The role of neuroimmune signaling in alcoholism

Abstract

Alcohol consumption and stress increase brain levels of known innate immune signaling molecules. Microglia, the innate immune cells of the brain, and neurons respond to alcohol, signaling through Toll-like receptors (TLRs), high-mobility group box 1 (HMGB1), miRNAs, pro-inflammatory cytokines and their associated receptors involved in signaling between microglia, other glia and neurons. Repeated cycles of alcohol and stress cause a progressive, persistent induction of HMGB1, miRNA and TLR receptors in brain that appear to underlie the progressive and persistent loss of behavioral control, increased impulsivity and anxiety, as well as craving, coupled with increasing ventral striatal responses that promote reward seeking behavior and increase risk of developing alcohol use disorders. Studies employing anti-oxidant, anti-inflammatory, anti-depressant, and innate immune antagonists further link innate immune gene expression to addiction-like behaviors. Innate immune molecules are novel targets for addiction and affective disorders therapies. This article is part of the Special Issue entitled "Alcoholism".

Keywords: Addiction; Alcohol; Azithromycin (PubChem CID: 447043); Cytokines; Glycyrrhizin (PubChem CID: 14982); HMGB1; Ibudilast (PubChem CID: 3671); Indomethacin (PubChem CID: 3715); Minocycline (PubChem CID: 54675783); Naltrexone (PubChem CID: 5360515); Pioglitazone (PubChem CID: 4829); Rapamycin (PubChem CID: 5284616); Rifampin (PubChem CID: 5381226); Simvastatin (PubChem CID: 54454); TLR; miRNA-let-7.

Figure 1. Immune Activation and the Development of Substance Use Disorders

A variety of common, naturally occurring stressors such as injury or infection cause immune activation. Immune activation occurs in both the periphery and central nervous system and leads to inflammatory cytokine production. Activation of the immune system causes adaptive behavioral changes known as sickness behavior, which includes anhedonia, listlessness, lethargy and decreased activity, poor concentration, somnolence/sleepiness, loss of appetite and reduced social interaction. These changes are adaptive, transient and facilitate the recovery of the organism by reducing activity for healing. However, under conditions of chronic repeated stress and/or alcohol abuse, increased brain innate immune activation can lead to pathological and persistent changes in mood, cognition and other physiological factors such as sleep. Many of the behavioral characteristics of substance abuse disorders have been linked to peripheral and central immune activation, including substance self-administration, negative affect, decreased cognitive function and social withdrawal.

Figure 2. Mechanisms of Stress- and Ethanol-induced Immune Activation

Stress and ethanol activate the peripheral and central immune systems in multiple ways. Both stress and ethanol can enhance gut leakiness. This causes increased translocation of bacterial products such as endotoxins from the intestinal lumen to the periphery. Leaked bacterial products make their way to the liver via the portal system where they induce an inflammatory response from resident macrophages. The production of peripheral inflammatory cytokines such as TNFα, IL-1β and IL-6 impact the brain and behavior through multiple mechanisms. One way is the neural route. The vagus nerve expresses cytokines receptors and is activated by peripheral inflammation. The signal of peripheral inflammation is transmitted to central brain regions involved in the regulation of sickness behavior. Another route is the humoral route. Peripheral cytokines can cross the blood brain barrier either by transport proteins or by diffusion in regions where the barrier is leaky. This can lead to a central immune response. Stress and ethanol also activate glia through more direct mechanisms. Glucocorticoids are important for the priming effect of stress on microglia. Also, ethanol exposure can directly activate glia.

Figure 3. Microglial Activation Following Stress and Ethanol

A. In the healthy brain, microglia normally exist in a “resting” or ramified state. However, in response to insults microglia undergo a process known as activation which involves changes in microglial morphology, gene expression and function. Microglial activation can be morphologically classified according to stages of increasing activation, including hyper-ramified, bushy and amoeboid (illustrations adapted from Beynon & Walker, 2012). Hyper-ramified microglial activation occurs in response to relatively mild insults and is thought to be associated with cytokine release. Bushy and amoeboid microglia are observed in more overt forms of brain damage, such as seizure, stroke or trauma. B. Hyper-ramified microglial activation has been observed in chronically stressed brains. It is also the predominant form of microglial activation following alcohol abuse. Repeated cycles of stress and ethanol abuse result in increasingly sensitized/activated hyper-ramified microglia, contributing to the neurobiology of substance use disorders.

Figure 4. Mechanisms of HMGB1 signaling

The HMGB1 protein is comprised of two similar Box structures, A and B, and a long C terminal negatively charged tail. There are several cysteines, some of which can form disulfide bonds. HMGB1 is secreted from activated or stressed cells or from dying necrotic cells and acts as an immune modulator. A. TLR4 Agonist HMGB1. The reduced Cys106 thiol/Cys23-Cys45 disulfide bond form of HMGB1 acts directly as an agonist at TLR4 receptors to cause innate immune activation. B. RAGE agonist-HMGB1. The fully reduced non-disulfide form of HMGB1 acts at RAGE receptors to cause innate immune induction and neurite outgrowth. C. Heterodimer HMGB1/IL-1 agonist: HMGB1 forms pro-inflammatory complexes with cytokines such as IL-1β to cause enhanced IL-1β signaling through the IL-1 receptor and greater innate immune induction. D. The fully oxidized form of HMGB1 is inactive at immune receptors. E. HMGB1-miRNA-chaperone: HMGB1 binds miRNA and activates the RNA sensing TLR7 receptor increasing immune activation.

Figure 5. Neuron-like SH-SY5Y cells are more sensitive to ethanol induction of TLR7 than microglia-like BV cells

Shown are TLR7 mRNA levels following 24 hr. of treatment with various concentrations of ethanol in cultures of microglia-like BV2 and neuron-like RA-differentiated SH-SY5Y cells. TLR7 mRNA was determined using RT-PCR. Data is represented as percent control (%CON) for each respective cell type, with CON set at 100%. Note neuron-like SHSY-5Y show a maximal response at about 15 mM ethanol, whereas microglia show little response at this concentration, but show far larger responses at higher concentrations of ethanol. (*p<0.05, #p<0.01 vs. CON.) Adapted from: (Lawrimore and Crews, 2017)

Figure 6. Autocrine and Paracrine Innate Immune Activation by Ethanol

Ethanol causes Toll-like Receptor (TLR) activation in both autocrine and paracrine fashions. This occurs through release of endogenous TLR agonists. HMGB1 acts at TLRs directly and also acts as a chaperone for miRNA such as let-7 in microvesicles, promoting TLR7 signaling. Cytokines such as IL-1β bind cytokine receptors. Immune mediators act in both autocrine and paracrine fashions on neurons and microglia to amplify neuroimmune responses.

Figure 7. β2-microglobulin is induced in adult hippocampus following adolescent intermittent ethanol treatment

Shown are levels of β2 microglobin immunoreativity (β2M+IR) determined by immunohistochemistry in hippocampal dentate gyrus. Rats were treated with ethanol on an intermittent schedule through adolescence, i.e. adolescent intermittent ethanol treatment as described previously (Liu and Crews, 2015). β2M+IR was assessed on post-natal day 57, 24 hours after the last AIE ethanol treatment, and in adulthood on post-natal day 95. Note the ethanol-triggered induction of β2M during abstinence, but not at P57, just after ethanol treatment ended. (**p<0.01 compared with control at P95; #p<0.05 compared with AIE at P57).