Fig 1: Verification of P-SEP production by addition of PR3 to recombinant human CD14.(a) Presepsin (P-SEP) levels in the supernatant were measured after addition of PR3 to rCD14. Conditions with PR3 inhibition using the inhibitors PMSF or elafin were also compared. (b) Western blot analysis of CD14 and P-SEP proteins, with band intensities quantified using ImageJ. Data are presented as means ± SDs (n = 3). Statistical analysis was performed using one-way analysis of variance followed by Tukey–Kramer’s honestly significant difference test. **p < 0.01.
Fig 2: Schematic illustration of the mechanism by which M1 MΦs produce P-SEP via PR3 following NET phagocytosis. Upon bacterial infection of blood vessels, neutrophils release neutrophil extracellular traps (NETs) as a host defense mechanism. M1 MΦs phagocytose CD14-rich NETs, and the engulfed CD14 is degraded by the proteolytic enzyme PR3 within the macrophages, resulting in the generation of presepsin (P-SEP). Subsequently, P-SEP is released extracellularly from M1 MΦs, leading to elevated blood levels of P-SEP.
Fig 3: Effect of PR3 inhibition in M1 MΦs on P-SEP production during NET phagocytosis.(a, b) Supernatant presepsin (P-SEP) levels after co-culture of M1 MΦs and PMA-neutrophil extracellular trap (NET), with PMSF or elafin added at various concentrations to inhibit PR3. (c) M1 MΦs were co-cultured with NET and subjected to immunofluorescence staining. Confocal microscopy confirmed that CD68-positive M1 macrophages phagocytosed Cit-H3-positive NETs and produced P-SEP (yellow arrows). However, PR3-inhibited M1 macrophages did not produce P-SEP even after phagocytosing NET. Scale bar: 10 μm. (d) P-SEP levels after PR3 inhibition with 10 µM PMSF or 0.5 µM elafin. (e) Western blot analysis of P-SEP protein in M1 MΦs after PR3 inhibition; protein levels were quantified using ImageJ and normalized to β-actin. Data are presented as means ± SDs (n = 3). Statistical analysis was performed using one-way analysis of variance followed by Tukey–Kramer’s honestly significant difference test. **p < 0.01, *p < 0.05.
Fig 4: Comparison of the proteolytic enzyme levels in phagocytes.Three types of blood-derived cells (neutrophils and Mos isolated from the peripheral blood of healthy participants, and M1 MΦ differentiated from Mos) were analyzed for intracellular levels of PR3, cathepsin D, and elastase by flow cytometry. Proteolytic enzyme levels were compared using mean fluorescence intensity. Lymphocytes were included as negative controls and are shown in gray, while phagocytic cells are shown in green.
Fig 5: Evaluation of PR3-mediated P-SEP production during NET phagocytosis by M1 MΦ. (a) M1 MΦs were co-cultured with neutrophil extracellular trap (NET) induced by E. coli DH5α or PMA, followed by May–Grünwald–Giemsa (MG) staining and observation using a biological microscope. M1 MΦs were observed phagocytosing NET induced by both E. coli DH5α and PMA. Scale bar: 10 μm. (b, c) M1 MΦs were co-cultured with PMA-induced NET (PMA-NET), and immunofluorescence staining was performed. Confocal microscopy revealed that CD68-positive M1 MΦs phagocytosed Cit-H3–positive NET, and the intracellular localization of PR3 and presepsin (P-SEP) was examined. PR3 and P-SEP were detected in regions of M1 MΦs that had phagocytosed PMA-induced NET. Scale bar: 10 μm. (d) The concentration of P-SEP in the culture supernatant following phagocytosis of PMA-induced NET by M1 MΦs was strongly correlated with the NET ratio.
Supplier Page from Abcam for FITC Anti-PR3 antibody [PR3G-2]