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Neuropharmacology 122 (2017) 56e73

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Invited review

The role of neuroimmune signaling in alcoholism

Fulton T. Crews, Colleen J. Lawrimore, T. Jordan Walter, Leon G. Coleman Jr. *

University of North Carolina, Chapel Hill School of Medicine, Bowles Center for Alcohol Studies, CB# 7178 UNC-CH, Chapel Hill, NC 27599, USA

a r t i c l e i n f o

Article history:Received 14 September 2016Received in revised form24 January 2017Accepted 29 January 2017Available online 1 February 2017

Chemical compounds studied in this article:Minocycline (PubChem CID: 54675783)Rapamycin (PubChem CID: 5284616)Azithromycin (PubChem CID: 447043)Rifampin (PubChem CID: 5381226)Indomethacin (PubChem CID: 3715)Simvastatin (PubChem CID: 54454)Glycyrrhizin (PubChem CID: 14982)Pioglitazone (PubChem CID: 4829)Ibudilast (PubChem CID: 3671)Naltrexone (PubChem CID: 5360515)

Keywords:HMGB1TLRCytokinesmiRNA-let-7AlcoholAddiction

* Corresponding author.E-mail address: leon_coleman@med.unc.edu (L.G.

http://dx.doi.org/10.1016/j.neuropharm.2017.01.0310028-3908/© 2017 Elsevier Ltd. All rights reserved.

a b s t r a c t

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 andtheir associated receptors involved in signaling between microglia, other glia and neurons. Repeatedcycles of alcohol and stress cause a progressive, persistent induction of HMGB1, miRNA and TLR receptorsin brain that appear to underlie the progressive and persistent loss of behavioral control, increasedimpulsivity and anxiety, as well as craving, coupled with increasing ventral striatal responses thatpromote reward seeking behavior and increase risk of developing alcohol use disorders. Studiesemploying anti-oxidant, anti-inflammatory, anti-depressant, and innate immune antagonists further linkinnate immune gene expression to addiction-like behaviors. Innate immune molecules are novel targetsfor addiction and affective disorders therapies.

This article is part of the Special Issue entitled “Alcoholism”.© 2017 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572. Alcohol and stress, microglia and neuroimmune signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573. Sensitization of glia in the stressed and addicted brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584. Stress, inflammation and affective disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595. Innate immune molecules mimic addiction-like behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616. HMGB1, Toll-like receptors and innate immune signaling in the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627. TLR4 - a key brain receptor involved in ethanol induced neuropathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 638. Induction of innate immune genes, and miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649. Autocrine and paracrine processes amplify NF-kB innate immune gene induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

10. New treatments strategies based on immune pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6511. Neuroimmune basis of addiction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Coleman).

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1. Introduction

Neuroimmune signaling is emerging as a contributor to thedevelopment of alcohol use disorders. Alcohol consumption andstress increase brain levels of known innate immune signalingmolecules. Microglia, the innate immune cells of the brain, play animportant role in brain development and function. As mesodermaltissue specific monocytes, microglia express signaling moleculesfirst characterized as “acute phase proteins” that increase in bloodrapidly in response to infections. More recently, Toll-like receptors(TLRs), members of a superfamily of receptors, i.e. - interleukin-1receptor/toll-like receptor superfamily, were found to initiate pro-inflammatory responses to infectious agents by sensing largemolecules with lipid, sugar, protein and nucleic acid components.Pro-inflammatory cytokines and their associated receptors sharecomplex signaling pathways with TLRs that converge upon NF-kB, akey innate immune transcription factor in monocyte-like cells, andother transcription factors regulated by TLR-kinase cascades.Recent studies in brain, which is generally sterile, indicate endog-enous TLR agonists, i.e. danger-associated molecular patterns(DAMPs), represent important signaling systems between micro-glia, other glia and neurons. Further, emerging studies find cyto-kines and DAMPs may signal directly between neurons. High-mobility group box 1 (HMGB1) is a cytokine-like molecule highlyexpressed in neurons that can directly activate TLRs as well asenhancing responses at other pro-inflammatory receptors. Alcohol-and stress-induced increases in Toll-like receptors, and other neu-roimmune signaling molecules in brain sensitize the brain tofurther innate immune gene induction, resulting in progressive andpersistent increases in HMGB1 and TLR signaling as evidenced byprefrontal cortical expression levels correlating with lifetimealcohol consumption across alcoholic and moderate drinking con-trols (Crews et al., 2013). Mechanistic studies of epilepsy findneuronal firing results in the rapid release of HMGB1 and IL-1bpreceding the seizure, inducing changes in excitatory receptors thatsensitize neurons to excitation, lowering seizure threshold andcontributing to the “kindling” progressive increase in seizure fre-quency and severity with each event (Maroso et al., 2011). Althoughdifferent brain regions and circuits distinguish addiction from ep-ilepsy, HMGB1 and TLR signaling and cycles of stress and drugabuse appear to share the common mechanisms of sensitization ofcircuitry through increased HMGB1 and TLR signaling.

Alcohol administration to mice and rats increases expression ofHMGB1 and multiple TLR receptors in brain that persist for longperiods of abstinence. Furthermore, human post-mortem alcoholicbrains show increased expression of microglial markers, cytokines,TLR receptors and HMGB1, with the latter two being correlatedwith lifetime alcohol consumption. Repeated alcohol, drug use andstress in rodents sensitize microglia toward hyperactivity, likelythrough induction of immune signaling (see Sections 2 and 5).Cytokine infusions into specific rodent brain regions initiate cravingand alcohol consumption, and human alcoholics have increasedplasma pro-inflammatory cytokines that correlate with alcoholcraving and severity of dependence (see Section 5). Further, pro-inflammatory cytokines TNFa, IL-6, and IL-1b play critical roles inthe pathologies of mood disorders (Bhattacharya et al., 2016;Bhattacharya and Drevets, 2017). These observations, detailedfurther below, support the hypothesis that stress and alcohol in-duction of neuroimmune signaling contribute to the progressive,persistent increase in craving and mood dysfunction in addiction.Recent discoveries indicate broad induction of TLR receptors inbrain as well as endogenous agonists for TLR receptors, includingmiRNA that activate nucleic acid sensing TLRs and HMGB1 thatactivates TLR4, a key signaling TLR in the brain. Although poorlyunderstood, studies suggest that activation and escalated signaling

of this system leads to progressive and persistent loss of behavioralcontrol, increased impulsivity and anxiety, as well as negative affectand craving coupled with increasing ventral striatal responses thatpromote reward seeking behavior and increase risk of developingalcohol use disorders. Studies employing anti-oxidant, anti-in-flammatory, anti-depressant, and innate immune antagonistsfurther link innate immune gene expression to addiction-like be-haviors. In this review we discuss both preclinical rodent and hu-man studies that examine the role of neuroimmune signaling inalcoholism. Unless otherwise noted the presented studies wereperformed in rodents. From these studies, we surmise that innateimmune molecules are novel targets for addiction and affectivedisorders therapies.

2. Alcohol and stress, microglia and neuroimmune signaling

Repeated cycles of alcohol, drugs of abuse and stress interact,contributing to a progressive sequence of stages leading to addic-tion (Volkow et al., 2016). Alcohol, drugs of abuse and stress alsotrigger neuroinflammatory responses. Inflammation may havebeneficial or maladaptive consequences on brain function (Fig. 1).Physical stressors, such as injury and infection, increase pro-inflammatory cytokines and other leukocyte derived proteins andprostaglandins as part of the acute inflammatory phase response.Stressor-induced inflammation also triggers adaptive behavioralchanges known as “sickness behavior.” Sickness behavior includessocial withdrawal, decreased activity, somnolence/sleepiness,anhedonia, etc. that help to conserve energy and facilitate recovery(Dantzer et al., 2008). Interestingly, even psychological stressors(e.g. e social stress) can cause inflammation (Steptoe et al., 2007).The acute pro-inflammatory response, often referred to as Th1,includes signals that activate a delayed wound healing and cellgrowth through “anti-inflammatory” Th2 innate immune signalingmolecules. While the benefit of the Th1 to Th2 progressionfollowing trauma or infection may be apparent, what happens inbrain is poorly understood. Therefore, both physical and psycho-logical stressors may enhance inflammation, initiating molecularand behavioral changes that facilitate recovery. While stress andinflammation can lead to the adaptive response of sicknessbehavior, intense or chronic stress and inflammation can becomemaladaptive and lead to neuropsychiatric disease such as depres-sion. Indeed, the similarities between sickness behavior anddepression have been noted (Maes et al., 2012). Models ofdepression include endotoxin treatments of rodents followedbeyond the 24 h “sickness behavior” time course (Dantzer et al.,2008). The inflammation caused by excessive alcohol consump-tion may have similar maladaptive consequences.

Studies have found similar mechanisms through which stressand alcohol contribute to inflammation (Fig. 2). Stress enhancesintestinal permeability and bacterial translocation from the gutlumen (Garate et al., 2013). The immune systemmounts a responseto the leaked bacteria, thereby causing peripheral inflammation.Alcohol also contributes to gut leakiness and translocation of bac-teria from the intestinal lumen to the periphery (Adachi et al.,1995). Leaked bacterial products cause inflammation in the liverand release of pro-inflammatory (Giraldo et al., 2010)cytokines intothe systemic circulation (Mayfield et al., 2013). Therefore, stress andalcohol can cause gut leakiness, leading to peripheral inflammation.Both stress and alcohol (Ellis, 1966) also increase glucocorticoids.Intriguingly, glucocorticoids have pro-inflammatory effects underthe right circumstances (Sorrells et al., 2009).While glucocorticoidsare acutely anti-inflammatory at high doses, glucocorticoids alsohave a priming effect on the inflammatory response. That is to say,prior exposure to glucocorticoids enhances the inflammatoryresponse to subsequent stimuli (Frank et al., 2010). Therefore, both

Fig. 1. Immune Activation and the Development of Substance Use Disorders. A variety of common, naturally occurring stressors such as injury or infection cause immuneactivation. Immune activation occurs in both the periphery and central nervous system and leads to inflammatory cytokine production. Activation of the immune system causesadaptive behavioral changes known as sickness behavior, which includes anhedonia, listlessness, lethargy and decreased activity, poor concentration, somnolence/sleepiness, loss ofappetite and reduced social interaction. These changes are adaptive, transient and facilitate the recovery of the organism by reducing activity for healing. However, under conditionsof chronic repeated stress and/or alcohol abuse, increased brain innate immune activation can lead to pathological and persistent changes in mood, cognition and other physio-logical factors such as sleep. Many of the behavioral characteristics of substance abuse disorders have been linked to peripheral and central immune activation, including substanceself-administration, negative affect, decreased cognitive function and social withdrawal.

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stress and alcohol may exert inflammatory/priming effects throughglucocorticoid signaling. Stressors also enhance the systemicrelease of cytokines (e.g. IL-1 and IL-6) (Merlot et al., 2002) as wellas danger-associated molecular patterns (DAMPs) such as highmobility group box 1 (HMGB1), uric acid and heat-shock protein 72(Hsp72) (Maslanik et al., 2013). Physical stressors such as traumacan release DAMPs directly via cellular damage. Immune activationdue to psychological stressors might involve multiple mechanismsincluding sympathetic nervous system activation, as noradrenalinecauses Hsp72 release from neutrophils (Giraldo et al., 2010) orendotoxin release due to increased intestinal permeability (Ait-Belgnaoui et al., 2012; Ferrier et al., 2003) that may involve CRFrelease (Overman et al., 2012; Saunders et al., 2002; Teitelbaumet al., 2008). Released DAMPs and cytokines can activate innateimmune cells, thereby increasing inflammatory signaling. Periph-eral inflammation can impact the brain and behavior in many ways(Fig. 2) (Miller and Raison, 2016). One route is the neural route: thevagus nerve can sense peripheral inflammatory cytokines such asIL-1b through IL-1 receptors (Ek et al., 1998). Activation of vagalafferents transmits signals to the central nervous system whereneural centers that promote sickness behavior are activated.Indeed, vagotomy reduces the sickness behavior response to a pe-ripheral injection of LPS (Bluthe et al., 1994). Another means bywhich peripheral inflammation can impact the brain and behavioris the humoral route. This includes diffusion of cytokines from theperiphery through leaky regions of the blood brain barrier, such asthe circumventricular organs and by transport of cytokines acrossthe blood brain barrier. Indeed, TNFa transporters on the blood

brain barrier are essential for systemic inflammation to cause braininflammation (Qin et al., 2007). A third route is the cellular route, bywhich activated immune cells such as monocytes traffic into thebrain. The brain recruits monocytes in response to peripheralinflammation (D'Mello et al., 2009). In each case, the peripheralinflammatory signal is transmitted to the brain, where it can impactcentral inflammation and behavior. Thus, alcohol abuse and stressshare common mechanisms of inducing neuroimmune signalingthat could contribute to the progression and persistence of theneuropathology of addiction by cycles of stress and substanceabuse.

3. Sensitization of glia in the stressed and addicted brain

Microglia are the resident macrophages of the brain and theprimary cells of the neuroimmune system. They are involved inmany physiological processes in the healthy brain, including syn-aptic pruning, debris clearance, immune surveillance/defense,neurogenesis and more (Kettenmann et al., 2011, 2013). Microglianormally exist in a “resting” state, but can become activated inresponse to insults. Activation of microglia has been categorizedaccording to either morphological or functional standards. Underthe morphological classification, “resting” microglia are called“ramified” microglia. Morphological activation of microglia pro-gresses through multiple states of increasing activation, includingthe hyper-ramified state, the bushy state and finally the amoeboidstate (Fig. 3) (Beynon and Walker, 2012). Fully activated amoeboidmicroglia are phagocytic cells with a rounded morphology. To date,

Fig. 2. Mechanisms of Stress- and Ethanol-induced Immune Activation. Stress andethanol activate the peripheral and central immune systems in multiple ways. Bothstress and ethanol can enhance gut leakiness. This causes increased translocation ofbacterial products such as endotoxins from the intestinal lumen to the periphery.Leaked bacterial products make their way to the liver via the portal systemwhere theyinduce an inflammatory response from resident macrophages. The production of pe-ripheral inflammatory cytokines such as TNFa, IL-1b and IL-6 impact the brain andbehavior through multiple mechanisms. One way is the neural route. The vagus nerveexpresses cytokines receptors and is activated by peripheral inflammation. The signalof peripheral inflammation is transmitted to central brain regions involved in theregulation of sickness behavior. Another route is the humoral route. Peripheral cyto-kines can cross the blood brain barrier either by transport proteins or by diffusion inregions where the barrier is leaky. This can lead to a central immune response. Stressand ethanol also activate glia through more direct mechanisms. Glucocorticoids areimportant for the priming effect of stress on microglia. Also, ethanol exposure candirectly activate glia.

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no known studies have observed alcohol causing an amoeboidmicroglial activation. Rather, alcohol seems to sensitize microglia,inducing a hyper-ramified activation state characterized by modestmorphological changes and increased release of cytokines andother signaling molecules (Fig. 3). Functional microglial activationis traditionally understood as occurring along a spectrum betweentwo extremes, although recent studies suggest functional activationis a more complex phenomenon (Xue et al., 2014). Functionalactivation states range from the pro-inflammatory, destructive M1state (comparable to systemic Th1) to the anti-inflammatory,reparative M2 state (comparable to systemic Th2). M1 microgliasecrete pro-inflammatory cytokines such as TNFa, IL-1b and IL-6

and produce reactive oxygen species through increased iNOS andNADPH oxidase expression. M2 microglia secrete anti-inflammatory cytokines such as IL-10 and synthesize extracellularmatrix products to assist with healing (Cherry et al., 2014). Theexact relationship between the morphological and functionalactivation states is currently unclear. Microglia also undergoanother phenomenon known as priming, or sensitization. Primingoccurs when an initial stimulus causes a greater microglialresponse to a subsequent stimulus (Perry and Holmes, 2014).Primed microglia produce a greater inflammatory response to im-mune stimuli than non-primed microglia.

Interestingly, astrocytes are also an important component of thebrain immune system (Farina et al., 2007). Astrocytes are involvedin numerous physiological processes, such as metabolic support ofneurons and modulation of synaptic transmission (Khakh andSofroniew, 2015). Astrocytes express multiple immune receptorsand cytokines that allow them to participate in immune processes(Jensen et al., 2013). In response to insults, astrocytes also undergoa process of activation or reactive gliosis that serves to limit tissuedamage (Pekny and Pekna, 2014). Some studies suggest that, likemicroglia, astrocytes are capable of adopting pro-inflammatory andanti-inflammatory states (Jang et al., 2013). Together, microglia andastrocytes are the primary components of the brain immune sys-tem. Both microglia and astrocytes interact with neuronal synap-ses, positioning these cells to regulate synaptic transmission.Therefore, changes in neuronal functioning may occur throughaltered glial functioning.

Both stress and alcohol can affect glia. Various types of acute andchronic stress, including restraint (Tynan et al., 2010), foot shock(Frank et al., 2007), social defeat (Wohleb et al., 2011) and chronicvariable stress (Kreisel et al., 2014), increase microglial markerssuch as CD11b and Iba1 across multiple brain regions, consistentwith microglial activation. Acute stress also primes microglia.Exposure to acute foot shock enhanced the ex vivo microglial in-flammatory response to the inflammogen lipopolysaccharide (LPS)(Frank et al., 2007). In addition to microglia, stress can also affectastrocytes. Sub-chronic stress increased the astrocyte marker GFAPin the hippocampus, suggestive of astrocyte activation (Lambertet al., 2000). Therefore, stress activates/primes glia. Variousalcohol treatment protocols also alter glia. Ethanol increasesmicroglial markers such as CD11b (Fernandez-Lizarbe et al., 2009)and Iba1 (Qin and Crews, 2012a) in vivo, consistent with microglialactivation. Alcohol exposure can also prime the microglial responseto peripheral inflammation (Qin and Crews, 2012a). Astrocytes arealso affected by alcohol, as evidenced by increased GFAP (Alfonso-Loeches et al., 2010b). Other studies show that alcohol consump-tion can initially increase astrocytic GFAP in the hippocampus, butdecrease GFAP after extended exposure (Franke, 1995). Therefore,alcohol can also impact glia. Repeated bouts of exposure to thesestimuli may lead to increasing glial activation that persists overtime. Indeed, two alcohol binge treatments activate microglia to agreater extent than a single binge treatment (Marshall et al.,2016b). Binge ethanol also increases microglial markers up tofour weeks following the binge, indicative of persistent activation(Marshall et al., 2013). Thus, repeated episodes of stress and/oralcohol abuse may cause escalating and persistent glial activation(Fig. 3), and this pathological activation/sensitization maycontribute to the neurobiology of alcohol use disorders.

4. Stress, inflammation and affective disorders

Stress and the resulting inflammation impact the neuroimmunesystem of the CNS. Stress sensitizes microglia to inflammation in anHMGB1-dependent manner (Weber et al., 2015). Chronic stressactivatesmicroglia inmultiple brain regions (Tynan et al., 2010) and

Fig. 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 insultsmicroglia undergo a process known as activation which involves changes in microglial morphology, gene expression and function. Microglial activation can be morphologicallyclassified according to stages of increasing activation, including hyper-ramified, bushy and amoeboid (illustrations adapted from Beynon and Walker, 2012). Hyper-ramifiedmicroglial activation occurs in response to relatively mild insults and is thought to be associated with cytokine release. Bushy and amoeboid microglia are observed in moreovert 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 predominantform of microglial activation following alcohol abuse. Repeated cycles of stress and ethanol abuse result in increasingly sensitized/activated hyper-ramified microglia, contributingto the neurobiology of substance use disorders.

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causes depression-like behavior. Administration of microglial-modulating agents can block the development of depression-likebehavior, suggesting that microglia may have a causal role in thedevelopment of depression (Kreisel et al., 2014). Human studiesalso support a role for chronic stress and inflammation in affectivedisorders. Administration of inflammatory agents (such as inter-feron-a for the treatment of hepatitis C) can cause depression inpreviously non-depressed patients (Bonaccorso et al., 2002). Hu-man studies find individuals with increased plasma levels of CRP,an acute phase marker of inflammation, have an increased risk fordepression (Young et al., 2014). Interestingly, increased CRP in ad-olescents has also been found to predict addiction later in life(Costello et al., 2013). Other studies find peripheral inflammationcan increase brain expression of innate immune signals andcontribute to depression and negative affect (Harrison et al., 2014).Imaging studies in humans show that a marker of microglial acti-vation, TSPO, is elevated in patients with depression (Setiawanet al., 2015). Depressed patients who committed suicide showedincreased expression of microglial markers Iba1 and CD45 and

increased levels of the inflammatory cytokine Ccl2 in the cingulatecortex (Torres-Platas et al., 2014). Studies also find changes inastrocyte markers in the brains of depressed patients (Rajkowskaand Stockmeier, 2013). These findings suggest that stress contrib-utes to increased brain innate immune signaling induction andpsychopathology.

Stress-induced CNS inflammation can contribute to affectivedisorders through many mechanisms. Acute inflammation altersthe activity of neural circuitry implicated in anxiety and depression.For example, acute inflammation changes activity in the cingulatecortex, medial prefrontal cortex and amygdala (Harrison et al.,2009). Acute inflammation can also impair spatial memory inhumans (Harrison et al., 2014), potentially contributing to thecognitive changes seen in affective disorders. Furthermore, studiesfind that a variety of cytokine receptors, such as those for TNFa, IL-1b, IL-6 and the interferons, are expressed on neurons (Khairovaet al., 2009), suggesting that cytokines act directly on neurons toinfluence their activity. Inflammation can also act on the seroto-nergic system to cause depression-like behavior. LPS increases

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activity of the serotonin transporter (SERT) and increasesdepression-like behavior. The ability of inflammation to causedepression-like behavior is lost in SERT knock-out animals (Zhuet al., 2010). Inflammation also increases the activity of anenzyme known as indoleamine 2,3-dioxygenase (IDO) in microglia.IDO generates the compound kynurenine, which has been shown toinduce depression-like behavior (O'Connor et al., 2009). Pharma-cological inhibition of IDO blocked the development of depression-like behavior (O'Connor et al., 2009) and blocks microglial activa-tion. The trafficking of peripheral inflammatory monocytes into theCNS has also been implicated in stress-induced behaviors. Stresscauses trafficking of peripheral monocytes into the brain that, inturn, promotes anxiety-like behavior (Wohleb et al., 2013). Thus,stress-induced microglial activation contributes to increaseddepression-like negative affect and anxiety-like morbidity thatoverlaps with and contributes to progressive stages of addiction.

Inflammatory cytokines such as TNFa and IL-1b can also impactneuronal plasticity. Interestingly, low physiological levels of TNFaand IL-1b seem to be important for long-term potentiation (LTP);however, high pathological levels of these cytokines disrupt theprocess. Blocking TNFa in hippocampal slices decreased synapticstrength (Beattie et al., 2002). However, excessive TNFa can inhibitLTP in hippocampal slices (Tancredi et al., 1992). The effects of cy-tokines on plasticity are behaviorally relevant, as TNFa over-expressing mice show decreased performance on spatial learningandmemory tasks (Aloe et al., 1999). Also, physiological doses of IL-1b are needed for memory. Blockade of IL-1b signaling by itsantagonist, IL-1ra, impairs memory. However, addition of excessiveIL-1 b also impairs memory (Goshen et al., 2007). Therefore, im-mune signaling may alter neurons and neural circuitry and changebehavioral phenotypes by impacting plasticity through changes incytokine levels.

Inflammation can also impact neurogenesis, which is implicatedin affective disorders. Neurogenesis occurs throughout adulthoodin discrete brain regions, including the forebrain subventricularzone and the subgranular zone of the hippocampal dentate gyrus.Hippocampal neurogenesis contributes not only to learning andmemory (Shors et al., 2001), but also mood and affective state(Malberg et al., 2000). Decreased neurogenesis contributes both toanxiety and depression (Hill et al., 2015). The ability of anti-depressants to alleviate depression-like behavior is dependent onhippocampal neurogenesis (Santarelli et al., 2003). Both inflam-mation (Ryan and Nolan, 2016) and chronic stress (Kreisel et al.,2014) reduce neurogenesis and cause depression-like behavior.Indeed, stress induces IL-1b in the hippocampus which decreasesneurogenesis and contributes to depression. Inhibition of IL-1bblocks stress-induced decreases in neurogenesis and depression-like behavior (Koo and Duman, 2008). Furthermore, enhancedneurogenesis is sufficient to reverse the anxiety-like anddepression-like behavior caused by chronic corticosterone treat-ment (Hill et al., 2015). Therefore, stress can increase CNS immunesignaling, which contributes to affective disorders.

5. Innate immune molecules mimic addiction-like behavior

Like stress, excessive alcohol use can impact the neuroimmunesystem, contributing to the development of alcohol use disordersand related psychopathology in many ways. There are multiplemechanisms through which this can occur. Alcohol is capable ofacting directly on immune cells to increase inflammation. Alcoholcan directly activate microglia in vitro, increasing expression of pro-inflammatory genes such as TNFa, IL-1b and iNOS (Fernandez-Lizarbe et al., 2009). Alcohol also increases neuroimmunesignaling molecules in vivo. These effects in vitro and in vivo aredependent on TLR4, which seems to play a pivotal role in alcohol-

induced neuroimmune signaling (Alfonso-Loeches et al., 2010b).Repeated binge alcohol treatment persistently increases neuro-immunemolecules such as TLR3, TLR4, HMGB1, RAGE, etc. (Vetrenoand Crews, 2012; Vetreno et al., 2013). Studies on the post-mortembrains of human alcoholics also find increased microglial markers(He and Crews, 2008; Rubio-Araiz et al., 2016) and increasedastrocyte markers (Rubio-Araiz et al., 2016). Post-mortem humanalcoholic brains show increased neuroimmune molecules, such asMCP-1, TL2, TLR3, TLR4 and HMGB1 (Crews et al., 2013; He andCrews, 2008). Furthermore, alcohol appears to cause a pro-inflammatory M1 activation of microglial cells, as alcohol treat-ment induces expression of pro-inflammatory cytokines, such asTNFa and IL-1b, and enzymes, such as NADPH oxidase (Qin andCrews, 2012a; Qin et al., 2008). Further, alcoholic hepatic enceph-alopathy patients have increased microglial proliferation (Denniset al., 2014). Thus, alcohol increases microglia and glial signalingcontributing to lasting changes.

Alcohol also sensitizes the neuroimmune system to subsequentinflammatory stimuli. Prior alcohol treatment enhanced brainlevels of pro-inflammatory cytokines to immune challenges such aslipopolysaccharide (LPS) or polyinosine-polycytidylic acid (poly I:C)(Qin and Crews, 2012a; Qin et al., 2008). It is important to note thatLPS itself does not enter the brain, but causes systemic release ofimmune mediators that do enter the brain that are required forcentral LPS responses such as TNFa (Qin et al., 2007). It is unclearwhether poly I:C crosses into the brain, but it does cause TNFarelease similar to LPS (Qin and Crews, 2012a). The sensitization orpriming of the neuroimmune response has important conse-quences for the pathogenesis of alcohol use disorders. Alcoholneuroimmune sensitization increases neurodegeneration and lossof neurogenesis following an immune challenge (Qin and Crews,2012a, b; Qin et al., 2008). Neurodegeneration and decreasedneurogenesis have been linked to a variety of neuropsychiatricdiseases. Therefore, repeated exposure to alcohol enhances andsensitizes neuroimmune signaling, thereby contributing to molec-ular and cellular causes of alcohol use disorders and co-morbiditiesof alcoholism, such as affective disorders.

Current theories of addiction posit a three-staged cycle thatcontributes to substance use disorders: binge/intoxication, with-drawal/negative affect and preoccupation/craving (Koob andVolkow, 2010). Interestingly, the neuroimmune system canimpact the neurobiology of addiction at multiple points in thisprocess. Regarding the binge/intoxication, multiple immune in-terventions have been found to regulate alcohol consumption. In-jection of the IL-1 receptor antagonist or the anti-inflammatorycytokine IL-1Qin and Crews, 2012a0 into the basolateral amygdalareduced alcohol self-administration (Marshall et al., 2016a, 2016c).Furthermore, microglial-inhibiting compounds blocked condi-tioned place preference to cocaine, suggesting a role for the neu-roimmune system in the subjective reward of drugs (Northcuttet al., 2015). Manipulations of the immune system have also beenfound to change alcohol consumption in experimental animals.Injectionwith the inflammogen LPS increases ethanol consumptionin mice (Blednov et al., 2011). Furthermore, deletion of certainneuroimmune genes, including cytokine genes encoding IL-6 andIL-1ra, decreased ethanol consumption (Blednov et al., 2012). Localknockdown of TLR4 in the central amygdala also decreased ethanolself-administration (Liu et al., 2011). Administration of the cytokineMCP-1 increased self-administration of alcohol in rats (Valenta andGonzales, 2016). These data suggest that neuroimmune signalingimpacts alcohol consumption. Regarding the craving/preoccupa-tion stage, which often precedes binge intoxication, blocking IL-1bsignaling in the VTAwas found to block cocaine-induced dopaminerelease in the nucleus accumbens (Northcutt et al., 2015).Furthermore, human alcoholics have increased plasma levels of

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multiple pro-inflammatory cytokines such as TNFa (Heberleinet al., 2014), which correlates with severity of alcoholism, IL-1b,IL-6 and IL-8 which correlate with alcohol craving (Heberlein et al.,2014; Leclercq et al., 2014). TNFa, IL-6, and IL-1b each cross theblood brain barrier and might contribute to central effects (Bankset al., 1994, 1995). Though the precise signaling mechanisms needto be further expounded, central pro-inflammatory cytokinesmodulate alcohol craving and consumption and peripheral cyto-kines are indices of craving and severity of alcoholism.

The immune system also impacts the withdrawal/negativeaffect stage. Indeed, a significant body of literature suggests thatsubstance abuse contributes to the development of negative af-fective states such as anxiety and dysphoria (Koob and Le Moal,2005). Additional substance abuse aimed at alleviating thesenegative states contributes to a cycle of abuse and dependence.Inflammation and cytokine induction are known to contribute tonegative affect. Withdrawal from alcohol increases inflammatorycytokines in the brain (Freeman et al., 2012). Also, intra-cerebroventricular injection of cytokines sensitized anxiety-likebehavior during withdrawal from ethanol (Breese et al., 2008).These data suggest that cytokines are involved in the ethanolwithdrawal process and contribute to the negative affect of alcoholuse disorders. Alcohol can also decrease neurogenesis, which isassociated with negative affect. Chronic voluntary alcohol con-sumption decreases neurogenesis during withdrawal and isaccompanied by the development of depression-like behavior(Stevenson et al., 2009). Both the decreased neurogenesis anddepression-like behavior were reversed by treatment with anti-depressants. Therefore, there are multiple mechanisms by whichalcohol increases CNS neuroimmune signaling, contributing to themolecular and cellular causes of alcohol use disorders.

6. HMGB1, Toll-like receptors and innate immune signaling inthe brain

Understanding the mechanism of signaling for many hormonesand neurotransmitters has benefited from manipulations inchemical structure allowing agonist and antagonist pharmacolog-ical distinction of responses. However, receptors that respond tolarger molecules are more difficult to fully characterize. In theinnate immune system, this has been the case. Initial studiescharacterized many receptors as responding to pathogens such asbacteria and virus components. These pattern recognition receptors(PRRs) and somewhat promiscuous and recently, studies involving“sterile” inflammation i.e. innate immune activation in the absenceof a foreign pathogen have identified additional roles for thesereceptors. This is especially true for the CNS, which is normally asterile environment, yet PRRs play critical roles in addiction pa-thology. PRRs have revolutionized the understanding of the innateimmune system signaling. PRRs recognize specific molecular pat-terns associated with either foreign pathogens (Pathogen Associ-ated Molecular Patterns-PAMPs) or endogenous moleculesassociated with cell death, damage or stress (Danger AssociatedMolecular Patterns-DAMPs). Five classes of PRRs have beendescribed: Toll-like receptors (TLRs), C-type lectin receptors,nucleotide binding domain receptors (leucine-rich repeat con-taining or NOD-like receptors), RIG-I-like receptors, and AIM 2-likereceptors (Brubaker et al., 2015). TLRs are the most well charac-terized PRRs, and have been implicated in addiction. TLRs arecharacterized by an N-terminal extracellular leucine-rich repeatsequence and an intracellular Toll/Interleukin-1 receptor/resistancemotif (TIR) (Takeuchi and Akira, 2010). These receptors recognizePAMPs and DAMPs that include a variety of molecules from bac-terial endotoxin to mammalian HMGB1 and heat shock proteins(Vabulas et al., 2002). To date ten TLRs have been identified in

humans and 12 inmice (Brubaker et al., 2015). Activation of TLRs byendogenous DAMPs has been implicated in several non-infectiousneurological disorders, including alcoholism (Table 1). Tran-scriptome analyses in Drosophila have implicated Toll genes in thealcohol response, as discussed by Park et al. in their review in thisspecial edition (Park et al., 2017). The expression of several TLRs areupregulated in the brains of human alcoholics. TLRs 2e4 as well asthe PRR RAGE (Receptor for advanced glycation End-products)were each increased in the frontal cortex of postmortem humanalcoholics (Crews et al., 2013). Ethanol treatment in vivo has alsobeen shown to increase the expression of TLRs 2e4 in cortex andcerebellum with subsequent NF-kb activation and cytokine induc-tion (Crews et al., 2013; Lippai et al., 2013b). Furthermore, ethanolsensitizes neuroimmune responses to TLR3 and TLR4 agonists (Qinand Crews, 2012a; Qin et al., 2008). Chronic ethanol enhanced re-sponses to systemic TLR3 and TLR4 agonists. This increased sys-temic and brain pro-inflammatory response is in part due to bingealcohol increasing intestinal permeability, resulting in endotoxin/LPS as well as bacterial RNA and other gut molecules crossing intothe blood inducing innate immune responses (Bala et al., 2014;Keshavarzian et al., 2009; Szabo et al., 2010). Thus, there are mul-tiple mechanisms by which ethanol leads to TLR activation andinduction in the brain.

High mobility group ss protein (HMGB1) has been identified asone such critical immune mediator in alcohol abuse. HMGB1 isincreased in the cortex of human alcoholics (Crews et al., 2013).Similar to TLRs 2e4, ethanol treatment in vivo increases HMGB1 incortex and cerebellum (Crews et al., 2013; Lippai et al., 2013b).HMGB1 is a nuclear chromatin binding protein that can also bereleased as an innate immune mediator (Muller et al., 2004).HMGB1 consists of two basic L-shaped ‘HMG box’ domains and anacidic 30 aa tail domain joined by a connecting linker (Stott et al.,2010). In the nucleus, HMGB1 stabilizes chromatin structure andacts as a chaperone for certain transcription factors (Thomas andStott, 2012). Extracellular HMGB1 activates innate immune re-sponses through direct binding to TLR4 or RAGE depending on itsredox state (Janko et al., 2014) (Fig. 4). The Cys106 thiol/Cys23-Cys45 disulfide bond form of HMGB1 acts at TLR4 receptors(Fig. 4a), while fully reduced HMGB1 is active at the RAGE receptor(Fig. 4b) (Venereau et al., 2012; Yang et al., 2012). Fully oxidizedHMGB1 is inactive at immune receptors (Fig. 4c) (Liu et al., 2012).Both ethanol and tobacco smoke extract cause HMGB1 trans-location from the nucleus with subsequent release in vitro (Chenet al., 2016; Zou and Crews, 2014). Thus, ethanol and possiblyother drugs of abuse lead to ‘sterile inflammation’ through therelease of HMGB1. The release of other endogenous DAMPs likelyalso occurs, with recent reports indicating ethanol-induced releaseof TLR7-activating miRNA. The complex structure of HMGB1 allowsit bind to a large number of molecules that also sensitizes otheragonist responses through their associated receptors in a complexmanner that is poorly understood. The double box and cysteine-disulfide structure of HMGB1 creates unique heterodimers withlipoglycans, endotoxin, and cytokines (e.g. IL-1b, Fig. 4d) andnucleic acids (e.g. miRNA, Fig. 4e) (Bianchi, 2009; Hreggvidsdottiret al., 2009). HMGB1 enhances the potency of cytokines at theirrespective receptors leading to greater immune responses (Fig. 4c).Furthermore, HMGB1 is required for immune responses to endo-somal nucleic acid binding TLRs (3, 7 and 9) (Yanai et al., 2009),though the mechanism is unclear. Recent evidence has found thatethanol causes increasing binding of HMGB1 to the miRNA let-7while reducing its association with the RISC complex proteinAgo2. HMGB1might aid in the presentation of nucleic acids to theirendosomal TLR receptors. Thus, the modulation of HMGB1 byethanol might activate signaling through multiple TLRs in additionto TLR4.

Table 1Selected Toll-like receptors (TLRs) implicated in neurological diseases.

TLR Foreign PAMP Endogenous DAMP Neurological condition

2 Bacterial di- and tri-aceylated polypeptides(Buwitt-Beckmann et al., 2006)Gram (þ) lipoglyans(Blanc et al., 2013)

a-synuclein Parkinson's Disease (Kim et al., 2013)

3 dsRNA stathmin Alzheimer's Disease (Jackson et al., 2006)Multiple Sclerosis (Bsibsi et al., 2010)

4 Bacterial endotoxinPeptidoglycans

HMGB1HSPs 60, 70/72 (Vabulas et al., 2002)

Stroke, Traumatic Brain InjuryChronic PainAlcoholism (Crews et al., 2013)

7 ssRNA (Lehmann et al., 2012b) Let-7, miR-21 Alcoholism (Coleman et al., 2017)Alzheimer's Disease (Lehmann et al., 2012a)Chronic Pain (Park et al., 2014)

Fig. 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 severalcysteines, 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 AgonistHMGB1. 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-1b to cause enhanced IL-1b signaling through the IL-1 receptor and greater innate immune induction. D. Thefully oxidized form of HMGB1 is inactive at immune receptors. E. HMGB1-miRNA-chaperone: HMGB1 binds miRNA and activates the RNA sensing TLR7 receptor increasing immuneactivation.

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7. TLR4 - a key brain receptor involved in ethanol inducedneuropathology

Ethanol likely affects the signaling of multiple TLRs. However,TLR4 in particular has been found to play a critical role in theneuroimmune activation by ethanol. TLR4 knockout mice are pro-tected from ethanol induced glial cell activation, NF-kB activation,and caspase-3 activation (Alfonso-Loeches et al., 2010a, b; Blancoet al., 2005; Fernandez-Lizarbe et al., 2009; Pascual et al., 2011;Valles et al., 2004). Cultured astrocytes with siRNA against TLR4 orMD-2 and CD14 (critical adaptor molecules for TLR4 signaling)were also protected from ethanol induced NF-kB induction. Micelacking TLR4 were also protected against ethanol withdrawalassociated anxiety-like behavior and memory impairment (Pascual

et al., 2011). Ethanol not only increases the expression of TLR4(Crews et al., 2013; Fernandez-Lizarbe et al., 2013), it increases theformation of TLR4/TLR2 heterodimers in lipid rafts of microglia,leading to iNOS induction and MAPK activation (Fernandez-Lizarbeet al., 2013). Importantly, both TLR4 and TLR2 KO microglia wereprotected in this study. In another genetic model, the alcoholpreferring p-rats have increased expression of TLR4 in the VTA thatwas related to binge ethanol responding (June et al., 2015). TLR4expression in the VTA was regulated by GABA(A)a2 receptor (Liuet al., 2011) and corticotropin-releasing factor (CRF) expression(June et al., 2015). Pharmacological studies also illustrate the role ofTLR4 in the immune pathology of ethanol. TLR4 stimulation withLPS leads to a prolonged increase in alcohol self-administration(Blednov et al., 2011). Mice lacking CD14, a critical adapter

Fig. 5. Neuron-like SH-SY5Y cells are more sensitive to ethanol induction of TLR7than microglia-like BV cells. Shown are TLR7 mRNA levels following 24 h of treat-ment with various concentrations of ethanol in cultures of microglia-like BV2 andneuron-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 about15 mM ethanol, whereas microglia show little response at this concentration, but showfar larger responses at higher concentrations of ethanol. (*p < 0.05, #p < 0.01 vs. CON.)Adapted from (Lawrimore and Crews, 2016).

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protein for TLR4, were protected from this effect. Naltrexone andNaloxone, opioid antagonists used to treat alcoholism, also haveanti-TLR4 actions. These antagonists reduce superoxide generation,cytokine production, and dopaminergic neurotoxicity by the TLR4agonist LPS (Liu et al., 2000a, 2000b). In Silico analyses indicate thatnaltrexone and naloxone fill the LPS binding site of the MD2 co-receptor for TLR4 (Hutchinson et al., 2010). Furthermore, theopioid inactive stereoisomers (þ)-naltrexone and (þ)-naloxonehave been shown recently to possess anti-TLR4 properties (Wanget al., 2016) indicating an immune mechanism that is indepen-dent of the opioid effects. These studies have identified a link be-tween neuroimmune activation by ethanol via TLR4 signaling. This,coupled with the aforementioned studies regarding the endoge-nous TLR4 agonist HMGB1, emphasizes the importance of studyingthe efficacy of targeted TLR4 antagonists for the treatment ofalcohol use disorders. Thus, significant opportunities exist foridentifying unique drug targets for neuroimmune pathology inalcoholism.

8. Induction of innate immune genes, and miRNA

Innate immune signaling is complex and involves multipleintracellular signaling cascades that lead to activation of key tran-scription factors. TLR signaling is mediated through key adapterproteins. Upon recognition of the ligand by the TLR, these adapterproteins initiate the signaling cascade. The TIRAP/MyD88 adapterprotein complex interacts with all the TLRs, except for TLR3. TIRAP/MyD88 complex formation leads to activation of IL-1 receptor-associated kinases (IRAKs) and TNF receptor-associated factor 6(TRAF6) which cause IkB and MAPK activation. IkB and MAPK ul-timately lead to activation of the transcription factors NuclearFactor kappa-light-chain-enhancer of Activated B cells (NF-kB) andActivated Protein-1 (AP-1) respectively. These transcription factorsregulate the expression of pro-inflammatory cytokines that prop-agate and magnify the immune response. Furthermore, thesetranscription factors are also linked to both stress and addictivebehaviors. For instance, NF-kB activation occurs in the nucleusaccumbens in response to cocaine, and was required for increasedconditioned place preference (Ang et al., 2001; Russo et al., 2009).Restraint stress increases the expression of NF-kB, cytokines,prostaglandin E2, and cyclooxygenase-2 (COX-2) in the CNS(Madrigal et al., 2002, 2003). Also, ethanol has been found to induceAP-1 transcription in primary cortical neurons (Qiang and Ticku,2005). Subcellular location and receptor trafficking alsocontribute to TLR responses, with nucleic acid sensing TLRs beingendosomal and cell surface trafficking to endosomal TLRs(including TLR4 after its endocytosis) also bind to the TRIF adapterprotein leading to transcription factor IRF3 activation and type Iinterferon gene induction. It is important to note that responsesvary across cell type and most signaling has been characterized inmonocyte and other immune cells that likely differ from that ofbrain cells.

The precise signaling pathways for the TLRs in each brain celltype have yet to be delineated. Bothmicroglia and astrocytes clearlyshow canonical TLR4 responses to ethanol leading to NF-kB acti-vation and further innate immune induction. However, TLR re-sponses in neurons are poorly understood. Some have suggestedthat neurons are not capable of activating NF-kB gene transcription(Mao et al., 2009). Others, however, find NF-kB activation in glu-tamatergic neurons that may regulate plasticity, learning andmemory (Kaltschmidt and Kaltschmidt, 2015). However, RAGE wasoriginally identified as a neuronal receptor that bound HMGB1,originally called amphoterin, involved in neurite outgrowth (Horiet al., 1995). Ethanol is known to activate NF-kB in neurons inbrain slice culture (Zou and Crews, 2006) and in vivo (Ward et al.,

1996). The activated p-65 NF-kB subunit has been shown tocolocalize with dorsal horn spinal neurons (Bai et al., 2014) andboth P19 and SH-SY5Y neuronal cell lines show NF-kB dependentregulation of m-opioid receptor expression (Borner et al., 2012;Wagley et al., 2013). These responses are complex in part due tothe release of HMGB1 by stimuli that can subsequently activatemultiple TLR and RAGE signals difficult to distinguish from theprimary stimuli. For example, ethanol responses in brain involvesignaling across microglia, astroglia and neurons. HMGB1 is one ofthe signals that contribute to neuroimmune activation and otherresponses. A comparison of ethanol activation of neuroimmunesignaling in neuron-like SH-SY5Y and microglia-like BV cells findsethanol induces TLR7mRNA in both cell types, but neurons respondat concentrations below binge drinking blood levels whereasmicroglia-like BV cells do not respond until high concentrations ofalcohol well above the binge drinking BAC, but had a more robustresponse (Fig. 5). Though the unique cellular roles need to befurther identified, it is clear that ethanol activates NF-kB signalingin brain. We have found that ethanol increases NF-kBeDNA bindingboth in in vivo in mice (Crews et al., 2006) and in vitro in rathippocampal-entorhinal cortex slice culture (Zou and Crews, 2006).Furthermore, we and others have found that ethanol causes in-duction of NF-kB target genes in brain, such as the chemokinemonocyte chemoattractant protein-1 (MCP-1, CCL2) (He and Crews,2008), proinflammatory cytokines (TNFa, IL-1b, and IL-6) (Qin et al.,2007), proinflammatory oxidases (inducible nitric oxide synthase

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(Alfonso-Loeches et al., 2010b; Zou and Crews, 2010), COX (Alfonso-Loeches et al., 2010b; Knapp and Crews, 1999), and NOX (Qin et al.,2008)), and proteases (TNFa-converting enzyme [TACE] and tissueplasminogen activator [tPA]) (Zou and Crews, 2010).

In addition to activation of TLRs via release of DAMPs, ethanolalso modulates immune function through release of microRNAs(miRNA). MicroRNAs are small non-coding RNAs (~22 nucleotides).Neuroimmune regulation of miRNAs is extensive with numerousmiRNAs involved (Thounaojam et al., 2013). These moleculesregulate immune function in two known fashions. First, theyregulate the stability of target mRNAs in the cytosol via formationof an RNA-induced silencing complex (RISC) and binding to the 3’-untranslated region of mRNA (Ambros, 2004; Bartel, 2004; Czechand Hannon, 2011). The role of microRNAs as transcriptional reg-ulators in alcoholism is discussed by Warden and Mayfield in theirarticle in this special edition (Warden and Mayfield, 2017). Anothernewly identified mechanism whereby ethanol modulates immuneactivation is through the extracellular release of miRNA(Turchinovich et al., 2013). Extracellular miRNA are containedeither in extracellular vesicles (EVs) or bound to either protein orlipoprotein chaperones. MicroRNA-containing vesicles are capableof being endocytosed by other cells where they can modulate theirfunction. Recently, microRNA in EVs from ethanol-treated mono-cytes were found to modulate the activation state of naïve mono-cytes (Saha et al., 2016). Further, the miRNAs let-7 andmiR-21 werefound to activate TLR7, leading to neuroimmune activation andneurodegeneration (Lehmann et al., 2012a; Yelamanchili et al.,2015). Thus, these miRNAs can have diverse actions to modulateneuroimmunity, both through regulation of mRNA stability and TLRactivation. Ethanol exposure causes significant alterations to themiRNA milieu in the brain. In both human alcoholics and mice (20day treatment), ethanol altered the expression of dozens of miRNAsin the frontal cortex (Lewohl et al., 2011; Nunez andMayfield, 2012;Nunez et al., 2013). Interestingly, members of the miRNA let-7family were induced across species. Recently, ethanol has beenfound to potentiate TLR7-mediated neurotoxicity via release of let-7 in MVs (Coleman et al., 2017). The miR-155 is also found in ves-icles and promotes TLR4 associated neuroimmune responses afterchronic ethanol (Lippai et al., 2013a). The miR-155 KO was pro-tected from ethanol-induction of cytokines. Thus, ethanol modu-lation of miRNA release is an important aspect of innate immuneinduction. These pathways represent new pathological mecha-nisms that are poorly understood, but are targets for potentialintervention in the pathology of alcoholism.

9. Autocrine and paracrine processes amplify NF-kB innateimmune gene induction

As has been discussed above, regulation of innate immune re-sponses in the brain is complex. This involves microglial primingand changes in activation state, microglia-neuronal interactions,and positive and negative feedback of intracellular signaling cas-cades. The transcription factor NF-kB is a critical modulator of im-mune function. Both stress and alcohol result in NF-kB activationthrough the immunemechanisms discussed above. Various modelsof stress in humans and rodents result in central and peripheral NF-kB activation in immune cells. Human psychosocial stress causesNF-kB activation in peripheral blood mononuclear cells (Bierhauset al., 2003). Restraint stress in rodents causes NF-kB trans-location into the nucleus with subsequent production of TNFa andpro-inflammatory prostaglandins (Bierhaus et al., 2003; Madrigalet al., 2002). Ethanol also activates NF-kB in rat and mouse brain(Qin and Crews, 2012b; Ward et al., 1996), as well as human as-trocytes (Davis and Syapin, 2004). The system itself is well-regulated. However, in the context of repeated or prolonged

activation, dysregulation occurs leading to many disease states (deJesus et al., 2015). Recurrent and persistent NF-kB activation byrepeated alcohol consumption appears to mimic a chronic inflam-matory state. This is evidenced by increased innate immunemarkers in the post-mortem brains of human alcoholics (Colemanet al., 2017; Crews et al., 2013) Further, NF-kB target genes areupregulated in the prefrontal cortex of alcoholics (Okvist et al.,2007). This persistent and chronic immune response has alsobeen seen after one dose of systemic LPS. One administration ofhigh dose LPS (5 mg/kg) resulted in persistent microglial activationwith ROS generation that led to dopaminergic neuron loss over aperiod of months (Qin et al., 2013). Though on a lower scale, binge-ethanol doses cause similar pathophysiology (Bala et al., 2014; Qinet al., 2008; Zou and Crews, 2010).

The prolonged amplification of neuroimmune responses occurson multiple levels. TLR activation in response to ethanol causes NF-kB activation, leading to increased TLR expression, cytokine pro-duction and DAMP release. This amplifies immune responses in anautocrine and paracrine fashion, as secreted DAMPs and cytokinesactivate both their cell of origin and surrounding cells (Fig. 6).Paracrine signaling induced by ethanol also proceeds through therelease of pro-inflammatory miRNA-containing microvesicles (Balaet al., 2011; Coleman et al., 2017; Szabo and Lippai, 2014). Thismethod of intercellular paracrine signaling has been implicated inmany conditions such as cancer, cardiovascular disease, and neu-rodegeneration (Hulsmans and Holvoet, 2013; Lehmann et al.,2012a; Ohshima et al., 2010). Also, ethanol induces ROS genera-tion that activates NF-kB and leads to further cytokine release andparacrine signaling (Pietri et al., 2005; Qin and Crews, 2012b;Thakur et al., 2007). A ‘smoldering’ level of inflammation due torepeated binge-ethanol, as is typical with alcoholism, might un-derlie the transition from alcohol abuse to addiction. Thus, phar-macological strategies should target the amplification andpersistence of the immune response, as this likely underlies diseasepathology.

10. New treatments strategies based on immunepharmacology

The discovery of immunemechanisms of alcohol presents a newapproach for the treatment of alcohol use disorders. It is currentlyunclear whether immune therapies would be of highest benefit forprevention or recovery from alcohol addiction. Neuroimmuneactivation can contribute to neurodegeneration in many diseasesettings (Crews and Vetreno, 2014; Qin et al., 2013; Rocha et al.,2015; von Bernhardi et al., 2015; Wang et al., 2015). Microglia andcytokines might also alter synaptic signaling (Lacagnina et al.,2017). For example, IL-1b reduces eIPSCs in the central amygdalato modulate ethanol effects on GABA receptors (Bajo et al., 2015).Other immune mediators might also impact synaptic signaling.Though immune therapies would likely not be of benefit in brainregions where neurodegeneration has already occurred, theremight yet still be benefit if they allow for recovery of normal syn-aptic function. Therefore, significant exploration of immune ther-apies in various models of alcohol abuse is warranted. Manycommercially available drugs have been found to have anti-inflammatory actions in the CNS. Certain antibiotics in particularhave immune actions in addition to their antimicrobial functions.Table 2 details some candidate drugs that have been found to bebeneficial for alcohol use disorders or other neuroimmune condi-tions. Minocycline, for example, is a tetracycline antibiotic andmicroglial inhibitor (Plane et al., 2010) that prevents ethanolinduced-microglial activation and reduces alcohol self-administration (Agrawal et al., 2011; Qin and Crews, 2012a).Phosphodiesterase 4 (PDE4) inhibitors have traditionally been used

Fig. 6. Autocrine and Paracrine Innate Immune Activation by Ethanol. Ethanol causes Toll-like Receptor (TLR) activation in both autocrine and paracrine fashions. This occursthrough 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. Cytokinessuch as IL-1b bind cytokine receptors. Immune mediators act in both autocrine and paracrine fashions on neurons and microglia to amplify neuroimmune responses.

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to treat psoriasis and psoriatic arthritis. These drugs are thought toreduce inflammation by increasing cAMP concentration resulting inreduced NF-kB activation (Jimenez et al., 2001). Recently, theseagents have been found to reduce ethanol intake in rodents (Bellet al., 2015; Blednov et al., 2014; Hu et al., 2011). A phase I clin-ical trial involving the PDE4 inhibitor ibudilast was recentlycompleted but has not yet been reported. A recent placebo-controlled trial found that ibudilast reduced some of the

subjective reward effects of methamphetamine (Worley et al.,2016). PPARg agonists, historically used for the treatment of typeII diabetes, might also be helpful in alcohol use disorders throughtheir anti-inflammatory activities. The PPARg agonist pioglitazoneis a microglial inhibitor (Storer et al., 2005) that reduces toxicity inmodels of fetal alcohol spectrum disorders (Drew et al., 2015; Kaneet al., 2011) andmay potentially play a beneficial role in alcoholism.Each of these drugs, and others with anti-inflammatory actions

Table 2Antibiotics and other drugs with anti-neuroinflammatory actions.

Drug MechanismPrimaryImmune

CNS activity

Minocycline Tetracycline antibioticMicroglial inhibitor

Reduces alcohol self-administration (Agrawal et al., 2011)Reduces ethanol microglia activation (Qin and Crews, 2012a)Prevents reinstatement of morphine and amphetamine seeking (Arezoomandan and Haghparast, 2016;Attarzadeh-Yazdi et al., 2014)

Rapamycin Macrolide antibioticmTORC1 inhibitor

Reduces binge ethanol intake in males (Cozzoli et al., 2016)Neuroprotection via Autophagy Promotion (Chen et al., 2012; Pla et al., 2016)

Azithromycin Macrolide antibioticMicroglial inhibitor

Promotes anti-inflammatory M2 microglial activation state (Zhang et al., 2015)

Rifampin Bacterial RNA polymeraseinhibitorTLR4 inhibition

Inhibits microglia activation to TLR4 (Bi et al., 2011; Wang et al., 2013)

Indomethacin COX-2 inhibitor Reduces alcohol self-administration (George, 1989)Reduces ethanol neurotoxicity (Pascual et al., 2007)

Simvastatin HMG-CoA ReductaseInhibitorNF-kB inhibition

Reduces inflammation and neurotoxicity to ischemia and injury (Lim et al., 2017; Sironi et al., 2006)

Glycyrrhizin HMGB1 inhibition Blocks ethanol-induced cytokine release (Zou and Crews, 2014)Reduces neuroinflammation after traumatic brain injury (Okuma et al., 2014)

Pioglitazone, DHA PPARg Agonists Reduce toxicity and pro-inflammatory cytokines in fetal alcohol spectrum disorder model (Drew et al.,2015; Kane et al., 2011)

Ibudilast, Mesopram, Rolipram,CDP 840

Phosphodiesterase 4inhibition

Reduce ethanol intake in C57BL/6J mice (Blednov et al., 2014)Reduces ethanol self-administration in rats (Bell et al., 2015)

Naltrexone/Naloxone m-opioid antagonistTLR4 inhibition

Reduces alcohol self-administrationBinds TLR4 adaptor protein MD2 (Hutchinson et al., 2010; Wang et al., 2016)

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should be investigated for their potential efficacy in alcohol usedisorders.

Fig. 7. b2-microglobulin is induced in adult hippocampus following adolescentintermittent ethanol treatment. Shown are levels of b2 microglobin immunoreativity(b2M þ IR) determined by immunohistochemistry in hippocampal dentate gyrus. Ratswere treated with ethanol on an intermittent schedule through adolescence, i.e.adolescent intermittent ethanol treatment as described previously (Liu and Crews,2015). b2M þ IR was assessed on post-natal day 57, 24 h after the last AIE ethanoltreatment, and in adulthood on post-natal day 95. Note the ethanol-triggered induc-tion of b2M 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).

11. Neuroimmune basis of addiction

A hypothesis of stress- and drug-induced neuroimmunesignaling that inactivates PFC and sensitizes limbic circuitry thatsuggest the progressive and persistent increases in HMGB1 and TLRwith cycles of stress and drug abuse are the mechanisms under-lying the progressive and persistent nature of addiction. Innateimmune signaling is known to have a major impact on cognitiveand emotive functioning, leading to dysfunction (Dantzer et al.,2008; Hanke and Kielian, 2011; Okun et al., 2010; Yirmiya andGoshen, 2011). Ethanol and other drugs promote innate immunegene induction (Crews and Vetreno, 2016; He and Crews, 2008; Qinet al., 2008) that are linked to changes in executive function,reinforcement-reward and negative affect-craving-anxiety thatpromote alcohol abuse and addiction (Vetreno and Crews, 2014).Indeed, innate immune activation and TLR signaling via ethanolappear to be essential for ethanol induction of addiction-like be-haviors. For instance, Guerri and colleagues (Pascual et al., 2011)demonstrated that ethanol-induced innate immune activation inmice impaired short- and long-term memory for object recogni-tion. This behavioral impairment was accompanied by a reductionof H3 and H4 histone acetylation as well as histone acetyl-transferase activity in the frontal cortex, striatum, and hippocam-pus. Interestingly, ethanol neither impaired behavioralperformance nor altered histone activity in TLR4 knockout mice.Another inflammatory pathway, mediated by the RAGE receptor,might be involved in the memory impairments associated withchronic alcohol exposure since neuroinflammation associated withincreased expression of this receptor is implicated in the memoryimpairments that accompany Alzheimer's disease (Arancio et al.,2004; Fang et al., 2010; Maczurek et al., 2008; Wilson et al.,2009). Adolescent intermittent ethanol treatment of rats has beenfound to increase expression of RAGE that persists into adulthood

and mimics the increase found in post-mortem human alcoholicbrain (Vetreno et al., 2013). Thus, HMGB1, TLRs, RAGE, and otherinnate immune signaling are likely to drive changes in neurobi-ology related to addiction.

The innate immune system can modulate alcohol consumptivebehavior. Analysis of genetically paired rats and mice that differprimarily in the amount of ethanol consumption reveal that NF-kBand other pro-inflammatory genes have high expression in highethanol drinking animals (Mulligan et al., 2006). Beta-2 micro-globulin, a NF-kB target gene involved in MHC immune signaling

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(Pahl, 1999), was elevated in high ethanol preferring brain tran-scriptomes (Mulligan et al., 2006). Interestingly, adolescent inter-mittent ethanol treatment results in increased expression of beta-2microglobulin in the adult hippocampus (Fig. 7). Beta-2 micro-globulin progressively increases following adolescent alcoholexposure (Fig. 7) in a manner similar to that found with HMGB1(Vetreno and Crews, 2012) and RAGE (Vetreno et al., 2013). Inaddition, work from Harris, Blednov and colleagues providedinteresting and novel data supporting the hypothesis that innateimmune genes regulate ethanol drinking behavior (Blednov et al.,2005, 2012). Multiple strains of mice with immune gene deletionuniversally drink significantly less ethanol than matched controlsacross multiple ethanol drinking paradigms. Recently, Blednov andcolleagues (Blednov et al., 2011) also demonstrated that innateimmune activation through LPS can cause long-lasting increases inethanol drinking. Indeed, strains of mice show varied innate im-mune responses to LPS that correspond to increases in the con-sumption of ethanol. Furthermore, a single injection of LPS iscapable of producing a delayed, but long-lasting increase in ethanolconsumption even in strains of high drinking mice. Recent data hasrevealed that the TLR family, especially TLR4, modulates ethanolintake. Administration of a GABAAa2 siRNA vector into the centralnucleus of the amygdala of alcohol-preferring rats diminishedbinge drinking, which was associated with reduced expression ofTLR4 (Liu et al., 2011). Interestingly, the neuronal location ofGABAAa2 receptors suggests that the influence of TLR4 on bingedrinking is at least partially mediated by neurons. Taken together,these findings are consistent with a modulatory role of innate im-mune genes in alterations in GABA signaling as well as alcoholpreference and consumption.

Frontal cortical executive functions include: motivation, plan-ning and goal setting, and behavioral flexibility. Among socialdrinkers the heaviest binge drinkers report more negative moodand performworse on executive functioning tasks (Townshend andDuka, 2003; Weissenborn and Duka, 2003). The frontal cortexregulates mood and cognition through reciprocal glutamatergicconnections with multiple brain regions. In astrocytes, ethanolexposure induces NF-kB transcription leading to increasedexpression of pro-inflammatory innate immune genes (Pascualet al., 2007; Zou and Crews, 2006, 2010) and reduced astrocyteglutamate transport (Zou and Crews, 2005). The increased extra-cellular glutamate levels lead to increased neuronal excitation,microglial activation, and excitotoxicity (Ward et al., 2009; Zou andCrews, 2006). Indeed, increased glutamate excitotoxicity contrib-utes to ethanol-induced increases of caspase-3 and COX-2 in thefrontal cortex that require TLR4 signaling (Alfonso-Loeches et al.,2010b; Knapp and Crews, 1999). Glutamatergic hyper-excitabilityhas also been demonstrated to occur in the cocaine- andstimulant-addicted brain (Reissner and Kalivas, 2010). Theinvolvement of innate immune genes in the reduction of glutamatetransporters and subsequent hyper-excitability reduces frontalcortical executive function that contributes to the neurobiology ofaddiction (Crews et al., 2006, 2011). Frontal cortical dysfunction iscommon in the alcoholic brain (Crews and Boettiger, 2009) and ismanifest in impulsivity and behavioral inflexibility. Reversallearning is an index of behavioral flexibility that is especially rele-vant to addiction. Reversal learning involves adjusting learned re-sponses to changes in reinforcement-reward (Stalnaker et al.,2009). Indeed, proper frontal cortex function is necessary toweigh the value of decisions and is important when new learningand/or responses are reinforced. The involvement of the frontalcortex in reversal learning is supported by lesion-induced reversallearning deficits similar in nature to chronic drug abuse-induceddysfunction (Schoenbaum et al., 2006). The entire reversallearning circuitry involves loops between the prefrontal cortex,

striatum and amygdala (Izquierdo et al., 2016). This paradigmmodels the changes that occur when the initial reinforcement,intoxication, and binge drinking stages of alcohol abuse progress toobsessive-compulsive stages that are not able to avoid negativeconsequences of drinking due to persistent perseveration andinability to learn new approaches to reinforcement when situationschange. Indeed, both human alcoholics (Fortier et al., 2008; Jokischet al., 2014) and cocaine addicts (Stalnaker et al., 2009) showreversal learning deficits. Using a rat model of binge drinking, wefound persistent deficits in reversal learning in rats (Obernier et al.,2002) and in adult mice following adolescent binge drinking(Coleman et al., 2011). Others have also seen reversal learningdeficits in adult mice after chronic ethanol (Badanich et al., 2011).Interestingly, these reversal learning deficits are also found in ratsexposed to self-administered cocaine or passive cocaine injections(Calu et al., 2007; Schoenbaum et al., 2004). Frontal corticaldysfunction is established as resulting in perseveration and repe-tition of previously learned behaviors due to failure to associatenew information (e.g. negative consequences) into decision-making. Alcohol-induced damage to other regions associatedwith reversal learning (i.e. striatum and amygdala) might alsocontribute to reversal learning deficits and should be investigated.Further, the efficacy of immune modulating therapies to treatcognitive dysfunction associate with alcohol abuse should beinvestigated. Considerable emerging evidence supports a role forHMGB1/TLR signaling, innate immune gene induction and alter-ations in epigenetics and neurotransmission as culminating in theneurobiology of addiction (Crews et al., 2011; Cui et al., 2015;Vetreno and Crews, 2014).

Acknowledgments

We would like to thank NIAAA for their financial support.(AA019767, AA11605, AA007573, and AA021040)

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