LPS or ethanol triggers clathrin- and rafts/caveolae-dependent endocytosis of TLR4 in cortical astrocytes
Article first published online: 5 JAN 2014
© 2013 International Society for Neurochemistry
Journal of Neurochemistry
Volume 129, Issue 3, pages 448–462, May 2014
How to Cite
J. Neurochem. (2014) 129, 448–462.
- Issue published online: 19 APR 2014
- Article first published online: 5 JAN 2014
- Accepted manuscript online: 18 DEC 2013 02:49AM EST
- Manuscript Revised: 12 DEC 2013
- Manuscript Accepted: 12 DEC 2013
- Manuscript Received: 16 JUL 2013
- Spanish Ministries of Science and Innovation. Grant Number: SAF-2009-07503
- Spanish Ministries of Economy and Competitivity. Grant Number: SAF-2012-33747
- Spanish Ministries of Health
- Carlos III Institute
- PNSD. Grant Number: 2010I037
- Generalitat Valenciana-Conselleria de Educación. Grant Number: PROMETEO/2009/072
- M. Lautenschaläger Award
- innate immune response;
- TLR4 signalling
Toll-like receptor 4 (TLR4) activation and signalling in glial cells play critical roles in neurological disorders and in alcohol-induced 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-β (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 TLR4GFP, 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.
The results demonstrate that ethanol or lipopolysaccharide (LPS) triggers toll-like receptor 4 (TLR4) endocytosis by caveolae and clathrin-dependent pathways in astrocytes. We proposed that while clathrin is the protein responsible for TLR4 internalization, caveolin-1/lipid rafts membrane microdomains are required for TLR4 signaling. The results provide new insights into the effects of ethanol on TLR4 signalling in astrocytes, events that may underlie neuroinflammation.