Fig. 5.

Flagellin enhanced bacterial phagocytosis and killing by macrophages. a, b Peritoneal macrophages from C57BL/6 mice (n = 5 per group) were pretreated with flagellin (100 ng/ml) in the presence or absence of anti-TLR5 antibodies (1 μg/ml) or TLR5 Fc Chimera (1 μg/ml) for 24 h, and then infected with E. coli (multiplicity of infection, 10) for 30 min. Cells were then washed in PBS and resuspended for 30 min in medium containing 100 μg/ml tobramycin to remove extracellular bacteria. Cells were lysed in PBS containing 0.1% Triton 100 for assessment of a phagocytosis (t = 0), and additional samples were incubated for 2 additional hours (t = 2 h) to assess b bacterial killing. Intracellular killing (t = 2 h) was determined as described in the “Methods” section. *p < 0.05, **p < 0.01, and ***p < 0.001 when compared between groups (denoted by the horizontal bracket; Kruskal–Wallis test followed by Dunn’s multiple comparisons post-test). c, d Peritoneal mouse neutrophils were pretreated with or without flagellin (100 ng/ml) for 12 h and then infected with E. coli (multiplicity of infection, 100) for 30 min. Cells were then washed in PBS and resuspended for 30 min in medium containing 100 μg/ml tobramycin to remove extracellular bacteria. Cells were lysed in PBS containing 0.1% Triton 100 for assessment of c phagocytosis (t = 0), and additional samples were incubated for 1 additional hour (t = 1 h) to assess d bacterial killing