Microglial cells are the primary immune effector cells in the brain and play a pivotal role in the neuroinflammatory processes associated with a variety of neurological and pathological disorders. Alcohol consumption induces brain damage, although the neuropathological processes are poorly understood. We previously suggested that ethanol promotes inflammatory processes in the brain, up-regulating inflammatory mediators and signaling pathways associated with IL-1RI/TLR4 receptors. In the present study we investigate whether ethanol induces microglia activation by stimulating TLR4 response and whether this response causes neuronal death and contributes to ethanol-induced neuroinflammatory damage. We demonstrate that ethanol activates microglía and stimulates NF-κB, MAPKs, and MyD88-independent (IFN regulatory factor-3, IFN-β) pathways to trigger the production of inflammatory mediators, causing neuronal death. The inflammatory response induced by ethanol is completely abrogated in microglia of TLR4-deficient mice (TLR4−/−), thus supporting the role of these receptors in microglia activation and neuronal death. In accord with the in vitro findings, acute ethanol administration induces microglia activation (CD11b+ cells) in cerebral cortex of TLR4+/+ mice, but not in TLR4−/− mice. Taken together, our results not only provide the first evidence of the critical role of the TLR4 response in the ethanol-induced microglia activation, but also new insight into the basic mechanisms participating in ethanol-induced neuroinflammatory damage.

Alcohol consumption can induce brain damage, demyelination, and neuronal death, although the mechanisms are poorly understood. Toll-like receptors are sensors of the innate immune system and their activation induces inflammatory processes. We have reported that ethanol activates and recruits Toll-like receptor (TLR)4 receptors within the lipid rafts of glial cells, triggering the production of inflammatory mediators and causing neuroinflammation. Since TLR2 can also participate in the glial response and in the neuroinflammation, we investigate the effects of ethanol on TLR4/TLR2 responses. Here, we demonstrate that ethanol up-regulates TLR4 and TLR2 expression in microglial cells, inducing the production of inflammatory mediators which triggers reactive oxygen species generation and neuronal apoptosis. Ethanol also promotes TLR4/TLR2 recruitment into lipid rafts-caveolae, mimicking their activation by their ligands, lipopolysaccharide, and lipoteichoic acid (LTA). Immunoprecipitation and confocal microscopy studies reveal that ethanol induces a physical association between TLR2 and TLR4 receptors, suggesting the formation of heterodimers. Using microglia from either TLR2 or TLR4 knockout mice, we show that TLR2 potentiates the effects of ethanol on the TLR4 response reflected by the activation of MAPKs and inducible NO synthase. In summary, we provide evidence for a mechanism by which ethanol triggers TLR4/ TLR2 association contributing to the neuroinflammation and neurodegeneration associated with alcohol abuse.

We have recently reported that ethanol‐induced inflammatory processes in the brain and glial cells are mediated via the activation of interleukin‐1 beta receptor type I (IL‐1RI)/toll‐like receptor type 4 (TLR4) signalling. The mechanism(s) by which ethanol activates these receptors in astroglial cells remains unknown. Recently, plasma membrane microdomains, lipid rafts, have been identified as platforms for receptor signalling and, in astrocytes, rafts/caveolae constitute an important integrators of signal events and trafficking. Here we show that stimulation of astrocytes with IL‐1β, lipopolysaccharide or ethanol (10 and 50 mM), triggers the translocation of IL‐1RI and/or TLR4 into lipid rafts caveolae‐enriched fractions, promoting the recruitment of signalling molecules (phospho‐IL‐1R‐associated kinase and phospho‐extracellular regulated‐kinase) into these microdomains. With confocal microscopy, we further demonstrate that IL‐1RI is internalized by caveolar endocytosis via enlarged caveosomes organelles upon IL‐1β or ethanol treatment, which sorted their IL‐1RI cargo into the endoplasmic reticulum–Golgi compartment and into the nucleus of astrocytes. In short, our findings demonstrate that rafts/caveolae are critical for IL‐1RI and TLR4 signalling in astrocytes, and reveal a novel mechanism by which ethanol, by interacting with lipid rafts caveolae, promotes IL‐1RI and TLR4 receptors recruitment, triggering their endocytosis via caveosomes and downstream signalling stimulation. These results suggest that TLRs receptors are important targets of ethanol‐induced inflammatory damage in the brain.

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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.