Healthy flounders of body length 5–6 cm or 30–32 cm were purchased from a fish farm in Qingdao, Shandong Province, China. The flounders were acclimated in aerated seawater at 22°C for 1 week and used for the subsequent studies. Fish were anesthetized with tricaine methanesulfonate (Sigma, St. Louis, MO, USA) prior to experiments involving injection and then were sacrificed. The animal experiments were approved by the Instructional Animal Care and Use Committee of the Ocean University of China. All possible endeavors were made to minimize suffering and maintain animal welfare.

The preparation of aluminum hydroxide was done following the protocol reported previously (Reithofer et al., 2020). In brief, 5% NaOH and 5% Al₂(SO₄)₃ were sterilized by passing through a 0.22-μm filter. After being heated at 60°C for 30 min, two volumes of 5% NaOH and five volumes of 5% Al₂(SO₄)₃ were mixed with stirring, followed by centrifugation at 10,000 g for 5 min. After being washed twice with sterile phosphate-buffered saline (PBS), the mixture was suspended in L-15 medium (HyClone, Logan, UT, USA) to give 8 mg ml⁻¹. For fluorescence-dyed Alum formulation, a total of 10 mg of fluorescein isothiocyanate (FITC) (MP Biomedicals, Solon, OH, USA) was added to Alum suspension and incubated for 24 h in the dark at room temperature on a rotator. Free FITC was removed from FITC-Alum by 12,000 g of centrifugation for 10 min and then washed 10 times or until no fluorescence was present in the supernatant. The pellets were resuspended in PBS and used to stimulate peritoneal cells. For OVA/Alum formulation, Alum was mixed with an equal volume of 10 mg ml⁻¹ of OVA (grade V; Sigma, Poole, UK) and incubated at room temperature for 20 min. After centrifugation at 14,000 g for 10 min, sedimented OVA/Alum was resuspended in PBS. For OVA/oil formulation, OVA was emulsified with Freund’s incomplete adjuvant (FIA; Sigma, St. Louis, MO, USA) at equal volume.

Flounders of 30–32 cm in length were intraperitoneally injected with 40 ml of L-15, and the abdomen was gently massaged to get peritoneal cells exuded. Peritoneal fluid was withdrawn using a syringe and thereafter centrifuged at 480 g for 10 min. The cell pellets were resuspended in 5 ml of L-15 medium and washed again by centrifugation at 480 g for 5 min. The purified peritoneal cells were resuspended in 5 ml of L-15 medium before seeding.

Since ETs were fragile, each step was done with maximal care to preserve the nanostructures. Peritoneal cells (1.5 × 10⁶) were seeded onto round glass coverslips (12 mm) that previously had been treated with 0.001% poly-l-lysine (Sigma, St. Louis, MO, USA) in 24-well cell culture plates (Corning Costar, Cambridge, MA, USA). After the cells had been attached, the peritoneal cells were stimulated with 250 nM ml⁻¹ of phorbol myristate acetate (PMA) (Sigma, St. Louis, MO, USA), 10 mg ml⁻¹ of OVA, 10 mg ml⁻¹ of Alum, or 10 mg ml⁻¹ of OVA/Alum for 3 h. Non-stimulated cells served as controls. For fluorescence microscopy, the peritoneal cells were fixed with 4% paraformaldehyde (Sigma, St. Louis, MO, USA). The following steps were performed in a humid chamber at room temperature. Washing of coverslips was performed three times by pH 7.4 PBS for 5 min, and then unspecific binding was blocked with 5% bovine serum albumin (BSA) for 60 min. The coverslips were incubated for 1 h with 1:500 mouse monoclonal antibody against histone 3 (H3) (EASYBIO, Beijing, China). After being washed three times, the coverslips were incubated for 1 h with 1:1,000 anti-rabbit IgG labeled by Alexa Fluor 649 (Sigma, St. Louis, MO, USA). DNA was stained by 4′,6-diamidino-2-phenylindole (DAPI; BioLegend, Santiago, Chile) for 5 min. After DAPI staining, the slides were observed by fluorescence microscopy (Zeiss, Jena, Germany).

The peritoneal cells were seeded at a density of 1.5 × 10⁶ cell ml⁻¹ on glass coverslips and stimulated with 10 mg ml⁻¹ of OVA/Alum for 3 h. After treatment, the cells were fixed with 2.5% glutaraldehyde (Hushi, Shanghai, China) in PBS for 2 h and dehydrated in a series of increased concentrations of ethanol (30%, 50%, 70%, 80%, 90%, and 100%) for 10 min at 4°C in each step. The cells were treated with isoamyl acetate for 10 min, critical point-dried (Hitachi-HCP, Hitachi, Tokyo, Japan), sputter-coated with platinum (MC1000, Hitachi, Tokyo, Japan), and examined with a scanning electron microscopy (SEM) (S-3400N, Hitachi, Tokyo, Japan).

For the transmission electron microscopy (TEM) study, the peritoneal cells were stimulated with 10 mg ml⁻¹ of OVA/Alum in centrifuge tubes for 3 h. After centrifugation at 480 g for 10 min, the pellets were fixed with 2.5% glutaraldehyde (Hushi, Shanghai, China) in PBS for 2 h and washed three times for 30 min in PBS at 4°C. Then, the cells were post-fixed with 1% osmium tetroxide (Ted Pella, CA, USA) in PBS for 2 h and washed three times with PBS at 4°C. Following this, the samples were dehydrated in alcohol (Hushi, Shanghai, China), infiltrated with acetone (Tieta, Laiyang, China) and epoxy resin (SPI-CHEM, California, USA) mixture, and embedded in polymerizing epoxy resin. Ultrathin sections were obtained by a Leica EM UC7 ultramicrotome (Leica Microsystems, Berlin, Germany) and were transferred onto copper grids covered with the Formvar membrane (Electron Microscopy China, Beijing, China); 2% uranyl acetate and lead citrate (Ted Pella Inc., California, USA) were used for contrast staining. The sections were photographed with a TEM (HT7700, Hitachi, Tokyo, Japan).

The quantification of ETs was performed as reported previously (Parker et al., 2012). Briefly, peritoneal cells (1 × 10⁶ cells ml⁻¹) were suspended in L-15 medium and seeded in a black 96-well plate (200 μl well⁻¹) (Cayman Chemical, Ann Arbor, MI, USA). The cells were treated with 500 μg ml⁻¹ of OVA, or 500 μg ml⁻¹ of OVA/Alum for 1, 2, or 4 h. The untreated cells served as control. For the concentration-dependent study of Alum adjuvant, 25, 50, or 100 μg ml⁻¹ of Alum was used to stimulate the cells. After treatment, 10 μl of Sytox Green with 80 mg ml⁻¹ was added to each well, 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 (POLARstar OPTIMA, Ortenberg, Germany).

Flounders of 3–5 cm in length were randomly divided into four groups (10 individuals for each group). On the day of immunization, 10 mg ml⁻¹ of OVA, 10 mg ml⁻¹ of OVA/FIA, and 10 mg ml⁻¹ of OVA/Alum or PBS were injected into the fish in each group (50 μl per individual). The dye SYTOX Green was intraperitoneally injected along with the formulations to stain extracellular DNA. Multiphoton imaging was performed on a small animal in vivo imaging instrument (Vilber Bio Imaging Fusion FX6, Collegien, France). Whole-body images were obtained at 0, 6, 12, 24, 36, 48, and 72 h after injection-using excitation and emission wavelengths of 488 and 523 nm, respectively. To determine ET degradation by DNaseI, flounders were intraperitoneally injected with 10 μl of PBS medium containing 100 U ml⁻¹ of DNase I at 12 h after 10 mg ml⁻¹ of OVA/Alum injection. Images were obtained every 1 min for up to 8 min.

Twelve hours after intraperitoneal injection of the formulations, Sytox Green was injected into the peritoneal cavity. After 15 min, the peritoneal fluid was obtained and seeded on glass coverslips. The evaluation of ET formation was done by fluorescence microscopy (Zeiss, Jena, Germany).

All experiments were performed more than three times, and statistical analyses were performed using the one-way ANOVA followed by least significant difference (LSD) multiple group comparisons in the SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± SD, and the difference was considered significant when p < 0.05.

Isolated peritoneal cells exposed to FITC-dyed Alum were investigated in vitro with special attention to whether they were associated with Alum aggregates. As shown in Figure 1 , most DAPI-stained cells were identified in or close to colloidal Alum particles at hour 3 after stimulation of FITC-dyed Alum. Some cells exhibited nuclear characteristics as producing ETs, including reniform indented nuclei, the release of fiber-like structures, and cell clustering. The peritoneal cells treated by PBS are shown in Figure 2A . Overall, the imaging data demonstrate that Alum rapidly induces recruitment of neutrophils like cells and that Alum induced “neutrophils swarm” cell aggregates in close connection to Alum deposits.

The formation of ETs was detected by using a fluorometric method at 3 h after stimulation with OVA, Alum, and OVA/Alum in vitro. Except for the control cells treated with PBS, extracellular DNA fibers were observed from the wells containing cells in all stimulated groups. DNA from disintegrated nuclei or web-like extracellular DNA fibers labeled by DAPI were co-localized with citrullinated histone H3 (CitH3) labeled by Alexa Fluor^® 649 goat anti-mouse IgG ( Figure 2A ).

As shown above, OVA and Alum induced apparent production of ETs in peritoneal cells. The quantitation of ETs in control and stimulated groups was measured by a fluorescence spectrophotometer. The DNA release of OVA- and OVA/Alum-stimulated cells was significantly higher than that of the control cells at all the time points (p < 0.05) and increased significantly with time after injection throughout the study period (p < 0.05) ( Figure 2B ). In addition, the DNA release in the OVA/Alum-stimulated group was significantly higher compared to that of the OVA-stimulated group at the corresponding time points (p < 0.05). In the Alum concentration study ( Figure 2C ), the presence of Alum increased in a dose- and time-dependent manner with the release of ETs. In the Alum-stimulated groups with 25, 50, and 100 μg ml⁻¹, ET release increased from hour 1 to hour 3, and a significant difference was observed at hour 4 compared to other time points (p < 0.05). The DNA released from 100 μg ml⁻¹ of Alum-stimulated cells was significantly higher than that from 25 μg ml⁻¹ of Alum-stimulated cells at all the time points (p < 0.05). The cells stimulated with 50 μg ml⁻¹ of Alum were significantly higher than the cells stimulated with 25 μg ml⁻¹ of Alum at hours 1 and 3. Alum-stimulated cells measuring 100 μg ml⁻¹ produced significantly more ETs as compared to 50 μg ml⁻¹ of Alum-stimulated cells at hour 1.

To identify the morphology and types of ETs releasing peritoneal cells stimulated with OVA/Alum, ultrastructural studies using EM were performed. High-resolution SEM showed that flounder peritoneal cells released long stretches of fibers in response to OVA/Alum ( Figures 3A, B ). TEM revealed that such fibers were mainly released from two types of peritoneal cells. One type was 5–10 μm in diameter, with the irregular cell membrane, while the cytoplasm possessed many peroxisomes containing fusiform filaments ( Figures 3C, D ). The other types of cells showed blunt pseudopods on the surface of the plasma membrane and round nucleus with some heterochromatin clumps. The cytoplasm contained many mitochondria, free ribosomes, but no peroxisomes ( Figures 3E, F ). Both types of cells seemed to release fibers through the pores in the cell membrane.

The observation of ET formation of peritoneal cells in vitro prompted us to develop a method to investigate such a process in vivo. After OVA, OVA/FIA, and OVA/Alum were injected into the peritoneal cavity, fluorescence signals were detected by using a small animal imaging instrument and quantified from the membrane-impermeable DNA dye (Sytox Green). The fluorescence signal could be detected from hour 6, reached a maximum at hour 12, and then gradually decreased till hour 72 after OVA, OVA/FIA, or OVA/Alum injection ( Figure 4 ). The fluorescence from PBS-injected fish was negligible throughout the study period at hour 6 to hour 72 after injection ( Figures 4A, B ). After OVA injection, fluorescence signals due to extracellular DNA were detected in the peritoneum, where the highest fluorescence intensity was 5.4 × 10⁷ photons/s/cm²/sr at hour 12 ( Figures 4C, D ). The treatment with OVA/FIA ( Figures 4E, F ) and OVA/Alum ( Figures 4G, H ) induced enhanced extracellular DNA release and the fluorescence intensity reached 7.8 × 10⁷ and 1.1 × 10⁸ photons/s/cm²/sr at hour 12, respectively. To verify that fluorometric determination reflected extracellular DNA, a group of fish received DNase I to degrade DNA, which will decrease the fluorescence intensity in the peritoneal cavity. After OVA/Alum-treatment at hour 12, the fish were injected with DNase I intraperitoneally. As shown in Figures 5A, B , the fluorescence intensity decreased time-dependently from 9.1 × 10⁷ to 6.2 × 10⁷ photons/s/cm²/sr within 480 s, which suggested that the fluorescence originated from extracellular DNA fibers.

Peritoneal exudates were observed by fluorescence microscopy at hour 12 after immunization to determine the reliability of the mode of detection of ETs in vivo. The cells obtained after vaccination with OVA, OVA/FIA, and OVA/Alum possessed typical features of ET formation, such as cell adherence, flattening, and DNA extrusion into the extracellular space ( Figure 6 ). The fluorescence intensity was the highest in extracellular DNA fibers, and dead small cells were encountered. The control cells from PBS-injected fish exhibited no DNA fiber, thus showing low fluorescence. We observed, similarly to above, some dead cells that were stained with the fluorescent dye. The released DNA fibers from OVA/Alum-stimulated immune cells were clustered together and located in or close to Alum aggregates, in line with the observation of in vitro study shown in Figure 1 .