Effects of Medically Relevant Concentrations of Ethanol on Cellular Responses to Ligands for Toll-Like Receptors 3 and 4.
Introduction: Excessive ethanol consumption suppresses the innate immune response. Current literature suggests that ethanol suppresses the pro-inflammatory cytokine response after pathogen challenge in part by interfering with Toll-like receptor signaling. The main focus of this study is to assess the effects of ethanol on the TLR3 and TLR4 mediated cytokine response, measured by expression and secretion of Tumor necrosis factor alpha (TNF-alpha) as a key cytokine in the regulation of the immune response. In addition, ethanol effects on NF-kappaB expression and TLR3 protein structure are investigated. Hypothesis: Ethanol in concentrations relevant for humans inhibits the TNF-alpha response to pathogens by interfering with the early steps of the TLR signaling cascade, resulting in decreased transcription of NF-kappaB dependant genes. Methods: RAW264.7 cells were treated with LPS or poly (I:C), and co-treated with ethanol and other alcohols, and/or inhibitors of protein synthesis or tumor necrosis factor alpha converting enzyme (TACE). TNF-alpha secretion or TNF-alpha standard protein were measured by ELISA. Transgenic mice were employed to assess NF-kappaB activation. TNF-alpha mRNA expression was measured by real time RT-PCR. Ethanol effects on TNF-alpha cell surface expression were assessed by flow cytometry. In circular dichroism studies, effects of ethanol on TLR3 ectodomain protein conformation were investigated. Results: Increasing carbon chain length of straight chain alcohols positively correlated with their ability to inhibit detection of TNF-alpha and IL-10, but not with the detection of IL-6, IL-8, and IL-12. Ethanol inhibits LPS and poly (I:C) induced NF-kappaB activation in vivo. TNF-alpha is synthesized de novo, rather than released from cellular stores. Ethanol inhibits LPS and poly (I:C) induced TNF-alpha mRNA expression in vitro. Ethanol does not function as a TACE inhibitor. A decrease in TNF-alpha surface expression through ethanol is caused mostly by decreased TNF-alpha production. Inhibition of LPS- or poly (I:C)-induced TNF-alpha production by ethanol starts at the signaling pathway and is not primarily mediated by post-transcriptional effects. Ethanol at relevant concentrations for humans causes conformational changes that occur when TLR3 binds a dsRNA ligand. Conclusions: Ethanol at concentrations relevant in humans inhibits TNF-alpha gene transcription by altering the conformation of TLR3, thus inhibiting signaling.