LPS or ethanol triggers clathrin‐ and rafts/caveolae‐dependent endocytosis of TLR4 in cortical astrocytes

Abstract

Toll-like receptor 4 (TLR4) activation and signalling in glial cells play critical roles in neurological disorders and in alcoholinduced brain damage. TLR4 endocytosis upon lipopolysaccharide (LPS) stimulation regulates which signalling pathway is activated, the MyD88-dependent or the TIR-domain-containing adapter-inducing interferon-b (TRIF)-dependent pathway. However, it remains elusive whether ethanol-induced TLR4 signalling is associated with receptor internalization and trafficking, and which endocytic pathway(s) are used in cortical astrocytes. Using the adenoviral over-expression of TLR4 GFP , confocal microscopy and the imagestream technique, we show that upon ethanol or LPS stimulation, TLR4 co-localizes with markers of the clathrin and caveolin endocytic pathways, and that this endocytosis is dependent on dynamin. Using chlorpromazin and filipin as inhibitors of the clathrin and rafts/ caveolae endocytic pathways, respectively, we demostrate that TRIF-dependent signalling relies on an intact clathrin pathway, whereas disruption of rafts/caveolae inhibits the MyD88-and TRIF-dependent signalling pathways. Immunofluorescence studies also suggest that lipid rafts and clathrin cooperate for appropriate TLR4 internalization. We also show that ethanol can trigger similar endocytic pathways as LPS does, although ethanol delays clathrin internalization and alters TLR4 vesicular trafficking. Our results provide new insights into the effects of ethanol or LPS on TLR4 signalling in cortical astrocytes, events that may underlie neuroinflammation and brain damage.

Induction of interferon-induced transmembrane protein 3 gene expression by lipopolysaccharide in astrocytes

“…In contrast, Lee et al reported that stimulation of astrocytes with LPS led to the induction of IFN-β through activation of NF-κB (Lee et al, 2005). A recent study also showed that IFN-β mRNA level was significantly increased 3 h after LPS treatment in astrocytes (Pascual-Lucas et al, 2014). In addition, LPS was found to induce IFN-β through p38 MAPK and NF-κB pathways in U373MG human astrocytoma cells (Imaizumi et al, 2013).…”

Molecular mechanisms underlying alcohol-drinking behaviours

“…Cholesterol content varies across brain regions, and thus alcohol may regulate a broad range of cell-surface receptor-induced signalling in a brain region-specific manner by changing the structure, fluidity and composition of membrane lipids, including cholesterol-enriched rafts. Several studies have started to address this possibility in cells 184, 185 and in vivo 30, 186, 187 . However, data from animal models of alcohol consumption are sparse 30 , in part owing to technical limitations.…”

Neuroimmune Pathways in Alcohol Consumption: Evidence from Behavioral and Genetic Studies in Rodents and Humans

“…2.1). Ethanol and LPS can promote TLR4 endocytosis through clathrin- and caveolae-dependent pathways, and in cortical astrocytes, TRIF-dependent signaling relies on the clathrin pathway, while disruption of rafts/caveolae inhibits both the MyD88- and TRIF-dependent signaling pathways (Pascual-Lucas, Fernandez-Lizarbe, Montesinos, & Guerri, 2014). …”