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Cannabinoids suppress dendritic cell-induced T helper cell polarization

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Title:
Cannabinoids suppress dendritic cell-induced T helper cell polarization
Physical Description:
ix, 105 leaves : ill. ; 29 cm.
Language:
English
Creator:
Lu, Tangying (Lily)
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
Publication Date:

Subjects

Subjects / Keywords:
Dendritic Cells -- drug effects   ( mesh )
Dendritic Cells -- physiology   ( mesh )
Tetrahydrocannabinol   ( mesh )
Cannabinoids -- immunology   ( mesh )
Legionella Pneumophila -- immunology   ( mesh )
Immunosuppressive Agents -- pharmacology   ( mesh )
Th1 Cells -- immunology   ( mesh )
Mice, Inbred BALB C   ( mesh )
Mice   ( mesh )
THC
DCs
Th
Legionella
Infection
Immunity
Dissertations, Academic -- Molecular Medicine -- Doctoral -- USF   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: Cannabinoids suppress Th1 immunity in a variety of models including infection with the intracellular pathogen Legionella pneumophila (Lp). To examine the cellular mechanism of this effect, mouse bone marrow-derived dendritic cells (DCs) were studied following infection and drug treatment. DCs produced high levels of IL-12p40 following Lp infection. THC suppressed this cytokine response in a concentration-dependent manner and the endocannabinoids 2-arachidonoyolglycerol and virodhamine less potently suppressed cytokine production. DCs expressed mRNA for cannabinoid receptor 1 (CB1), CB2, and transient receptor potential vanilloid type 1 (TRPV1); furthermore, inhibition of Gi signaling by adding pertussis toxin completely attenuated the suppression induced by low concentrations of THC but not at high concentrations.In addition, the THC suppression was partially attenuated in DC cultures from CB1 and CB2 knockout mice and in cultures from normal mice co-treated with THC and cannabinoid receptor antagonists. Cytokine suppression was not attenuated by pretreatment with the TRPV1 antagonist capsazepine, suggesting that Gi signaling and cannabinoid receptors, but not TRPV1, are involved in THC-induced suppression of DC potential to polarize the development of naïve T cells to be Th1 cells. Besides IL-12, THC suppressed other DC polarizing characteristics such as the expression of MHC class II and co-stimulatory molecules CD86 and CD40, as well as the Notch ligand Delta 4. However, THC treatment did not affect other DC functions such as intracellular killing of Lp and Lp-induced apoptosis.Testing the capacity of THC to suppress DC polarizing function with T cells showed that DCs infected in vitro with Lp were able to immunize mice when injected prior to a lethal Lp infection; however, the immunization potential along with Th1 cytokine production was attenuated by THC treatment of the cells at the time of in vitro infection. In addition, THC-treated and Lp-infected DCs poorly stimulated primed splenic CD4 T cells in culture to produce IFN-gamma (IFN-y); however, this stimulating deficiency was reversed by adding recombinant IL-12p40 protein to the cultures. In conclusion, the data suggest that THC inhibits Th1 polarization by targeting essential DC functions such as IL-12p40 secretion and the maturation and expression of co-stimulatory and polarizing molecules.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2006.
Bibliography:
Includes bibliographical references (leaves 86-105).
Additional Physical Form:
Also available online.
Statement of Responsibility:
by Tangying (Lily) Lu.
General Note:
Includes vita.

Record Information

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University of South Florida Library
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University of South Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001967147
oclc - 263388197
usfldc doi - E14-SFE0001790
usfldc handle - e14.1790
System ID:
SFS0026108:00001


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ABSTRACT: Cannabinoids suppress Th1 immunity in a variety of models including infection with the intracellular pathogen Legionella pneumophila (Lp). To examine the cellular mechanism of this effect, mouse bone marrow-derived dendritic cells (DCs) were studied following infection and drug treatment. DCs produced high levels of IL-12p40 following Lp infection. THC suppressed this cytokine response in a concentration-dependent manner and the endocannabinoids 2-arachidonoyolglycerol and virodhamine less potently suppressed cytokine production. DCs expressed mRNA for cannabinoid receptor 1 (CB1), CB2, and transient receptor potential vanilloid type 1 (TRPV1); furthermore, inhibition of Gi signaling by adding pertussis toxin completely attenuated the suppression induced by low concentrations of THC but not at high concentrations.In addition, the THC suppression was partially attenuated in DC cultures from CB1 and CB2 knockout mice and in cultures from normal mice co-treated with THC and cannabinoid receptor antagonists. Cytokine suppression was not attenuated by pretreatment with the TRPV1 antagonist capsazepine, suggesting that Gi signaling and cannabinoid receptors, but not TRPV1, are involved in THC-induced suppression of DC potential to polarize the development of nave T cells to be Th1 cells. Besides IL-12, THC suppressed other DC polarizing characteristics such as the expression of MHC class II and co-stimulatory molecules CD86 and CD40, as well as the Notch ligand Delta 4. However, THC treatment did not affect other DC functions such as intracellular killing of Lp and Lp-induced apoptosis.Testing the capacity of THC to suppress DC polarizing function with T cells showed that DCs infected in vitro with Lp were able to immunize mice when injected prior to a lethal Lp infection; however, the immunization potential along with Th1 cytokine production was attenuated by THC treatment of the cells at the time of in vitro infection. In addition, THC-treated and Lp-infected DCs poorly stimulated primed splenic CD4 T cells in culture to produce IFN-gamma (IFN-y); however, this stimulating deficiency was reversed by adding recombinant IL-12p40 protein to the cultures. In conclusion, the data suggest that THC inhibits Th1 polarization by targeting essential DC functions such as IL-12p40 secretion and the maturation and expression of co-stimulatory and polarizing molecules.
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Cannabinoids Suppress Dendritic Cell-I nduced T Helper Cell Polarization by Tangying (Lily) Lu A dissertation submitted in partial fulfillment of the requirement s for the degree of Doctor of Philosophy Department of Molecular Medicine College of Medicine University of South Florida Major Professor: Thomas W. Klein, Ph.D. Peter G. Medveczky, M.D. Kenneth E. Ugen, Ph.D. Marzenna Wiranowska, Ph.D. Herman Friedman, Ph.D. Date of Approval: October 24, 2006 Keywords: THC, DCs, Th, legionella, infection, immunity Copyright 2006, Tangying Lu

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i TABLE OF CONTENTS LIST OF T ABLES.................................................................................................iv LIST OF FI GURES...............................................................................................v ABSTRACT ........................................................................................................v iii INTRODUC TION..................................................................................................1 Cannabis Products and Cannabi noids .......................................................1 Cannabinoid Re ceptors.............................................................................2 Endocannabinoids and Sy nthesized Canna binoids ...................................3 Receptors Involved in C annabinoid E ffects...............................................5 Effects of Cannabinoid s on Innate I mmunity..............................................6 Effects of Cannabinoids on Adaptive Immunity..........................................8 Effects of Cannabi noids on DCs..............................................................10 Project Signifi cance.................................................................................12 OBJECTIV ES.....................................................................................................14 MATERIALS AND METHODS............................................................................18 Mice ........................................................................................................18 Reagents.................................................................................................18 Bacteria ....................................................................................................19 Preparation and Treatm ent of Bone Marrow Derived DC s.......................20

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ii Cell Surface Marker Analysi s by Flow Cy tometry....................................21 Bacteria Growth Determi ned by CFU A ssay............................................22 Cell Viability and Apop tosis Detect ion......................................................22 Cytokine Detection by ELIS A...................................................................23 Cell-based EL ISA....................................................................................24 Reverse Transcription Polymerase Chain Reaction (RT-PCR)................25 Animal Injections and Tissue Samp ling...................................................26 Statistical A nalysis ...................................................................................27 RESULTS ...........................................................................................................28 Lp Infection of DCs Induc ed IL-12p40 Prod uction...................................28 Suppression by THC of Lp-i nduced IL-12p40 Se cretion..........................29 No Suppressive Effect by THC on LPS-induced IL-12p40 Production ........................................................................................29 Other Non-selective Agonists Su ppressed IL-12p40 Production.............30 THC Suppressed the Expression of DC Maturation and Polarizing Markers............................................................................................31 THC Treatment Did Not Affect Lp Survival in DCs or Enhance Apoptosis of In fected DCs ...............................................................32 Expression of Cannabinoid and Vanill oid Receptor mRNA in DCs..........33 Pertussis Toxin Attenuated THC-i nduced Suppression of IL-12p40........34 Role of Cannabinoid Receptors in THC-induced Suppression of IL12p40...............................................................................................34 TRPV1 Was Not Involved in THC E ffect..................................................36

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iii The Activation of P38 MAP Kinas e was Modulated by THC....................36 THC Treatment Impaired the Imm unization Potential of Lp-loaded DCs..................................................................................................38 THC Treatment of Lp-loaded DCs Inhibited Th1 Activity in Splenocytes from Re cipient Mi ce.....................................................39 IL-12p40 Addition Restored the Pola rizing Function of THC-treated DCs..................................................................................................40 DISCUSSI ON.....................................................................................................69 DCs are Potential Target s of Cannabino ids.............................................69 THC Suppressed IL-12p40 Producti on in Lp-infec ted DCs ......................70 THC Did Not Suppress LPS-indu ced IL-12p40 Se cretion........................71 THC Suppressed DC Maturation and Polarizing Mole cules.....................72 THC Did Not Affect Lp Survival and Apoptosis in DCs............................73 The Involvement of Cannabinoid Receptors and MAP Kinases in THC Effe ct.......................................................................................75 TRPV1 Was Not Involved in THC E ffect..................................................77 THC Impaired the Immunization Potential of Lploaded DCs ...................78 THC Inhibited Th1 activity Induced by Lp-lo aded DCs ............................79 THC Suppression of DC IL-12p40 Pr oduction Mediated Loss of Th1 Polarizati on......................................................................................81 SUMMARY .........................................................................................................83 LIST OF RE FERENCES....................................................................................86 ABOUT THE AUTH OR............................................................................E nd Page

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iv LIST OF TABLES Table 1. THC Treatment Suppressed DC Maturation Ma rkers...................48 Table 2. Attenuation Effect of SR Compounds on THC-induced Suppression of IL-1 2p40 in Lp-infected Bone Marrowderived DCs From Cannabino id Receptor Knockout Mice............57

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v LIST OF FIGURES Figure 1. THC Suppressed the Production of IL-12p40 and IL-6 in Bone Marrow-deriv ed DCs............................................................42 Figure 2. Lp Infection Induced IL-12p40 Production in Bone Marrowderived DCs from BALB/c Mi ce.....................................................43 Figure 3. THC, in A Concent ration-dependent Manner, Suppressed IL12p40 Production in Lp-infected BM-DCs from BALB/c Mice.......44 Figure 4. No Significant Effe ct of THC on LPS-induced IL-12p40 from DCs...............................................................................................45 Figure 5. Cannabinoid Receptor Agonists 2-AG and Virodhamine in A Concentration-dependent M anner, Suppressed IL-12p40 Production in Lp-infected B one Marrow-derived DCs from BALB/c Mi ce.................................................................................46 Figure 6. THC Suppressed the Expr ession of Maturation Markers on Lp infectedDCs............................................................................47 Figure 7. THC Suppressed the Expr ession of Delta 4 in Lp-infected DCs (LpDC/THC) as Compared to Infected DCs Treated with DMSO (Lp DC/DMSO) ............................................................49

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vi Figure 8. Lp Uptake and Survival Were Not Affected by THC Treatment of Lp InfectedDCs.......................................................50 Figure 9. Apoptosis and Cell D eath Were Not Affected by THC Treatment ......................................................................................51 Figure 10. Demonstration by RT-PCR of Cannabinoid Receptor, TRPV1 and -actin Message in RNA from Bone MarrowDerived DC s..................................................................................53 Figure 11. Pertussis Toxin, the Gi signaling Inhibitor, Attenuated the Suppression Effect of THC on IL12p40........................................54 Figure 12. THC Suppressed IL-12p40 Pr oduction in Lp-infected Bone Marrow-derived DCs from C57BL/6 mice......................................55 Figure 13. Vanilloid Receptor Inhi bitor Capsazepine Did Not Antagonize the Suppression Effect of THC on IL -12p40.................................. 58 Figure 14. P38 MAP Kinase, but Not JNK or ERK Was Required for IL12p40 Production in Lp -infected DCs............................................59 Figure 15. THC Modulated P38 MAP Kinase Ac tivation ................................60 Figure 16. THC Impaired Immunizati on Potential of Lploaded DCs..............61 Figure 17. THC Treatment of Lploaded DCs Inhibited Immunizing Potential as Evidenced by In creased Bacteria l Burden .................62 Figure 18. THC Treatment of Lp-l oaded DCs Inhibited the Expression of Th1 Cytokines in Splenocyt es from Immunized Mice................63 Figure 19. THC Suppression of DC IL-12p40 Production Mediated Loss of Th1 Polarization of Lpprimed CD4+ T Cells ............................65

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vii Figure 20. THC Suppresses Th1 Activation Signals......................................67 Figure 21. Postulated Signaling Pathways Involved in THC Suppression Effect on DCs................................................................................68

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viii Cannabinoids Suppress Dendritic Cell-I nduced T Helper Cell Polarization Tangying (Lily) Lu ABSTRACT Cannabinoids suppr ess Th1 immunity in a variety of models including infection with the intracellular pathogen Legionella pneumophila (Lp). To examine the cellular mechanism of this effect, mouse bone marrow-derived dendritic cells (DCs) were studied follo wing infection and drug treatment. DCs produced high levels of IL-12p40 following Lp infection. THC suppressed this cytokine response in a concentration-dependent manner and the endocannabinoids 2-arachidonoyolglycer ol and virodhamine less potently suppressed cytokine production. DCs expressed mRNA for cannabinoid receptor 1 (CB1), CB2, and transient recept or potential vanilloi d type 1 (TRPV1); furthermore, inhibition of Gi signaling by adding pertussis toxin completely attenuated the suppression induced by low concentrations of THC but not at high concentrations. In addition, the THC s uppression was partially attenuated in DC cultures from CB1 and CB2 knockout mice and in cultures from normal mice cotreated with THC and cannabinoi d receptor antagonists. Cytokine suppression was not attenuated by pretreatment with the TRPV1 antagonist capsazepine,

PAGE 10

ix suggesting that Gi signaling and cannabinoid rec eptors, but not TRPV1, are involved in THC-induced suppression of DC potential to polarize the development of nave T cells to be Th1 cells. Besides IL-12, THC suppressed other DC polarizing characteristics such as the expression of MHC class II and co-stimulatory molecules CD86 and CD40, as well as the Notch ligand Delta 4. However, THC treatment did not affect ot her DC functions such as intracellular killing of Lp and Lp-induced apoptosis. Te sting the capacity of THC to suppress DC polarizing function with T ce lls showed that DCs infected in vitro with Lp were able to immunize mice when injected prior to a lethal Lp infection; however, the immunization potential along with Th1 cytokine production was attenuated by THC treatment of the ce lls at the time of in vitro infection. In addition, THCtreated and Lp-infected DCs poorly stimul ated primed splenic CD4 T cells in culture to produce IFN-gamma (IFN); however, this stimulating deficiency was reversed by adding recombinant IL-12p40 protein to the cultures. In conclusion, the data suggest that THC inhibits Th1 pol arization by targeting essential DC functions such as IL-12p40 secretion and the maturation and expression of costimulatory and polariz ing molecules.

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1 INTRODUCTION Cannabis products and cannabinoids Cannabis is one of the oldest psychotr opic drugs known in human history and has been used from the earliest re cords. In 2737 BC, Shen Nung, an emperor of ancient China, had described the properti es and therapeutic uses of cannabis in his compendium of Chinese medicinal compounds (98). Two main preparations derived from cannabis are marijuana and hashish. Marijuana is a green, brown, or gray mixt ure of dried leaves, stems, seeds, and flowers of the hemp plant (cannabis sativa) while hashish is the viscous resin of the Indian hemp plant (97). Despite the fact that cannabis and its products have been widely noted for their effects as an anal gesic, appetite stimulant, antiemetic, muscle relaxant and anticonvulsant for cent uries (178), it was not until the 1940s that scientists were able to purify and defi ne the structures of the cannabis plant, including more than 60 dibenzpyrene comp onents known as cannabinoids. The major psychoactive ingredient of cannabis is delta-9-tetrahydrocannabinol, usually termed as THC. Other cannabi noids in cannabis such as delta-8tetrahydrocannabinol ( 8THC), cannabinol (CBN), cannabidiol (CBD), cannabicyclol (CBL), cannabichrom ene (CBC) and cannabigerol (CBG) are

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2 present in small quantities and have littl e psychoactive effects compared to THC (14); however, it has been suggested t hey may have synergistic effects in combination with THC (8). Cannabinoid receptors Cannabinoids usually exert their actions by binding to specific receptors and two types of cannabinoid receptor have been decribed to date. These receptors are found in mammals, birds, fish, and reptiles (49). Cannabinoid receptor type 1 (CB1), originally cloned by Matsuda et al. in 1990 from a rat brain cDNA library by a probe derived from the sequence of bovine substance-K receptor, exhibits 97 to 99% amino acid sequence identity across species (30, 57, 114, 115). CB1 has been demonstrated in high levels in the central nervous system (CNS) and is predominantly found presynaptically expressed, and associated with the behavioral effects follo wing cannabinoid usage, such as loss of short-term memory, dizziness, at axia and sedation (34). Peripheral expression of CB1 has also been found in many peripheral tissues including heart, vascular endothelium, small intestine, liver (144, 176) and in the cells of immune system such as splenocytes (78, 136), mast cells (163) and DCs (43, 113). Cannabinoid receptor type 2 (CB2) cloned in 1993 by Munro and associates from a human HL60 promyelocyt ic cell line library, exhibits 48% homology with CB1 (129). CB2 expression differs from CB1 in that it is relatively undetectable in the CNS (129, 164) except for microglia that express both CB2

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3 and CB1 (54, 183) (26). CB2 is expressed in a high level in the tissues of lymphoid system including the thymus, tonsils, bone marrow and spleen (56, 87, 105). Both receptors are coupled to Gi proteins to negatively regulate adenylyl cyclase and cAMP accumulation. This Gi protein-induced signal can be blocked by pertussis toxin (71). Endocannabinoids and synthesized cannabinoids Discovery of the cannabinoid receptors in humans and animals led first to the prediction and later to the act ual findings of endogenous ligands termed endocannabinoids. Anandamide (Arachi donoylethanolamide; AEA) (40) and 2arachidonylglycerol (2-AG) (119), both deriv atives of arachidonic acid, are the most studied endocannabinoids. These endocannabinoids participate in the regulation of neurotransmi ssion (10, 41, 69) and m any biological effects associated with marijuana conna binoids. 2-AG is a full and potent agonist for both CB1 and CB2, while AEA is more selective to CB1 than to CB2 (70). AEA has also been shown to bind vanilloid receptors that are heat-gated, cation channels, sensitive to the vanilloid com pound capsaicin and its analogues (196). Many studies have shown that upon stim ulation with agents such bacteriaderived lipopolysaccharide (LPS), immune cells, including macrophages (42), DCs (113) and peripheral-bl ood mononuclear cells (PBM Cs) (107) release the endocannabinoids 2-AG and AEA that may act as chemot actants for leukocytes and take part in immune regulation (83). A novel endocannabi noid, Virodhamine,

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4 also a derivative of arachidonic acid, has been described recently as an agonist acting on both CB1 and CB2. Virodhamine was shown to be highly produced in spleen tissues suggesting its potential ro le in immunomodulat ion (148). Other endogenous cannabinoids including Narachidonyldopamine (NADA) and Docosatetraenylethanolamide (DEA) hav e also been described. NADA is produced in mammalian nervous tissue and acts at vanilloid and cannabinoid receptors with more selectivity toward CB1 than CB2 (16, 36, 73). DEA is an endogenous ligand selectively activating CB1, and produced by astrocytes. It acts as a cannabimimetic in vivo causing hypothermia, analgesia, motor activity inhibition and catalepsy (12, 61). In addition to these naturally occurring cannabinoids and endocannabinoids, also many structur al analogues of cannabinoids have been synthesized in a number of laboratorie s. Some of these analogues include arachidonyl-2-chloroethylamide (ACAE), ajulemic acid (AJA), methyl arachidonyl fluorophosphonate (MAFP), JWH-133 and CP55, 940. Among these analogues, ACEA has high selectivity for CB1 (64, 70). AJA is a nonpsychoactive, synthetic analog of a metabolite of THC with relatively low affinity for cannabinoid receptors but some demonstrated efficacy in animal models of chronic pain (46) and inflammatory diseases (24). MAFP is a potent, irreversible inhibitor of AEA amidase, the enzyme responsible for AEA hydrolysis, and a selective ligand for CB1 (39, 110). JWH-133 is a highly selective agonist for CB2 while CP55, 940 has high affinity for both CB1 and CB2 (71). Some of these analogues of cannabinoids possess low CB1 binding resulting in low p sychoactivity. Therefore,

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5 these analogues may have potent ial therapeutic usage in immune inflammatory diseases associated with dysregulation of cannabinoid receptor signaling. Receptors involved in cannabinoid effects Since both CB1 and CB2 are Gi protein-coupled rec eptors, the signaling mechanisms involved in cannabinoid effect s are associated with activation of Gi proteins. It is well known that these het erotrimeric proteins are activated by ligand binding to a seven-transmembrane G protein-coupled receptor (GPCR) causing a conformational change, prom oting an exchange of GDP for GTP by the G subunit, and the dissociation of G from the G dimmer. Activated G and G subunits subsequently modulate spec ific downstream signaling pathways, and therefore relay information intracellularl y in response to various extracellular receptor stimulants (117). Dysregulated Gprotein signaling leads to pathologies in numerous organ systems and many important classes of medications can modify GPCR signaling pathways either dire ctly or indirectly (155). A number of studies suggested that Gi signaling was involved in cannabinoid effects mainly through binding to either CB1 or CB2 (69). However, alt hough currently there are only two known cannabinoid receptors, other known or yet to be identified receptors or non-receptor mediated mec hanisms may be involved in cannabinoid effects. For example, the synthetic cannabinoid AJA, which has potent antiinflammatory effects but low affinity to CB2, is known to bind directly to and activate the peroxisome proliferator-act ivated receptor gamma (PPAR-gamma), a

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6 pharmacologically important member of the nuclear re ceptor superfamily (24, 101). Similarly, the plant-derived cannabinoid CBD has immunosuppressive effects and binds weakly to both CB1 and CB2. One recent study demonstrated that the CBD effect could be reversed by an A2a adenosine receptor antagonist and abolished in A2a receptor knockout mice providing a non-cannabinoid receptor mechanism mediated by cannabi noids (27). Also, it has been shown that the endocannabinoid AE A can activate TRPV1 receptors (102, 108). However, some cannabinoid effect s seemed to be mediated by nonCB1, nonCB2 and non-TRPV1 receptor mechanisms that remain to be elucidated (65, 124, 143). Overall, the signa ling pathway involved in cannabinoids is more diverse than originally speculated and some effe cts are not mediated by the already known cannabinoid receptors. Effects of cannabinoid s on innate immunity Innate immunity is critical in i mmune surveillance against pathological infection agents (188). Macrophages and ne utrophils are mediators of innate immunity and can recognize, phagocytize and kill microbes through the activation of several enzymes including oxidases and inducible nitric oxide synthase (iNOS); these enzymes produce the toxic reactive oxygen intermediates (ROI) and nitric oxide (NO) that not only k ill microbes but cause inflammation and tissue damage (162). It has been shown that THC, through inhibition of cAMP signaling, inhibits iNOS and NO producti on by macrophages stimulated with LPS

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7 (77). In similar studies, lung alve olar macrophages collected from marijuana smokers exhibited limited antimicrobial activity. However, treatment with granulocyte/macrophage colony-stimulati ng factor (GM-CSF) or IFNrestored these cells to produce NO and antibacterial efficiency (159, 165) In addition, the endogenous cannabinoid AEA has been show n to suppress, though not as strong as THC, the expression of cyt okines such as IL-1, IL-6 and TNF by rat microglial cells, the macrophage cell type in brain (149). Gongora et al. recently showed that synthetic c annabinoid CP55, 940 block ed the expression of MHC class II molecules induced by IFNon the surface of microgl ial cells (58). Also CP55, 940, but not AEA treatment, inhibit ed superoxide production in neutrophils (94), while macrophage proteolytic and lysosome processing could be suppressed by THC (116). It was recent ly reported that JW H-133 inhibited the production of IL-12p40 and enhanced IL-10 by LPSor Theiler's virus -activated macrophages (32). In addi tion, it has been demonstr ated that the main nonpsychoactive component of marijuana cannabidiol (CBD) significantly modulated murine macrophage cytokine produc tion and chemotaxis (160). Natural killer (NK) cells, which are a class of innate immune cells, can rapidly respond to intracellular infections with viruses or bacteria by direct killing of the infected cells (60). It has b een shown that THC can suppress NK cell function in both animal models (85, 142) and humans (169). A recent study using marijuana users demonstrated t hat cannabis induced a significant decrease in the absolute number of NK cells as well as T and B cells in peripheral blood (48). In other studies, CB1 and CB2 antagonists were shown to

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8 partially reverse the THCinhibited NK cytolytic activity in mice (111). In addition, the endogenous cannabinoid 2-A G, but not AEA, has been shown to induce the migration of KHYG-1 cells an NK leukemia cell line, and human peripheral blood NK cells (82). Thes e studies demonstrated that various cannabinoids are capable to significantly modulate (mostly suppress) the innate immune cell functions which include mi gration, phagocytosis and processing foreign pathogens, cytokine production, and killing of target cells. Innate immunity can also highly impact t he development of adaptive immunity. Therefore cannabinoid modul ation of innate immune cells might also modulate the activation and development of T cells and B cells. Effects of cannabinoid s on adaptive immunity T helper cells (Th) are CD4+ T cells t hat through a variety of mechanisms provide help for activating adaptive immunity Th cells generate their effects by releasing cytokines and/or by direct cellcell interactions. T helper cytokines and co-stimulatory molecules interact with ma crophages, B cells and CD8+ killer cells to produce the effector mechanisms of adaptive immunity such as activated macrophages, antibodies and killer T cells to clear the invading pathogens (137). Based on the types of cytokines Th cells produce, they are classified into two subtypes, i.e., T helper cell type 1 (Th1) and type 2 (Th2). Th1 cells produce IL2, IFNand TNF, which promote the development of cell-mediated immunity, while Th2 cells produce IL-4, IL-5, IL10 and IL-13, and can activate humoral

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9 immunity, mainly directed against extracellu lar infections (4, 45) Recent studies show that many immune disorders are at tributable to the collapse of the system controlling the proportion of Th1 and Th2 cell s. For example, allergy, multiple sclerosis, and organ-specific autoimmune disease have pathology associated with aberrant Th1 and Th2 polarization (92, 100, 128). Moreover restoration of the proper balance between T h1 and Th2 cells is generally considered essential in the treatment of tumors, which are generated when cellular immunity is affected by immunosuppressive factors (93). Marijuana smoking increases susceptibilit y to infections (84) and is a risk factor in cancers of the respiratory syst em (174). Many studies indicate that cannabinoids have a Th biasing effect t hat shifts Th1 to Th2 response. Our group previously examined the effect of THC on host immune resistance to infection with Legionella pneumophila (Lp) (89, 134). Lp is a facultative, Gramnegative, intracellular bacterial pathogen that causes Legionnaires’ disease in healthy as well as especially immunoc ompromised individuals (55). Host resistance to this pathogen depends on acti vation of Th1 cells, cell-mediated immunity, and acute phase cytoki ne mobilization (17, 133, 175), and THC was shown to suppress Th1 immunity and concomitantly to enhance Th2 development that could not mediate the pr otection against Lp infection (89, 134). These studies suggested that cannabinoid s may have the unique character of biasing immune responses away from T h1 and toward Th2. The mechanism of the T helper biasing effect is unclear, but in mice involves activation of cannabinoid receptors, suppression of se rum interleukin-12 (IL-12) and splenic

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10 IL-12 receptor expression, s uppression of serum interferon (IFN) (89), and an increase in the Th2 biasing tr anscription factor GATA3 (86). Also the effect of THC on Th basi ng has been observed in other animal models. Zhu et al. demonstrated THC decreased t he production of Th1 cytokine IFNand increased the immunosuppressi ve cytokines IL-10 and TGF, disrupted host anti-tumor immunity and promoted lung tumor growth (192). Similarly, a recent paper by Mckallip et al. demonstrated THC enhanced breast cancer growth and metastasis along with increased Th2 but decreased Th1 related gene expression (118). It was not clear in this study if the suppression of cytokines was due to the enhanced Th2 response or the activation of T regulatory cells, which are characteri zed as CD4+CD25+Foxp3+ and able to produce IL-10 and TGF(146). The alteration effect of THC on the balance of Th1 and Th2 cytokines was also observ ed in human T cell cultures stimulated with allogeneic DCs (191) and in peripheral blood mon onuclear cells (PBMCs) isolated from marijuana smokers (140). Therefore, it has been implicated that cannabinoids have T helper biasing effect. However, the precise molecular and cellular mechanisms for these effects are far from defined; also the involvement of cannabinoid receptors (CBRs) remains unclear. Effects of cannabinoids on DCs DCs are professional anti gen-presenting cells (APCs) that are generated in the bone marrow and migrate as precurso r cells to sites of potential entry of

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11 pathogens. During the past decade, intens ive studies have demonstrated that DCs are central to the in tegration of innate and adapt ive immunity (11). In contrast to B and T lymphocytes, DCs express many pattern recognition receptors including various Toll-like rec eptors (TLRs) and ar e therefore uniquely able to sense stimuli such as bacterial and viral infection as well as tissue damage and necrosis (47). Immature DCs residing in tissues respond to antigenic signals in the environment, leading to their maturation and migration to lymphoid organs. During this process, the phenotypic characteristics and functions of these cells change, in cluding reduced phagocytic capacity and increased secretion of high levels of imm unostimulatory cytokines such as IL-12 and expression of MHC and co-stimulatory molecules (79). Also, expression of the polarizing Notch ligands, Jagged and/or De lta, is increased (6). Matured DCs then acquire the ability to direct the devel opment of adaptive immunity including shaping the type of Th cell response (79). In addition, it is becoming evident that DCs also play a critical role in amp lifying the innate immune response, either directly by stimulating NK cells and other innate immune cells (37) or indirectly through orchestrating Th development. Studi es have delineated the role of DCs in immune responses to a variety of pat hogens, including bacteria, viruses, and protozoan parasites as well as to tumors (13, 126). Little is known, however, concerning the role of cannabinoids on DCs. Only recently it has been shown that both CB1 and CB2 receptors are expressed on human and murine DCs (43, 113); and several endocannabi noids including AEA and 2-AG were found to be present in lipid extracts from immature DCs

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12 (113). These findings suggest the possibl e involvement of DCs in cannabinoid modulatory effects on immunity including THC induced shift from Th1 to Th2 effect. The current projec t, therefore, studys the i mmunomodulatory effect of cannabinoids on mouse bone marrow-der ived DCs during Lp infection. Project significance The current study examines a model of infection using Lp infected DCs which were treated with THC and related pharmacological agents. This study uncovers detailed mechanisms of the imm unomodulatory effects of cannabinoids on DCs especially during the primary stage s of infection and biasing toward T helper immunity. Moreover, even though a Th shift might be detrimental in the case of Lp and other intracellular pathogenic infections in mice and human because Th1 immunity is critical to reco very from these infections, in certain autoimmune diseases, for example systemic lupus erythematosus (SLE), the enhanced expression of Th1 immunity to se lf proteins can be a major cause of the development of disease (125). In this instance, cannabinoids might have therapeutic potential by suppressing Th1 immunity. The traditional focus for immunosuppressive drugs has been on ly mphocytes as the primary cellular target. However, it is now underst ood that several classical and newly established immunosuppressive drugs in terfere with immune responses in the early stages by suppressing DC differentia tion, maturation and activation (59). Moreover, DCs have been suggested as i mmunotherapy for a number of cancers

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13 (21, 132) and as adjuvants to induce Th1 and Th2 immunity (81). This study provides a close examination of cannabi noids on DC biology and clues to the use of these drugs in the pharmacologica l manipulation of immune responses. Overall, the results gathered will better def ine the public health risk of smoking marijuana and exposure to other cannabinoid agents as well as provide potential uses for these agents as immunomodulating and anti-inflammatory therapeutics.

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14 OBJECTIVES The studies to be conducted will inve stigate the impact of cannabinoids on mouse bone marrow-derived DCs. Pr evious studies suggested that cannabinoids bias Th polarization and we believe that cannabinoids influence the functions of DCs which are key to the pol arizing event. Prelim inary results from our laboratory showed that THC treatment of murine bone marrow-derived DCs, infected with Lp, suppressed the production of IL-12p40 (Figure 1), a key protein involved in Th1 polarization. These findings led to the hypothesis that cannabinoids, such as THC, suppress immu nity against Lp infection by inhibiting the Th1 polarization function of DCs. In order to verify this hypothesis, the following aims are proposed. Aim 1. To determine the effect of cannabinoids on the polarizing phenotype of DCs infected with Lp Evidence is accumulating that endoc annabinoids are physiologically essential molecules in various biol ogical systems including the immune system (83, 87). However, the specific imm unomodulatory effects of cannabinoids on DCs have not been fully investigated (84). DCs are the major source of IL-12

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15 production in the early stages of an infect ion, and IL-12 is the key cytokine to direct Th naive cell development to Th1 cells (137). We will st udy the kinetics of IL-12p40 production in Lp-infected DC cult ures and will determine the effect of THC treatment on the quantitative and qualitative aspects of IL-12p40 production. Similar experim ents will be done using LPS as stimulant. In addition, we will examine the effect of other c annabinoid receptor agonists on IL-12p40 including the endocannabinoids 2-AG and Virodhamine, as well as CB1-selective agonists ACEA, AJA, Methanandamide, MAFP, DEA and NADA, and the CB2selective agonist, JWH-133. A variety of DC characteristics in addition to IL12p40 production are known to influence T cell subset development (79). Therefore, we will also examine the effect of THC on the expression of DC maturation markers including MHC class II and the co-stimulatory molecules CD86 and CD40, and the Notc h ligand Delta 4 (6). In addition, DC functions such as Lp intracellular killing abilit y and Lp induced-apoptosis will also be studied. Aim 2. To determine the role of canna binoid receptors in drug effects on DC polarization Both CB1 and CB2 have been reported to be involved in the THC-induced attenuation of IL-12 production in THC-tr eated mice infected with Lp (89). We detected the mRNA expression of both c annabinoid receptors in DCs by using

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16 semi-quantitative RT-PCR. CB1 and CB2 are Gi protein-coupled receptors. Gi signaling is sensitive to pertussis toxi n inhibition and has been suggested to be able to suppress IL-12 production (19, 89). We therefore will ex amine if the role of Gi-mediated signaling pathways in the suppression of IL-12p40 by THC in experiments using the Gi inhibitor, pertussis toxin. Also, experiments will be performed using CB1 -/mice (194) and CB2 -/(23) mice in combination with receptor antagonists to fully examine the involvement of cannab inoid receptors. Moreover, other receptors, such as TR PV1, have also been reported to mediate cannabinoid effects (180). We, therefore, will examine its role in THC effects on IL-12p40 production by pretreatment with the TRPV1 antagonist capsazepine. In addition, the intracellular activation of MAP kinases has been shown to regulate IL-12 production (18, 179, 186); by us ing specific antagonists MAP kinase involvement in Lp induced IL-12p40 and suppressi on byTHC will be stud ied. Aim 3. To determine the e ffect of THC treatment on the T helper polarizing function of DCs Effective protection from infections with intracellular microorganisms needs the induction of cell-mediated immune responses requiring the activation of Th1 cells stimulated by the intera ction between nave T cells and polarized DCs (123). To examine the mechanism of the effect of THC on the polarizing function of DCs with antigen (Ag)-specific T cells, Lp-infected DCs will be treated with THC and then co-cultur ed with Lp-primed splenic CD4+ T cells to detect the

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17 protein levels of IFNand IL-12p40 in supernatants. Furthermore, we will examine the immunization effe ct of Lp-infected DCs treat ed in culture with either DMSO or THC to induce protective immuni ty in mice when injected prior to Lp. In these experiments, mice mortalit y will be monitored and Th1 and Th2 cytokines measured in cultures of sp lenocytes from mice administrated DCs treated under different conditions.

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18 MATERIALS AND METHODS Mice BALB/c and C57BL/6 mice, 7 week of age, were obtained from NCI (Fredericksburg, MD). Cannabinoid CB1 receptor and CB2 receptor gene deficient mice (CB1 -/and CB2 -/-) on C57BL/6 background were bred by USF animal facility staff from stocks provided by Dr. Andreas Zimmer (CB1-/-, University of Bonn) and Dr. Nancy Buckley (CB2 -/-, California State Polytechnic U.). The mice were housed and cared for in University of South Florida Health Sciences animal facility, which is fully accredited by the American Association for Accreditation of Laboratory Animal Care. Reagents THC, cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4chlorophenyl)-1-(2,4-dichl orophenyl)-4-methyl-1H-py razole-3-carboxamide hydrochloride (SR141716A), and cannabinoid CB2 receptor antagonist N-[(1S)endo-1,3,3-trimethyl bicycle [2.2.1] heptan-2-yl]-5-(4 -chloro-3-methylphenyl)-1-(4methylbenzyl)-pyrazole-3-carboxamide (SR144528) were obtained from the

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19 Research Technology Branch of the National Institute on Drug Abuse (Rockville, MD). SR141716A, SR1445 28 and THC were first diluted in dimethyl sulfoxide (DMSO) at 20 mg/ml and then in 5% feta l calf serum RPMI 1640 medium to a concentration of 0.01-10 M. 2-Arachi donoylglycerol (2-AG), Virodhamine, Methanadamide, ACEA, MAFP, DEA, NA DA and CP55,940 were purchased from Tocris (Bristol, UK). AJA wa s obtained from Dr. Sumner Burstein (University of Massachusetts, MA) and JWH-133 from Dr. John Huffman (Clemson University, Clemson, South Carolina). Pertussis toxin (Gi signaling inhibitor) was purchased from Sigma (St. Louis, MO). C apsazepine (TRPV1 antagonist) was purchased from ALEXIS (San Diego, CA). Pertussis toxin was dissolved in medium to a concentration of 0.01-1.0 ng/ml. Other cannabinoids and capsazepine were dissolved in medium to a concentration of 0.01-10 M. MAP kinases antagonists UO126, SP600125 and SB203580 were purchased from Tocris (Bristol, UK). Bacteria A virulent strain of Lp (M124), serogr oup 1, was obtained from a case of Legionellosis from Tampa General Hosp ital (Tampa, FL) and cultured on BCYE medium (Difco, Detroit, MI) as described pr eviously (89). Bacteria from colonies of 48 hr cultures were suspended in pyrogen free saline and adjusted spectrophotometrically to a working concentration.

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20 Preparation and treatment of bone marrow derived DCs Bone marrow cells were collected from femurs and tibias of the BALB/c and C57BL/6 wildtype as well as CB1 -/and CB2 -/mice at 8 to 12 weeks age. Cells were suspended at 1.0 x 106/ml and cultured overnight in 6-well cell culture plates (GIBCO-Costar, Cambridge, MA) in RPMI1640 medium supplemented with 5 M 2-mercaptoetham ol, 2 mM L-glutamine, 1% antibiotic/antimycotic solution (Sigma), 5% heat-inactivated fe tal calf serum (HyClone, Logan, UT) and 10ng/ml granulocyte/macrophage colony-s timulating factor (GM-CSF) (BDPharmingen, San Diego, CA). Nonadherent cells were removed and the adherent cells were incubated with fres h GM-CSF-containing medium for an additional 7-9 days, during which ti me the bone marrow-derived DCs became nonadherent and were harvested. T he purity of the obtained DCs was determined by flow cytometry st aining using fluorochrome-conjugated monoclonal antibodies to CD11b and CD 11c (BD-Pharmingen). The purity was about 100% CD11b+ and greater than 75% CD11c+ cells. These DCs were either uninfected or infected with Lp at ratio 10:1 for 30 to 35 min. DCs were then washed two times to remove non-internalized Lp and re-suspended to106 cells/ml. The cells were treated wit h different cannabinoids at various concentrations or with the highest conc entration of DMSO or ethanol (vehicle control). To study the mechanisms in volved in THC effect, the DCs were pretreated with SR141 716A or SR144528 at 0.01, 0.05, 0.1 and 0.5 M or with capsazepine at 0. 01, 0.1, and 1.0 M. In the study with pertussis toxin, cells

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21 were cultured for 18 hr with 0.01-1.0 ng/ ml pertussis toxin before infected with Lp. When LPS (Sigma) was used as stim ulator, DCs were incubated with LPS 0.01-1 g/ml and supernatants were collect ed for cytokine detection at different time points as indicated. In studies with CD4 T cells, cells were obtained from mice intravenously (iv) infected (primed) with a sub-lethal dosed of Lp (7 x 106) and the spleens were removed 5 days post-in fection. The T cells were isolated from the splenocytes by mouse T ce ll Enrichment Columns (R&D system, Minneapolis, MN) and CD4+ T cells were ne gatively selected from the purified T cells with CD4 enrichment magnetic bead ki ts (BD-Pharmingen). Isolated CD4 T cells were then dispensed in 24-well cell culture plates (GIBCO-Costar) and cocultured with DCs (CD4: DC = 10:1) in either the absence or presence of recombinant IL-12p40 (BD-Pharmingen) for 24 hr followed by cytokine analysis. Cell surface marker analysis by flow cytometry To evaluate the effects of THC on MHC class II, CD86 and CD40 expression on DCs, cells, either uninfected or infected with Lp, were treated with DMSO or THC 10 M for 48 hr. Following inc ubation, the cells were treated with fluorochrome-conjugated mAbs (BD-Pha rmingen) at 4C for 30 min, and then washed in PBS containing 2% BGS, and fixed in 1% paraformaldehyde. Cells were analyzed using FACScan (Becton Dic kinson, Mountain View, CA). The instrument is equipped with lasers tuned to 488 nm and to 635 nm. The following fluorochrome-conjugated mouse mA bs were used for DC surface marker

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22 staining: PE-conjugated anti-MHC class II; PE-conjugated ant i-CD86; and PEconjugated anti-CD40 (BD Pharmingen). Bacteria growth determined by CFU assay After 24 hr infection, spleens from infected mice were homogenized in Hanks balanced salt solution (HBSS). In studies of Lp growth in DC cultures, cells were lysed by with 0.1% saponin (Sigma) and diluted in HBSS. Homogenized spleens or lysed DCs were plated on BCYE agar plates and incubated at 37C for 72 hr. CFU counts were determined on an AutoCount apparatus (Dynatech Labs Chantilly, Va.). Cell viability and apoptosis detection DC viability and apoptosis were detect ed using the Annexin V-FITC kit ( BD-Pharmingen). Briefly, uninfected or infected cells (105), treated with DMSO (LpDC/DMSO) or THC 10 M for 24 hr (LpDC/THC), were washed twice with PBS, and incubated with Annexin V-FITC (5 l) and pr opidium iodide (5 l) in binding buffer for 15 min. Early apoptotic ce lls (Annexin V positive and propidium negative) and late apoptotic or dead ce lls (Annexin V positive and propidium positive) were quantitated by flow cytometry.

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23 Cytokine detection by ELISA IL-12p40, IL-4, IL-23 and IL-10 were determined using a sandwich ELISA with antibody pairs from BD Pharmi ngen. In 96-well enzyme immunoassay plates (GIBCO-Costar), each well was coat ed with 50 l of anti-murine antibody in 0.1 M NaHCO3, pH 8.2 (ant i-IL-12 p40 for IL-12p40 and IL-23; 5 g/ml) or in PBS (anti-IL-4 and anti-IL-10; 2 g/ml) overnight at 4oC. The wells of the plate were blocked with 150 l of 3% BSA/ 0.05% Tween 20 in PBS (IL-12p40 and IL23) or 0.5% BSA/0.05% Tween 20 in PBS (IL-4, IL-10) and incubated for 1 hr. The culture supernatants or serial dilu tions of cytokine standards were added and incubated for 1-2 hr, followed by bi otinylated detection antibodies (2ug/ml, 50ul) for 1 hr, and streptavidin-horseradish peroxidase (HRP) (1:1000 in 50ul) for 30 min. The plates were washed betw een each addition. The tetramethyl benzidine (TMB; Sigma) substrates were developed for 5-30 min; the reaction was stopped with 1 N sulfuric acid and read at 450 nm on an Emax microplate reader (Molecular Devices; Menlo Park, CA). The concentrations of sample cytokines were calculated from standard cu rves that were done for each plate. The levels of IFNand IL-12p70 in supernatants were measured using BD OptEIATM Sets (BD Pharmingen) according to the manufacturer instructions.

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24 Cell-based ELISA Phosphorylation of p38, JNK and ER K1/2 were measured by Fast Activated Cell-Based ELISA kits from Acti ve Motif (Carlsbad, CA). Briefly, 96well culture plates were treated with 100 l 10 g/ml poly-L-Lysine for 30 min at 37oC and then washed twice with PBS. uninf ected DCs or Lp-infected DCs were then seeded into 96-well plates with cult ure medium containing THC (6M) for indicated time. Cells were fixed by replacing the cu lture medium with 100 l of 8% formaldehyde in PBS. After 20 min in cubation at room temperature, plates were washed three times with wash buffe r (PBS containing 0.1% Triton X-100). Cells were then incubated with quenchi ng buffer (wash buffer containing 1% H2O2 and 0.1% Azide) to inactivate the cells endogenous peroxidase activation. After 20 min, cells were washed twice and incubated with antibody blocking buffer 100 l for 1 hr and incubated over night with primary antibody for phosphoMAP kinase protein or total -MAP kinase at 4C. Next day, cells were washed three times incubated with horseradish peroxidase-conjugated secondary antibody for 1 hr at room temperatur e and washed three times with wash buffer and twice with PBS. Subsequently the ce lls were incubated with 100 l developing solution for 2-20 min at room temperature; the reactions were stopped by adding 100 l stop solution and absorbance was read on an Emax microplate reader (Molecular Devices). Levels of MAP kinase activation were expressed as the ratios of phosphoryl ated MAP kinase to total MAP kinase.

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25 Reverse Transcription Polymer ase Chain Reaction (RT-PCR) Total RNA was extracted from de ndritic cell cultures by standard techniques using TriReagent (Sigma ) and quantitated using RiboGreen RNA Quantitation Kit (Molecular Probes, Eugene, OR). The extracted RNA was treated with DNase using DNA-free kit from Ambion (Austin, TX). 1g of total RNA were used for cDNA synthesis at 42oC for 45 min by priming with 0.5 g oligo (dT)15 primer, 20 nmol each deoxynucleoside tripho sphate, 0.5 U RNase inhibitor, and 15 U avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) in a total volume of 25 l. 2 l of the reverse transcriptase product was used for PCR, which was carried out in PCR buffer (TaKaRa, Fisher; Atlanta, GA) containing 250 M dNTP, 1.0 M each primer and 2.5 U Tag DNA polymerase (TaKaRa). The primer pai rs used were as follows: cannabinoid CB1 receptor forward primer, 5'-TCA CCACAGACCTCCTCCTCTAC-3'; reverse primer, 5'-CTCCTGCCGTCATCTTTTC-3 (149 bp product); cannabinoid CB2 receptor forward primer, 5'-GTACATG ATCCTGAGCAGTGG-3'; reverse primer 5'-TGAACAGGTACGAGGGCTTTCT-3' (147 bp product); TRPV1 forward primer, 5'-AATTTGGGATGTGGAGCAAG-3'; reverse primer, 5GATCCCCCGAGTATCCATTT-3' (176 bp product); -actin forward primer, 5'GGGAATGGGTCAGAAGAACT-3'; reverse primer, 5'AGGTGTGGTGCCAGATCTTC-3' (133 bp produc t); Jagged1, forward primer, 5'AGAAGTCAGAGTTCAGAGGCGTCC-3' reverse primer, 5'AGTAGAAGGCTGTCACCAGCAAC-3' ( 113 bp product); Delta4, forward primer,

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26 5'-AGGTGCCACTTCGGTTACACAG3', reverse primer 5'CAATCACACACTCGTTCCTCTCTTC-3' (123 bp product); and -actin, forward primer, 5'-ATGGATGACGATATCGCT-3', reverse primer, 5'ATGAGGTAGTCTGTCAGGT-3' (530 bp product) All PCR were performed in a Mastercycler (Eppendorf, We stbury, N.Y.) at 60oC for annealing. The number of cycles in each PCR was as follows: CB1, CB2, TRPV1 and -actin (133bp), 40 cycles; Jagged1 and Delta4, 35 cycles; and -actin (530bp), 28 cycles. PCR products were analyzed on ethidium brom ide-stained, 2% agarose gels. RTnegative amplifications were also done to control for contaminating genomic DNA. Animal injections and tissue sampling Mice were immunized iv with 0.3-0.5 x 106 treated DCs suspended in PBS, and 7-9 days later spleens were isolated fr om mice. Single-cell suspensions of splenocytes (2 x 106 cells/ml) were cultured with formalin-killed Lp (107/ml) for 24 hr and supernatants collected for cytokine detection. Or, DC-treated mice were challenged iv with live Lp (sublethal dose, 7 x 106) diluted in pyrogen free saline. Spleens were obtained after 24 hr and CF Us of Lp were counted. In other experiments, mice were immunized iv with DCs (0.5 x 106) for two or three times at 7 day interval, challenged iv with a lethal dose of Lp (1.7-2.0 x 107) and survival of mice was monitored.

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27 Statistical analysis Data were analyzed by one-way analysis of variance with Dunnett’s test for comparing individuals using SigmaStat (Jandel Scientific, San Rafael, CA), or using the two-tailed Student’s t test. A value of p < 0.05 was accepted as indicating significance.

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28 RESULTS Aim 1. To determine the effect of cannabinoids on the polarizing phenotype of DCs infected with Lp Lp infection of DCs induced IL-12p40 production IL-12 is a heterodimer produced by DCs and formed by the association of a 35-kDa light chain (p35) and a 40-kDa heavy chain (p40). Interestingly, microbial components alone seem to induc e primarily p40 with relatively low amounts of IL-12p70 (28). Based on this the kinetics of the IL-12p40 subunit production was investigated to determine the bone marrow-derived dendritic cell response to Lp infection. Dendritic ce ll cultures were infected, supernatants harvested at indicated time points, and supernatant IL-12p40 was measured by ELISA. As shown in Figure 2, there was a rapid increase in the secretion of the p40 protein reaching a peak by 44 hr following infection. These data indicate that cultured bone marrow-derived DCs produc e IL-12p40 following infection with Legionella

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29 Suppression by THC of Lp-induced IL-12p40 secretion To determine if cannabinoids can modulat e the ability of DCs to produce IL-12p40, infected DCs were treated with in creasing concentrations of THC (0, 1, 3, 6 and 10 M) and incubated for 24 hr followed by IL-12p40 measurements. Figure 3 A shows that THC treatment led to a significant decrease in IL-12p40 at a concentration of 3 M and higher while drug vehicle (DMSO) had no effect. In addition, Figure 3 B shows that the T HC (10M) suppression effect on DC IL12p40 was observed as early as 6 hr afte r treatment and Lp infection. These data demonstrated THC consistently and si gnificantly suppressed DC secretion of IL-12p40. No suppressive effect by THC on LPS-induced IL-12p40 production To examine if the THC effect is relat ed to TLR4 signaling, we used LPS, a ligand for TLR4, as a stimulant in DC cu ltures. Compared with Lp infection, LPS induced a rapid increase in IL-12p40 (Figur e 4 A) that reached maximum at 12 hr after stimulation. However, unlike with Lp infection, THC treatment had no significant effect on LPS-induced IL12p40 production (Figure 4 B). These data suggest that Lp infection stimulates IL -12p40 through receptors other than TLR4 and that the THC suppressive effect is not generalized for all stimuli but selective for mechanisms related to Lp infection rather than the TLR4/LPS pathway.

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30 Other non-selective agonists suppressed IL-12p40 production In addition to the effect of THC on DC function, we also examined the effect of the endocannabinoids 2-AG and Virodhamine. 2-AG has been shown to be produced in DC cultures (113) and Virodhamine has been reported in rat peripheral tissues including immune organs such as spleen in higher levels than AEA, another endocannabinoid (148). 2AG and Virodhamine are potent agonists for both CB1 and CB2 (119, 148, 172) and there are no reports examining their functional effects on DCs We therefore tr eated Lp-infected DCs with various concentrations of 2-AG and Virodhamine. We found that 2-AG treatment significantly suppressed IL-12p40 at concentrations of 1 M and 10 M (Figure 5 A). No effect was observed wit h ethanol as the drug vehicle control. Virodhamine also showed significant s uppression at 10 M and the suppression level was comparable to 2-AG, though not as strong as THC used at the same concentration (Figure 5 B). In addi tion, we tested other endogenous ligands, such as NADA and DE A, that are both CB1 selectvie and produced in nervous tissues (12, 16, 36, 61, 73). We also tested the synthetic cannabinoids including the CB1 selective agonists ACEA, AJA, M-AEA (1, 46, 64, 80), and the CB2 agonist JWH-133 (74). The results show ed these agents did not significantly suppress IL-12p40 in Lp infected DCs (dat a not shown). Thus, nonselective cannabinoid receptor agonist s, including THC, 2-AG and Virodhamine, but not other agonists, led to a significant inhi bition of IL-12p40 production in our culture system.

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31 THC suppressed the expression of DC maturation and polarizing markers Upon exposure to microbes, DCs are ac tivated to go through a maturation process characterized by an increase in surface expression of MHC class II proteins and co-stimulatory molecules c ontributing to initiation of an effective adaptive immune response (76). To determi ne if THC modulates this polarizing DC phenotype, we treated infected and non -infected DCs with either DMSO or THC and assessed the expression of thes e surface markers. After 48 hr, we observed by flow cytometry that Lp infection increased the surface expression of CD86 and CD40; however, THC treatm ent significantly suppressed the expression of both markers (Figure 6 and T able 1). Regarding MHC class II, we observed that although expression was hi gh in all three groups the intensity per cell of the marker was enhanced in the Lp infected-DCs but was significantly decreased by THC treatment (Table 1). Fr om these results, it is possible that drug suppression of T helper polarization is due in part to a down-modulation of these phenotypic markers. Ot her surface proteins such as Notch receptors are known to regulate T cell development (151) Recently it was shown that the Notch ligands, Delta4 or Jagged1 on DCs prom ote induction of either Th1 or Th2 activity, respectively (6). We, therefore, examined t he relative mRNA expression of these ligands in DCs loaded with Lp and tr eated with THC or vehicle for 18 hr. We observed that mRNA for both ligands was increased in DCs after Lp infection (LpDC/DMSO group; Fi gure 7) but that the Delta 4 band intensity relative to -

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32 actin was decreased following THC treatme nt suggesting that the message level of this Th1 polarizing ligand was decreased by drug treatment. THC treatment did not affect Lp survi val in DCs or enhance apoptosis of infected DCs THC has been observed to induce apoptosis in macrophages and lymphocytes (193) and also in DCs (43); t herefore, it could be argued that drug suppression of IL-12 production and mark er expression could be due to a toxic effect on DCs. Also, antigen presentation to CD4 T cells by DCs requires internalization and procession of infe ctious agents by the DCs and drug-induced suppression of phagocytosis, therefore, mi ght contribute to suppression of T cell activation. In order to test the THC e ffect on these other relevant DC functions, we studied the drug effect on the survival of Lp in DCs and the induction of apoptosis in Lp-infected DCs. DCs were infected with Lp for 30 min followed by washing to remove non-internalized bacteria. Infected cultur es were then treated with THC or DMSO for 0, 24 and 48 hr and the number of cell-associated CFUs, as a measure of phagocytized bacteria, wa s determined by cell lysis and viable bacteria colony counts. The results (Figure 8) showed that the bacteria internalizing function as measured by intr acellular survival was unaffected by THC treatment. Both drugtreated and vehicle treated cells restricted the growth of Lp in an equivalent manner over time To determine if THC induced apoptosis in Lp-infected DCs, staini ng with propidium iodide an d annexin V in treated DCs

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33 was analyzed by flow cytometry. Com pared with uninfected DCs, the percentage of apoptotic cells, assessed as single positive for annexin V, was enhanced after Lp infection (Figure 9), and treatment with THC did not in crease the annexin positivity. Furthermore, analysis of propidi um iodide staining, as indicative of necrotic cells, was similar in infected and infected plus THC treated cells. The data suggest THC treatment did not affe ct the degree of apoptosis or processing of bacteria in DCs following Lp infection. Aim 2. To determine the role of canna binoid receptors in drug effects on DC polarization Expression of cannabinoid and vanilloid receptor mRNA in DCs The cannabinoid receptors identified so far are CB1 and CB2. Both have been reported to be involved in the THC-in duced attenuation of IL-12 production in drug-treated mice infected with Legionella (89). Moreover, TRPV1, has recently been shown to bind AEA and other endocannabinoids an d mediate their effects (96, 180, 196). To examine receptor expression in dendritic cell cultures, RNA was isolated from DCs and analyzed by RT-PCR for CB1, CB2, and TRPV1 messages. Figure 10 shows mRNA of both cannabinoid re ceptors and TRPV1 was readily detected in DCs and the level of cannabinoid CB2 receptor and TRPV1 messages appeared to be more abundant than CB1 receptor (Ratios of

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34 target genes to -actin are: CB2 receptor 0.76, TRPV1 0.84, and CB1 receptor 0.48). Pertussis toxin attenuated THCinduced suppression of IL-12p40 Cannabinoid receptors are Gi protein-coupled receptors. Gi signaling is suppressed by pertussis toxin and the to xin has also been reported to suppress IL-12 production (19, 89). To examine if THC -induced suppression of IL-12p40 is through Gi protein-coupled mechanisms, DCs were pretreated with different concentrations of pertussis toxin (0.01, 0.1, or 1.0 ng/ml) for 18 hr followed by Lp infection and THC (3, 6 or 10 M) treatment for 24 hr. Pertussis toxin (at 1.0 ng/ml) completely reversed the suppression effect of THC at low concentrations (3 or 6 M) (Figure 11). However, the e ffect of THC at a higher concentration (10 M) was only partially attenuated by pertu ssis toxin (Figure 11). This finding suggests the involvement of Gi protein-coupled mechanis ms in the suppression of IL-12p40 at low THC concentrations, but other suppressive mechanisms at higher concentrations. Role of cannabinoid receptors in THC-induced suppression of IL-12p40 To fully examine if cannabinoid recept ors were involved in suppression of IL-12p40, experiments were performed using CB1 -/(194) and CB2 -/(23) mice. Because the knockout mice are on t he C57BL/6 background and our previous

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35 studies had been done with BALB/c mice, we first tested the response of bone marrow-derived DCs from wild-type C57BL/6 mice in terms of IL-12p40 production and suppression by THC. Comparing Figure 12 A, to Figure 3 A shows that cells from C57BL/6 mice displayed a comparabl e suppressive response to those from BALB/c. We next studied the response of DCs from CB1 -/and CB2 -/mice and the results showed that both were suppress ed by THC similar to wild-type cells (Figure 12, panels B and C). Since THC is a non-selective receptor agonist and can bind both CB1 and CB2 receptors (51), it is possible that the drug could have a relatively unimpeded effect in cells from single receptor knockout mice. To examine this possibility, t he specific antagonists for CB1 (SR141716A) and CB2 (SR144528) were used in combination with cells from knockout mice. CB1 -/DCs, after Lp addition, were pr etreated with SR144 528 at different concentrations for 30 min prior to THC treatment and the reci procal experiment was performed using CB2 -/and SR141716A pretreat ment. After 24 h, supernatant IL-12p40 was assessed by ELISA. As a control, cultur es were treated with ei ther SR141716A or SR144528 only followed by infection and no effect on IL-12p40 was observed (not shown). Table 2, shows the SR co mpounds attenuated the THC effect in knockout mice, especially at the lower drug concentrations. For example, the attenuating effect of SR144528 (at 0.1 M) in CB1 -/cells is nearly 70% of control at 3 M THC but only 28% of control at 10 M THC. A higher concentration of SR144528 (0.5 M) had the i dentical attenuating effect (data not shown). Similar decreases in receptor antagonist effi cacy with increasing receptor agonist concentration were seen using CB2 -/cells (Table 2). These results suggest that

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36 cannabinoid receptors are involved in the THC-induced suppression of p40, especially at the lower drug concentrati ons, but that other mechanisms become involved as the THC concentration is increased. TRPV1 was not involved in THC effect From the above, it appears that mechanisms other than cannabinoid receptors are involved in THC suppression of IL-12p40. Theref ore, we tested for a possible role of TRPV1 in the THC effe ct by pre-treating with capsazepine, the specific receptor antagonist for TRPV1. De ndritic cell cultures were infected with Lp and treated with either THC alone (3 and 10 M) or in combination with capsazepine at 0. 01, 0.1, and 1.0 M. As shown in Figure 13, there was no attenuating effect of capsazepine on the THC effect suggesting that TRPV1 receptors were not involved in the response. The activation of p38 MAP ki nase was modulated by THC It is reported that MAP kinases can be ac tivated in response to ligands for G protein-coupled receptors, and many st udies have indicated kinase activation affects either positively or negatively IL -12 production (18, 179, 186). There are three major groups of MAP kinases in ma mmalian cells: the extracellular signalregulated protein kinases (ERK), t he p38 MAP kinases, and the c-Jun-NH2terminal kinases (JNK)(44). Many studies have suggested the modulation effect

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37 of various cannabinoids on MAP kinase activi ty (69). However, a role for these kinases in the THC effect on DC cyt okine production has not been reported (158); therefore we examined t he role of kinase activity in our system. Initially we treated Lp-infected DCs with specific antagon ists for different kinases for 18 hr and then measured IL-12p40. We found that only the p38 inhibitor, SB203580, but not Erk inhibitor, UO 128, nor JNK inhibitor, SP 600125, was able to suppress IL-12p40 production suggesting only p38 kinase activation is required for Lpinduced IL-12p40 secretion (Figure 14). Fr om this finding, we next examined the effect of THC on p38 activation in DCs dur ing Lp infection. As shown in Figure 15, Lp infection induced an increase in phosphorylated p38 protein within 10 min after infection indicating an up-regulat ion in p38 activity. THC treatment, interestingly, initially increased phosphorylated p38 but then caused a drop in the level by 3 hr after infection. From thes e studies it is clear that p38 kinase is an important signaling com ponent of the Lp induction of IL-12p40 and that modulation of p38 phosphorylation by THC mi ght be a critical mechanism of drug action.

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38 Aim 3. To determine the e ffect of THC treatment on the T helper polarizing function of DCs THC treatment impaired the immuniza tion potential of Lp-loaded DCs Due to the pivotal role in stimulat ing T cells, DCs loaded with specific antigens have been utilized as immunizing vehicles in numerous studies of tumor therapies (29, 195) and infectious di seases (126). To test whether DCs loaded with Lp would induce a specific immune response and if THC would impair this ability, we treated Lp-infe cted DCs in culture with 10 M THC (LpDC/THC) or drug vehi cle DMSO (LpDC/DMSO) fo r 24 hr. DCs without infection and drug treatment were incubated for the same time as controls. Following treatment, DCs were injected iv in to mice two to th ree times at 7 day intervals, and seven days after the last in jection, the mice were challenged with a lethal dose (1.7-2.0x107) of bacteria and survival monitored. The results in Figure 16 showed that uninfected DCs failed to induce protection as none of the mice survived; however, Lp-loaded DCs (LpDC/DM SO) induced significant protection with a survival ratio of 66 percent (6/9 ). However, mice receiving loaded DCs treated with THC (LpDC/THC) showed no surviv al after 25 hr indicating a lack of immunizing potential similar to mice injected with non-loaded DCs. In other experiments to examine immunizing pot ential, mice were injected with DCs, LpDC/DMSO or LpDC/THC (0.3-0.5x106) and seven to nine days later challenged with a sublethal dose (7x106) of Lp rather than a lethal dose as in the

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39 above experiments. After 24 hr, spleens were isolated and homogenized, and bacterial burdens measured by CFU analy sis. The data showed that spleens from mice receiving Lp-loaded DCs had mu ch lower CFUs than spleens from mice receiving either unloaded DCs or l oaded DCs treated with THC (Figure 17). These findings together dem onstrated that mice imm unized with Lp-loaded DCs were able to induce immunization agains t Lp infection and that THC treatment significantly attenuated this effect. THC treatment of Lp-loaded DCs inhibi ted Th1 activity in splenocytes from recipient mice Type 1 cytokines, including IL-12 and IFN, can be measured in immune organs and are a measure of the develop ment of protective immunity against intracellular microbial infections (150) To determine polarization toward Th1 immunity, the cytokine profiles in splenocyt es of mice immunized with the various DC populations were analyzed to dete rmine whether THC treatment of DCs suppressed an upregulation of Th1 activity in recipient mice. As in the transfer experiments above, mice were immuniz ed with DCs only, Lp-loaded DCs, or loaded DCs treated with THC, and seven to nine days later splenocytes were harvested from the recipi ent mice and stimulated in vitro for 24 hr with specific Lp antigens. Supernatants from these cult ures were collected and analyzed for type1-associated cytokines by ELISA. As shown in Figure 18, the splenocytes from mice receiving Lp-loaded DCs tr eated with DMSO produced 1.5-2 fold

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40 increases in IL-12p40 and IFNas compared to splenocytes from mice treated with unloaded DCs. This suggested an upregulat ion of Th1 activity in the spleens of mice immunized with Lp-loaded DCs accounting for their enhanced resistance to Lp infection (see Figure 16 and 17). However, THC treatment of the DCs inhibited this upregulation of Th1 acti vity, suggesting an attenuation of the immunizing potential of these cells (F igure 18 A and B). IL-4 production by splenocytes was also examined and we observed it was suppressed following injection of Lp-loaded DCs either treated or not with THC (Figure 18 C). This suggested that the increase in Th1 acti vity in the spleens coincided with a decrease in the Th2 cytokine, IL-4; furthermore, it s uggested that THC suppressed Th1 cytokines by mechanisms other than the upregulation of IL-4. The data overall suggest ed that THC treatment of antigen-loaded DCs can suppress the immunizing and Th1 polariz ing potential of these cells when subsequently injected into mice. IL-12p40 addition restored the polar izing function of THC-treated DCs We have shown above that THC suppresses the production of IL-12p40 in Lp-infected DC cultures. Therefore, to ex amine if this attenuat ion is responsible for the impaired Th1 polarizing function of these cells, co-cultures of DCs with T cells from both unprimed and Lp-primed ani mals were prepared to examine the reconstitution efficacy of exogenously added IL-12p40. Figure19 A shows results from co-cultures of Lp-loaded DCs and unpr imed CD4 T cells. Lp loading of DCs

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41 induces the production of IL-12p40 as det ected in culture supernatants by ELISA and THC treatment of the ce lls suppressed this response. The addition of unprimed T cells had little effect on IL-12 production (Figure 19 A) and in studies not shown, no IFNwas detected in these cultures. We next examined the accessory cell potential of drug treated DCs in cultures containing Lp-primed T cells and supplied with various concentrations of IL-12p40. Figure 19 B shows that DCs plus primed T cells (LpCD4) prod uced a relatively small amount of IL12p40; however, when DCs were loaded with Lp (LpDC/DMSO), a robust IL12p40 response was evident and this was significantly attenuated by THC treatment (LpDC/THC + LpCD4). Of intere st was the finding that the addition of recombinant IL-12p40 protein to the cu ltures increased t he IL-12 supernatant concentrations above the amounts added (Fi gure 19 B). For example, addition of 0.5ng/ml recombinant IL-12p40 result ed in an increase of supernatant IL-12 from 2ng to 6ng/ml. Furthermore in contrast to co-cultures containing unprimed CD4 T cells, cultures cont aining primed T cells, produced in addition to IL-12p40, robust amounts of IFNbut only in the presence of Lp-loaded DCs (Figure 19 C) and this effect was attenuated by THC tr eatment of the DCs. However, the addition of recombinant IL12p40 completely restored IFNproduction suggesting a restoration of T h1 polarization by IL-12. In addition to IL-12p40, we also tested for the presence of IL12p70, IL-23, and IL-10 in the culture supernatants. These cytokines were not detected suggesting that the suppression of IL-12p40 by THC treatm ent was primarily responsible for the reduced Th1 polarization.

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42 Figure 1. THC suppressed the production of IL-12p40 and IL-6 in bone marrow-derived DCs. L. pneumophila (Lp) infected DCs were treated with DMSO (Lp), or THC 6 M for 24 hr. Supernatants were collected for cytokines detection. Data represent the mean of 3 experiments S.E.M.. *P< 0.05, compared to Lp group.

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43 Figure 2. Lp infection induced IL12p40 production in bone marrow-derived DCs from BALB/c mice. Immature bone marrow-derived DCs were infected as indicated in the methods. Supernatants we re collected at indicated time points and IL-12p40 measured by ELISA. Data r epresent the mean of 4 experiments S.E.M..

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44 A B Figure 3. THC, in a concentrationdependent manner, suppressed IL-12p40 production in Lp-infected BM-DCs from BALB/c mice. (A) Lp-infected BMDCs were treated with different conc entrations of THC or the highest concentration of DMSO (vehicle contro l) for 24 hr. (B) Using THC at 10 M, THC suppresses IL-12p40 production at 6, 12, and 21 hr. Data repres ent the mean of 3-5 experiments S.E.M.. *P< 0.05, compared to Lp group (THC 0 M).

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45 A B Figure 4. No significant effect of THC on LPS-i nduced IL-12p40 from DCs. (A) immature BM-DCs were incubated with LPS (1 g/ml). Supernatants were collected at indicated time points and IL-12p40 measured by ELISA. (B) DCs were stimulated with LPS (10 ng/ml) and tr eated with different concentrations of THC or the highest concentration of DMSO (vehicle control) for 24 hr.

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46 A B Figure 5. Cannabinoid receptor a gonists 2-AG and Virodhamine in a concentration-dependent manner, s uppressed IL-12p40 production in Lpinfected bone marrow-derived DCs from BALB/c mice. Lp-infected DCs were treated with different concentrations of 2-AG or the highest concentration of Ethanol (ETOH, vehicle control) (A) or Virodhamine with different concentrations or THC or 2-AG at 10 M (B) for 24 hr. IL-12p40 was detected by ELISA and the data represent the mean of 3-5 experiments S.E.M.. *P< 0.05, compared to 2AG 0 M (A) or Vi rodhamine 0 M (B).

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47 Figure 6. THC suppressed the expr ession of maturation markers on Lp infected-DCs. Cell surface markers were dete rmined by flow cytometry on DCs treated for 48 hr in various ways: uninf ected (DC); Lp-infect ed and DMSO treated (LpDC/DMSO); and Lp-infect ed and THC (10 M) treated (LpDC/THC). Data are expressed as percent expression (%) of the surface marker and mean fluorescence intensity (MFI) of the population for the ma rker. Data are representative of 4 experiments. DC LpDC/ DMSO LpDC/ THC 86% 42% 57% MFI: 1033 MFI: 156 MFI: 58 80% 36% 47% MFI: 721 MFI: 146 MFI: 65 92% 73% 80% MFI: 2940 MFI: 95 MFI: 157 I-A MHC II CD86 CD40 Relative cell number

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48 Table 1. THC treatment s uppressed DC maturation markers. Cell surface markers were determined in uninfec ted DCs (DC), Lp-infected and DMSO treated cells (LpDC/DMSO) or THC (10 M) treated cells (LpDC/THC) by flow cytometry after 48 hr treatment. Percent Expression Fluorescent intensity per cell a = Percent +/SEM, n=4 b =Mean fluorescence intensity +/SEM; n=4 # = p <0.05 versus the uninfected DC control = p <0.05 versus LpDC/DMSO group MHC class II 2212.1 499.5 b 2693.7 313.1 1433.5 348.6* CD86 147.1 8.6 183.7 22.1 133.2 12.7 CD40 83.4 21.4 130.3 29.6 81.0 9.2 DC LpDC/DMSO LpDC/ THC MHC class II 82.3 11.2a 87.2 6.0 79.4 4.8 CD86 63.8 3.1 81.7 2.8# 53.7 5.4* CD40 37.7 7.9 70.1 5.6# 36.5 9.6* DC LpDC/DMSO LpDC/ THC

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49 Figure 7. THC suppressed the expression of Delta 4 in Lp-infected DCs (LpDC/THC) as compared to inf ected DCs treated with DMSO (LpDC/DMSO). DCs were uninfected or infe cted with Lp and treated with DMSO or THC (10 M) for 18 hr. Jagged1, Delta4, and -actin mRNAs were amplified by RT-PCR. Data are represent ative of 3 experiments. 1.1* 1.8 2.1 0.3 0.7 0.2 -actin Jagged1 Delta4 DC LpDC/ DMSO LpDC/ THC Target to –actin ratio

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50 Figure 8. Lp uptake and surv ival were not affected by THC treatment of Lp infected-DCs. DCs infected with Lp for 30 min, washed twice to remove noninternalized bacteria and treat ed with DMSO or THC (10 M) for 0, 24, 48 hr. At various time post-infection, cell lysates were harvested and plated on agar medium, and CFUs of Lp determined by plat e counts at 72 hr. Data represent the mean of 3 experiments +/SEM.

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51 Figure 9 Apoptosis and cell death were not affected by THC treatment. Cultures of DCs were untreated (DC) or treated for 24 hr with either DMSO (LpDC/DMSO) or THC at 10 M (LpDC/ THC) and apoptosis and cell death were analyzed by staining with Anne xin V and propidium iodide, respectively. (A) Dot plot of propidium iodide and Annexin V staining; representat ive of 3 similar experiments. (B) Percent of apoptotic cells (Annexin V+; propidium iodide-) and dead cells (propidium iodide+); mean +/ SEM n=3. #, p <0.05 versus DC control.

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52 B DC LpDC/ DMSO LpDC/ THC A 12 20 1 18 40 1 14 40 1 Annexin V Propidium iodide

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53 Figure 10. Demonstration by RT-PCR of cannabinoid receptor, TRPV1 and -actin message in RNA from bone marrow-derived DCs. Negative RT represents PCR results when the reverse tran scriptase is left out of reaction mix. 200 bp

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54 Figure 11. Pertu ssis toxin, the Gi signaling inhibitor, attenuated the suppression effect of THC on IL-12p40. Bone marrow-derived DCs were preincubated with pertussis toxin at 0.01, 0.1, 1.0 ng/ml 18 hr prior to Lp infection and treated with THC 0, 3, 6 and 10 M. Cells were incubated for 24 hr and data represent the mean of 3 ex periments S.E.M.. *P< 0.05, compared to THC + Lp group.

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55 Figure 12. THC suppressed IL-12p 40 production in Lp-infected bone marrow-derived DCs from C57BL/6 mice. (A) THC suppresses IL-12p40 in DCs from wild type (WT) mice. (B and C) THC suppresses IL-12p40 in cannabinoid CB1 receptor knockout (CB1-/-) and CB2 receptor knockout (CB2-/-) mice. Lp-infected DCs were treated with different concentrations of THC or the highest concentration of DMSO (vehicle contro l). Cells were cultured for 24 hr. Data represent the mean of 3 experiment s S.E.M.. *P< 0.05, compared to THC 0 M.

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56 A B C

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57 Table 2. Attenuation effect of SR compounds on THC-induced suppression of IL-12p40 in Lp-infected bone marrow-derived DCs from cannabinoid receptor knockout mice. Lp-infected DCs were pretreated with SR144528 (CB1 -/DCs) or SR141716A (CB2 -/DCs) at 0.01, 0.05, or 0.1 M for 30 min prior to THC (3, 6 or 10 M). Cells were cu ltured for 24 hr and the data represent the mean of 4 experiments S.E.M.. SR al one at these doses had no effect on IL12p40 production (not shown). The percentage of attenuation is computed from 1-[IL-12p40(Lp-SR/THC/Lp)/Lp ]/[IL-12p40(Lp-THC/Lp)/Lp]. SR144528 Attenuation (%) (M) 0.01 20.0 6.0 18.2 8.8 8.4 1.5 0.05 59.5 8.7 37.0 5.7 15.8 5.0 0.1 69.6 7.0 44.7 2.5 27.9 2.0 THC (M) 3 6 10 CB1-/SR141716A Attenuati on (%) (M) 0.01 24.1 7.8 18.9 4.2 3.0 1.6 0.05 38.0 5.1 33.2 4.1 14.7 5.8 0.1 61.1 8.5 39.9 7.0 24.2 5.7 THC (M) 3 6 10 CB2-/

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58 Figure 13. Vanilloid receptor inhibito r Capsazepine did not antagonize the suppression effect of THC on IL-12p40. Lp-infected DCs we re pretreated with Capsazepine at 0.01, 0.1 and 1 M for 30 min prior to THC (3 or 10 M). Cells were cultured for 24 hr and the data r epresent the mean of 3 experiments S.E.M..

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59 Figure 14. p38 MAP kinase, but not JNK or ERK was required for IL-12p40 production in Lp-infected DCs. Lp-infected DCs were treated with ERK inhibitor UO126 (5 M), or JNK inhibitor SP600125 (5 M) or p38 inhibitor SB203580 (5 M) for 18 hr. IL-12p40 in supernatants was detected by ELISA and the data represent the m ean of 3 experiments S.E.M. *P< 0.05, compared to Lp group.

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60 Figure 15. THC modulated p38 MAP kinase activation. DCs were either uninfected or infected with Lp or treated with THC (6 M) following infection. Cells were fixed at indica ted time points and the levels of p38 kinase expression were measured by cell-based ELISA. Data are expressed as the ratio of phosphorylated p38 to total p38 in DCs. Data represent the mean of 3 experiments S.E.M.. *P< 0. 05, compared to Lp group.

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61 Figure 16. THC impaired immuniza tion potential of Lp-loaded DCs. Nave mice were iv immunized with DCs (0.5x106 cells/mouse) two to three times at 7 day intervals prior to being challenged with a lethal dose of Lp (1.7 2.0 x107/mouse). The DCs were either not loaded with Lp (DC gr oup) or loaded with Lp and treated for 24 hr with either DMSO (LpDC/DMSO group) or THC, 10 M (LpDC/THC group). Mice were monitor ed for survival and the data represent 9 mice per group from 3 experiments.

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62 Figure 17. THC treatment of Lp-loaded DCs inhibi ted immunizing potential as evidenced by increased bacterial burden. Mice were iv injected with 0.30.5x106 DCs loaded or not in vitro with Lp and treated with DMSO (LpDC/DMSO), or THC at 10 M (LpDC/ THC) for 24 hr. Then, mice were challenged 7-9 days later with a s ublethal dose of Lp (7x106 Lp/mouse), spleens isolated 24 hr post-infecti on, and colonies forming units (CFU) determined by plate counts. Data pr esented as the mean CFU +/SEM for 4 mice per group. # and (p <0.05) versus the uninfected DC control and LpDC/DMSO group, respectively.

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63 Figure 18. THC treatment of Lp-loaded DCs inhibi ted the expression of Th1 cytokines in splenocytes from immunized mice. Mice were iv injected with control DCs (0.3-0.5x106), Lp-loaded and DMSO trea ted DCs (LpDC/DMSO), and Lp-loaded and THC treated DCs (LpDC/T HC) as in Figure17. Seven to 9 days post-injection, splenocytes were harvested and stimulated in vitro with killed Lp (107/ml) for 24 hr and cytokines (IL12p40, IFN-gamma and IL-4) detected in supernatants by ELISAs (A, B and C) Data represent the mean of 5 experiments +/SEM. # and (p <0.05) versus t he control DC and Lp/DMSO group, respectively.

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64 A B C

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65 Figure 19. THC suppr ession of DC IL-12p40 produc tion mediated loss of Th1 polarization of Lp-primed CD4+ T cells. Cytokines were measured in 24 hr supernatants of co-cultures containi ng DCs and either Lp-primed or unprimed CD4 T cells. Primed T cells were obtained 5 days post-infection from the spleens of mice infected with a sublethal dose of Lp. (A) IL-12p40 measured in cocultures containing Lp-loaded DCs treat ed with DMSO or T HC (10 M) (LpDC/ DMSO or LpDC/THC) co-cultured wit h unprimed CD4 T cells. (B) IL-12p40 measured in co-cultures treated as in panel A and containing primed T cells; recombinant IL-12p40 was added in in creasing amounts. (C) IFN-gamma measured in co-cultures as in panel B and treated with IL-12p40. Data are representative of 4 experiments.

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66 A B C

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67 Figure 20. THC suppresses Th1 activation signals. Signals delivered by DCs are required for Th1 activation (2, 152). THC suppressed Th1 immune response is associated with THC im paired DC function leading to the insufficiency of (pointed by dot arrows): 1. Polarizing signals including suppre ssed IL-12p40 production and Notch ligand Delta 4 expression; 2. Antigen-specific signal through suppressed MHC class II expression; 3. Co-stimulatory signal as low expression of CD86 and CD40. Activated DC Th1 activation TLR Lp MHC Notch Costimulator IFNIL-12p40 THC THC

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68 Figure 21. Postulated signaling pathways involved in THC suppression effect on DCs. Lp infection, through TLRs and activation of NF-kappaB and p38, leads to DC activation and maturation. THC may suppress these effects via Gi/PI3K/AKT pathway and subsequent r egulatory JNK activation; and PPAR stimulation to suppress NF-kappaB trans activation. Normal arrow represents positive regulation; dot arrow repr esents negative regulation. THC CBR IL-12 MHC Co-stimulatory molecules Pertussis toxin GiPI3K/AKT JNK regulatory MAP kinase Lp TLR p38, NFB PPAR THC

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69 DISCUSSION DCs are potential targets of cannabinoids Cannabinoids, such as THC, have been shown to be immunosuppressive and anti-inflammatory (83) and can polarize adaptive immunity in mice away from Th1 immunity and toward Th2 immunity ( 89, 134). Polarization by cannabinoids has recently been reviewed (87, 158) and Th1 suppression occurs following treatment with endocannabinoi ds (112, 139), in drug-treated T cell cultures stimulated with allogenic DCs (191), following LPS and drug injection into C. parvum -primed mice (168), and in immune respons es to tumor cells (192). In at least one of these studies (168), c annabinoids were shown to suppress production of the DC cytokine, IL-12. Be cause DCs are a primary link between innate and adaptive immunity, we postulat ed that DCs might be a target of cannabinoid action and that suppressing t he function of these cells could be a key event in the drug-induced suppr ession of Th1 polarization. Intracellular bacterial pathogens can modulate the host cell intracellular meilu thus providing an environment suppor tive of intracellular growth and survival. Lp, for example, a microbe ubiquitous in the aquatic environments and the causative agent for Legionnaires’ dis ease in humans, can replicate within

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70 eukaryotic host cells such as protozoa and human phagocytic and epithelial cells (68, 131). It has been demonstr ated that T helper cell cytokines such as the type 1 cytokine IFNcan activate host cells to control Lp infection and other intracellular pathogens (25, 91, 130). In vivo experiments have shown that Lp causes severe infection in IFN-deficient mice and IFNtreatment results in increased clearance of the bacteria from the lungs (166). Suppression of immunity to Lp in mice has also been repor ted by our group following injection of THC along with a decrease in Th1 acivity and associated cytokines such as IFNand IL-12 (88). DCs are c entral in the orchestration of the formation of Th1 immunity from nave CD4+ by providing mult iple signals after pathogen priming. One set of these signals is provided by members of t he IL-12 family shown to play a pivotal polarizing role in Th1 differentiation (106, 138, 145). IL-12p40 protein, which is a subunit shared by two IL-12 family members, IL-12p70 and IL23, has been shown to be indispensabl e in Th1 development either by combining with other subunits or on its own (31, 66, 147, 184). THC suppressed IL-12p40 production in Lp-infected DCs DCs are a major cell source of IL-12. These cells can be purified from various mouse tissues such as spleen and bone marrow and studies with cultures of these cells hav e shown that a number of different bacteria induce IL12p40 (79). We speculated that cultur ed DCs would also produce IL-12 in response to Lp infection since these bacteria have been shown to increase

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71 serum levels of IL-12 in infected mice ( 89). We also wanted to study dendritic cell cultures in order to see if THC added to the cultures directly suppressed IL12 production by these cells. Initially, we observed (Figure 2) Lp infection of DC cultures triggered a high level of IL -12p40 production that was significantly inhibited by THC treatment in a conc entration-dependent manner (Figure 3). Thus, it appears that Lp like other microbes directly induces DCs to produce IL12p40 (79). Also, because DCs are a major source for serum IL-12 levels in infected animals, the current in vitro results suggest THC is suppressing DC function resulting in decreased serum IL -12 production in treated animals (89). THC did not suppress LPS-induced IL-12p40 secretion The immediate recognition of mi crobes by the innate immune system plays a crucial role in host defense (121). This recognition process is based on the detection of uni que pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors of the host such as TLRs. Activation of TLRs not only plays a critical role in activation of innate immunity but also in the activation of adaptive immune responses (120) through the production of IL-12p40 and other polarizing factors. One of the most commonly st udied TLRs is TLR4 and we found that the TLR4 agonist LPS induce d a significant amount of IL-12p40 in our DC cultures, which is consistent with previous reports (127). However, the LPS effect was not suppressed by THC as was the case with Lp infection (Figure 4). This finding is not surprising in light of recent studies showing that production

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72 of IL-12p40 after infection with Lp was in hibited in macrophages from TLR2(-/-) but not inTLR4(-/-) mice suggesting that TLR2, but not TLR4, is involved in cytokine production by Lp (5). Archer et al. also reported that TLR2 but not TLR4 is involved in MyD88-dependent responses to Lp infection in mice (7). Besides TLR2, other TLRs might be stimulated by Lp, such as TLR9, which recognizes bacterial unmethylated CpG motifs and has been shown to be involved in the IL12 response to Lp (135) and other bacteria such as Brucella abortus (72) and Mycobacterium tuberculosis (9). Thus, our data sugges ts that the mechanism of THC suppression of IL-12p40 does not involve the signaling cascades associated with all of the TLRs, for exam ple TLR4, but might be associated with others such as TLR2 and 9. THC suppressed DC maturation and polarizing molecules Besides producing polarizing cytokines such as IL-12, DCs also promote the maturation of T helper cells by the production of helper cell-surface proteins such as MHC class II and co-stimulatory mo lecules (79). To further explore the basis of THC modulation of DC functi on, we examined THC effects on the expression of these surface markers. The results showed THC treatment markedly reduced the expression of M HC class II and the co-stimulatory molecules CD86 and CD40 (Figure 6 and Tabl e 1). The mechanism for this is unclear at this time; however, signalin g through cannabinoid receptors could be involved because ligation of similar receptors (i.e., Gi-linked) has been shown to

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73 modulate DC maturation from human PBM Cs (33). In addition to the above markers, Notch ligand expression on DCs has been reported to be critical for T cell differentiation and various stimuli hav e been shown to induce the expression of either Jagged1 (Th2 polar izing) or Delta4 (Th1 polar izing) (6). Our data showed mRNAs for Jagged1 and Delta4 were induced in DCs after Lp infection; however, THC treatment significantly suppressed the expression of the Th1 polarizing Delta4 ligand but had little e ffect on Jagged1 (Figure 7). These results, coupled with our other findings t hat the THC-treated DCs are deficient in promoting Th1 polarizitation, support t he previous conclusion that the Delta ligands induce Th1 cells (6). The THC effect could involve cannabinoid receptors because G protein-coupled receptors activated through Gs have been shown to increase Jagged ligand expression and pol arize to Th2 (6); therefore, cannabinoid receptors, coupled to Gi, might be expected to suppress Delta ligands and Th1 polarization as seen in our study along with inhibiting IL-12 as shown by others (95). THC did not affect Lp survival and apoptosis in DCs To initiate T helper activation, DCs must in addition to producing cytokines and co-stimulatory molecules, take up and process the bacteria for pathogenrelated antigen presentation. The cells mu st also survive the bacterial infection and we wanted to see if antigen uptake by and survival of DC was also affected by cannabinoids. In contrast to macrophag es, wherein intracellular growth of Lp

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74 was observed (161), we observed that DCs restricted Lp growth with moderate killing over time (Figure 8). THC-tr eated DCs showed a similar restriction and time course of intracellular infecti on, suggesting Lp handling was the same in both groups. THC has been reported to induce apoptosis under certain conditions in mouse DCs (43) and Lp infection has also been shown to be apoptotic in macrophages and other cells (3 53). To see if these treatments together were adversely affecting DC survival, we examined necrosis and apoptosis induction. Our results with Anne xin V staining (Figur e 9) showed that Lp infection induced limited apoptotic activity in DCs similar to that observed in other cell types (3, 53). THC treatment did not increase the level of apoptosis and both treatments had no effect on cell necrosis as measured by propidium iodide staining (Figure 9). The minimal effect of THC on apoptosis we observed is at variance with that observed previous ly. We used the drug at 10 M and this concentration was shown previously to i nduce Annexin V positivity in 80% of the cells (43). However, the previous st udies were done using serum-free medium while ours were done with medium containing fetal calf serum which is known to reduce the potency (and toxicity) of t he added cannabinoids (90). From these results, it is concluded that THC treatment is not suppressing T helper polarization by either altering the intr acellular life cycle of Lp or by causing enhanced apoptosis and death of the DCs.

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75 The involvement of cannabinoid receptors and MAP kinases in THC effect The previous studies showed that c annabinoid treatment of DCs altered the polarizing phenotype of the cells in terms of cytokine and co-stimulatory production. To uncover the mechanism of these effects, we wanted to see if cannabinoid receptors might be involved. One previouos report showed that CB1 and CB2 mRNA and protein were expressed in human blood-derived DCs but the function of these recept ors was not studied (113) In the current study, we showed mRNA for both receptors was also expressed in mouse bone marrowderived DCs and that CB2 message appeared to be more abundant than CB1 in these cells (Figure 10). Furthermore, usi ng DCs from receptor knockout mice as well as using CB1 and CB2 antagonists, we observ ed that these receptors mediated suppression but only at the lo werTHC concentrations used in these studies (Figure 12 and Table 2). CB1 and CB2 are Gi protein-coupled receptors and activate genes by means of the heter otrimeric G protein complex containing the -GTP subunit and the dimer (19). Stimulation of cannabinoid receptors with agonists has been shown to modulate various signaling and transcription factors; for example, cAMP (164) and AP1 and MAP kinase (50) were decreased in splenocytes following treatment with cannabinoids, and drug effects were attenuated by pertussis toxin which inactivates Gi (78). In the current study, we showed that pertussis toxin treatment co mpletely attenuated the effect of low THC concentrations; however, complete attenuation could not be achieved at

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76 higher drug concentrations (Figure 11). This finding along with the partial attenuation observed with cells from receptor knockout mice treated with receptor antagonists suggested that, at the low concentration of THC, the drug is suppressing cytokine production through activation of a Gi-coupled mechanism linked to both cannabinoid receptor and non-receptor pathways. The Gi-coupled mechanism might involve the dimer of the Gi that is reported to suppress IL-12 through MAP kinases (19, 52). Many studies have shown cannabinoids modulate the activation of MAP kinases (38). These protein kinases are widely used throughout evolution in many physiol ogical processes and are involved in all aspects of immune responses, includ ing the regulation of nave Th cells differentiation into Th1 or Th2 cells (167) and the production of IL-12 either directly or indirectly through production of Th2 type cytokines (103, 179, 189). MAP kinase activation and IL-12 produc tion have been shown to be induced by G protein-coupled receptors in response to ligands such as bacterial toxins, neuropeptides and chemoattractants (19) Our data showed that p38 MAP kinase, but not Erk or JNK, was in volved in IL-12p40 production upon Lp infection (Figure 14). Furthemore, T HC treatment enhanced p38 phosphorylation initially followed by suppression with ti me (Figure 15). Recently, using human monocytes, MAP kinase phosphorylation was observed to accompany the inhibition of LPS/IFN-induced IL-12 produc tion by the mediator C5a and this was shown to involve the PI3K/Akt signalin g pathway (95). Together, these data suggest THC and other Gi ligands suppress IL-12 through MAP kinase phosphorylation.

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77 TRPV1 was not involved in THC effect Besides cannabinoid receptors, our dat a suggest that THC might also be working through other moieties (CBRX?) with affinity for THC but a relatively low sensitivity to cannabinoid receptor ant agonist. For example, it has been reported that a non-CB1 G protein-coupled receptor in mouse brain was activated by AEA and WIN55212-2 but not other cannabinoid re ceptor agonists and was relatively insensitive to antagonism with SR141716A (20). In addition, the attenuating activity of SR141716A on the antinociceptive effect of cannabinoids in mice was found to be receptor agonist-dependent (185), being most potent following CP55,940 injection and least potent following AEA injection. Vanilloid receptors, such as TRPV1, might also be involved because they respond to endocannabinoid treatment but are relatively insensitive to SR141716A (96, 180, 196). There are no previous reports of TRPV1 expression in DCs and we are the first to show in the current study that DCs readily express TRPV1 mRNA (Figure 10). Because we could find no reports t hat THC activates TRPV1 receptors on immune cells, we tested the possibi lity that TRPV1 was mediating the suppression of IL-12p40 in our system DCs, pretreated with the selective TRPV1 antagonist, capsazepine, were st ill completely suppressed by THC treatment suggesting that TRPV1 was not in volved in the drug effect on IL-12p40 production (Figure 13). The above results show that Lp infe ction of bone marrow-derived DCs

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78 leads to an increase in IL-12p40 and that co-treatment with THC significantly suppresses this effect. We al so showed that inhibition of Gi signaling completely attenuates the THC effect at low cannabi noid concentrations; however, inhibiting CB1 and CB2 at low doses only partially attenuat es the THC effect and inhibition of TRPV1 has no effect. These data s uggest that THC is working through Gi signaling in DCs to suppress IL-12p40 and is also working partially through cannabinoid receptors in addition to possibl y a third receptor with sensitivity to the effects of THC but low s ensitivity to the action of SR compounds. In addition, we found that besides THC, two endocan nabinoids, 2-AG and Virodhamine had a significant suppression effect on IL -12p40 production from Lp-DCs (Figure 5) suggesting the possible involvement of endocannabinoids in host antiinflammatory responses. Other cannabi noids, either endogenous or synthetic, however, did not seem to have signific ant effect on DC IL-12p40 production. These observations support the speculatio n that other targets besides CB1 and CB2 may exist and that other signaling mechanisms may be involved in different cannabinoid-mediated actions. THC impaired the immunization potential of Lp-loaded DCs The results so far show that T HC treatment can c hange the polarizing phenotype of DCs to one that is non-supportive of Th1 pol arization. To further examine the drug effects and to functionally test the polarizing potential of the DCs, we utilized a cellular immune reconstitu tion model of infection with Lp. DCs

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79 are potent antigen-presenting cells and loading them with microbial antigens has been shown to immunize mice against infection with various pathogens (104, 171, 181). In the current st udy, we showed that DCs loaded in vitro with Lp and injected into mice immunized and protect ed the mice to a subsequent lethal Lp infection (Figure 16). Evidence of imm unity to Lp was documented because the number of CFUs in the spleens of the animals was reduced after injection with Lp-loaded DCs (Figure 17). Thus, it appears, that bone marrow-derived DCs have good immunizing potential when loaded or infected with Lp in culture and then injected into recipient mice. The immunity generated was probably Th1 because replication of Lp in the mice is known to depend on the activity of these helper cells (134). The next question invo lved what effect THC treatment had on the immunizing potential of the loaded DCs These results showed that THC treatment attenuated the DC function as evidenced by the reduced ability to protect against infection and to reduce t he number of CFUs in the spleens of infected mice (Figure 16 and 17). THC inhibited Th1 activity induced by Lp-loaded DCs The above results suggested that THC treatment suppressed the Th1 polarizing potential of Lp-loaded DCs. To test this more di rectly, splenocytes from immunized mice were analyzed for Th1 and Th2 cytokine production in vitro in response to Lp antigens. The results in Figure 18 showed that, as expected, immunization with Lp loaded DCs (LpDC/ DMSO group) caused an increase in

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80 the Th1 polarizing cytokines IL-12p40 and IFN, probably produced by the antigen-presenting cells and lymphocytes in the splenocyte cultures; however, this effect was attenuated in splenocyt es from animals immunized with THCtreated DCs (LpDC/THC group), suggesting drug treatment suppressed their polarizing potential. The mechanism surro unding the regulation of Th1 activity is controversial. IL-4, a key cytokine in promoting Th2 cells (173), was originally proposed to also inhibit Th1 development; how ever, more recently this has been challenged and IL-4 has been shown to ac tually promote Th1 development by inducing DCs to produce IL-12 (15). Because of these uncertainties and because we had previously observed a decrease in IL-12 and increase in IL-4 production in THC-treated and Lp-infected mice (89), we examined for IL-4 production by splenocytes from imm unized mice. Figure 18 shows that immunization suppressed IL-4 production; furthermore, immunization with THCtreated cells had no effect on this suppre ssion. These findings suggest several things. First, as expect ed, immunization by Lp led to a decrease in IL-4 producing splenocytes as confirmation of Th1 polarization in response to this agent (134). Second, it appears that immunization with drug-treated and Lploaded DCs, causes a decrease in Th1 ac tivity with no concomitant increase in Th2 activity, at least as measured by IL -4 producing splenocyt es. Third, these results would appear to be at odds with our previous finding that THC injection along with Lp infection led to an increase in IL-4. However, there are a several differences in the two models and the incr ease in IL-4 observed in our previous study (89) occurred within hours after infection and was transient in nature

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81 whereas, in the current study, IL-4 was measured in splenocytes taken 7 9 days following immunization. Also, in the pr evious study, THC was injected into Lpinfected mice, whereas in the current model, mice were immunized with THCtreated and Lp-loaded DCs. It is perhaps no t surprising that IL-4 production is regulated differently under these varying conditions of THC administration; furthermore, the studies suggest that Lp immunization under these conditions results in primarily a Th1 response and t hat the suppression of this response by THC is mediated by mechanisms independent of IL-4 production. THC suppression of DC IL-12p40 pr oduction mediated loss of Th1 polarization The previous studies suggested that THC treatment of DCs suppressed their Th1 polarizing function and we want ed to test this directly using an in vitro co-cultivation paradigm. Because IL12 is potent in directing Th1 cell differentiation (109) and because we found t hat THC suppressed this cytokine in Lp-infected DC cultures, we evaluated the Th1 promoting potential (as measured by IFNproduction) of both Lp-loaded DCs and drug-treated DCs in co-culture with Lp-primed CD4 T cells as well as the role of IL-12p40 in the response. The data showed that co-culturing Lp-loade d DCs with Lp-primed T cells led to enhanced IL-12p40 and IFNproduction compared to co -culture with unprimed T cells, and that THC treatment of the DCs attenuated the production of polarizing cytokines (Figure 19). Fu rthermore, the addition of exogenous IL-

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82 12p40 to the THC-treated cultures rest ored the robust production of both IL12p40 and IFN, suggesting that drug suppression of the p40 protein was responsible for inhibiting Th1 polarizat ion in the cultures. IL-12p70 and IL-23 have also been shown to polarize toward Th1 (75), but drug effects on these cytokines were not involved because t hey could not be detected in the supernatants (data not shown). In addi tion, IL-10 has been shown to suppress Th1 polarization under various conditions (1 87), but again it was not detected in the supernatants so was probably not involv ed in the drug effect. These results show that IL-12p40 can be a major T h1 polarizing protein and that its suppression by THC is a key factor in the drug-induced inhibition of Th1 cell development. In this regard, severa l other reports have shown that the p40 protein has Th1 polarizing potential and affini ty for IL-12 receptor s (22, 31, 66). In conclusion, our results show that a major cellular target of THC-induced immune suppression of Th1 immunity is the dendritic cell and that the drug attenuates polarizing function by s uppressing IL-12p40 production and the expression of MHC class II co-stimulatory molecules and Notch ligand Delta4 (Figure 20). Although, THC might compromi se the host’s ability to fight infection it also might be of use in the treatment of chronic inflammatory diseases such as coeliac disease and Crohn's disease (63, 153), rheumatoid arthritis (177, 182) and systemic lupus (62). The data repor ted here suggest that THC and other cannabinoids may belong to this gr oup of anti-inflammatory drugs.

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83 SUMMARY The legalization of marijuana has been debated for years because many people consider it a relatively beni gn substance with possible beneficial medicinal properties. Indeed, it is not as addictive as are other drugs such as heroin, cocaine or nicotine (122). Howe ver, in the aspect of health impact, the potential risks and benefits of marijuana use remain to be further explored. The current study investigated the role of cannabinoids in the immune system and found multiple effects of THC on t he cellular immune function of DCs. Based on the data presented in this study, it appears that during Lp infection, immature DCs are capable of capturing Lp and its associated antigen. This leads to maturation and activation of DCs with up-regulation of MHC class II and other costimulatory molecules and the production of proinflammatory cytokines. Secretion of IL-12p40 by DCs pl ays a critical role in activation and differentiation of Th1 cells which are required for control and elimination of infection; activation of macrophages and B cells and the production of pathogen specific neutralizing antibodies such as IgG2a, which also contribute to host resistance and clearing of the infecti on. THC, however, suppresses IL-12p40 production in DCs after Lp infection and down-regulates DC MHC class II and several costimulatory molecules includ ing CD86, CD40 and Notch ligand, leading

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84 to the depressed Th1 development and immunosuppression against Lp challenge. Current data suggest the involvement of Gi signaling a nd cannabinoid receptors in THC suppression of IL-12p40 and probably also MHC (67) and costimulatory molecules. The mechani sm may involve the activation of phosphatidylinositol 3-kinas e-protein 3 kinase B/Akt pathway and its downstream MAP kinases activation. It was recently reported that inhibition of IL-12 by ligands for Gi-protein-coupled receptors was mediated by the activation of PI3K/Akt signaling and the MAP kinase JNK (95). Gi receptors for THC may also stimulate Akt and JNK and consequently suppress the activation of NF-kappaB and p38 required for DC activation and matura tion (190) (Figure 21). Our results also suggest the existence of mechanisms other than Gi signaling. One possible candidate is PPAR, which belongs to the nuclear receptor superfamily and can be activated by cannabinoid ligands such as 2-AG (154) and AJA (24). Recent studies showed that activation of PPAR or PPAR may help to control inflammatory responses through inhibiti ng NF-kappaB transactivation, which is critical for DC immunostimulatory func tions (141, 156, 190) (Figure 21). Due to their immunomodulation effect s, cannabinoids have been recently used in the treatment of several chronic inflammatory disorder s. For example, diabetes is associated with the aut oimmune destruction of pancreatic cells and insulin deficiency. Studies show that T HC attenuates the severity of disease in an animal model and is associated with the suppression of IFN, TNFand IL12 mRNA expression in pancreatic tissues from mice (99). More recently,

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85 Steffens et al. reported that oral administration of T HC significantly inhibited the progression of atherosclerosis in a murine model, suggesting a beneficial effect of cannabinoids in this chronic inflammato ry disease of the vessels (157, 170). Moreover, in a model of multiple scl erosis, an autoimmune inflammatory disease of the CNS, the cannabinoid, Win55,212, showed suppression of disease associated with the suppression of T cell proliferation and IFNsecretion and other pro-inflammatory, Th1-ty pe cytokines, such as IL-1 and TNF(35). All of these studies suggest the potential t herapeutic uses of cannabinoids. Our studies reported here addre ss possible cellular mechanisms of cannabinoidinduced suppression of Th1 immunity. By in vestigating the effect of cannabinoids on DCs controlling antigen-driven T-cell polarization, our results not only serve to expand our understanding of cannabinoid effe cts in the immune system, but also provide clues to the cellular mechanism involved and therefore offer more specific targets for future drug devel opment in the treatment of chronic inflammatory diseases.

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ABOUT THE AUTHOR Tangying (Lily) Lu was born and rais ed in Southern Chin a. She earned her bachelor’s degree in Medi cine from Fujian Medical University in 1998 and became a pathological surgeon at the First Affiliated Hosp ital of Fujian Medical University. She entered the Doctor of Philosophy program in the department of Medical Microbiology and Immunology/Molecul ar Medicine at USF in 2001. Lily has conducted research with her mentor Dr. Thomas Klein since 2001. The data in this research were published in European Journal of Pharmacology and Journal of Pharmacology and Ex perimental Therapeutics in 2006.