Ethanol inhibits LPS-induced signaling and modulates cytokine production in peritoneal macrophages in vivo in a model for binge drinking.

Previous reports indicate that ethanol, in a binge drinking model in mice, inhibits the production of pro-inflammatory cytokines in vivo. However, the inhibition of signaling through TLR4 has not been investigated in this experimental model in vivo. Considering evidence that signaling can be very different in vitro and in vivo, the present study was conducted to determine if effects of ethanol on TLR4 signaling reported for cells in culture or cells removed from ethanol treated mice and stimulated in culture also occur when ethanol treatment and TLR4 activation occur in vivo. Phosphorylated p38, ERK, and c-Jun (nuclear) were quantified with kits or by western blot using samples taken 15, 30, and 60 min after stimulation of peritoneal macrophages with lipopolysaccharide in vivo. Effects of ethanol were assessed by administering ethanol by gavage at 6 g/kg 30 min before administration of lipopolysaccharide (LPS). Cytokine concentrations in the samples of peritoneal lavage fluid and in serum were determined at 1, 2, and 6 hr after lipopolysaccharide administration. All of these data were used to measure the area under the concentration vs time curve, which provided an indication of the overall effects of ethanol in this system. Ethanol suppressed production of most pro-inflammatory cytokines to a similar degree as it inhibited key TLR4 signaling events. However, NF-kappaB (p65) translocation to the nucleus was not inhibited by ethanol. To determine if NF-kappaB composed of other subunits was inhibited, transgenic mice with a luciferase reporter were used. This revealed a reproducible inhibition of NF-kappaB activity, which is consistent with the observed inhibition of cytokines whose expression is known to be NF-kappaB dependent. Overall, the effects of ethanol on signalling in vivo were similar to those reported for in vitro exposure to ethanol and/or lipopolysaccharide. However, inhibition of the activation of NF-kappaB was not detected as translocation of p65 to the nucleus but was detected using transgenic reporter mice. The observation that ethanol given 24 hr before dosing with LPS modulated production of some cytokines indicates a persistent effect which does not require continued presence of ethanol.