Abstract
Neuroimmune gene induction is involved in many brain pathologies including addiction. Although increased expression of proinflammatory cytokines has been found in ethanol-treated mouse brain and rat brain slice cultures as well as in post-mortem human alcoholic brain, the mechanisms remain elusive. High-mobility group box 1 (HMGB1) protein is a nuclear protein that has endogenous cytokine-like activity. We previously found increased HMGB1 in post-mortem alcoholic human brain as well as in ethanol treated mice and rat brain slice cultures. The present study investigated the mechanisms for ethanol-induced release of HMGB1 and neuroimmune activation in a model of rat hippocampal-entorhinal cortex (HEC) brain slice cultures. Ethanol exposure triggered dose-dependent HMGB1 release, predominantly from neuronal cells. Inhibitors of histone deacetylases (HDACs) promoted nucleocytoplasmic mobilization of HDAC1/4 and HMGB1 resulting in increased total HMGB1 and acetylated HMGB1 release. Similarly, ethanol treatment was found to induce the translocation of HDAC1/4 and HMGB1 proteins from nuclear to cytosolic fractions. Furthermore, ethanol treatment reduced HDAC1/4 mRNA and increased acetylated HMGB1 release into the media. These results suggest decreased HDAC activity may be critical in regulating acetylated HMGB1 release from neurons in response to ethanol. Ethanol and HMGB1 treatment increased mRNA expression of proinflammatory cytokines TNFα and IL-1β as well as toll-like receptor 4 (TLR4). Targeting HMGB1 or microglial TLR4 by using siRNAs to HMGB1 and TLR4, HMGB1 neutralizing antibody, HMGB1 inhibitor glycyrrhizin and TLR4 antagonist as well as inhibitor of microglial activation all blocked ethanol-induced expression of proinflammatory cytokines TNFα and IL-1β. These results support the hypothesis that ethanol alters HDACs that regulate HMGB1 release and that danger signal HMGB1 as endogenous ligand for TLR4 mediates ethanol-induced brain neuroimmune signaling through activation of microglial TLR4. These findings provide new therapeutic targets for brain neuroimmune activation and alcoholism.
Highlights
Neuroimmune activation in brain has been hypothesized to contribute to brain damage and behavioral changes associated with alcohol consumption
Slices were placed onto a 30 mm diameter membrane tissue insert and cultured with medium containing 75% MEM with 25 mM HEPES and Hank’s salts +25% horse serum (HS) +5.5 g/L glucose +2 mM L-glutamine in a humidified 5% CO2 incubator at 36.5uC for 7 days in vitro (DIV), followed by 4 DIV in medium containing 12.5% HS and in serum-free medium supplemented with N2 till the end of experiment
high mobility group box 1 (HMGB1) release, hippocampal-entorhinal cortex (HEC) slices were treated with various ethanol concentrations
Summary
Neuroimmune activation in brain has been hypothesized to contribute to brain damage and behavioral changes associated with alcohol consumption. Many studies have reported that chronic alcohol consumption can increase proinflammatory cytokines and innate immune gene expression in the brain [1,2]. Recent studies suggest activation of brain neuroimmune signaling induces changes in mood and drinking behavior and increases risk of alcoholism as well as alcoholic neurodegeneration [1]. Studies have demonstrated that Toll-like receptor 4 (TLR4) is critical for ethanol-induced neuroimmune activation, neurodegeneration and behavioral pathology [2,6]. Recent studies support the hypothesis that high mobility group box 1 (HMGB1) protein, an endogenous cytokine that can activate toll-like receptors including TLR4, is linked to ethanol-induced increase in expression of brain neuroimmune genes [12]. It is conceivable that ethanol exposure may trigger release of endogenous TLR4 ligand HMGB1 contributing to ethanol-induced neuroimmune signaling through TLR4 receptor activation
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