Autophagy in pulmonary macrophages mediates lung inflammatory injury via NLRP3

22 The inflammatory response is a primary mechanism in the pathogenesis of ventilator-induced 23 lung injury. Autophagy is an essential, homeostatic process by which cells break down their own 24 components. We explored the role of autophagy in the mechanisms of mechanical ventilation25 induced lung inflammatory injury. Mice were subjected to low (7 ml/kg) or high (28 ml/kg) tidal 26 volume ventilation for 2 h. Bone marrow-derived macrophages transfected with a scrambled or 27 autophagy-related protein 5 small interfering RNA were administered to alveolar macrophage28 depleted mice via a jugular venous cannula 30 min before starting ventilation protocol. In some 29 experiments, mice were ventilated in the absence and presence of autophagy inhibitors 330 methyladenine (15 mg/kg, i.p.) or trichostatin A (1 mg/kg, i.p.). Mechanical ventilation with a 31 high tidal volume caused rapid (within minutes) activation of autophagy in the lung. 32 Conventional transmission electron microscopic examination of lung sections showed that 33 mechanical ventilation-induced autophagy activation mainly occurred in lung macrophages. 34 Autophagy activation in the lungs during mechanical ventilation was dramatically attenuated in 35 alveolar macrophage-depleted mice. Selective silencing of autophagy-related protein 5 in lung 36 macrophages abolished mechanical ventilation-induced nucleotide-binding oligomerization 37 domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and lung 38 inflammatory injury. Pharmacological inhibition of autophagy also significantly attenuated the 39 inflammatory responses caused by lung hyperinflation. The activation of autophagy in 40 macrophages mediates early lung inflammation during mechanical ventilation via NLRP3 41 inflammasome signaling. Inhibition of autophagy activation in lung macrophages may therefore 42 provide a novel and promising strategy for the prevention and treatment of ventilator-induced 43 lung injury. 44