Experiments involving live animals were conducted in accordance with the “Regulations for the Administration of Affairs Concerning Experimental Animals” promulgated by the State Science and Technology Commission of Shandong Province. The study was approved by the Ethics Committee of Institute of Oceanology, Chinese Academy of Sciences.

Clinically healthy tongue sole (average 250 g) were purchased from a local fish farm. Isolation of neutrophils was performed as reported previously (17, 21, 22) with modifications as follows. Head kidney was aseptically collected from tongue sole and placed into a 50 ml test tube containing 30 ml of Hank’s balanced salt solution with Ca²⁺ and Mg²⁺ but without phenol red (HBSS) (Mediatech-CellGro, AK, USA). The tissue was processed by being passed through a 100 μm nylon Falcon cell strainer (BD Falcon, Lexington, KY, USA). The cells were isolated using 51% percoll (GE Healthcare, Uppsala, Sweden), with the buffy coat being removed. The remaining red blood cell/granulocyte pellet was collected, and the red blood cells were separated using a specific gravity of 61% percoll. The granulocytes/macrophages were collected and pooled. The cells were washed twice in HBSS and seeded into 25 cm³ polystyrene cell culture flasks (Costar, Tewksbury, MA, USA) containing L15 medium (HyClone, Logan, UT, USA) supplemented with 2% fetal bovine serum (HyClone, Logan, UT, USA) and cultured at 22°C with 5% CO₂ for overnight. The non-adherent neutrophil-like cells were harvested, and a portion of the cells were used for total cell count, and cytochemical staining as reported previously (17). As shown in Figure S1 in Supplementary Material, most of the prepared cells were neutrophils.

The inducers of NETosis used in this study were PMA and three fish bacterial pathogens. PMA was used because it is known to be a very effective inducer of NETosis in mammalian models (2), and therefore served in this study as a positive control for the three bacteria pathogens. Microscopic observation of NETs was performed as reported previously (17) with modifications. Briefly, the fish bacterial pathogens Pseudomonas fluorescens, Vibrio harveyi, and Edwardsiella tarda (23–25) were cultured in Luria–Bertani broth (LB) medium at 28°C to an OD₆₀₀ of 0.8; the cells were collected by centrifugation and washed with PBS. Neutrophils (~10⁶) were seeded onto a glass coverslip that had already been treated with 0.001% polylysine (Sigma, St. Louis, MO, USA) and placed in a 12-well cell culture plate. The cells were allowed to settle for 2 h and then treated with PMA (1 μg ml⁻¹) (Sigma, St. Louis, MO, USA), P. fluorescens (1 × 10⁶ CFU), V. harveyi (1 × 10⁶ CFU), or E. tarda (1 × 10⁶ CFU) at 22°C for 2 h. The control cells were left untreated. For fluorescent microscopy, Sytox Green was added to the cells, and after incubation for 5 min, the cells were washed three times with PBS. Then the cells were fixed with 4% paraformaldehyde (Sigma, St. Louis, MO, USA) for 25 min and stained with 4′,6-diamidino-2-phenylindole (DAPI) according to the instructions of the manufacturer (Bioss, Beijing, China). Scanning electron microscopy (SEM) was performed as reported (10).

The survival of NETs-entrapped bacteria was examined as reported previously (17). Briefly, bacterial cells were cultured in LB medium to an OD₆₀₀ of 0.8 and harvested by centrifugation. The bacterial cells were washed with PBS for three times and resuspended in PBS. Neutrophils (2 × 10⁵ cells/well in a 200 μl volume) were seeded in 96-well cell culture plates and allowed to adhere for 60 min at 22°C. The cells were then stimulated with 1 μg ml⁻¹ PMA for 2 h and centrifuged at 400 g for 5 min, and 150 μl of supernatant was discarded. Then, 50 μl L15 medium containing or not containing 100 U ml⁻¹ DNase I was added to the cells. The cells were maintained at 22°C for 20 min, then cytochalasin D (20 μg ml⁻¹) (Invitrogen, Carlsbad, CA, USA) and 2,000 CFU bacteria (P. fluorescens, V. harveyi, or E. tarda) were added to the plates. The plates were centrifuged at 800 g for 10 min to allow intimate contact of the bacteria with NETs/neutrophils. The plates were then incubated at 22°C for 2, 4, 6, and 8 h. After incubation, the content of each well (bacteria plus neutrophils and NETs) was taken out and serially diluted, and the dilutions were plated on LB agar plates. The plates were incubated at 28°C for 24 h, and the colonies that emerged on the plates were counted. The genetic nature of the colonies was verified by PCR and sequence analysis of the PCR products.

To quantify NETs, the method of Parker et al. (11) was adopted. Briefly, neutrophils (5 × 10⁶ cells/well in a 200 μl volume) were suspended in HBSS (Mediatech-CellGro, USA) and seeded in a black 96-well plate (Cayman Chemical, Ann Arbor, MI, USA). The cells were treated with PMA (1 μg ml⁻¹), P. fluorescens (1 × 10⁶ CFU), V. harveyi (1 × 10⁶ CFU), or E. tarda (1 × 10⁶ CFU) for 1, 2, 3, or 4 h. The control cells were untreated. After treatment, the membrane-impermeable DNA-binding dye, Sytox Green (5 μM), was added to the cells, followed by incubation for 5 min. Fluorescence was then quantified as relative fluorescence units (RFU) at 485 nm excitation and 530 nm emission using a fluorescence spectrophotometer (Infinite M1000, Tecan, Switzerland). For NETs inhibition assay, neutrophils were pre-incubated with the following inhibitors for 30 min at 22°C: 100 μM ROS scavenger Trolox (Sigma, St. Louis, MO, USA), 1 mM nitric oxide (NO) inhibitor N(G)-nitro-L-arginine methylester L-NAME (Sigma, St. Louis, MO, USA), or 100 μM MPO inhibitor 4-aminobenzoic acid hydrazide (ABAH) (Calbiochem, San Diego, CA, USA). NETs production and measurements were then performed as above.

To measure ROS and NO production, the methods described by Patel et al. and Lim et al. (26, 27) were adopted, respectively. Briefly, neutrophils were suspended in HBSS and seeded in a black 96-well plate (5 × 10⁶ cells/well). The cells were incubated with 10 μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Sigma, USA) at 22°C for 20 min for ROS quantification and with 5 μM 4,5-diaminofluorescein diacetate (DAF-2DA) (Sigma, USA) for NO quantification, respectively. The plates were centrifuged for 10 min at 400 g, and the supernatant was replaced with fresh HBSS containing PMA (1 μg ml⁻¹), P. fluorescens (1 × 10⁶ CFU), V. harveyi (1 × 10⁶ CFU), or E. tarda (1 × 10⁶ CFU). The control cells were untreated. The plates were incubated at 22°C for 20, 40, 80, 100, and 120 min. After incubation, ROS and NO were measured as RFU at 485 nm excitation and 525 nm emission and at 495 nm excitation and 515 nm emission, respectively, using a fluorescence spectrophotometer (Infinite M1000, Tecan, Switzerland).

Myeloperoxidase release was measured as reported previously (18). Briefly, neutrophils were suspended in HBSS and seeded in a 96-well plate (5 × 10⁶ cells/well). The cells were treated with PMA (1 μg ml⁻¹), P. fluorescens (1 × 10⁶ CFU), V. harveyi (1 × 10⁶ CFU), or E. tarda (1 × 10⁶ CFU). The control cells were untreated. The cells were incubated at 22°C for 60 and 120 min, and 50 μl of 3,3′,5,5′-tetramethylbenzidine hydrochloride (TMB) (Sigma, USA) was added to the cells, followed by immediately adding 50 μl of hydrogen peroxide. The color change reaction was allowed to proceed for 3 min, and then 50 μl 2 M sulfuric acid was added to stop the reaction. The plates were centrifuged at 400 g for 15 min, and 200 μl of the supernatant from each well was transferred to another plate and the optical density in each well was determined at 405 nm.

All experiments were performed three times, and statistical analyses were performed using analysis of variance with SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Statistical significance was determined with Student’s t-test. In all cases, significance was defined as P < 0.05.

Scanning electron microscopy showed that when tongue sole kidney neutrophils were treated with PMA, abundant NETs structures were produced, which contained long stretches of fibers dotted with spherical objects similar to the 25–50 nm globular protein domains observed in mammalian NETs (3) (Figure 1). To examine whether the NETs-producing neutrophils were alive, the cells were stained with Sytox Green, which is impermeable to live cells, and DAPI after PMA treatment. The results indicated that DAPI was associated with both NETs and the cells, whereas Sytox Green was excluded from the cells (Figure 2A), suggesting that NETs were produced by live cells. Consistent with this observation, when PMA-induced NETs was incubated with DNase I, the NETs structure gradually disappeared as the incubation time increased (Figure 2B; Video S1 in Supplementary Material), suggesting that DNA was an essential component of NETs.

The bacteria E. tarda, P. fluorescens, and V. harveyi are common pathogens to marine fish including tongue sole. To investigate their effects on NETosis, tongue sole neutrophils were incubated with each of these bacteria, and NETs production was subsequently determined microscopically. The results showed that SEM detected NETs formation in all groups of neutrophils treated with the bacteria, and that the produced NETs were able to trap the bacterial cells (Figure 3). Quantitative analysis revealed that bacteria-induced NETs production in a time-dependent manner, with the amounts of NETs increasing from 1 to 4 h after bacterial treatment (Figure 4).

To examine the fate of the bacteria trapped by NETs, NETs-positive neutrophils and NETs-negative neutrophils were incubated with E. tarda, P. fluorescens, and V. harveyi for 0, 2, 4, 6, or 8 h, and viable bacteria were recovered at each time point. The results showed that for P. fluorescens and V. harveyi, the numbers of viable bacteria after 2 and 4 h incubation with NETs-positive neutrophils were comparable to those after incubation with NETs-negative neutrophils; however, after 6 and 8 h incubation, the bacterial recoveries from NETs-positive neutrophils were significantly lower than those from NETs-negative neutrophils (Figures 5A,B). By contrast, for E. tarda, bacterial recoveries from NETs-positive neutrophils were similar to those from NETs-negative neutrophils at all time points (Figure 5C).

Reactive oxygen species analysis showed that in neutrophils treated with PMA, P. fluorescens, and V. harveyi, ROS levels increased with time, whereas in neutrophils treated with E. tarda, no apparent change in ROS was observed (Figure 6A). For NO and MPO, their productions were enhanced significantly in a time-dependent fashion in neutrophils treated with all tested stimulants, i.e., PMA, E. tarda, P. fluorescens, and V. harveyi, with E. tarda being the strongest inducer among all bacteria for NO production (Figure 6B), and V. harveyi for MPO production (Figure 6C).

Given the above observation, we wondered whether ROS, NO, and MPO production was required for NETosis. To investigate this question, neutrophils were treated with PMA, E. tarda, P. fluorescens, and V. harveyi in the presence of the inhibitor against the production/accumulation of ROS, NO, or MPO, and the amount of NETs produced by the cells was subsequently determined. The results showed that the presence of Trolox, L-NAME, and ABAH, which are inhibitors of ROS, NO, and MPO, respectively, caused significant NETs reduction in all groups of neutrophils (Figure 7).