Activation of inflammatory signaling by lipopolysaccharide produces a prolonged increase of voluntary alcohol intake in mice

https://doi.org/10.1016/j.bbi.2011.01.008Get rights and content

Abstract

Previous studies showed that mice with genetic predisposition for high alcohol consumption as well as human alcoholics show changes in brain expression of genes related to immune signaling. In addition, mutant mice lacking genes related to immune function show decreased alcohol consumption (Blednov et al., 2011), suggesting that immune signaling promotes alcohol consumption. To test the possibility that activation of immune signaling will increase alcohol consumption, we treated mice with lipopolysaccaride (LPS; 1 mg/kg, i.p.) and tested alcohol consumption in the continuous two-bottle choice test. To take advantage of the long-lasting activation of brain immune signaling by LPS, we measured drinking beginning one week or one month after LPS treatment and continued the studies for several months. LPS produced persistent increases in alcohol consumption in C57BL/6 J (B6) inbred mice, FVBxB6F1 and B6xNZBF1 hybrid mice, but not in FVB inbred mice. To determine if this effect of LPS is mediated through binding to TLR4, we tested mice lacking CD14, a key component of TLR4 signaling. These null mutants showed no increase of alcohol intake after treatment with LPS. LPS treatment decreased ethanol-conditioned taste aversion but did not alter ethanol-conditioned place preference (B6xNZBF1 mice). Electrophysiological studies of dopamine neurons in the ventral tegmental area showed that pretreatment of mice with LPS decreased the neuronal firing rate. These results suggest that activation of immune signaling promotes alcohol consumption and alters certain aspects of alcohol reward/aversion.

Highlight

► Alcohol consumption is regulated by inflammatory signaling suggesting new drug treatments for reduction of excessive alcohol consumption.

Introduction

Genes encoding proteins involved in “immune/stress responses” are one of the most prominent functional groups that exhibit differential gene expression in the frontal cortex between human alcoholics and non-alcoholics (Liu et al., 2006). Expression of these genes in brain is also related to genetic predisposition for alcohol consumption in mice, indicating a role for pro-inflammatory mediators in regulating alcohol intake (Mulligan et al., 2006). Analysis of these gene-expression data sets led to the selection of six genes related to neuroimmune pathways, and behavioral testing of genes selected from these studies showed that null mutant mice lacking selected neuroimmune signaling components drink less alcohol (Blednov et al., 2011). Other evidence linking brain neuroimmune or pro-inflammatory signaling to alcohol action includes the finding that long-lasting increases in levels of several pro-inflammatory cytokines were found in rat brain after chronic treatment with high doses of ethanol followed by injection of lipopolysaccharide (LPS) (Qin et al., 2008). Interestingly, one of the cytokines that increased in rat brain, MCP-1 (Ccl2), was also increased in the brain of human alcoholics (He and Crews, 2008). Recently, Kong et al. (2010) found up-regulation of genes in the Toll and Imd innate immune signaling pathways in Drosophila exposed to ethanol, extending the link between alcohol and immune mediators to invertebrates.

We hypothesized that the normal functioning of the immune system maintains drinking at low to moderate levels, whereas activation of the neuroimmune system could promote excessive alcohol consumption. We tested this by using LPS to activate neuroimmune signaling before measuring drinking. LPS is a bacterial endotoxin normally confined to the gut, but it can leak from the gut as a result of chronic alcohol abuse (Mandrekar and Szabo, 2009). LPS produces activation of the immune system when administered systemically by binding to toll-like receptor 4 (TLR4) found on macrophages, Kupffer and stellate cells in the liver, and endothelial cells (Andonegui et al., 2003, Mandrekar and Szabo, 2009, Suzumura et al., 2006, Zanoni and Granucci, 2010). TLR4 is also found in the brain on neurons, astrocytes, microglia and endothelial cells (Tang et al., 2007, Mallard et al., 2009). Some publications suggest that LPS elicits TLR4 signaling in the brain by interactions with these receptors (Chakravarty and Herkenham, 2005, Gosselin and Rivest, 2008), but others have failed to find LPS within the brain parenchyma after systemic administration (Singh and Jiang, 2004). It is clear that LPS binds to TLR4 on cerebral endothelial cells and this may have a role in actions of LPS on brain function (Singh and Jiang, 2004, Verma et al., 2006).

Activation of TLR4 by LPS produces release of a number of immune mediators, including cytokines, and a “sickness” response characterized by decreased food and water intake, loss of weight, lethargy and anhedonia (Dantzer, 2001). Most of these effects of LPS are due to actions on the brain, and are generally attributed to cytokines that are released peripherally and are transported across the blood–brain barrier (Wisse et al., 2007). The increased levels of serum cytokines produced by LPS are transient (hours) and the sickness response lasts only a day or two. However, there are persistent actions of LPS exposure on brain neuroinflammatory signaling, including elevation of certain brain cytokines such as TNFα, IL-1 and CCl2 for up to 10 months (Qin et al., 2007, Qin et al., 2008).

To determine if inflammatory activation by LPS might produce a long-lasting increase in alcohol consumption, we gave mice one or two injections of LPS, waited one week (to allow normalization of body weight and water intake following the sickness response), then began tests of alcohol consumption. Because alcohol drinking in mice is highly dependent on genetic background, we used several different strains of mice with different levels of alcohol consumption. We also tested phenotypes that might be related to alcohol consumption or reward: conditioned place preference (CPP), conditioned taste aversion (CTA), firing activity of midbrain dopamine neurons, consumption of saccharin and quinine, and olfactory detection of ethanol.

Section snippets

Animals

Studies were conducted in drug-naïve C57BL/6 J (B6), FVB/NJ (FVB), NZB/B1NJ (NZB), and F1 hybrid mice derived from these three progenitors (FVBxB6 F1, maternal strain x paternal strain; B6xNZB F1). B6, FVB, and NZB breeders as well as Cd14 (B6.129S-cd14tm1Frm/J; Stock #003726) null mice were purchased from Jackson Laboratories (Bar Harbor, ME) and mated at the age of 8 weeks in the Texas Genetic Animal Core of the Integrated Neuroscience Initiative on Alcohol (INIA) at the University of Texas at

Ethanol intake

In a two-bottle, free-choice paradigm in which mice could drink either water or an ascending series of ethanol concentrations, pretreatment with LPS significantly increased the amount of ethanol consumed in B6 male mice after one injection of LPS as well as after two LPS injections (Fig. 2A and D). A similar increase of alcohol intake was seen in both experimental groups after the first period (one week) of alcohol deprivation (Fig. 2B and E). However, after a second period (one month) of

Discussion

Our results clearly show that inflammatory activation (treatment with LPS) can produce long-lasting (up to 3 months, including several periods of alcohol deprivation) increases of ethanol intake, although there are differences in the size and persistence of the effect among different genetic backgrounds and between male and female mice. Both B6 and FVBxB6F1 mice showed the increased consumption. It should be noted that B6 show the highest level of ethanol preference and intake among all

Acknowledgments

This research was supported by grants from the National Institute of Alcohol Abuse and Alcoholism (AA U01 13520 - INIA West Project, AA06399, AA015521). The authors would like to thank Danielle Walker and Virginia Bleck for excellent technical assistance.

References (72)

  • A.B. Kampov-Polevoy et al.

    Initial acceptance of ethanol: gustatory factors and patterns of alcohol drinking

    Alcohol

    (1990)
  • S.W. Kiefer

    Alcohol, palatability, and taste reactivity

    Neurosci. Biobehav. Rev.

    (1995)
  • S.J. Larson

    Lipopolysaccharide and interleukin-1beta decrease sucrose intake but do not affect expression of place preference in rats

    Pharmacol Biochem Behav.

    (2006)
  • C. Mallard et al.

    The role of toll-like receptors in perinatal brain injury

    Clin. Perinatol.

    (2009)
  • P. Mandrekar et al.

    Signalling pathways in alcohol-induced liver inflammation

    J. Hepatol.

    (2009)
  • D. Martinez et al.

    Alcohol dependence is associated with blunted dopamine transmission in the ventral striatum

    Biol. Psychiatry

    (2005)
  • J. McAfoose et al.

    Evidence for a cytokine model of cognitive function

    Neurosci. Biobehav. Rev.

    (2009)
  • K. Mori et al.

    Peripherally injected lipopolysaccharide induces apoptosis in the subventricular zone of young adult mice

    Neurosci. Lett.

    (2010)
  • B.P. Nathan et al.

    Olfactory function in apoE knockout mice

    Behav. Brain Res.

    (2004)
  • A.K. Singh et al.

    How does peripheral lipopolysaccharide induce gene expression in the brain of rats?

    Toxicology

    (2004)
  • T.M. Tzschentke

    Measuring reward with the conditioned place preference paradigm: a comprehensive review of drug effects, recent progress and new issues

    Prog. Neurobiol.

    (1998)
  • S. Verma et al.

    Release of cytokines by brain endothelial cells: a polarized response to lipopolysaccharide

    Brain Behav. Immun.

    (2006)
  • N. Yoneyama et al.

    Voluntary ethanol consumption in 22 inbred mouse strains

    Alcohol

    (2008)
  • I. Zanoni et al.

    Differences in lipopolysaccharide-induced signaling between conventional dendritic cells and macrophages

    Immunobiology

    (2010)
  • G. Andonegui et al.

    Endothelium-derived toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs

    J. Clin. Invest.

    (2003)
  • A.A. Bachmanov et al.

    Chemosensory factors influencing alcohol perception, preferences, and consumption

    Alcohol. Clin. Exp. Res.

    (2003)
  • J.K. Belknap et al.

    Voluntary consumption of ethanol in 15 inbred mouse strains

    Psychopharmacology

    (1993)
  • H.O. Besedovsky et al.

    Immune-neuro-endocrine interactions: facts and hypotheses

    Endocr. Rev.

    (1996)
  • Y.A. Blednov et al.

    GABAA receptor alpha 1 and beta 2 subunit null mutant mice: behavioral responses to ethanol

    J. Pharmacol. Exp. Ther.

    (2003)
  • Y.A. Blednov et al.

    Hybrid C57BL/6JxFVB/NJ mice drink more alcohol than do C57BL/6J mice

    Alcohol. Clin. Exp. Res.

    (2005)
  • Y.A. Blednov et al.

    Perception of sweet taste is important for voluntary alcohol consumption in mice

    Genes Brain Behav.

    (2008)
  • Y.A. Blednov et al.

    Hybrid mice as genetic models of high alcohol consumption

    Behav. Genet.

    (2010)
  • Y.A. Blednov et al.

    Mice lacking Gad2 show altered behavioral effects of ethanol, flurazepam and gabaxadol

    Addict. Biol.

    (2010)
  • Blednov, Y.A., Ponomarev, I., Geil, C., Bergeson, S., Koob, G.F., Harris, R.A., 2011. Neuroimmune regulation of alcohol...
  • J. Broadbent et al.

    Ethanol-induced conditioned taste aversion in 15 inbred mouse strains

    Behav. Neurosci.

    (2002)
  • L.A. Brown et al.

    Acute and chronic alcohol abuse modulate immunity

    Alcohol. Clin. Exp. Res.

    (2006)
  • Cited by (201)

    View all citing articles on Scopus
    View full text