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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.
Microglial cells are considered the resident macrophage-like population within the CNS and they are the prime component of the brain immune system (Streit and Xue 2009). Microglia detects danger signals through various receptors, including Toll-like receptors (TLRs), which play a crucial role in innate immunity by recognizing microbial pathogens and ligands from injured cells (Lehnardt 2010). Activation of TLRs stimulates glial cells to prevent infections or tissue damage. However, a heightened inflammatory state can lead to unnecessary damage and neuroinflammation (Rivest 2009; Amor et al. 2010). Activation of TLR2 and TLR4 is involved in neurodegeneration. Mice deficient in these TLRs exhibit reduced levels of pro-inflammatory cytokines and milder clinical disease following traumatic brain injury (Koedel et al. 2007; Ziegler et al. 2011). The levels of TLR4 and TLR2 have been reported to increase in Parkinson's disease, stroke, and amyotrophic lateral sclerosis (Letiembre et al. 2009; Okun et al. 2009).
Alcohol is a neurotoxic compound and its abuse can cause brain damage (Pfefferbaum 2004; Harper and Matsumoto 2005). Nevertheless, the mechanism by which ethanol induces neurodegeneration remains largely elusive. Previous work done in our laboratory demonstrates the critical role of TLR4 in alcohol-induced microglia (Fernandez-Lizarbe et al. 2009) and astroglial activation (Alfonso-Loeches et al. 2010), demyelination, and neuronal damage (Fernandez-Lizarbe et al. 2009; Alfonso-Loeches et al. 2012), indicating that activation of the TLR4 response by ethanol can be an important mechanism of ethanol-induced neuroinflammation and neurodegeneration. We also demonstrate that ethanol is capable of activating TLR4 signaling by promoting translocation and the clustering of TLR4 and signaling molecules (IL-1 receptor-associated kinase, myeloid differentiation primary response protein 88, extracellular-signal-regulated kinase) into the membrane microdomains lipid rafts (Blanco et al. 2008; Fernandez-Lizarbe et al. 2008). Accordingly, different studies have demonstrated that, upon activation, TLRs are recruited into lipid rafts microdomains, acting as signaling platforms for several TLRs and leading to innate immune activation (Triantafilou et al. 2011).
Among TLRs, TLR2 seems to be the most promiscuous TLR receptor capable of recognizing the most diverse set of pathogens. TLR2 complexes with TLR1 or TLR6 are involved in the recognition of bacterial lipoproteins (Akira and Takeda 2004; Gay and Gangloff 2007). TLR2 can also interact with other molecules such as CD36 (Triantafilou et al. 2006) or CD14 (Yang et al. 1999; Flo et al. 2002) and can induce multimerization in response to different microbial ligands (Triantafilou et al. 2006).
We propose that ethanol, through its interaction with lipid rafts, can recruit several receptors like TLR4 and TLR2, triggering innate immune activation and leading to neuroinflammation. Here, we report that ethanol not only up-regulates TLR4 and TLR2 receptors in microglial cells but also promotes the recruitment and association of both receptors into rafts-caveolae, leading to TLRs signaling production and cytokine release. These events might contribute to the neuroinflammation and neuronal cell death associated with alcohol abuse.
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Glial cells and TLRs are vital players in CNS immune responses. TLRs activation is a major inducer of neuroinflammation, contributing to both infectious and non-infectious CNS injury. Indeed, several studies have demonstrated the role of TLR2 and TLR4 in neurodegeneration (Lehnardt et al. 2008; Kawai and Akira 2010) and neuroinflammation (Babcock et al. 2006; Buchanan et al. 2010). We previously demonstrated that ethanol induces TLR4 activation in glial cells (Blanco et al. 2005; Fernandez-Lizarbe et al. 2009) causing neuroinflammation and brain damage (Alfonso-Loeches et al. 2010). This study extends previous findings by showing that ethanol, by interacting with LR membrane microdomains, can not only recruit TLR4 and TLR2 receptors within the LR-caveolae in microglial cells, but also can promote a physical association of these receptors by triggering TLRs signaling and leading to the induction of pro-inflammatory cytokine formation. These effects could contribute to ethanol-induced neuroinflammation and neuronal damage.
Different studies demonstrated that upon activation, TLRs are recruited with other receptors into membrane microdomains rich in sphingolipid and cholesterol, called lipid rafts (LR), and that these microdomains stabilize TLRs and form signaling platforms, which transduce signals leading to innate immune activation (Triantafilou et al. 2011). This combinational use of cell surface receptors determines the character of the immune response. Our previous studies demonstrate that while low/moderate ethanol concentrations (10-50 mM) induce clustering and the recruitment of TLR4 into LR-caveolae (Blanco et al. 2008; Fernandez-Lizarbe et al. 2008), high ethanol concentrations (100 mM) inhibit TLR4 signaling through the disruption of the LR and, consequently, receptor clustering (Fernandez-Lizarbe et al. 2008). Here, we further report that ethanol triggers the recruitment of TLR4 and TLR2 into LR-caveolae, leading to TLRs signaling, and acts as their natural ligands, such as LPS for TLR4 and LTA for TLR2. Significantly, we found that the recruitment of both receptors into the LR-caveolae promotes TLR4 and TLR2 interaction. This notion is supported by the results revealing that upon ethanol treatment, TLR4 and TLR2 co-localize within the rafts-caveolae and physically interact, as demonstrated by the co-immunoprecipitation experiments and the FRET studies. Disruption of LR with filipin abolishes ethanol-induced TLR4 and TLR2 recruitment.
Although the molecular mechanisms of the TLR4 and TLR2 interaction are not yet clear, several reports have shown that TLR2 can interact with other TLRs and that both TLR4 and TLR2 form clusters composed for several receptors (Triantafilou et al. 2006). For instance, bacterial lipopeptides through their acyl chains can directly crosslink TLR2 with TLR6 or TLR1 (Jin et al. 2007). TLR2 can also form complexes with various molecules, such as CD14 (Brightbill et al. 1999) or CD36 (Hoebe et al. 2005; Triantafilou et al. 2006). H pylori, P gingivalis, and LPS can induce receptor clusters comprising TLR2, TLR1, CD36, and CD11b/CD18 on vascular endothelial cells (Triantafilou et al. 2007), and CD14 has been implicated in facilitating TLR1/2-mediated responses to bacterial lipopeptides by enhancing the physical proximity of the ligand to TLR1/2 heterodimers (Manukyan et al. 2005; Nakata et al. 2006). An extensive hydrogen-bonding network, as well as hydrophobic interactions, between TLR2 and TLR4 could further stabilize the heterodimer, as demonstrated with the TLR2 and TLR1 receptors (Jin et al. 2007). Therefore, we propose that, by interacting with membrane lipid rafts, alcohol can induce TLR4/TLR2 recruitment within lipid rafts-caveolae, and can promote the formation of the TLR4/TLR2 heterodimer, causing the interaction of their toll-interleukin 1 receptor domains and initiating the cascade to endorse microglia activation.
Emerging evidence indicates the role of TLRs and other immune receptors in neural damage associated with various neurodegenerative diseases (Okun et al. 2009, 2011). Here, we demonstrate that ethanol induces microglia activation, leading to the release of pro-inflammatory cytokines, promotes ROS generation in neurons, which in turn causes oxidative stress and neuronal apoptosis, and that these effects are mediated by TLR4 and TLR2. These results extend previous findings demonstrating the potential role of TLR4 in ethanol-induced microglia activation and neuronal apoptosis (Fernandez-Lizarbe et al. 2009), and suggest that both receptors participate in ethanol-induced neuroinflammation and neural damage.
To support our hypothesis, ethanol abuse not only induces microglial markers and chemokine-MCP1 (He and Crews 2008) but also up-regulates the protein expression of the plasma membrane receptors TLR2 and TLR4 and the intracellular receptors TLR3 in post-mortem human alcoholic brain and in mice brain. Indeed, these effects have been correlated with lifetime alcohol consumption (Crews et al. 2013). These results suggest that chronic alcohol intake triggers brain neuroimmune activation through TLRs signaling (Crews et al. 2013). Alcohol consumption also increases the gene expression of TLR2 and TLR4 in the liver (Oliva et al. 2011), while it diminishes the ability of splenic macrophages to respond to TLR4 and TLR2 ligands (Goral and Kovacs 2005). Although the effects of ethanol on TLR4/TLR2 depend on the stimuli, ethanol concentration, and cell type or tissue analyzed, our results suggest that, in the brain, alcohol triggers neuroimmune activation by stimulating microglial cells and TLRs signaling response. Ethanol-induced neuroimmune signaling has also been linked with alcohol-induced drinking behavior, anxiety, and depression-like behaviors (Pascual et al. 2011; Crews et al. 2013).
The activation of TLR4 and TLR2 also contributes to the ischemic brain injury (Ziegler et al. 2007), experimental autoimmune encephalomyelitis (Zekki et al. 2002), or Alzheimer's disease (Liu et al. 2012) and the deficiency of these receptors reduces the expression of proinflammatory genes and decreased pain hypersensitivity after spinal nerve transection (Tanga et al. 2005; Kim et al. 2007). Elimination of TLR2 also reduces cell death induced by kainic acid administration (Hong et al. 2010), and attenuates leukocyte and microglial infiltration and neuronal death induced by focal ischemia (Ziegler et al. 2011). In agreement with these findings, our results demonstrate that both TLR4 and TLR2 participate in ethanol-induced microglia activation since ethanol not only activates several kinases (extracellular-signal-regulated kinase, p38, c-Jun N-terminal kinase) but also induces inflammatory cytokines (TNF-α, IL-1β) and mediators (iNOS) in WT microglia. These events were not observed in the ethanol-treated microglia from TLR4−/− and TLR2−/− mice. Therefore, by considering the role of both receptors in neuroinflammation, we propose that TLR4/TLR2 are important targets for ethanol-induced microglia activation, and that the elimination of one of the receptors might abolish the ethanol-induced recruitment and heterodimerization of TLR4/TLR2 by diminishing the action of TLRs signaling and neuroinflammation.
In summary, this study shows for the first time that ethanol, by triggering TLR4 and TLR2 recruitment into LR-caveolae, promotes TLR4 and TLR2 interactions and signaling in microglial cells, leading to the production of pro-inflammatory cytokines, which causes ROS generation and neuronal apoptosis. The results extend our previous findings (Fernandez-Lizarbe et al. 2009) and reveal the participation of TLR4 and TLR2 receptors in microglia activation, neuroinflammation, and neuronal apoptosis induced by ethanol.