Are the effects of alcohol on the CNS influenced by Toll-like receptor signaling?

Keywords::

• alcohol
• CNS
• MyD88
• NF-κB
• TLR4

Acute ethanol exposure produces a range of well-known behavioral effects including ataxia of gait, slurred speech, prolonged reaction time, poor memory consolidation, impaired emotional modulation and compromised judgment [1]. With chronic use, deficits in perceptual motor-skills and problem solving are noted, accompanied by changes in brain morphology including atrophy of subcortical areas, the thalamus, corpus callosum and cerebellum [2]. Alcohol is an addictive substance, with alcoholism characterized by excessive intake of alcohol, the development of tolerance and withdrawal with impairment in social and occupational functioning [3]. Traditionally, these effects of alcohol have been attributed to changes in neurotransmitter systems, including the GABAergic and glutamatergic systems [4]. However, recent research has indicated that activation of the innate immune system, particularly Toll-like receptor (TLR)4, also plays an integral role in the neurological response to alcohol.

Role of the innate immune system in mediating the CNS effects of alcohol

Microglia and astrocytes, the immunocompetent cells within the CNS, contribute to the acute and chronic effects of alcohol use; with research over the past decade making it increasingly clear that ethanol induces a neuroinflammatory response. Both acute and chronic ethanol consumption lead to an increase in cytokine production experimentally, including, TNF-α, IL-1β and IL-6 [5], as well as CCL2, which is also known as MCP-1 [6]. Clinically, alcoholism is associated with microglial activation, seen as an increase in the protein Iba-1, and increased levels of CCL2 in several brain regions when compared with healthy controls [7]. Inhibition of proinflammation, through either minocycline or IL-1Ra, reduced acute alcohol induced sedation [8], while the anti-inflammatory drug indomethecin reduced the chronic induction of brain innate immune genes, neuronal cell death and addictive-like behavioral dysfunction [9]. Indeed mice deficient in CCL2, CCL3 or CCR2 drink less alcohol than wild-type mice, with knockout of CCL2 or CCL3 also inducing a longer loss of righting reflex [10]. The importance of proinflammatory signaling in response to alcohol is evidenced by changes in NF-κB, a transcription factor that is integral for induction of proinflammatory cytokine gene expression in glia. NF-κB DNA binding is increased in response to ethanol treatment both in vivo[11] and in vitro[12], with the human NF-κB gene linked to alcoholism [13]. Critically, activation of TLR4 appears to be responsible for many of these downstream effects that have been observed following ethanol exposure, including activation of NF-κB and promotion of inflammatory cytokine release.

Toll-like receptors

The TLR family consists of approximately 12 single transmembrane receptors that recognize a diverse range of ‘patterns’ on exogenous and endogenous danger signals. TLR4 is the most extensively studied as it binds the endotoxin lipopolysaccharide (LPS), with recent research uncovering an extensive list of exogenous and endogenous molecules such as heat shock proteins and opioids that also have TLR4-binding properties [14]. Indeed, TLR4 is proposed to play a role in a number of acute and chronic neurological conditions including Alzheimer’s disease and stroke [14]. The TLR4 signal is produced through two adaptor proteins, MyD88 and TRIF, with the MyD88 pathway leading to NF-κB activation and the TRIF pathway, to Toll/IL-1 receptor-containing adaptor-inducing IFN-β activation [14].

Role of TLRs in mediating the acute CNS effects of alcohol

Prevention of TLR4 signaling through either genetic knockout or administration of the TLR4 antagonist, (+)- or (-)-naloxone, was found to attenuate the acute sedative and motor effects of ethanol, as detected by sleep-time and rotarod performance, respectively [15]. This supported previous in vitro work that demonstrated that ethanol rapidly activated TLR4 signaling in astrocytes [16] and microglia [17], leading to an increase in the production of proinflammatory cytokines and eventual cell death. Furthermore, priming of the TLR4 response with 24 h of LPS exposure worsened the motor ataxic effects of alcohol [18]. This appears to be a predominantly MyD88 pathway-dependent effect, as MyD88 knockout mice were also protected from the acute sedative and motor effects of ethanol exposure [15], with increased levels of MyD88 expression also noted following chronic ethanol intake [5]. Indeed, the downstream effects of TLR4 activation are mediated by NF-κB signaling, with translocation of NF-κB to the nucleus noted after 30 min of ethanol stimulation in vitro[5,17], an effect that was prevented by knockdown of TLR4 expression with siRNA [5]. Acute changes in the protein levels of IκBα, the main inhibitory protein of NF-κB have also been found, with IκBα levels increased after 15–30 min post-exposure, a pattern not seen in TLR4 or MyD88 knockout mice [15]. Although the final outcome of TLR4 activation involves the initiation of NF-κB signaling, the intermediate pathways remain unclear. In neonatal astroyctic [5] and microglial cultures [17], ERK, JNK and p38 phosphorylation were noted, but in ex vivo hippocampal and cerebellar samples, as well as mixed in hippocampal cells in vitro, no changes in the phosphorylation state of these proteins were seen [15]. Thus the relative involvement of the MAPK or alternative pathways such as the PI3K/AKT pathways have yet to be determined. Nonetheless, it is evident that acute ethanol exposure rapidly activates TLR4 signaling with the resultant translocation of NF-κB to the nucleus and production of proinflammatory cytokines. How proinflammatory cytokines modulate neuronal activity is just beginning to be elucidated. It has been suggested that proinflammatory mediators may facilitate the activation of GABA_A receptors [4]. Indeed an interaction between TLR4 and GABA_A-α2 has recently been demonstrated, although in this case knockdown of GABA_A-α2 in the central nucleus of the amygdala led to a reduction in TLR4 expression, with the reverse situation not examined [19]. Further studies are needed to determine how the production of cytokines promotes the motor and sedative effects noted with alcohol consumption.

Role of TLRs in mediating the chronic CNS effects of alcohol

The activation of TLR4 is not only involved in the acute effects of alcohol; chronic ethanol exposure over a 5-month period results in TLR4-dependent astrocyte and microglia activation [5], which is still evident following 2 weeks of withdrawal from alcohol [20]. This is associated with persistent nuclear activation of the NF-κB-p65 subunit and increases in the proinflammatory cytokines, TNF-α, IL-1β and IL-6 that were not seen in TLR4 knockout mice [5]. Knockout of TLR4 was also able to protect against conditional learning and memory deficits in the object recognition task following chronic alcohol consumption in mice [20]. This implies that prolonged activation of TLR4 with chronic ethanol exposure may promote neurodegeneration with the associated development of cognitive deficits.

Role of TLRs in alcohol addiction

Proinflammatory signaling triggered by TLR4 activation also plays a role in the addictive nature of alcohol. Repeated LPS administration before 5 days of chronic ethanol exposure led to withdrawal-induced anxiety, which was not seen in vehicle-treated mice [21]. This cannot be attributed to the administration of LPS alone, as no anxiety-like behaviors were observed in mice who did not receive ethanol [21]. This was supported by a study by Pascual et al., which demonstrated that wild-type, but not TLR4 knockout mice exhibited anxiety-related behavioral impairments during withdrawal [20]. The presence of TLR4 was also found to enhance consumption of ethanol in alcohol-preferring rats, as knockdown of TLR4 within the central nucleus of the amygdale with siRNA decreased binge drinking in these animals [19]. However, it should be noted that no genotypic differences in preference to alcohol intake during voluntary consumption were seen between wild-type and TLR4 knockout mice [20]. Thus a predisposing state that promotes reward from alcohol drinking may be potentiated by TLR4 activation, while the presence of TLR4 alone is not sufficient. Despite this, it appears that TLR4 activation potentiates and maintains anxiety-like behavior induced by ethanol during withdrawal, and may also play a role in activating reward pathways, promoting excessive alcohol consumption.

Implications of CNS TLR activation by alcohol

The discovery that TLR4 plays a role in the neurological response to alcohol opens a wide range of avenues for exploration. Given that TLR4 is proposed to enhance neurodegeneration in conditions such as Parkinson’s disease, stroke and Alzheimer’s [14], it is possible that moderate-to-heavy alcohol consumption at any time in life could worsen these conditions via increased neuroinflammation priming or aberrant signaling. Indeed ten daily doses of ethanol enhanced the response to LPS within the brain, with an increase in proinflammatory genes including MCP-1, TNF-α and IL-1β still evident 8 days after LPS exposure [6]. Activation of TLR4 receptors may also provide a potential link between the high rate of comorbidity between alcohol and mood disorders, particularly depression. As reviewed by Kelley and Dantzer, both depression and alcohol use are associated with a neuroinflammatory state and decreased neuroplasticity [22]. It is unclear whether depression predisposes to alcohol abuse or vice versa, with this most likely a bidirectional interaction [22]. Indeed, induction of TLR4 signaling in a susceptible individual may promote alcohol abuse, while the production of proinflammatory cytokines caused by chronic alcohol ingestion will predispose to depression. Finally, alcohol may also act synergistically with other exogenous agents that have TLR4 agonist properties, such as opioids [23], to amplify proinflammatory signaling and worsen their observed neurological effects. Indeed, alcohol increases the risk of heroin-related deaths and as this is not due to pharmacokinetic effects [24], it may relate to enhanced TLR4 activation.

Conclusion

Activation of the TLR4 signaling cascade has been shown to be integral for the acute and chronic neurological effects of alcohol, as well as promoting addiction. This interaction has provided further evidence for the role of immune signaling in modifying neuronal function and will allow greater understanding of the effects of alcohol on a range of other neurological conditions including depression.

Financial & competing interests disclosure

The authors are employees of The University of Adelaide and have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

No writing assistance was utilized in the production of this manuscript.