Depletion of PTEN delays neutrophil death in bacterial pneumonia. (A) Apoptotic neutrophils in the inflamed lungs. Mice were infected by intratracheal instillation of 106 CFU of E coli and killed 24 hours later. The viability of accumulated neutrophils was determined by TUNEL assay. Green arrows indicate apoptotic neutrophils. (B) Quantification of apoptotic neutrophils. Data shown are mean (± SD; n = 4). *P < .05 versus WT. (C-E) Examination of in vivo death of adoptively transferred neutrophils. Neutropenia in receipt mice was induced by a widely used chemotherapeutic drug, Cy (Figure S5). The mature WT or PTEN-null neutrophils were purified from the inflamed peritoneal cavity and labeled with intracellular fluorescent dye CFSE or 5-(and -6)-chloromethyl SNARF-1 acetate (SNARF-1). Labeled cells were mixed (1:1) and intraperitoneally injected to WT mice challenged with 3% thioglycollate (TG). Peritoneal lavage was collected 15 hours after cell injection, and the ratios of adoptively transplanted WT and PTEN-null neutrophils were analyzed using a FACSCanto II flow cytometer and FACSDiva software (D). Relative death rates were quantified and expressed as the ratio of adoptively transplanted PTEN-null neutrophils to WT neutrophils in the inflamed peritoneal cavity (E). Data shown are mean (± SD; n = 3 mice in each group). *P < .05 versus WT.
