Figure 3.

A1ATM₃₈₃S-CF inhibits NET formation by PMNs isolated from healthy adults. A1ATM₃₈₃S-CF as well as its SCR peptide control were synthesized for use in in vitro assays of NET formation. (A) PMNs were pretreated with A1ATM₃₈₃S-CF (1.2 μM) or nNIF (1.2 μM) or their SCR control peptides for 1 hour before stimulation with PMA (20 nM; 2 hours). NET formation was assessed by live cell imaging using confocal microscopy with DNA stains, one that is cell permeable (SYTO Green [Thermo Fisher Scientific], nuclear DNA, green) and the other that is cell impermeable (SYTOX Orange [Thermo Fisher Scientific], NETs, magenta). Images are representative of 4 different experiments in PMNs isolated from 4 different donors. (B) NET formation was quantified by using a high-throughput DNA fluorescence assay for PMNs treated as in panel A. The y-axis represents NET formation with relative fluorescent intensity ± SEM. (C) We performed a concentration curve of A1ATM₃₈₃S-CF or its SCR peptide control (241 pM to 12 μM) and assessed NET formation in PMNs isolated from healthy adults using live cell imaging as in panel A. PMNs were pretreated for 1 hour with either A1ATM₃₈₃S-CF or its SCR peptide control before stimulation with LPS (100 ng/mL; 2 hours). Images are representative of 3 separate experiments using PMNs from 3 different donors. (D) A standardized grid system was used to semiquantitate NET formation by PMNs treated as in panel C. The y-axis depicts NET formation with NETs crossing standardized grid lines/high-power field (hpf) (± SEM). (E) We pretreated PMNs with recombinant HTRA1 (rHTRA1) alone (1 nM) or full-length A1AT alone (1 nM) for 1 hour before stimulation with LPS (100 ng/mL; 2 hours). NET formation was assessed by live cell imaging as in panel A. Images are representative of 3 different experiments using PMNs isolated from 3 different donors. (F) We pretreated PMNs with nNIF (0.5 or 1.2 μM), A1ATM₃₈₃S-CF (0.5 or 1.2 μM), or full-length human recombinant A1AT (0.5 or 1.2 μM) for 1 hour before stimulation with LPS (100 ng/mL; 2 hours). NET formation was quantified by using a high-throughput DNA fluorescence assay for PMNs treated as in panel A. The y-axis represents NET formation with relative fluorescent intensity over baseline, arbitrarily set at 1 (±SEM). (G) PMNs were pretreated with the cell-free incubation reaction products of placental extracted or rHTRA1 (0.08 mg/mL) with full-length A1AT (0.8 mg/mL) for 1 hour before stimulation with LPS (100 ng/mL; 2 hours). NET formation was assessed by live cell imaging as in panel A. Images are representative of 3 different experiments using PMNs isolated from 3 different PMN donors. Scale bars, 50 μm for all images in this figure. *P < .05, **P < .01, ***P < .001.