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 , Bowles Center for Alcohol Studies, CB# 7178 UNC-CH, Chapel Hill, NC 27599, USA article info abstract

Article history: Alcohol consumption and stress increase brain levels of known innate immune signaling molecules. Received 14 September 2016 , the innate immune cells of the brain, and respond to alcohol, signaling through Toll- Received in revised form like receptors (TLRs), high-mobility group box 1 (HMGB1), miRNAs, pro-inflammatory and 24 January 2017 their associated receptors involved in signaling between microglia, other and neurons. Repeated Accepted 29 January 2017 cycles of alcohol and stress cause a progressive, persistent induction of HMGB1, miRNA and TLR receptors Available online 1 February 2017 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 Chemical compounds studied in this article: Minocycline (PubChem CID: 54675783) promote reward seeking behavior and increase risk of developing alcohol use disorders. Studies fl Rapamycin (PubChem CID: 5284616) employing anti-oxidant, anti-in ammatory, anti-depressant, and innate immune antagonists further link Azithromycin (PubChem CID: 447043) innate immune gene expression to -like behaviors. Innate immune molecules are novel targets Rifampin (PubChem CID: 5381226) for addiction and affective disorders . Indomethacin (PubChem CID: 3715) This article is part of the Special Issue entitled “Alcoholism”. Simvastatin (PubChem CID: 54454) © 2017 Elsevier Ltd. All rights reserved. Glycyrrhizin (PubChem CID: 14982) Pioglitazone (PubChem CID: 4829) Ibudilast (PubChem CID: 3671) Naltrexone (PubChem CID: 5360515)

Keywords: HMGB1 TLR Cytokines miRNA-let-7 Alcohol Addiction

Contents

1. Introduction ...... 57 2. Alcohol and stress, microglia and neuroimmune signaling ...... 57 3. Sensitization of glia in the stressed and addicted brain ...... 58 4. Stress, and affective disorders ...... 59 5. Innate immune molecules mimic addiction-like behavior ...... 61 6. HMGB1, Toll-like receptors and innate immune signaling in the brain ...... 62 7. TLR4 - a key brain receptor involved in ethanol induced ...... 63 8. Induction of innate immune genes, and miRNA ...... 64 9. Autocrine and paracrine processes amplify NF-kB innate immune gene induction ...... 65 10. New treatments strategies based on immune pharmacology ...... 65 11. Neuroimmune basis of addiction ...... 67 Acknowledgments ...... 68 References ...... 68

* Corresponding author. E-mail address: [email protected] (L.G. Coleman). http://dx.doi.org/10.1016/j.neuropharm.2017.01.031 0028-3908/© 2017 Elsevier Ltd. All rights reserved. F.T. Crews et al. / Neuropharmacology 122 (2017) 56e73 57

1. Introduction of this system leads to progressive and persistent loss of behavioral control, increased impulsivity and anxiety, as well as negative affect Neuroimmune signaling is emerging as a contributor to the and craving coupled with increasing ventral striatal responses that development of alcohol use disorders. Alcohol consumption and promote reward seeking behavior and increase risk of developing stress increase brain levels of known innate immune signaling alcohol use disorders. Studies employing anti-oxidant, anti-in- molecules. Microglia, the innate immune cells of the brain, play an flammatory, anti-depressant, and innate immune antagonists important role in brain development and function. As mesodermal further link innate immune gene expression to addiction-like be- tissue specific monocytes, microglia express signaling molecules haviors. In this review we discuss both preclinical rodent and hu- first characterized as “acute phase proteins” that increase in blood man studies that examine the role of neuroimmune signaling in rapidly in response to infections. More recently, Toll-like receptors alcoholism. Unless otherwise noted the presented studies were (TLRs), members of a superfamily of receptors, i.e. - -1 performed in rodents. From these studies, we surmise that innate receptor/toll-like receptor superfamily, were found to initiate pro- immune molecules are novel targets for addiction and affective inflammatory responses to infectious agents by sensing large disorders therapies. molecules with lipid, sugar, protein and nucleic acid components. Pro-inflammatory cytokines and their associated receptors share 2. Alcohol and stress, microglia and neuroimmune signaling complex signaling pathways with TLRs that converge upon NF-kB, a key innate immune transcription factor in monocyte-like cells, and Repeated cycles of alcohol, drugs of abuse and stress interact, other transcription factors regulated by TLR-kinase cascades. contributing to a progressive sequence of stages leading to addic- Recent studies in brain, which is generally sterile, indicate endog- tion (Volkow et al., 2016). Alcohol, drugs of abuse and stress also enous TLR agonists, i.e. danger-associated molecular patterns trigger neuroinflammatory responses. Inflammation may have (DAMPs), represent important signaling systems between micro- beneficial or maladaptive consequences on brain function (Fig. 1). glia, other glia and neurons. Further, emerging studies find cyto- Physical stressors, such as injury and infection, increase pro- kines and DAMPs may signal directly between neurons. High- inflammatory cytokines and other leukocyte derived proteins and mobility group box 1 (HMGB1) is a -like molecule highly prostaglandins as part of the acute inflammatory phase response. expressed in neurons that can directly activate TLRs as well as Stressor-induced inflammation also triggers adaptive behavioral enhancing responses at other pro-inflammatory receptors. Alcohol- changes known as “sickness behavior.” Sickness behavior includes and stress-induced increases in Toll-like receptors, and other neu- social withdrawal, decreased activity, somnolence/sleepiness, roimmune signaling molecules in brain sensitize the brain to anhedonia, etc. that help to conserve energy and facilitate recovery further innate immune gene induction, resulting in progressive and (Dantzer et al., 2008). Interestingly, even psychological stressors persistent increases in HMGB1 and TLR signaling as evidenced by (e.g. e social stress) can cause inflammation (Steptoe et al., 2007). prefrontal cortical expression levels correlating with lifetime The acute pro-inflammatory response, often referred to as Th1, alcohol consumption across alcoholic and moderate drinking con- includes signals that activate a delayed and cell trols (Crews et al., 2013). Mechanistic studies of epilepsy find growth through “anti-inflammatory” Th2 innate immune signaling neuronal firing results in the rapid release of HMGB1 and IL-1b molecules. While the benefit of the Th1 to Th2 progression preceding the seizure, inducing changes in excitatory receptors that following trauma or infection may be apparent, what happens in sensitize neurons to excitation, lowering seizure threshold and brain is poorly understood. Therefore, both physical and psycho- contributing to the “kindling” progressive increase in seizure fre- logical stressors may enhance inflammation, initiating molecular quency and severity with each event (Maroso et al., 2011). Although and behavioral changes that facilitate recovery. While stress and different brain regions and circuits distinguish addiction from ep- inflammation can lead to the adaptive response of sickness ilepsy, HMGB1 and TLR signaling and cycles of stress and drug behavior, intense or chronic stress and inflammation can become abuse appear to share the common mechanisms of sensitization of maladaptive and lead to neuropsychiatric disease such as depres- circuitry through increased HMGB1 and TLR signaling. sion. Indeed, the similarities between sickness behavior and Alcohol administration to mice and rats increases expression of depression have been noted (Maes et al., 2012). Models of HMGB1 and multiple TLR receptors in brain that persist for long depression include endotoxin treatments of rodents followed periods of abstinence. Furthermore, human post-mortem alcoholic beyond the 24 h “sickness behavior” time course (Dantzer et al., brains show increased expression of microglial markers, cytokines, 2008). The inflammation caused by excessive alcohol consump- TLR receptors and HMGB1, with the latter two being correlated tion may have similar maladaptive consequences. with lifetime alcohol consumption. Repeated alcohol, drug use and Studies have found similar mechanisms through which stress stress in rodents sensitize microglia toward hyperactivity, likely and alcohol contribute to inflammation (Fig. 2). Stress enhances through induction of immune signaling (see Sections 2 and 5). intestinal permeability and bacterial translocation from the gut Cytokine infusions into specific rodent brain regions initiate craving lumen (Garate et al., 2013). The mounts a response and alcohol consumption, and human alcoholics have increased to the leaked bacteria, thereby causing peripheral inflammation. plasma pro-inflammatory cytokines that correlate with alcohol Alcohol also contributes to gut leakiness and translocation of bac- craving and severity of dependence (see Section 5). Further, pro- teria from the intestinal lumen to the periphery (Adachi et al., inflammatory cytokines TNFa, IL-6, and IL-1b play critical roles in 1995). Leaked bacterial products cause inflammation in the liver the of mood disorders (Bhattacharya et al., 2016; and release of pro-inflammatory (Giraldo et al., 2010)cytokines into Bhattacharya and Drevets, 2017). These observations, detailed the systemic circulation (Mayfield et al., 2013). Therefore, stress and further below, support the hypothesis that stress and alcohol in- alcohol can cause gut leakiness, leading to peripheral inflammation. duction of neuroimmune signaling contribute to the progressive, Both stress and alcohol (Ellis, 1966) also increase glucocorticoids. persistent increase in craving and mood dysfunction in addiction. Intriguingly, glucocorticoids have pro-inflammatory effects under Recent discoveries indicate broad induction of TLR receptors in the right circumstances (Sorrells et al., 2009). While glucocorticoids brain as well as endogenous agonists for TLR receptors, including are acutely anti-inflammatory at high doses, glucocorticoids also miRNA that activate nucleic acid sensing TLRs and HMGB1 that have a priming effect on the inflammatory response. That is to say, activates TLR4, a key signaling TLR in the brain. Although poorly prior exposure to glucocorticoids enhances the inflammatory understood, studies suggest that activation and escalated signaling response to subsequent stimuli (Frank et al., 2010). Therefore, both 58 F.T. Crews et al. / Neuropharmacology 122 (2017) 56e73

Fig. 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 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 physio- logical factors such as . 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.

stress and alcohol may exert inflammatory/priming effects through brain barrier are essential for systemic inflammation to cause brain glucocorticoid signaling. Stressors also enhance the systemic inflammation (Qin et al., 2007). A third route is the cellular route, by release of cytokines (e.g. IL-1 and IL-6) (Merlot et al., 2002)aswell which activated immune cells such as monocytes traffic into the as danger-associated molecular patterns (DAMPs) such as high brain. The brain recruits monocytes in response to peripheral mobility group box 1 (HMGB1), uric acid and heat-shock protein 72 inflammation (D'Mello et al., 2009). In each case, the peripheral (Hsp72) (Maslanik et al., 2013). Physical stressors such as trauma inflammatory signal is transmitted to the brain, where it can impact can release DAMPs directly via cellular damage. Immune activation central inflammation and behavior. Thus, alcohol abuse and stress due to psychological stressors might involve multiple mechanisms share common mechanisms of inducing neuroimmune signaling including sympathetic nervous system activation, as noradrenaline that could contribute to the progression and persistence of the causes Hsp72 release from neutrophils (Giraldo et al., 2010)or neuropathology of addiction by cycles of stress and substance endotoxin release due to increased intestinal permeability (Ait- abuse. Belgnaoui et al., 2012; Ferrier et al., 2003) that may involve CRF release (Overman et al., 2012; Saunders et al., 2002; Teitelbaum et al., 2008). Released DAMPs and cytokines can activate innate 3. Sensitization of glia in the stressed and addicted brain immune cells, thereby increasing inflammatory signaling. Periph- eral inflammation can impact the brain and behavior in many ways Microglia are the resident of the brain and the (Fig. 2)(Miller and Raison, 2016). One route is the neural route: the primary cells of the neuroimmune system. They are involved in vagus can sense peripheral inflammatory cytokines such as many physiological processes in the healthy brain, including syn- IL-1b through IL-1 receptors (Ek et al., 1998). Activation of vagal aptic pruning, debris clearance, immune surveillance/defense, afferents transmits signals to the where and more (Kettenmann et al., 2011, 2013). Microglia “ ” neural centers that promote sickness behavior are activated. normally exist in a resting state, but can become activated in Indeed, vagotomy reduces the sickness behavior response to a pe- response to insults. Activation of microglia has been categorized ripheral injection of LPS (Bluthe et al., 1994). Another means by according to either morphological or functional standards. Under fi “ ” which peripheral inflammation can impact the brain and behavior the morphological classi cation, resting microglia are called “ fi ” is the humoral route. This includes diffusion of cytokines from the rami ed microglia. Morphological activation of microglia pro- periphery through leaky regions of the blood brain barrier, such as gresses through multiple states of increasing activation, including fi fi the circumventricular organs and by transport of cytokines across the hyper-rami ed state, the bushy state and nally the amoeboid the blood brain barrier. Indeed, TNFa transporters on the blood state (Fig. 3)(Beynon and Walker, 2012). Fully activated amoeboid microglia are phagocytic cells with a rounded morphology. To date, F.T. Crews et al. / Neuropharmacology 122 (2017) 56e73 59

and produce through increased iNOS and NADPH oxidase expression. M2 microglia secrete anti- inflammatory cytokines such as IL-10 and synthesize extracellular matrix products to assist with healing (Cherry et al., 2014). The exact relationship between the morphological and functional activation states is currently unclear. Microglia also undergo another phenomenon known as priming, or sensitization. Priming occurs when an initial stimulus causes a greater microglial response to a subsequent stimulus (Perry and Holmes, 2014). Primed microglia produce a greater inflammatory response to im- mune stimuli than non-primed microglia. Interestingly, are also an important component of the brain immune system (Farina et al., 2007). Astrocytes are involved in numerous physiological processes, such as metabolic support of neurons and modulation of synaptic transmission (Khakh and Sofroniew, 2015). Astrocytes express multiple immune receptors and cytokines that allow them to participate in immune processes (Jensen et al., 2013). In response to insults, astrocytes also undergo a process of activation or reactive gliosis that serves to limit tissue damage (Pekny and Pekna, 2014). Some studies suggest that, like microglia, astrocytes are capable of adopting pro-inflammatory and anti-inflammatory states (Jang et al., 2013). Together, microglia and astrocytes 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 through altered glial functioning. Both stress and alcohol can affect glia. Various types of acute and chronic stress, including restraint (Tynan et al., 2010), foot shock (Frank et al., 2007), social defeat (Wohleb et al., 2011) and chronic variable stress (Kreisel et al., 2014), increase microglial markers such as CD11b and Iba1 across multiple brain regions, consistent with 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 affect astrocytes. Sub-chronic stress increased the marker GFAP Fig. 2. Mechanisms of Stress- and Ethanol-induced Immune Activation. Stress and ethanol activate the peripheral and central immune systems in multiple ways. Both in the hippocampus, suggestive of astrocyte activation (Lambert stress and ethanol can enhance gut leakiness. This causes increased translocation of et al., 2000). Therefore, stress activates/primes glia. Various bacterial products such as endotoxins from the intestinal lumen to the periphery. alcohol treatment protocols also alter glia. Ethanol increases Leaked bacterial products make their way to the liver via the portal system where they microglial markers such as CD11b (Fernandez-Lizarbe et al., 2009) induce an inflammatory response from resident macrophages. The production of pe- ripheral inflammatory cytokines such as TNFa, IL-1b and IL-6 impact the brain and and Iba1 (Qin and Crews, 2012a) in vivo, consistent with microglial behavior through multiple mechanisms. One way is the neural route. The activation. Alcohol exposure can also prime the microglial response expresses cytokines receptors and is activated by peripheral inflammation. The signal to peripheral inflammation (Qin and Crews, 2012a). Astrocytes are of peripheral inflammation is transmitted to central brain regions involved in the also affected by alcohol, as evidenced by increased GFAP (Alfonso- regulation of sickness behavior. Another route is the humoral route. Peripheral cyto- Loeches et al., 2010b). Other studies show that alcohol consump- kines 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 tion can initially increase astrocytic GFAP in the hippocampus, but and ethanol also activate glia through more direct mechanisms. Glucocorticoids are decrease GFAP after extended exposure (Franke, 1995). Therefore, important for the priming effect of stress on microglia. Also, ethanol exposure can alcohol can also impact glia. Repeated bouts of exposure to these directly activate glia. stimuli may lead to increasing glial activation that persists over time. Indeed, two alcohol binge treatments activate microglia to a greater extent than a single binge treatment (Marshall et al., 2016b). Binge ethanol also increases microglial markers up to no known studies have observed alcohol causing an amoeboid four weeks following the binge, indicative of persistent activation microglial activation. Rather, alcohol seems to sensitize microglia, (Marshall et al., 2013). Thus, repeated episodes of stress and/or inducing a hyper-ramified activation state characterized by modest alcohol abuse may cause escalating and persistent glial activation morphological changes and increased release of cytokines and (Fig. 3), and this pathological activation/sensitization may other signaling molecules (Fig. 3). Functional microglial activation contribute to the neurobiology of alcohol use disorders. is traditionally understood as occurring along a spectrum between two extremes, although recent studies suggest functional activation 4. Stress, inflammation and affective disorders is a more complex phenomenon (Xue et al., 2014). Functional activation states range from the pro-inflammatory, destructive M1 Stress and the resulting inflammation impact the neuroimmune state (comparable to systemic Th1) to the anti-inflammatory, system of the CNS. Stress sensitizes microglia to inflammation in an reparative M2 state (comparable to systemic Th2). M1 microglia HMGB1-dependent manner (Weber et al., 2015). Chronic stress secrete pro-inflammatory cytokines such as TNFa, IL-1b and IL-6 activates microglia in multiple brain regions (Tynan et al., 2010) and 60 F.T. Crews et al. / Neuropharmacology 122 (2017) 56e73

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 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 and 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.

causes depression-like behavior. Administration of microglial- increased levels of the inflammatory cytokine Ccl2 in the cingulate modulating agents can block the development of depression-like cortex (Torres-Platas et al., 2014). Studies also find changes in behavior, suggesting that microglia may have a causal role in the astrocyte markers in the brains of depressed patients (Rajkowska development of depression (Kreisel et al., 2014). Human studies and Stockmeier, 2013). These findings suggest that stress contrib- also support a role for chronic stress and inflammation in affective utes to increased brain innate immune signaling induction and disorders. Administration of inflammatory agents (such as inter- psychopathology. feron-a for the treatment of hepatitis C) can cause depression in Stress-induced CNS inflammation can contribute to affective previously non-depressed patients (Bonaccorso et al., 2002). Hu- disorders through many mechanisms. Acute inflammation alters man studies find individuals with increased plasma levels of CRP, the activity of neural circuitry implicated in anxiety and depression. an acute phase marker of inflammation, have an increased risk for For example, acute inflammation changes activity in the cingulate depression (Young et al., 2014). Interestingly, increased CRP in ad- cortex, medial prefrontal cortex and amygdala (Harrison et al., olescents has also been found to predict addiction later in life 2009). Acute inflammation can also impair spatial memory in (Costello et al., 2013). Other studies find peripheral inflammation humans (Harrison et al., 2014), potentially contributing to the can increase brain expression of innate immune signals and cognitive changes seen in affective disorders. Furthermore, studies contribute to depression and negative affect (Harrison et al., 2014). find that a variety of cytokine receptors, such as those for TNFa, IL- Imaging studies in humans show that a marker of microglial acti- 1b, IL-6 and the interferons, are expressed on neurons (Khairova vation, TSPO, is elevated in patients with depression (Setiawan et al., 2009), suggesting that cytokines act directly on neurons to et al., 2015). Depressed patients who committed suicide showed influence their activity. Inflammation can also act on the seroto- increased expression of microglial markers Iba1 and CD45 and nergic system to cause depression-like behavior. LPS increases F.T. Crews et al. / Neuropharmacology 122 (2017) 56e73 61 activity of the transporter (SERT) and increases induced neuroimmune signaling (Alfonso-Loeches et al., 2010b). depression-like behavior. The ability of inflammation to cause Repeated binge alcohol treatment persistently increases neuro- depression-like behavior is lost in SERT knock-out animals (Zhu immune molecules such as TLR3, TLR4, HMGB1, RAGE, etc. (Vetreno et al., 2010). Inflammation also increases the activity of an and Crews, 2012; Vetreno et al., 2013). Studies on the post-mortem enzyme known as indoleamine 2,3-dioxygenase (IDO) in microglia. brains of human alcoholics also find increased microglial markers IDO generates the compound kynurenine, which has been shown to (He and Crews, 2008; Rubio-Araiz et al., 2016) and increased induce depression-like behavior (O'Connor et al., 2009). Pharma- astrocyte markers (Rubio-Araiz et al., 2016). Post-mortem human cological inhibition of IDO blocked the development of depression- alcoholic brains show increased neuroimmune molecules, such as like behavior (O'Connor et al., 2009) and blocks microglial activa- MCP-1, TL2, TLR3, TLR4 and HMGB1 (Crews et al., 2013; He and tion. The trafficking of peripheral inflammatory monocytes into the Crews, 2008). Furthermore, alcohol appears to cause a pro- CNS has also been implicated in stress-induced behaviors. Stress inflammatory M1 activation of microglial cells, as alcohol treat- causes trafficking of peripheral monocytes into the brain that, in ment induces expression of pro-inflammatory cytokines, such as turn, promotes anxiety-like behavior (Wohleb et al., 2013). Thus, TNFa and IL-1b, and enzymes, such as NADPH oxidase (Qin and stress-induced microglial activation contributes to increased Crews, 2012a; Qin et al., 2008). Further, alcoholic hepatic enceph- depression-like negative affect and anxiety-like morbidity that alopathy patients have increased microglial proliferation (Dennis overlaps with and contributes to progressive stages of addiction. et al., 2014). Thus, alcohol increases microglia and glial signaling Inflammatory cytokines such as TNFa and IL-1b can also impact contributing to lasting changes. neuronal plasticity. Interestingly, low physiological levels of TNFa Alcohol also sensitizes the neuroimmune system to subsequent and IL-1b seem to be important for long-term potentiation (LTP); inflammatory stimuli. Prior alcohol treatment enhanced brain however, high pathological levels of these cytokines disrupt the levels of pro-inflammatory cytokines to immune challenges such as process. Blocking TNFa in hippocampal slices decreased synaptic lipopolysaccharide (LPS) or polyinosine-polycytidylic acid (poly I:C) strength (Beattie et al., 2002). However, excessive TNFa can inhibit (Qin and Crews, 2012a; Qin et al., 2008). It is important to note that LTP in hippocampal slices (Tancredi et al., 1992). The effects of cy- LPS itself does not enter the brain, but causes systemic release of tokines on plasticity are behaviorally relevant, as TNFa over- immune mediators that do enter the brain that are required for expressing mice show decreased performance on spatial learning central LPS responses such as TNFa (Qin et al., 2007). It is unclear and memory tasks (Aloe et al., 1999). Also, physiological doses of IL- whether poly I:C crosses into the brain, but it does cause TNFa 1b are needed for memory. Blockade of IL-1b signaling by its release similar to LPS (Qin and Crews, 2012a). The sensitization or antagonist, IL-1ra, impairs memory. However, addition of excessive priming of the neuroimmune response has important conse- IL-1 b also impairs memory (Goshen et al., 2007). Therefore, im- quences for the pathogenesis of alcohol use disorders. Alcohol mune signaling may alter neurons and neural circuitry and change neuroimmune sensitization increases and loss behavioral phenotypes by impacting plasticity through changes in of neurogenesis following an immune challenge (Qin and Crews, cytokine levels. 2012a, b; Qin et al., 2008). Neurodegeneration and decreased Inflammation can also impact neurogenesis, which is implicated neurogenesis have been linked to a variety of neuropsychiatric in affective disorders. Neurogenesis occurs throughout adulthood diseases. Therefore, repeated exposure to alcohol enhances and in discrete brain regions, including the forebrain subventricular sensitizes neuroimmune signaling, thereby contributing to molec- zone and the subgranular zone of the hippocampal dentate gyrus. ular and cellular causes of alcohol use disorders and co-morbidities Hippocampal neurogenesis contributes not only to learning and of alcoholism, such as affective disorders. memory (Shors et al., 2001), but also mood and affective state Current theories of addiction posit a three-staged cycle that (Malberg et al., 2000). Decreased neurogenesis contributes both to contributes to substance use disorders: binge/intoxication, with- anxiety and depression (Hill et al., 2015). The ability of anti- drawal/negative affect and preoccupation/craving (Koob and depressants to alleviate depression-like behavior is dependent on Volkow, 2010). Interestingly, the neuroimmune system can hippocampal neurogenesis (Santarelli et al., 2003). Both inflam- impact the neurobiology of addiction at multiple points in this mation (Ryan and Nolan, 2016) and chronic stress (Kreisel et al., process. Regarding the binge/intoxication, multiple immune in- 2014) reduce neurogenesis and cause depression-like behavior. terventions have been found to regulate alcohol consumption. In- Indeed, stress induces IL-1b in the hippocampus which decreases jection of the IL-1 receptor antagonist or the anti-inflammatory neurogenesis and contributes to depression. Inhibition of IL-1b cytokine IL-1Qin and Crews, 2012a0 into the basolateral amygdala blocks stress-induced decreases in neurogenesis and depression- reduced alcohol self-administration (Marshall et al., 2016a, 2016c). like behavior (Koo and Duman, 2008). Furthermore, enhanced Furthermore, microglial-inhibiting compounds blocked condi- neurogenesis is sufficient to reverse the anxiety-like and tioned place preference to cocaine, suggesting a role for the neu- depression-like behavior caused by chronic corticosterone treat- roimmune system in the subjective reward of drugs (Northcutt ment (Hill et al., 2015). Therefore, stress can increase CNS immune et al., 2015). Manipulations of the immune system have also been signaling, which contributes to affective disorders. found to change alcohol consumption in experimental animals. Injection with the inflammogen LPS increases ethanol consumption 5. Innate immune molecules mimic addiction-like behavior in mice (Blednov et al., 2011). Furthermore, deletion of certain neuroimmune genes, including cytokine genes encoding IL-6 and Like stress, excessive alcohol use can impact the neuroimmune IL-1ra, decreased ethanol consumption (Blednov et al., 2012). Local system, contributing to the development of alcohol use disorders knockdown of TLR4 in the central amygdala also decreased ethanol and related psychopathology in many ways. There are multiple self-administration (Liu et al., 2011). Administration of the cytokine mechanisms through which this can occur. Alcohol is capable of MCP-1 increased self-administration of alcohol in rats (Valenta and acting directly on immune cells to increase inflammation. Alcohol Gonzales, 2016). These data suggest that neuroimmune signaling can directly activate microglia in vitro, increasing expression of pro- impacts alcohol consumption. Regarding the craving/preoccupa- inflammatory genes such as TNFa, IL-1b and iNOS (Fernandez- tion stage, which often precedes binge intoxication, blocking IL-1b Lizarbe et al., 2009). Alcohol also increases neuroimmune signaling in the VTA was found to block cocaine-induced dopamine signaling molecules in vivo. These effects in vitro and in vivo are release in the nucleus accumbens (Northcutt et al., 2015). dependent on TLR4, which seems to play a pivotal role in alcohol- Furthermore, human alcoholics have increased plasma levels of 62 F.T. Crews et al. / Neuropharmacology 122 (2017) 56e73 multiple pro-inflammatory cytokines such as TNFa (Heberlein humans and 12 in mice (Brubaker et al., 2015). Activation of TLRs by et al., 2014), which correlates with severity of alcoholism, IL-1b, endogenous DAMPs has been implicated in several non-infectious IL-6 and IL-8 which correlate with alcohol craving (Heberlein et al., neurological disorders, including alcoholism (Table 1). Tran- 2014; Leclercq et al., 2014). TNFa, IL-6, and IL-1b each cross the scriptome analyses in Drosophila have implicated Toll genes in the blood brain barrier and might contribute to central effects (Banks alcohol response, as discussed by Park et al. in their review in this et al., 1994, 1995). Though the precise signaling mechanisms need special edition (Park et al., 2017). The expression of several TLRs are to be further expounded, central pro-inflammatory cytokines upregulated in the brains of human alcoholics. TLRs 2e4aswellas modulate alcohol craving and consumption and peripheral cyto- the PRR RAGE (Receptor for advanced glycation End-products) kines are indices of craving and severity of alcoholism. were each increased in the frontal cortex of postmortem human The immune system also impacts the withdrawal/negative alcoholics (Crews et al., 2013). Ethanol treatment in vivo has also affect stage. Indeed, a significant body of literature suggests that been shown to increase the expression of TLRs 2e4 in cortex and substance abuse contributes to the development of negative af- cerebellum with subsequent NF-kb activation and cytokine induc- fective states such as anxiety and dysphoria (Koob and Le Moal, tion (Crews et al., 2013; Lippai et al., 2013b). Furthermore, ethanol 2005). Additional substance abuse aimed at alleviating these sensitizes neuroimmune responses to TLR3 and TLR4 agonists (Qin negative states contributes to a cycle of abuse and dependence. and Crews, 2012a; Qin et al., 2008). Chronic ethanol enhanced re- Inflammation and cytokine induction are known to contribute to sponses to systemic TLR3 and TLR4 agonists. This increased sys- negative affect. Withdrawal from alcohol increases inflammatory temic and brain pro-inflammatory response is in part due to binge cytokines in the brain (Freeman et al., 2012). Also, intra- alcohol increasing intestinal permeability, resulting in endotoxin/ cerebroventricular injection of cytokines sensitized anxiety-like LPS as well as bacterial RNA and other gut molecules crossing into behavior during withdrawal from ethanol (Breese et al., 2008). the blood inducing innate immune responses (Bala et al., 2014; These data suggest that cytokines are involved in the ethanol Keshavarzian et al., 2009; Szabo et al., 2010). Thus, there are mul- withdrawal process and contribute to the negative affect of alcohol tiple mechanisms by which ethanol leads to TLR activation and use disorders. Alcohol can also decrease neurogenesis, which is induction in the brain. associated with negative affect. Chronic voluntary alcohol con- High mobility group ss protein (HMGB1) has been identified as sumption decreases neurogenesis during withdrawal and is one such critical immune mediator in alcohol abuse. HMGB1 is accompanied by the development of depression-like behavior increased in the cortex of human alcoholics (Crews et al., 2013). (Stevenson et al., 2009). Both the decreased neurogenesis and Similar to TLRs 2e4, ethanol treatment in vivo increases HMGB1 in depression-like behavior were reversed by treatment with anti- cortex and cerebellum (Crews et al., 2013; Lippai et al., 2013b). depressants. Therefore, there are multiple mechanisms by which HMGB1 is a nuclear chromatin binding protein that can also be alcohol increases CNS neuroimmune signaling, contributing to the released as an innate immune mediator (Muller et al., 2004). molecular and cellular causes of alcohol use disorders. HMGB1 consists of two basic L-shaped ‘HMG box’ domains and an acidic 30 aa tail domain joined by a connecting linker (Stott et al., 6. HMGB1, Toll-like receptors and innate immune signaling in 2010). In the nucleus, HMGB1 stabilizes chromatin structure and the brain acts as a chaperone for certain transcription factors (Thomas and Stott, 2012). Extracellular HMGB1 activates innate immune re- Understanding the mechanism of signaling for many hormones sponses through direct binding to TLR4 or RAGE depending on its and has benefited from manipulations in redox state (Janko et al., 2014)(Fig. 4). The Cys106 thiol/Cys23- chemical structure allowing agonist and antagonist pharmacolog- Cys45 disulfide bond form of HMGB1 acts at TLR4 receptors ical distinction of responses. However, receptors that respond to (Fig. 4a), while fully reduced HMGB1 is active at the RAGE receptor larger molecules are more difficult to fully characterize. In the (Fig. 4b) (Venereau et al., 2012; Yang et al., 2012). Fully oxidized innate immune system, this has been the case. Initial studies HMGB1 is inactive at immune receptors (Fig. 4c) (Liu et al., 2012). characterized many receptors as responding to pathogens such as Both ethanol and tobacco smoke extract cause HMGB1 trans- bacteria and virus components. These pattern recognition receptors location from the nucleus with subsequent release in vitro (Chen (PRRs) and somewhat promiscuous and recently, studies involving et al., 2016; Zou and Crews, 2014). Thus, ethanol and possibly “sterile” inflammation i.e. innate immune activation in the absence other drugs of abuse lead to ‘sterile inflammation’ through the of a foreign pathogen have identified additional roles for these release of HMGB1. The release of other endogenous DAMPs likely receptors. This is especially true for the CNS, which is normally a also occurs, with recent reports indicating ethanol-induced release sterile environment, yet PRRs play critical roles in addiction pa- of TLR7-activating miRNA. The complex structure of HMGB1 allows thology. PRRs have revolutionized the understanding of the innate it bind to a large number of molecules that also sensitizes other immune system signaling. PRRs recognize specific molecular pat- agonist responses through their associated receptors in a complex terns associated with either foreign pathogens (Pathogen Associ- manner that is poorly understood. The double box and cysteine- ated Molecular Patterns-PAMPs) or endogenous molecules disulfide structure of HMGB1 creates unique heterodimers with associated with cell death, damage or stress (Danger Associated lipoglycans, endotoxin, and cytokines (e.g. IL-1b, Fig. 4d) and Molecular Patterns-DAMPs). Five classes of PRRs have been nucleic acids (e.g. miRNA, Fig. 4e) (Bianchi, 2009; Hreggvidsdottir described: Toll-like receptors (TLRs), C-type lectin receptors, et al., 2009). HMGB1 enhances the potency of cytokines at their nucleotide binding domain receptors (leucine-rich repeat con- respective receptors leading to greater immune responses (Fig. 4c). taining or NOD-like receptors), RIG-I-like receptors, and AIM 2-like Furthermore, HMGB1 is required for immune responses to endo- receptors (Brubaker et al., 2015). TLRs are the most well charac- somal nucleic acid binding TLRs (3, 7 and 9) (Yanai et al., 2009), terized PRRs, and have been implicated in addiction. TLRs are though the mechanism is unclear. Recent evidence has found that characterized by an N-terminal extracellular leucine-rich repeat ethanol causes increasing binding of HMGB1 to the miRNA let-7 sequence and an intracellular Toll/Interleukin-1 receptor/resistance while reducing its association with the RISC complex protein motif (TIR) (Takeuchi and Akira, 2010). These receptors recognize Ago2. HMGB1 might aid in the presentation of nucleic acids to their PAMPs and DAMPs that include a variety of molecules from bac- endosomal TLR receptors. Thus, the modulation of HMGB1 by terial endotoxin to mammalian HMGB1 and heat shock proteins ethanol might activate signaling through multiple TLRs in addition (Vabulas et al., 2002). To date ten TLRs have been identified in to TLR4. F.T. Crews et al. / Neuropharmacology 122 (2017) 56e73 63

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

TLR Foreign PAMP Endogenous DAMP Neurological condition

2 Bacterial di- and tri-aceylated polypeptides a-synuclein Parkinson's Disease (Kim et al., 2013) (Buwitt-Beckmann et al., 2006) Gram (þ) lipoglyans (Blanc et al., 2013) 3 dsRNA stathmin Alzheimer's Disease (Jackson et al., 2006) (Bsibsi et al., 2010) 4 Bacterial endotoxin HMGB1 Stroke, Traumatic Brain Injury Peptidoglycans HSPs 60, 70/72 (Vabulas et al., 2002) Chronic Alcoholism (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 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-1b to cause enhanced IL-1b 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.

7. TLR4 - a key brain receptor involved in ethanol induced et al., 2011). Ethanol not only increases the expression of TLR4 neuropathology (Crews et al., 2013; Fernandez-Lizarbe et al., 2013), it increases the formation of TLR4/TLR2 heterodimers in lipid rafts of microglia, Ethanol likely affects the signaling of multiple TLRs. However, leading to iNOS induction and MAPK activation (Fernandez-Lizarbe TLR4 in particular has been found to play a critical role in the et al., 2013). Importantly, both TLR4 and TLR2 KO microglia were neuroimmune activation by ethanol. TLR4 knockout mice are pro- protected in this study. In another genetic model, the alcohol tected from ethanol induced glial cell activation, NF-kB activation, preferring p-rats have increased expression of TLR4 in the VTA that and caspase-3 activation (Alfonso-Loeches et al., 2010a, b; Blanco was related to binge ethanol responding (June et al., 2015). TLR4 et al., 2005; Fernandez-Lizarbe et al., 2009; Pascual et al., 2011; expression in the VTA was regulated by GABA(A)a2 receptor (Liu Valles et al., 2004). Cultured astrocytes with siRNA against TLR4 or et al., 2011) and corticotropin-releasing factor (CRF) expression MD-2 and CD14 (critical adaptor molecules for TLR4 signaling) (June et al., 2015). Pharmacological studies also illustrate the role of were also protected from ethanol induced NF-kB induction. Mice TLR4 in the immune of ethanol. TLR4 stimulation with lacking TLR4 were also protected against ethanol withdrawal LPS leads to a prolonged increase in alcohol self-administration associated anxiety-like behavior and memory impairment (Pascual (Blednov et al., 2011). Mice lacking CD14, a critical adapter 64 F.T. Crews et al. / Neuropharmacology 122 (2017) 56e73 protein for TLR4, were protected from this effect. Naltrexone and 1996). The activated p-65 NF-kB subunit has been shown to Naloxone, opioid antagonists used to treat alcoholism, also have colocalize with dorsal horn spinal neurons (Bai et al., 2014) and anti-TLR4 actions. These antagonists reduce superoxide generation, both P19 and SH-SY5Y neuronal cell lines show NF-kB dependent cytokine production, and dopaminergic neurotoxicity by the TLR4 regulation of m-opioid receptor expression (Borner et al., 2012; agonist LPS (Liu et al., 2000a, 2000b). In Silico analyses indicate that Wagley et al., 2013). These responses are complex in part due to naltrexone and naloxone fill the LPS binding site of the MD2 co- the release of HMGB1 by stimuli that can subsequently activate receptor for TLR4 (Hutchinson et al., 2010). Furthermore, the multiple TLR and RAGE signals difficult to distinguish from the opioid inactive stereoisomers (þ)-naltrexone and (þ)-naloxone primary stimuli. For example, ethanol responses in brain involve have been shown recently to possess anti-TLR4 properties (Wang signaling across microglia, astroglia and neurons. HMGB1 is one of et al., 2016) indicating an immune mechanism that is indepen- the signals that contribute to neuroimmune activation and other dent of the opioid effects. These studies have identified a link be- responses. A comparison of ethanol activation of neuroimmune tween neuroimmune activation by ethanol via TLR4 signaling. This, signaling in -like SH-SY5Y and microglia-like BV cells finds coupled with the aforementioned studies regarding the endoge- ethanol induces TLR7 mRNA in both cell types, but neurons respond nous TLR4 agonist HMGB1, emphasizes the importance of studying at concentrations below binge drinking blood levels whereas the efficacy of targeted TLR4 antagonists for the treatment of microglia-like BV cells do not respond until high concentrations of alcohol use disorders. Thus, significant opportunities exist for alcohol well above the binge drinking BAC, but had a more robust identifying unique drug targets for neuroimmune pathology in response (Fig. 5). Though the unique cellular roles need to be alcoholism. further identified, it is clear that ethanol activates NF-kB signaling in brain. We have found that ethanol increases NF-kBeDNA binding 8. Induction of innate immune genes, and miRNA both in in vivo in mice (Crews et al., 2006) and in vitro in rat hippocampal-entorhinal cortex slice culture (Zou and Crews, 2006). Innate immune signaling is complex and involves multiple Furthermore, we and others have found that ethanol causes in- intracellular signaling cascades that lead to activation of key tran- duction of NF-kB target genes in brain, such as the scription factors. TLR signaling is mediated through key adapter monocyte chemoattractant protein-1 (MCP-1, CCL2) (He and Crews, proteins. Upon recognition of the ligand by the TLR, these adapter 2008), proinflammatory cytokines (TNFa, IL-1b, and IL-6) (Qin et al., proteins initiate the signaling cascade. The TIRAP/MyD88 adapter 2007), proinflammatory oxidases (inducible nitric oxide synthase protein 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 Nuclear Factor kappa-light-chain-enhancer of Activated B cells (NF-kB) and Activated Protein-1 (AP-1) respectively. These transcription factors regulate the expression of pro-inflammatory cytokines that prop- agate and magnify the immune response. Furthermore, these transcription factors are also linked to both stress and addictive behaviors. For instance, NF-kB activation occurs in the nucleus accumbens in response to cocaine, and was required for increased conditioned 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 induce AP-1 transcription in primary cortical neurons (Qiang and Ticku, 2005). Subcellular location and receptor trafficking also contribute to TLR responses, with nucleic acid sensing TLRs being endosomal and cell surface trafficking to endosomal TLRs (including TLR4 after its endocytosis) also bind to the TRIF adapter protein leading to transcription factor IRF3 activation and type I interferon gene induction. It is important to note that responses vary across cell type and most signaling has been characterized in monocyte and other immune cells that likely differ from that of brain cells. The precise signaling pathways for the TLRs in each brain cell type have yet to be delineated. Both microglia and astrocytes clearly show 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 suggested that neurons are not capable of activating NF-kB gene transcription fi Fig. 5. Neuron-like SH-SY5Y cells are more sensitive to ethanol induction of TLR7 (Mao et al., 2009). Others, however, nd NF-kB activation in glu- than microglia-like BV cells. Shown are TLR7 mRNA levels following 24 h of treat- tamatergic neurons that may regulate plasticity, learning and ment with various concentrations of ethanol in cultures of microglia-like BV2 and memory (Kaltschmidt and Kaltschmidt, 2015). However, RAGE was neuron-like RA-differentiated SH-SY5Y cells. TLR7 mRNA was determined using RT- originally identified as a neuronal receptor that bound HMGB1, 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 originally called amphoterin, involved in neurite outgrowth (Hori 15 mM ethanol, whereas microglia show little response at this concentration, but show et al., 1995). Ethanol is known to activate NF-kB in neurons in far larger responses at higher concentrations of ethanol. (*p < 0.05, #p < 0.01 vs. CON.) brain slice culture (Zou and Crews, 2006) and in vivo (Ward et al., Adapted from (Lawrimore and Crews, 2016). F.T. Crews et al. / Neuropharmacology 122 (2017) 56e73 65

(Alfonso-Loeches et al., 2010b; Zou and Crews, 2010), COX (Alfonso- activation, dysregulation occurs leading to many disease states (de Loeches et al., 2010b; Knapp and Crews, 1999), and NOX (Qin et al., Jesus et al., 2015). Recurrent and persistent NF-kB activation by 2008)), and proteases (TNFa-converting enzyme [TACE] and tissue repeated alcohol consumption appears to mimic a chronic inflam- plasminogen activator [tPA]) (Zou and Crews, 2010). matory state. This is evidenced by increased innate immune In addition to activation of TLRs via release of DAMPs, ethanol markers in the post-mortem brains of human alcoholics (Coleman also modulates immune function through release of microRNAs et al., 2017; Crews et al., 2013) Further, NF-kB target genes are (miRNA). MicroRNAs are small non-coding RNAs (~22 nucleotides). upregulated in the prefrontal cortex of alcoholics (Okvist et al., Neuroimmune regulation of miRNAs is extensive with numerous 2007). This persistent and chronic immune response has also miRNAs involved (Thounaojam et al., 2013). These molecules been seen after one dose of systemic LPS. One administration of regulate immune function in two known fashions. First, they high dose LPS (5 mg/kg) resulted in persistent microglial activation regulate the stability of target mRNAs in the cytosol via formation with ROS generation that led to dopaminergic neuron loss over a of an RNA-induced silencing complex (RISC) and binding to the 3’- period of months (Qin et al., 2013). Though on a lower scale, binge- untranslated region of mRNA (Ambros, 2004; Bartel, 2004; Czech ethanol doses cause similar pathophysiology (Bala et al., 2014; Qin and Hannon, 2011). The role of microRNAs as transcriptional reg- et al., 2008; Zou and Crews, 2010). ulators in alcoholism is discussed by Warden and Mayfield in their The prolonged amplification of neuroimmune responses occurs article in this special edition (Warden and Mayfield, 2017). Another on multiple levels. TLR activation in response to ethanol causes NF- newly identified mechanism whereby ethanol modulates immune kB activation, leading to increased TLR expression, cytokine pro- activation is through the extracellular release of miRNA duction and DAMP release. This amplifies immune responses in an (Turchinovich et al., 2013). Extracellular miRNA are contained autocrine and paracrine fashion, as secreted DAMPs and cytokines either in extracellular vesicles (EVs) or bound to either protein or activate both their cell of origin and surrounding cells (Fig. 6). lipoprotein chaperones. MicroRNA-containing vesicles are capable Paracrine signaling induced by ethanol also proceeds through the of being endocytosed by other cells where they can modulate their release of pro-inflammatory miRNA-containing microvesicles (Bala function. Recently, microRNA in EVs from ethanol-treated mono- et al., 2011; Coleman et al., 2017; Szabo and Lippai, 2014). This cytes were found to modulate the activation state of naïve mono- method of intercellular paracrine signaling has been implicated in cytes (Saha et al., 2016). Further, the miRNAs let-7 and miR-21 were many conditions such as cancer, cardiovascular disease, and neu- found to activate TLR7, leading to neuroimmune activation and rodegeneration (Hulsmans and Holvoet, 2013; Lehmann et al., neurodegeneration (Lehmann et al., 2012a; Yelamanchili et al., 2012a; Ohshima et al., 2010). Also, ethanol induces ROS genera- 2015). Thus, these miRNAs can have diverse actions to modulate tion that activates NF-kB and leads to further cytokine release and neuroimmunity, both through regulation of mRNA stability and TLR paracrine signaling (Pietri et al., 2005; Qin and Crews, 2012b; activation. Ethanol exposure causes significant alterations to the Thakur et al., 2007). A ‘smoldering’ level of inflammation due to miRNA milieu in the brain. In both human alcoholics and mice (20 repeated binge-ethanol, as is typical with alcoholism, might un- day treatment), ethanol altered the expression of dozens of miRNAs derlie the transition from alcohol abuse to addiction. Thus, phar- in the frontal cortex (Lewohl et al., 2011; Nunez and Mayfield, 2012; macological strategies should target the amplification and Nunez et al., 2013). Interestingly, members of the miRNA let-7 persistence of the immune response, as this likely underlies disease family were induced across species. Recently, ethanol has been pathology. found to potentiate TLR7-mediated neurotoxicity via release of let- 7 in MVs (Coleman et al., 2017). The miR-155 is also found in ves- 10. New treatments strategies based on immune icles and promotes TLR4 associated neuroimmune responses after pharmacology chronic ethanol (Lippai et al., 2013a). The miR-155 KO was pro- tected from ethanol-induction of cytokines. Thus, ethanol modu- The discovery of immune mechanisms of alcohol presents a new lation of miRNA release is an important aspect of innate immune approach for the treatment of alcohol use disorders. It is currently induction. These pathways represent new pathological mecha- unclear whether immune therapies would be of highest benefit for nisms that are poorly understood, but are targets for potential prevention or recovery from alcohol addiction. Neuroimmune intervention in the pathology of alcoholism. activation can contribute to neurodegeneration in many disease settings (Crews and Vetreno, 2014; Qin et al., 2013; Rocha et al., 9. Autocrine and paracrine processes amplify NF-kB innate 2015; von Bernhardi et al., 2015; Wang et al., 2015). Microglia and immune gene induction cytokines might also alter synaptic signaling (Lacagnina et al., 2017). For example, IL-1b reduces eIPSCs in the central amygdala As has been discussed above, regulation of innate immune re- to modulate ethanol effects on GABA receptors (Bajo et al., 2015). sponses in the brain is complex. This involves microglial priming Other immune mediators might also impact synaptic signaling. and changes in activation state, microglia-neuronal interactions, Though immune therapies would likely not be of benefit in brain and positive and negative feedback of intracellular signaling cas- regions where neurodegeneration has already occurred, there cades. The transcription factor NF-kB is a critical modulator of im- might yet still be benefit if they allow for recovery of normal syn- mune function. Both stress and alcohol result in NF-kB activation aptic function. Therefore, significant exploration of immune ther- through the immune mechanisms discussed above. Various models apies in various models of alcohol abuse is warranted. Many of stress in humans and rodents result in central and peripheral NF- commercially available drugs have been found to have anti- kB activation in immune cells. Human psychosocial stress causes inflammatory actions in the CNS. Certain antibiotics in particular NF-kB activation in peripheral blood mononuclear cells (Bierhaus have immune actions in addition to their antimicrobial functions. et al., 2003). Restraint stress in rodents causes NF-kB trans- Table 2 details some candidate drugs that have been found to be location into the nucleus with subsequent production of TNFa and beneficial for alcohol use disorders or other neuroimmune condi- pro-inflammatory prostaglandins (Bierhaus et al., 2003; Madrigal tions. Minocycline, for example, is a tetracycline antibiotic and et al., 2002). Ethanol also activates NF-kB in rat and mouse brain microglial inhibitor (Plane et al., 2010) that prevents ethanol (Qin and Crews, 2012b; Ward et al., 1996), as well as human as- induced-microglial activation and reduces alcohol self- trocytes (Davis and Syapin, 2004). The system itself is well- administration (Agrawal et al., 2011; Qin and Crews, 2012a). regulated. However, in the context of repeated or prolonged Phosphodiesterase 4 (PDE4) inhibitors have traditionally been used 66 F.T. Crews et al. / Neuropharmacology 122 (2017) 56e73

Fig. 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-1b bind cytokine receptors. Immune mediators act in both autocrine and paracrine fashions on neurons and microglia to amplify neuroimmune responses.

to treat psoriasis and psoriatic arthritis. These drugs are thought to subjective reward effects of (Worley et al., reduce inflammation by increasing cAMP concentration resulting in 2016). PPARg agonists, historically used for the treatment of type reduced NF-kB activation (Jimenez et al., 2001). Recently, these II diabetes, might also be helpful in alcohol use disorders through agents have been found to reduce ethanol intake in rodents (Bell their anti-inflammatory activities. The PPARg agonist pioglitazone et al., 2015; Blednov et al., 2014; Hu et al., 2011). A phase I clin- is a microglial inhibitor (Storer et al., 2005) that reduces toxicity in ical trial involving the PDE4 inhibitor ibudilast was recently models of fetal alcohol spectrum disorders (Drew et al., 2015; Kane completed but has not yet been reported. A recent placebo- et al., 2011) and may potentially play a beneficial role in alcoholism. controlled trial found that ibudilast reduced some of the Each of these drugs, and others with anti-inflammatory actions F.T. Crews et al. / Neuropharmacology 122 (2017) 56e73 67

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

Drug Mechanism CNS activity Primary Immune

Minocycline Tetracycline antibiotic Reduces alcohol self-administration (Agrawal et al., 2011) Microglial inhibitor 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 antibiotic Reduces binge ethanol intake in males (Cozzoli et al., 2016) mTORC1 inhibitor Neuroprotection via Autophagy Promotion (Chen et al., 2012; Pla et al., 2016) Azithromycin Macrolide antibiotic Promotes anti-inflammatory M2 microglial activation state (Zhang et al., 2015) Microglial inhibitor Rifampin Bacterial RNA polymerase Inhibits microglia activation to TLR4 (Bi et al., 2011; Wang et al., 2013) inhibitor TLR4 inhibition Indomethacin COX-2 inhibitor Reduces alcohol self-administration (George, 1989) Reduces ethanol neurotoxicity (Pascual et al., 2007) Simvastatin HMG-CoA Reductase Reduces inflammation and neurotoxicity to ischemia and injury (Lim et al., 2017; Sironi et al., 2006) Inhibitor NF-kB inhibition 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, Phosphodiesterase 4 Reduce ethanol intake in C57BL/6J mice (Blednov et al., 2014) CDP 840 inhibition Reduces ethanol self-administration in rats (Bell et al., 2015) Naltrexone/Naloxone m-opioid antagonist Reduces alcohol self-administration TLR4 inhibition Binds TLR4 adaptor protein MD2 (Hutchinson et al., 2010; Wang et al., 2016)

should be investigated for their potential efficacy in alcohol use disorders.

11. Neuroimmune basis of addiction

A hypothesis of stress- and drug-induced neuroimmune signaling that inactivates PFC and sensitizes limbic circuitry that suggest the progressive and persistent increases in HMGB1 and TLR with cycles of stress and drug abuse are the mechanisms under- lying the progressive and persistent nature of addiction. Innate immune signaling is known to have a major impact on cognitive and emotive functioning, leading to dysfunction (Dantzer et al., 2008; Hanke and Kielian, 2011; Okun et al., 2010; Yirmiya and Goshen, 2011). Ethanol and other drugs promote innate immune gene induction (Crews and Vetreno, 2016; He and Crews, 2008; Qin et al., 2008) that are linked to changes in executive function, Fig. 7. b2-microglobulin is induced in adult hippocampus following adolescent reinforcement-reward and negative affect-craving-anxiety that intermittent ethanol treatment. Shown are levels of b2 microglobin immunoreativity (b2M þ IR) determined by immunohistochemistry in hippocampal dentate gyrus. Rats promote alcohol abuse and addiction (Vetreno and Crews, 2014). were treated with ethanol on an intermittent schedule through adolescence, i.e. Indeed, innate immune activation and TLR signaling via ethanol adolescent intermittent ethanol treatment as described previously (Liu and Crews, appear to be essential for ethanol induction of addiction-like be- 2015). b2M þ IR was assessed on post-natal day 57, 24 h after the last AIE ethanol haviors. For instance, Guerri and colleagues (Pascual et al., 2011) treatment, and in adulthood on post-natal day 95. Note the ethanol-triggered induc- demonstrated that ethanol-induced innate immune activation in 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). mice impaired short- and long-term memory for object recogni- tion. This behavioral impairment was accompanied by a reduction of H3 and H4 histone acetylation as well as histone acetyl- transferase activity in the frontal cortex, striatum, and hippocam- and mimics the increase found in post-mortem human alcoholic pus. Interestingly, ethanol neither impaired behavioral brain (Vetreno et al., 2013). Thus, HMGB1, TLRs, RAGE, and other performance nor altered histone activity in TLR4 knockout mice. innate immune signaling are likely to drive changes in neurobi- fl Another in ammatory pathway, mediated by the RAGE receptor, ology related to addiction. might be involved in the memory impairments associated with The innate immune system can modulate alcohol consumptive fl chronic alcohol exposure since neuroin ammation associated with behavior. Analysis of genetically paired rats and mice that differ increased expression of this receptor is implicated in the memory primarily in the amount of ethanol consumption reveal that NF-kB impairments that accompany Alzheimer's disease (Arancio et al., and other pro-inflammatory genes have high expression in high 2004; Fang et al., 2010; Maczurek et al., 2008; Wilson et al., ethanol drinking animals (Mulligan et al., 2006). Beta-2 micro- 2009). Adolescent intermittent ethanol treatment of rats has been globulin, a NF-kB target gene involved in MHC immune signaling found to increase expression of RAGE that persists into adulthood 68 F.T. Crews et al. / Neuropharmacology 122 (2017) 56e73

(Pahl, 1999), was elevated in high ethanol preferring brain tran- striatum and amygdala (Izquierdo et al., 2016). This paradigm scriptomes (Mulligan et al., 2006). Interestingly, adolescent inter- models the changes that occur when the initial reinforcement, mittent ethanol treatment results in increased expression of beta-2 intoxication, and binge drinking stages of alcohol abuse progress to microglobulin in the adult hippocampus (Fig. 7). Beta-2 micro- obsessive-compulsive stages that are not able to avoid negative globulin progressively increases following adolescent alcohol consequences of drinking due to persistent perseveration and exposure (Fig. 7) in a manner similar to that found with HMGB1 inability to learn new approaches to reinforcement when situations (Vetreno and Crews, 2012) and RAGE (Vetreno et al., 2013). In change. Indeed, both human alcoholics (Fortier et al., 2008; Jokisch addition, work from Harris, Blednov and colleagues provided et al., 2014) and cocaine addicts (Stalnaker et al., 2009) show interesting and novel data supporting the hypothesis that innate reversal learning deficits. Using a rat model of binge drinking, we immune genes regulate ethanol drinking behavior (Blednov et al., found persistent deficits in reversal learning in rats (Obernier et al., 2005, 2012). Multiple strains of mice with immune gene deletion 2002) and in adult mice following adolescent binge drinking universally drink significantly less ethanol than matched controls (Coleman et al., 2011). Others have also seen reversal learning across multiple ethanol drinking paradigms. Recently, Blednov and deficits in adult mice after chronic ethanol (Badanich et al., 2011). colleagues (Blednov et al., 2011) also demonstrated that innate Interestingly, these reversal learning deficits are also found in rats immune activation through LPS can cause long-lasting increases in exposed to self-administered cocaine or passive cocaine injections ethanol drinking. Indeed, strains of mice show varied innate im- (Calu et al., 2007; Schoenbaum et al., 2004). Frontal cortical mune responses to LPS that correspond to increases in the con- dysfunction is established as resulting in perseveration and repe- sumption of ethanol. Furthermore, a single injection of LPS is tition of previously learned behaviors due to failure to associate capable of producing a delayed, but long-lasting increase in ethanol new information (e.g. negative consequences) into decision- consumption even in strains of high drinking mice. Recent data has making. Alcohol-induced damage to other regions associated revealed that the TLR family, especially TLR4, modulates ethanol with reversal learning (i.e. striatum and amygdala) might also intake. Administration of a GABAAa2 siRNA vector into the central contribute to reversal learning deficits and should be investigated. nucleus of the amygdala of alcohol-preferring rats diminished Further, the efficacy of immune modulating therapies to treat binge drinking, which was associated with reduced expression of cognitive dysfunction associate with alcohol abuse should be TLR4 (Liu et al., 2011). Interestingly, the neuronal location of investigated. Considerable emerging evidence supports a role for GABAAa2 receptors suggests that the influence of TLR4 on binge HMGB1/TLR signaling, innate immune gene induction and alter- drinking is at least partially mediated by neurons. Taken together, ations in epigenetics and neurotransmission as culminating in the these findings are consistent with a modulatory role of innate im- neurobiology of addiction (Crews et al., 2011; Cui et al., 2015; mune genes in alterations in GABA signaling as well as alcohol Vetreno and Crews, 2014). preference and consumption. Frontal cortical executive functions include: motivation, plan- Acknowledgments ning and goal setting, and behavioral flexibility. Among social drinkers the heaviest binge drinkers report more negative mood We would like to thank NIAAA for their financial support. and perform worse on executive functioning tasks (Townshend and (AA019767, AA11605, AA007573, and AA021040) Duka, 2003; Weissenborn and Duka, 2003). The frontal cortex regulates mood and cognition through reciprocal glutamatergic References connections with multiple brain regions. In astrocytes, ethanol exposure induces NF-kB transcription leading to increased Adachi, Y., Moore, L.E., Bradford, B.U., Gao, W., Thurman, R.G., 1995. Antibiotics expression of pro-inflammatory innate immune genes (Pascual prevent liver injury in rats following long-term exposure to ethanol. Gastro- et al., 2007; Zou and Crews, 2006, 2010) and reduced astrocyte enterology 108, 218e224. glutamate transport (Zou and Crews, 2005). The increased extra- Agrawal, R.G., Hewetson, A., George, C.M., Syapin, P.J., Bergeson, S.E., 2011. Mino- cycline reduces ethanol drinking. Brain Behav. Immun. 25 (Suppl. 1), cellular glutamate levels lead to increased neuronal excitation, S165eS169. microglial activation, and excitotoxicity (Ward et al., 2009; Zou and Ait-Belgnaoui, A., Durand, H., Cartier, C., Chaumaz, G., Eutamene, H., Ferrier, L., Crews, 2006). Indeed, increased glutamate excitotoxicity contrib- Houdeau, E., Fioramonti, J., Bueno, L., Theodorou, V., 2012. 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