Dataset Information

Transketolase and vitamin B1 influence on ROS-dependent neutrophil extracellular traps (NETs) formation.

ABSTRACT: Neutrophil extracellular traps (NETs) are a recently identified, web-like, extracellular structure composed of decondensed nuclear DNA and associated antimicrobial granules. NETs are extruded into the extracellular environment via the reactive oxygen species (ROS)-dependent cell death pathway participating in inflammation and autoimmune diseases. Transketolase (TKT) is a thiamine pyrophosphate (vitamin B1)-dependent enzyme that links the pentose phosphate pathway with the glycolytic pathway by feeding excess sugar phosphates into the main carbohydrate metabolic pathways to generate biosynthetic reducing capacity in the form of NADPH as a substrate for ROS generation. In this work, TKT was selected as a lead candidate from 24 NET-associated proteins obtained by literature screening and knowledge gap assessment. Consequently, we determined whether TKT influenced NET formation in vitro. We firstly established that the release of ROS-dependent NETs was significantly decreased after purified human PMNs were pretreated with oxythiamine, a TKT inhibitor, and in a concentration dependent manner. As a cofactor for TKT reaction, we evaluated the release of NET formation either in vitamin B1 treatment or in combined use of oxythiamine and vitamin B1, and found that those treatments also exerted a significant suppressive effect on the amount of NET-DNA and ROS production. The regulation of TKT by oxythiamine and/or vitamin B1 may therefore be associated with response to the modulation of NET formation by preventing generation of excessive NETs in inflammatory diseases.

SUBMITTER: Riyapa D

PROVIDER: S-EPMC6695114 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Transketolase and vitamin B1 influence on ROS-dependent neutrophil extracellular traps (NETs) formation.

Riyapa Donporn D Rinchai Darawan D Muangsombut Veerachat V Wuttinontananchai Chayanin C Toufiq Mohammed M Chaussabel Damien D Ato Manabu M Blackwell Jenefer M JM Korbsrisate Sunee S

PloS one 20190815 8

Neutrophil extracellular traps (NETs) are a recently identified, web-like, extracellular structure composed of decondensed nuclear DNA and associated antimicrobial granules. NETs are extruded into the extracellular environment via the reactive oxygen species (ROS)-dependent cell death pathway participating in inflammation and autoimmune diseases. Transketolase (TKT) is a thiamine pyrophosphate (vitamin B1)-dependent enzyme that links the pentose phosphate pathway with the glycolytic pathway by f ...[more]

PMID: 31415630

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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:The formation of neutrophil extracellular traps (NETs) is an immune defense mechanism of neutrophilic granulocytes. Moreover, it is also involved in the pathogenesis of autoimmune, inflammatory, and neoplastic diseases. For that reason, the process of NET formation (NETosis) is subject of intense ongoing research. In vitro approaches to quantify NET formation are commonly used and involve neutrophil stimulation with various activators such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), or calcium ionophores (CaI). However, the experimental conditions of these experiments, particularly the media and media supplements employed by different research groups, vary considerably, rendering comparisons of results difficult. Here, we present the first standardized investigation of the influence of different media supplements on NET formation in vitro. The addition of heat-inactivated (hi) fetal calf serum (FCS), 0.5% human serum albumin (HSA), or 0.5% bovine serum albumin (BSA) efficiently prevented NET formation of human neutrophils following stimulation with LPS and CaI, but not after stimulation with PMA. Thus, serum components such as HSA, BSA and hiFCS (at concentrations typically found in the literature) inhibit NET formation to different degrees, depending on the NETosis inducer used. In contrast, in murine neutrophils, NETosis was inhibited by FCS and BSA, regardless of the inducer employed. This shows that mouse and human neutrophils have different susceptibilities toward the inhibition of NETosis by albumin or serum components. Furthermore, we provide experimental evidence that albumin inhibits NETosis by scavenging activators such as LPS. We also put our results into the context of media supplements most commonly used in NET research. In experiments with human neutrophils, either FCS (0.5-10%), heat-inactivated (hiFCS, 0.1-10%) or human serum albumin (HSA, 0.05-2%) was commonly added to the medium. For murine neutrophils, serum-free medium was used in most cases for stimulation with LPS and CaI, reflecting the different sensitivities of human and murine neutrophils to media supplements. Thus, the choice of media supplements greatly determines the outcome of experiments on NET-formation, which must be taken into account in NETosis research.

Dataset Information

Transketolase and vitamin B1 influence on ROS-dependent neutrophil extracellular traps (NETs) formation.

Publications

Transketolase and vitamin B1 influence on ROS-dependent neutrophil extracellular traps (NETs) formation.

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