TLR4 (toll-like receptor 4) activation suppresses autophagy through inhibition of FOXO3 and impairs phagocytic capacity of microglia

Autophagy. 2019 May;15(5):753-770. doi: 10.1080/15548627.2018.1556946. Epub 2018 Dec 13.

Abstract

Macroautophagy/autophagy is a lysosome-dependent catabolic process for the turnover of proteins and organelles in eukaryotes. Autophagy plays an important role in immunity and inflammation, as well as metabolism and cell survival. Diverse immune and inflammatory signals induce autophagy in macrophages through pattern recognition receptors, such as toll-like receptors (TLRs). However, the physiological role of autophagy and its signaling mechanisms in microglia remain poorly understood. Microglia are phagocytic immune cells that are resident in the central nervous system and share many characteristics with macrophages. Here, we show that autophagic flux and expression of autophagy-related (Atg) genes in microglia are significantly suppressed upon TLR4 activation by lipopolysaccharide (LPS), in contrast to their stimulation by LPS in macrophages. Metabolomics analysis of the levels of phosphatidylinositol (PtdIns) and its 3-phosphorylated form, PtdIns3P, in combination with bioinformatics prediction, revealed an LPS-induced reduction in the synthesis of PtdIns and PtdIns3P in microglia but not macrophages. Interestingly, inhibition of PI3K, but not MTOR or MAPK1/3, restored autophagic flux with concomitant dephosphorylation and nuclear translocation of FOXO3. A constitutively active form of FOXO3 also induced autophagy, suggesting FOXO3 as a downstream target of the PI3K pathway for autophagy inhibition. LPS treatment impaired phagocytic capacity of microglia, including MAP1LC3B/LC3-associated phagocytosis (LAP) and amyloid β (Aβ) clearance. PI3K inhibition restored LAP and degradation capacity of microglia against Aβ. These findings suggest a unique mechanism for the regulation of microglial autophagy and point to the PI3K-FOXO3 pathway as a potential therapeutic target to regulate microglial function in brain disorders. Abbreviations: Atg: autophagy-related gene; Aβ: amyloid-β; BafA1: bafilomycin A1; BECN1: beclin 1, autophagy related; BMDM: bone marrow-derived macrophage; CA: constitutively active; CNS: central nervous system; ZFYVE1/DFCP1: zinc finger, FYVE domain containing 1; FOXO: forkhead box O; ELISA:enzyme-linked immunosorbent assay; HBSS: Hanks balanced salt solution; LAP: LC3-associated phagocytosis; MAP1LC3B: microtubule-associated protein 1 light chain 3; LPS: lipopolysaccharide; LY: LY294002; MTOR: mechanistic target of rapamycin kinase; Pam3CSK4: N-palmitoyl-S-dipalmitoylglyceryl Cys-Ser-(Lys)4; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PLA: proximity ligation assay; Poly(I:C): polyinosinic-polycytidylic acid; qRT-PCR: quantitative real-time polymerase chain reaction; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; TLR: Toll-like receptor; TNF: tumor necrosis factor; TFEB: transcription factor EB; TSPO: translocator protein.

Keywords: Amyloid; FOXO3; LC3-associated phagocytosis; PI3K; PtdIns3K; microglia.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amyloid beta-Peptides / pharmacology
  • Animals
  • Animals, Newborn
  • Autophagy / drug effects
  • Autophagy / genetics*
  • Cells, Cultured
  • Down-Regulation / drug effects
  • Down-Regulation / genetics
  • Forkhead Box Protein O3 / genetics*
  • Forkhead Box Protein O3 / metabolism
  • Lipopolysaccharides / pharmacology
  • Macrophages / drug effects
  • Macrophages / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Microglia / physiology*
  • Phagocytosis / genetics*
  • Phosphatidylinositol 3-Kinases / metabolism
  • RAW 264.7 Cells
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / metabolism
  • Toll-Like Receptor 4 / physiology*

Substances

  • Amyloid beta-Peptides
  • Forkhead Box Protein O3
  • FoxO3 protein, mouse
  • Lipopolysaccharides
  • Tlr4 protein, mouse
  • Toll-Like Receptor 4

Grants and funding

This work was supported by the National Research Foundation of Korea (NRF) grants (2013M3C7A1056099, 2016M3C7A1905074, 2017R1A2B4004289 and 2017R1A2B2004483) and the DGIST Convergence Science Center Program (18-BD-04) of the Ministry of Science and ICT of Korea.