Fig. 5.

Repletion of bone marrow-derived macrophages (BMDMs) with decreased ATG5 aggravates lung inflammatory injury in AM-depleted mice during mechanical ventilation. A: effects of mechanical ventilation on ATG5 protein expression in lung tissues and lung macrophages (MΦ). Mice were ventilated with a low (7 ml/kg) or high tidal volume (28 ml/kg) for 2 h. Lung macrophages were then isolated from the whole lung homogenates. Lysates from lung homogenates and macrophages were analyzed by Western blot. Data are representative of 3 independent experiments. B: silencing of ATG5 in BMDMs with a specific siRNA. BMDMs isolated from donor mice were transfected with a scrambled siRNA (siSc) or ATG5 siRNA (siAtg5). After 48 h, the efficiency of transfection was evaluated by Western blot. Representative data of Western blot showed the depletion of ATG5 protein expression from BMDM cell lysates. C: effects of in vivo injection of BMDMs treated with scrambled siRNA or ATG5 siRNA on reconstitution of macrophages in the lung. BMDMs were transfected with scrambled siRNA or ATG5 siRNA. After 48 h, BMDMs transfected with a scrambled siRNA or ATG5 siRNA (2 × 10⁶ cells, 200 μl total volume each) were labeled with PKH26 (red) and then given to AM-depleted mice via a jugular venous cannula. After 2 h, lung frozen sections (8 μm) were performed immediately. Photomicrographs were obtained with an Nikon Eclipse E400 microscope with a ×20 objective. D: effects of repletion of BMDMs with decreased ATG5 on mechanical ventilation (28 ml/kg)-induced autophagy activation in the lung. Mouse AMs were depleted with clodronate liposomes (CLOD) as described in materials and methods. BMDMs transfected with siSc or siAtg5 were intravenously injected into AM-depleted mice. Left, representative Western blots for LC3-I and LC3-II; right, bar graph showing the relative abundance of LC3-II protein (normalized to that of GAPDH) from 3 experiments. E: neutrophils from bronchoalveolar lavage (BAL) were enumerated to evaluate lung air space inflammation. F: total cell count from BAL fluid. G: pulmonary vascular protein permeability as determined by protein concentration of BAL fluid. H: pulmonary edema formation measured by extravascular lung water (ELW). I: histological analysis of lung tissue by hematoxylin and eosin staining (×40 magnification). *P < 0.05 compared with control group (nonventilated normal lungs); †P < 0.05 compared with MV alone group; ‡P < 0.05 compared with siSc group; n = 6/each group.