Acute ethanol treatment modulates Toll-like receptor-4 association with lipid rafts

Angela Dolganiuc, Gennadiy Bakis, Karen Kodys, Pranoti Mandrekar, Gyongyi Szabo

Research output: Contribution to journalArticle

75 Citations (Scopus)

Abstract

Background: Alcohol, a substance that is most frequently abused, suppresses innate immune responses to microbial pathogens. The host senses pathogens via Toll-like receptors (TLRs). Recent studies indicate that alcohol affects TLR signaling. Methods: Here, we hypothesized that acute alcohol treatment may interfere with early steps of membrane-associated TLR2 and TLR4 signaling at the level of lipid rafts. Human monocytes and Chinese hamster ovary (CHO) cells, transfected with human TLR2, TLR4, or CD14, were stimulated with peptidoglycan (PGN, TLR2 ligand) or lipopolysaccharide (LPS, TLR4 ligand) with or without alcohol (50 mM) and analyzed for cytokine production (enzyme-linked immunosorbent assay), nuclear factor-κB (NF-κB) activation (electrophoretic mobility shift assay), membrane fluidity (fluorescent pyrene eximer formation), and partition of cellular membrane into cholesterol-rich, detergent-resistant domains (DRMs; Western blot). Results: We determined that both TLR2 and TLR4 were located outside the rafts; flotillin, a DRM marker, was resident in the rafts, while CD14 was equally distributed in and outside the rafts in a steady-state condition. PGN forced TLR2 to migrate into DRMs. Engagement of TLR4 and CD14 with LPS induced their migration into the rafts. Alcohol prevented TLR4 partitioning; however, it did not affect TLR2 migration into the rafts. Furthermore, alcohol downregulated TLR4-induced, but not TLR2-induced, NF-κB activation and cytokine production in monocytes. We found that alcohol increased membrane fluidity and depleted cellular cholesterol in CHO cells without affecting cell viability. Conclusions: These data demonstrate for the first time that alcohol disturbs TLR4 and CD14 association with lipid rafts. We propose that alcohol-induced effects on lipid rafts may contribute to modulation of TLR4-CD14-triggered early cellular responses.

Original languageEnglish (US)
Pages (from-to)76-85
Number of pages10
JournalAlcoholism: Clinical and Experimental Research
Volume30
Issue number1
DOIs
StatePublished - Jan 2006
Externally publishedYes

Fingerprint

Toll-Like Receptor 4
Ethanol
Alcohols
Association reactions
Lipids
Membranes
Membrane Fluidity
Therapeutics
Fluidity
Toll-Like Receptors
Pathogens
Cricetulus
Monocytes
Ovary
Assays
Chemical activation
Cholesterol
Cells
Cytokines
Ligands

Keywords

  • Cholesterol
  • Ethanol
  • Fluidity
  • NF-κB
  • Rafts
  • TLR

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Toxicology

Cite this

Acute ethanol treatment modulates Toll-like receptor-4 association with lipid rafts. / Dolganiuc, Angela; Bakis, Gennadiy; Kodys, Karen; Mandrekar, Pranoti; Szabo, Gyongyi.

In: Alcoholism: Clinical and Experimental Research, Vol. 30, No. 1, 01.2006, p. 76-85.

Research output: Contribution to journalArticle

Dolganiuc, Angela ; Bakis, Gennadiy ; Kodys, Karen ; Mandrekar, Pranoti ; Szabo, Gyongyi. / Acute ethanol treatment modulates Toll-like receptor-4 association with lipid rafts. In: Alcoholism: Clinical and Experimental Research. 2006 ; Vol. 30, No. 1. pp. 76-85.
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AB - Background: Alcohol, a substance that is most frequently abused, suppresses innate immune responses to microbial pathogens. The host senses pathogens via Toll-like receptors (TLRs). Recent studies indicate that alcohol affects TLR signaling. Methods: Here, we hypothesized that acute alcohol treatment may interfere with early steps of membrane-associated TLR2 and TLR4 signaling at the level of lipid rafts. Human monocytes and Chinese hamster ovary (CHO) cells, transfected with human TLR2, TLR4, or CD14, were stimulated with peptidoglycan (PGN, TLR2 ligand) or lipopolysaccharide (LPS, TLR4 ligand) with or without alcohol (50 mM) and analyzed for cytokine production (enzyme-linked immunosorbent assay), nuclear factor-κB (NF-κB) activation (electrophoretic mobility shift assay), membrane fluidity (fluorescent pyrene eximer formation), and partition of cellular membrane into cholesterol-rich, detergent-resistant domains (DRMs; Western blot). Results: We determined that both TLR2 and TLR4 were located outside the rafts; flotillin, a DRM marker, was resident in the rafts, while CD14 was equally distributed in and outside the rafts in a steady-state condition. PGN forced TLR2 to migrate into DRMs. Engagement of TLR4 and CD14 with LPS induced their migration into the rafts. Alcohol prevented TLR4 partitioning; however, it did not affect TLR2 migration into the rafts. Furthermore, alcohol downregulated TLR4-induced, but not TLR2-induced, NF-κB activation and cytokine production in monocytes. We found that alcohol increased membrane fluidity and depleted cellular cholesterol in CHO cells without affecting cell viability. Conclusions: These data demonstrate for the first time that alcohol disturbs TLR4 and CD14 association with lipid rafts. We propose that alcohol-induced effects on lipid rafts may contribute to modulation of TLR4-CD14-triggered early cellular responses.

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