Toll-like receptor signaling and stages of addiction

Abstract

Background: Athina Markou and her colleagues discovered persistent changes in adult behavior following adolescent exposure to ethanol or nicotine consistent with increased risk for developing addiction. Building on Dr. Markou's important work and that of others in the field, researchers at the Bowles Center for Alcohol Studies have found that persistent changes in behavior following adolescent stress or alcohol exposure may be linked to induction of immune signaling in brain.

Aim: This study aims to illuminate the critical interrelationship of the innate immune system (e.g., toll-like receptors [TLRs], high-mobility group box 1 [HMGB1]) in the neurobiology of addiction.

Method: This study reviews the relevant research regarding the relationship between the innate immune system and addiction.

Conclusion: Emerging evidence indicates that TLRs in brain, particularly those on microglia, respond to endogenous innate immune agonists such as HMGB1 and microRNAs (miRNAs). Multiple TLRs, HMGB1, and miRNAs are induced in the brain by stress, alcohol, and other drugs of abuse and are increased in the postmortem human alcoholic brain. Enhanced TLR-innate immune signaling in brain leads to epigenetic modifications, alterations in synaptic plasticity, and loss of neuronal cell populations, which contribute to cognitive and emotive dysfunctions. Addiction involves progressive stages of drug binges and intoxication, withdrawal-negative affect, and ultimately compulsive drug use and abuse. Toll-like receptor signaling within cortical-limbic circuits is modified by alcohol and stress in a manner consistent with promoting progression through the stages of addiction.

Keywords: Alcohol use disorder; Cytokines; HMGB1; miRNA let-7.

Fig. 1

High-mobility group box 1 (HMGB1) signaling involvement in addiction neuropathology. HMGB1 is actively and/or passively released from neurons and other cells leading to the activation of multiple innate immune signaling pathways. Extracellularly, HMGB1 can directly interact with toll-like receptors (TLRs) or form complexes with various ligands to enhance immune responses at various pattern recognition receptors (PPRs). In addition, HMGB1 can form complexes with nucleic acids and microRNAs (miRNAs; e.g., let-7) that are endocytosed into the cell-activating intracellular TLRs. Activation of TLRs and other PRRs leads to activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and subsequent induction of proinflammatory cytokines and oxidases that are released into the extracellular space. Evidence implicates these cascades in contributing to the progression through the stages of addiction. LPS lipopolysaccharide, RAGE receptor for advanced glycation end-products, IL-1β interleukin-1beta

Fig. 2

Lifetime alcohol consumption and HMGB1-TLR expression in the human postmortem orbitofrontal cortex. Expression of toll-like receptors (TLRs) 2, 3, and 4 and the TLR endogenous agonist high-mobility group box 1 (HMGB1) are positively correlated with lifetime alcohol consumption (kg) (Crews et al. 2013). Moderate drinking controls are clustered along the left of the graph due to low lifetime levels of alcohol consumption and concomitant low levels of TLR-HMGB1 expression. Alcoholic subjects all consumed more alcohol than moderate drinking controls but show a 10-fold variation in lifetime alcohol consumption. Note that the x axis is broken to allow for visualization of the moderate drinking control data. Repeated cycles of binge drinking are hypothesized to induce a progressive and persistent shift in the allostatic set point for negative affect that contributes to the neurobiology of addiction (Koob and Le Moal 2005). Depicted are correlations for individual TLRs and HMGB1 and overall grouped correlations with lifetime alcohol consumption

Fig. 3

Adolescent intermittent ethanol (AIE) treatment upregulates expression of toll-like receptors (TLRs) in the adult cerebellum. From postnatal day (P)25 to P55, AIE-treated male Wistar rats received a single daily intragastric dose of ethanol (5.0 g/kg, 20% ethanol, w/v) on a 2-day on/off schedule, and CON subjects received comparable volumes of water as described previously (Vetreno et al. 2016). Following a 25-day abstinence period, cerebellar tissue samples were collected on P80. Tissue samples were processed for qPCR and TLR mRNAs assessed as previously described (Vetreno and Crews 2012). Data are presented as mean ± SEM. *p < 0.05

Fig. 4

Simplified schematic depicting mechanisms of alcohol-induced alterations to brain plasticity. Alcohol binge drinking and abuse increases expression of the endogenous innate immune receptor agonist high-mobility group box 1 (HMGB1) leading to activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and subsequent induction of proinflammatory cytokines and oxidases (Crews et al. ; Qin and Crews ; Vetreno and Crews , ; Vetreno et al. 2016). This also leads to increased expression of toll-like receptors (TLRs) and the receptor for advanced glycation end-products (RAGE) (Crews et al. ; Vetreno and Crews , ; Vetreno et al. 2013), which are receptors for HMGB1. Activation of this signaling pathways leads to the establishment of positive loops of amplification that persist during abstinence from alcohol (Crews and Vetreno 2016) and alter synapse formation through increased expression of thrombospondins (Risher et al. 2015) and alterations in extracellular matrix proteins (Coleman et al. 2014). Concomitantly, innate immune induction causes epigenetic modifications that induce further release of HMGB1 (Zou and Crews 2014) that contribute to positive loops of amplification as well as alterations in synaptic plasticity molecules (Pandey et al. ; Sakharkar et al. 2016) that contribute to alterations in cognitive and emotive functioning. Innate immune driven epigenetic modifications might also lead to microglial alterations resulting in the reprogramming of the innate immune system. Innate immune induction also causes neurodegeneration (Braun and Crews ; Crews et al. 2000) and the loss of neuron-specific cell populations (Boutros et al. ; Vetreno et al. ; Vetreno and Crews ; Vetreno et al. 2016) that might also contribute to the dysfunctional cognitive and affective states observed in addiction (Crews and Vetreno 2016)

Fig. 5

Adolescent intermittent ethanol (AIE) treatment impairs reversal learning and increases risky decision-making in adulthood. a From postnatal day (P)25 to P55, AIE-treated male Wistar rats received a single daily intragastric (i.g.) dose of ethanol (5.0 g/kg, 20% ethanol, w/v) on a 2-day on/off schedule, and CON subjects received comparable volumes of water as previously described (Vetreno and Crews 2012). Following a 25-day abstinence period, spatial and reversal learning were assessed on the Barnes maze. While AIE treatment did not impair spatial learning, reversal learning, which provides a measure of behavioral flexibility, was impaired in adulthood as evidenced by increased latency to the reversal goal and increased perseveration. Following behavioral testing, brain tissue was collected and tissue stained for innate immune markers. Expression of toll-like receptor 4 (TLR4) was positive correlated with latency to the reversal goal, indicating that innate immune induction by AIE contributes to deficits in adult deficits in behavioral flexibility. Adopted from Vetreno and Crews (2012). Data are presented as mean ± SEM. *p < 0.05. b From P28 to P53, male Wistar rats received three daily i.g. doses of ethanol (5.0 g/kg, 25% ethanol, w/v) on a 2-day on/off schedule, and CON subjects received comparable volumes of water. Following an abstinent period on P63, training and testing were performed on the probability-discounting task. Prior AIE treatment significantly increased risky responses at reducing reward probabilities, relative to CON subjects. Expression of choline acetyltransferase (ChAT), a marker of cholinergic neurons, was reduced in the basal forebrain of adult AIE-treated animals that was negatively correlated with probability-discounting behavior. Adopted from Markou and colleagues (Boutros et al. 2015). Data are presented as mean ± SEM. *p < 0.05

Fig. 6

Mechanisms of stress- and ethanol-induced innate immune activation. Alcohol and stress activate the peripheral and central immune systems in multiple ways. Alcohol and stress cause microbiome dysbiosis and disrupt gut tight junctions, leading to permeability of the gut and release of bacteria and endotoxin that enter portal circulation-inducing inflammatory responses in the liver. The consequent release of inflammatory cytokines from the liver activates the innate immune system in brain through direct transport via cytokine receptors and activation of the vagus nerve. Stress and alcohol exposure leads to innate immune activation in brain that induces sickness-like behaviors that contribute to the stages of addiction. These pathways of ethanol- and stress-induced activation of HMGB1-LTR signaling likely contribute to the persistent and progressive stages of addiction