Anti ne

Human sputum NE was acquired from Elastin Product Company (Owensville, MO, USA). The NE was dissolved in a solution containing 50% 0.02 M NaOAc (pH 5) and 50% glycerol. LPS, phenylmethylsulfonyl fluoride (PMSF), and rosiglitazone were obtained from Sigma-Aldrich (St. Louis, MO, USA). The selective elastase inhibitor Elaspol was purchased from Ono Pharmaceutical Co., Ltd. (Osaka, Japan). The anti-IκBα antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-peroxisome proliferator-activated receptor gamma (PPARγ), anti-TLR4, anti-p65, anti-NE, and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

NETs were induced with E. histolytica trophozoites on the Chamber Slide™ System as described above, and the cells were fixed with 4% paraformaldehyde for 10 min. The fixed cells were then permeabilized by adding 0.2% Triton X-100 in PBS for 5 min. Detergent was washed out three times with cold PBS and unspecific protein binding was blocked with a solution of 1% BSA, 0.3 M glycine, 0.1% Tween 20 in PBS for 30 min at 37°C. The samples were incubated with primary anti-NE (Santa Cruz Biotechnology), anti-myeloperoxidase (Abcam), anti-histone H4 (Abcam), or anti-citrulline (Abcam) antibodies diluted 1:100 in 1% BSA, 0.1%Tween 20 in PBS during 1 h at room temperature. The cells were gently washed with cold PBS and incubated with secondary anti-mouse IgG-FITC (Sigma) or anti-rabit IgG-TRITC (Zymax) antibodies diluted 1:50 in the same solution that primary antibody for 1 h at room temperature in darkness. The cells were then washed with PBS and stained with 5 μg/mL DAPI. The coverslips were mounted with Fluoroshield (Sigma) before observation in a fluorescence microscope (Olympus BX51).

To investigate the cross-talk between the TF-bearing NETs and endothelial cells, HAoECs were treated with in vitro–generated NET structures (0.5 μg DNA/mL; ref. 22 (link)). To destroy the DNA scaffold, NET structures were incubated with DNase I (1 U/mL; EN0525, Thermo Fisher Scientific), anti-MPO (sc-52707, Santa Cruz Biotechnology Inc), anti-NE (sc-25621, Santa Cruz Biotechnology Inc), anti-H4Cit3 (07-596, Merck KGaA), or anti-LL37 (sc-166770, Santa Cruz Biotechnology Inc), according to manufacturer’s instructions. To hinder PAR1 signaling, HAoECs were treated with the FLLRN peptide (500 μM; AS-60678, Anaspec). To inhibit TF signaling, NETs were treated with an IgG1 goat anti–human TF polyclonal antibody (10 μg/mL; 4501, Sekisui Diagnostics), having a neutralizing effect. In inhibition studies, HAoECs or NET structures were pretreated with the abovementioned inhibitory agents for 30 minutes. For mRNA studies and in-cell ELISA, HAoECs were incubated with NETs for 3 or 6 hours, respectively, at 37°C, 5% CO₂. For collagen assay, cells were stimulated with NETs for 24 hours, at 37°C, 5% CO₂, and cell culture supernatants were then collected. The concentrations and time points used to examine neutrophils and HAoECs were optimized before the experiments. All substances used in the study were endotoxin free, as determined by a Limulus amebocyte assay (E8029, MilliporeSigma).