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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 ? (A?) clearance. PI3K inhibition restored LAP and degradation capacity of microglia against A?. 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; A?: amyloid-?; 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.

SUBMITTER: Lee JW

PROVIDER: S-EPMC6526818 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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

Lee Ji-Won JW Nam Hyeri H Kim Leah Eunjung LE Jeon Yoonjeong Y Min Hyunjung H Ha Shinwon S Lee Younghwan Y Kim Seon-Young SY Lee Sung Joong SJ Kim Eun-Kyoung EK Yu Seong-Woon SW

Autophagy 20181213 5

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 ...[more]

PMID: 30523761

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Project description:Background Microglia plays crucial roles in Alzheimer’s disease (AD) development. Triggering receptor expressed on myeloid cells 2 (TREM2) in association with DAP12 mediates signaling affecting microglia function. Here we study the negative regulation of TREM2 functions by leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), an inhibitory receptor bearing ITIM motifs. Methods To specifically interrogate LILRB2-ligand (oAβ and PS) interactions and microglia functions, we generated potent antagonistic LILRB2 antibodies with sub-nanomolar level activities. The biological effects of LILRB2 antagonist antibody (Ab29) were studied in human induced pluripotent stem cell (iPSC)–derived microglia (hMGLs) for migration, oAβ phagocytosis, and upregulation of inflammatory cytokines. Effects of the LILRB2 antagonist antibody on microglial responses to amyloid plaques were further studied in vivo using stereotaxic grafted microglia in 5XFAD mice. Results We confirmed the expression of both LILRB2 and TREM2 in human brain microglia using immunofluorescence. Upon co-ligation of the LILRB2 and TREM2 by shared ligands oAβ or PS, TREM2 signaling was significantly inhibited. We identified a monoclonal antibody (Ab29) that blocks LILRB2/ligand interactions and prevents TREM2 signaling inhibition mediated by LILRB2. Further, Ab29 enhanced microglia phagocytosis, TREM2 signaling, migration, and cytokine responses to the oAβ-lipoprotein complex in hMGL and microglia cell line HMC3. In vivo studies showed significantly enhanced clustering of microglia around plaques with a prominent increase in microglial amyloid plaque phagocytosis when 5XFAD mice were treated with Ab29. Conclusions This study revealed for the first time the molecular mechanisms of LILRB2-mediated inhibition of TREM2 signaling in microglia and demonstrated a novel approach of enhancing TREM2-mediated microglia functions by blocking LILRB2-ligand interactions. Translationally, a LILRB2 antagonist antibody completely rescued the inhibition of TREM2 signaling by LILRB2, suggesting a novel therapeutic strategy for improving microglial functions. Supplementary Information The online version contains supplementary material available at 10.1186/s13024-022-00550-y.

Project description:Prion diseases are neurodegenerative disorders characterized by the accumulation of misfolded prion protein, spongiform changes in the brain, and brain inflammation as a result of the wide-spread activation of microglia. Autophagy is a highly conserved catabolic process for the clearance of cytoplasmic components, including protein aggregates and damaged organelles; this process also eliminates pathological PrPSc as it accumulates during prion infection. The NALP3 inflammasome is a multiprotein complex that is a component of the innate immune system and is responsible for the release of pro-inflammatory cytokines. Our previous study showed that the neurotoxic prion peptide PrP106-126 induces NALP3 inflammasome activation and subsequent IL-1β release in microglia. Autophagy is involved in the regulation of the immune responses and inflammation in many diseases including neurodegenerative diseases. However, the relationship between autophagy and NALP3 inflammasome in prion diseases has not been investigated. In this study, we demonstrated that the processing and release of mature IL-1β is significantly enhanced by the inhibition of autophagy. Conversely, gene-silencing of the NALP3 inflammasome promotes autophagy. Suppression of TRIF or TLR4 by siRNA attenuated PrP106-126-induced autophagy, which is indicating that the TLR4-TRIF signaling pathway is involved in PrP106-26-induced autophagy. Caspase 1 directly cleaved TRIF to diminish TLR-4-TRIF mediated autophagy. Our findings suggest that the inhibition of autophagy by NALP3 inflammasome is probably mediated by activated Caspase-1-induced TRIF cleavage. This is the first study reporting that the NALP3 inflammasome complex negatively regulates autophagy in response to PrP106-126 stimulation in microglia, and partly explains the mechanism of autophagy inhibition by Caspase-1 in PrP106-126-induced BV2 cell activation. Our findings suggest that autophagy up-regulation and inhibition of Caspase-1 may protect against prion-induced neuroinflammation and accelerate misfolded protein degradation and are potential therapeutic approaches for prion diseases.

Dataset Information

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

Publications

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

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