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Updated: Jul 5, 2025
Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation
Published on: July 6, 2019
MLKL Protects Pulmonary Endothelial Cells in Acute Lung Injury
Ying Li1,2, Yingxiang Liu1,2, Xueya Yao1,2
1Department of Anesthesiology, Renji Hospital, Shanghai Jiao Tong University School of Medicine; and.
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 β; 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.
Insights
Mixed lineage kinase domain-like pseudokinase (MLKL) facilitates autophagy in lung endothelial cells during acute lung injury. Activated MLKL accelerates phagophore closure via CHMP2A, protecting cells and reducing injury. This reveals a novel role for MLKL in cell survival.
Area of Science:
- Cell Biology
- Immunology
- Pulmonary Medicine
Background:
- The role of autophagy in LPS-induced acute lung injury (ALI) within pulmonary microvascular endothelial cells (PMVECs) remains debated.
- Mixed lineage kinase domain-like pseudokinase (MLKL) is known for necroptosis but may also promote cell survival by facilitating autophagic flux under starvation conditions.
Purpose of the Study:
- To investigate the role of MLKL in autophagy within PMVECs during LPS-induced ALI.
- To elucidate the mechanism by which MLKL influences autophagic flux and its impact on ALI.
Main Methods:
- Utilized a mouse PMVEC model and an LPS-induced ALI mouse model.
- Assessed autophagic flux markers (LC3-II, SQSTM1/p62) and autophagosome-lysosome fusion using pHluorin-mKate2-human LC3 assay.
- Investigated MLKL's role using MLKL inhibitors and siRNA, and its interaction with CHMP2A.
Main Results:
- Short-term LPS stimulation (1-3h) induced MLKL phosphorylation (p-MLKL) and accelerated autophagic flux in PMVECs.
- MLKL inhibition or knockdown reversed these effects, increasing vascular permeability and worsening ALI in mice.
- p-MLKL formed multimers to facilitate phagophore closure, a process involving CHMP2A, which was upregulated by LPS.
- CHMP2A knockdown inhibited LPS-induced autophagic flux, while its overexpression enhanced flux and attenuated ALI.
Conclusions:
- Activated MLKL by short-term LPS exposure accelerates autophagic flux in PMVECs by promoting phagophore closure through CHMP2A.
- This mechanism protects PMVECs and alleviates LPS-induced ALI, suggesting a novel cytoprotective role for MLKL in this context.
