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. 2015 Jun;145(2):213-24.
doi: 10.1111/imm.12437.

Metabolic requirements for neutrophil extracellular traps formation

Oscar Rodríguez-Espinosa  1 , Oscar Rojas-Espinosa, María Maximina Bertha Moreno-Altamirano, Edgar Oliver López-Villegas, Francisco Javier Sánchez-García
Affiliations

Metabolic requirements for neutrophil extracellular traps formation

Oscar Rodríguez-Espinosa et al. Immunology. 2015 Jun.

Abstract

As part of the innate immune response, neutrophils are at the forefront of defence against infection, resolution of inflammation and wound healing. They are the most abundant leucocytes in the peripheral blood, have a short lifespan and an estimated turnover of 10(10) to 10(11) cells per day. Neutrophils efficiently clear microbial infections by phagocytosis and by oxygen-dependent and oxygen-independent mechanisms. In 2004, a new neutrophil anti-microbial mechanism was described, the release of neutrophil extracellular traps (NETs) composed of DNA, histones and anti-microbial peptides. Several microorganisms, bacterial products, as well as pharmacological stimuli such as PMA, were shown to induce NETs. Neutrophils contain relatively few mitochondria, and derive most of their energy from glycolysis. In this scenario we aimed to analyse some of the metabolic requirements for NET formation. Here it is shown that NETs formation is strictly dependent on glucose and to a lesser extent on glutamine, that Glut-1, glucose uptake, and glycolysis rate increase upon PMA stimulation, and that NET formation is inhibited by the glycolysis inhibitor, 2-deoxy-glucose, and to a lesser extent by the ATP synthase inhibitor oligomycin. Moreover, when neutrophils were exposed to PMA in glucose-free medium for 3 hr, they lost their characteristic polymorphic nuclei but did not release NETs. However, if glucose (but not pyruvate) was added at this time, NET release took place within minutes, suggesting that NET formation could be metabolically divided into two phases; the first, independent from exogenous glucose (chromatin decondensation) and, the second (NET release), strictly dependent on exogenous glucose and glycolysis.

Keywords: ATP synthase; cell metabolism; glycolysis; neutrophil extracellular traps; neutrophils.

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Figures

Figure 1
Figure 1
Dose–response and kinetics of PMA-induced neutrophil extracellular trap (NET) formation. (a) Polymorphprep-enriched neutrophils were stimulated with PMA (10, 50 and 100 nm) fixed with 4% paraformaldehyde in PBS for 15 min, washed with PBS, treated with DAPI for DNA staining, and then analysed by fluorescence microscopy (formation of NETs). (b) Neutrophils were activated with 100 nm PMA and then at the indicated times post-PMA (30 min, 1, 2, 3, 4, 5 hr) analysed for the formation of NETs, Bar = 20 μm. (c) Neutrophils stimulated with PMA (100 nm) for 3 hr were fixed with 4% paraformaldehyde and stained for histones. (d) Neutrophils were stimulated with 1 μm fMLP, as an additional control of NET formation. (e) Flow cytometry-based assessment of neutrophil enrichment by polymorphprep on the basis of forward and side scatter and CD16+ cells. Results are representative of at least three independent experiments.
Figure 2
Figure 2
Dose–response and kinetics the formation of neutrophil extracellular traps (NETs), and reactive oxygen species (ROS) production. (a) Isolated neutrophils were cultured in specific formulated media (conditioned media), containing: glucose and glutamine, glucose but no glutamine, glutamine but no glucose, or no glucose and no glutamine. Cells were left untreated or stimulated with PMA (100 nm) for 3 hr; thereafter, cells were fixed with 4% paraformaldehyde in PBS for 15 min, washed with PBS, and stained for DNA with DAPI. (b and c) DNA area quantification was performed from fluorescence microscopy images using ImageJ software, to quantify the formation of NETs, as described in the Materials and methods. (d) ROS production was determined by CM-H2DCFDA in non-stimulated and PMA-stimulated neutrophils, cultured in the different conditioned media as shown. Results are from three independent experiments. Raw data were analysed by one-way analysis of variance.
Figure 3
Figure 3
Pharmacological inhibition of glycolysis and to a lesser extent of ATP synthase inhibits the formation of neutrophil extracellular traps (NETs). (a) Isolated neutrophils were cultured in complete RPMI-1640 medium, incubated with 2-deoxyglucose (2-DOG; a glycolysis inhibitor), with oligomycin (an ATP synthase inhibitor), or medium alone for 15 min at 37°. Thereafter, a set of cells was left untreated (non-stimulated) and another set of cells was stimulated with 100 nm PMA. After 3 hr at 37°, the cells were fixed with 4% paraformaldehyde in PBS for 15 min, washed with PBS and stained for DNA with DAPI for fluorescence microscopy, or fixed with 2·5% glutaraldehyde and treated for scanning electron microscopy. Images are representative of multiple microscopic fields from three independent experiments. (b) Quantification of NET formation (DNA area) using ImageJ software. Data obtained from ImageJ were analysed by one-way analysis of variance.
Figure 4
Figure 4
PMA stimulation of neutrophils increases Glut-1 cell membrane expression, glucose uptake and glycolysis rate. (a) Glut-1 expression was analysed by staining neutrophils with an anti-Glut-1 fluorochrome-labelled monoclonal antibody and flow cytometry, the upper panels depict a representative set of flow cytometry data and the lower panel depicts the normalized data of Glut-1 cell membrane expression from three independent experiments. (b) Glucose uptake was assessed by the uptake of the fluorescent glucose analogue 2-NBDG and flow cytometry. The upper panels depict a representative set of flow cytometry data, and the lower panel depicts the fold change of glucose uptake, from 15 to 120 min by non-stimulated and PMA-stimulated neutrophils, as compared with the glucose uptake by non-stimulated neutrophils at 15 min (with a given value of 1). (c) Glycolytic rate was assessed by measuring the lactate production in the supernatant of cultured neutrophils and dividing the amount of lactate at one time-point by that in the previous time-point. Glycolytic rate is expressed as the fold change in lactate production. Depicted is the mean ± SD of three independent experiments. Discontinued lines represent linear regression and the slope for each line. Differences in slopes and, therefore, in glycolytic rate were statistically significant (P < 0·05).
Figure 5
Figure 5
PMA activation of neutrophils, even in the absence of glucose and glutamine, induces chromatin decondensation. Neutrophils (1 × 106) in glucose- and glutamine-containing medium or in glucose- and glutamine-free medium were cultured for 3 hr with or without 100 nm PMA, after which, (a) chromatin condensation was assessed using the Nuclear-ID™ Green chromatin condensation detection kit and flow cytometry, results are expressed as the percentage of neutrophils with decondensed chromatin, and (b) as the fold change (non-stimulated versus PMA) in the percentage of neutrophils with decondensed chromatin for the two culture conditions. Results are from five independent experiments. Data were analysed by one-way analysis of variance. (c) Neutrophils were prepared for transmission electron microscopy as described in the Materials and methods, images are representative from multiple microscopic fields from two independent experiments.
Figure 6
Figure 6
Addition of glucose and to a lesser extent of pyruvate to PMA-stimulated neutrophils in glucose-free medium induces a rapid release of neutrophil extracellular traps (NETs). (a) Neutrophils were suspended in glucose- and glutamine-free medium and then stimulated with PMA (100 nm) for 3 hr at 37°. After this time, when no evident release of NETs was observed, 4 mm glucose or 2 mm pyruvate was added. A set of cells was fixed after 2 min, and another set of cells was fixed after 10 min of glucose or pyruvate addition, with 4% paraformaldehyde in PBS for 15 min, washed with PBS and then treated with DAPI for DNA staining. Cell preparations were analysed by fluorescence microscopy. Images are representative of multiple microscopic fields from three independent experiments. (b) To test if the release of NETs upon glucose addition to pre-activated neutrophils in glucose-free medium is due to glycolysis, the glycolysis inhibitor 2-deoxyglucose (2-DOG) was added before glucose.
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