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

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 ß (Aß) clearance. PI3K inhibition restored LAP and degradation capacity of microglia against Aß. 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; Aß amyloid-ß; 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; ELISAenzyme-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(IC) 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.