Fig. 6.

Flagellin promoted phagosome formation and increased reactive oxygen species (ROS) levels in macrophages. a Microscopy analysis of zymosan phagocytosis by untreated and flagellin-treated peritoneal macrophages. 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 challenged with zymosan (20 particles/M_) for 30 min at 37 °C. Scanning electron microscopy showed that zymosan particles were no longer seen on the cell surface of flagellin-treated macrophages, because most have been internalized by macrophages (TEM data). Moreover, TEM showed that fewer zymosan particles were internalized in flagellin-treated macrophages after TLR5 blockade using anti-TLR5 antibodies or TLR5 Fc Chimera when compared with macrophages treated with flagellin alone. 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 were incubated with heat-inactivated E. coli, and ROS levels were assayed at the indicated time points. Representative examples were shown for the production of ROS at 60 min, and the data were also shown as the mean ± standard deviation (SD) and were compared to respective control group at each time point by Student’s t test. ***p < 0.001 when compared with control macrophages