The content and execution of the current study were approved by The Ethical Committee of the Kagawa Prefectural University of Health Sciences, Japan (No.327). All methods were carried out in accordance with the guidelines and regulations of The Ethical Committee of the Kagawa Prefectural University of Health Sciences. Written informed consent was obtained from the participants before the study.
Healthy donor samples and cell lines culture.
The study was approved by the ethics committee of Kagawa Prefectural University of Health Sciences, and consent was obtained from the participating students and teaching staff (n = 7). U937 cells (American Type Culture Collection: CRL-1593.2) and THP-1 cells (American Type Culture Collection: TIB-202) were grown in RPMI1640 medium (Cat, No. R8758; Sigma-Aldrich, St. Louis, MO, USA) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) in a humidified atmosphere with 5% CO2 at 37°C.
Antibodies, proteins, and chemicals.
The details of reagents used in this study and their sources are as follows: diphenyleneiodonium chloride (DPI; Cat, No.81050; Cayman Chemical Company, Ann Arbor, MI, USA), cytochalasin D (Cat, No.11330; Cayman Chemical Company), sivelestat (Cat, No.17779; Cayman Chemical Company), phorbol 12-myristate 13-acetate (PMA; Cat, No.P8139; Sigma-Aldrich), 1α, 25-dihydroxyvitamin D3 (VD3; Cat, No.71820; Cayman Chemical Company), May–Grünwald’s stain solution (Cat, No.15053; Mutokagaku, Tokyo, Japan), Gimsa’s stain solution (Cat, No.15002; Mutokagaku), 1/15M phosphate buffer solution (pH 6.4) (Cat, No.15612; Mutokagaku), DAB Stain Kit ( Cat, No.15712; Mutokagaku), anti-human myeloperoxidase mouse monoclonal antibody (Cat, No.sc-52707; Santa Cruz Biotechnology, Dallas, TX, USA), anti-human neutrophil elastase mouse monoclonal antibody (Cat, No.sc-55549; Santa Cruz Biotechnology), anti-human citrullinated histone H3 rabbit polyclonal antibody (Cat, No.ab5103; Abcam, Cambridge, UK), anti-human presepsin mouse monoclonal antibody (F1106-13-3 antibody; Mochida Seiyaku, Tokyo, Japan), anti-human CD14 mouse monoclonal antibody (Cat, No.14-0149-82; Invitrogen, Carlsbad, CA, USA), FITC-conjugated anti-human CD14 mouse monoclonal antibody (Cat, No.6603511; Beckman Coulter, Brea, CA, USA), Alexa Flour 405-conjugated goat anti-rabbit IgG (Cat, No.ab175652, Invitrogen), Rhodamine (TRITC) -conjugated goat anti-mouse IgG (Cat, No.SA00007-1; Cosmobio, Tokyo, Japan), and E. Coli DH5α Competent Cells (Cat, No.9057; Takara Bio Shiga, Japan).
Isolation of neutrophils.
Neutrophils were isolated at room temperature from the EDTA-anticoagulated peripheral blood of healthy volunteers by density gradient centrifugation using Polymorphprep (Cat, No.1114683; Axis-Shield, Dundee, Scotland). After centrifugation for 30 min at 500 × g, the lower cellular fraction containing neutrophils was collected, serum-free RPMI 1640 medium was added, and the neutrophils were washed by centrifugation for 10 min at 400 × g.
Isolation of monocytes from PBMCs.
After density gradient centrifugation using Polymorphprep, the upper cell fraction containing peripheral blood mononuclear cells (PBMCs) was collected, serum-free RPMI 1640 medium was added, and the cells were washed twice by centrifugation for 10 min at 100 × g to remove platelets. Thereafter, monocytes were isolated from purified PBMCs using the EasySep Human Monocyte Isolation Kit (Cat. No. 19359; Stemcell Technologies, Vancouver, BC, Canada) according to the manufacturer’s instructions.
Immobilization and optical density of E. coli DH5α competent cells.
Escherichia coli DH5α competent cells (DH5α) were used after formaldehyde fixation. To standardize the amount of immobilized DH5α, the optical density (OD) was measured and adjusted. The number of DH5α bacteria at OD 1.0 was equivalent to 3.0 × 105/µL, as determined from the colony assay. For the induction of NET formation, 100 µL of the bacterial solution was added to 900 µL of the neutrophil suspension.
Induction of NETs and measurement of the NET ratio.
Purified neutrophils (1.8 × 106 cells/well) were seeded in 24-well plates (Cat. No. SIAL0526; Sigma-Aldrich) and stimulated using an OD-adjusted DH5α bacterial solution (DH5α-NETs) or 50 nM PMA (PMA-NETs). After stimulation with OD 1.0 DH5α for 4 h at 37°C or 50 nM PMA for 2 h at 37°C, SYTOX Green was added to 100 µL of the cell suspension and incubated for 20 min at room temperature, and the cells were analyzed using a flow cytometer (Cell Lab Quanta SC; Beckman Coulter) without washing. The NET area was defined as the fraction of cells with high side and forward scatter and positive extracellular SYTOX Green nuclear staining was compared with untreated neutrophils. The SYTOX Green positivity rate was evaluated as the NET ratio [38, 45].
Cellular morphology in NETs.
Purified neutrophils were seeded in 24-well plates with submerged glass discs at the bottom, and NETs were induced using DH5α and PMA. The induced NETs were May–Giemsa-stained, myeloperoxidase-stained, and observed under a microscope (OLYMPUS Corporation, BX50, Tokyo Japan) at 1,000× magnification. Staining was performed according to the manufacturer’s instructions.
Measurement of CD14, MPO, and NE expression in NETs.
The expression levels of CD14, myeloperoxidase (MPO), and neutrophil elastase (NE) in neutrophils (untreated) and NET areas after 4 h of DH5α addition were compared using mean fluorescence intensity.
Evaluation of Presepsin levels by co-culturing with monocytes after NET induction.
Purified monocytes (5.0 × 105 cells per well) were seeded in 24-well plates, and the same number of purified neutrophils (control) and NETs from the peripheral blood of the same subject were added after incubation for 3 h at 37°C, and presepsin in the supernatant was measured using PATHFAST (LSI Medience Corporation, Tokyo, Japan).
Immunofluorescence imaging of NETs.
Purified neutrophils were seeded in 24-well plates with submerged glass discs at the bottom, and NETs were induced using DH5α and PMA. After stimulation, the medium was removed, and the remaining cells were washed with phosphate-buffered saline (PBS). Cells on the glass discs were fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, cells were incubated in PBS containing 5% rat serum for 60 min to block non-specific antibody binding. The samples were then incubated for 60 min with the following primary antibodies: anti-human citrullinated histone H3 (Cit-H3) rabbit polyclonal antibody (dilution 1:1,000), anti-human MPO mouse monoclonal antibody (dilution 1:2,000) or anti-human NE mouse monoclonal antibody (dilution 1:2,000) or anti-human CD14 mouse monoclonal antibody (dilution 1:500). After washing in PBS, each primary antibody binding was visualized using secondary antibodies coupled to Alexa Fluor 405-conjugated goat anti-rabbit IgG (dilution 1:1,000) and rhodamine (TRITC)-conjugated goat anti-mouse IgG (dilution 1:2,000), and the samples were stained for DNA (Sytox Green; dilution 1:2,000). After incubation for 60 min, samples were washed with PBS and embedded in 80% glycerol. All the procedures were performed at room temperature. Images were obtained using a confocal microscope (FLUOVIEW FV10i; Olympus Corporation, Tokyo, Japan).
Immunofluorescence imaging of monocytes that had phagocytosed NETs.
Purified monocytes were seeded in 24-well plates with glass discs submerged at the bottom and co-cultured with monocytes after induction of NET formation. After incubation for 3 h at 37°C, the medium was removed, and the remaining cells were washed with PBS. Cells on the glass discs were fixed with 4% paraformaldehyde. After washing with PBS, samples were incubated in PBS containing 5% rat serum for 60 min. Thereafter, the samples were incubated for 60 min with the following primary antibodies: anti-human cit-H3 rabbit polyclonal antibody (dilution, 1:1,000) and anti-human presepsin mouse monoclonal antibody (dilution, 1:5,000). After washing in PBS, each primary antibody binding was visualized using secondary antibodies coupled to Alexa Fluor 405-conjugated goat anti-rabbit IgG (dilution 1:1,000) and rhodamine (TRITC)-conjugated goat anti-mouse IgG (dilution 1:2,000). Subsequently, the samples were stained for DNA (Sytox Green; dilution 1:2,000) or CD14 using FITC-conjugated anti-human CD14 mouse monoclonal antibody. After incubation for 60 min, samples were washed with PBS and embedded in 80% glycerol. All the procedures were performed at room temperature. Images were acquired using a confocal microscope (FLUOVIEW FV10i).
NET ratio and presepsin evaluation after inhibitor treatment.
NETs were treated with DPI, which inhibits NADPH oxidase activity. Before DH5α or PMA stimulation, purified neutrophils (1.8 × 106 cells/well) were exposed to 10 µM DPI for 30 min at 37°C. After NET inhibition, the NET ratio was analyzed by flow cytometry, and presepsin levels were measured after monocyte phagocytosis. Cytochalasin D was used as a phagocytosis inhibitor, and sivelestat was used as an neutrophil elastase inhibitor. Before phagocytosis of NETs by monocytes, purified monocytes (5.0 × 105 cells/well) were exposed to 50 µM cytochalasin D or 50 µM sivelestat for 30 min at 37°C. After co-culture with NETs, the presepsin levels were measured.
NET phagocytosis by macrophages increases presepsin levels.
To differentiate U937 and THP-1 cells into active macrophage-like cells, the cells were resuspended in a culture medium containing 100 nM VD3 to a density of 2.0 × 105 cells/mL and incubated for 48 h at 37°C. After differentiation, the cells were co-cultured with PMA-NETs derived from neutrophils obtained from healthy volunteers for 3 h, and presepsin in the supernatant was measured.
Statistical analyses.
All statistical analyses were performed using SPSS version 24.0 (SPSS Inc, Chicago, IL, USA). The data are presented as the mean ± standard deviation (SD), and Student’s t-test was used for comparisons between two groups. A p-value of less than 0.05 was considered statistically significant. In the graphically represented data, *, and ** denote p values of less than 0.05, 0.01, respectively.