and 5.5) and intracellular acidification inhibit the release of ROS-dependent NETs upon stimulation of neutrophils with phorbol myristate acetate and immobilized immune complexes. Moreover, our findings suggest that the diminished NET release is a consequence of reduced ROS production and diminished glycolysis of neutrophils under acidic conditions. It was suggested previously that neutrophils can sense the border of inflamed tissue by the pH gradient and that a drop in pH serves as an indicator for the progress of inflammation. Following this hypothesis, our data indicate that an acidic inflammatory environment results in inhibition of extracellular operating effector mechanisms of neutrophils such as release of ROS and NETs. This way the release of toxic components and tissue damage can be avoided. However, we observed that major antimicrobial effector mechanisms such as phagocytosis and the killing of pathogens by neutrophils remain functional under acidic conditions.

Project description:Trypanosoma brucei brucei trypomastigotes are classical blood parasites of cattle, these stages might become potential targets for circulating polymorphonuclear neutrophils (PMN). We here investigated NETs extrusion and related oxygen consumption in bovine PMN exposed to motile T. b. brucei trypomastigotes in vitro. Parasite exposure induced PMN activation as detected by enhanced oxygen consumption rates (OCR), extracellular acidification rates (ECAR), and production of total and extracellular reactive oxygen species (ROS). Scanning electron microscopy (SEM) showed that co-cultivation of bovine PMN with motile trypomastigotes resulted in NETs formation within 120 min of exposure. T. b. brucei-induced NETs were confirmed by confocal microscopy demonstrating co-localization of extruded DNA with neutrophil elastase (NE) and nuclear histones. Immunofluorescence analyses demonstrated that trypomastigotes induced different phenotypes of NETs in bovine PMN, such as aggregated NETs (aggNETs), spread NETs (sprNETs), and diffuse NETs (diffNETs) with aggNETs being the most abundant ones. Furthermore, live cell 3D-holotomographic microscopy unveiled detailed morphological changes during the NETotic process. Quantification of T. b. brucei-induced NETs formation was estimated by DNA and nuclear area analysis (DANA) and confirmed enhanced NETs formation in response to trypomastigote stages. Formation of NETs does not result in a decrease of T. b. brucei viability, but a decrease of 26% in the number of motile parasites. Referring the involved signaling pathways, trypomastigote-induced NETs formation seems to be purinergic-dependent, since inhibition via NF449 treatment resulted in a significant reduction of T. b. brucei-triggered DNA extrusion. Overall, future studies will have to analyze whether the formation of aggNETs indeed plays a role in the outcome of clinical disease and bovine African trypanosomiasis-related immunopathological disorders, such as increased intravascular coagulopathy and vascular permeability, often reported to occur in this disease.

Dataset Information

Classical ROS-dependent and early/rapid ROS-independent release of Neutrophil Extracellular Traps triggered by Leishmania parasites.

Publications

Classical ROS-dependent and early/rapid ROS-independent release of Neutrophil Extracellular Traps triggered by Leishmania parasites.

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