Fig 2: PSMα4-induced HBP release increases HVUEC monolayer permeability.(A) PSMα4 did not cause cytotoxicity in HUVECs. HUVECs (104/well) were seeded in 24-well plates and incubated for 24 h. The HUVECs were then incubated with PSMα4 at 10, 20, or 30 μg/mL at 37 °C for 30 min. The supernatant was collected and cytotoxicity was analyzed by LDH assay. (B) PSMα4-induced HBP release increased HVUEC monolayer permeability. The HUVEC monolayer on the transwell insert chamber was incubated with d-mannitol (1.4 mM), culture supernatant of whole blood treated with PSMα4 (10 μg/mL), the supernatant + functional blocking antibody against HBP, the supernatant + the isotype control IgG (50 μg/mL), PSMα4 (10 μg/mL), or PBS at 37 °C for 30 min. Lucifer yellow was also added to the incubation media. The fluorescence intensity in the lower chamber of the transwells was determined. d-mannitol and PBS were used as the positive and negative control, respectively.

Fig 3: PSMα4 stimulates HBP release from PMNs via FPR2 and PI3K signaling pathway.(A) FPR2 antagonist WRW4 significantly reduced HBP release from whole blood. Whole blood was pre-incubated with WRW4 (50 μg/mL) or the scramble all d-amino acid control wwrw3 (50 μg/mL) at 37 °C for 15 min30 and then treated with PSMα4 (10 μg/mL) at 37 °C for 30 min. HBP in the supernatant was analyzed by ELISA. (B) Formyl PSMα4 peptide induced HBP release dose-dependently. Human whole blood was incubated with 10, 20, or 30 μg/mL non-formyl or formyl PSMα4 at 37 °C for 30 min. PBS was used as the negative control. (C) The critical amino acid residues of PSMα4 to induce HBP release were screened by alanine substitution. The concentration of the peptides was 10 μg/mL. Replacement of I3, V4, G5, T6, I11, I15, I17, or F18 with alanine significantly abolished HBP release. (D) PI3K-specific inhibitor wortmannin completely abolished HBP release. (E) Rac-specific inhibitor NSC23766 completely abolished HBP release. Whole blood was pre-incubated with 1 μM wortmannin or 50 μM NSC23766 at 37 °C for 1 h and then treated with PSMα4 (10 μg/mL) for 30 min. HBP in the supernatant was analyzed by ELISA. (F) EGTA in culture media significantly reduced HBP release. Human whole blood was pre-treated with 20 mM EGTA at 37 °C for 15 min and then stimulated with PSMα4 (10 μg/mL). (G) PSMα4 induced Ca2+ influx into PMNs. Fluo-3/AM was loaded to PMNs before stimulation with PSMα4. fMLP (1 μM) was used as a positive control for Ca2+ influx.

Fig 4: PSMα4 stimulates primary granule exocytosis, which is free of the blocking effect of serum lipoprotein.(A) Synthetic PSMα4 appeared to be less cytotoxic than PSMα1–3. Cytotoxicity of PSMα1–4 on PMNs was evaluated LDH test. PMNs were incubated with PSMα1–3 at 10 μg/mL, PSMα4 at 10, 20, 50, or 100 μg/mL at 37 °C for 30 min. The control peptide was added at 10 μg/mL. (B) Human serum (10%) significantly reduced PSMα1–3-induced HBP release from PMNs but did not affect PSMα4-induced HBP release from PMNs. (C) Serum significantly reduced PSMα1–3-induced PMN lysis but had no effects on PSMα4-induced PMN lysis. (D) PSMα4 peptide (10 μg/mL) increased MPO release from whole blood. (E) PSMα4 peptide (10 μg/mL) increased elastase release from whole blood. MPO and elastase in the supernatant were analyzed by ELISA. (F) PSMα4-induced cell surface expression of CD63 was not affected by human serum (10%). PMNs were incubated with PSMα4 peptides (10 μg/mL) with 10% human serum at 37 °C for 30 min. The PMNs were stained with PE-labeled anti-CD63 antibody (1:100 dilution) and 5 μM DRAQ5. ***P < 0.001, **P < 0.01, *P < 0.05.