Donald T. Lysle - US grants

A good deal of evidence suggests an interaction between the opioid and immune systems. The studies proposed here examine the effects of several opioids on a large battery of immune measures with particular emphasis on the effects of chronic administration, the type of opioid administered, the particular immune measure examined and the mechanism whereby the opioids alter immune function. The first specific aim provides background information for these investigations by examining the effects of acute administration of opioids with predominant activity at mu opioid receptors, i.e., methadone, 1-alpha-acetylmethadol (LAAM) and buprenorphine and comparing them to effects already obtained for morphine. Additional data regarding their mu-opioid mechanism of action will he obtained by examining methadone, LAAM and buprenorphine in combination with the mu-selective opioid antagonist naltrexone. Immune function will be assessed with several different rodent in vitro assays, including mitogen and superantigen-induced stimulation of splenic, lymph node and whole-blood lymphocytes, a natural-killer cell assay, assessment of interleukin-2, interferon and nitric oxide production. The second specific aim explores the effects of opioid administration on immune function in rats that are maintained on a regimen in which their drinking water is adulterated with morphine, methadone, LAAM or buprenorphine. In these studies, the effects of chronic opioid administration will he assessed with the measures of immune function outlined in Specific Aim l and with emphasis on the contribution of factors such as type of opioid administered, dose and duration of administration. The analgesic effects of these drugs also will be assessed prior to, during and subsequent to termination of the regimen of chronic opioid administration. The third specific aim investigates the mechanisms which underlie opioid-induced alterations in immune function. In these experiments, we will extend our investigations of the role of the CNS in morphine's immunomodulatory effects by focusing on four specific brain regions, the periaqueductal gray (PAG), nucleus accumbens (NA), ventral tegmental nucleus (VTA) and the basolateral amygdala (BLA). The first series of studies will examine the effects of morphine on immune status when injected directly into those sites. A second series of experiments will determine whether naltrexone can block the effects of systemic morphine when it is injected into the same four sites.

DESCRIPTION: (Applicant's Abstract) The proposed studies will expand upon new data from the Principal Investigator's laboratory which demonstrate that in vivo morphine administration produces robust reductions in T-cell function which appear to be mediated by macrophage-derived nitric oxide. Since both macrophages and T-cells are key immunoregulatory cells, and the target of HIV-1 in humans, understanding the mechanisms by which opioids exert these actions has important human health implications. Specific Aim 1 will test the hypothesis that morphine, acting at the level of the central nervous system, induces pharmacologically-specific alterations in nitric oxide production by splenic macrophages. It is proposed to compare the dose-dependent effects of morphine administered systemically, intracerebroventrically (ICV), and in vitro on the expression of nitric oxide by splenic macrophages. Antagonism studies also will be performed. Tests will be conducted on splenocytes to identify the subtype and number of macrophages expressing inducible nitric oxide synthase (iNOS). The level of nitric oxide activity produced by splenocytes will be assessed by measuring iNOS protein using western blotting and by measuring the concentration of nitrate/nitrite in vitro. The relationship between morphine-induced alterations in nitric oxide production and changes in the function of lymphocytes will be established. Specific Aim 2 will determine the subtype(s) of opioid receptors involve in opioid-induced alterations of nitric oxide production. To distinguish the role of mu, kappa, and delta opioid receptor subtypes, the investigators will compare the effects of ICV and in vitro administration of selective opioid agonists and antagonists on splenic nitric oxide production. Specific Aim 3 will characterize the effect of opioid administration on nitric oxide production in vivo. These experiments will assess the dose-dependent effects of morphine, and selective opioid agonists and antagonists. To measure alterations of nitric oxide production in vivo, a complementary approach which uses RT-PCR and western blotting to directly measure alterations of splenic iNOS mRNA and protein expression will be applied. Serum nitrite/nitrate levels will also be measured. Collectively, these investigations could provide important new information concerning the site of action and the subtype of receptor involved in opioid-induced modulation of nitric oxide production.

This is an application for renewal of an Independent Scientist Award (KO-2) to support Dr. Donald T. Lysle's research activities. The specific aim of this award is to provide Dr. Lysle with some release time from his classroom teach responsibilities so that he can devote the majority of his time to research and the training of doctoral students and post-doctoral level fellows. The additional time will allow Dr. Lysle to enhance his range of scientific training and expertise. More specifically, Dr. Lysle will acquire skills in the development of animal models of infection suitable for the study of opioid-immune interactions. These goals will be accomplished within the framework of two existing NIDA funded projects. The first project (DA-7481), entitled Determinants of Opioid/Immune Interactions, provides the first direct comparisons of the impact of heroin, morphine, and their metabolites on immune status, as measured by alterations of inflammatory immune responses. The project assesses the effect of heroin, 6-monoacetylmorphine, the active metabolite of heroin, morphine and its major metabolites, morphine- 3beta-glucuronide, and morphine-6beta-glucuronide, on several models of inflammation. The second project (DA13371), entitled Behavioral Factors in Heroin's Effect on Nitric Oxide, will test the hypothesis that heroin induces wide-spread alterations in the in vivo expression inducible nitric oxide synthase (iNOS). The experiments establish dose- effect relationships for heroin, as well as the antagonism of those effects. The research plan also tests the hypothesis that heroin-induced alterations of iNOS production are conditional to environmental stimuli paired with drug administration, and that the effects are influenced by the manner of drug administration. The studies will determine whether heroin is differentially efficacious in altering nitric oxide production depending upon whether the drug is self-administered or passively infused which has important implications for the health consequences of heroin use. These projects provide important new information about how opioids induce alterations of inflammatory responses, including nitric oxide production. The HO-2 aw2ard will allow Dr. Lysle to achieve a major goal of integrating our knowledge about opioid-induced alterations of inflammatory responses with the clinical outcomes of models of bacterial infection. This integration will provide a unique and comprehensive understanding of the health consequences of opioid use.

There is a high incidence of infectious disease in heroin-dependent individuals. In spite of the major health issues surrounding heroin use, few studies have examined the impact of heroin on immune status. A wealth of recent data has revealed that the induction of nitric oxide plays a critical role in the immune response to infectious diseases. New data from our laboratory has provided the first evidence that heroin administration produces alterations of inducible nitric oxide synthase (iNOS), the enzyme responsible for the production of nitric oxide. Specific Aim I extensively tests the hypothesis that heroin induces widespread alterations in the in vivo expression of NOS. This specific aim fully evaluates the effect of heroin on lipopolysaccaride (LPS)-induced iNOS expression. The experiments establish dose-effect relationships for heroin, as well as the antagonism of those effects with naltrexone. To measure NOS production in vivo, we employ both RT-PCR and western blotting to measure alterations of iNOS mRNA expression and protein in spleen, lung, and liver tissue. We also measure nitrite/nitrate in plasma. Specific Aim II tests the hypothesis that heroin-induced alteration of NOS production is conditioned to environmental stimuli paired with drug administration. The demonstration that immune alterations can be induced by the stimuli associated with heroin administration is important for it indicates that the detrimental health consequences of heroin use are conditioned to environmental stimuli and not solely the pharmacological property of the drug. The proposed studies determine whether heroin's effect on nitric oxide production can be elicited by environmental stimuli paired with heroin administration. Control procedures are used to assure that these conditioned alterations are due to conditioning processes per se, and not the result of ancillary factors involved in the conditioning procedure. Tests of extinction and latent inhibition will establish that the effects follow the principles of Pavlovian conditioning. Specific AimIII tests the hypothesis that drug self-administration is an important behavioral variable controlling heroin's effect on the production of nitric oxide. The self-administration paradigm provides a clinically relevant methodology for the assessment of heroin's effects on nitric oxide production. The study compares the effects of heroin self-administration to those of passive drug administration. Investigation of the effects of self-administered heroin on nitric oxide production is driven by evidence showing that control over drug administration greatly influence it's physiological effects. The demonstration that heroin is differentially efficacious in altering nitric oxide production depending upon whether the drug is self-administered or passively infused has important implications for the health consequences of heroin use. Collectively, the characterization of heroin-induced modulation of inducible nitric oxide synthase production provides a greater understanding of how opioids impact the immune system and health.

DESCRIPTION (provided by applicant): Recent research has produced a wealth of information on the immunomodulatory effects of opioids, but little is known about how the sex of the individual impacts opioid-induced immunomodulation. Given the widespread clinical use of opioids and their high abuse potential, an understanding of the interaction of sex with opioid-induced immune alterations is critical. Specific Aim I provides a pharmacological analysis of the effects of morphine on the contact hypersensitivity (CHS) response in males and females, with an emphasis on clinical outcome measures (i.e., swelling), as well as the immunological and receptor mechanisms that mediate these effects. Our initial findings indicate that morphine enhances CHS in both males and females, but in females, morphine is more than twice as potent, has a greater maximal effect, and the effects persist for a longer period of time. The proposed studies will determine the specific immune mechanisms that account for these dramatic sex differences by evaluating the role of immunologic mediators at the site of CHS, including IL-1-beta, TNF-alpha, IFN-gamma, IL-4, IL-6, IL-10, and nitric oxide expression. Studies will also test hypotheses that morphine activates different central and peripheral opioid receptor types in males and females. Specific Aim II will determine if the gonadal (or sex) hormones mediate sex differences in morphine-induced alterations of contact hypersensitivity (CHS). Given the ample evidence that gonadal hormones contribute to observed sex differences in both immune function and opioid sensitivity, depleting these hormones represents a logical and critical first step in the analysis of the hormonal mechanisms underlying the profound sex differences in opioid-induced immunomodulation. The proposed studies test if gonadal hormone depletion in males and females impacts morphine-induced alterations of CHS and the specific immunologic mediators of this sexually differentiated response. Specific Aim III determines the generality of sex differences across clinically relevant opioids, and whether the magnitude of the sex differences is related to the relative efficacy (i.e., ability to stimulate the mu opioid receptor) of the opioid. Our plan is to evaluate sex differences in opioid-immunomodulation with a series of clinically important opioids that differ along a continuum of efficacy. Our hypothesis is that the sex differences will be apparent with opioids other than morphine, and that the magnitude of the sex-related differences will be inversely related to their ability to stimulate the mu opioid receptor. Given that virtually nothing is known about how the sex of the individual interacts with the immunomodulatory actions of opioids, the proposed studies are the first to advance our understanding of the regulatory role of sex in opioid-induced immunomodulation. These studies have clinical importance and will influence the selection of opioids for patient care, as well as enhance our understanding of potential sex differences in the adverse consequences of opioid use and abuse.

There is high incidence of bacterial, viral, and fungal opportunistic infections among long-term heroin users. Our laboratory was the first to demonstrate that, remarkably, environmental stimuli paired with heroin administration acquire heroin-like immunomodulatory properties as a result of associative learning processes. Thus, the detrimental health consequences of opiates can be conditioned to environmental stimuli predictive of drug administration. The proposed studies are the first to characterize the role of dopamine in the nucleus accumbens and amygdala in the conditioned immunomodulatory effects of heroin. The studies will assess the effects of intra-nucleus accumbens and intra-amygdala administration of dopamine receptor antagonists on heroin-conditioned alterations in the expression of proinflammatory immune responses. Lipopolysaccharide (LPS) will be used to challenge the immune system. LPS is an immunogenic component of Gram-negative bacteria that induces robust proinflammatory immune responses, and it is well established that heroin-paired stimuli attenuate the expression of these immune responses. Specific Aim I tests the hypothesis that stimulation of dopamine receptors in the nucleus accumbens is necessary for the expression of conditioned opioid-induced reductions in the production of in vivo proinflammatory cytokines and nitric oxide. The proposed investigations provide a comprehensive assessment of the role of nucleus accumbens dopamine receptors in the expression of heroin-conditioned immunomodulation. Specific Aim II tests the hypothesis that dopamine receptor stimulation in the amygdala is necessary to produce the expression of conditioned opioid-induced reductions in the in vivo proinflammatory cytokines and nitric oxide. The proposed investigations determine the role of amygdalar dopamine receptors in the expression of heroin-conditioned immunomodulation using selective dopaminergic receptor antagonists. Specific Aim III tests the hypothesis that sequential information processing by the amygdala and nucleus accumbens is necessary for heroin-induced conditioned immunomodulation. The proposed experiments will evaluate whether communication between the amygdala and nucleus accumbens is necessary for expression of heroin-induced conditioned immunomodulation. These experiments are especially important for they are the first to examine systems-level interaction between brain regions mediating this important function. Collectively, these studies provide new insights into the neuroanatomical substrates and dopamine receptor mechanisms of conditioned opioid/immune interactions, and make important advancements in our understanding of the health consequences of opioid use.

DESCRIPTION (provided by applicant): Drugs of abuse, such as heroin, have devastating effects on immune function and lead to addictive behavior. Remarkably, these effects of heroin are conditioned to environmental stimuli. The resulting conditioned immunomodulatory and motivational effects are robust, but the neural mechanisms of these effects are poorly understood. We have made major contributions to the understanding of the neural circuitry underlying conditioned immunomodulation and motivation. Building on these findings, this R01 proposal will test the overarching hypothesis that heroin-conditioned immunomodulation and motivation to seek drug rely on altered central nervous system signaling mediated by the cytokine, interleukin-1beta (IL-1beta). Specific Aim I will test the hypothesis that exposure to heroin-conditioned stimuli is associated with IL-1beta expression in distinct brain regions and cell types within the CNS. We will evaluate changes in IL-1beta expression across time in the dorsal hippocampus (DH), basolateral amygdala (BLA), nucleus accumbens shell (NACs), and ventral tegmental area (VTA), after exposure to a heroin-conditioned context or after control manipulations designed to determine whether these alterations are due to conditioning per se. We will identify specific cell types involved in these effects. As follow-up, in the brain regions that exhibit conditioned IL-1beta expression, we will explore whether IL-1beta is sufficient to induce immunosuppression in the absence of heroin conditioning, by examining the effects of site-specific recombinant IL-1beta infusion. Specific Aim II will test the hypothesis that IL-1beta gene expression is necessary in specific brain regions for the expression of heroin-conditioned immunomodulation. To demonstrate that IL-1beta expression is necessary for heroin-conditioned immunomodulation, we will examine the effects of inhibiting IL-1beta expression in specific brain regions on heroin-conditioned immunomodulation. IL-1beta gene expression in the DH, BLA, NACs, or VTA will be inhibited selectively using small interfering RNA (siRNA) or the IL-1 receptor will be blocked using IL-1 receptor antagonist in the same brain regions. Specific Aim III will be to test the hypothesis that IL-1beta gene expression in the DH is necessary for the conditioned motivational effects of a heroin-paired context that facilitate drug seeking. We will test whether IL-1beta expression is necessary for the motivational effects of drug-paired contextual stimuli using the conditioned place preference (CPP) paradigm, given its similarity to the heroin-conditioned immunosuppression procedure. We will also evaluate whether IL-1beta expression is necessary for drug context-induced reinstatement of heroin-seeking behavior using the contextual reinstatement paradigm, a model of drug relapse with exceptional face and predictive validity. Overall, this project takes an innovative approach to reveal novel neuroimmunological mechanisms by which drug-conditioned stimuli impact immune function and drug relapse, thereby fundamentally enhancing our understanding of the health consequences of opioid use and informing the development of novel neuroimmunological therapies to restore immune function and prevent drug relapse.

Project Summary/Abstract SPIRE (Seeding Postdoctoral Innovators in Research and Education) is a postdoctoral research and career development training program built as a partnership between UNC/Chapel Hill and four partner institutions in North Carolina, each of which has an historical mission to educate students from backgrounds underrepresented in the biomedical research enterprise. Through this partnership, the SPIRE program prepares scholars with the research and teaching skills to inspire students from underrepresented groups to pursue science careers. The current proposal maintains many successful components introduced over SPIRE?s 17-year history and adds several innovations to benefit the partner institutions and the scholars? preparation for academic careers. Throughout its history, SPIRE scholars have received training in research- based, student-centered approaches in science education. The current proposal expands upon these approaches by providing scholars with formalized training in Classroom-based Undergraduate Research Experiences (CUREs). Moreover, in order to increase the number of students from our partner institutions who can benefit from one-on-one research interactions and to provide additional mentoring experience for the scholars, we propose to increase the number of undergraduates from our partners that can take advantage of the very effective SPIRE Summer Research Program, piloted during the current grant cycle. In order to broaden exposure to research disciplines at partner institutions and promote potential collaborations, we propose to launch a Traveling Research Symposium, which will take place each year at one of our partner institutions. As the SPIRE program enhances the effectiveness of science education at its partner institutions through the initiatives described above, we will also insure that the scholars obtain high quality training within research laboratories at UNC/Chapel Hill by matching scholars with productive scientists whose training records continue to reveal their commitment to mentorship. Taken together, by providing scholars with cutting-edge training in research, teaching and professional development, the SPIRE program strives to continue its record of training scholars who attain academic positions and continue to advance science education.

Project Summary/Abstract Individuals that have been exposed to a traumatic event are at risk for developing a set of symptoms known as post-traumatic stress disorder (PTSD). Evidence suggests that there is a high comorbidity between PTSD and alcohol use disorders (AUDs), with a three-fold increased risk for experiencing an AUD in sufferers of PTSD. While it is generally thought PTSD proceeds, and is a risk factor for, AUDs, there is also evidence that a prior history of AUDs may leave individuals biologically more vulnerable to the impact of severe stress and thus more likely to develop PTSD. Despite converging evidence of co-morbidity between PTSD and AUDs, our understanding of the underlying neuronal substrates mediating these comorbid disorders, as well as available pharmaceutical treatments, are limited. This application brings together a team of investigators to address this scientific question in a convergent manner with the expertise of the neural immune mechanisms that underlie PTSD-like behavior (Lysle), the neurobiology of excessive alcohol (ethanol) intake (Thiele), and the understanding of astrocyte physiology (Reissner). Interestingly, a comparison of the present team?s research suggests that common overlapping neuroimmune mechanisms may underlie the development of each pathology. Dr. Lysle has discovered that severe stress induces a time-dependent increase in dorsal hippocampal (DH) interleukin-1? (IL-1?), and that directly blocking IL-1 signaling in the DH after severe stress (repeated unpredictable foot shock) prevents stress-enhanced fear learning (SEFL), an animal model of PTSD. Consistently, the present research team has found that withdrawal following chronic ethanol exposure increases hippocampal IL-1? mRNA, and a recent collaborative pilot project between the research team revealed that ethanol withdrawal potentiates the magnitude of SEFL. These observations support our overarching hypothesis, that hippocampal IL-1? represents a cellular mechanism for exacerbated stress response in alcohol- withdrawn/dependent individuals. Specific Aim 1 will test the hypothesis that withdrawal-induced potentiation of SEFL is associated with (A) a potentiation of IL-1? signaling specifically in astrocytes that correlates with the magnitude of SEFL, (B) pharmacological blockade of DH IL-1R during withdrawal will protect against withdrawal- induced potentiation of PTSD-like phenotypes, and (C) that DH-infusion of exogenous IL-1? will substitute for the effects of ethanol withdrawal. Specific Aim 2 will test the hypothesis that changes in the morphometric properties of astrocytes, and/or alterations of astrocyte/neuron interactions, correlate with increased astrocyte IL-1? levels stemming from ethanol withdrawal and the severe stress used in SEFL. The studies proposed here are appropriate for the R21 grant mechanism because they are high-reward, potentially filling a gap in our understanding of the role that astrocyte-derived cytokines play in co-morbid PTSD and AUD disorders. They are also high-risk because we currently do not have direct evidence that IL-1? signaling in the DH is a mechanism for withdrawal-induced potentiation of PTSD-like phenotypes.

Project Summary/Abstract Opiate withdrawal and post-traumatic stress disorder (PTSD) frequently present together in clinical settings. Clinical data also suggests that opiate abuse is a potential risk factor for PTSD. Understanding the mechanism for co-occurrence and vulnerability to PTSD in opiate users will provide information about the consequences of opiate abuse and inform the development of interventions that mitigate both the symptoms of heroin withdrawal, a known risk factor for relapse, and heroin?s long-term effects on stress disorders. Our laboratory has identified the hippocampal mechanisms required for development of stress enhanced fear learning (SEFL), an animal model of PTSD. We established that the severe stressor in the SEFL paradigm induces dorsal hippocampal (DH) interleukin-1? (IL-1?) in astrocytes, and that directly blocking DH IL-1 signaling with IL-1 receptor antagonist (IL- 1RA) after severe stress prevents subsequent enhanced fear learning. We have developed a pre-clinical model to investigate the effect of heroin withdrawal on enhanced fear learning. We found that withdrawal from escalating heroin administration substitutes for a severe stressor in the SEFL paradigm and that heroin withdrawal similarly increases DH IL-1? and GFAP expression, a marker of astrocyte activation. Accordingly, the plan is to test the innovative unique hypothesis that heroin withdrawal also acts through hippocampal IL-1? and alterations in astrocytes that result in enhanced fear learning. Specific Aim 1 will test the hypothesis that heroin withdrawal- induced enhancement of fear learning is mediated by IL-1? in the DH. Specifically, we will determine (A) whether withdrawal-induced enhanced fear learning is associated with potentiation of IL-1? signaling specifically in astrocytes, (B) whether blockade of DH IL-1 receptors with IL-1RA during withdrawal will protect against the development of enhanced fear learning following heroin withdrawal, (C) whether DH infusion of exogenous IL- 1? will substitute for the effects of stress and/or withdrawal to induce enhanced fear learning, and (D) whether pharmacological blockade of DH IL-1 receptors during heroin withdrawal will prevent withdrawal symptoms. Predicted results are that heroin withdrawal will increase IL-1? levels, primarily in astrocytes, and that the effect of heroin withdrawal on enhanced fear learning will be blocked by IL-1RA and mimicked by infusion of IL-1? in the DH. Also, the symptoms of withdrawal will be attenuated or blocked by the administration of IL-1RA. Collectively, the results elucidate the critical role of DH IL-1 signaling in heroin withdrawal and enhanced fear learning. Specific Aim 2 will use advanced 3-D reconstruction of individual cells to conduct morphometric analysis in combination with assessment of synaptic markers (postsynaptic density 95 and Synapsin 1) to test innovative hypotheses that changes in the morphometric properties of astrocytes, and/or alterations of astrocyte/neuron interactions are associated with the effects of both the heroin withdrawal and the severe stressor used in SEFL, and contingent on IL-1 signaling. These studies test unique hypotheses regarding the underlying mechanism of the effects of heroin withdrawal, and provide new targets for mitigating the long-term effects of heroin abuse.