Mouse neutrophil extracellular traps in microbial infections

Abstract

Neutrophil extracellular traps (NETs) play an important role in innate immunity to microbial infections. NETs have been described in several species, but the molecular details of NET formation and their role in infection has not been addressed, partly because we lack optimal experimental models. Here we describe tools to investigate NET formation in neutrophils isolated from mice. Upon in vitro stimulation of wild-type mouse neutrophils with PMA, we analyzed 3 important steps in the process of NET formation: reactive oxygen species (ROS) production, NET cell death and NET release. As expected, neutrophils from NADPH oxidase-deficient mice failed to produce ROS and did not die nor release NETs upon stimulation. We found that neutrophils from several mouse strains produced NETs with different efficiency and that NET formation correlated with the amount of ROS produced. Activation with Candida albicans also resulted in ROS production and NET cell death. The hyphal form of this fungus induced NETs more effectively than the yeast form. With this work, we provide tools to study in vitro NET assembly in the mouse system.

Fig. 1

Mouse neutrophils release extracellular traps in vitro. Neutrophils were purified from C57Bl/6 (wild type) and gp91–/– mice (KO). a ROS production by wild-type and KO neutrophils activated with 100 nM PMA was measured with a luminometric assay. Wild-type (b) or KO (c) neutrophils were activated with 100 nM PMA and cell death was analyzed as described in Materials and Methods. NET cell death was calculated by subtracting the fluorescence of unstimulated neutrophils from the stimulated neutrophils at each time point and dividing it by the lysis control. d Cumulative data of cell death induced in wild-type and KO neutrophils. Fig. 1. e Wild-type and KO (f) neutrophils were activated for 16 h with 100 nM PMA, stained for DNA (blue), histone/DNA complexes (red) and MPO (green) and visualized by confocal microscopy. Only wild-type neutrophils show NET formation (arrows in e indicate NETs). High-resolution SEM analysis of wild-type (g, g’, g”) and KO (h) neutrophils. Cells were activated for 16 h with 100 nM PMA. Wild-type, but not KO neutrophils show formation of NETs. a–d Data are representative of at least 5 independent experiments [error bars (b, c), SEM of 5 samples]. f Scale bar = 10 μm. g, h Scale bars = 5 μm. g’ Scale bar = 500 nm. g” Scale bar = 100 nm. RLU = Relative luminescence units; AU = arbitrary units.

Fig. 2

Microscopical analysis of NET formation in murine neutrophils. Neutrophils were purified from C57Bl/6 (wild type, WT) or gp91–/– (KO) mice and stimulated with 100 nM PMA for the indicated time. Confocal immunomicroscopy of wild-type (a–d) and KO (e–h) neutrophils stained for DNA (blue), MPO (green) and histone/DNA complexes (red). a’–d’ and e’–h’ represent a higher magnification of the corresponding pictures a–d and e–h, respectively Changes of nuclear morphology were visualized by TEM of wild-type (i–l) and KO (m–p) neutrophils. Both techniques show that 4 h after stimulation, the nuclei of wild-type neutrophils delobulate and there is colocalization of MPO and chromatin. NET formation is observed 12 h after activation (arrows in d and l indicate NETs, arrowhead in o indicates apoptotic cell). Data are representative of at least 3 independent experiments. Scale bars = 10 μm.

Fig. 3

Neutrophils were purified from C57Bl/6 (wild type) or gp91–/– (KO) mice and stimulated with 100 nM PMA. To quantify the morphological changes, neutrophils were stained for histone/DNA complexes and DNA at the indicated time points. Wild-type (a) and KO (b) neutrophils were assigned to the different morphological stages (error bars, SEM of 5 independent areas per time point). Data are representative of 3 independent experiments.

Fig. 4

ROS production by activated neutrophils correlates with NET cell death in different mouse strains. Neutrophils from the indicated mouse strains were activated with 100 nM PMA for 10 h. a ROS production was measured with a luminometric assay. Total amounts of produced ROS were obtained by analyzing the area under the curve (AUC) and amounts were normalized to the strain C57Bl/6 (= 100). b NET cell death was analyzed 10 h after stimulation with the cell death assay described in figure 1. Values were normalized to the C57Bl/6 strain (= 100; error bars, SEM of at least 3 independent experiments per strain). c Values from the cell death assay and the ROS production were set in relationship and analyzed with a test for monotonous relationships (Spearman). Rank correlation coefficient is 0.9429 with a p value of 0.0167. Neutrophils from all tested strains produce ROS and die. The amount of produced ROS significantly correlates to NET cell death.

Fig. 5

Mouse NETs trap C. albicans hyphae. Neutrophils purified from wild-type C57Bl/6 (wild type, red) produce more ROS when stimulated with C. albicans hyphae (a) than with yeast forms (b). gp91–/– mice (KO, blue) were used as negative controls. c Neutrophils purified from wild-type mice (red) underwent NET cell death when stimulated with C.albicans hyphae but not with the yeast form. Neutrophils from KO mice (blue) were used as negative controls and PMA-activated wild-type neutrophils as positive controls. NET cell death was assessed after 10 h (error bars, SEM of at least 4 independent experiments). ** p < 0.01, *** p < 0.001, significantly different from unpaired t test. To analyze NET release, activated wild-type (d) and KO (e) neutrophils were stained for DNA (blue), hyphae (green) and histone/DNA complexes (red) and analyzed by confocal microscopy. Both wild-type and KO neutrophils entwine the hyphae, but only wild-type neutrophils make NETs (arrowheads in d and f indicate NETs). High-resolution SEM analysis of wild-type (f) or KO (g) neutrophils stimulated with C. albicans hyphae (arrows in f and g indicate C. albicans hyphae) for 16 h shows that murine neutrophils make NETs. Scale bars = 10 μm, insert scale bar = 1 μm. RLU = Relative luminescence units.

Fig. 6

Mouse NETs trap L. monocytogenes. Neutrophils purified from C57Bl/6 mice were stimulated with L. monocytogenes (multiplicity of infection = 1) for 16 h. a–a” SEM shows that L. monocytogenes is trapped in NETs. a Scale bar = 2 μm. a’, a” Scale bars = 300 nm.