Autophagy in pulmonary macrophages mediates lung inflammatory injury via c-Src tyrosine kinase pathway activation during mechanical ventilation

OBJECTIVE: It has been clearly demonstrated that autophagy plays a critical role in mechanical ventilation-Induced lung injury (VILI). Herein, we first evaluated the mutual effects of autophagy and c-Src signaling on the lung inflammatory response to mechanical ventilation.

MATERIALS AND METHODS: Mice were respectively subjected to a lower or higher lung stretch induced by mechanical ventilation with low (7 mL/kg) or high (28 mL/kg) tidal volume, before measuring the activation of autophagy and c-Src signaling through LC3 lipidation and c-Src phosphorylation, respectively. Bone marrow-derived macrophages (BMDMs) were transfected with Atg5 siRNA and administered to AM-depleted mice to generate an autophagy-deficient phenotype, and c-Src signaling was evaluated by Western blot assay to determine the impact of autophagy on c-Src activation during VILI. Afterwards, the c-Src pathway was then blocked using PP2, prior to the evaluation of polymorphonuclear neutrophils (PMN), total cell counts in BAL fluid, and lung injury scores, in order to elucidate the role of the c-Src pathway in autophagy-mediated VILI.

RESULTS: Both LC3-II and p-c-Src were remarkably increased after mechanical ventilation, in a time-dependent and tidal volume-dependent manner. Moreover, c-Src phosphorylation induced by ventilation was significantly compromised in autophagy-deficient mice. On the other hand, LC3-II expression did not change due to c-Src signaling abolishment. But the inflammatory response induced by injurious ventilation was markedly attenuated by PP2 or AM-abolishment, shown by PMN and total cell counts in BAL fluid, as well as lung injury scores.

CONCLUSIONS: Our results suggested that autophagy caused VILI via regulating c-Src activation, which implies that c-Src may serve as a promising therapeutic target in VILI.

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