Figure 4: mTOR activity is restricted to neutrophils, and is required for survival and efficient bacterial clearance in burned mice following infection.
Burn or sham mice treated with rapamycin (4mg/kg/day) underwent an intra-tracheal inoculation with PAK at 14 dpi. A) 48 hours following infection with 1 × 106 CFU of PAK, bacterial burden was determined in whole lung homogenate. B) Following infection with 5 × 106 CFU of PAK, mice were monitored for morbidity for 72h, and moribund or deceased animals were recorded. C) Blood from the submandibular vein was collected and NO levels were directly measured. D) Total number of neutrophils in the lung is determined as (%neutrophils as live cells x live cells counted at harvest). E) Cells from whole lung tissue were collected at 14 d post burn injury and stimulated with FBS in vitro with (Rapa) or without (FBS) Rapamycin for 30 minutes. After gating on neutrophils, we show intracellular pS6 staining in three representative replicates from three repeat experiments. FMO refers to the Fluorescence Minus One control, which is the fluorescence signal using every antibody apart from anti-pS6. E-F) pS6 staining of gated neutrophils were measured and used to calculate F) MFI of cellular pS6 and G) pS6+ percentage of gated neutrophils. Data shown are +/−SEM. *p<0.05, **p<0.01 and representative of three repeated experiments. Numbers of mice in representative figures; A, C) sham, n=4; burn, n=6; B) sham, n=8; burn, n=8; D) sham, n=6; burn, n=6; F,G) sham, n=5; burn, n=5.