MLKL Protects Pulmonary Endothelial Cells in Acute Lung Injury.

ABSTRACT

The role of autophagy in pulmonary microvascular endothelial cells (PMVECs) is controversial in LPS-induced acute lung injury (ALI). Mixed lineage kinase domain-like pseudokinase (MLKL) has recently been reported to maintain cell survival by facilitating autophagic flux in response to starvation rather than its well-recognized role in necroptosis. Using a mouse PMVEC and LPS-induced ALI model, we showed that in PMVECs, MLKL was phosphorylated (p-MLKL) and autophagic flux was accelerated at the early stage of LPS stimulation (1-3 h), manifested by increases in concentrations of lipidated MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 ß; LC3-II), decreases in concentrations of SQSTM1/p62 (sequestosome 1), and fusion of the autophagosome and lysosome by pHluorin-mKate2-human LC3 assay, which were all reversed by either MLKL inhibitor or siRNA MLKL. In mice, the inhibition of MLKL increased vascular permeability and aggravated mouse ALI upon 3-hour LPS stimulation. The p-MLKL induced by short-term LPS formed multimers to facilitate the closure of the phagophore by HaloTag-LC3 autophagosome completion assay. The charged multivesicular body protein 2A (CHMP2A) is essential in the process of phagophore closure into the nascent autophagosome. In agreement with the p-MLKL change, CHMP2A concentrations markedly increased during 1-3-hour LPS stimulation. CHMP2A knockdown blocked autophagic flux upon LPS stimulation, whereas CHMP2A overexpression boosted autophagic flux and attenuated mouse ALI even in the presence of MLKL inhibitor. We propose that the activated MLKL induced by short-term LPS facilitates autophagic flux by accelerating the closure of the phagophore via CHMP2A, thus protecting PMVECs and alleviating LPS-induced ALI.