MLKL Protects Pulmonary Endothelial Cells in Acute Lung Injury

The role of autophagy in pulmonary microvascular endothelial cell (PMVEC) are controversial in lipopolysaccharide (LPS)-induced ALI. Mixed lineage kinase domain like pseudokinase (MLKL) has been recently reported to keep cell survival by facilitating autophagic flux in response to starvation, rather than its well-recognized role in necroptosis. Using mouse PMVEC and LPS-induced ALI model, we showed that in PMVEC, MLKL was phosphorylated (p-MLKL) and autophagic flux was accelerated at the early stage of LPS stimulation (1-3 h), manifested by increases in levels of lipidated MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta; LC3-II), decreases in levels of SQSTM1/p62 (sequestosome 1), and fusion of 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-h LPS stimulation. The p-MLKL induced by short-term LPS formed multimers to facilitate the closure of phagophore by HaloTag-LC3 autophagosome completion assay. The charged multivesicular body protein 2A (CHMP2A) is essential in the process of phagophore closure into nascent autophagosome. Agreeing with the p-MLKL change, CHMP2A levels markedly increased during 1-3 h 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 phagophore via CHMP2A, thus protecting PMVEC and alleviating LPS-induced ALI.