Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Edwardsiella tarda (E. tarda), Vibrio anguillarum (V. anguillarum), and hirame rhabdovirus (HIRRV) strain were previously isolated from diseased flounder and stored in our laborotory (23, 24). Bacteria were cultured in Luria-Bertani (LB) medium up to an OD600 of 0.5 and harvested by centrifugation before use. Healthy flounders (15-35 cm in length) were purchased from a fish farm in Rizhao, Shandong Province, China, and were acclimatized in recirculating seawater at 21 °C for two weeks. During the acclimatization, fish were fed daily with commercial dry food pellets. BALB/c mice were obtained from Qingdao Animal Experimental Center of Shandong Province, China, and used for antibody production. The protocols for animal care and handling were approved by the Animal Care and Use Committee of Ocean University of China (Permit Number: 20180101). All possible efforts were dedicated to minimizing suffering.

For infection, flounders were randomly divided into four groups (30 fish per group). Fish in each group were intraperitoneally (i.p.) injected with V. anguillarum, S. aureus (1 × 10⁶ CFU per individual), or HIRRV (1 × 10⁷ TCID₅₀ per individual). The fish in the control group was injected with the same volume of PBS. At 0, 12, 24, 48, and 96 h post-injection, the head kidney and spleen of five fish from each group was sampled for expression profile analysis. All samples were immediately frozen in liquid nitrogen and then stored at -80 °C until RNA isolation.

Total RNA was extracted from tissues or cells of flounder by a Trizol method reported previously (25). The cDNA strand was synthesized using a Hiscript III RT SuperMix kit for PCR (Vazyme, Nanjing, China). The full-length open reading frame (ORF) of the PoMPO gene was amplified using primers PoMPO-OF/PoMPO-OR ( Table 1 ). The gene sequences and location in the genome of MPO were analyzed in the NCBI database. The SMART, IEDB-AR, and ExPASy Molecular Biology Server were used to analyze the protein structure, antigenic epitopes, and physicochemical properties. MEGA 5.0 software was used to construct and analyze a phylogenetic tree using the neighbor-joining method with 1,000 bootstrap trials (26, 27).

The sequence (427-1155 bp) with strong antigenicity of PoMPO was amplified using primers PoMPO-F/PoMPO-R ( Table 1 ) and cloned into the pET32a vector to construct recombinant pET32a-PoMPO expression plasmid. E. coli BL 21 (TransGen Biotech, Beijing, China) was transformed by pET32a-PoMPO and cultured with LB medium at 37 °C. After growth to log phase (OD 600 = 0.6-0.8), the bacteria were induced with IPTG (Isopropyl β-D-Thiogalactoside, 250 µg mL^-1) and incubated overnight at 16 °C. Then the recombinant protein of PoMPO (rPoMPO) was obtained by affinity chromatography using His Trap™ HP Ni-Agarose (GE healthcare China, Beijing, China) (28).

The concentration of rPoMPO was determined to be 1 mg mL^-1 by using the Bradford kit (P0006C, Beyotime, Shanghai, China). BALB/c mice were immunized with 100 µg rPoMPO according to previously reported methods (29). After two boosters with a mixture of rPoMPO and incomplete Freund’s adjuvant (Sigma, St. Louis, MO, USA), the mouse serum was collected and purified the antibodies with protein G-agarose column (Pierce/Thermo Scientific, Waltham, Massachusetts, USA). The anti-rPoMPO Abs titer was tested by enzyme-linked immunosorbent assay, and then the specificity was analyzed using Western blotting and Mass spectrometry analysis (Sangon Biotech, Shanghai, China).

The leukocytes in the head kidney, peripheral blood and peritoneal cavity of flounder (30-35 cm) were isolated according to the method described previously (30). In brief, the peripheral blood was drawn from the caudal vein and diluted in solution (65% RPMI-1640 containing 20 IU mL⁻¹ heparin, 0.1% w/v NaN3, and 1% w/v BSA). The head kidney was passed through a nylon falcon filter while grinding and adding L-15 medium to form cell suspension. Then the leukocytes were isolated by percoll (GE Healthcare, Uppsala, Sweden) on a discontinuous density gradient (1.020-1.070 g mL^-1). Finally, the interfacial cells were collected and resuspended in L-15 medium at a density of 1×10⁶ cells mL^-1. For peritoneal cell isolation, L-15 medium was intraperitoneally (i.p.) injected into flounder and then gently massaged for 5 min. The peritoneal fluid was collected along the edge of the peritoneal cavity by a syringe and centrifugation at 450 × g for 10 min. The cell pellet was resuspended and adjusted to a concentration of 1×10⁶ cells mL^-1.

The peripheral blood leukocytes (PBLs), head kidney leukocytes (HKLs) and peritoneal cells (PerCs) were obtained from healthy or LPS (1mg mL^-1) -stimulated flounder (i.p injection of LPS). After being adjusted to 5 × 10⁶ cells mL^-1 in PBS, the potentially unspecific staining was reduced by adding 5% BSA and incubated with antibodies (rabbit anti-PoMHCII or anti-PoGCSFR Abs) for 1 hour at room temperature (BD Cytofix/Cytoperm kit; BD Biosciences) (31, 32). Afterwards, the cells were incubated with Abs [mixture of mouse anti-rPoMPO and rabbit anti-Zap-70 (Cell Signal Technology, Danvers, Massachusetts, USA)] for 1 hour at room temperature (33). Mouse anti-Trx Abs and rabbit anti-Trx Abs were used as negative controls. The cells were subsequently incubated with Alexa Fluor 649-conjugated goat anti-mouse IgG or 488-conjugated goat anti-rabbit IgG Abs for 45 min at 37°C in the dark. Flow cytometry was performed using FACSCalibur (BD Biosciences) flow cytometers with acquisition enabled by CellQuest Pro software.

The leukocytes (1×10⁶ cells mL^-1) were seeded onto circular coverslips (14 mm, Solarbio, Beijing, China) pretreated with 0.01% polylysine (Sigma, St. Louis, MO, USA), and placed on 24-well cell culture plates (Corning Costar, Cambridge, MA, USA) to settle for 1 h. The cells were fixed with 4% paraformaldehyde (Solarbio, Beijing, China) and permeabilized by 0.25% TritonX-100 in 5% BSA. Then cells were incubated with mouse anti-rPoMPO Abs, or the mixture of antibodies - mouse anti-rPoMPO Abs together with rabbit anti-Zap-70 Abs, rabbit anti-MHCII Abs, or rabbit anti- GCSFR Abs overnight at 4 °C, followed by incubation with Alexa Fluor 649-conjugated goat anti-mouse IgG or 488-conjugated goat anti-rabbit IgG secondary Abs for 1 h at 37 °C. After washing, the cells were counterstained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Thermo Scientific, Waltham, Massachusetts, USA) and visualized by fluorescence microscopy (ZEISS, Oberkochen, Germany). The mouse anti-Trx and rabbit anti-Trx Abs were used as negative controls.

According to the user-instruction of the RIPA Lysis Buffer Kit (Beyotime, Shanghai, China), 100 µL of lysis buffer was added to 1 × 10⁶ peritoneal cells pellet or 10 mg tissue. After ultrasonication for 20 min, the lysate was centrifuged at 12000 × g for 30 min, and then the protein concentration in the supernatant was adjusted to 1mg mL^-1 by using the BCA kit (EpiZyme, Shanghai, China). Twenty microliters of the cell or tissue (head kidney, spleen, gill, skin, muscle, and liver) lysates went through SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany). Then the membranes were blocked with 5% BSA and incubated with anti-rPoMPO, anti-Trx, or anti-GAPDH Abs (latter ab: Abclonal, Wuhan, China). After washing three times with PBST, the membranes were incubated with goat anti-mouse IgG-HRP or goat anti-rabbit IgG-HRP (Sigma, St. Louis, Mo, USA) diluted with 5% BSA at 37 °C for 45 min. Finally, the bands were detected with ECL Enhanced Kit (Abclonal, Wuhan, China) in a chemiluminescence detection instrument (Vilber, Ile-de-France, France). The semi-quantification of protein bands from homogenized PerCs priority subjected to LPS (100 µg per individual (N = 3)), Sigma, St. Louis, Mo, USA) at different time points was analyzed using Image J software (34).

The Real-time quantitative PCR (qPCR) reaction system contained 10 µL 2 × Universal SYBR Green Fast qPCR Mix (Abclonal, Wuhan, China), 10 ng sample DNA, 0.4 µL detection primer qPCR-F/qPCR-R or 18S-F/18S-R ( Table 1 ) and sterile distilled water for a final volume of 20 µL. The qPCR amplification reaction program was set up as follows: 95 °C for 3 min, followed by 40 cycles at 95 °C for 5 s, 60 °C for 30 s. The relative transcriptional levels of PoMPO were calculated by normalizing the abundance of 18S as an internal control. The relative expression of PoMPO was analyzed using the 2^-ΔΔCt method based on the Ct (cycle threshold) values generated by the software automatically.

The leukocytes (1×10⁶ cells mL^-1) from the head kidney on circular coverslips were stimulated with PMA (100 ng mL^-1), E. coli, E.tarda, or S. aureus (1×10⁸ CFU mL^-1) for 3 h respectively, and then fixed with 2.5% glutaraldehyde (Hushi, Shanghai, China) at 4 °C in the dark for 2 h. Next, the samples were dehydrated by adding ethanol (Hushi, Shanghai, China) by a graded series (30%, 50%, 70%, 80%, 90%, 100%) for 15 min of each at room temperature. Samples were treated with isoamyl acetate (Sigma, St. Louis, Mo, USA) for 20 min, subjected to critical point drying for 3 h (Hitachi-HCP, Hitachi, Tokyo, Japan), coated with gold (MC1000, Hitachi, Tokyo, Japan) and viewed under SEM (S-3400N, Hitachi, Tokyo, Japan).

The measurement of fluorescence intensity changes of ETs was examined as the methods reported previously (35). Briefly, to quantify ETs, HKLs were suspended in phenol red-free L-15 medium and seeded in black 96-well plates (Cayman Chemical, Ann Arbor, MI, USA). The cells were stimulated with PMA (100 ng mL^-1), live E. coli, E. tarda, or S. aureus (1 × 10⁵ CFU mL^-1) for 1, 2, and 3 h, while the containing cytochalasin D in each group (20 µg mL⁻¹, Absin, Shanghai, China) functioned as control. After stimulation, Sytox Green (5 µM, Thermo Scientific, Waltham, Massachusetts, USA) was added to the wells to dye the extracellular DNA. Fluorescence was then quantified as relative fluorescence units (RFU) at 485 nm excitation and 520 nm emission using a fluorescence spectrophotometer (Infinite M26000, FLUOstar Omega, Germany).

To determine whether anti-rPoMPO Abs inhibits MPO activity, the supernatant of HKLs was incubated alone, or with anti-rPoMPO Abs (1:1000) for 45 min. The activity of MPO was measured by the MPO colorimetric activity assay kit (Nanjing Jiancheng Co., Ltd., China) according to the manufacturer’s instructions. Finally, the absorbance was measured at 460 nm to evaluate the MPO activity.

The survival of ETs-trapped bacteria was examined as the methods reported previously (35–37). Briefly, HKLs (1×10⁶ cells mL^-1) were suspended in L-15 medium and seeded in 96-well plates to adhere for 60 min at 22 °C. The cells were stimulated with PMA for 2 h to induce the ET formation, while control cells were added with the same volume of L-15 medium. Then, L-15 medium containing cytochalasin D (20 µg mL⁻¹) was added to the wells of the plates. In addition, some wells were added anti-rPoMPO Abs (1:1000). After treatment for 45 min, 2,000 CFU E. coli, E. tarda, or S. aureus in 100 µL L15 medium were added to wells, respectively. After incubation at 0, 2, 4, and 8 h, the content from each well were taken out, diluted, and plated on LB agar plates. Finally, after incubation at 30°C for 24 h, the colonies appearing on the plates were counted.

Student’s t-test or the one-way analysis of variance (ANOVA) and Duncan’s multiple comparisons were performed by using Statistical Product and Service Solution (SPSS) software (Version 19.0; SPSS, IBM, BY, USA). In all cases, P < 0.05 was considered significant.

The PoMPO cDNA sequence (GenBank accession no. XM_020082562.1) consisted of an ORF of 2313 bp encoding a protein of 770 amino acids ( Supplementary Figure 1A ). The putative protein had a molecular mass of 86.928 kDa and an estimated pI of 9.21. Secondary structure analysis exhibited that PoMPO comprised signal peptides and a peroxidase domain ( Supplementary Figure 1B ). The multiple amino acid sequence alignment results show several conserved domains such as the signal peptide (1-18 aa), propeptide (19-104 aa), light (105-248 aa), and heavy chains (249-770 aa). Other important sites for regulation of MPO activity, including propeptide cleavage site (98-104 aa), haem cavity, and Ca²⁺-binding motif (312-319 aa), are also present in PoMPO. The similarity of MPO between flounder and other animals ranged from 62.4% to 87.4%. ( Supplementary Figure 2 ). A phylogenetic tree was built with MPO and EPO from fish, frog, chicken, and mammals showed that the PoMPO formed a separate clade with MPO sequences of teleost and its closest relationship is confirmed to be with the turbot ( Figure 1 ). Furthermore, the motifs of at least ten genes at adjacent positions of the MPO in five teleost species were analyzed to obtain more information about the origin of the PoMPO gene. The analysis revealed a conserved set of genes (nfkbil1, slc35e4, snx24, ggcx, gmcl1, fam136a, pcyox1) in the vicinity of the MPO gene in teleost ( Figure 2 ).

The SDS-PAGE analysis showed that rPoMPO-Trx (46 kDa) was successfully expressed in E. coli BL 21. The recombinant proteins of PoMPO-Trx with high purity were obtained and used for the production of antibodies ( Supplementary Figure 3A ). Western blotting verified that the anti-rPoMPO Abs could specifically recognize rPoMPO (46 kDa) and endogenous PoMPO (75 kDa) ( Supplementary Figure 3B ). The mass spectrometry results showed that the protein sequence of this 75 kDa protein similar to the theoretical PoMPO - with 46% sequence identity ( Supplementary Figures 3C, D ). From the Western blot experiment, the anti-PoMPO Abs specifically recognized PoMPO - this polyclonal antibody can be used to target PoMPO in functional studies.

The PoMPO gene was widely expressed in all detected tissues. The highest expression levels of PoMPO mRNA were found in the head kidney, followed by peritoneal cells, gill, spleen, skin, muscle, and liver ( Figure 3A ). In agreement with the mRNA expression pattern, PoMPO protein was found mainly in the head kidney, peritoneal cells, gill, and spleen. In contrast, no clear bands were detected in skin, muscle, and liver ( Figure 3B ). To assess the involvement of PoMPO in the response of V. anguillarum, S. aureus, and HIRRV infection, its transcriptional level was examined in the head kidney and spleen at different time points. The expression of PoMPO mRNA in samples taken from infected fish was significantly lower compared with the expression in samples taken from control fish at all time points examined (P < 0.05) ( Figure 3C ). In the spleen, PoMPO expression was significantly upregulated after infection with V. anguillarum and peaked at 96 h (P < 0.05). However, after infection with S. aureus or HIRRV, the expression of PoMPO was not significantly different from the PBS group in the spleen ( Figure 3C ). For further verification, quantitative analysis of HKLs by western blot showed that the content of PoMPO protein was decreased after stimulated by V. anguillarum for 12 h. However, the content of PoMPO protein in spleen showed no significant change compared to the control group ( Supplementary Figure 4B ).

After stimulation with LPS in vivo, the content of PoMPO protein in PerCs was found to be elevated at all sampling time points compared with 0 h ( Figure 4A ). The analysis showed that the content of PoMPO was increased approx two-fold after LPS stimulation (p = 0.2350) ( Figure 4B ). By immunofluorescence staining and analysis, the number of MPO⁺ cells increased in the peritoneal cavity and in the peripheral blood following LPS stimulation, while the number was reduced in the head kidney after LPS stimulation ( Supplementary Figure 4A ). Flow cytometric analysis showed that the percentage of MPO⁺ cells increased from 15.1 ± 1.47% to 43.6 ± 1.45% in the peritoneal cavity and from 4.7 ± 1.21% to 14.5 ± 2.86% in peripheral blood after LPS stimulation. The percentage of head kidney MPO⁺ cells was, however, reduced (from 42.0 ± 3.0% to 12.3 ± 0.46%) ( Figure 4C ) following i.p. injection of LPS.

For the identification of PoMPO⁺ cell types, double immunofluorescence staining of PerCs was performed. The results revealed also the presence of MHCII and GCSFR proteins in PoMPO⁺ cells. However, by immunocytochemical analysis, the cells did not stain dually positive using both anti-PoMPO and anti-Zap-70. No fluorescence was observed in the negative controls ( Figure 5A ). The flow cytometric analysis of PerCs showed that the percentage of MPO⁺/MHCII⁺ cells and MPO⁺/GCSFR⁺ cells were 6.0 ± 1.17% and 5.6 ± 1.58%, respectively. The proportion of MPO⁺/Zap70⁺ cells was very low ( Figure 5B ).

Leukocytes from the peritoneal cavity and head kidney were isolated and stimulated by PMA to induce the formation of extracellular DNA scaffold containing PoMPO. As shown in Figure 6 , the extracellular DNA released by MPO⁺ cells from the peritoneal cavity ( Figures 6A, B ) and head kidney ( Figures 6C, D ) were stained blue by DAPI. In addition, PoMPO proteins were also detected in the ETs ( Figures 6B, D ). The control group used mouse anti-Trx Abs that were not observed positive cells (data not shown).

To examine whether PMA and bacteria could induce the formation of ETs in flounder, the HKLs were incubated with PMA, E. coli, E. tarda, or S. aureus. The quantity of ET-formation (fluorescence) from stimulated cells was significantly higher than from control cells at all examined time points after stimulation (P < 0.05) ( Figure 7B ). To identify the morphology of ET-formation and entrapment of bacteria, ultrastructural studies using SEM were performed and revealed that netlike structures were formed as long fibers alone or coalesced into bundles with some small particles attached. In addition, the entrapment of coccoid- or rod-shaped bacteria on the extracellular fibers was clearly observed ( Figures 7A, C, E, G ).

Under in vitro conditions, the neutralization of anti-rPoMPO Abs was determined. The results showed that the MPO´s enzymatic activity was significantly reduced after adding anti-rPoMPO Abs (p = 0.0167) ( Supplementary Figure 5 ). To examine whether PoMPO had any effect on the viability of the trapped bacteria, ETs-positive cells, ETs-negative cells, ETs-positive cells incubated with anti-rPoMPO Abs were incubated with E. coli, E. tarda, or S. aureus for 0 h, 2 h, 4 h, or 8 h. Then, viable bacteria were quantified. The recoveries of E. coli and S. aureus from the ETs-positive cells groups were significantly lower than ETs-negative cells at 2 h and 8 h of incubation. The number of recovered live E. coli and S. aureus in ETs-positive cells treated with anti-rPoMPO Abs were significantly higher compared to the ETs-positive cells at 8 h. ( Figures 7D, H ). However, for E. tarda, bacterial recoveries were not significantly different among the three groups at the same examined time points ( Figure 7F ).