Disrupting PTEN enhances the bacteria-killing capability of neutrophils. (A) Bacterial killing in inflamed lungs. Mice were intratracheally instilled with 106 CFU of E coli and killed at the indicated times. Whole lungs were homogenized and serially diluted with sterile cold water. Aliquots were spread on Luria-Bertani agar plates and incubated overnight at 37°C. Live bacteria were quantified as CFU/lung. (B) In vitro bacterial killing assay. Purified bone marrow WT or PTEN-null neutrophils were incubated with E coli for 2 hours. Diluted aliquots were spread on agar plates and incubated overnight at 37°C. (C) In vitro bacterial killing capabilities were reflected by the decrease of CFU after indicated incubation periods. (D,E) In vitro phagocytosis assay. FITC-labeled zymosans (D) or E coli bioparticles (E) were opsonized with mouse serum and incubated with neutrophils at 4°C (control) or 37°C for 1 hour. Extracellular fluorescence was quenched by trypan blue. Phagocytosis index (PI) was expressed as the number of bioparticles engulfed by 100 neutrophils. Binding index was expressed as the number of bioparticles bound to 100 neutrophils. More than 200 neutrophils were counted in each group. (F) Phagosome maturation. WT or PTEN-null neutrophils were incubated with mouse serum-opsonized latex beads for 60 minutes. Phagosomes were stained with LysoTracker and visualized under fluorescent microscopy. (G) Intracellular reactive oxygen species (ROS) production during phagocytosis. Bone marrow–derived neutrophils from WT and PTEN KO mice were incubated with (or without) mouse serum–opsonized E coli or zymosan bioparticles in the presence of superoxide dismutase (SOD; 50 U/mL) and catalase (2000 U/mL). ROS production was monitored in a luminometer at 37°C. Chemiluminiscence (arbitrary light units) was recorded every 150 seconds for 1 hour. (H) Intracellular killing assay. Bone marrow–derived neutrophils from WT and PTEN KO mice were incubated with mouse serum-opsonized live E coli for 1 hour and then with 100 μg/mL gentamicin for an additional hour. Viable intracellular bacteria were quantified by subsequent plating the lysed samples on Luria-Bertani agar. The results were normalized by phagocytosis indexes. All data are presented as mean (± SD; n = 3-4 mice in each group). *P < .05, **P < .01, and ***P < .001 versus WT.
