Induction of Innate Immune Gene Expression Cascades in Brain Slice Cultures by Ethanol: Key Role of NF‐κB and Proinflammatory Cytokines

Abstract

Postmortem human alcoholic brain has increased expression of proinflammatory cytokines (He and Crews, 2007). Nuclear factor kappaB (NF-kappaB) is a transcription factor known to induce proinflammatory cytokine expression. Ethanol exposure increases NF-kappaB-DNA binding in rat brain (Crews et al., 2006) and in brain slice cultures in vitro (Zou and Crews, 2006). Using hippocampal-entorhinal cortex (HEC) brain slice cultures, we explored the effect of ethanol on NF-kappaB-DNA binding, proinflammatory gene expression, and sensitivity to glutamate neurotoxicity. The HEC brain slice cultures are prepared from rats on P7 and used after 2 weeks in culture. NF-kappaB-DNA binding is determined by EMSA, NF-kappaB subunit-DNA binding by ELISA and mRNA by RT-PCR. Multiple antibody immunohistochemistry and confocal microscopy are used to characterize cell types expressing ethanol-induced genes. Ethanol treatment results in a progressive increase in NF-kappaB-DNA binding that includes large increases in NF-kappaB subunit p50 protein-DNA binding. The expression of NF-kappaB proinflammatory target genes progressively increased with time of ethanol treatment. Ethanol induces proinflammatory cytokines TNFalpha, MCP-1, and IL-1beta, proinflammatory proteases TACE, and tissue plasminogen activator (tPA) as well as inducible nitric oxide synthase. Blockade of NF-kappaB by using NF-kappaB p65 siRNA and BHT reduces ethanol induction of proinflammatory genes. Neutralizing antibody to proinflammatory cytokine TNFalpha reduces ethanol induction of proinflammatory genes, suggesting cytokine propagation of proinflammatory gene induction. Furthermore, neutralizing antibodies to proinflammatory cytokines and protease tPA inhibitors blunt ethanol sensitization to glutamate neurotoxicity. These findings indicate that ethanol treatment increases NF-kappaB-DNA binding and proinflammatory gene expression in brain slices. Ethanol-induced innate immune proinflammatory gene induction alters neurotransmission and likely contributes to alcoholic neurodegeneration.