Lipid rafts regulate ethanol-induced activation of TLR4 signaling in murine macrophages.
Toll-like receptors (TLRs) response is critical in innate resistance to infection. Alcohol consumption has been shown to suppress the inflammatory response mediated through TLR4, down regulating the production of inflammatory cytokines. We recently reported that low concentrations of ethanol activate TLR4 signaling in astrocytes and triggers neuroinflammation. Because macrophages are important cells in innate immunity, we investigate whether low concentrations of ethanol could stimulate the TLR4 signaling response in murine RAW 264.7 macrophages, and the mechanism involved in the ethanol-induced TLR4 activation. Our results show that while ethanol, at high concentrations (100mM) or in the presence of the LPS, suppresses the TLR4 response, low to moderate levels (10-50mM) activate the TLR4 response and triggers the stimulation of the mitogen-activated protein kinases (MAPKs) and the transcription factor NF-kappaB pathways, leading to the production of nitric oxide (NO) and inflammatory cytokines. Pre-treatment with anti-TLR4 Abs abolishes the effects of ethanol on the production of cytokines. We also present evidence that stimulation with either ethanol or LPS induces translocation and clustering of TLR4 and signaling molecules (IRAK and MAPKs) into lipid rafts. Treatment with either streptolysin-O or saponin, lipid rafts disrupting agents, abolishes the ethanol-induced activation of the TLR4/IL-1RI signaling pathway. In summary, the present results demonstrate that low to moderate concentrations of ethanol are capable of stimulating TLR4/IL-1RI response, and provide evidence of a novel mechanism by which ethanol, through its interaction with membrane rafts, can promote TLR4/IL-1RI recruitment and signaling.
Department of Cellular Pathology, Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16, 46013 Valencia, Spain.
Sara
S
Fernandez-Lizarbe
Maria
M
Pascual
M Soledad
MS
Gascon
Ana
A
Blanco
Consuelo
C
Guerri
D000818:Animals; D002460:Cell Line; D051546:Cyclooxygenase 2 / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism; D016753:Interleukin-10 / Q000096:biosynthesis; D053583:Interleukin-1beta / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D021962:Membrane Microdomains / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D017472:Receptors, Interleukin-1 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D017472:Receptors, Interleukin-1; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D000431:Ethanol; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D053592:Interleukin-1 Receptor-Associated Kinases; D048049:Extracellular Signal-Regulated MAP Kinases