Mechanisms of neuroimmune gene induction in alcoholism

Abstract

Rationale: Alcoholism is a primary, chronic relapsing disease of brain reward, motivation, memory, and related circuitry. It is characterized by an individual's continued drinking despite negative consequences related to alcohol use, which is exemplified by alcohol use leading to clinically significant impairment or distress. Chronic alcohol consumption increases the expression of innate immune signaling molecules (ISMs) in the brain that alter cognitive processes and promote alcohol drinking.

Objectives: Unraveling the mechanisms of alcohol-induced neuroimmune gene induction is complicated by positive loops of multiple cytokines and other signaling molecules that converge on nuclear factor kappa-light-chain-enhancer of activated B cells and activator protein-1 leading to induction of additional neuroimmune signaling molecules that amplify and expand the expression of ISMs.

Results: Studies from our laboratory employing reverse transcription polymerase chain reaction (RT-PCR) to assess mRNA, immunohistochemistry and Western blot analysis to assess protein expression, and others suggest that ethanol increases brain neuroimmune gene and protein expression through two distinct mechanisms involving (1) systemic induction of innate immune molecules that are transported from blood to the brain and (2) the direct release of high-mobility group box 1 (HMGB1) from neurons in the brain. Released HMGB1 signals through multiple receptors, particularly Toll-like receptor (TLR) 4, that potentiate cytokine receptor responses leading to a hyperexcitable state that disrupts neuronal networks and increases excitotoxic neuronal death. Innate immune gene activation in brain is persistent, consistent with the chronic relapsing disease that is alcoholism. Expression of HMGB1, TLRs, and other ISMs is increased several-fold in the human orbital frontal cortex, and expression of these molecules is highly correlated with each other as well as lifetime alcohol consumption and age of drinking onset.

Conclusions: The persistent and cumulative nature of alcohol on HMGB1 and TLR gene induction support their involvement in alcohol-induced long-term changes in brain function and neurodegeneration.

Keywords: Alcohol use disorder; Amphoterin; Cytokines; Ethanol; Frontal cortex; Gut permeability; HMGB1; Innate immune; Microglia; RAGE; TLR4.

Fig. 1

Activated morphology of microglia. Representative schematics and photomicrographs of human brain microglia (Iba1 immunohistochemistry) depicting morphological stages of microglial activation. Ramified or “resting” microglia are characterized by long, ramified processes with comparatively small cell bodies. Mildly activated hyper-ramified microglia are characterized by increased branching of processes as well as lengthening of processes and the secretion of proinflammatory cytokines (Beynon and Walker 2012). Bushy morphology is intermediate activation and is characterized by swollen, truncated processes, and enlarged cell bodies. Amoeboid or “phagocytic” microglia are characterized rounded macrophage-like morphology with no or few processes and are associated with maximal proinflammatory activation, oxidative-free radicals, and microglial apoptosis (Kreutzberg ; Raivich et al. 1999). Post-mortem human brains from controls and alcoholics contain all of these subtypes likely due in part to age-related changes in all individuals. Alcoholics have more microglial markers indicative of hyper-ramified morphology in alcoholics increasing staining compared to control ramified less dense staining. Figure adapted from He and Crews (2008) and Beynon and Walker (2012)

Fig. 2

High-mobility group box 1 (HMGB1) is actively and/or passively released leading to multiple signaling pathways. Actively released HMGB1 from brain slice cultures found histochemical evidence of release from neurons by ethanol (Zou and Crews 2014), although HMGB1 release likely occurs from most brain cell types. Neurons and glia release HMGB1 during glutamate excitation (Maroso et al. 2010, 2011). HMGB1 is also released during necrotic cell death activating innate immune signaling. HMGB1 has multiple signaling mechanisms regulated by oxidation of cysteines. Fully oxidized HMGB1 (blue, left) does not activate proinflammatory signaling, although it may contribute to resolution of the proinflammatory state. HMGB1 in the all-thiol form (yellow, middle) is an agonist at the receptor for advanced glycation end-products (RAGE; Allette et al. 2014). All-thiol-HMGB1 also forms heterodimers with proinflammatory molecules such as interleukin-1beta (IL-1β; Wahamaa et al. 2011) with the HMGB1-IL1 heterodimer synergistically enhancing stimulation of the IL-1β receptor leading to proinflammatory gene induction through activation of NF-κB transcription (Venereau et al. 2012). Disulfide-HMGB1 (red, right) activates Toll-like receptor 4 (TLR4) also leading to nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induction of proinflammatory cytokines. Adapted from Tang et al. (2012)

Fig. 3

Ethanol in the gut causes leakage of bacterial products into the portal vein increasing hepatic TNFα release into the blood which induces neuroimmune gene expression in the brain. High doses of consumed alcohol in the gut (i.e., at least 2–3 g/kg ETOH intragastric doses [Ferrier et al. 2006]) increases permeability allowing bacterial products such as endotoxin-lipopolysaccharide (LPS) to enter portal circulation. Alcohol and LPS enter portal circulation leading to induction of liver tumor necrosis factor-alpha (TNFα) and other proinflammatory cytokines that are released into the blood and enter the brain through cytokine-specific receptor transport (e.g., the TNFα receptor) (See Qin et al. for details). This activates positive loops of proinflammatory gene induction in the brain that all converge upon nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) that amplify through autocrine and paracrine positive loops. Further, ethanol causes nuclear release of high-mobility group box 1 (HMGB1) and other danger associated molecular patterns (DAMPs) that enters the extracellular space activating Toll-like receptors (TLRs) and the receptor for advanced glycation end products (RAGE) in an autocrine and paracrine fashion further amplifying NF-κB transcription. In parallel, proinflammatory oxidases form reactive oxygen species (ROS) also amplify activation of NF-κB transcription that can contribute to inhibition of neurogenesis and cause neuronal death

Fig. 4

Neuronal excitability releases HMGB1, which increases cytokine secretion by microglia that activate astrocytes that increase glutamate increasing neuronal excitability through glutamate, HMGB1, and other signals. Glutamate, alcohol, and other factors release HMGB1 from neurons causing microglia to become hyper-ramified, resulting in their release of high-mobility group box 1 (HMGB1) and other proinflammatory cytokines. These innate immune signaling molecules stimulate astrocytes reducing glutamate uptake (Zou and Crews 2005) and increasing astrocyte release of glutamate that induce neuronal excitability causing further increases in HMGB1 release. Increased neuronal hyperexcitability can contribute to potentiation of specific neuronal connectivity and/or to excitotoxic neuronal cell death. Figure adapted from Kettenmann et al. (2013)

Fig. 5

Ethanol releases high-mobility group box 1 (HMGB1) leading to activation of neuroimmune signaling. Ethanol causes the release of HMGB1 into the media from hippocampal-entorhinal cortex (HEC) slice culture. a Ethanol causes dose-dependent increase of HMGB1 release into culture media, relative to controls (* p < 0.05, n = 3). b Western blot analysis of the whole cell lysate revealed that HMGB1 protein content increased progressively over time in response to ethanol treatment (100 mM EtOH). c RT-PCR analysis found that ethanol treatment (100 mM) for 4 days significantly increased Toll-like receptor (TLR) 2, TLR4, and HMGB1 mRNA in HEC slice culture (* p < 0.05, n = 3). Data are adapted from Zou and Crews (2014)

Fig. 6

Adolescent intermittent ethanol (AIE) treatment leads to a persistent induction of neuroimmune genes in the adult brain. a Male Wistar rats were treated with ethanol (5 g/kg/day, i.g., w/v, 2 days on/2 days off) or comparable volumes of water from postnatal day (P)25 to P55. Brain tissue was collected either 24 h (P56) or 25 days after the last ethanol treatment (P80) to assess the persistent expression of neuroimmune markers. Toll-like receptor 4 (TLR4) immunoreactivity was upregulated 24 h after ethanol treatment and remained elevated for 25 days following the conclusion of ethanol treatment. In contrast, there was no change in receptor for advanced glycation end-product (RAGE) expression immediately following the conclusion of ethanol treatment but was elevated 25 days after the conclusion of ethanol treatment. Expression of high-mobility group box 1 (HMGB1), an endogenous TLR4 and RAGE agonist, was increased both 24 h and 25 days following the conclusion of ethanol treatment. b In a separate cohort of subjects, frontal cortex tissue samples were collected from CON- to AIE-treated animals on P80 (25 days following the conclusion of ethanol treatment) and neuroimmune gene mRNA levels were assessed. Adolescent binge ethanol treatment led to long-term upregulation of proinflammatory cytokines (tumor necrosis factor-alpha [TNFα] and monocyte chemotactic protein-1 [MCP-1]) and oxidases (cyclooxygenase [COX-2] and gp91^PHOX [NOX2]). **p < 0.01, relative to CONs. Data are adapted from Vetreno and Crews (2012) and Vetreno et al. (2013)

Fig. 7

Risk of alcoholism and induction of innate immune genes correlate with age of drinking onset in humans. a Toll-like receptor 4 (TLR4) and high-mobility group box 1 (HMGB1) expression in the post-mortem human brain is negatively correlated with age of drinking onset adapted from Vetreno et al. (2013). b An earlier age of drinking onset is predictive of an increased likelihood of developing an alcohol use disorder during an individual’s lifetime. Adapted from Grant (1998)