Fig. 5

Effects of NMN on phagocytic and bactericidal activity and ATP production. A, B, C A single dose of NMN (500 mg/kg, i.p.) was administrated into mice after an hour of FIP. Twenty-four hours later, the peritoneal lavage fluids and blood were assayed for bacterial loads. A Upper panels: representative pictures of bacterial colonies from peritoneal lavage (A1) and blood on plates (A2); Lower panel: colony forming units (CFUs) in each group. Data are mean ± SD, n = 8 mice in each group. *P < 0.05 vs vehicle. B and C In vivo neutrophil phagocytosis in peritoneum was analyzed at 6 h (B) and 24 h after FIP, respectively. Left panels: representative flow cytometry analysis of neutrophil uptake of pHrodo Green E. coli Bioparticles from 5–6 mice in each group; Right panels: quantification of fluorescence intensity. Data are mean ± SD, n = 5–6 mice in each group. *P < 0.05 vs vehicle. D, E, F, G, H In vitro phagocytosis and bacterial killing.(D Phagocytosis was determined in neutrophils (D1) and macrophages (D2) at different time points (0.5, 1 and 2 h) by analyzing the uptake of pHrodo Green E. coli Bioparticles. E and F Neutrophils and macrophages were pretreated with NMN (500 µM) and then incubated with living E. coli bacteria for 5 min and one hour, respectively. Upper panels: representative pictures of engulfed E. coli on the tryptic agar plates; Lower panel: CFUs of E. coli in each group (E for neutrophils and F for macrophages). G and H E. coli killing assay. Neutrophils and macrophages were pretreated with NMN (500 µM) and then incubated with living E. coli bacteria for 5 min or one hour, respectively. After washing neutrophils or incubating macrophages with gentamicin for 30 min to remove extracellular E. coli bacteria, the intracellular living bacteria were determined as described above. After that, the neutrophils and macrophages were maintained in normal cultured medium at 37℃ for additional 2 and 5 h, respectively. The intracellular living bacteria were then analyzed (G for neutrophils and H for macrophages). MFI, median fluorescence intensity. I, J ATP production in neutrophils and macrophages. Cells were pretreated with NMN and then challenged with E. coli. ATP production was determined in neutrophils (I) and macrophages (J). Data are mean ± SD from 5 to 8 independent experiments. *P < 0.05 vs vehicle or saline + vehicle and # P < 0.05 vs E. coli + vehicle (One-way ANOVA followed by Newman–Keuls test)