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, 15 (5), 753-770

TLR4 (Toll-Like Receptor 4) Activation Suppresses Autophagy Through Inhibition of FOXO3 and Impairs Phagocytic Capacity of Microglia

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TLR4 (Toll-Like Receptor 4) Activation Suppresses Autophagy Through Inhibition of FOXO3 and Impairs Phagocytic Capacity of Microglia

Ji-Won Lee et al. Autophagy.

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.

Figures

Figure 1.
Figure 1.
Autophagy is suppressed by LPS in microglia. (a) Analysis of autophagic flux in primary microglia after treatment with the indicated dosages of LPS for 24 h. Graph, quantification of MAP1LC3B-II after normalization to ACTB (n = 3). (b) Analysis of autophagic flux in BMDMs after treatment with the indicated dosages of LPS for 24 h. Graph, quantification of MAP1LC3-II after normalization to ACTB (n = 3). (c) Analysis of autophagic flux in BV-2 microglial cells after treatment with LPS (1 μg/mL) for 6 or 24 h. (d) Analysis of autophagic flux in Raw264.7 macrophages after treatment with LPS (1 μg/mL) for 6 and 24 h. (e) Quantification of ZFYVE1 puncta following LPS treatment in BV-2 cells (n > 30 cells per condition). (f) Quantification of ZFYVE1 puncta following LPS treatment in Raw264.7 cells (n > 40 cells per condition). (g) Quantification of GFP-MAP1LC3B puncta in BV-2 cells 24 h after LPS (1 μg/mL) treatment (n > 30 cells per condition). (h) Time course analysis of MAP1LC3B-II level in primary microglia. Cells were treated with LPS (1 μg/mL) for 1, 2, 6, 12, or 24 h. Graph, quantification of MAP1LC3B-II after normalization to ACTB (n = 4). In all experiments where BafA1 was used, BafA1 (20 nM) was added 2 h before sampling. Scale bars: 10 μm. All data are mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the control (Con) unless indicated otherwise.
Figure 2.
Figure 2.
LPS decreases the expression of Atg genes in primary microglia. (a) Time course analysis of the mRNA levels of Atg genes by qRT-PCR following LPS (1 μg/mL) treatment (n = 3). (b) Comparison of relative mRNA levels of Atg genes between primary microglia (n = 5) and BMDMs (n = 4) 24 h after LPS (1 μg/mL) treatment. (c) Comparison of relative mRNA levels of Atg genes, between microglia and peritoneal macrophages acutely isolated from the same LPS-injected mice (5 mg/kg, intraperitoneally), 24 h post-injection (n = 3). In all experiments, mRNA levels were normalized to Actb. All data are mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the control (Con).
Figure 3.
Figure 3.
Suppression of autophagy by LPS is TLR4-dependent. (a) The genotyping of tlr4−/− mice by RT-PCR. (b) Autophagic flux analysis in tlr4−/− microglia after treatment with LPS (1 μg/mL) for 24 h. BafA1 (20 nM) was added 2 h before sampling. (c) Analysis of the mRNA levels of Atg genes in tlr4−/− microglia by qRT-PCR 24 h after LPS (1 μg/mL) treatment (n = 4).
Figure 4.
Figure 4.
Bioinformatics analysis of RNA sequencing data for microglia activated by LPS. LogFC (fold change) values from 0 to 1 (red) indicate upregulation of gene expression and values below 0 (green) indicate downregulation.
Figure 5.
Figure 5.
LPS reduces the synthesis of PtdIns and PtdIns3P in microglia. (a, b) LC/MS metabolomics analysis of PtdIns 38:4 amounts in primary microglia (a) and BMDMs (b) 16 h after LPS (1 μg/mL) treatment. (c, d) ELISA analyses of PtdIns3P amounts in BV-2 (c) and Raw264.7 cells (d) 16 h after LPS (1 μg/mL) treatment. All data are mean ± SEM. **P < 0.01 and ***P < 0.001 compared to the control (Con).
Figure 6.
Figure 6.
LPS suppresses autophagy via the PI3K-AKT1 signaling pathway in primary microglia. (a) Western blotting analyses of phosphorylation of AKT1 (T308, S473), MTOR (S2448), MAPK1/3 (T203/Y205), and RPS6KB1 (T389) following LPS treatment (1 μg/mL) in primary microglia. (b) Western blotting analysis of MAP1LC3B-II level after inhibition of MAPK1/3 (with PD98059), MTOR (with rapamycin or Torin-1) or PI3K (with LY294002; LY or wortmannin; Wort) in primary microglia treated with LPS (1 μg/mL) for 12 h. (c) Analysis of autophagic flux after inhibition of PI3K with LY (20 μM) in primary microglia treated with LPS (1 μg/mL) for 12 h. (d) Quantitative analysis of MAP1LC3B-II levels in primary microglia. In all experiments, the blots shown are representative of at least 3 experiments with similar results. All inhibitors were added 1 h prior to LPS treatment. All data are mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the control (Con) unless indicated otherwise. #P < 0.05 and ##P < 0.01 compared to the control treated with BafA1 only.
Figure 7.
Figure 7.
Phosphorylation of FOXO3 by PI3K-AKT1 suppresses autophagy in primary microglia. (a) Time course analysis of phosphorylation of FOXO3 (S253) in primary microglia following LPS treatment (1 μg/mL). (b) Phosphorylation of FOXO3 (S253) in primary microglia after LY294002 (LY, 20 μM) treatment at 6 and 12 h following LPS (1 μg/mL) treatment. The blots shown are representative of 3 experiments with similar results. (c, d) Analyses of nuclear localization of FOXO3 in primary microglia (n > 30 cells per condition) (c) and BMDMs (n = 30 cells per condition) (d) 12 h after LPS (1 μg/mL) treatment with 1 h pretreatment of LY (20 μM). Endogenous FOXO3 (green) was visualized by immunocytochemistry. Scale bars: 10 μm. Graphs, quantitative analysis of FOXO3 nuclear localization. (e) Analysis of the mRNA levels of Atg genes in primary microglia treated with LPS (1 μg/mL) for 12 h with 1 h pretreatment of LY (20 μM). Relative mRNA levels were quantified by qRT-PCR and normalized to ACTB (n = 3). (f) Time-course analyses of phosphorylation of FOXO3 (S253) and AKT1 (T308, S473) in BMDMs following LPS treatment (1 μg/mL). All data are mean ± SEM. n.s., not significant. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the control (Con) unless indicated otherwise. #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to the LPS-treated cells.
Figure 8.
Figure 8.
Overexpression of constitutively active FOXO3 (FOXO3-CA) increases the number of MAP1LC3B puncta under basal conditions and following LPS treatment. (a) Nuclear localization of FOXO3-CA-GFP in BV-2 cells. (b) Increased number of MAP1LC3B dots in FOXO3-CA-GFP-transfected BV-2 cells compared to control cells transfected with the GFP-only control vector. LPS (1 μg/mL) was added for 12 h and MAP1LC3B puncta (red) were visualized by immunocytochemistry with an antibody against endogenous MAP1LC3B. Scale bar: 10 μm. Graph, quantification of the puncta from at least 30 cells per condition, using an LSM700 confocal microscope (Carl Zeiss). BafA1 (20 nM) was added 2 h before sampling. All data are mean ± SEM. n.s., not significant. *P < 0.05 compared to the control in the absence of BafA1. #P < 0.05 and ###P < 0.001 compared to the control in the presence of BafA1.
Figure 9.
Figure 9.
LPS suppresses MAP1LC3B-associated phagocytosis (LAP) and Aβ degradation via the PI3K-AKT1 signaling pathway in microglia. (a) Analysis of LAP in primary microgliawhere stimulated with LPS (1 μg/mL) for 12 h and then incubated with zymosan for another 2 h. LY (20 μM) was added 1 h prior to LPS. Immunocytochemistry images show MAP1LC3B (green), zymosan (red), and nucleus (blue). Graph, quantitative analysis of MAP1LC3B-positive phagosomes from 3 independent experiments (n = 45 to 113 cells). (b) Analysis of LAP in Atg7 knockdown BV-2 cells (n = 35 to 63 cells). BV-2 cells were infected with lentivirus expressing Atg7-targeting (shAtg7) or control (shCon) shRNA for stable knockdown. Experimental conditions were the same as (a). (c) Analysis of PLA signal between MAP1LC3B and Aβ in primary microglia after treatment with LPS (1 µg/mL) for 12 h. LY (20 µM) was added 1 h prior to LPS treatment. Graph, quantification of PLA signal (n = 82 to 135 cells). (d) Measurement of Aβ degradation in LPS-stimulated primary microglia. FITC-conjugated Aβ1−42 fibrils were added to the cells and the fluorescence intensity of the remaining FITC-Aβ1−42 was measured using a fluorescence microscope 6, 12, or 24 h later. Graph, quantification of Aβ fibril intensities from 3 independent experiments (n = 82 to 106 cells). *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the control (Con) unless indicated otherwise.

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Grant support

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.
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