Spontaneous formation of neutrophil extracellular traps in serum-free culture conditions

Abstract

Neutrophils play a critical role in the innate immune response. Recently, a new neutrophilic biological defense mechanism, termed neutrophil extracellular traps (NETs), has been attracting attention. Neutrophils have been observed to release both lysosomal enzymes and their nuclear contents, including unfolded chromatin, which together trap and inactivate bacteria. The environment in tissues where neutrophils act is thought to be different from that of the blood serum. In this study, we assessed the effect of serum on NET formation. We found that neutrophils spontaneously form NETs in serum-free cultivation conditions at early times. These NETs functioned properly to trap bacteria. Furthermore, we demonstrated that reactive oxygen species play a critical role in the spontaneous formation of NETs. These results suggest that the serum condition must be considered in studies on neutrophils, including the formation and mechanism of action of NETs.

Keywords: neutrophil; neutrophil extracellular traps; serum.

Figure 1

Neutrophils form NETs after cultivation in a serum‐free condition for 1 h. Neutrophils were cultured for 1 h in RPMI 1640 in the presence of 2% human serum or in a serum‐free condition. (A) Fixed neutrophils were stained with anti‐neutrophil elastase antibody (NE; red). DNA was stained with DAPI (blue). Stained cells were visualized with fluorescence microscopy. Scale bar = 50 μm. (B) Extracellular DNA was stained with SYTOX Green and fluorescence was quantified using a microplate reader. Where indicated, neutrophils were cultured in the presence of DNase I. Data are shown as the mean ± SD; n ≥ 3 per group. ***P < 0.001 relative to the serum‐free condition. Results are representative of three or more independent experiments (using neutrophils from different donors).

Figure 2

Effect of serum concentration and medium or buffer on the formation of NETs. (A) Neutrophils were cultured for 1 h in RPMI 1640 containing 2, 1, 0.2% human serum, or 2% FBS. Cells were fixed and stained with DAPI (blue) and observed under a fluorescence microscope. Scale bar = 50 μm. (B) Neutrophils were cultured for 1 h in RPMI 1640, DMEM, HBSS, or PBS in the presence of 2% human serum or in a serum‐free condition. Extracellular DNA was stained with SYTOX Green and fluorescence was quantified using a microplate reader. Data are shown as the mean ± SD; n ≥ 3 per group. ***P < 0.001 relative to the serum‐free condition. Results are representative of three or more independent experiments (using neutrophils from different donors).

Figure 3

Kinetics of NET formation induced by serum‐free culture conditions. (A) Neutrophils were cultured for 3 or 5 h in RPMI 1640 containing 2% human serum or in a serum‐free condition. Cells were fixed and stained with DAPI (blue) and visualized under a fluorescence microscope. Scale bar = 50 μm. (B) Extracellular DNA was stained with SYTOX Green at 1, 3 and 5 h of culture and fluorescence was quantified using a microplate reader. The fluorescence rate was expressed relative to neutrophils in 2% serum at the corresponding time point (set as 1.0). Data are shown as the mean ± SD; n ≥ 3 per group. ***P < 0.001 relative to neutrophils in 2% serum. Results are representative of three or more independent experiments (using neutrophils from different donors).

Figure 4

Comparison of NET formation induced by serum‐free conditions or PMA. Neutrophils were cultured for 1 or 3 h in RPMI 1640 containing 2% human serum, serum‐free, 2% serum with PMA stimulation, or serum‐free with PMA stimulation. Extracellular DNA was stained with SYTOX Green and fluorescence was quantified using a microplate reader. The fluorescence rate was expressed relative to neutrophils in 2% serum at the corresponding time point (set as 1.0). Data are shown as the mean ± SD; n ≥ 3 per group. ***P < 0.001. Results are representative of three or more independent experiments (using neutrophils from different donors).

Figure 5

Functionality of NETs induced by serum‐free culture conditions. (A) Neutrophils were cultured for 1 h in a serum‐free condition to induce the formation of NETs. Escherichia coli (green) was added to the NETs and cultured for 15 min. After washing, cells were fixed and stained with DAPI (blue). Cells were observed under a fluorescence microscope. Arrows indicate E. coli trapped in NETs. Scale bar = 20 μm. (B) Neutrophils were cultured for 1 h in a serum‐free condition to induce NET formation. E. coli was added to the well and cultured for 1 h. The culture supernatants were spread onto LB agar plates and colonies were counted to determine the CFU of surviving bacteria. Data are shown as the mean ± SD; n ≥ 3 per group. *P < 0.05. Results are representative of three or more independent experiments (using neutrophils from different donors).

Figure 6

Effect of serum components on the formation of NETs. Neutrophils were cultured for 1 h in RPMI 1640 containing 40 mg·mL⁻¹ BSA, 40 mg·mL⁻¹ human serum albumin, 2% complement‐inactivated serum (by heat treatment; Complement (−)), 2% immunoglobulin‐depleted serum (by protein G/A sepharose; Ig (−)), or EDTA (10 mm) in the presence or absence of serum. (A) Cells were fixed and stained with DAPI (blue) and visualized under a fluorescence microscope. Scale bar = 50 μm. (B) Extracellular DNA was stained with SYTOX Green and fluorescence was quantified using a microplate reader. Data are shown as the mean ± SD; n ≥ 3 per group. **P < 0.01, ***P < 0.001 relative to the serum‐free condition. Results are representative of three or more independent experiments (using neutrophils from different donors).

Figure 7

Effect of ROS on the formation of NETs induced by serum‐free culture conditions. Neutrophils were cultured for 1 h in RPMI 1640 containing 2% human serum or in a serum‐free condition. Where indicated, neutrophils were cultured in the presence of 5 or 20 mm NAC. (A) Cells were stained with CellROX Deep Red Reagent (red) and DAPI (blue), and visualized under a fluorescence microscope. Scale bar = 50 μm. (B) Extracellular DNA was stained with SYTOX Green and fluorescence was quantified using a microplate reader. Data are shown as the mean ± SD; n ≥ 3 per group. ***P < 0.001 relative to serum‐free cultivation (−). Results are representative of three or more independent experiments (using neutrophils from different donors).