One of the major focuses for our lab involves examining the immunosuppressive properties of various agents and receptors in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). This dissertation encompasses an investigation of cannabidiol in the EAE model, the endocannabinoid CB1 receptor in the EAE model, staphylococcal superantigens (SAgs) as immunosuppressive agents, and various aspects of the EAE model. The first chapter covers the existing literature pertinent to these studies, the second and third chapters cover the material, methods, and results from the studies, and the fourth chapter is a discussion of how those results fit into the existing body of literature. A fifth chapter has also been included which covers two additional studies designed to develop alternative EAE models for our lab; however, both studies turned out differently than expected. One of the most interesting developments from this final chapter was the discovery of an age dependent difference in the memory T cell response of older mice, which allows for more robust disease to be induced when cells from 6 month old mice are used in the passive EAE (P-EAE) model as opposed to mice 10 weeks of age.

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The human immunodeficiency virus type-1 (HIV-1) is the major etiological agent of acquired immunodeficiency syndrome (AIDS), the cause of over 30 million deaths worldwide. Highly active antiretroviral therapy (HAART) has demonstrated great efficacy at suppressing viral load and is therefore the standard therapeutic treatment for HIV-1 infection. Noncompliance due to severe HAART-associated side effects significantly undermines therapeutic efficacy. Dronabinol, the synthetic form of the cannabinoid THC found in marijuana, is FDA-approved for countering some of these side effects. Studies have reported that cannabinoids restrict HIV-1 replication, although no mechanism has yet been proposed. Thus the purpose of this study was to characterize the effects of cannabinoids on HIV-1 infection and to determine the molecular basis of cannabinoid-induced viral suppression. By transcriptomic sequencing of T cells treated with cannabinoids, we have found that the expression of BAG6, a protein uncharacterized within the context of HIV-1 infection, was downregulated. To identify the role of this protein during infection, we knocked down BAG6 and were able to recapitulate the protective effects of cannabinoids by observing reduced severity of viral challenge. Moreover, we have also identified BAG6 to be a binding partner of two HIV-1 viral accessory proteins, Vif and Vpr. Importantly, we have discovered that Vpr mediates targeted degradation of BAG6 by leveraging the host proteasome during the early stages of the viral lifecycle, revealing a hitherto unknown function of this poorly-understood viral protein. We thus establish modulation of BAG6 expression as a novel mediator of the effects of cannaninoids on HIV-1 infection. / Microbiology and Immunology

Despite the ongoing political debate regarding the legality of medical marijuana, clinical investigations of the therapeutic use of cannabinoids are now more prevalent than at any time in history. Cannabinoids have been shown to have analgesic, anti-spasmodic, anticonvulsant, anti-tremor, anti-psychotic, anti-inflammatory, anti-oxidant, anti-emetic and appetite-stimulant properties. There are mainly two well-known cannabinoid receptors, CB1 and CB2, located in the central (CB1) and peripheral (CB2) nervous systems as well as the immune system. More recently, endocannabinoids (ligands) and their receptors have also been found in the skeleton which appear as the main body system and physiologically regulated by CB2. This study aimed to examine the effect of both CB1 and CB2 receptor stimulation on wound closure response of MG-63 osteoblast bone cell monolayers using different treatments with cannabinoid such as Winn55,212-2, URB602 and HU308. Also, cell adhesion, cell proliferation and cell length was investigated. The study also aimed to examine the effect of HU308 treatments in combination with TGF-β3 (transforming growth factor beta -3) on wound healing, cell adhesion and extracellular matrix up regulation (collagen type I, fibronectin and protien S-100A6) as well as other biological factors such as secretion of matrix metalloproteinase (MMP-2) and nitric oxide (NO). Finally, this study investigated HU308/TGF-β3 combination treatment on the regulation of extracellular matrix (collagen type I, fibronectin and protien S-100A6) in a 3D multilayer system of MG-63 osteoblast bone cells. Wound healing assays of MG-63 monolayers revealed accelerated wound repair as well as increased cell proliferation mainly regulated through CB2 receptors, and that treatments with HU308 and HU308/TGF-β3 achieved minimum closure timings compared with control groups (P<0.05). Our finding suggested that proliferation rate with 500nM HU308 was significantly higher than control and TGF-β3/HU308 combination groups (P<0.05). Interestingly, percentage of wound remained open after 15 hours for combination groups was 17.6%±1.32 whereas treatment with 500nM HU308 had 20%±2.25 indicating that the combination groups took the lead throughout wound healing. It was also observed that bridge formation in all treatment groups was taking place between 15 to 20 hour periods whereas within control treatments bridge formation started to take place after 25 hours. Cell surface attachment was examined via the trypsinization assay in which the time taken to trypsinize cells from the surface provided a means of assessing the strength of attachment. The results indicated that higher concentrations of HU308 (2μM), induced significant force of cell attachment compared with control and concentrations of 500nM and 1μM (P<0.05). However, groups treated with TGF-β3 and combination HU308/TGF-β3 indicated reduced cell surface attachment compared with control groups, indicating enhanced cell migration. Immunofluorescence staining as well as Elisa based semi-quantification technique indicated that both collagen type I and fibronectin were unregulated using higher concentrations of HU308 with decreased cell proliferation compared to lower concentrations. Nevertheless, protein S-100A6 was up-regulated in treatments with HU308, TGF-β3 and their combination HU308/TGF-β3 (P<0.05), indicating the positive role of these treatments in promoting cell differentiation. MMP-2 levels in the current study were also shown to be concentration-dependent, i.e. higher concentrations of HU308 significantly reduced MMP-2 secretion leading to decreased cell migration, while HU308/TGF-β3 combination treatment increased MMP-2 levels, indicating an increase in cell migration. The current study also examined levels of nitric oxide synthesis in relation to different treatments with HU308, TGF-β3 and HU308/TGF-β3 combination. It was found that nitric oxide up-regulation influences rate of MG-63 osteoblast wound healing in a concentration dependent manner. Lastly, UpCell culture dishes proved to have efficacy in obtaining a multilayer model of MG-63 osteoblast system in-vitro through changes in cell morphology. It was also found that treatments with HU308, TGF-β3 and HU308/TGF-β3 combination influenced collagen type I, fibronecton and protein S-100A6 secretion. These findings supported the earlier Elisa based semi-quantification results obtained for monolayer cultures.

Les effets de la marijuana, un médicament utilisé par l’homme depuis des millénaires, sur le système visuel sont peu connus. Une meilleure connaissance de la distribution du système endocannabinoïde (eCB) de la rétine pourrait expliquer comment cette drogue affecte la vision. Cette étude vise à caractériser la distribution du récepteur cannabinoïde CB1 (CB1R) et de l’enzyme de dégradation FAAH (“fatty acid amide hydrolase”) des ligands du CB1R dans la rétine du singe Vert (Chlorocebus sabaeus). De plus, elle vise à déterminer quelles sous-populations cellulaires de la rétine expriment ces composantes. La plupart des études à ce jour ont été conduites surtout sur les rongeurs et peu de travaux ont été réalisés chez le singe. Notre étude vient donc combler cette carence. Par le biais de méthodes immunohistochimiques, nous avons investigué la localisation du CB1R et de l’enzyme FAAH à différentes excentricités rétiniennes, de la fovéa centralis vers la périphérie. Nos résultats, en accord avec notre hypothèse de travail, démontrent que CB1R et FAAH sont exprimés à travers toute la rétine mais avec, cependant, des différences notoires. Au niveau de la couche des photorécepteurs, CB1R est exprimé préférentiellement dans les cônes et ce patron d’expression suit la distribution des photorécepteurs centre-périphérie. De plus, CB1R se retrouve surtout dans les pédicules des cônes de la couche plexiforme externe. CB1R et FAAH sont abondants dans les cellules bipolaires tant au centre qu’en périphérie. Le soma et l’axone des cellules ganglionnaires expriment aussi CB1R et FAAH. Ces données suggèrent que le système eCB est présent à travers toute la rétine du primate et pourrait expliquer les perturbations visuelles entrainées par la marijuana, telles la photosensibilité et la vision des couleurs. / The effects of marijuana, a drug that has been used by men for millennia, on the visual system are poorly understood. A better understanding of the distribution of the endocannabinoid system in the retina will help us explain how this drug affects vision. This study aims at characterizing the distribution of the endocannabinoid receptor CB1 (CB1R) and the enzyme degrading CB1R ligands, fatty acid amide hydrolase (FAAH) throughout the Green monkey retina (Chlorocebus sabaeus). In addition, it seeks to determine which sub-population of neurons expresses CB1R and the degrading enzyme FAAH. Most data on the endocannabinoid system have been acquired in rodents and studies on monkeys are rather scarce. We attempted to fill this void by using immunohistochemical methods to locate CB1R and FAAH at various eccentricities of the monkey retina, from the center to the far periphery. Our results, in agreement with our hypothesis, demonstrate that CB1R and FAAH are expressed throughout the retina. At the level of the photoreceptors, CB1R is expressed preferentially in cones rather than in rods, and this expression pattern follows the photoreceptors distribution. In the outer plexiform layer, CB1R immunoreactivity is predominantly concentrated in the cone pedicles. Although foveal cones are the main expressers of both CB1R and FAAH, these are also found in rod bipolar cells. The ganglion cell axons strongly express the CB1 receptor and the enzyme FAAH. These data suggest that the presence of CB1R throughout the retina may be responsible for the visual effects commonly reported by cannabis users, such as the increase in photosensitivity and alterations in color discrimination.

Pain of various etiologies (e.g., visceral, inflammatory) can be a debilitating disorder that presents a problem of clinical relevance. While it is known that ∆9-tetrahydrocannabinol (THC) the primary psychoactive constituent found in marijuana produces analgesia in various rodent models of pain, its pharmacological properties are overshadowed by its psychomimetic effects. THC is the primary phytocannabinoid found in marijuana though other prevalent constituents such as the phytocannabinoids (e.g., cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC), tetrahydrocannabivarin (THCV)) may possess antinociceptive actions without the psychomimetic effects associated with THC. Indeed, these phytocannabinoids act upon the endocannabinoid system (ECS) that is comprised of the CB1 and CB2 cannabinoid receptors, endogenous ligands (anandamide (AEA), 2-arachidonoyolglycerol (2-AG)), and endocannabinoid biosynthetic and catabolic enzymes. We hypothesize that phytocannabinoids as well as endocannabinoid catabolic enzyme inhibitors reduce nociception preclinical models of pain. In the first series of studies, the antinociceptive effects of prevalent phytocannabinoids were evaluated in the acetic acid stretching test, a rodent visceral pain model. While CBN and THC both produced antinociceptive effects via a CB1 mechanism of action, CBC, and CBD had no effect on nociception. Conversely, THCV antagonized the antinociceptive effects of THC. These results suggest that various constituents of marijuana may interact in a complex manner to modulate pain. Since the THC and CBN displayed their effects via specific endogenous cannabinoid receptors, we investigated whether increasing endocannabinoids block nociceptive behavior. Blockade of the catabolic enzyme fatty acid amide hydrolase (FAAH) elevates AEA levels and elicits antinociceptive effects, without psychomimetic issues associated with THC. Similarly, blockade of another endocannabinoid catabolic enzyme monoacylglycerol lipase (MAGL) elevates (2-AG) and elicits antinociceptive effects. Therefore, we tested the hypothesis that FAAH and/or MAGL inhibition blocks nociception in the acetic acid abdominal stretching model, and the LPS-induced allodynia (i.e. painful response to a non-noxious stimuli) model of inflammation. Genetic deletion or pharmacological blockade of FAAH or pharmacological blockade of MAGL significantly reduced the total number of abdominal stretches in the visceral pain model. Additionally, blockade of both enzymes simultaneously produced an enhanced antinociceptive effect versus blocking the enzymes individually. These effects were mediated through CB1 receptors. However, in the LPS-induced allodynia model, FAAH inhibited anti-allodynic effects through a CB1 and CB2 receptor mechanismn. In both assays other potential targets of FAA substrates (i.e., mu-opioid, TRPV1, and PPAR-alpha receptors) did not play an apparent role in FAAH inhibited antinociceptive responses. Taken together, these results illustrate that targeting the endocannabinoid system via direct acting agonists such as the phytocannabinoids, or indirect methods (i.e. inhibiting degradative enzymes of the endogenous cannabinoids), represents a promising strategy to treat pain.

Exogenous and endogenous cannabinoids have been reported to modulate functional activities of macrophages. It is recognized that macrophages express primarily the CB2 cannabinoid receptor, but recent studies indicate that its expression is differential in relation to activation state with maximal levels occurring when cells are in "responsive" and "primed" states. The functional activities of macrophages when in these states of activation are the most susceptible to the action of cannabinoids, at least in terms of a functional linkage to the CB2. To assess the effect of cannabinoid treatment on macrophage chemotaxis and test the hypothesis that cannabinoids inhibit the chemotactic response of macrophages and microglia to endogenous and exogenous, pathogen-derived stimuli, primary murine peritoneal macrophages and neonatal rat microglia were used. Chemotaxis assays and scanning electron microscopy studies demonstrated that cannabinoids inhibit chemotaxis, a signature activity attributed to "responsive" macrophage-like cells, to the endogenous chemokine RANTES (Regulated upon Activation Normal T-cell Expressed and Secreted) and to Acanthamoeba conditioned medium containing secreted proteases. The partial agonist delta-9-tetrahydrocannabinol (THC), administered in vitro, inhibited the chemotactic response of peritoneal macrophages to the chemokine RANTES and to Acanthamoeba conditioned medium. In vivo treatment with THC also resulted in inhibition of the in vitro chemotactic response of murine peritoneal macrophages to RANTES and amoebic conditioned medium. Pharmacological studies employing cannabinoid receptor agonists and antagonists demonstrated the involvement of CB2 in cannabinoid-mediated inhibition of peritoneal macrophage chemotaxis to RANTES and Acanthamoeba conditioned medium, implying that signaling through cannabinoid receptors may desensitize chemokine receptors. Treatment with cannabinoids had no apparent effect on chemokine receptor mRNA levels, but did enhance CCR5 protein phosphorylation. Macrophage migration to Acanthamoeba conditioned medium may involve activation and signaling through protease activated receptors (PARs), as pathogen-derived proteases have been shown to activate PARs and initiate cellular migration; however, further studies are required to demonstrate PAR activation by amoebic conditioned medium and to assess the effects of cannabinoids on PAR signaling. Acanthamoeba are opportunistic pathogens that cause Granulomatis amoebic encephalitis, an infection of the CNS that is often fatal. THC treatment has been shown to increase mortality to Acanthamoeba infections and is characterized by an absence of granuloma formation. We hypothesize that inhibitory effect of THC on macrophage migration may be a key factor in cannabinoid-mediated immunosuppression. To assess the effect of cannabinoids on microglial migration to Acanthamoeba conditioned medium, chemotaxis assays were performed using primary rat microglia treated with cannabinoids. These studies demonstrated that cannabinoids inhibit microglial chemotaxis to amoebic conditioned medium. Furthermore, the studies demonstrate that cannabinoids, acting through cannabinoid receptors, may cross-talk with a diverse array G-protein coupled receptors so as to modulate responsiveness of macrophage and macrophage-like cells.

HIV-Associated Neurocognitive Disorders (HANDs) are becoming one of the largest problems in patients infected with HIV-1. The ability of infected cells such as monocytes and microglial cells to act as viral reservoirs causes extreme inflammation in the CNS and has led to several different types of neurocognitive problems. Specifically these HIV-1 infected monocytes are able to secrete inflammatory factors such as the regulatory protein Tat which acts as a chemoattractant for monocytes while also promoting the adhesion of leukocytes to the extracellular matrix (ECM). We have shown that one of the major features of the Tat protein is that it promotes cytoskeletal rearrangement resulting in increased adhesion. Specifically integrin and actin visualization was performed using confocal immunofluorescence while cytoskeletal morphology was shown with light and SEM. This microscopy work showed the Tat protein resulted in altered β1-integrin expression and distribution as well as changes in polymerized actin. These cytoskeletal changes resulted in increased adhesion to the ECM. Similarly we have also shown that these cytoskeletal changes of β1-integrin distribution and polymerized actin can be modulated through select cannabinoids THC and CP55940 that bind through the CB2 receptor which inhibits this adhesion as well as the morphological changes. The modulation of this reorganization is characteristic of a signal transduction pathway where a novel convergent point between extracellular Tat and the select cannabinoids THC and CP55940 exists. The aim of this project was to show the cytoskeletal reorganization using different microscopy techniques including light and scanning electron microscopy. This was followed by identifying and characterizing the convergent point along the signal transduction pathway linked to these changes. Different techniques were utilized in order to identify the putative convergent point in the signal transduction cascade including antibody arrays, RT-PCR, and western immunoblotting. The cytoskeletal rearrangements of β1-integrin and actin polymerization were shown successfully via light and scanning electron microscopy in the context of treatment with Tat in the presence and absence of select cannabinoids THC and CP55940. Several different pathways were identified as possibly linked to cannabinoid-mediated inhibition of signal transductional activation consequent of attachment to extracellular matrix proteins. However, the exact molecules implicated in specific signal transductional pathways as targets of cannabinoid-mediated action remain to be defined.

Cysteine cathepsins B, L and S are endosomal/lysosomal proteases that participate in numerous physiological systems. Cathepsin expression and activity are altered during various inflammatory diseases, including rheumatoid arthritis, atherosclerosis, neurodegenerative diseases and cancers. Early immune responses to microbial pathogens are mediated by pattern-recognition receptors, including Toll-like receptors (TLR). Signaling through TLR causes cell activation and release of inflammatory mediators, which can contribute to the severity of chronic inflammatory diseases. The impact of TLR cell activation on cathepsins B, L and S activities was investigated using live-cell enzymatic assays. Individual ligands of TLR4, TLR2 and TLR3 increased intracellular activities of the three cathepsins indicating the involvement of both MyD88-dependent and -independent pathways. To investigate the role of inflammatory cytokines in regulating these proteases, a lipopolysaccharide (LPS) non-responsive cell line was utilized. LPS non-responsive cells co-cultured with LPS responsive macrophages upregulated cathepsin activities. Furthermore, culture supernatants from LPS-stimulated macrophages increased cathepsin activities in LPS non-responsive cells, which could be reduced by neutralizing antibodies to TNF-α or IL-1β. These findings indicate cytokines regulate cathepsin activities during macrophage responses to TLR stimulation. Using LPS as a model for inflammation, the ability of the cannabinoids, delta9-tetrahydrocannabinol (THC), and CP55940 to suppress cysteine cathepsins during an inflammatory response was investigated. Cannabinoids, including the major psychoactive component of marijuana THC, modulate a variety of immune responses and have been proposed as possible therapeutics to control chronic inflammation. Cannabinoids may mediate their effects through receptor-dependent or independent mechanisms. Cannabinoid receptor subtype 1 (CB1) and receptor subtype 2 (CB2) have differential expression in leukocytes. Dose response studies showed that 1 nM THC was sufficient to inhibit cathepsin enhancement in LPS-stimulated cells. P388D1 macrophages expressed CB2 mRNA, but had no detectable CB1 mRNA indicating a role for the CB2 receptor. Utilizing a CB2-/- macrophage cell line, the role of CB2 receptor participation in THC inhibition of cysteine cathepsin upregulation was explored. THC did not affect cathepsin activity in LPS-stimulated cells lacking CB2 expression. These findings support the possibility of receptor selective agonists as therapeutic treatment during inflammatory diseases to prevent cathepsin involvement in pathological tissue destruction.

ROLE OF MAGL INHIBITION IN NICOTINE WITHDRAWAL AND REWARD. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. by Pretal Ishvarlal Patel Muldoon Director: M. Imad Damaj, PhD Professor, Department of Pharmacology and Toxicology Tobacco use is one of the leading causes of preventable deaths worldwide. Nicotine, the main psychoactive component of tobacco, sustains and initiates tobacco addiction. Cessation of nicotine induces a dependence withdrawal syndrome. Recent in vivo studies indicate that the endocannabinoid (EC) system modulates both nicotine reward and withdrawal. The purpose of this proposal is to investigate the role of enhancing endogenous 2-arachidonoylglycerol (2-AG) and by blocking its degradative enzyme, monoacylglycerol lipase (MAGL) enzyme, in nicotine reward and dependence. The selective MAGL inhibitor JZL184 dose-dependently reduced both precipitated and spontaneous somatic and aversive withdrawal signs in mice. These effects were blocked by rimonabant indicating a CB1 receptor mechanism. Furthermore, repeated administration of JZL184 for 6 days did not produce tolerance to the alleviation of withdrawal and the treatment did not induce alterations in CB1 receptor levels or receptor-mediated G-protein activity in various brain regions. In addition, a decrease in 2-AG levels was found in the nucleus accumbens in nicotine-dependent mice undergoing precipitated withdrawal, suggesting that a dysregulation of this EC signaling system occurs during nicotine withdrawal. Lastly, we tested the effectiveness of a combination of low-dose JZL184 and high dose of the FAAH inhibitor PF-3845 on spontaneous nicotine withdrawal. Indeed, the combination of low-dose JZL184 and PF-3845 significantly attenuated nicotine spontaneous withdrawal signs. MAGL inhibition by JZL184 dose-dependently caused a significant blockade of nicotine reward as measured in the mouse conditioned place preference (CPP). In contrast to withdrawal, JZL184’s effect on nicotine CPP was not CB1 mediated. In addition, JZL184 treatment did not cause significant alterations in CB1 receptor levels or receptor-mediated G-protein activity in several brain regions involved in nicotine reward. The effects of JZL184 on nicotine CPP was selective since the drug failed to alter food-induced CPP and LiCl-induced conditioned place aversion in the mouse. Interestingly, active doses of JZL184 did not only cause an increases in 2-AG levels but also induced a concomitant decrease in arachidonic acid (AA) levels in various brain regions suggesting an AA cascade dependent-mechanism. In line of these changes, a cox-2 inhibitor, valdecoxib, dose-dependently blocked nicotine preference.