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Neuropharmacology 76 (2014) 218e227

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Neuropharmacology

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

Invited review

Why is neuroimmunopharmacology crucial for the future of addictionresearch?

Mark R. Hutchinson a,*, Linda R. Watkins b

aDiscipline of Physiology, School of Medical Sciences, University of Adelaide, Level 5, Medical School South, Frome Rd, Adelaide, South Australia 5005,AustraliabDepartment of Psychology & Neuroscience, University of Colorado at Boulder, Boulder, CO 80309, USA

a r t i c l e i n f o

Article history:Received 5 February 2013Received in revised form13 May 2013Accepted 23 May 2013

Keywords:GliaCytokineChemokineInnate immuneToll-like receptor

Abbreviations: TLR, Toll-like receptor; LPS, Lipopo* Corresponding author. Tel.: þ61 8 8313 0322; fax

E-mail address: mark.hutchinson@adelaide.edu.au

0028-3908/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.neuropharm.2013.05.039

a b s t r a c t

A major development in drug addiction research in recent years has been the discovery that immunesignaling within the central nervous system contributes significantly to mesolimbic dopamine rewardsignaling induced by drugs of abuse, and hence is involved in the presentation of reward behaviors.Additionally, in the case of opioids, these hypotheses have advanced through to the discovery of thenovel site of opioid action at the innate immune pattern recognition receptor Toll-like receptor 4 as thenecessary triggering event that engages this reward facilitating central immune signaling. Thus, thehypothesis of major proinflammatory contributions to drug abuse was born. This review will examinethese key discoveries, but also address several key lingering questions of how central immune signalingis able to contribute in this fashion to the pharmacodynamics of drugs of abuse. It is hoped that bycombining the collective wisdom of neuroscience, immunology and pharmacology, into Neuro-immunopharmacology, we may more fully understanding the neuronal and immune complexities ofhow drugs of abuse, such as opioids, create their rewarding and addiction states. Such discoveries willpoint us in the direction such that one day soon we might successfully intervene to successfully treatdrug addiction.

This article is part of a Special Issue entitled ‘NIDA 40th Anniversary Issue’.� 2013 Elsevier Ltd. All rights reserved.

1. Drugs of abuse: where to begin?

The neurocircuitries that contribute to the adaptive and bene-ficial aspects of hedonia, learning, and memory are crucial for anorganism’s long-term survival. In contrast to activation of thesemulti-nuclei networked/patterned response systems by naturalrewards (palatable food, salt, sex, etc.), several classes of foreigncompounds (xenobiotics) are capable of directly “high-jacking”these systems, creating states of pharmacologically inducedeuphoria and reward. Repeated exposure to xenobiotics with thesepharmacological properties can lead to neuronal and behavioraladaptations that produce states of addiction and dependence to thexenobiotic, which is self reinforcing leading to escalation of druguse, and in the absence of drug produces states of withdrawal;hence these agents are collectively termed drugs of abuse. Theseabused xenobiotics have diverse structures and pharmacologies of

lysaccharide.: þ61 8 8224 0685.(M.R. Hutchinson).

All rights reserved.

both biologically-derived and fully synthetic origins. And yet, theaction of many drugs of abuse converge on the mesolimbic dopa-mine reward pathway, in which exposure to these xenobiotics re-sults in activation of the dopamine neurons projecting from theventral tegmental area to the nucleus accumbens shell and/orelevation of extracellular dopamine within the nucleus accumbensshell itself (Ikemoto, 2007). These xenobiotic effects occur viabypassing classical adaptive signaling pathways and directlymanipulating neurotransporter function, altering activation andinhibition pathways and vesicular displacement of neurotrans-mitters. Additional neuronal-mediated complexities to this systemhave also been observed (Laviolette et al., 2002; Vargas-Perez et al.,2009).

These xenobiotics can be from both legal and illicit drug origins,but, irrespective of origin, when these drugs are administeredpurely for their rewarding properties their use causes a profoundsocial and economic burden on the individual and the communityaround them. The state of addiction and dependence leads the in-dividual to repeatedly administer these xenobiotics, resulting inextensive exposure of the central nervous system to the parentxenobiotic and/or its metabolites. This xenobiotic exposure has a

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variety of consequences for the central nervous system, includingneuronal adaptation and toxicity (Büttner, 2011; Cappon et al.,1998; Fantegrossi et al., 2008; Salazar et al., 2008; Tilleux andHermans, 2007; Weber et al., 2006). These xenobiotic-induced al-terations in central nervous system homeostasis often lead toincreased allosteric load. Thus, in the absence of the xenobiotic, orwhen its pharmacological action is blocked, behavioral signs ofdependence and relapse are precipitated. However, these responsesare varied across abused xenobiotics, thus demonstrating speci-ficity of adaptation (Hyman et al., 2006).

Owing to the profound, abundant neuronal actions of theseabused xenobiotics, much of the research focus over several decadeshas been on understanding the neurocircuitry, neuronal receptors,intra-neuronal signalingpathways andneuronal-sensitizationeventsthat lead to the physiological state of addiction and dependence.However, in the past two decades, a trickle ofmanuscripts examiningthe non-neuronal central nervous system immune consequences ofdrugs of abuse hasnowswollen to a significantbodyofwork. Initially,these studies reported correlative evidence of central nervous systemproinflammation resulting from exposure to the drugs of abusedemonstrating key implications for neurotoxicity and disease pro-gression associated with, for example, HIV infection (Coller andHutchinson, 2012). However, more recently, this drug-induced acti-vation of central immune signaling is now understood to contributesubstantially to the pharmacodynamic actions of drugs of abuse, byenhancing the engagement of classicalmesolimbic dopamine rewardpathways and withdrawal centers. Thus the hypothesis of majorproinflammatory contributions to drug abuse was formed throughthe unification of the collective wisdoms of neuroscience, immu-nology and pharmacology; hence, Neuroimmunopharmacology.

Such discoveries of central nervous system immune involve-ment in themesolimbic dopamine reward pathway have significantimplications for how we understand reward to be modulated inbeneficial adaptive situations versus maladaptive pathogen- andxenobiotic-induced reward conditions. However, whilst exciting inits implications, the hypothesis of major proinflammatory contri-butions to drug abuse also presents a series of quandaries whichhave rightly been raised by the addiction neuroscience establish-ment. None are less critical than the question: How can proin-flammatory immune signaling be involved in drug reward andaddiction when we don’t like being sick?

Thus, the aim of this review is to introduce and review the litera-ture of the central immunology targets of drugs of abuse, highlightingthe common mediators and mechanisms and the exciting opportu-nities these new targets have in identifying ‘at risk’ individuals andnovel therapeutic opportunities. Additionally, some of the key con-ceptual and intellectual stumbling blocks that have impeded theproinflammatory hypothesis of drug abuse will be highlighted andexplained in detail. Finally, the future of addiction research through“Neuroimmunopharmacology tinted glasses”will be surveyed to paint apicture of what the future of addiction research might hold.

Owing to the breadth of xenobiotics abused it will be difficult tocover all the developments in Neuroimmunopharmacology foreach class. For a detailed review of these topics for abused xeno-biotics where central immune signaling involvement has beenestablished see our recent review (Coller and Hutchinson, 2012).Instead, here we will focus in this review on the evidence that we,and others, have generated over the past decade for opioid acti-vation of central immune signaling and the impact this has onopioid reward and dependence.

2. An introduction to central immune signaling

Given that the hypothesis of major proinflammatory contribu-tions to drug abuse requires both the knowledge of, and an

appreciation for, neuroscience, immunology and pharmacology, afew key concepts need to be introduced in order to make thisfascinating area optimally accessible. Firstly, the concept ofimmune-to-brain communication, which result in central immunesignaling and subsequent altered behavior via neuronal-dependentadaptations will be examined.

It is very uncommon in an Immunology 101 course for anyreferences to the central nervous system to be included, exceptperhaps when referring to immune involvement in neuro-inflammatory diseases, such as Alzheimer’s and Multiple Sclerosis.The predominant focus of most basic immunology courses, and infact the collective wisdom held by the general public, is that theimmune system’s role is to defend the host organism from invadingpathogens and to fight off infections. Whilst this host defensedogma is correct, the immune system has a far more nuanced rolethanwe inwestern medicine and medical research currently give itcredit for. The potential impact of peripheral immune cells, or im-mune signaling factors on brain function is commonly not dis-cussed. However, this limited view of immune function is rapidlychanging owing to a wealth of literature over more than 50 years.

Few of us will be unfamiliar with feeling sick at some point inour lives. But how do we feel sick and why don’t we like it? Astandard systemic immune response to an insult such as endotoxin(lipopolysaccharide from gram negative bacteria) causes a pro-found alteration in behavior, termed sickness behavior or the illnessresponse. The anhedonic qualities associated with the illnessresponse have been well established in multiple domains such asanimal husbandry as well as the clinic (Yirmiya et al., 2000). Clearlyanhedonia is only one facet of the complex sickness response,which also includes lethargy, depression, anxiety, anorexia,heightened pain states (hyperalgesia and allodynia), and cognitiveimpairment (Dantzer et al., 1999). Many of these behaviors requiresignificant central nervous system engagement, demonstratingthat this peripheral immune response is capable of profoundlymodifying behavior and thusmust have the capacity to alter centralnervous system function. These discoveries of immune-to-braincommunication are a cornerstone of Psychoneuroimmunology(Besedovsky and Rey, 2007).

The cause of the altered behavior induced by illness had beenpostulated to be due to changes inmetabolic reserves resulting fromthe full activation of the immense power, but energy hungry, im-mune system. But when it was discovered that a blood borne factorresulting from endotoxin exposure was capable of altering behav-ioral function without the need for endotoxin to cross the bloodbrain barrier (Holmes andMiller,1963), the age of immune-to-brainsignaling was born. These immune derived cytokines have beencharacterized to act by various humoral and neuronal routes to altercentral nervous system neuronal function (Capuron and Miller,2011; Miller et al., 2009). But this is not the only alteration in thebrain during an illness response. The non-neuronal cells of thecentral nervous system, glia, also respond following a similar pe-ripheral immune challenge (LaflammeandRivest,1999), causing thegeneration of proinflammatory cytokines and a myriad of otherneuronal adaptations and sensitizations within the brain and spinalcord. Hence, illness-induced immune responses are capable ofprofoundly altering central nervous system function via multipleparallel routes.

3. But is this too non-specific? Are all brain nuclei altered tothe same extent? How can this peripheral immune responsecause a specific behavioral phenotype such as the illnessresponse?

The key concepts of bioavailability, neuroanatomy and immuneheterogeneity come to the forefront. Firstly, much of the central

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nervous system is protected from the primary factors of an invadingorganism, owing to the blood brain barrier, thus rendering factors,such as endotoxin, with poor central nervous system bioavailabilityand immediately restricting the triggers of central immunesignaling. Secondly, discrete central nervous system nuclei arecapable of responding to the immune challenge owing to theirneural networks and structural characteristics. The circum-ventricular organs are sensitive to peripherally restricted factorslike endotoxin and circulating cytokines, but likely choose thisleaky blood brain barrier phenotype in order to sense peripheralchallenges. The ascending sensory neural projects that sense pe-ripheral immune responses via the sensory vagus nerve project tothe nucleus of the tractus solitarius and area postrema and fromthere can secondarily recruit additional centers through specific,targeted pathways the activation of which leads to the generationof each element of the sickness response: fever, increased sleep,adipsia/aphagia, anhedonia, etc. Therefore, the majority of thecentral nervous system is not influenced by peripheral immuneresponses. Finally, even within these peripheral immune-influenced nuclei there is significant immune heterogeneity. Justas a dopamine neuron is as distinct in its neuroanatomical location,transcriptome and proteome from a serotonin neuron, it isapparent that the immune cells and immune signalingmolecules ofthe central nervous system have similar regional specificity (Adlerand Rogers, 2005). Thus, a peripheral immune response does notsimply cause pan proinflammatory glial reactivity within the cen-tral nervous system. Rather discrete nuclei are sensitive to suchimmune stimuli and become activated (Laflamme and Rivest, 1999;Park et al., 2008). Moreover, the immune signals within the centralnervous system may not be “inflammatory” per se, and rather canact at sub-inflammation levels functioning in a fashion akin toneurotransmitters (Adler and Rogers, 2005). Interestingly, therange of factors that have been identified as capable of triggeringcentral immune signals has expanded to include endogenousdanger signals (DAMPs; danger associated molecular patterns) thatelicit responses from the innate immune system, such as viapattern recognition receptors (Buchanan et al., 2010).

By examining a combination of these neuronal and non-neuronal adaptations following peripheral illness, a series of hy-potheses have been put forward to explain the altered behaviorcaused by the initial immune response. These include, but are notlimited to, active transport of cytokines into CNS (Erickson et al.,2012), induction of neuroinflammatory mediators in brain as aconsequence of blood-borne cytokines (Dantzer et al., 2008), acutedepletion of serotonin owing to microglial upregulation of indol-amine-2,3-oxygenase which causes a direction of tryptophan awayfrom serotonin synthesis and instead into the kynurenic acidpathway; and alterations in glutamate homeostasis due to proin-flammatory signaling induced down regulation of astrocyticglutamate transporters which cause acute loss of extracellularglutamate control (Capuron and Miller, 2011; Miller et al., 2009;Yirmiya and Goshen, 2011).

4. So where do drugs of abuse fit in and how isproinflammatory immune signaling within the centralnervous system involved? How can proinflammatory immunesignaling be involved in drug reward and addiction when wedon’t like being sick? If illness is something we don’t enjoyshouldn’t illness counteract the rewarding pharmacodynamicactions of drugs of abuse?

Examples of alterations of central nervous system immunologyby drugs of abuse began surfacing in the early 1990s with profoundopioid-induced changes in glial cellular morphology and pheno-typic receptor/immunohistological marker expression. For

example, after long-term systemic morphine administration, asignificant increase in astrocytic GFAP (glial fibrillary acidic protein;a cell surface marker of astrocyte reactivity) expression wasobserved in the ventral tegmental area but not in the substantianigra, locus ceruleus, cerebral cortex, or spinal cord, thereby dis-playing significant regional heterogeneity (Beitner-Johnson et al.,1993), perhaps in part reflective of known heterogeneities in as-trocytes (Chaboub and Deneen, 2012). Moreover, these opioid-induced differences in GFAP expression were strain specific(Beitner-Johnson et al., 1993). It is noteworthy that Beitner-Johnsonet al. (1993) hypothesized a possible role for these opioid-inducedGFAP adaptations in response to drugs of abuse, because theyoccurred primarily in the mesolimbic dopamine system and straindifferences were correlated with preference for drugs of abuse.However, this line of research fell silent until the turn of themillennium.

Immune involvement, in general, was first demonstrated byDafny et al. (1990) and Dougherty et al. (1990) who found thatseveral non-selective immunosuppressive treatments amelioratedmorphine withdrawal behaviors in rats. However, these studieswere conducted prior to an appreciation of the importance of glialcells in the behaviorally relevant functioning of the central nervoussystem. Additionally, concerns about the animals’ ability to displaywithdrawal behaviors under such generalized immunosuppressantregimens led to the end of the work (Berthold et al., 1989; Dantzeret al., 1987). However, one intriguing aspect to Dafny’s researchwasthat his use of systemically administered immunosuppressantswould likely have drastically altered immune-to-brain and centralimmune signaling capacities.

The first implication of central immune signaling modulation ofmorphine reward behaviors was accomplished by direct injectionof astrocyte-conditioned medium (that likely contained importantsoluble co-factors such as cytokines and chemokines, but notendotoxin) into the nucleus accumbens (Narita et al., 2006). Thiscaused heightened morphine conditioned place preference, a pre-clinical behavioral index of reward that relies upon Pavlovianconditioning of neutral environmental stimuli and pairing with theadministration of a drug (Narita et al., 2006). Through the use ofpharmacological proinflammatory glial attenuating drugs, thisstudy (Narita et al., 2006), and others to follow, demonstrated thatblockade of proinflammatory glial activation could: blockmorphineand oxycodone conditioned place preference (Hutchinson et al.,2009, 2008b, 2012; Narita et al., 2006); block drug-induced rein-statement of morphine conditioned place preference (Schwarzet al., 2011); attenuate remifentanil self administration(Hutchinson et al., 2012); block morphine and oxycodone sponta-neous and precipitated withdrawal (Hutchinson et al., 2009,2010b), and simultaneously prevent brain increases or causedbrain decreases of cytokines and chemokines in various brain re-gions shown to mediate withdrawal (e.g. in the ventral tegmentalarea and in the nucleus accumbens)(Hutchinson et al., 2009); andblock morphine-induced elevations of dopamine in the nucleusaccumbens (Bland et al., 2009; Hutchinson et al., 2012). Collec-tively, this body of work from multiple laboratories has entwinedcentral immune signaling in the complexities of the mesolimbicdopamine reward neurocircuitry.

Despite opioid-induced central immune signaling being foundto modify opioid-induced dopamine release in the nucleusaccumbens, much of the prior and perhaps parallel events that leadto the elevated behavioral reward and dependence endpointsremain poorly characterized. Clearly, opioid-induced central im-mune signaling must have neuronal consequences in order toprecipitate these changes in behavior. The neuronal consequence ofopioid-induced proinflammatory immune signaling that have beencharacterized to date suggest that the response is dependent on

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proinflammatory-induced down-regulation of glutamate trans-porter expression, which can lead to a dysregulation of extracel-lular glutamate (Nakagawa et al., 2005; Nakagawa and Satoh, 2004;Ozawa et al., 2004, 2001; Wang et al., 2003), potentially contrib-uting at minimum to opioid withdrawal (Nakagawa and Satoh,2004). Additionally, proinflammatory cytokines themselves havebeen proposed as potential contributors to opioid reward (Collerand Hutchinson, 2012) via their neuroexcitatory effects (Watkinset al., 2009), GABA receptor down-regulation (Stellwagen et al.,2005), upregulation of AMPA/NMDA expression and function (Deet al., 2003), and enhancement of neurotransmitter release (Younet al., 2008). The opioid-induced modulation of glial derived neu-rotrophic factors such as GDNF are also of significant interest owingto the established behavioral significance for GDNF on nucleusaccumbens dopamine levels and the expression of reward behav-iors (Airavaara et al., 2006, 2004). However, the significance andconsequences of the regional heterogeneity of opioid-inducedcentral immune signaling in the very early Beitner-Johnson et al.(1993) studies are at present unclear.

Whilst evidence for thesemajorproinflammatorycontributions todrugabusehasbeenpresentedhere formorphineandsimilaropioids,similar central proinflammatory responses have been observed for adiverse range of abuse xenobiotics, such as alcohol, cocaine, meth-amphetamine, and 3,4-methylenedioxymethamphetamine (seeColler and Hutchinson, 2012 for review). Interestingly, as time passesand the proinflammatory hypothesis of drug abuse becomes morewidelyknownandaccepted, increasingnumbersofmanuscriptshavebeen published that transition this associative relationship betweenxenobiotic exposure and proinflammatory central immune re-sponses, to a causative pharmacodynamic relationship. To date, evi-dence has now been published demonstrating central immunesignaling involvement in behavioral responses to alcohol, cocaine,methamphetamine, and 3,4-methylenedioxymethamphetamine(Coller and Hutchinson, 2012). However, the mechanistic link be-tween the xenobiotic-induced central immune signaling and alteredmesolimbic dopamine reward neurocircuitry remains to be charac-terized and contrasted for the different classes of abused substances.

5. How do structurally diverse xenobiotics have a similarcentral immune proinflammatory phenotype but such diverseneuropharmacologies?

The apparent non-specificity of the activation of the centralimmune response has raised concerns that this event is a byproductof direct neuronal actions of the drugs of abuse rather than a spe-cific triggering of immune signaling and their activation pathways.Whilst this is a very valid hypothesis supported by generic neuro-toxicity events triggered for example by xenobiotic-inducedglutamate excitotoxicity, recent evidence suggests an entirelynew site of xenobiotic action. Whilst first characterized for opioids,this new non-opioid receptor site of opioid action likely has im-plications for a very diverse range of xenobiotics, both of abusedand non-abused pharmacologies (Coller and Hutchinson, 2012;Hutchinson et al., 2012, 2011).

Opioid research has long focused on opioid ligand action atclassical neuronal opioid receptors and their role in disinhibition ofthe mesolimbic dopamine reward pathway and modification ofseveral other nuclei in the presentation of acute reward, develop-ment of dependence and presentation of withdrawal. Classicalneuronal opioid receptors and their associated pharmacodynamicactions are well characterized as stereoselective, that is the natu-rally occurring (�)-isomer of opioid ligands has far greater activitythan the unnatural, fully synthetic, (þ)-isomer (Takagi et al., 1960).However, the early work of Takagi et al. (1960) demonstrated thepharmacodynamic relevance of non-classical non-stereoselective

opioid actions. Since this early work, only a handful of papers haveexamined the pharmacological and behavioral significance ofopioid actions at these non-stereoselective sites (Hutchinson et al.,2010a, 2011, 2008c). Several sites have been proposed, such asFilamin A (Burns and Wang, 2010; Wang et al., 2008) and NADPHoxidase (Wang et al., 2012a). However, our interest has focused on afascinating class of innate immune receptors called Toll-like re-ceptors (TLR), owing to their unique and diverse detection capacityand downstream signaling events, some of which had already beenimplicated in opioid pharmacodynamics. Interestingly, we havegenerated evidence that some of the other sites of potential actionof opioids may lie downstream of TLR engagement increasing theimportance of TLRs to opioid pharmacodynamics (Hutchinsonet al., 2012).

As introduced previously, structurally diverse drugs of abuse,including opioids can be viewed as xenobiotics; that is, chemicalswhich are foreign to the organism. The body has a range of well-characterized systems to detect these foreign chemicals such asthe liver’s pregnane X receptor (Matic et al., 2007). The questionweposed was whether there were other receptors that would recog-nize chemicals as substances “foreign” to the central nervous sys-tem and trigger a central immune signaling response, and thus bethe entity upstream of the proinflammatory central immune sig-nals contributing to opioid reward and dependence behaviors.

Within the central nervous system, pattern recognition re-ceptors, such as TLRs, can serve this sentinel role identifying “mo-lecular patterns” as “non-self” or “danger” signals (Buchanan et al.,2010). These receptors are expressed by the innate immune systemcells of the central nervous system, including endothelial cells,microglia, and some astrocytes, but rarely by adult neurons undernon-pathological conditions (Buchanan et al., 2010; Chen andNunez, 2010; Rivest, 2009). However, in some cases neuronal TLRexpression has been reported (Li et al., 2009; Wadachi andHargreaves, 2006), although the behavioral role of such expres-sion is yet to be clarified. Unlike neuronal receptors for neuro-transmitters, which display ligand selectivity and specificity,pattern recognition receptors have explicitly evolved to recognizemultiple diverse conserved pathogen-associated molecular pat-terns (PAMPs) that are associated with microbial pathogens orcellular signals of danger or stress (danger-associated molecularpatterns; DAMPs). Harking back to the original psychoneuroim-munology work in the 1960s, TLR4 has been characterized as theinnate immune receptor responsible for detecting the presence ofendotoxin (Hoshino et al., 1999). Binding of agonist ligands to TLR4and its accessory molecules such as MD2 and CD14 activatesdownstream intracellular signaling pathways similar to thoseactivated by interleukin-1b (O’Neill, 2008), and in fact belongs tothe interleukin-1 receptor/TLR superfamily, inducing the produc-tion and release of proinflammatory, neuroexcitatory mediators viaMyD88-dependent intracellular pathways (Yirmiya and Goshen,2011). Therefore, TLRs may play a pivotal sensing role for theinnate immune system within the central nervous system bydetecting the presence of xenobiotics, such as opioids, and trans-lating their presence into a central immune signal.

Over recent years, supported by National Institute on DrugAbuse funding, we have examined the xenobiotic-mediated TLR4actions of opioids using a collection of in vivo, in vitro, molecularand in silico strategies, demonstrating that various opioids,including morphine, activate TLR4 signaling (Hutchinson et al.,2012, 2010b; Wang et al., 2012b) through binding to an accessoryprotein of TLR4, MD2, thereby inducing TLR4 oligomerization andtriggering immune signaling within the central nervous system(Hutchinson et al., 2012; Wang et al., 2012b). With specific rele-vance to opioid reward we have demonstrated that pharmacolog-ical blockade of TLR4 or genetic deletion of MyD88-TLR4-

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dependent signaling suppresses opioid-induced conditioned placepreference; reduces remifentanil self-administration and reducesmorphine-induced elevations of extracellular dopamine in thenucleus accumbens (Hutchinson et al., 2012). Importantly, theseactions of opioids at TLR4 do not produce a unidirectional influenceon opioid pharmacodynamics, as blockade of opioid action at TLR4or reduction of opioid-induced proinflammatory central immunesignaling enhances acute and chronic opioid analgesia (Hutchinsonet al., 2007, 2008a,b, 2010a,b, 2009, 2012, 2011; Thomas andHutchinson, 2012; Wang et al., 2012b). Thus we have establishedat minimum opioid-induced TLR4 as an initiating site of proin-flammatory central immune signaling which occurs parallel to ac-tions at opioid receptors, collectively creating the behavioral opioidphenotypic responses.

6. Whose fault is it, the parent or the “child”? The effect ofdrug metabolites on central immune signaling

In order for the body to clear xenobiotics from the system, theycan undergo a myriad of biotransformations, often catalyzed byspecific enzymatic reactions, such as those carried out by thefamilies of Cytochrome P450s and glucuronyltransferases, just toname two. Traditionally, these reactions are considered bio-inactivation steps that serve the dual purpose to also facilitate thehydrophilic clearance of the xenobiotic. However, in some cases theopposite is true and bioactivation can occur through the unmaskingof an active functional group, thus metabolism of the parentxenobiotic gives birth to an active metabolite “child”, throughwhich the pharmacological activity of the parent xenobiotic con-tinues. This pharmacological activity of the metabolite can be equalto, or greater than the parent, and critically, can share the same,lose or gain pharmacological characteristics.

Themainmetabolites of morphine, morphine-3- andmorphine-6-glucuronide are intriguing beasts. Owing to the establishedstructure activity relationship of the morphine 4,5-epoxymorphianstructure established with simpler structures such as codeine/morphine and hydrocodone/hydromorphone, one can immediatelyhypothesize that morphine-6-glucuronide will have maintainedopioid activity, since the critical 30 hydroxyl group is maintained,whilst morphine-3-glucuronide will have profound loss of opioidfunction (Chen et al., 1991).What is not anticipated is the newfoundneuroexcitatory activity of morphine-3-glucuronide (Labella et al.,1979), which does not involve direct morphine-3-glucuronide ac-tivity at NDMA receptors (Hemstapat et al., 2009). Thus, despiteextensive searchers for direct neuronal targets, the mechanism ofmorphine-3-glucuronide action remained unclear.

Until recently the acknowledgment of the immune modulatorycapacity of opioids had been limited to their parentmolecules, withlittle to no attention paid to the much longer lasting and abundantmetabolites. We identified that morphine-3-glucuronide displayedprofound TLR4 activity, greater than that of its parent morphine,and was capable of inducing in vitro microglial proinflammatoryactivation (isolated from the spinal cord) and release of Interleukin-1b, with similar in vivo effects also observed and noted to produceprofound nociceptive TLR4-dependent behavioral consequences(Lewis et al., 2010). This discovery of immune-biasing of morphinevia 3-glucuronidation has profound ramifications as it opens thedoor for other glucuronidated xenobiotics to follow a similar pathto become recognized as xenobiotic associated molecular patterns(XAMPs) by pattern recognition receptors such as TLR4. The firstextension of this hypothesis is born out in our recent work thatdemonstrates that a major ethanol metabolite, ethyl-glucuronide,possesses TLR4 activity and associated in vitro molecular andin vivo behavioral consequences (Lewis et al., 2013; Schäfer, 2013).How many other xenobiotics of either licit or illicit use, naturally

occurring or in the Pharmacopoeia undergo similar immuno-bioactivation steps? Only time and further research will tell.

The reason why xenobiotic metabolism has such an immuno-bioactivation action may be found in early immunogenicityresearch of haptenation (Landsteiner, 1936). A hapten is a smallchemical functional group, which alone possesses minimal im-mune activity, but when combined with a carrier molecule, such asa protein, now gains immunogenicity to which the immune systemcan respond. This strategy has been commercialized to create, forexample, many of the antibodies used for immunohistochemistryor other research purposes today. Haptenization can be employedby the host organism to unmask the presence of an otherwiseimmunologically invisible parasite (Palm and Medzhitov, 2009).Interestingly, many of the haptens which were discovered early lastcentury have been forgotten, instead only the most active agentsare still employed to engender strong acquired immune antibodyresponses (Palm and Medzhitov, 2009). Of note is that glucur-onidation is among the haptenation that Landsteiner (1936) char-acterized to possess immunogenicity. Recently, additionaladvancements in understanding the immune response to haptenswas clarified by Palm and Medzhitov (2009), which has furtherimplicated the innate immune system and TLRs in the complexitiesof hapten detection and subsequent innate and acquire immuneresponses. Therefore, could it be that biotransformation enzymesand the innate immune system are joining forces to detect thepresence of an evolutionarily ancient foe, similar to TLR4 detectionof endotoxin? This is an area with wider implications than the drugabuse field and disserves significant attention.

7. But hang on, we have just said endotoxin and hence TLR4activation produces sickness behaviors and anhedonia. Howcan xenobiotic-induced TLR4 activation contribute to drugreward, but pathogen-induced TLR4 signaling causes sicknessbehaviors? Aren’t these contradictory?

Rewarding behaviors, such as the ingestion of palatable foods,are altered by sickness induced TLR4 signaling. This signaling isinitiated out in the body as a consequence of pathogen/endotoxinrecognition by TLR4-expressing peripheral immune cells. Alter-ation in palatable food intake is the behavioral culmination of thecomplex balance of both anhedonic and hedonic neurocircuitries(Baldo and Kelley, 2007; Berridge and Kringelbach, 2013). Impor-tantly, a distinction here needs to be made when comparing thecentral immune signaling following administration of a xenobioticof abuse and exposure to a peripheral immune stimulant likeendotoxin or gram negative bacteria. Xenobiotics of abuse, unlikeendotoxin or gram negative bacteria from which endotoxin isderived, can broadly access brain nuclei including those associatedwith drug reward owing to their penetrance across the blood brainbarrier. Xenobiotics of abuse directly engage TLR4-dependentcentral immune signaling, rather than relying on indirect humor-al and neuronal pathways activated as a consequence of strictlyperipheral activation of TLR4. Thus a distinct pattern of activationshould occur following CNS-penetrant xenobiotic exposure versusfollowing peripherally-restricted endotoxin/gram negative bacte-ria, and hence each sums to produce substantially differentbehavioral responses.

Whilst, behaviorally, illness associated TLR4 signaling gives riseto reduction in palatable food intake, this effect is not solely causedby a reduction in the hedonic value of the food but is also due tocombined influences of illness-induced aphagia, fatigue, anxietyand other illness associated outcomes as well (Borowski et al., 1998;Park et al., 2008). Therefore, even during peripherally triggeredTLR4-dependent illness responses, were an ill organism toencounter a rewarding stimulus, activation of the reward

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neurocircuitries by such stimuli can still occur, despite the behav-ioral response appearing as anhedonia (Borowski et al., 1998; Parket al., 2008). A key example of this is the activation of the caudalnucleus accumbens by palatable food consumption under bothcontrol and peripheral endotoxin-induced sickness conditions(Park et al., 2008). The caudal nucleus accumbens is a key nucleicontributing to the positive hedonic value of numerous pharma-cological and non-pharmacological stimuli (McBride et al., 1999).Importantly, the behavioral anhedonia reported by Park et al.(2008) likely resulted from endotoxin-induced activation of therostral nucleus accumbens, an area linked with anhedonia(Berridge and Kringelbach, 2013; McBride et al., 1999).

But can a peripheral TLR4-dependent illness response or centralimmune signaling event alone engage any part of the rewardneurocircuitry? This has been examined by Borowski et al. (1998),who demonstrated that systemic endotoxin (lipopolysaccharide;LPS) administration caused significant elevations in nucleusaccumbens dopamine release, a neurochemical response classicallyassociated with hedonic rewarding experiences, despite this beingassociated with decreased intra-cranial self stimulation onascending but not descending rate-intensity functions. As nucleusaccumbens dopamine release can also be induced by non-rewarding stimuli, like shock or other stressors, the interpretationof dopamine release in response to sickness remains to be defined.

However, given the fact that either central immune signalingalone (caused by centrally administered astrocyte conditionedmedia (Narita et al., 2006)) or TLR4 activation alone (produced bycentral endotoxin administration (Holmes and Miller, 1963)) fail topresent behaviorally as rewarding experiences indicates, additionalengagement of other reward pathways is require. And yet, as out-lined previously, opioid reward in TLR4 null mutant mice, or whenTLR4 is pharmacologically blocked by an antagonist, fail to producebehavioral reward (Hutchinson et al., 2012). Therefore, the twosystems are interdependent and activation of both appears to benecessary to produce behavioral reward. However, the precisemechanism of this fascinating relationship requires furtherexamination.

This does raise a quizzical point of methodological contention:does decreased intracranial self stimulation, palatable food con-sumption or self administration of a xenobiotic of abuse mirrordiscounting of their rewarding qualities thus rendering responsesinherently less valued, or alternatively, does this reflect anincreased rewarding value of the stimuli and hence reduced re-sponses are required to reach hedonic satisfaction? Sure enough,examples of both interpretations can be found in the literature(Park et al., 2008; Ranaldi and Beninger, 1994), emphasizing thevalue of testing multiple preclinical models. However, futuremethodological complications presented by examining the role ofcentral immune signaling in reward behavior and neurochemistryneed to be examined carefully using multiple compatible meth-odologies. Collectively, these data open a fascinating window intothe potential, and yet extremely complex relationship betweenillness responses and drug reward, and thus the likely involvementof TLR4 and central immune signaling in both.

Thus, opioids together with their newly identified TLR4 actions,affect the mesolimbic dopamine system to amplify opioid-inducedelevations in extracellular dopamine levels, with especial attentionbeing drawn to the caudal nucleus accumbens. This means that onecould expect that activation of TLR4 or pharmacologicallymimicking its downstream proinflammatory central immune sig-nals would potentiate drug reward. This has already been demon-strated for alcohol (Blednov et al., 2011), and as discussed andreviewed previously (Coller and Hutchinson, 2012), blockade ofTLR4 and related immune signaling pathways drastically alters thebehavioral response to multiple drugs of abuse. However, why

behaviorally alcohol consumption under these TLR4-burdenedconditions presents as an increase in response rate, whilst palat-able food consumption does not, needs greater examinationacknowledging a role of the central immune signaling and rewardneurochemistry events.

Thus, to reiterate, it is hypothesized that if proinflammatorycentral immune signaling is triggered in key central nervous systemnuclei combined with activation of mesolimbic dopamine rewardpathways the “perfect storm” of neuronal and immune signals arecreated to produce the full range of reinforcing signals that presentbehaviorally as drug reward (see schematic in Fig. 1). However, itmust be emphasized that a great deal of additional work is requiredto consolidate these hypotheses with established neural pathwaysand temporal signaling events.

8. Do we have any human data to support theproinflammatory hypothesis of drug abuse?

Currently there are no clinically applicable pharmacologicaltherapies that target the central nervous system immune signalingpathways. Even the range of immunomodulatory agents that areclinically prescribed have been designed to alter peripheral notcentral immune function and as such often have very poor centralnervous system bioavailability. As such, pharmacological testing ofthis proinflammatory hypothesis is currently difficult, althoughpromising data are on the horizon (www.clinicaltrials.gov; searchibudilast; studies NCT01740414 and NCT00723177). Nevertheless,given that up to 60% of drug dependence is heritable (Tsuang et al.,1998), if central immune signaling were amajor contributor to drugabuse one would expect that genetic variability in immune path-ways would be associated with drug dependent populations.Moreover, if the TLR4 immunobioactivation hypothesis above iscorrect, onewould hypothesize that similar immunogenetic relatedtraits would be associated with multiple drugs of abuse. Addi-tionally, such a hypothesis would also aid in the scaling of theladder of drugs of abuse from the perceived less harmful drugs ofabuse to those whose consumption is profoundly detrimental.

To date we have examined functionally important singlenucleotide polymorphisms in the Interleukin-1b gene and theirassociation with dependence to opioid or alcohol in two separatepopulations. Here we positively linked�511C/T and �31T/C in bothpopulations (Liu et al., 2009). Mechanistically how these genesagree with the central hypothesis of proinflammation linked to riskof dependence, the Interleukin-1B wild-type sequence at �511and �31 is associated with increased Interleukin-1b release, henceit is hypothesized that carriers of the wild-type allele have greatercentral immune signaling and proinflammatory responsesfollowing opioid exposure (Liu et al., 2009) and hence greater riskof dependence which is supported by preclinical data (Liu et al.,2011). Examination of a wider range of genetic polymorphisms inimmune genes is near completionwith exciting implications for theidentification of at risk individuals and the personalization ofpharmacological dependence treatment to a central immune tar-geted therapy when immunogenetic screening indicates probableproinflammatory involvement in a case of drug abuse.

9. Can we pharmacologically or non-pharmacologicallyintervene to reduce drug abuse by targeting central immunesignaling pathways?

Abstinence programs and substitution based maintenancetherapies are commonly employed, but unfortunately are ineffec-tive in the long run, and in some countries face stiff political op-position. Non-pharmacological therapies such as cognitive basedtherapy are widely used with some success. Thus the newly

Fig. 1. Hypothesized central immune signaling contributions to opioid reward. a) The classic view of opioid reward suggests that opioids bind to neuronal opioid receptors resultingin the eventual activation of the cortico-mesolimbic-dopamine reward pathway and the presentation of associated reward behaviors. In this model, glia, such as astrocytes andmicroglia, are not accounted for, beyond their homeostatic roles. b) The recent appreciation of non-neuronal actions of opioids and opioid-induced proinflammatory glial activationnecessitates additions to the solely neuronal hypotheses of opioid reward. The wealth of information of neuronal signaling and adaptations that occurs following opioid exposureand neuronal opioid receptor activation can now be complimented by the parallel TLR4-dependent activation of central immune signaling, and alteration of neuronal excitabilitydue to a collection of opioid-TLR4 dependent glial adaptations. Collectively, the neuronal opioid receptor-dependent and complementary opioid-TLR4 dependent mechanismsproduce a heightened opioid reward signal and ensuing presentation of altered behavior.

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discovered role of central immune signaling in drug reward anddependence provides an exciting novel target to achieve a drugaddiction treatment and supplement existing treatment regimens.

However, as mentioned previously, there are no specificallydesigned or developed pharmacotherapies for the targeted indi-cation of intervening in a proinflammatory signaling event withinthe central nervous system, either one associated with drug abuseor any other neuroimmune pathology. However, several agents arein various stages of development. The relative success of cognitivebased therapy in treating drug addiction is intriguing. Has the “talkand thought” based therapy altered the hedonic and anhedonicneurocircuitry thereby breaking the maladaptive rewarding asso-ciationwith drugs of abuse? Or has the therapy strengthened otherlife skill coping mechanisms providing an alterative outlet otherthan drug abuse? Whilst not the focus of our research, a significantpsychoneuroimmunology based literature of therapy modulationof immune function has developed. Could it be that “talk andthought” based therapies have activity, at least in part due to theirinfluence on brain-to-immune and immune-to-brain communica-tion? This is a fascinating as yet unexplored hypothesis.

As we are still at the infancy of drug development pipelines,especially when examining the treatment of drug addiction it is

opportune to raise several key questions. What should a new drugtarget and pharmacodynamically achieve if it were to intervene inthis central immune signaling process associated with drugreward? Pan blockade of glial activation? Create an anti-inflammatory environment within the central nervous system,thus reducing active or limiting subsequent proinflammatory sig-nals? Specifically target a key secondary signaling kinase(s)? Blocka proinflammatory protein receptor or neutralize the moleculebefore it can act? Block the initial detection step the immuneresponse? Should it be an antagonist, agonist, partial agonist orinverse agonist? Will it be able to reverse an established centralimmune signaling event and hence an established drug addiction?If multiple drugs of abuse are shown to rely on central immunesignaling to produce their pharmacodynamic response should apharmacological therapy be specific for a particular drug of abuseor capable of blocking multiple drugs of abuse?

Our preclinical investigations, and those of our peers, havefocused on several pharmacological agents that have been oppor-tunistically identified historically to have the right pharmacokineticand pharmacodynamic profiles to attenuate, or block, various formsof central immune signaling. These include the exploitation of theanti-inflammatory properties of minocycline, propentofylline and

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more recently, ibudilast. In the cases where bioavailability issues,owing to hindrances such as the impermeability of the blood brainbarrier, direct central administration has been employed. However,when looking to the future clinical application of pharmacother-apies and imagining the perfect drug to counter addiction adifferent profile immerges.

Oral bioavailability would be beneficial, with the option of suf-ficient functional potency to allow for the application of indwellinglong-termdeliverymethods, such as subcutaneous rods or pellets toaid compliance in the traditionally non-compliant or treatmentmandated populations. The drug’s pharmacokinetics would need tobe sufficient to allow for once or at most twice daily administration,again to aid in the compliance of treatment. The best precise phar-macological target requires gazing into an as yet still cloudy crystalball. Panblockade of all glial functions, as occurswith glialmetabolicinhibitors, is not plausible as this would produce catastrophic sei-zures owing to loss of homeostatic glial functions (Watkins andMaier, 2003). Pan blockade of glial activation, per se is not specificenough, as such activation may be beneficial in an anti-inflammatory or proinflammatory direction. Proinflammatoryblockade by either neutralization of proinflammatory cytokinemechanisms or intracellular cascadesmay have unwanted off targetconsequences, as central immune signaling in all its diversity playskey roles in a myriad of normal health central nervous systemfunctions. Creation of an anti-inflammatoryenvironmentwithin thecentral nervous systemmay be beneficial, but howwould one directthis to the nuclei most in need for altering drug reward behaviors?No, in the case of opioids, we believe the best target to pharmaco-logically block lies at the first step of the central immune response,TLR4. The long-term safety consequences of such a treatment areunclear, but seem less harmful than the likely immunosuppressionimplications of pan blockade of immune responses generally or in-hibition of pan monocyte-like cell function. In this case it is fortu-itous that TLRs are evolutionarily and old system, thus muchredundancyhas been built around them. In fact, a significant portionof the population has a genetic deficiencies in TLR4 rendering themwith diminished responsiveness, and yet these individuals thrive(e.g. Koch et al. (2011)). However, the association of similar geneticpolymorphisms in drug abuse has yet to be examined.

To this end we have capitalized on our discovery that the(þ)-isomers of opioid receptor antagonists, such as (þ)-naloxoneand (þ)-naltrexone, which we have characterized as functionalantagonists of the MD2/TLR4 activity of opioid ligands. Critically,(þ)-isomers have little to no opioid receptor activity owing to thestereoselectivity of neuronal opioid receptors. To date we haveestablished that (þ)-naloxone and (þ)-naltrexone are capable ofthe range of anti-opioid reward and dependence characteristicsoutlined previously, and yet maintain and in some cases potentiateacute beneficial opioid analgesia. These characteristics of reducingabuse liability of opioids whilst potentiating their indicated clinicalutility suggest formulations of existing opioids with such drugscould beneficially impact the clinical safety and efficacy of theexisting repertoire of opioid agonists.

An alternative future approach would be to design an opioidthat either lacks TLR4 activity or actively blocks it in the onemolecule. Interestingly, a novel opioid agonist, PTI-609, developedby Pain Therapeutics (Burns and Wang, 2010), that combines Fila-min A inhibitor functions and is also capable of reduced TLR4signaling has been found in animal models to engender limitedconditioned place preference, whilst maintaining opioid analgesia.Collectively, these are key data demonstrating that the beneficialanalgesic actions of opioids can be separated from the unwantedrewarding properties, by either co-administration of glial-targetedactivation inhibitors or rationale drug design to avoid xenobiotic-induced activation of central immune signaling. Further

refinement of these approach promises great things in the effortsfor improved drug abuse treatment and avoiding drug abuse lia-bility of clinically diverted agents.

10. What does “glial activation” actually mean?

At this juncture, it is appropriate to note that the phrase “glialactivation”, one which we have used on numerous occasions, is nolonger sufficiently specific enough to render it useful in describingthe responses of the immunocompetent cells of the central nervoussystem. Nor is it sufficient to class glially targeted drugs as glialactivation inhibitors or attenuators. Rather we propose the use ofthe phrase “central immune signaling” in the future. The reason forthis change is terminology is that it is quite apparent that whilstglia are the predominant and classically held immune-like cells ofthe central nervous system, who are the probable sources of manycentral immune signals, they are by no means the only origins ofthese signals, nor may they be the key population that affects apharmacodynamic response. There is evidence that nearly everycell of the central nervous system, either resident like neurons,oligodendrocytes and blood brain barrier endothelial cells, ortransient populations such as migrating peripheral immune cellsare all capable of contributing and modifying this central immunesignal. Also residing at the heart of this change in terminology is theacknowledgment that whilst glia are the new kids on the block anddisserve scrutiny, the established neuronal networks must betested for their non-classical immune signaling contributions to theproinflammatory hypothesis of drug addiction and other neuro-immune related pathologies.

11. Viewing addiction through Neuroimmunopharmacologytinted glasses: what does the future hold?

“Very lame and imperfect theories are sufficient to suggestuseful experiments which serve to correct those theories andgive birth to others more perfect. These, then occasion fartherexperiments which bring us still nearer to the truth; and in thismethod of approximation we must be content to proceed, andwe ought to think ourselves happy if, in this slow method, wemake any real progress”.

Joseph Priestley (1733e1804)

The discoveries and their associated hypotheses outlined herefor the proinflammatory hypothesis of addiction have been built onthe established wealth of addiction neuroscience, with a touch ofre-examination and re-experimentation. However, as beautifullyput by Priestley, even the hypotheses here are imperfect and requirefurther refinement and sharpening. Nevertheless, the groundswellof supporting evidence for further Neuroimmunopharmacologicalinvestigations into drug addiction mechanisms is immense.

In order to capitalize on these exciting developments, predomi-nantly in the preclinical domain, every effort should be made totranslate them to the clinical setting. The development, testing andapproval of new central immune signaling targeted pharmacother-apies is a key step in causally implicating these hypothesizedmechanisms in drug reward and abuse, and therefore moving for-ward to more efficacious treatments. Less expensive and mechanis-tically critical studies to characterize immunological predispositionsto drug abuse should also be examined, such as the immunogeneticdifferences between drug abuse and healthy populations.

Examination of the full clinical landscape of drug dependentpopulations and those at high risk of drug abuse should be fullyassessed in the future for factors that may be associated withenhanced central immune signaling. For example, exciting

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developments in examining early life stressors that trigger later lifedrug abuse propensity have begun to mechanistically implicatecentral immune signaling (Schwarz and Bilbo, 2013; Schwarz et al.,2011), and can be reversed by immune targeted pharmacologicalinterventions. Another example would be to minimize or eliminatechronic peripheral inflammation that may facilitate detrimentaldrug-reward enhancing central immune signaling. Liver damage,associated with a life of substance abuse, left untreated during anaddiction treatment regimen may maintain central immunesignaling patterns in their drug abuse primed states. Our recentclinical work developing a novel clinical neuroimmune model ofpain by combining intravenous endotoxin and intradermal capsa-icin (Hutchinson et al., 2013) demonstrates that modeling of thesecomplex neuroimmune interactions is possible in healthy volun-teer patients. Many other such systems level changes may provecumulatively beneficial in normalizing peripheral immunologythus minimizing the detrimental impact of central immune signalsand their action in drug abuse.

On the flip side of our efforts to understand the mechanisms ofdrug reward and addiction that lead to drug abuse, we need to bemindful of the efforts by some to increase the rewarding actions ofexisting and novel drugs of abuse through targeting these centralimmune system signaling pathways. As we have yet to fullyappreciate the complexity of this multi system response thisconcern is not on our horizon, but legislative efforts should be atthe ready to intervene and to provide the disincentives to partieswho may wish to exploit this route.

In closing, appreciating the existence and impact of xenobiotic-induced central immune signaling combined with the establishedknowledge of neuronal actions of drug of abuse is key to fullyappreciate the complete pharmacodynamic actions of multipledrugs of abuse. Whilst our research to date has focused extensivelyon opioids, glimpses of data suggest this area could be a hot bed ofactivity for multiple drugs of abuse. We hope in the near future thatsuch an appreciation has a meaningful impact on avoiding drugaddiction the treatment of dependent populations.

Acknowledgments

This work was supported in part by NIH Grants DA023132,DA024044, and DE017782 and an Australian Research CouncilResearch Fellow (DP110100297).

References

Adler, M.W., Rogers, T.J., 2005. Are chemokines the third major system in the brain?J. Leukoc. Biol. 78, 1204e1209.

Airavaara, M., Mijatovic, J., Vihavainen, T., Piepponen, T.P., Saarma, M., Ahtee, L.,2006. In heterozygous GDNF knockout mice the response of striatal dopami-nergic system to acute morphine is altered. Synapse 59, 321e329.

Airavaara, M., Planken, A., Gäddnäs, H., Piepponen, T.P., Saarma, M., Ahtee, L., 2004.Increased extracellular dopamine concentrations and FosB/DeltaFosB expres-sion in striatal brain areas of heterozygous GDNF knockout mice. Eur. J. Neu-rosci. 20, 2336e2344.

Baldo, B.A., Kelley, A.E., 2007. Discrete neurochemical coding of distinguishablemotivational processes: insights from nucleus accumbens control of feeding.Psychopharmacology (Berl.) 191, 439e459.

Beitner-Johnson, D., Guitart, X., Nestler, E.J., 1993. Glial fibrillary acidic protein andthe mesolimbic dopamine system: regulation by chronic morphine and Lewis-Fischer strain differences in the rat ventral tegmental area. J. Neurochem. 61,1766e1773.

Berridge, K.C., Kringelbach, M.L., 2013. Neuroscience of affect: brain mechanisms ofpleasure and displeasure. Curr. Opin. Neurobiol. 23, 294e303.

Berthold, H., Borel, J.F., Flückiger, E., 1989. Enigmatic action of ciclosporine A on thenaloxone-precipitated morphine withdrawal syndrome in mice. Neuroscience31, 97e103.

Besedovsky, H.O., Rey, A.D., 2007. Physiology of psychoneuroimmunology: a per-sonal view. Brain Behav. Immun. 21, 34e44.

Bland, S.T., Hutchinson, M.R., Maier, S.F., Watkins, L.R., Johnson, K.W., 2009. The glialactivation inhibitor AV411 reduces morphine-induced nucleus accumbensdopamine release. Brain Behav. Immun. 23, 492e497.

Blednov, Y.A., Benavidez, J.M., Geil, C., Perra, S., Morikawa, H., Harris, R.A., 2011.Activation of inflammatory signaling by lipopolysaccharide produces a pro-longed increase of voluntary alcohol intake in mice. Brain Behav. Immun. 25,S92eS105.

Borowski, T., Kokkinidis, L., Merali, Z., Anisman, H., 1998. Lipopolysaccharide, centralin vivo biogenic amine variations, and anhedonia. Neuroreport 9, 3797e3802.

Buchanan, M.M., Hutchinson, M.R., Watkins, L.R., Yin, H., 2010. Toll-like receptor 4in CNS pathologies. J. Neurochem. 114, 13e27.

Burns, L.H., Wang, H.-Y., 2010. PTI-609: a novel analgesic that binds Filamin A tocontrol opioid signaling. Recent Patents CNS Drug Discov. 5, 210e220.

Büttner, A., 2011. Review: the neuropathology of drug abuse. Neuropathol. Appl.Neurobiol. 37, 118e134.

Cappon, G.D., Morford, L.L., Vorhees, C.V., 1998. Enhancement of cocaine-inducedhyperthermia fails to elicit neurotoxicity. Neurotoxicol. Teratol. 20, 531e535.

Capuron, L., Miller, A.H., 2011. Immune system to brain signaling: neuro-psychopharmacological implications. Pharmacol. Ther. 130, 226e238.

Chaboub, L.S., Deneen, B., 2012. Developmental origins of astrocyte heterogeneity:the final frontier of CNS development. Dev. Neurosci. 34, 379e388.

Chen, G.Y., Nunez, G., 2010. Sterile inflammation: sensing and reacting to damage.Nat. Rev. Immunol. 10, 826e837.

Chen, Z.R., Irvine, R.J., Somogyi, A.A., Bochner, F., 1991. Mu receptor binding of somecommonly used opioids and their metabolites. Life Sci. 48, 2165e2171.

Coller, J.K., Hutchinson, M.R., 2012. Implications of central immune signaling causedby drugs of abuse: mechanisms, mediators and new therapeutic approaches forprediction and treatment of drug dependence. Pharmacol. Ther. 134, 219e245.

Dafny, N., Dougherty, P.M., Drath, D., 1990. Immunosuppressive agent modulatesthe severity of opiate withdrawal. NIDA Res. Monogr. 105, 553e555.

Dantzer, R., O’Connor, J.C., Freund, G.G., Johnson, R.W., Kelley, K.W., 2008. Frominflammation to sickness and depression: when the immune system subjugatesthe brain. Nat. Rev. Neurosci. 9, 46e56.

Dantzer, R., Satinoff, E., Kelley, K.W., 1987. Cyclosporine and alpha-interferon do notattenuate morphine withdrawal in rats but do impair thermoregulation.Physiol. Behav. 39, 593e598.

Dantzer, R., Wollman, E., Vitkovic, L., Yirmiya, R., 1999. Cytokines and depression:fortuitous or causative association? Mol. Psychiatry 4, 328e332.

De, A., Krueger, J.M., Simasko, S.M., 2003. Tumor necrosis factor alpha increasescytosolic calcium responses to AMPA and KCl in primary cultures of rat hip-pocampal neurons. Brain Res. 981, 133e142.

Dougherty, P.M., Pellis, N.R., Dafny, N., 1990. The brain and the immune system: anintact immune system is essential for the manifestation of withdrawal in opiateaddicted rats. Neuroscience 36, 285e289.

Erickson, M.A., Dohi, K., Banks, W.A., 2012. Neuroinflammation: a common pathwayin CNS diseases as mediated at the blood-brain barrier. Neuro-immunomodulation 19, 121e130.

Fantegrossi,W.E., Ciullo, J.R.,Wakabayashi, K.T., de La Garza, R., Traynor, J.R.,Woods, J.H.,2008. A comparison of the physiological, behavioral, neurochemical and microglialeffects of methamphetamine and 3,4-methylenedioxymethamphetamine in themouse. Neuroscience 151, 533e543.

Hemstapat, K., Le, L., Edwards, S.R., Smith, M.T., 2009. Comparative studies of theneuro-excitatory behavioural effects of morphine-3-glucuronide and dynor-phin A(2-17) following spinal and supraspinal routes of administration. Phar-macol. Biochem. Behav. 93, 498e505.

Holmes, J.E., Miller, N.E., 1963. Effects of bacterial endotoxin on water intake, foodintake, and body temperature in the Albino rat. J. Exp. Med. 118, 649e658.

Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K.,Akira, S., 1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice arehyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps geneproduct. J. Immunol. 162, 3749e3752.

Hutchinson, M.R., Bland, S.T., Johnson, K.W., Rice, K.C., Maier, S.F., Watkins, L.R.,2007. Opioid-induced glial activation: mechanisms of activation and implica-tions for opioid analgesia, dependence, and reward. ScientificWorldJournal 7,98e111.

Hutchinson, M.R., Buijs, M., Tuke, J., Kwok, Y.H., Gentgall, M., Williams, D., Rolan, P.,2013. Low-dose endotoxin potentiates capsaicin-induced pain in man: evidencefor a pain neuroimmune connection. Brain Behav. Immun. 30, 3e11.

Hutchinson, M.R., Coats, B.D., Lewis, S.S., Zhang, Y., Sprunger, D.B., Rezvani, N.,Baker, E.M., Jekich, B.M., Wieseler, J.L., Somogyi, A.A., Martin, D., Poole, S.,Judd, C.M., Maier, S.F., Watkins, L.R., 2008a. Proinflammatory cytokines opposeopioid-induced acute and chronic analgesia. Brain Behav. Immun. 22,1178e1189.

Hutchinson, M.R., Northcutt, A.L., Chao, L.W., Kearney, J.J., Zhang, Y.,Berkelhammer, D.L., Loram, L.C., Rozeske, R.R., Bland, S.T., Maier, S.F.,Gleeson, T.T., Watkins, L.R., 2008b. Minocycline suppresses morphine-inducedrespiratory depression, suppresses morphine-induced reward, and enhancessystemic morphine-induced analgesia. Brain Behav. Immun. 22, 1248e1256.

Hutchinson, M.R., Zhang, Y., Brown, K., Coats, B.D., Shridhar, M., Sholar, P.W.,Patel, S.J., Crysdale, N.Y., Harrison, J.A., Maier, S.F., Rice, K.C., Watkins, L.R., 2008c.Non-stereoselective reversal of neuropathic pain by naloxone and naltrexone:involvement of toll-like receptor 4 (TLR4). Eur. J. Neurosci. 28, 20e29.

Hutchinson, M.R., Lewis, S.S., Coats, B.D., Skyba, D.A., Crysdale, N.Y.,Berkelhammer, D.L., Brzeski, A., Northcutt, A., Vietz, C.M., Judd, C.M., Maier, S.F.,Watkins, L.R., Johnson, K.W., 2009. Reduction of opioid withdrawal andpotentiation of acute opioid analgesia by systemic AV411 (ibudilast). BrainBehav. Immun. 23, 240e250.

Hutchinson, M.R., Lewis, S.S., Coats, B.D., Rezvani, N., Zhang, Y., Wieseler, J.L.,Somogyi, A.A., Yin, H., Maier, S.F., Rice, K.C., Watkins, L.R., 2010a. Possible

M.R. Hutchinson, L.R. Watkins / Neuropharmacology 76 (2014) 218e227 227

involvement of toll-like receptor 4/myeloid differentiation factor-2 activity ofopioid inactive isomers causes spinal proinflammation and related behavioralconsequences. Neuroscience 167, 880e893.

Hutchinson, M.R., Zhang, Y., Shridhar, M., Evans, J.H., Buchanan, M.M., Zhao, T.X.,Slivka, P.F., Coats, B.D., Rezvani, N., Wieseler, J.L., Hughes, T.S., Landgraf, K.E.,Chan, S., Fong, S., Phipps, S., Falke, J.J., Leinwand, L.A., Maier, S.F., Yin, H.,Rice, K.C., Watkins, L.R., 2010b. Evidence that opioids may have toll-like re-ceptor 4 and MD-2 effects. Brain Behav. Immun. 24, 83e95.

Hutchinson, M.R., Shavit, Y., Grace, P.M., Rice, K.C., Maier, S.F., Watkins, L.R., 2011.Exploring the neuroimmunopharmacology of opioids: an integrative review ofmechanisms of central immune signaling and their implications for opioidanalgesia. Pharmacol. Rev. 63, 772e810.

Hutchinson, M.R., Northcutt, A.L., Hiranita, T., Wang, X., Lewis, S.S., Thomas, J., vanSteeg, K., Kopajtic, T.A., Loram, L.C., Sfregola, C., Galer, E., Miles, N.E., Bland, S.T.,Amat, J., Rozeske, R.R., Maslanik, T., Chapman, T.R., Strand, K.A., Fleshner, M.,Bachtell, R.K., Somogyi, A.A., Yin, H., Katz, J.L., Rice, K.C., Maier, S.F., Watkins, L.R.,2012. Opioid activation of Toll-like receptor 4 contributes to drug reinforce-ment. J. Neurosci. 32, 11187e11200.

Hyman, S.E., Malenka, R.C., Nestler, E.J., 2006. Neural mechanisms of addiction: therole of reward-related learning and memory. Annu. Rev. Neurosci. 29, 565e598.

Ikemoto, S., 2007. Dopamine reward circuitry: two projection systems from theventral midbrain to the nucleus accumbens-olfactory tubercle complex. BrainRes. Rev. 56, 27e78.

Koch, A., Hamann, L., Schott, M., Boehm, O., Grotemeyer, D., Kurt, M., Schwenke, C.,Schumann, R.R., Bornstein, S.R., Zacharowski, K., 2011. Genetic variation of TLR4influences immunoendocrine stress response: an observational study in cardiacsurgical patients. Crit. Care 15, R109.

Labella, F.S., Pinsky, C., Havlicek, V., 1979. Morphine derivatives with diminishedopiate receptor potency show enhanced central excitatory activity. Brain Res.174, 263e271.

Laflamme, N., Rivest, S., 1999. Effects of systemic immunogenic insults and circu-lating proinflammatory cytokines on the transcription of the inhibitory factorkappaB alpha within specific cellular populations of the rat brain. J. Neurochem.73, 309e321.

Landsteiner, K., 1936. The Specificity of Serological Reactions. C.C. Thomas,Springfield, IL; Baltimore, MD.

Laviolette, S.R., Nader, K., van der Kooy, D., 2002. Motivational state determines thefunctional role of the mesolimbic dopamine system in the mediation of opiatereward processes. Behav. Brain Res. 129, 17e29.

Lewis, S.S., Hutchinson, M.R., Rezvani, N., Loram, L.C., Zhang, Y., Maier, S.F., Rice, K.C.,Watkins, L.R., 2010. Evidence that intrathecal morphine-3-glucuronide maycause pain enhancement via toll-like receptor 4/MD-2 and interleukin-1beta.Neuroscience 165, 569e583.

Lewis, S.S., Hutchinson, M.R., Zhang, Y., Hund, D.K., Maier, S.F., Rice, K.C.,Watkins, L.R., 2013. Glucuronic acid and the ethanol metabolite ethyl-glucuronide cause toll-like receptor 4 activation and enhanced pain. BrainBehav. Immun. 30, 24e32.

Li, Y., Sun, X., Zhang, Y., Huang, J., Hanley, G., Ferslew, K.E., Peng, Y., Yin, D., 2009.Morphine promotes apoptosis via TLR2, and this is negatively regulated bybeta-arrestin 2. Biochem. Biophys. Res. Commun. 378, 857e861.

Liu, L., Coller, J.K., Watkins, L.R., Somogyi, A.A., Hutchinson, M.R., 2011. Naloxone-precipitated morphine withdrawal behavior and brain IL-1b expression: com-parison of different mouse strains. Brain Behav. Immun. 25, 1223e1232.

Liu, L., Hutchinson, M.R., White, J.M., Somogyi, A.A., Coller, J.K., 2009. Association ofIL-1B genetic polymorphisms with an increased risk of opioid and alcoholdependence. Pharmacogenet. Genomics 19, 869e876.

Matic, M., Mahns, A., Tsoli, M., Corradin, A., Polly, P., Robertson, G.R., 2007. PregnaneX receptor: promiscuous regulator of detoxification pathways. Int. J. Biochem.Cell. Biol. 39, 478e483.

McBride, W.J., Murphy, J.M., Ikemoto, S., 1999. Localization of brain reinforcementmechanisms: intracranial self-administration and intracranial place-conditioning studies. Behav. Brain Res. 101, 129e152.

Miller, A.H., Maletic, V., Raison, C.L., 2009. Inflammation and its discontents: therole of cytokines in the pathophysiology of major depression. Biol. Psychiatry65, 732e741.

Nakagawa, T., Fujio, M., Ozawa, T., Minami, M., Satoh, M., 2005. Effect of MS-153, aglutamate transporter activator, on the conditioned rewarding effects ofmorphine, methamphetamine and cocaine in mice. Behav. Brain Res. 156, 233e239.

Nakagawa, T., Satoh, M., 2004. Involvement of glial glutamate transporters inmorphine dependence. Ann. N. Y. Acad. Sci. 1025, 383e388.

Narita, M., Miyatake, M., Narita, M., Shibasaki, M., Shindo, K., Nakamura, A.,Kuzumaki, N., Nagumo, Y., Suzuki, T., 2006. Direct evidence of astrocyticmodulation in the development of rewarding effects induced by drugs of abuse.Neuropsychopharmacol. Off. Publ. Am. Coll. Neuropsychopharmacol. 31, 2476e2488.

O’Neill, L.A.J., 2008. The interleukin-1 receptor/Toll-like receptor superfamily: 10years of progress. Immunol. Rev. 226, 10e18.

Ozawa, T., Nakagawa, T., Sekiya, Y., Minami, M., Satoh, M., 2004. Effect of genetransfer of GLT-1, a glutamate transporter, into the locus coeruleus by

recombinant adenoviruses on morphine physical dependence in rats. Eur. J.Neurosci. 19, 221e226.

Ozawa, T., Nakagawa, T., Shige, K., Minami, M., Satoh, M., 2001. Changes in theexpression of glial glutamate transporters in the rat brain accompanied withmorphine dependence and naloxone-precipitated withdrawal. Brain Res. 905,254e258.

Palm, N.W., Medzhitov, R., 2009. Immunostimulatory activity of haptenated pro-teins. Proc. Natl. Acad. Sci. U. S. A. 106, 4782e4787.

Park, S.-M., Gaykema, R.P.A., Goehler, L.E., 2008. How does immune challengeinhibit ingestion of palatable food? Evidence that systemic lipopolysaccharidetreatment modulates key nodal points of feeding neurocircuitry. Brain Behav.Immun. 22, 1160e1172.

Ranaldi, R., Beninger, R.J., 1994. Rostral-caudal differences in effects of nucleusaccumbens amphetamine on VTA ICSS. Brain Res. 642, 251e258.

Rivest, S., 2009. Regulation of innate immune responses in the brain. Nature 9,429e439.

Salazar, M., Pariente, J.A., Salido, G.M., González, A., 2008. Ethanol induces gluta-mate secretion by Ca2þ mobilization and ROS generation in rat hippocampalastrocytes. Neurochem. Int. 52, 1061e1067.

Schäfer, M., 2013. The painful Toll of ethanol and its metabolites: a new molecularpattern of recognition by Toll like receptors? Brain Behav. Immun. 30, 22e23.

Schwarz, J.M., Bilbo, S.D., 2013. Adolescent morphine exposure affects long-termmicroglial function and later-life relapse liability in a model of addiction.J. Neurosci. 33, 961e971.

Schwarz, J.M., Hutchinson, M.R., Bilbo, S.D., 2011. Early-life experience decreasesdrug-induced reinstatement of morphine CPP in adulthood via microglial-specific epigenetic programming of anti-inflammatory IL-10 expression.J. Neurosci. 31, 17835e17847.

Stellwagen, D., Beattie, E.C., Seo, J.Y., Malenka, R.C., 2005. Differential regulation ofAMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha.J. Neurosci. 25, 3219e3228.

Takagi, K., Fukuda, H., Watanabe, M., Sato, M., 1960. Studies on antitussives. III.(þ)-Morphine and its derivatives. J. Pharm. Soc. Japan 80, 1506e1509.

Thomas, J., Hutchinson, M.R., 2012. Exploring neuroinflammation as a potentialavenue to improve the clinical efficacy of opioids. Expert Rev. Neurother. 12,1311e1324.

Tilleux, S., Hermans, E., 2007. Neuroinflammation and regulation of glial glutamateuptake in neurological disorders. J. Neurosci. Res. 85, 2059e2070.

Tsuang, M.T., Lyons, M.J., Meyer, J.M., Doyle, T., Eisen, S.A., Goldberg, J., True, W.,Lin, N., Toomey, R., Eaves, L., 1998. Co-occurrence of abuse of different drugs inmen: the role of drug-specific and shared vulnerabilities. Arch. Gen. Psychiatry55, 967e972.

Vargas-Perez, H., Ting-A-Kee, R., Walton, C.H., Hansen, D.M., Razavi, R., Clarke, L.,Bufalino, M.R., Allison, D.W., Steffensen, S.C., van der Kooy, D., 2009. Ventraltegmental area BDNF induces an opiate-dependent-like reward state in naiverats. Science 324, 1732e1734.

Wadachi, R., Hargreaves, K.M., 2006. Trigeminal nociceptors express TLR-4 andCD14: a mechanism for pain due to infection. J. Dent. Res. 85, 49e53.

Wang, H.-Y., Frankfurt, M., Burns, L.H., 2008. High-affinity naloxone binding to fil-amin a prevents mu opioid receptor-Gs coupling underlying opioid toleranceand dependence. PLoS One 3, e1554.

Wang, Q., Zhou, H., Gao, H., Chen, S.-H., Chu, C.-H., Wilson, B., Hong, J.-S., 2012a.Naloxone inhibits immune cell function by suppressing superoxide productionthrough a direct interaction with gp91phox subunit of NADPH oxidase.J. Neuroinflammation 9, 32.

Wang, X., Loram, L.C., Ramos, K., de Jesus, A.J., Thomas, J., Cheng, K., Reddy, A.,Somogyi, A.A., Hutchinson, M.R., Watkins, L.R., Yin, H., 2012b. Morphine acti-vates neuroinflammation in a manner parallel to endotoxin. Proc. Natl. Acad.Sci. U. S. A. 109, 6325e6330.

Wang, Z., Pekarskaya, O., Bencheikh, M., Chao, W., Gelbard, H.A., Ghorpade, A.,Rothstein, J.D., Volsky, D.J., 2003. Reduced expression of glutamate transporterEAAT2 and impaired glutamate transport in human primary astrocytes exposedto HIV-1 or gp120. Virology 312, 60e73.

Watkins, L.R., Hutchinson, M.R., Rice, K.C., Maier, S.F., 2009. The “toll” of opioid-induced glial activation: improving the clinical efficacy of opioids by target-ing glia. Trends Pharmacol. Sci. 30, 581e591.

Watkins, L.R., Maier, S.F., 2003. Glia: a novel drug discovery target for clinical pain.Nat. Rev. Drug Discov. 2, 973e985.

Weber, M., Modemann, S., Schipper, P., Trauer, H., Franke, H., Illes, P., Geiger, K.D.,Hengstler, J.G., Kleemann, W.J., 2006. Increased polysialic acid neural celladhesion molecule expression in human hippocampus of heroin addicts.Neuroscience 138, 1215e1223.

Yirmiya, R., Goshen, I., 2011. Immune modulation of learning, memory, neuralplasticity and neurogenesis. Brain Behav. Immun. 25, 181e213.

Yirmiya, R., Pollak, Y., Morag, M., Reichenberg, A., Barak, O., Avitsur, R., Shavit, Y.,Ovadia, H., Weidenfeld, J., Morag, A., Newman, M.E., Pollmächer, T., 2000.Illness, cytokines, and depression. Ann. N. Y. Acad. Sci. 917, 478e487.

Youn, D.-H., Wang, H., Jeong, S.-J., 2008. Exogenous tumor necrosis factor-alpharapidly alters synaptic and sensory transmission in the adult rat spinal corddorsal horn. J. Neurosci. Res. 86, 2867e2875.