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The Role Of Curcumin In Human D endritic Cell Maturation And Function by Shawna A. Shirley, M.S. 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 Co-Major Professor: Shyam S. Mohapatra, Ph.D. Co-Major Professor: Richard Heller, Ph.D. Thomas Klein, Ph.D. Mark Glaum, M.D., Ph.D Date of Approval: October 02, 2008 Keywords: immunology, turmeric, cyt oskeleton, inflammation, immune suppression Copyright 2008, Shawna A. Shirley
DEDICATION This dissertation is dedicated to my family, my parents Byron and Lowalean, my sister Leisle and my husband Karl, for their sacrifice and unconditional support. Their confidenc e in me and their encouragement motivated me to persevere.
i TABLE OF CONTENTS LIST OF FIGURES v LIST OF TABLES vii LIST OF ABBREVIATIONS viii ABSTRACT xii PREFACE xiv INTRODUCTION 1 A Natural History of Curcumin 2 Curcumin Classification and Stru cture 3 Molecular Targets and Receptors of Curcumin 3 Anti-inflammatory Activity of Cu rcumin 5 Pharmacology and Pharmacokinetics of Curcumin 6 Curcumin in the Clinic 8 Dendritic Cell Biology 8 The Actin Cytoskeleton 11 Curcumin and the Actin Cytoskeleton 12 Curcumin and Dendritic Cells 13 Significance of the Study 14 GOALS AND OBJECTIVES 16
ii Purpose 16 Hypothesis 16 Specific Aims 17 Aim 1: Curcumin Prevents Dendrit ic Cell Maturation 17 Aim2: Curcumin Prevents Dendritic Cell Function 18 Aim 3: Curcumin Modulates the Ac tin Cytoskeleton 19 MATERIALS AND METHODS 20 Materials 20 Monocyte Isolation and Cultur e 21 T Cell Isolation and Culture 24 Curcumin Treatment and Cell St imulation 24 Cell Viability by Flow Cyto metry 24 Surface Marker Analysis by Flow Cytometry 25 Multiplex Bead Assay 26 Homotypic Clustering 27 Western Blotting 27 Endocytosis Assay 28 Chemotaxis Assay 28 Mixed Lymphocyte Reaction 29 Immunofluorescence Labeling 30 Microscopy 31 RNA Extraction from Human DC s 31 Microarray 31
iii Statistical Data Analysis 33 RESULTS 35 Aim 1: Curcumin Prevents Dendritic Cell Maturation 35 Curcumin Affects DC Morphology 35 Curcumin Prevents Homotypic Clu ster Formation 36 Curcumin Reduces Surface Marker Expression 38 Curcumin Reduces Cytokine Pr oduction 44 Aim 2: Curcumin Prevents Dendritic Cell Function 49 Curcumin Reduces Endocytosis 49 Curcumin Reduces Chemokine Se cretion 51 Curcumin Prevents DC Chemot axis 53 Curcumin Reduces DC-induced T Ce ll Proliferation 56 Curcumin Induces a CD4+CD25+ T Regulatory Cell Population 59 Aim 3: Curcumin Modulates the Acti n Cytoskeleton 63 Microarray Analysis of To tal RNA Reveals the Effects of Curcumin on Gene Expression in Dendritic Cells 63 Curcumin Alters the Actin Cyto skeleton 66 Curcumin-Induced Reduction in Endocytosis May be Due to Actin Inhibi tion 70 DISCUSSION 71 CONCLUSION 79 LIMITATIONS OF THE STUDY 80
iv FUTURE DIRECTIONS 81 LIST OF REFERENCES 82 APPENDICES 100 Appendix A: List of Definitions 101 Appendix B: List of Publications by the Author 107 Appendix C: First Author Pub lication 111 ABOUT THE AUTHOR End Page
v LIST OF FIGURES Figure 1 Structure of Curcumin 4 Figure 2 Aim 1: Experimental Ou tline 17 Figure 3 Aim 2: Experimental Ou tline 18 Figure 4 Aim 3: Experimental Ou tline 19 Figure 5 Dendritic Cell Morphology Gating 22 Figure 6 Surface Marker Expr ession on Cultured Immature and Mature DCs 23 Figure 7 Cell Viability 25 Figure 8 Experimental Outline for Micr oarray Analysis 33 Figure 9 Curcumin Affects Cell Mo rphology 36 Figure 10 Curcumin Prevents Homoty pic Clustering 37 Figure 11 Curcumin Reduces Surface Ma rker Expression 40 Figure 12 Curcumin Reduces Surface Marker Fluorescence Intensity 41 Figure 13 Curcumin Reduces the Expression of Antigen Presentation Molecules 43 Figure 14 Curcumin Reduces Cyto kine Production 46 Figure 15 Curcumin Reduces Endocytosis 50
vi Figure 16 Curcumin Reduces Chem okine Production 52 Figure 17 Curcumin Prevents Chemotaxis 53 Figure 18 Curcumin Does Not Affect CCR7 Expression 55 Figure 19 Curcumin Reduces DC-Induced Proliferation of Allogeneic CD4+ T Cells (Representative Donor) 57 Figure 20 Curcumin Reduces DC-Induced Proliferation of Allogeneic CD4+ T Cells (Averaged) 58 Figure 21 Curcumin Induces Regulatory T Cells in Allogeneic ML R (Average) 59 Figure 22 Curcumin Induces Regulatory T Cells in Allogene ic MLR 60 Figure 23 Curcumin-DCs Reduce T Helper Cytokine Production After Allo geneic MLR 61 Figure 24 Curcumin Interferes With Ce ll Attachment and Mot ility 67 Figure 25 Curcumin Changes Ce ll Morphology 68 Figure 26 Curcumin Reduces Cell Adhesion 69 Figure 27 Curcumin Reduces the Activity of Rac1/Cdc42 GTPases 69 Figure 28 Curcumin-Induced Disrupt ion of the Actin Cytoskeleton Results in Reduced Endocytos is in Immature Cells 70
vii LIST OF TABLES Table 1 Age and Gender of Donors 21 Table 2 Significance: Effect of Curcumin on Surface Marker Expression 42 Table 3 Significance: Effect of Curcumin on Cytokine Production 47 Table 4 Significance: Effect of Stimulants on Cytokine Producti on 48 Table 5 Significance: Effects of Curc umin on Endocytosis 51 Table 6 Significance: Effects of Stimul ants on Endocytosis 51 Table 7 Significance: Effects of Curcumin on Chemokine Producti on 52 Table 8 Significance: Effects of Curcum in on Chemotaxis 54 Table 9 Significance: Effects of Curcumin on DC-Induced Allogeneic CD4+ T Cell Proliferation 58 Table 10 Significance: Effects of Stimulated DCs on Allogeneic CD4+ T Cell Proliferat ion 58 Table 11 Significance: Effects of Curcumin-DCs on T Helper Cytokine Production afte r Allogeneic MLR 62 Table 12 Pathways Affected by Curcumin 64 Table 13 Actin Cytoskeleton Genes Modul ated by Curcumin 65
viii LIST OF ABBREVIATIONS 5-LOX: 5-lipoxygenase 7-AAD: 7-amino-actinomycin M: micro molar AP-1: activator protein 1 APC: allophycocyanin Arp: actin related protein BCA: bicinchoninic acid BSA: bovine serum albumin CCL19: chemokine (c-c motif) ligand 19 (MIP-3 ) CCL21: chemokine (c-c motif) ligand 21 (Exodus-2) CCR7: chemokine (c-c motif) receptor 7 CFSE: carboxyfluorosce in succinimidyl ester CD: cluster of differentiation Cdc42: cell division cycle 42 cDNA: complementary deoxyribonucleic acid COX-2: cyclooxygenase 2 CX3CL1: chemokine (c-x3-c motif) ligand 1 (fractalkine) CytoB: cytochalasin B DAPI: 4',6-diamidino-2-phenylindole
ix DC: dendritic cell DMSO: dimethyl sulfoxide Exodus-2: chemokine (c-c motif) ligand 21 (CCL21) FITC: fluorescein isothiocyanate Foxp3: forkhead box p3 GAPDH: glyceradehy de-3-phosphate dehydorgenase GDP: guanosine diphosphate GM-CSF: granulocyte macrophage co lony stimulating factor GTPase: guanosine triphosphate hydrolase enzyme HLA-DR: human leukocyte derived antigen HRP: horseradish peroxidase HTS: high throughput system ICAM1: intracellular adhes ion molecule 1 (CD54) iDC: immature dendritic cell IFN : interferon gamma IP-10: interferon-i nducing protein 10 (CXCL10) IPA: ingenuity pathways analysis IL: interleukin LITAF: lipopolysaccharide-induced tumor necrosis factor Log: logarithm base 10 LPS: lipolysaccharide g: microgram MFI: mean fluorescence intensity
x MIP-3 : chemokine (c-c motif) ligand 19 (CCL19) MHC: major histocompatibility complex ml: milliliter MLR: mixed lymphocyte reaction mdDC: monocyte-deriv ed dendritic cells NF B: nuclear factor kappa B ng: nano gram NSAID: non-steroidal anti-inflammatory drug PAK: p21 activated kinase PBMC: peripheral bl ood mononuclear cells PBS: phosphate buffered saline PE: phycoerythrin PFA: paraformaldehyde pg: pico gram PHA: phytohemmaglutinin Poly I:C: polyinosinic-polycytidylic acid Rho: Ras homolog family member RNA: ribonucleic acid ROS: reactive oxygen species SDS-PAGE: sodium dodecyl sulfate poly acrylamide gel electrophoresis SEM: standard error of the mean STAT: signal transducer and activator TNF: tumor necrosis factor alpha
xi WASP: Wiskott-Aldrich syndrome protein
xii THE ROLE OF CURCUMIN IN HUMAN DENDNRITIC CELL MATURATION AND FUNCTION Shawna A. Shirley, M.S. ABSTRACT Curcumin is the yellow pigment found in the Indian spice curry. It has antiinflammatory, ant-oxidant, anti-cancer anti-viral, anti-bacterial and wound healing properties. It is widely used in i ndustry for its flavor as a spice and as a coloring agent because of its brilliant yellow color. It is also used as a dye for textiles and as an additive to cosmetics. D endritic cells (DCs) ar e the sentinels of the immune system and functions as the bridge between the innate and adaptive immune response. The effect of curcum in on DCs is poorly understood. A study shows curcumin prevents the immunostimulatory function of bone marrowderived murine DCs, but no study exami nes the effects on human DCs. This study investigates the effects of cu rcumin on immature human DC maturation and function in response to immune st imulants lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly I:C). Human CD14+ monocytes isolated fr om the peripheral blood of donors are cultured with GM-CSF and IL-4 suppl emented media to generate immature DCs. The cultures are treated with curcumin, stimulated with the above
xiii mentioned stimulants then f unctional assays performed. These assays include homotypic cluster formation, surface marker expression, cytokine production, chemotaxis, endocytosis, DC-induced allo geneic CD4+ T cell proliferation after mixed lymphocyte reaction, gene expre ssion analysis and immuno-fluorescence labeling and imaging. Curcumin-induced ch anges in gene expression indicate the actin cytoskeleton signaling pathway is a target. Immuno-fluorescence labeling and imaging of f-actin was carried out. Curcumin reduces DC maturation in response to the stimulants used in the study. Expression of su rface markers, cytokines an d chemokines is reduced as well as DC-induced stimul ation of allogeneic CD4+ ce lls after MLR. Curcumin prevents chemotaxis without affecting chemokine receptor expression and significantly reduces endocytosis in non-st imulated cells. Curcumin-treated DCs do not induce a Th1 or Th2 populati on in allogeneic MLR but induces a CD25+Foxp3+ regulatory ce ll population. Immuno-fluor escence imaging shows curcumin causes the cell to become more rounded. These data imply that curcumin inhibits f-actin polymerizati on and thereby prevents DC maturation and function in response to stimulation. This outlines a novel role for curcumin as an immune suppressant and show s its therapeutic potential as an anti-inflammatory agent.
xiv PREFACE I would first like to acknowledge T he Creator; through him all things are possible. I would like to t hank my major professor, Dr. Shyam S. Mohapatra for his continued guidance, support and encouragement and for giving me the wonderful opportunity to explore the bas ic sciences. Thanks to my co-major professor, Dr. Richard Heller, for is adv ice, encouragement and motivating spirit. When I needed someone to give me another perspective, his door was always open. To my committee members, Dr. Thom as Klein and Dr. Mark Glaum, thanks for your invaluable advice and support th roughout the process. I would like to extend a special thank you to Dr. Richard F. Lockey. His faith in my abilities has meant a lot to me. I acknowledge the Joy McCann Culverhouse endowment to the University of South Florida as we ll as the Mabel and Ellsworth Simmons professorship to Dr. S.S. Mohapatra for funding the research. Thanks to wonderful researchers and staff at the Joy McCann Culverhouse Center for Airway Disease and Nanomedicine. You were always happy to help me and answer my questions. Special thanks to Bobby, Weidong, Sonya, P.K. and Sandyha. I would like to extend special thanks to Homero San-Juan Vergara for his friendship, support and advice.
xv I would like to thank Dr. Maureen Groer at the USF College of Nursing for her encouragement and for allowing me to use her lab and equipment to complete some of my expe riments. Thanks to Karoly Szekeres (Charlie) for his friendship and for helping me with all my fl ow cytometry experiments. I would like to acknowledge the Analytic microscopy co re and the Microarray core facilities at the H. Lee Moffitt Cancer Cent er and Research Institute. I would like to acknowledge Dr. Alison Montpetit, my lab partner and friend. Her quiet determination and encouragement made many late night experiments bearable. I also acknowledge my friends Karen Corbin and Thomas Lendrihas, fellow grad students, who were always there to listen and offer support when things were not going as pl anned. Thank you to my parents Byron and Lowalean, who always believed in me. Their unconditional love, support and patience has been invaluable. I also acknow ledge my sister Leisle, my family and friends for all their support, prayers and wo rds of inspiration. To my wonderful husband, Karl Gilman, my rock and my confidant, your love and support has inspired me.
1 INTRODUCTION Turmeric is a bright yellow spice deriv ed from the root of the perennial Curcumin longa a member of the ginger family. Curcumin, also called diferuloylmethane, is the most biologic ally active compound turmeric and belongs to a family of compounds called curcumi noids which usually comprises about 3% of turmeric powder. The spice is widely us ed for its culinary flavor as it gives curry its characteristic taste. Its use also extends to me dicine. For centuries it has been used in Asian cultures in Ayurv edic systems of medicine most likely because of its properties as an antisept ic, analgesic, appetite suppressant, antiinflammatory agent, antioxid ant, anti-malarial and insect repellant (3, 49). In industry it is commonly used as a food pr eservative, a yellow dye or coloring for food and textiles and as an ingredient in pharmaceuticals and cosmetics. Research shows curcumin has anti-inflammatory, antioxidant, antiparasitic, anti-viral and ant i-cancer properties (12, 74, 88, 91). It targets transcription factors, cytokines, cell adh esion molecules, su rface receptors, growth factors and kinases, among other mole cules (9, 72, 78, 152), and directly binds to a variety of surface and intrace llular proteins causing direct cellular pathway inhibition or activation of secondary cellular responses (3, 48).
2 A Natural History of Curcumin Curcumin was first isolated from tu rmeric in 1815 and its 1910 its chemical structure was determined as C21H20O6 by Vogel and Pellatier (145). The history of curcumin however, dates back over 5000 years to the time of Ayurveda. Ayurveda, or the science of good life, is an ancient system of healthcare practiced in India. In this system of medicine curcumin was used for wound healing, blood cleansing and to cure stom ach illnesses. Also known as Haldi, curcumin has been used for centuries wit hout known side effects as a food preservative or additive for coloring and flav or. It can be ingested to treat a host of internal ailments or mixed into a pas te and applied topically to treat wounds, bruises, boils pains, sprains, swellings and other disorders of the skin (61). Curcumin is a valuable export crop and is wi dely cultivated by Asian countries. In addition to its culinary and medicinal uses, it is also commonly used in industry as a food preservative, a yellow dye or coloring for food and textiles and as an ingredient in pharmaceuticals and co smetics (128). In Hindu religious ceremonies it is mixed with sandalwood powder and applied to the forehead (61). The earliest entry about curcumin in PubMed is from 1949, a study published in Nature explored the antibacteri al action of curcumin (115). To date there are 2438 articles in P ubMed related to curcumin.
3 Curcumin Classification and Structure Taxonomically, turmeric belongs to Class Liliopsida; Subclass Commelinids; Order Zingiberales; Family Zingiberaceae; Genus Curcuma ; Species Curcuma longa (ref). It is a hydrophobic, polyphenolic compound that is soluble in ethanol, dimethyl sulfoxide, acetone, chloroform and oils. Curcumin exists in both keto and enol forms, but the ke to form is more energetically stable (Figure 1). Its absorption maxima is ar ound 420 nm and therefore it fluoresces at the wavelength of FITC. Mo st commercially available preparations of curcumin contain other compounds such as its analogs demethoxycurcumin and bisdemethoxycurcumin (3). Though curcumin is thought to be the most potent of the three, it is unclear whether the other analogs have similar activity (4, 127). It is suggested that the combination of all three is more potent than each individually (58). Molecular Targets and Receptors of Curcumin The lipophilic property of curcumin allows it to rapidly permeate cell membranes and enter the cytoplasm (62). Th ere are proteins to which curcumin binds and initiates secondary cellular re sponses. These include, among others, 5-LOX, serum albumin, iron, IKK, PKC, PKA, GST and autophosphorylationactivated protein kinase (3). Due to its size and chemical composition, it is possible that curcumin enters cells by pa ssive diffusion though the lipid bi-layer. Its anti-inflammatory properties are ascri bed to its ability to inhibit transcription factors such as NF B, AP-1 and STATs, its ability to reduce COX-2 and 5-LOX
4 expression and its ability to reduce the expression of surface adhesion molecules. Its role as a potent antioxi dant may also contribute to its antiinflammatory actions (3, 15, 118, 143). An tioxidants are compounds that delay or arrest disease progression. Curcumin func tions as an antioxidant by inducing the expression of reactive oxygen specie s (ROS) and binding iron (39, 155). The structure of curcumin is rich in hydr oxyl and methoxyl groups. There is some debate over whether it is the methylen ic group at center or the phenol groups that contribute the hydrogen atom that c onfer its antioxidant property (16, 65, 107). Figure1. Structure of curcumin (diferuloylmethane) in both keto and enol forms (reprinted from Wikipedia) Keto Enol
5 Anti-inflammatory Acti vity of Curcumin The anti-inflammatory property of cu rcumin is thought to be responsible for many of the activities associated with curcumin. It has been suggested that this property is directly related to the structure of the molecule, though there is debate over which portion is responsible; the diene bonds in t he center or the phenol groups at either end (12, 27, 28, 70). Another notable property of curcumin related to its role as an anti-infl ammatory agent is its antitumor activity. Curcumin can alter multiple signaling pat hways by interacting with a number of molecular targets (3, 10) and function as a chemopreventative agent. It suppresses multiple forms of cancer includi ng cancers of the breast, colon, liver, oral cavity and prostate. Curcumin inhibits proliferation of an array of tumor cell types in vitro (2) and prevents metastasis in an in vitro model of mouse melanoma (93). It interferes with angiogenesis by inhibiting fibroblast gr owth factor (FGF)-induced neovascularization and inhibits vascula r endothelial growth factor (VEGF) expression (13, 52, 94). Adhesion molecu le expression is important for tumor metastasis. Curcumin may mediate its ant i-tumor effects partially by reducing adhesion molecule expression (19, 138). Curcumin also modulates matrix metalloproteinases (MMPs) that r egulate endothelial cell migration and attachment (82). Curcumin affects mu ltiple signaling pathways including key pathways regulated by transcription factors NFB, AP-1, Akt and Nrf2 (2, 54). These pathways control the production of cytokines and other inflammatory
6 mediators, cell prol iferation and apoptosis. Modulat ion of these pathways by curcumin has a direct impact on tumor progression and survival. Pharmacology and Pharmacokinetics of Curcumin Turmeric has been classified by The Food and Drug Administration among substances Generally Recognized as Safe (GRAS). It has been shown to be safe at high doses in humans, rats, mice m onkeys and guinea pigs (26, 116, 117). It has also been tested for mutagenicity us ing the Ames test and has been shown to be nonmutagenic (97). Curcumin has been safely used as a dietary spice for centuries without adverse effects. An esti mated 200mg of curcumin is ingested daily by Indian adults (29, 49). The only adv erse effects noted in the literature are rare cases of allergic contact dermatitis (46, 53). No adverse drug interactions have been reported (49). Turmeric contains turmerin, essent ial oils (turmerones, altanones and zingiberene), curcumin, flavanoids, resins, proteins and sugars. Curcumin is the most biologically active compound found in turmeric and comprises about 2 to 8 percent of turmeric preparations (137). It is estimated that about 40-85% of curcumin remains unaltered after ingestion in the gastrointestinal tract where it is absorbed by the intestinal mucosa (109, 146) The oral toxicity of curcumin is low. Human clinical trials indicate no to xicity at doses as high as 12g/day (11). The oral bioavailability of curcumin is al so very low due to its rapid metabolism in the intestinal mucosa and liver. Humans given about 3g/day had undetectable or very low serum levels (119, 122). One study reports a patient with a serum
7 concentration of 58ng/ml two hours and 51ng/ml four hours after receiving 12g of curcumin. Another patient had serum levels of 51ng/ml four hours after receiving a 10g dosage (79). The degradation kinetics and stability of curcumin in physiological conditions vary. Degradation is pH dependent. At neutral or basic pH, the degradation is rapid, while more acidic conditions promote slower degradation. In 0.1M phosphate buffer and serum free medi a curcumin is degraded by about 90% over 30 minutes. In culture medium c ontaining 10% fetal calf serum and in human blood only 20% of curcumin is degraded after 1 hour. After 8 hours 50% still remained (148). Byproducts of curcumin metabolism differ based on the route of delivery; when given oral ly, curcumin sulfonate and curcumin glucuronide are produced but when giv en intraperitoneally or systemically tetrahydrocurcumin is produced. It is unc lear whether these metabolites are biologically active, though tetrahydrocurcu min is active in some systems (103, 104, 130) but not others (60, 98). The rapid metabolism and poor bioavailabi lity of curcumin impedes its use as an orally delivered dr ug. A study by Shoba et al showed that combining piperine, a known inhibi tor of hepatic and intestinal glucuronidation, with curcumin significantly increases its oral bioavailability in humans and rats (122). A more recent approach is to use polym eric nanoparticle-encapsulated curcumin or nanocurcumin as a novel formula tion to deliver curcumin (20).
8 Curcumin in the Clinic The ability of curcumin to modulate multiple molecular targets, coupled with its pharmacological safety and low cost make it attractive for clinical research. There are currently about twentyfive clinical trials examining the therapeutic potential and efficacy of curcumin (www.clinicaltrials.gov). These are outlined in a few review articles (10, 45, 54). Initial results are positive in some subsets of patients when cu rcumin is used to treat cancer and inflammatory conditions including idiopathic infla mmatory orbital pseudo tumors, postoperative inflammation, external cancer ous lesions and pancreatic cancer (33, 75-77, 112). Other disease targets being considered include psoriasis and Alzheimers disease. Dendritic Cell Biology Dendritic cells are the sentinels of the immune syst em and regulate the immune response. They are widely dist ributed throughout the body and exist in two functionally distinct states; immatu re and mature. Immature or resting dendritic cells reside in per ipheral organs where they monitor the surrounding tissue for invading microorganisms. T hey alert the immune system to the presence of pathogens by engulfing them, processing the foreign proteins and presenting the peptide fragments on their surface. Afte r DCs are activated, they mature and migrate to the lymphoid tissue where they prime nave T lymphocytes and stimulate a specific or adaptive response (50, 92, 129). Immature DCs express rela tively low levels of co-stimulatory and antigen
9 presenting molecules but have a high end ocytic capacity while mature DCs express higher levels of these ma rkers and a reduced en docytic capacity. Maturation of dendritic cells involves c hanges in gene expression, activation of signaling pathways and substantial cytopl asmic reorganization. mDCs extend long dendritic processes that increase t he cell surface area that enhances the opportunity for T cell interaction (92). The changes that occur in the DC during the maturation process are regulated by actin assembly and disassembly mediated by the Rho family of GTPases which include Rho, Rac and Cdc42 (24, 43, 100, 124, 133, 151). Maturation of DCs is a key step in t he initiation of immunity. Decreased endocytosis, increased migration and t he increased ability to stimulate proliferation and differentia tion of T cells are characteristics of mature DCs. Another characteristic feat ure of mature DCs is thei r ability to form cellular aggregates or homotypic clusters (37) Cluster formation has been observed in vivo with cutaneous Langerhans cells as well as in vitro (31, 84, 150). It has been shown that cluster formation is not an accidental encounter between migrating cells, but rather has a physi ological function to enhance maturation. Clustering results in increased CD86, CD80 and CD54 marker expression, and a modest increase in the ability to stimul ate T cells in a syngeneic MLR (31). The authors also suggest clustering facilitates antigen transfer between maturing DCs. Immature DCs are quite di fferent from mature cells. They can take up particles, antigen and microorganisms by phagocytosis and they express
10 receptors that mediate endocytosis (64, 132). Once iDCs have captured antigens or particles, their ability to capture mo re quickly decreases. The antigens enter the endocytic pathway where they are processed and presented on the surface of the cell in the context of MHCII molecules (25, 106). Primed DCs will migrate to secondar y lymphoid organs and present the antigen-peptide-MHC comple xes to nave CD4+ T cells and cytotoxic CD8+ T cells which induce differentiate into memory and effector cells. The mixed lymphocyte reaction (MLR) can be used as an in vitro model of DC-TC interaction. DCs will stimulat e proliferation of the T cells in co-culture and drive a specific phenotype based on the cyt okine environment and the type and activation state of the DCs (32, 135). The ratio of DCs to TCs will influence the phenotype of T cells produced. A low ratio induces a Th2 population, while a high ratio induced mixed Th1/Th2 cell development (135). DC migration is regulated by chem okine and chemokine receptor interactions with the aid of accessory proteins (38). The chemokine receptor CCR7 plays and important role in DC migr ation. Mature DCs upregulate CCR7 expression to improve their responsiv eness to its ligands CCL21 and CCL19. CCL21 is important in guiding the matu ring DCs to the lymphatic vessels and CCL19 guides cells to the T-cell zones of the lymphoid tissues (30, 35, 36, 51, 85).
11 The Actin Cytoskeleton The actin cytoskeleton provides the scaffolding of the cell that helps to maintain its shape. Most dendritic cell f unctions are controlled by cytoskeletal rearrangement. Individual units of actin, globular actin (g-a ctin), assemble in long polymer filaments to from filamentous actin (f-actin). Two parallel strands of factin twist around each other to form the microfilaments of the cytoskeleton. Antigen capture, antigen pr esentation, cell adherence and cell migration is regulated by the Rho family of GTPa ses which regulate actin cytoskeleton organization (14, 43, 95, 100). The engagement of T cells by DCs is also dependent on cytoskeletal rearrangement fo r formation of the immunological synapse (5, 6). The Rho family of GT Pases belongs to the larger Ras superfamily of GTP-binding proteins. T he Rho subfamily consist of more than twenty distinct proteins including RhoA, RhoB, RhoC RhoD, RhoE, Rac1, Rac2 and Cdc42 (124). The Rho GTPases func tion by cycling between the active GTP-bound state and the inactive GD P-bound state (22). Regulation of endocytosis is in part due to the levels of activated Cdc42 (43). Cdc42, Rac and Rho are involved in antigen presentation in DCs as well as motility, adhesion and chemotaxis (7, 8, 124). Regulation of the DC cytoskeleton is largely developmentally regulated (24). The Wiskott-Aldrich syndrome protein (W ASP) is the specific effector of Cdc42 (141). It is expressed mainly in hematopoietic cells and its functions as a signal transducer to the actin cytoskelet on (140). WASP is also important in filapodia formation, adhesion marker expr ession and DC chemotaxis (7, 8, 140).
12 WASP binds the actin related protein (Arp ) 2/3 complex. Together they regulate the actin cytoskeleton by nucleating the actin filament assembly to create a branching network at podosomes that govern the direct ional movement of DCs (21, 96, 154). Nexilin is an f-actin binding protein localized at the ce ll-matrix adherens junction that was first described in 1998 in rat brain and fibroblasts by Ohtsuka et al (102). Nelin (nexilin-lik e protein) is the hum an homolog of nexilin found primarily in the heart, skeletal muscle, artery and vein. Based on structural analysis, it can regulate the formation of stress fibers and focal adhesions (156). In HeLa cells it stimulates migration and adhesion and so mediates cell motility (147). The role nelin plays in dendritic cell function is unknown. Curcumin and the Actin Cytoskeleton Little is know about how curcumin affects cytoskeleton of the cell. A few studies have examined the effects of cu rcumin on the actin cytoskeleton in neurons, hepatic cells and cancer cells but none have outlined these effects in dendritic cells. In an in vitro study using prostate cancer cells, curcumin shows profound effects on actin-based motility and microfilament organization (56). The actin inhibitor chytochalasin B was used in this study as a control. Curcumin shows similar inhibitory effects. Cyclindependent kinase 1A (p21) functions as a regulator of cell cycle progression at the S phase. p21-activated kinases (PAKs) also participate in the r egulation actin filaments al ong with the Rho GTPases. Curcumin suppresses PAK translocation in aged Tg2576 transgenic mice with
13 Alzheimer amyloid pathology (86). Although this was an Alzheimers study and curcumin was not the focus, it demonstr ates the role curcumin plays in the regulation of actin organi zation. In another study, curcumin affected the formation of actin stress fibers which helps to suppress the intra-hepatic metastasis in an orthotopi c implantation model (101). Curcumin and Dendritic Cells Outside of the work published from this study, there is only one article to date that explores the role of curc umin dendritic cells. The authors show curcumin inhibits the immuno-stim ulatory function of murine bone marrowderived dendritic cells (69) The authors show that at non-toxic concentrations, curcumin is a potent inhibitor of DC maturation. Curcumin suppresses the expression of surface maturation mark ers CD80, CD86 and MHC class II in a concentration dependent manor and reduces the production of IL-12 and other pro-inflammatory cytokines IL-6, IL-1 and TNFin LPS-matured DCs. Studies have chronicled the effects of curcumin on antigen presenting cells other than dendritic cells. An in vivo model of murine latex allergy shows CD80 and CD86 levels are reduced on lung B cells treated with curcumin (73). Another study reports reduced CD80 and CD86 ex pression on macrophages treated with curcumin (121). The immunomodulatory properti es of curcumin also extend to its effects on cytokine production in dendritic cells and other anti gen presenting cells (1, 44, 66, 69, 152). Curcumin reduc ed levels of IL-12, IL-6, IL-1 TNFin murine DCs, monocytes and macrophages. Pr e-treatment with curcumin also
14 inhibits transcription of IL-1 IL-1 IL-2, IL-6, IL-10 and TNFmRNA in rat liver (40). In this study we investigate the effect of curcumin on human dendritic cell maturation by pre-treating the cells with curcumin and then inducing maturation with immune stimulants. Significance of the Study A study by Kim et al reveals that curcumin im pairs the immunostimulatory function of murine dendritic cells (69), but the effe cts of curcumin on human dendritic cells remain unknown. In this study we investig ate the effects of curcumin on human monocyte-derived DC maturation and function. Dendritic cells direct the adaptive immune res ponse to pathogens and allergens so we hypothesize they play a critical role in mediating curcumins systemic effects. We examine effects of curcumin on human dendritic cell maturation and function. Modulating the DC response could provi de an effective approach to treat and control unwanted inflammation and could provide an effective approach to treating inflammatory diseases. Eluci dation of the underlying mechanism of curcumin modulation will have a direct im pact on allergic disease control as recent studies point to its great potential for protecti on against lung diseases and allergic asthma (71, 73, 110, 131, 144). Curcumin shows promise as an immunomodulatory compound for the treatment of allergic diseases. Kobayashi et al. reports that curcumin inhibits IL-5, GM-CSF and IL-4 production and inhibits the proliferation and IL-2 production in lymphocytes obtained from atopic asthmatics in response to Dermatophagoides farinea (71). Curcumin diminishes
15 the Th2 response, reduces lung inflammation and reduces eosinophilia in a murine model of latex allergy (73) and al so reduces histamine release from rat basophilic leukemia cells (131). In guinea pi gs it is shown to attenuate airway hyper-responsiveness (110). Curcumin present s itself as an interesting molecule for further investigation. The findings of this study provide novel treatment and control strategies for allerg ic diseases. Due to its low toxicity, curcumin would offer itself as a safe alternative to nonsteroidal anti-inflammatory drugs (NSAIDs) and other inflammatory drugs currently available.
16 GOALS AND OBJECTIVES Purpose The purpose of this study is to examine the effect of curc umin on immature human dendritic cell development and function in response to external stimulants that mimic infection and stimulate cell matu ration or activation. This study utilizes in vitro cultures of primary dendritic ce lls obtained from a number of donors assumed to be in good health. Lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C ) or tumor necrosis factor alpha (TNF) were used to stimulate or induce DC maturation through independent cellular pathways. Hypothesis Curcumin has potent anti-inflamma tory properties and prevents the immunostimulatory function of murine dendr itic cells. We hypothesize that the same is true for human dendritic cells. We expect curcumin will prevent DC maturation in response to a variety of stimulants and im pede cell function.
17 Specific aims Aim 1: To determine the effect of curc umin on human mdDC maturation in response to immune stimulants. Does curcumin affect visual si gns of DC maturation such as cell morphology and homotypic clustering? Can curcumin alter the expressi on of the surface markers on iDCs? Does curcumin affect the level of expression of the su rface markers on DCs in response to immune stimulants? If so, is it dependent on the pathway propagated by the stimulant? Can curcumin alter cytokine pr oduction in stimulated cells? Figure 2. Aim 1 experimental outline. Designed to investigate the effects of curcumin on human mdDCs with and without stimulation. Dash ed boxes represent independent variables and solid boxes represent dependent variables. mdDC primary culture Model Dependent Variables Independent Variables Cell morphology Homotypic cluster formation Surface marker expression: ( CD86, CD83, HLA-DR, CD40, CD54) Cytokine secretion: (IL-12p70, IL-10, IL-6, TNF, IFN) LPS stimulation (24 hrs) poly I:C stimulation (24hrs) TNFstimulation (24hrs) No stimulation (24 hrs) Curcumin pre-treatment (1hr) : 0 M (DMSO) 20 M (7.4g/ml) 30 M (11.1g/ml)
18 Aim 2: To determine the effect of curc umin on human mdDC function in the presence of immune stimulants Does curcumin affect iDC endocytosis? Does curcumin affect endo cytosis of stimulated DCs? Does curcumin affect DC chemoki ne secretion and chemokine receptor expression? Does curcumin affect DC chemotaxis? Does curcumin affect the ability of DCs to induce proliferation of allogeneic donor CD4+ T cells in co-culture? How does curcumin affect the phenoty pe of proliferated T cells in coculture? Figure 3. Aim 2 experimental outline. Designed to examine the effects of curcumin on stimulated DC function. Model Dependent Variables Curcumin pre-treatment (1hr): 0 M (DMSO) 20 M (7.4g/ml) 30 M (11.1g/ml) LPS stimulation (24 hrs) poly I:C stimulation (24hrs) Endocytosis Chemotaxis Chemokine and chemokine receptor expression Allogeneic DC-TC co-culture T cell proliferation T cell phenotype DC primary culture No stimulation (24 hrs) Independent Variables
19 Aim 3: To determine the effect of curcumin on actin rearrangement in human mdDCs. Does curcumin affect the expr ession of actin and actin pathwayassociated proteins in human DCs? Does curcumin affect actin polymerization and cytoskeleton rearrangement in human DCs? Does the inhibition of actin result in similar functional observations to that of curcumin? Figure 4. Aim 3 experimental outline. Designed to examine the effect of curcumin on the actin cytoskeleton in dendritic cells. Model Dependent Variables Curcumin pre-treatment (1hr): 0 M (DMSO) 20 M (7.4g/ml) 30 M (11.1g/ml) LPS stimulation (24 hrs) Microarray analysis of total RNA Confocal imaging of actin Confocal imaging of actin pathwayassociated proteins Western blot of actin pathway molecules Functional assay usi ng actin inhbitor ( endoc y tosis assa y) DC primary culture No stimulation (24 hrs) Independent Variables
20 MATERIALS AND METHODS Materials Curcumin (from Curcuma longa) was obtained from Sigma Aldrich (St. Louis, MO) and dissolved in DMSO ( 11mg/ml). Buffy coats were obtained from Florida Blood Services (St. Petersburg Flori da). Donors in good health and ranging in age from 18 to 50 were used for the study (Table 1). The cell isolation reagents CD14 microbeads and nave CD4+ T cell isol ation kit were obt ained from Miltenyi Biotec (Auburn, CA). For ce ll isolation and culture, Histopaque-1077 and was obtained from Sigma Aldrich and recombinant human cytokines GM-CSF and IL4 were obtained from PeproTech (Rocky Hill NJ). All other cell culture reagents were obtained from GIBCO Invitrogen (Car lsbad, CA). LPS, poly I:C and PHA were obtained from Sigma Aldr ich (St. Louis, MO). CFSE and Alexa-647 conjugated dextran (molec ular weight 10,000) were obtained from Molecular Probes Invitrogen (Carlsbad, CA). LINCOplex Multiplex cytokine assay kits were purchased from Millipore (Tem ecula, CA). All antibodies used for flow cytometry CD11c, HLA-DR, CD40, CD86, CD83 and CD54 were obtained from BD Biosciences (San Jose, CA). The antibodies used for we stern blotting: CD83, CD86 and HLA-DR were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Chemokines CCL19 and CCL21 were obt ained from PeproTech (Rocky Hill, NJ).
21Table 1. Age and gender of donors used in the study Donor ID Age Gender 13 31 Female 14 24 Female A 61 Female B 26 Male C 20 Female D 21 Female E 21 Male F 21 Male G 42 Male H 28 Male I 59 Female J 50 Female K 42 Female Monocyte Isolation and culture In the human body DCs are generated via multiple pathways. They can be derived from CD34+ stem cell precursors or derived from blood monocytes (123). Monocyte-derived DCs generated from CD 14 + cells obtained from peripheral blood are phenotypically and functionally si milar to circulating DCs in the human body (47, 134). For the purposes of th is study mdDCs provide a suitable in vitro model. CD14+ monocytes were isolated and cultured as described by Picki et al. (105) Leukocytes were extracted from buffy coats using Histopaque-1077. Monocytes expressing CD14 were positivel y selected with magnetic microbeads.
22 Purity (>90%) was verified by staining with anti-CD14 antibodies and analyzing by flow cytometry. Cells were cultured at 1 106 cells/ml in complete RPMI (2% L-Glutamin,10% fetal bovine serum, 1% penicillin/streptomycin, 10mM Hepes, non-essential amino acids and 5mM sodium pyruvate) with 20 ng/ml each rh IL-4 and GM-CSF for five days at 37 C in an atmosphere of 5% CO2 and 95% air, (supplementing at day three with fresh medi um). On day 5, most cells express an immature DC phenotype expressing high CD11c, low CD40, CD86, CD83 and HLA-DR (113) (Figure 6). Mature DCs we re induced by adding either LPS, Poly I:C or the cytokine TNFto the culture for 24hours (23). The mature phenotype was confirmed by surface marker expr ession (high CD11c, CD40, CD86, CD83 and HLA-DR) measured by flow cytometry (Figure 6). DCs were identified by forward and side scatter morphology; those cells were gated and subjected to further analysis to quantify marker expression (Figure 5). Figure 5. Dendritic cell morphology gating based on fo rward and side scatter from flow cytometry after culture with GM-CSF and IL-4. 93.6 percent of the total events recorded were assumed to be DCs.
23 Figure 6. Surface marker expression on cultured immature and mature DCs. Cells display both immature and mature dendritic cell phenotype under the specified culture conditions. Surface marker expression determined by antibody staining of cell surface markers and flow cytometry analysis. The mature DCs express higher intens ity surface marker expression than immature DCs. Immature DC Phenotype: CD11c+ HLA-DR+ (low) CD83CD86+ (low) C40+ (low) Mature DC Phenotype: CD11c+ HLA-DR+ (high) CD83+ CD86+ (high) CD40+ (high) Un-stimulated Cells Stimulated Cells CD83 CD86 CD40 CD11c HLA-DR Unstained cells Stained cells
24 T cell isolation and culture CD4+ T cells were isolated from t he non-CD14 expressing fraction remaining after monocyte depletion and cultured in complete RPMI. The untouched cells were negatively selected using m agnetic beads (Miltenyi Biotec). Curcumin treatment and cell stimulation Curcumin, an ingredient of Indian curry powder, supplied as a powder (Sigma) is a polyphenolic compound insoluble in wate r. Dimethylsulfoxide (DSMO) is used as a solvent in this study and therefor e used as a control. Curcumin was added to iDC culture (1 106 cells/ml and 3 ml/well in 6-well plates) at the indicated concentrations (20M or 30M in most experiments). Cu ltures were incubated for 1 hour at 37 C in an atmosphere of 5% CO2 and 95% air after which a stimulant (LPS, Poly I:C or TNF) was added to the appropriate wells. Control wells received no stimulants. Cultures were incubated overnight at 37C and 5% CO2 and 95% air. Curcumin toxicity was assessed by 7AAD incorporation and measured by flow cytometry. Cells were found to be 95% ( 0.06) viable after 24 hours of culture under all conditions listed above. Cell viability by flow cytometry In order to assess the toxicity of curc umin in DC culture after a prolonged period, cell viability was assessed. Cells we re collected and stained with 7-aminoactinomycin D (7AAD), a nuclear dye, and analyzed by flow cytometry.7AAD is used to discriminate living cells from dead ce lls. Live cells with intact membranes
25 will exclude the dye, while damaged cells will allow the dye to enter the cell (114). The gating strategy was simila r to that mentioned above (Figure 5). Measurements were taken at three time points using six concentrations of curcumin. The 30M concentration is the highest at which viability does not fall below 90% after 12 hr culture (Figure 7). The 10M concentration did not show significant changes in preliminary studies and so was not included in this work. Only the non-toxic 20 M or 30 M concentrations of curcum in were used for the experiments in this study. Figure 7. Dendritic cell viability measured by 7AAD staining and flow cytometry after 3 hrs, 12 hrs and 24 hrs of culture with curcum in at various concentrations. Surface marker analysis by flow cytometry Cells were collected, washed, re-suspended (1 106 cells/ml) and stained with fluorochrome-conjugated antibodies spec ific for DC surface markers CD11c, HLA-DR, CD40, CD86, CD83 and CD54. Afte r staining, cells were washed and fixed with 4% paraformaldehyde (PFA ) then re-suspended in staining buffer, protected from light and stored at 4 C until flow cytometry analysis. Cells were 3 hr 010203050100 0 10 20 30 40 50 60 70 80 90 100 Curcumin Concentration ( M)Percent Viable DCs 12 hr 010203050100 0 10 20 30 40 50 60 70 80 90 100 Curcumin concentraion ( M)Percent Viable DCs 24 hr 010203050100 0 10 20 30 40 50 60 70 80 90 100 Curcumin Concentraion ( M)Percent Viable DCs
26 analyzed using the Becton Dickenson (BD) Canto II with HTS sampler and BD FACSDiva software. Figures were generat ed using FlowJo software (Tree Star Inc.) Multiplex bead assay Cytokines produced by DCs in culture were measured by multiplex bead assay (Millipore). Culture supernatant was co llected, centrifuged to remove any particulates and stored at -20 C. Cytokine levels meas ured from the supernatant using the LINCOplex human mult iplex assay. Six cytokines (IL-12p70, IL-10, IL6, IL-8, TNF and IFN ) and two chemokines (IP-10 and fractalkine) were measured from DC supernatant using this me thod. Six cytokines (IL-2, IL-4, IL-6, IL-10, IL-13 and IFN ) were measured from DC TC co-culture supernatant after the mixed lymphocyte reaction (MLR). Assays were performed in duplicate according to the manufacturers instructi ons. In summary, samples were diluted with an equal volume of medium and 25 l aliquots were used per assay well. Culture medium was used as the blank fo r the assay. Samples were incubated with antibody coated capture beads for 1hr, wells were washed and the cocktail of biotin labeled anti-human cytokine antibodies were added to all wells. After a 2hr incubation at room temperature str eptavidin-phycoerythrin was added for 30 minutes. Samples were analyzed using t he Luminex 100 IS system and IS 2.3 software (Luminex, Austin TX). Data was generated as mean fluorescence intensity (MFI) for each cytokine. Standard curves were generated using 5 parameter logistic regression based on k nown concentrations of the recombinant
27 cytokines provided by the manufacturer. This was used to calculate the concentration (pg/ml) for the sa mples that were assayed. Homotypic clustering Dendritic cell clustering is a hallmark of ac tivated or mature cells. It allows cells to communicate with each other as well as responder cells such as T cells and B cells Cell to cell contact is critical for antigen presentatio n and the propagation of the adaptive immune response (31). The si ze and density of the cluster may be indicative of the activation state of the cells. Strongly activated cells form larger and more dense clusters than weakly activa ted or immature cells. Clusters were observed by light microscopy at low power magnification (4x). Images were recorded using an Olympus digital camera. Western Blotting Cells were collected, washed and lys ed using NP-40 lysis buffer containing protease and phosphatase inhibitors. Pr oteins were quantified using a BCA protein assay kit and 100g loaded onto an SDS-PAGE gel. After electrophoresis, proteins were tran sferred onto a PVDF membrane by electrophoresis. The membranes were blocked using 5% non-fat milk for 1 hr at room temperature and subs equently probed with the a ppropriate antibodies in 5% bovine serum albumin (BSA) buffer overnight at 4 C. The membranes were washed and incubated with horseradish -peroxidase (HRP)-conjugated secondary antibodies. The bands were visualized by incubating the membranes in West
28 Pico chemiluminescent reagent (Thermo Sci entific) for 5 minutes protected from light and exposing the membranes to x-ray film (Kodak). Endocytosis assay Immature DCs have the intrinsic ability to capture foreign materials by endocytosis. Stimulated or mature DCs do not possess this ability (113). This inherent property is used as a measur e of DC maturity. Treated and stimulated cells were collected, washed and incubated with 1mg/ml (per 1 106 cells) Alexa 647 conjugated dextran at ei ther 4C or 37C for 1 ho ur. Cells were washed with cold PBS and either analyzed by flow cytom etry or plated on gelatin coated cover slips and imaged by confocal microscopy. The change in mean fluorescence intensity (MFI) is calculated as the di fference between the MFI of 37C and 4C cultures. Chemotaxis assay Another measure of DC function is it s ability to migrate towards chemoattractants (81). Mature DCs are more motile than iDCs. Tr eated and stimulated cells were collected, counted and re-s uspended at a concentration of 1 x 106 cells/ml. 50l of cell suspension was pl aced in the upper chambers of 5m pore size polycarbonate filter inserts in a 96 well microchemotaxis plate (Chemicon). The lower chambers contained 40l of ei ther CCL19 or CCL21 in 150l of medium. Control wells had medium only. In put wells (in triplicate) contained 1 x 104 cells in the lower chambers without chemokines. Cells were incubated at
29 37C and 5% CO2/95% air overnight. Migration was stopped by the removal of the inserts. 1 x 104 polystyrene beads were added to each well (lower chamber) and analyzed by flow cytometry. The numbe r of cells in each sample and input was calculated using the following equation: Number of cells/well = (number of cell events number of bead events) x 104. Input cells = average number of input cells/well x 5 (dilution factor) The percentage migration for each sample (% input) is determined by the following equation: Percent migration = (migrating cells input cells) x 100. Mixed lymphocyte reaction Mature DCs are able to stimulate prolifer ation of allogeneic T cells and induce a helper response. In order to determine the effect of curcumin on DC function after stimulation, T cell proliferation and polarization was assayed using a mixed lymphocyte reaction. In order to measure proliferati on, T cells were loaded with an intracellular dye carboxyfluorosce in succinimidyl ester (CFSE). CFSE passively diffuses into cells and reacts with amines in the cytoplasm forming highly fluorescent conjugates. As the cells divide the conjugates are distributed to the daughter cells. Fluorescence intensity was measured by flow cytometry, with each generation of cells emitting approximat ely half the fluorescence intensity of the parent. CFSE labeling of CD4+ T cells was carried out according to published procedures (108). Cells were suspended in 1ml PBS containing 5% (v/v) FBS. 1.1l of the CFSE stock (5M) was d iluted in 110l of PBS and then quickly mixed with the cell suspension. After a 5 minute incubation at room temperature,
30 the reaction was stopped by adding ten volumes of room temperature PBS containing 5% (v/v) FBS and centrifuging at 300 g for 5 minutes at 20C. Cells were washed twice and re-suspended in complete medium (1 106 cells/ml). The DC-T cell co-culture was set up at a ratio of 1:16. Curcumin treated and stimulated DCs were removed from cult ure and placed in 96 well plates in triplicate (6.25 103 cells in 100l per well). 100l of T cells were added to each well and cultures incubated at 37C and 5% CO2 /95% air for 5 days. Unstimulated T cells were used as the negative control, mitogen (phytohemagultinin: PHA at a concentration of 5 g/ml) stimulated T cells were used as the positive control. CFSE fluore scence intensity was measured by flow cytometry using the BD Canto II wit h HTS attachment and BD FACS Diva software. Immunofluorescence labeling Cells were collected, washed and fixed with 4% PFA. For intracellular staining, cells were permeablized with CytoFix/CytoPerm solution (BD Pharmingen). Nonspecific antigens were blocked by usi ng staining buffer containing FBS. The staining buffer also contained saponin (B D PermWash buffer) which maintains cell permeabilization. Cells were inc ubated with the approp riate antibodies, washed thoroughly and mounted in a gl ycerol-based mounting medium that contains DAPI. Slides were stored at 4C, protected from light until imaging.
31 Microscopy All bright field images were captured using the 4x objective of an Olympus IX71 inverted fluorescent microscope with an attached DP70 camera. Fluorescent images were captured using either the 63x or the 40x objective of a Leica scanning confocal microscope. RNA extraction from human DCs Monocyte-derived DCs obtai ned from human peripheral blood were placed in experimental groups, treated with curcum in and stimulated wit h LPS (Figure 8). Total RNA was extracted from cells usi ng the RNeasy isolation kit (Qiagen) as per the manufacturers instructions. RNA was quantified by optical density measurements and its pur ity and integrity dete rmined by agarose gel electrophoresis of samples stored at room temperature and samples heated to 42C for 1 hr and 70C for 10 minutes. Microarray All microarray experiments and analysis were carried out at the H. Lee Moffitt Cancer Center Microarray Core Lab. Affymetrix HG U133 Plus 2 array GeneChips were used for the experimen t. A separate chip was used for each of the four samples (Figure 8). Isolated RNA was biotinylated as described in the Affymetrix GeneChip Expression Analysis Manual (Affymetrix). 5 g total RNA was converted to double stranded DNA usin g 100pmol of an oligo-dT primer that contains a T7 promotor. The resulting cDNA was used in a transcription reaction
32 with biotinylated nucleotides. The produc t of this reaction was fragmented and hybridized to the GeneChips. After 16 hours of hybridization, the chip was washed and stained with streptavidin -phycoeryhtrin and then read using an Affymetrix GeneChip scanner. Data was processed using the GeneChip Operating Software (GCOS) Microarray Suit e 5.0 (Affymetrix). Based on criteria set by Affymetrix GC OS software, only genes that ar e considered to be present will be used for further analysis. Present calls are made by comparing the 11 perfect matches and mismatches fo r each probe set. The data generated was normalized to control genes and filtered by present and absent calls or by gene increase or decrease calls. Genes were annotated using software provided by the Microarray Core Facility. To identify cellular pathways affected by curcumin treatment and LPS stimulati on, the data was examined using Ingenuity Systems Pathways Analysis 6.3 (Ingenuity System s, www.ingenuity.com) software which categorizes identified genes based on biol ogical function and signaling pathways. Canonical Pathways Analysis identif ied the pathways from the Ingenuity Pathways Analysis library of canonical pathw ays that were most significant to the dataset. The significance of the a ssociation between the dataset and the canonical pathway was measured in 2 ways: 1) A ratio of the number of genes from the dataset that m ap to the pathway divided by the total number of molecules that exist in the canonical pathw ay is displayed. 2) Fischers exact test was used to calculate a p -value determining the probability that the association between the genes in the dataset and the canonical pathway is explained by chance alone.
33 Figure 8. Experimental outline for microarray analysis Statistical analysis In this study a 4 x 3 factorial design wa s used. All values are reported as mean and standard error of the mean (SEM). Most data was transformed using a base 10 logarithm to ensure normal distributi on, with the exception of viability and proliferation values in which case the raw data was reported. All donors displayed similar trends in response to tr eatment and stimulatio n, however due to individual variation there were differences in the magnitude of the values. Paired ttests (repeated measures) for planned comparisons were conducted. Significance was determined using a modifi ed Bonferroni correction (55). Each test of significance was r anked and the range of observed p values were compared to critical alpha values a ( a = alpha 0.05 divided by the number of Add DMSO for 12hr md-iDCs Add curcumin (20M) dissolved in DMSO for 12hr Add curcumin (20M) dissolved in DMSO for 1hr then add LPS for 11hrs Add DMSO for 1hr then add LPS for 11hrs RNA isolation Microarray Model Independent variable Dependent variable
34 planned comparisons). Values are present ed as the average of six donors. Error bars represent SEM. All statistical ca lculations were performed using the Statistical Package for Social Scienc es (SPSS version 15). Figures were generated using GraphPad Prisim (versi on 3.03). A concentration dependent reduction in values was observed in almost all cases. Though significance was calculated for only the 20M concentrations the change in values of the 30M concentration compared with the control we re therefore thought to be significant as well.
35 RESULTS Aim 1: Curcumin Prevents Dendritic Cell Maturation Curcumin Affects DC Morphology Dendritic cells have a characteristic mo rphology. They are slightly irregular in shape and have finger-like projections or dendrites that increase their surface area to maximize cell-cell contact. Curcumin appears to cause the cells to become more spherical and to lose their dendrites (Figure 9). This change is evident in both the absence and presence of LPS. Similar effects were noted for poly I:C and TNFstimulated cells (data not shown).
36 Figure 9 Curcumin affects cell morphology Cells were cultured for 24hs with or without curcumin (20M) and LPS (1g/ml). Images were captured using an inverted light microscope with a 40x objective lens. Red arrows indicate cell dendrit es. Images are from a single representative of eight donors. Curcumin Prevents Homotypic Cluster Formation Homotypic cluster formation correlates directly with phenotypic DC maturation. Stimulated or mature DCs wi ll form clusters due to their increased marker expression and enhanced motility. In order to determine the effects of curcumin on DC clustering, cells were cult ured in the presence of curcumin for 24 hrs and then examined under an inverted light microscope using a low power objective (4x). Curcumin prevents DCs from forming large, dense clusters characteristic of mature DCs in response to stimulants such as LPS, poly I:C and TNF(Figure 10). This effect is c oncentration dependent as the 30M DMSO LPS DMSO Cur 20 M
37 concentration abrogates cluster formation completely. There are some clusters formed in the experimental groups t hat received 20M curcumin and were stimulated with poly I:C and TNF, but these were much smaller than those formed in the similarly stimulated c ontrol groups. The clusters formed in response to LPS were larger and more dens e that those formed in response to poly I:C and TNF. Figure 10. Curcumin prevents homotypic clusteri ng of stimulated DCs. Cells were cultured for 24 hrs with or without curcumin (20M or 30M) and LPS, poly I:C or TNFadded to the appropriate wells. Images were captured using an in verted light microscope with a 4x objective lens. Images are of a single representative of eight donors. No Stim LPS Pol y I:C DMSO Cur 20 M Cur 30 M TNF-
38 Curcumin Reduces Surface Marker Expression Immunofluorescence labeli ng and flow cytometry was used to determine the effects of curcumin on surface mark er expression. Cells were evaluated using two concentrations of curcumin (20M and 30M) and three immune stimulants (LPS, poly I:C and TNF). Data is reported either as percentage positive cells or mean fluorescenc e intensity (MFI) of all donors ( n = 8 ). Table 2 shows p values of significance for each ex perimental group assessed. A similar number of cells were st ained for the surface mark ers CD11c, HLA-DR, CD83, CD86 and CD40 and analyzed in each experimental group. In order to determine the effects of surface ma rker expression on iDCs, cells were treated with curcumin for 24 hrs and no stimulant was introduced into culture. There was no significant change in surface marker ex pression for all donors (Figure 12). In figure 11 the unstimulated cells treated with 30M curcumin show reduced CD86 expression, this was only seen in this donor and not the trend across all donors. In the presence of all three stimulants, t he cells that were tr eated with curcumin had reduced expression of CD83, CD 86, CD54 and CD40 when compared to stimulated controls (Figures 11, 12) HLA-DR surface expression was not significantly affected by the 20M conc entration of curcumin; however the 30M concentration of curcumin showed significant results (Figure 11). Immunofluorescece staining shows that curcumin-treated DCs retain some HLADR in the cytoplasm (Figure 13a). Wester n blot shows curcumin-treated DCs have a lower level of expression of the antigen presenting molecules CD86, CD83 and HLA-DR (Figure 13b). All cells expressed high levels of CD11c, but by
39 measure of fluorescence in tensity, the LPS and TNFstimulated cells treated with 30M concentrations and the L PS stimulated cells 20M showed significantly reduced expression of CD 11c compared to stimulated controls.
40 Figure 11. Curcumin reduces dendritic cell surfac e marker expression in stimulated cells. Flow cytometry histograms are a single representativ e of 6 donors. The yellow-green line represents the unstained cells and used to designate the negative population. CD86 CD83 CD40 CD54 CD11c HLA-DR No Stim LPS Poly I:C TNF Unstained DMSO 20M Curcumin 30M Curcumin
41 Figure 12. Curcumin reduces dendritic cell surfac e marker fluorescence intensity in stimulated cells. The y-axis represents the average log10 mean fluorescence intensity (MFI) SEM for 8 donors. indicates significance by one-tailed t -test of planned comparisons p < critical alpha value. CD86 No StimLPSPoly I:CTNF 2.5 3.5 4.5 5.5 6.5Log MFI CD54 No StimLPSPoly I:CTNF 2.5 3.5 4.5 5.5 6.5Log MFIDMSO Cur 20 M Cur 30 M CD11c No StimLPSPoly I:CTNF 2.5 3.5 4.5 5.5 6.5Log MFI HLA-DR No StimLPSPoly I:CTNF 2.5 3.5 4.5 5.5 6.5Log MFI CD83 No StimLPSPoly I:CTNF 2.5 3.5 4.5 5.5 6.5Log MFI CD40 No StimLPSPoly I:CTNF 2.5 3.5 4.5 5.5 6.5Log MFI* ** * * * * * * * *
42Table 2. Significance of curcumin effects on surface marker expression (mean fluorescence intensity): Comparison of DMSO vs. Curcumin (Cur 20M) Mean (DMSO) Mean (Cur 20 M) Critical p value Observed p value Significance No Stimulation CD11c 4.830 0.122 4.715 0.134 0.0500 0.0051 Yes HLA-DR 3.285 0.350 3.279 0.346 0.0500 0.4518 No CD86 3.916 0.120 4.010 0.170 0.0500 0.0762 No CD83 2.872 0.235 2.730 0.277 0.0500 0.0099 Yes CD40 3.071 0.488 3.132 0.442 0.0500 0.0623 No CD54 4.532 0.190 4.377 0.246 0.0500 0.0078 Yes LPS CD11c 5.024 0.042 4.842 0.075 0.0085 0.0009 Yes HLA-DR 3.544 0195 3.542 0.211 0.0170 0.1326 No CD86 5.013 0.102 4.317 0.145 0.0085 0.0000 Yes CD83 3.868 0.152 3.260 0.208 0.0085 0.0001 Yes CD40 3.793 0.222 3.399 0.336 0.0085 0.0005 Yes CD54 5.052 0.087 4.792 0.107 0.0085 0.0000 Yes Poly I:C CD11c 4.920 0.076 4.621 0.105 0.0170 0.0019 Yes HLA-DR 3.412 0.167 3.319 0.197 0.0085 0.0013 Yes CD86 4.644 0.514 4.120 0.446 0.0170 0.0000 Yes CD83 3.542 0.088 2.873 0.111 0.0170 0.0004 Yes CD40 3.854 0.595 3.454 0.589 0.0170 0.0011 Yes CD54 4.659 0.516 4.307 0.615 0.0170 0.0011 Yes TNFCD11c 4.926 0.093 4.744 0.076 0.0250 0.0022 Yes HLA-DR 3.415 0.198 3.409 0.190 0.0250 0.4483 No CD86 4.173 0.523 4.006 0.490 0.0250 0.0024 Yes CD83 3.445 0.106 2.995 0.106 0.0250 0.0006 Yes CD40 4.463 0.633 4.282 0.569 0.0250 0.0188 Yes CD54 3.884 0.799 3.679 0.819 0.0250 0.0033 Yes observed p values were less than 0.0001
43 Figure 13. Curcumin reduces the expression of the anti gen presentation molecules. Immunofluorescence labeling of fixed and permeabili zed DCs (A) and western blot of whole cell lysate (B) showing reduced expression of marker expression in the presence of curcumin and LPS. DAPI CD86 DAPI HLA-DR CD83 DMSO Cur 20M LPS Cur 20M LPS CD86 CD83 HL A -DR GAPDHDMSO LPS Cur 20M Cur 20M +LPS A B
44 Curcumin Reduces Cytokine Production Cytokine production by dendritic cells is indicative of maturation. Cytokines produced will begin the cascade of immune reaction and induce cell cell interactions which initiate the adapt ive response. In order to assess the effects of curcumin on cytokine pr oduction, iDCs were treated with two concentrations of curcumin and stimulat ed with either LPS or poly I:C. Culture supernatants were collected and analyze d by multiplex bead assay. IFN IL12p70, the immunomodulatory cytokine IL-1 0 as well as pro-inflammatory cytokines IL-6, IL-8 and TNFwere measured. The assay was carried out in duplicate and the average of 6 donors is r epresented in figure 14. Due to the innate variations in donor response to the stimulants, the data are averaged and represented as log10 concentration. Table 3 shows the p value significance for the curcumin effects noted in this assay. Table 4 shows the p value significance of the stimulant effects. Characteristic ally low levels of all cytokines were produced by non-stimulated cells. These levels were not affected by curcumin at either concentration. Stim ulation with LPS and poly I:C resulted in substantial production of all cytokines. IL-10, IL-6 and IL-12p70 were significantly reduced in the curcumin treated cells stimulated wit h LPS and poly I:C in a concentration dependent manor. These levels were more or less reduced to the levels of the iDC cells that received no stimulation (Fi gure 14). In cells stimulated with LPS, higher levels of IL-8 were produced t han the poly I:C stimulated groups. Some values were outside of the dynamic r ange of the assay. The 20M curcumin concentration significantly increased IL-8 pr oduction in poly I:C stimulated cells
45 above the stimulated controls. Due to t he fact that the L PS-stimulated 20M curcumin treated cells were out of the r ange of the assay, t he curcumin effects were not significant. The 30M concentration did not reduce levels below the untreated control in response to either st imulant. There was a significant increase in IL-8 levels however in the unstimu lated control group treated with curcumin (20M). The level of IFN produced in this assay was mi nimal. In LPS-stimulated cells, curcumin caused a significant reduc tion in the amount of this cytokine that was produced. No significant reduction wa s observed in the poly I:C stimulated group. Stimulated DCs showed significantly reduced TNFlevels in when curcumin was present. Though this reduction was concentration dependent, unlike with the other cytokines, the TNFlevels were not reduced to that of the unstimulated controls (Figure 14).
46 Figure 14. Curcumin reduces cytokine production by human DCs in response to stimulants. Cells were cultured in the presence of curcumin (20 M and 30M) and stimulated with either LPS or poly I:C. Data is represented as log (base 10) concentration and the average of 6 donors. Error bars are SEM and indicates significance (p < critical alpha) IL-6 No StimLPSPoly I:C 0 1 2 3 4 5Log Concentration IL-8 No StimLPSPoly I:C 0 1 2 3 4 5Log Concentration IL-12p70 No StimLPSPoly I:C 0 1 2 3 4 5Log Concentration TNF No StimLPSPoly I:C 0 1 2 3 4 5Log Concentration IFN No StimLPSPoly I:C 0 1 2 3 4 5Log ConcentrationDMSO Cur 20 M Cur 30 M IL-10 No StimLPSPoly I:C 0 1 2 3 4 5Log Concentration * * * * *
47 Table 3. Significance of curcumin effects on cytokine production: Comparison of DMSO vs. Cur 20 M Mean (DMSO) Mean (Cur 20 M) Critical p Observed p Significant No Stimulation IL-10 1.196 0.4540.977 0.459 0.025 0.003 Yes IL-12p70 0.477 0.0000.477 0.000 IL-6 1.194 0.6761.099 0.831 0.05 0.199 Yes IL-8 2.022 0.4282.553 0.899 0.025 0.0078 Yes TNF1.234 0.1130.869 0.548 0.025 0.0665 No IFN 0.627 0.4230.477 0.000 0.05 0.1753 No LPS IL-10 3.453 0.8252.078 1.011 0.017 0.0005 Yes IL-12p70 2.626 0.9830.964 0.596 0.025 0.002 Yes IL-6 4.132 0.2573.372 0.926 0.025 0.009 Yes IL-8 4.162 0.3064.314 0.022 0.05 0.097 No TNF4.157 0.3883.623 0.398 0.017 0.0065 Yes IFN 1.557 0.7640.832 0.612 0.017 0.0074 Yes Poly I:C IL-10 1.705 0.3941.151 0.585 0.05 0.001 Yes IL-12p70 2.467 1.2960.865 0.809 0.05 0.003 Yes IL-6 3.309 0.4972.285 0.808 0.017 0.001 Yes IL-8 3.309 0.4973.630 0.282 0.017 0.0007 Yes TNF2.839 0.9182.362 0.702 0.05 0.0145 Yes IFN 0.780 0.4240.557 0.225 0.025 0.1376 No
48Table 4. Significance of stimulant effects: Co mparison DMSO (No stim) vs. Stimulant (Stim) Mean (No Stim) Mean (Stim) Critical p value Observed p value Significance LPS* IL-10 1.196 0.454 3.453 0.825 0.025 0.0000 Yes IL-12p70 0.477 0.00 2.626 0.983 0.025 0.0000 Yes IL-6 1.194 0.676 4.132 0.257 0.025 0.0000 Yes IL-8 2.022 0.428 4.162 0.306 0.025 0.0000 Yes TNF 1.234 0.113 4.157 0.388 0.025 0.0000 Yes IFN 0.627 0.423 1.557 0.764 0.025 0.0210 Yes Poly I:C IL-10 1.196 0.454 1.705 0.394 0.05 0.0010 Yes IL-12p70 0.477 0.00 2.467 1.296 0.05 0.0015 Yes IL-6 1.194 0.676 3.309 0.497 0.05 0.0005 Yes IL-8 2.022 0.428 3.309 0.497 0.05 0.0035 Yes TNF 1.234 0.113 2.839 0.918 0.05 0.0010 Yes IFN 0.627 0.423 0.780 0.424 0.05 0.2719 No Observed p values were less than 0.0001
49 Aim 2: Curcumin Prevents Dendritic Cell Function Curcumin Reduces Endocytosis Endocytosis is a feature of immatu re dendritic cells which are usually found in the periphery. Maturing migrati ng DCs lose their ability to edocytose. Therefore, the test of endocytic capacity is thought of as the gold standard in assessing DC maturity as a measure of func tion. In order to assess the effects of curcumin on iDC endocytosis, cells were cu ltured in the presence of curcumin for 24hrs and then cultured with fluorescently labeled dextran (MW 10,000). Dextran uptake was measured by flow cytometry and confocal microscopy. Data is represented as the average c hange in mean fluorescence intensity (MFI) of six donors (Figure 15b). Confocal microscope imaging of a representative donor is shown in figure 15a. The significance of the effects of curcumin are shown in table 4 and the significance of stimul ant effects are shown in table 5. Curcumin prevents iDCs from taki ng up dextran in a concentration dependent manner (Figure 15b). In the curcumin treated cells, dextran accumulates along the cell membrane and not throughout the cytoplasm as in the untreated control (Figure 15a). LPS st imulated cells show characteristically reduced dextran uptake. The level of endocytosis for both LPS and poly I:C stimulated DCs were significantly reduc ed when compared to the unstimulated control (open bars in figure 15b). Pre-tr eated stimulated DCs show dextran uptake similar to that of the unstimula ted pre-treated controls (black and hatched bars in Figure 15b). Similar to curcumin treated DCs, LPS stimulated DCs show
50 dextran accumulated along the cell me mbrane and not throughout the cytoplasm (Figure 15a). There was no significant difference between curcumin-treated and untreated DCs in the LPS and poly I:C tr eated experimental groups (Table 5). Figure 15. Curcumin reduces endocytosis in human dendritic cells. Confocal microscopy image (a) and change in mean fluorescence intensity (M FI) as measured by flow cytometry (B). Confocal images were captured using a 64x objec tive lens. Change in MFI was calculated by subtracting the MFI at 4C from the MFI at 37 C. indicates significant curcumin effects p < 0.05; indicates significant stimulant effects p < 0.05. No StimLPSPoly I:C 10000 20000 30000Change MFIDMSO Cur 20 M Cur 30 M B * No stim LPS DMSO Cur 20 M A
51Table 5. Significance of curcumin effects on endocytosis: Comparison of DMSO vs. Cur 20 M Mean (DMSO) Mean (Cur 20 M) Critical p value Observed p value Significance No Stim 23,729 6196 13,596 6353 0.017 0.0126 Yes LPS 12,726 12,237 9,850 8,543 0.025 0.1165 No Poly I:C 14,242 9,198 12,964 6,060 0.050 0.2960 No Table 6. Significance of the effects of stimulant s on endocytosis: Comparison of DMSO (No Stim) vs. Stimulant (Stim) Mean (No Stim) Mean (Stim) Critical p value Observed p value Significance LPS 23,729 6196 12,726 12,237 0.050 0.0418 Yes Poly I:C 23,729 6196 14,242 9,198 0.050 0.0382 Yes Curcumin Reduces Chemokine Secretion Chemokines are cytokines that i nduce cellular migration. Two key cytokines produced by DCs to attract inflammatory cells that are fractalkine (CX3CL1) and IP-10 (CXCL10). The super natant of DC exper imental cultures were analyzed for these chemokines by mu ltiplex bead assay. Data are reported as the average log10 transformed concentration of f our donors. The significance of the curcumin effects are shown in tabl e 7. A lower concentration of curcumin (20M) was used in this experiment, sinc e for previous experiments the changes at this concentration were found to be significant. LPS was the only stimulant used as curcumin effects do not s eem to depend on the stimulant used.
52 Curcumin reduces the levels of IP-10 and fractalkine produc ed in LPS-stimulated DCs (Figure 16). The reduction in IP-10 production was found to be significant, however, due to the low levels of frac talkine produced, the reduction was not found to be significant (Table 7). Figure 16. Curcumin reduces chemokine production by dendritic cells. Error bars represent SEM and indicates p values < critical value. Table 7. Significance of curcumin effects on c hemokine production: Comparison DMSO vs. Cur 20 M Mean (DMSO) Mean (Cur 20 M) Critical p Observed p Significant No Stimulation IP-10 1.588 0.7691.144 0.726 0.05 0.0828 No Fractalkine 0.486 0.0150.382 0.166 0.05 0.2113 No LPS IP-10 4.278 0.0363.008 0.705 0.025 0.0009 Yes Fractalkine 0.980 0.4560.486 0.015 0.025 0.0970 No Fractalkine No StimLPS 0.0 0.5 1.0 1.5Log ConcnetrationDMSO Cur 20 M IP-10 No StimLPS 0 1 2 3 4 5Log Concentration*
53 Curcumin Prevents DC Chemotaxis DC chemotaxis is a characteristic f unction of mature DCs. After they acquire antigen they migrate toward s lymphoid organs to stimulate T lymphocytes. In order to assess the e ffects of curcumin on DC chemotaxis, treated and stimulated cells were placed in a chemot axis chamber and allowed to migrate towards chemo-attractants CCL19 and CCL21. Data is reported as the average percent cell migration of two dono rs. The significance of the effects of curcumin are shown in table 8. The sa mples were normalized to the percentage of cells migrating towards medium onl y. Curcumin treated cells are unable to migrate towards the chemo-attractant s CCL19 and CCL21 in response to LPS (Figure 17). The chemokine receptor CCR 7, expressed on the surface of DC binds both the CCL21 and CCL19 ligands. The expression of CCR7 is not affected by curcumin (Figure 18). CCR7 expression on DCs was evaluated by surface marker immunofluorescence staini ng followed by flow cytometry (Figure 18a,b) and by western blot of w hole cell lysate (Figure 18c).
54 Figure 17. Curcumin prevents chemot axis of dendritic cells in re sponse to LPS. Cell migration was normalized using percent cell migration towards culture medium. Error bars represent SEM and indicates p values < critical value. CCL21 chemokine ligand 21 or exodus-2; CCL19 chemokine ligand 19 or macrophage inflammatory protien-3-beta (MIP-3) Table 8. Significance of curcumin on dendritic cell chemotaxis: Comparison of DMSO vs. Cur 20 M Mean (DMSO) Mean (Cur 20 M) Critical p value Observed p value Significance CCL21 No Stim 0.599 0.765 0.160 0.089 0.050 0.2999 No LPS 25.219 1.457 0.176 0.083 0.025 0.0123 Yes CCL19 No Stim 0.780 0.052 0.117 0.087 0.025 0.0120 Yes LPS 22.431 3.225 0.319 0.439 0.050 0.0371 Yes DMSO Cur 20 M CCL21 0 10 20 30 40 50Percent Cell Migration CCL19 0 10 20 30 40 50Percent Cell MigrationNo Stim LPS No Stim LPS ** *
55 Figure 18. Expression of the c hemokine receptor CCR7 is not affected by curcumin. CCR7 expression is determined both on the cell surf ace by immunofluorescence labeling and flow cytometry (a and b) and intracellularly by wester n blot of whole cell lysate of a representative donor (c). Histograms shown are from a single d onor (a) and the bar chart shows the average mean fluorescence intensity (MFI) of four dono rs with error bars representing SEM (b). CCR7 chemokine receptor 7; GAPDH gylceraldehyde 3phos phate dehydrogenase. Cur 20M + LPS CCR7 GAPDHDMSO Cur 20M LPS Cur 20M DMSO Cur 20M DMSO Cur 20M DMSO No StimLPSPoly I:C 0 1 2 3 4Log10 MFIDMSO Cur 20mM A B CNo stim LPS Poly I:C
56 Curcumin Reduces DC-induced T Cell Pr oliferation in an Allogeneic Mixed Lymphocyte Reaction The mixed lymphocyte reaction c an be used as a measure of DC maturation. Although iDCs can weakly stimul ate T cell proliferation, mDCs initiate a more robust proliferation of the T cell population in a co-cul ture. In order to study the effects of curcumin in this pr ocess, iDCs were treated with curcumin, stimulated with either LPS or poly I:C and then co-cultured wit h allogeneic CD4+ T helper cells from mismatched donors. T he T cells were pre-loaded with CFSE, an intracellular dye and proliferation was assessed after 5 days of co-culture by flow cytometry. Flow cytometry data is represented as either the average mean fluorescence intensity of six donors or of a representative donor. The significance of curcumin effects and stimulant effect s on the experimental populations are shown in tables 8 and 9 respectively. For the flow cytometry analysis, gates were set based on side scatter vs. forward scatte r morphology (figure 19a) and those cells were evaluated for CFSE expressi on. Proliferated cells have lower fluorescence intensity of CFSE than unpr oliferated cells. Mature DCs induce significantly higher proliferation by CD4 + T cells than iDCs (Figure 20). Though iDCs induce some proliferation in t he T helper cell population, curcumin significantly impairs this ability as the num ber of proliferated ce lls is reduced from 25.28 ( 10.84) % to 4.83 ( 4.20) % on average. Even in the presence of the stimulants LPS or poly I:C curcumin -treated DCS are unable to induce proliferation of T helper cells at levels greater than 6.03 ( 3.50) % (Figure 19).
57 Figure 19. Curcumin reduces DCs ability to induce proliferation in allogeneic CD4+ helper T cells in a MLR. Data is representative of six donors. The gating strategy is shown in panel (a) and the histograms of gated cell proliferation based on CFSE fluorescence is show in panel (b). CFSE carboxyfluorescein succinimdyl ester; MLR mixed lymphocyte reaction. Proliferated T cells Non-proliferated T cells DMSO Cur 20M Cur 30M No stim LPS Poly I:C A B
58 Figure 20. Curcumin-treated DCs show reduced CD 4+ T cell proliferation in allogeneic MLR. Data represents the average of six donors. Table 9. Significance of curcumin effects on induci ng proliferation of CD4+ T cells: Comparison of DMSO vs. Cur 20 M Mean (DMSO) Mean (Cur 20 M) Critical p value Observed p value Significance No Stim 25.275 10.838 4.825 4.195 0.050 0.0215 Yes LPS 38.750 10.376 4.700 1.337 0.017 0.0040 Yes Poly I:C 36.900 13.422 6.025 3.491 0.025 0.0055 Yes Table 10. Significance of stimulated DCS induc ing T cell proliferation: Comparison DMSO (No stim) vs. Stimulated cells (Stim) Mean (No Stim) Mean (Stim) Critical p value Observed p value Significance LPS 25.275 10.838 38.750 10.376 0.05 0.0045 Yes Poly I:C 25.275 10.838 36.900 13.422 0.05 0.0570 No No StimLPSPoly I:C 0 10 20 30 40 50 * *% CD4+ TC Proliferation
59 Curcumin Induces a CD4+ CD25+ T Regulatory Cell Population Mature DCs will induce T helper cell differentiation. In order to assess the effects of curcumin on the phenotype of pr oliferated T cells in co-culture, cells were immunoflourescently labeled for Fo xP3 and CD25. Prolifer ated T cells were gated and only that population was analyze d for surface marker expression (Figure 22a). Curcumin-treated DCs, bot h stimulated and unstimulated induce a CD4+ CD25+ FoxP3+ population from the proliferated T cell population (Figures 21 and 22b). Culture supernatant was also assessed for T helper cytokines IL-2, Il-4, IL-5, IL-10, IL-13 and IFN Data is reported as average log10 concentration of four donors. Curcumin -treated DCs induced significantly lower cytokine production from the T cells in co-culture. Figure 21. Curcumin-treated DCs induce regulatory T cells in MLR. Data represents the average percentage of FoxP3+ CD25+ T cells of four do nors. Only the proliferated cell population was analyzed. Error bars represent the SEM. No StimLPSPoly I:C 0 20 40 60 80 100 DMSO Cur 20 M Cur 30 M % FoxP3+ CD25+ cells
60 Figure 22. Curcumin induces CD4+ CD25+ FoxP3+ regulatory T cells. After 5 days of co-culture with curcumin-treated and stimulated DCs in an allogeneic MLR, T cells were immunofluorescently labeled and analyzed by flow cytometr y. The gating strategy is outlined in panel (a); cells are gated based on morphology and then proliferated T cells are gated and analyzed for FoxP3 and CD25 expression. The numbers are the re lative percentages of proliferated cells (b) No stimulus LPSDMSO Cur 20M Cur 30M B Poly I:C Morphology gate Proliferated cells FoxP3 vs. CD25 A
61 Figure 23. Curcumin reduces T helper cytokine pr oduction after five days of co-culture in allogeneic MLR with DCs. Cytokines were measur ed from the supernatant after T cell co-culture with curcumin-treated, stimulated DCs. Data is reported as average log10 concentration SEM. DMSO Cur 20 M Cur 30 M IL-2 0.0 0.5 1.0 1.5 2.0 2.5Log Concentration IL-10 0 1 2 3 4Log Concentration IL-4 0.0 0.5 1.0 1.5 2.0 2.5Log Concentration IL-13 0 1 2 3 4Log Concentration IL-5 0.0 0.5 1.0 1.5 2.0 2.5Log Concentration IFN 0 1 2 3Log ConcnetrationNo Stim LPS Pol y I:CNo StimLPSPol y I:C No Stim LPS Pol y I:CNo StimLPSPol y I:C No Stim LPS Pol y I:CNo StimLPSPol y I:C
62Table 11. Significance of the effects of curc umin on DC-induced T cell cytokine production Mean (DMSO) Mean (Cur 20 M) Critical p Observed p Significant No Stimulation IL-2 1.270 0.201 0.477 0.000 0.017 0.0021 Yes IL-10 2.941 0.226 0.925 0.383 0.017 0.0013 Yes IL-4 1.886 0.217 1.425 0.146 0.050 0.0320 Yes IL-13 2.463 0.433 0.843 0.486 0.025 0.0075 Yes IL-5 1.485 0.286 0.477 0.000 0.025 0.0029 Yes IFN 2.213 0.142 0944 0.464 0.025 0.0027 Yes LPS IL-2 1.777 0.350 0.477 0.000 0.025 0.0025 Yes IL-10 3.100 0.279 1.213 0.517 0.025 0.0034 Yes IL-4 2.054 0.251 1.452 0.142 0.017 0.0061 Yes IL-13 2.792 0.407 0.826 0.394 0.017 0.0013 Yes IL-5 1.979 0.319 0.477 0.000 0.017 0.0013 Yes IFN 2.722 0.230 0.933 0.121 0.017 0.0001 Yes Poly I:C IL-2 1.516 0.715 0.477 0.000 0.050 0.0311 Yes IL-10 2.571 1.003 1.403 0.440 0.050 0.0154 Yes IL-4 1.904 0.343 1.458 0.136 0.025 0.0219 Yes IL-13 2.230 1.185 0.815 0.394 0.050 0.0390 Yes IL-5 1.506 0.705 0.477 0.000 0.050 0.0308 Yes IFN 2.086 0.926 0.921 0.268 0.050 0.0408 Yes
63 Aim 3: Curcumin Modulates the Actin Cytoskeleton Microarray Analysis of Total RNA Reve als the Effects of Curcumin on Gene Expression in Dendritic Cells Curcumin has anti-inflammato ry, anti-oxidant and anti-viral properties It is reasonable to speculate that changes in gene expression as well as protein expression are in part responsible for t hese properties. In order to assess the effect of curcumin on human dendritic cells, DCs were cultured with curcumin and then stimulated with LPS. Total RNA was analyzed using an Affymetrix HG U133 plus 2 genome array. Ingenuity pathw ays analysis (IPA) software was used to identify pathways that may be affect ed. Table 12 shows some pathways that were most significantly a ffected by curcumin treatment as identified by IPA software. Many of the pathways affect ed are associated with cell structure, motility and function. These pathways also have many of the same molecules in common. Upon closer inspection of the ac tin cytoskeleton pathway, though many genes are unaffected, there are some genes that show an increase or decrease in expression in response to curc umin-treated and stimulated cells when compared to LPS-stimulated cells (Table 13). Of interest are those genes that are down-regulated in curcumin-treated and stimulated cells such as nexilin which decreased 129 fold. A few GTPases and Rho family members were downregulated along with WASP interacting prot ein, plexin and a tubulin-specific chaperone.
64Table 12. Some pathways affected by curcumin as determined by Ingenuity pathways analysis software. Pathway -Log(P-value) Axonal Guidance Signaling 0.01540 Purine Metabolism 0.01220 Glucocorticoid Receptor Signaling 0.01060 Xenobiotic Metabolism Signaling 0.00966 Huntington's Disease Signaling 0.00904 Actin Cytoskeleton Signaling 0.00868 G-Protein Coupled Receptor Signaling 0.00828 Integrin Signaling 0.00810 Protein Ubiquitination Pathway 0.00801 Leukocyte Extravasation Signaling 0.00779 LPS/IL-1 Mediated Inhibition of RXR Function 0.00766 ERK/MAPK Signaling 0.00766 NRF2-mediated Oxidative Stress Response 0.00735 Calcium Signaling 0.00735 Ephrin Receptor Signaling 0.00726 Acute Phase Response Signaling 0.00717 RAR Activation 0.00703 Wnt/ 2-catenin Signaling 0.00681 cAMP-mediated Signaling 0.00663 PPAR /RXR Activation 0.00659
65Table 13. Actin cytoskeleton pathway genes affected by curcumin. Gene Symbol Gene description Fold change Cur 20M + LPS vs LPS RHOBTB1 Rho-related BTB domain containing 1 11.761 PLEC1 plectin 1, intermediate filament binding protein 5.332 SVIL supervillin 4.815 FGD6 FYVE, RhoGEF and PH domain containing 6 4.408 MAP7 microtubule-associated protein 7 4.292 CDC42EP3 CDC42 effector protein 3 4.081 ARHGDIB Rho GDP dissociation inhibitor (GDI) beta 3.836 RHOBTB2 Rho-related BTB domain containing 2 3.727 SPTAN1 spectrin, alpha, non-erythr ocytic 1 (alpha-fodrin) 3.4278 SPTBN1 spectrin, beta, non-erythrocytic 1 2.3971 RASA4 RAS p21 protein activator 4 1.994 PAK1 p21/Cdc42/Rac1-activ ated kinase 1 1.172 VCL vinculin 1.155 FGD4 FYVE, RhoGEF and PH domain containing 4 -1.074 CDC42SE1 Cdc42 small effector 1 -1.408 RND3 Rho family GTPase 3 -1.442 PHACTR2 phosphatase and actin regulator 2 -1.454 PHACTR4 phosphatase and actin regulator 4 -1.582 RAB12 RAB12, member RAS oncogene family -1.659 MYOZ3 myozenin 3 -1.687 ARHGAP25 Rho GTPase activating protein 25 -1.835 RAB30 RAB30, member RAS oncogene family -1.888 TBCD tubulin-specific chaperone d -2.161 WASPIP Wiskott-Aldrich syndrome pr otein interacti ng protein -2.214 MASTL microtubule associated serine/threonine kinase-like -2.885 PLXNA1 plexin A1 -3.426 ARHGAP25 Rho GTPase activating protein 25 -4.224 RHOH ras homolog gene family, member H -4.681 SCIN scinderin -4.695 RABGAP1L RAB GTPase activating protein 1-like -4.976 NEXN nexilin (F-actin binding protein) -129.149
66 Curcumin Alters the Actin Cytoskeleton in Human DCs The actin cytoskeleton of DCs is re sponsible for its shape and function. We hypothesize that curcumin can alter the conformation of the cytoskeleton and so provide a mechanism for its observed ant i-inflammatory effects. Fluorescently labeled phalloidin was used to visualize actin in this experiment. In order to assess the effect of curcumin on the ce ll architecture, DCs were treated with curcumin, washed, fixed with 4% paraf ormaldehyde and stained with Alexa 555phalloidin in suspension. The cell nuc leus was visualized using mounting medium containing the nuclear dye 4' 6-diamidino-2-phenylindole (DAPI). Images were captured using a confocal microscope and representative z slices are shown in figure 24. Cytochalasin B (CytoB) prevents actin polymer elongation and causes cells to become more rounded. It was used as a control in this study. Increasing concentrations of curcumin causes DCs to become more rounded and less filamentous. The rounding and loss of protrusions is similar to what was observed in the presence of the ac tin inhibitor CytoB (Figure 25). The actin cytoskeleton plays an impor tant role in cell adhesion and motility. In order to assess curcumin effects, iDCs were allowed to adhere to gelatin coated cover slips for 2 hrs at 37C (5%CO2 and 95% air). iDCs were either treated with curcumin or stimulated. Actin accumulates at podosomes towards the leading edge of the iDCs (Fi gure 24, left). Curcumin-treated cells show podosome formation, but the cells are not elongated (Figure 24, center). Maturing LPS-stimulated DCs do not show podosomes but rather stress fiber formation (Figure 24, right). In order to assess the effect of curcumin on cell
67 attachment, DCs are treated with curc umin and stimulated before they are allowed to attach to poly L-lysine c oated slides. Curcumin -treated DCs have a rounded morphology and do not adhere tightl y to the substrate (Figure 26, center). iDCs and LPS-stimulated DCs adhere and are polar ized with leadingedge lamellapodia (Figure 26, left). DCs treated with an actin inhibitor, cytochalasin B, were rounded and loosely adherent (Figure 26, right). The Rho GTPases play an essential role in cellu lar organization. Curcumin reduces the activity of Rac1 and Cdc42 GTPases evid enced by western blotting of whole cell lysate using phospho-antibodies. The ex pression of Cdc42 was not affected (Figure 27). Figure 24. Curcumin interferes with DC motilit y and attachment. DCs were allowed to adhere to gelatin for 2 hrs at 37C, 5% CO2 and 95% air. Curcumin and LPS were added to appropriate wells. After 1hour, cells were fixed with 4% paraformaldehyde and stained with phalloidin to visualize f-actin. Images were captured with a co nfocal microscope using a 63x objective lens. Red Alexa 555-phalloidin; blue 4', 6-diamidino-2-phenylindole (DAPI); green curcumin fluorescing in FITC channel. DMSO Cur 20M LPS
68 Figure 25. Curcumin changes DC morphology in a concentration dependent manor. DCS were fixed, stained and imaged in suspension to main tain their three-dimensional shape. Curcumin inhibits actin similar to cytochalasin B. The nucl eus if the cell is represented by the blue color 4', 6-diamidino-2-phenylindole (DAPI), the actin is st ained red (Alexa 555), curcumin fluoresces at about 435nm and is detected in the FITC channel. This accounts for the increasing green color in the cytoplasm of the cells as the co ncentration of curcumin increases. iDC immature DC (untreated); DMSO dimethyl sulfoxide; Cytoc halasin B an actin inhibitor; Cur curcumin iDC DMSO Cytochalasin B (20M) Cur 10M Cur 30M Cur 50M Cur 80M Cur 100M Cur 20M
69 Figure 26. Curcumin affects DC adhesion. DCs we re cultured with curcumin and stimulated with LPS overnight, washed and allowed to adhere to pol y L-lysine coated slides for 2 hrs. Cells were fixed with 4% paraformaldehyde and stained for actin (Alexa 555-phalloidin). Images were captured with a confocal microscope using a 40x objective lens. Figure 27. Curcumin reduces the activity of t he Rac1/Cdc42 kinases. Western blots from whole cell lysate of curcumin-treated stimulated DCs. DMSO CytoB LPS No Stim Cur 20M Cur 20M LPS Cdc42 phospho-Rac1/cdc42 DMSO Cur 20M + LPS
70 Curcumin-Induced Reduction in Endocytosis May be Due to Actin Inhibition. The actin cytoskeleton plays a key ro le in iDC endocytosis. In order to assess whether the curcumin -induced reduction in phago cytosis is mediated by actin inhibition, a known actin inhibito r, cytoB, was used as a control. CytoBtreated cells showed a similar reduction in phagocytosis to curcumin treated cells (Figure 28). Figure 28. Disruption of the actin cytoskeleton ca uses reduced endocytosis in immature DCs. DCs cultured with curcumin for 24hrs an d then endocytosis assay was performed. DMSOCur 20 MCBLPSCur 20+LPS 0 25000 50000 75000 Change MFI
71 DISCUSSION This is the first study to exami ne the effects of curcumin on human dendritic cells in vitro Donors for the study were selected at random and supplied by Florida Blood Services, St. Petersburg, Florida. The concentrations of curcumin used were based on those previously found efficacious in the literature and confirmed not to be toxic to the cells by viability assays. All cultures remained more than 90% viable up to 24hrs after curcumin addition. The pharmacokinetics and pharmacodymanics of curcumin have been more extensively studied in rodents than in humans (120). From the limited human data available, the low bioav ailability of curcumin limit s its clinical usefulness when administered orally. High doses can be administered without adverse effects but the systemic distributi on may not be sufficient to exert pharmacological activity. Combining cu rcumin with other compounds, or using drug delivery systems such as li posomes and nanoparticles provide an alternative approach to overcome these issues (20, 122, 142). The immunostimulants lipopolysacchaide (LPS) and polyinosinic:polycytidylic acid (poly I:C) were used in this study to independently stimulat e DC activation. LPS via the toll-like receptor 4 (TLR4) pathway and poly I:C mimics viral infections through TLR3. TNFsignals through its receptor TNF-R, which is a TLR-
72 independent pathway. These compounds were chosen to ensure the immunostimulatory effects were not pathway specific or TLR dependent. The inhibition of transcription factors NF B and AP-1 and other cell signaling pathways by curcumin explains some of the observations made in this study, but curcumin may be targeting other essential cellular pathways as well. The observations of this study suggest curcumin functions as an inhibitor of actin signaling. Since f-actin reorganization is responsible for dendritic cell maturation and function, its inhibition may be the me chanism by which curcumin prevents DC response to stimulants. Elucidation of the underlying mechanism of curcumin immunosuppression could lead to clinical applications of this novel antiinflammatory agent. DCs aggregate in clusters in response to stimuli as a visual sign of maturation (31). Cluster formation co rrelates with increased CD86, CD54 and CD80 expression. Here we show curc umin impairs homotypic DC cluster formation in response to LPS poly I:C and TNFin a concentration-dependent manner. Adhesion molecules such as ICA M-1 (CD54) are important in cellular interactions and in generating T cell re sponse. Murine antigen presenting cells (APCs) deficient in ICAM-1 have an im paired ability to induce T cell responses (41, 126). CD11c, a member of the integrin family of pr oteins, is also important for cell attachment and found in high le vels on DCs. Curcumin significantly reduces expression of both markers on the DC surface. The reduced CD11c could be the result of curcumin -induced AP-1 inhibition (99).
73 Mature or activated dendritic cells express elevated levels of costimulatory and antigen pres enting molecules on their surface such as CD86, CD83 and HLA-DR. If the ant igen presenting machiner y of DCs are impaired, they can not effectively engage the T cells to initiate a response. HLA-DR surface expression is only significantly inhibited at the 30M concentration of curcumin in LPS and TNFstimulated cells but in both concentrations stimulated with poly I:C show a signifi cant reduction in HLA-DR surface expression. These data suggest curcumin may be interfering with the antigen presenting machinery of DCs by affecti ng the expression of key presentation molecules. Mature monocyte-derived DCs secret e IL-12, IL-10 and other proinflammatory cytokines. Stimulated curc umin-treated DCs produce significantly lower levels of IL-12, IL-10, IL-6 and TNFwhen compared to the controls creating a Th2 permissive environment. Though the reduction of TNFwas significant, they were not reduced to the levels of the unstimulated controls. These findings correlate with those from the study by Kim et al (69) which shows curcumin prevents immunostimulatory function of murine bone marrow-derived cells. They along with others show cu rcumin is a potent inhibitor of NF B and AP-1 activation as well as MAPK signaling (59, 149). This provides a reasonable explanation for the observed reduction of IL-12 and IL-10 levels in this study. This is the first study to report that curcumin decreases IL-10 in human DCs. TNFexpression is controlled by other transcription factors such as lipopolysaccharide-induced TNF factor (L ITAF) (136) or interferon regulatory-
74 factor 3 (IRF3) (111) that may not be affected by curcumin, allowing the transcription of some TNFindependent of the NF B pathway. Capture and presentation of antigen is an important feature of DC biology. This provides the link between innate and adaptive immunity. Immature DCs are highly endocytic, a feature which is lost when cells become mature. We find curcumin reduces endocytosis in non-st imulated DCs. There is a significant decrease in dextran uptake by non-stimul ated cells treated with curcumin similar to stimulated cells, but not in stimulated cells. There are conflicting reports on the effects of curcumin on antigen captur e; a few studies show increased endocytosis, while others show suppression (44). Our findings indicate curcumin interferes with antigen handling in human DCs. Mature DCs travel to the lymph nodes where they present processed antigen to T cells. Migration towards chem o-attractants is a feature of mature DCs (81). They also secrete chemokines to attract responder cells to the site of injury or inflammation. Monocyte-deriv ed DCs migrate in response to CCL19 or macrophage-inflammatory protein-3beta (MIP-3 ) and CCL21 or exodus-2, which are expressed in the lymph nodes. Both chemokines bind to the CCR7 receptor on the DC surface. Though CCR7 expression is not affected, curcumin prevents migration towards CCL19 and CCL21 in a c hemotaxis assay and also reduces the levels of chemokines fractalkine (CX3CL1) and interferon producing factor (IP-10). Both fractalkine and IP-10 attr act inflammatory cells to sites of inflammation. Fractalkine attracts T cells, monocytes and microglia and mediates firm cell adhesion (17, 87). It also i nduces actin polymerization in human
75 dendritic cells (34). IP-10 is produced in response to IFN and LPS. This chemokine plays an important role in effe ctor T cell trafficking (68). Poly I:C stimulated cells did not migrate in res ponse to the chemokines, even in the absence of curcumin. By preventing DC migration, curcumin reduces the probability of the DC encounterin g T cells to initiate a specific immune response. Reduced chemokine secretion will stem the flow of inflammatory cell traffic to sites of inflammation. The mixed lymphocyte reaction (MLR) is used as the basic test of DC function since it measures their ability to st imulate proliferation of an allogeneic T cell population. Studies show curcumin can inhibit MLR (42, 57, 125, 153). Immature DCs will weakly stimulate proliferation, while the mature DCs will induce a significantly more robust response. This was observed in this study as there was a significant increase in the amount of T cell pro liferation in the stimulated groups compared to the nonstimulated group. Increased expression of co-stimulatory markers on the surface of DCs is essential for T cell interaction and proliferation. Curcumin-treated DCs, both stimulated and non-stimulated, show muted T cell proliferation (Figures 19 and 20). Curcumin inhibits the Th1 profile in antigen-primed CD4+ T cells while promoting the Th2 profile by suppressing IL-12 production in macr ophages (66, 67). We observe that curcumin suppresses IL-12 production in DCs, but the cytokines produced after the MLR were mixed Th1/Th2. There was no clear delineation in either direction and the levels were very low. The low level of expression is most likely due to the reduced number of prolifer ated T cells generated by the co-culture. The mixed
76 phenotype could also be due to the ratio of DC to T cells used in the reaction (135). Curcumin-treated DCs induce a low level of proliferation and those proliferated cells expre ss CD25 and Foxp3. This im plies that curcumin is conferring a tolerogenic property to DCs A measure of this propensity is the capacity to expand Foxp3 expressing lympho cytes (83, 139). It is suggested that an environment containing IL-10 can induc e tolerogenic DCs (63, 89, 90) but in this study, the levels of IL-10 are only slightly (not significantly) elevated above control levels in both DC culture and co-culture, suggesting there may be an alternative mechanism at play. We show that curcumin is able to exert profound effects on the expression of genes involved with multiple signaling pathways associated with cytoskeleton organization and function. These pathwa ys include axonal guidance signaling, glucocorticoid receptor signaling, actin cytoskeleton signaling. Immunofluorescence staining reveals curc umin interferes with cell actin based cell motility, attachment and microf ilament organization on human dendritic cells in vitro The actin inhibitor cytochalasin B was used as a control in most experiments to confirm the effects noted ar e most likely due to curcumins effects on f-actin organization. The regulation of the DC cytoskele ton is important in DC T cell interactions (18). Little is known about the effect of curcumin on cytoskeletal rearrangement. One study reveals curcum in significantly alters the actin cytoskeleton in prostate cancer cells ( 56). Based on this premise, curcumininduced alterations in DC cytoskeleton could account for our observations. DC
77 migration involves regulation of the actin assembly. The cells must form protrusions such as filapodia and lamellapo dia, form adhesions and retract its tail (80). Curcumin reduces expression of adhesion molecules and though lamellapodia and podosome formation appear s to be unaffected, the cells are more rounded and we conclude less motile as there is the absence of the trailing edge seen in the non curcumin-treated ce lls and these cells do not seem to polarize in any particular direction. Cells were allowed to adhere to coated slides before curcumin treatment and LPS stimulation. For a more three-dimens ional viewpoint, the cells that were imaged in suspension show a dramatic change in mo rphology. As the concentration of curcumin increases, the surface of the cell becomes more rounded and smooth and less elongated projections are visible. Th is results in an overall decrease in the cell surface area and so will affect the ability of the cell to interact with other cells such as T cells. The changes obser ved are similar to those induced by CytoB therefore we can infer that curcum in is inhibiting f-ac tin polymerization. Curcumin-treated DC show reduced attachmen t. They did not polarize or adhere strongly to the surface of the poly L-lysine coated slides as the non-treated cells did. The morphology was similar to that of the CytoB treated cells which clearly indicates the reduced attachment is due to inhibition of f-actin. The Rho family of GTPases is centra l to the reorganization of the actin cytoskeleton. Two key members are Rac1 and Cdc42. They participate in the control of cell migratio n, endocytosis and antigen presentation (43, 124). As determined by western blot analysis, curc umin reduces the activity of these
78 GTPases and so modulates the function of DCs in part through inhibition of actin signaling. The reduction in endocytosis obs erved in curcumin-treated cells may be due in part to the inhibition of actin pathway signaling. This is evidenced in figure 28 in which curcumin-treated cells show a similar level of endocytosis to that of CytoB-treated cells.
79 CONCLUSION Curcumin reduces the DC response to immune stimulants by reducing cell maturation and preventing normal cell functi on. It causes reduced expression of the surface markers CD86, CD84, HLADR, CD40 as well as the adhesion molecules CD11c and CD54 in response to a variety of external stimulants when compared to the untreated controls. St imulant-induced cytok ine and chemokine production was also reduced as a result of curcumin treatment. Functional aspects of DC maturation were also affe cted by curcumin. The high endocytic capacity of iDCs was significantly reduced in curcumin culture. Chemotaxis and DC-induced T cell proliferation was al so abrogated. The few T cells that proliferated in co-culture expressed CD25 and Foxp3 indicating a regulatory population and curcumin may induce a to lerogenic DC. Curcumin affects cell architecture by inhibiting f-actin.
80 LIMITATIONS OF THE STUDY The major limitation of this study wa s the use of donors from Florida blood services. Without access to medical record s or individual medica l history, we are left to assume the participants were in good health. There is also the aspect of human subject variability. We elected to use a repeated measures analysis approach for data analysis in an effort to cont rol for this variability. There is no established standard for the response of a donor to curcumin. The data was log transformed to ensure a normal distribut ion for statistical analysis. The age, gender and ethnicity of the donors may have played a role in the noted variation in responses. A larger sample size would be needed to address these aspects of the study. The concentrations of curcumin used in the study, though non-toxic, are not physiological. Due to the pharmacoki netic properties of curcumin and low bioavailability, the experimental concentra tions can not be achieved through daily ingestion. In order to achieve these levels in the circulation, a special formulation may need to be engineered to prevent bi odegradation. The method of delivery needs to be taken into account as well. When administered systemically curcumin will affect multiple cell types To reproduce the DC-specific effects observed here, a formulation needs to be developed that will target curcumin specifically to DCs.
81 FUTURE DIRECTIONS Further investigation into the generat ion of tolerogenic DCs by curcumin would be important in elucidating the immu nosuppressive role of curcumin. This would include further characterizati on of the regulatory T cell population generated in co-culture. The DC to T cell ratio can be adjusted to examine the effects on the phenotype of these cells. This phenomenon needs to be examined in vivo using a mouse model of allergic asthma. If curcumin-DCs induce a regulatory T cell population, then the Th2 type response should be reduced. Using curcumin to target DCs and induc e immune tolerance would make it an effective treatment for inflammation. Further investigation into the effects of curcumin on the actin cytoskeleton needs to be carried out as well. It would be important to dete rmine if curcumin binds to any members of the actin cyto skeleton pathway. Disruption of f-actin organization affects cell attachment and f unction. Elucidation of the underlying mechanism is important for the de velopment on novel curcumin based therapeutics.
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101 Appendix A: List of Definitions Adaptive immunity : host defenses that are medi ated by T and B cells after exposure to antigen that generate a specific or memory response or self/non-self recognition. Allergen : non-infectious antigen that induces a hypersensitivity or allergic reaction. Allogeneic: individuals of the same spec ies that differ genetically. Antigen: any molecule that can bind to an antibody. Some have the ability to stimulate antibody production. Chemokine: small proteins that induce that mediate chemotaxis by regulating the expression of leukocyte integrins. Chemo-attractant: a substance that attracts leucocytes Chemotaxis: directional movement of cells. CD4: cluster of differentiation 4 is present on thymocytes, monocytes, macrophages and is a co-receptor of MHC Class II CD11c : cluster of differentiation 11c is present on myeloid cells and binds fibrinogen. It can be used to identify dendritic cells. CD80: cluster of differentiation 80 is pres ent on dendritic cells and is a ligand for CD28 on T cells. It is important in the activation of nave T cells through costimulation (signal 2).
102 Appendix A (Continued) CD83: cluster of differentiation 83 is present on dendritic, B and Langerhans cells. It is a marker for activated dendr itic cells and may assist in antigen presentation or cellular interactions that follow lymphocyte activation CD86: cluster of differentiation 86 is present on dendritic, monocytes and activated B cells and it is a ligand for CD 28 on T cells. It is important in the activation of nave T through co-stimulation (signal 2). CD54: cluster of differentiation 54 is present on hematopoietic and nonhematopoietic cells. Also known as inte rcellular adhesion molecule (ICAM-1) and is involved in adhesion of neutrophils and is a receptor for rhinovirus. Cluster of differentiation (CD): a monocolonal antibody t hat identifies surface molecules, used to identify various immune cells. Cytokine: low molecular weight proteins t hat regulate the intensity and duration of the immune response by affecting the functions on other immune cells as well as the cell that produces it. Dendritic cell (DC): professional antigen present ing cell and the most potent activator of T and B cells. They are link the innate and adaptive immune systems and reside in tissues exposed to the extern al environment such as the skin, lungs and intestines. Dimethyl sulfoxide (DMSO): a chemical compound that is a polar aprotic solvent. It is an excellent solvent and cryoprotectant.
103 Appendix A (Continued) Flow cytometry: a technique used to count, examine and sort microscopic particles or cells suspended in a fluid str eam. It uses the principles of light scattering, light excitation, and emission of fluorochrome molecules to generate specific multi-parameter data. Homotypic cluster formation: clustering of dendritic cells with each other that is an indicator of increased expression of adhesion molecules, chemokines and chemokine receptors. Human leukocyte antigen DR (HLA-DR): human leukocyte antigen -DR, the term for MHC in humans is required fo r antigen presentation to T cells. Innate immunity: non-specific host defense to invading pathogens such as bacteria, virus and allergens that incl udes various recognition systems such as toll-like receptors, endocytic, phagocytic and inflammatory mechanisms. The innate immune response does not increase with repeated exposure. Interferon gamma (IFN ): cytokine that can induce cells to resist viral replication. It is produced by CD4+ Th1 ce lls and is a type II interferon and has antiviral, immunoregulatory, and anti-tumor properties. Interferon-inducing protein 10 (IP-10): chemokine that selectively attracts Th1 lymphocytes and monocytes, and inhibits cytokine-stimulated hematopoietic progenitor cell proliferati on. Also called CXCL10.
104 Appendix A (Continued) Interleukin 2 (IL-2): a T cell derived cytokine t hat stimulates growth and differentiation of T cells, B cells, NK cells, monocytes, macrophages. A central cytokine in the development of an adaptive immune response. Interleukin 4 (IL-4): a cytokine that is secreted by Th2 cells. It enhances both secretion and cell surface expression of IgE and IgG1. An important cytokine in allergic disease and has overlapping functions with IL-13. Interleukin 5 (IL-5): secreted by Th2 and mast cells that is a key mediator in eosinophil activation. An important cytokine in allergic disease. Interleukin 6 (IL-6): both a pro-inflammatory and anti-inflammatory cytokine. Important mediator of fe ver and of the acute phase re sponse and secreted in response to activation of the innate i mmune system. High IL-6 It is associated with both Th1 and Th2 responses and low I L6 is associated with Treg responses. Interleukin 8 (IL-8): inflammatory chemokine produced by many cell types. Mainly functions as a neutrophil chemoattractant. Interleukin 10 (IL-10): secreted by Th0 cells blocks cytokine synthesis by Th1 cells. Interleukin 12 p70 (IL-12p70): Th1 polarizing type cytokine secreted mainly by dendritic cells. Induces IFN production and proliferatio n/differentiation of Th1 cells. Interleukin 13 (IL-13): secreted by Th2 cells, involved in the up-regulation of IgE secretion by B cells. An important cytokine in allergic disease.
105 Appendix A (Continued) Ligand: general term for a molecule recognized by a receptor. MHC Class II: major histocompatibi lity complex class II present on antigen presenting cells with the primary functi on to present peptide antigens, both self and non-self, to lymphocytes (T cells) fo r the purpose of eliciting an immune response. Mixed lymphocyte reaction (MLR): lymphocytes from two individuals are cultured together for several days in or der to induce T ce ll proliferation. Monocyte: mononuclear phagocytic leukocyte Monocyte-derived dendritic cells (mdDCs): dendritic cells obtained by culturing CD14+ monocytes with granul ocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL4). Phagocytosis: the engulfment of particles, bacteria, cell debris, etc. by cells. A characteristic function of ma crophages and dendritic cells. Polyinosinic-polycytidylic acid (Poly I:C): a commercially available synthetic double stranded RNA. A toll-like receptor 3 ligand that mimics viral infection and induces a Th1 response. T cells (TC): thymic lymphocytes that developed in the thymus. T cell proliferation: The reproduction of a T cell to produce two daughter cells. Nave T helper lymphocyte (Th0): T lymphocytes that have never engaged a specific antigen.
106 Appendix A (Continued) T helper type 1 lymphocyte (Th1): T lymphocyte characterized by the cytokines they produce (especially IFN ). An increase in Th1 cytokines are associated with autoimmune diseases. T helper type 2 lymphocyte (Th2): T lymphocyte characterized by the cytokines they produce (especially I L4, IL5, IL13). An increas e in Th2 cytokines are associated with allergic diseases. Toll-like receptor (TLR): recognize pathogen-associated molecular patterns (PAMPs) that are expressed on infecti ous agents. They mediate the production of cytokines necessary for the dev elopment of effective immunity. Toll-like receptor 3 (TLR3): one of the toll-like recept ors that recognizes double stranded RNA of viruses. Toll-like receptor 4 (TLR4): one of the toll-like receptor s that recognize bacterial lpipoplysachharide on Gram-negative bacteria. T regulatory lymphocyte (Treg): T lymphocyte characterized by the cytokines they produce (IL10, TGF). They have the ability to inhibit T cell responses and induce tolerance. Tumor necrosis factor alpha (TNF): a pleiotropic inflammatory cytokine involved in apoptotic cell death/ proliferation, differentia tion, inflammation, tumor growth, and viral replication. Viability: ability of the cell to survive.
107 Appendix B: List of Pub lications by the Author Primary Articles Shirley, S.A. Montpetit, A.J., Lockey, R.F., M ohapatra, S.S., Curcumin prevents human dendritic cell respons e to immune stimulants, Biochem Biophys Res Commun 2008 Sep 26; 374(3):431-6. Epub 2008 Jul 17. Lee, D., Zhang, W., Shirley, SA. Kong, X., Hellermann, GR., Lockey, RF., Mohapatra, SS. Thiolated chitosan/DNA nanocomplexes exhibit enhanced and sustained gene delivery, Pharm Res 2007 Jan;24(1):157-67. Epub 2006 Nov 14. Lee, DW., Shirley, SA ., Lockey, RF., Mohapatra, SS. Thiolated chitosan nanoparticles enhance anti-inflammatory ef fects of intranasally delivered theophylline. Respir Res 2006 Aug 24(7):112. Review Articles Mohapatra, SS., Lockey, RF., Shirley, S. Immunobiology of Grass Pollen Allergens, Current Allergy and Asthma Reports 2005 Sept: 5(5) 381-7.
108 Appendix B (Continued) Abstracts/Posters Conference Proceedings S. Shirley A.J. Montpetit, R.F. Lockey, S. S. Mohaptra, Curcumin Modulates LPS-Induced Inflammation in Human Dendritic Cells T he Journal of Allergy and Clinical Immunology, February 2008 (Vol. 121, Issue 2, Page S10) A.J. Montpetit, S.A. Shirley R.F. Lockey, S.S. Mohapatra, Cigarette Smoke Condensate affects Dendritic Cell (DC) Maturation by Modula ting DC-Epithelial Cell Cross-talk, The Journal of Allergy and Clinical Immunology February 2008 (Vol. 121, Issue 2, Page S234) X. Kong, S. Song, X. Wang, W. Xu, S. Shirley R.F. Lockey, S.S. Mohapatra, Bone Marrow Derived Stem Cells Reduce Lung Inflammation in a Mouse Asthma Model The Journal of Allergy and Clinical Immunology February 2008 (Vol. 121, Issue 2, Page S132) Shawna Shirley Alison Montpetit, Richard Lockey and Shyam Mohapatra, Curcumin modulates human dendritic cell function USF Health Research Day 2008
109 Shyam S. Mohapatra; Xiaoyuan Kong; Xi aoqin Wang; Weidong Xu; Jia-Wang Wang; Gary Hellermann; Raji Singham; Shawna Shirley ; Prasanna Jena; Weidong Zhang; Subhra Mohapatra; Richard F. Lockey; William Gower, A critical role for atrial natriuretic peptide re ceptor signaling in allergic disease Abstracts of the XX World Allergy Congress (TM) 2007 December 2-6, 2007, Bangkok, Thailand: ORAL ABSTRACT SESSION S: MECHANISMS OF ASTHMA I: 26 Vanesa Fal-Miyar, Arun Kumar, Shyam Mohapatra, Shawna Shirley, Natalie A. Frey, Jos M. Barandiarn, and Galina V. Kurlyandskaya, Giant Magnetoimpedance for Biosens ing in Drug Delivery AIP Conf. Proc.,Biomagnetism and magn etic biosystems based on molecular recognition processes June 19, 2008,Volume 1025, pp. 131-138 X. Wang, W. Xu, X. Kong, S. Shirley R. Lockey, S. Mohapatra, siRNA Targeting the Natriuretic Peptide Receptor-A Pr events Airway Inflammation in a Mouse Model of Allergic Asthma, AAAAI Annual Meeting San Diego, California, 2007 Shawna Shirley Arun Kumar, Prassanna Jenna, Sumita Behera, Richard F. Lockey and Shyam S. Mohapatra Multifunctional Nanoparticl es for Specific Cell Targeting and Their Commercialization, Micro and Nanotechnology Commercialization and E ducation Foundation (MANCEF) Commercialization of Micro and Nano Systems Conference St. Petersburg, Florida, 2006
110 Appendix B (Continued) Shawna Shirley Xiaoyuan Kong, Rajeswari Singham, Weidong Xu, Xiaoqin Wang, Richard F. Locke y and Shyam S. Mohapatra Modulation of Immune Response by a Novel Kaliuretic Peptide, AAAAI Annual Meeting Miami, Florida 2006 D. Lee, S. Shirley X. Kong, R. F. Lockey, S. Mohapatra, Thiolated Chitosan Nanoparticles Enhanced Anti-Inflammatory Effects of Intranasally Delivered Theophylline, AAAAI Annual Meeting Miami, Florida, 2006 Shawna Shirley Weidong Zhang, Xiaoyuan K ong, Richard F. Lockey and Shyam S. Mohapatra, Development of a Murine Model of Chronic Airway Disease, University of South Florida Health Sciences Center Research Day 2005
111 Appendix C: First Author Publication Curcumin prevents human dendritic cel l response to immune stimulants Shawna A. Shirley, M.S.a, Alison J. Montpetit, M.S.b, R.F. Lockey, M.D.c and Shyam S. Mohapatra Ph.D.c a Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612 b College of Nursing, University of South Florida, Tampa, FL33612 c Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida and VA Hospital Medical Center, Tampa, Florida 33612. Corresponding Author: Shyam S. Mohapatra, PhD., Division of Allergy and Immunology, Department of Internal Medicine, College of Medicine, University of South Florida, MDC 19, 12901 Bruce B. Downs Blvd., Tampa, Florida 33612, USA. Tel: (813) 974-8568 Fax: (813) 974-8575 Email: firstname.lastname@example.org
112 Appendix C (C ontinued) ABSTRACT Curcumin, a compound found in the Indian spice turmeric, has antiinflammatory and immunomodulatory proper ties, though the mechanism remains unclear. Dendritic cells (DCs) are im portant to generating an immune response and the effect of curcumin on human DCs has not been explored. The role curcumin in the DC response to bacterial and viral infection was investigated in vitro using LPS and Poly I:C as models of infection. CD14+ monocytes, isolated from human peripheral blood, were cu ltured in GM-CSFand IL-4-supplemented medium to generate immature DCs. Cu ltures were incubated with curcumin, stimulated with LPS or Poly I:C and func tional assays were performed. Curcumin prevents DCs from responding to imm unostimulants and inducing CD4+ T cell proliferation by blocking maturation mark er, cytokine and chem okine expression and reducing both migration and endocytosis. These dat a suggest a therapeutic role for curcumin as an immune suppressant.
113 Appendix C (C ontinued) INTRODUCTION Curcumin is a biologically active compound found in the Indian spice turmeric. It belongs to a family of compounds called curcuminoids and usually comprises about 3% of turmeric powder. The spice is commonly used as a preservative for foods and a yellow dye or coloring for textiles and as an ingredient in pharmaceuticals and cosmeti cs. For many centuries curcumin has been used as an antiseptic, analgesic, appet ite suppressant, anti-inflammatory agent, anti-oxidant, anti-malarial, and insect repellant [1,2] The pharmacological potential of curcumin is under investigati on. Researchers found that it has antiinflammatory, anti-oxidant, anti-parasitic anti-viral, and anti-cancer properties [3 6] It targets transcription factors, cytok ines, cell adhesion molecules, surface receptors, growth factors, and kinases, among other molecules  and directly binds to a variety of surface and intracellular proteins causing direct cellular pathway inhibition or activa tion of secondary cellular responses [2,11] Dendritic cells are the sentinels of the immune system and regulate the immune response. Immature or resting dendr itic cells reside in peripheral organs where they monitor the surrounding tiss ue for invading microorganisms. They alert the immune system to the pres ence of pathogens by engulfing them, processing the foreign proteins and presenting the pepti de fragments on their surface. After DCs are activated, they mature and migrate to the lymphoid tissue where they prime T lymphocytes and stim ulate a specific or adaptive response
114 Appendix C (C ontinued) [12,13] Maturation of dendritic cells invo lves changes in gene expression and activation of signaling pathways, and it is reasonable to hypothesize that curcumin can modulate some of t hese pathways and thereby prevent DC maturation and alter function. While a study by Kim et al. reveals that curcumin impairs the immunostimulatory function of murine dendritic cells  its effects on human dendritic cells remain unknow n. Curcumins effects on human DC stimulation are examined in this st udy. Modulating the DC response could provide an effective approach to treat and control unwanted inflammation.
115 Appendix C (C ontinued) MATERIALS AND METHODS Reagents. Curcumin (from Curcuma longa ) was obtained from Sigma Aldrich (St. Louis, MO) and dissolved in DMSO. Buffy coats were obtained from Florida Blood Services (St. Petersburg, Florida). Six donors, four males and two females, in good health and ranging in age from 18 to 50 were used for the study. Cell isolation reagents CD14 micr obeads and CD4+ T cell isolation kit were obtained from Miltenyi Biotec (Auburn, CA). Histopaque_-1077 and was obtained from Sigma Aldrich and recombinant human cytokines GM-CSF and IL4 were obtained from PeproTech (Rocky Hill NJ). All other cell culture reagents were obtained from GIBCO Invitrogen (C arlsbad, CA). LPS, poly I:C and PHA were obtained from Sigma Aldrich (St. Louis, MO). CFSE and Alexa-647 conjugated dextran (molecular weight 10,000) were obtained from Molecular Probes Invitrogen (Carlsbad, CA). LINCOplex Multiplex cytokine assay kits were purchased from Millipore (Tem ecula, CA). All CD11c, HLA-DR, CD86, CD83, and CD54 antibodies were obtained from BD Biosciences (San Jose, CA). CCL19 and CCL21 were obtained from P eproTech (Rocky Hill, NJ). Cell Isolation and Culture. CD14+ monocytes were isolated and cultured as described by Picki et al.  Briefly, leukocytes we re extracted from buffy coats using Histopaque-1077. Monocytes expressing CD14 were positively selected with magnetic microbeads. Purity (> 90%) was verified by staining with anti-CD14 antibodies and analyzi ng by flow cytometry. Ce lls were cultured at 1
116 Appendix C (C ontinued) 106 cells/ml in complete RPMI (10% FBS, 1% pen/strep, 10 mM Hepes, nonessential amino acids and 5 mM sodium py ruvate) with 20 ng/ml each rh IL-4 and GM-CSF for 56 days, (supplementing at day three with fresh medium). Nonadherent and loosely adherent cells were removed on day five for analysis or stimulation. On day 5, more than 90% of the harvested cells expressed CD11c and HLA-DR. CD4+ T cells were isolated from the CD14+ fraction remaining after monocyte depletion and cultured in co mplete RPMI. Purity was confirmed by flow cytometry after CD4 staining. Cell Treatment and Stimulation Curcumin was added to cell culture (1 106 cells/ml and 3 ml/well in 6-well plates ) at concentrations of 20 or 30 M. DSMO was used as a control. After a 1 h incubation, LPS (1 g/ml) or Poly I:C (25 g/ml) was added to the appropriate we lls. Control wells received neither. Cultures were incubated overnight at 37 C and 5% CO2/95% air. Cell viability was 95% 0.06 after 24 h of culture under all conditions listed above as determined by a viability assay using 7AAD incorporation. Flow Cytometry Cells were collected, washed and stained with fluorochrome-conjugated antibodies specific for DC surface markers. Cells were analyzed using the Becton Dickenson (BD) Canto II with HTS sampler and BD FACSDivaTM software.
117 Appendix C (C ontinued) Cytokine Assay Culture supernatant was co llected and cytokine levels measured using the LINCOplex multiple x assay. Assays were performed in duplicate according to the m anufacturers instructions. Chemotaxsis Assay Treated and stimulated cells were collected, counted and re-suspended at a concentration of 1 106 cells/ml. Fifty microliters of cell suspension was placed in the upper cham bers of 5 m pore size polycarbonate filter inserts in a 96-well microchemotax is plate (Chemicon). The lower chambers contained 40 l of either CCL19 or CCL21 in 150 l of medium. Control wells had medium only. Input wells (in triplicate) contained 1 104 cells in the lower chambers without chemokines. Cells were incubated at 37 C and 5% CO2/95% air overnight. Migration was stopped by t he removal of the inserts. Polystyrene beads (1 104) were added to each well (lower chamber) and analyzed by flow cytometry. The number of ce lls in each sample and input was calculated using the following equation: Number of ce lls/well = (number of cell events number of bead events) 104. Input cells = average [number of input cells/well 5 (dilution factor)]. The percentage migration for eac h sample (% input) is determined by the following equation: Percent migration = (migrating cells input cells) 100. Mixed Lymphocyte Reaction CFSE labeling of CD4+ T cells was carried out by resuspending cells in 1ml PBS cont aining 5% (v/v) FBS. 1.1 l of the CFSE stock (5 M) was diluted in 110 l of PBS and quickly mixed with the cell suspension. After a 5 min incubation at room temperature, the reaction was
118 Appendix C (C ontinued) stopped by adding ten volumes of room te mperature PBS containing 5% (v/v) FBS and centrifuging at 300g for 5 min at 20 C. Cells were washed twice and resuspended in complete medium (1 106 cells/ml). The dendritic cell-T cell coculture was set up at a ratio of 1:16. Curcumin treated and stimulated DCs were removed from culture and placed in 96-well plates in triplicate (6.25 103 cells in 100 l per well). T cells (100 l) were added to each well and cultures incubated at 37 C and 5% CO2 /95% air for 5 days. CFSE fluorescence intensity was measured by flow cytometry using the BD Canto II with HTS attachment and BD FACS Diva software. Endocytosis Assay Cells were collected, washed and incubated with 1 mg/ml (per 1 106 cells) Alexa-647 conjugated dextran at either 4 C or 37 C for 1 h. Cells were washed with cold PBS and either analyzed by flow cytometry or plated on gelatin coated cover slips and imaged by confocal microscopy. The change in mean fluorescence intensity (MF I) is calculated as the difference between the MFI of 37 C and 4 C cultures. Microscopy All bright field images were captured using the 4 objective of an Olympus IX71 inverted fluorescent microscope with an attached DP70 camera. Fluorescent images were captur ed using the 63x objective of a Leica scanning confocal microscope. Statistical Analysis Data was log transformed to ensure normal distribution. Significance was dete rmined using paired t tests (repeated
119 Appendix C (C ontinued) measures) for planned comparisons with m odified Bonferroni correction. Data are presented as the average of six do nors. Error bars represent SEM with p values less than 0.05 consider ed statistically significant.
120 Appendix C (C ontinued) RESULTS AND DISCUSSION This is the first study to exami ne the effects of curcumin on human dendritic cells in vitro. Donors for t he study were selected at random and supplied by Florida Blood Services, St. Petersburg, Florida. The concentrations of curcumin used were based on those previously found efficacious in the literature and confirmed not to be toxic to the cells by viability assays (data not shown). All cultures remained more than 90% viable up to 24 h after curcumin addition. The pharmacokinetics and pharmacodymanics of curcumin have been more extensively studied in rodents than in humans  From the limited human data available, the low bioav ailability of curcumin limit s its clinical usefulness when administered orally. High doses can be administered without adverse effects but the systemic distributi on may not be sufficient to exert pharmacological activity. Combining cu rcumin with other compounds, or using drug delivery systems such as lip osomes and nanoparticles provide an alternative approach to overcome these issues  The immunostimulants lipopolysacchaide (LPS) and polyinosinic:polycytidylic acid (poly I:C) were used in this study to independently stimulate DC activation. LPS via the toll like receptor 4 (TLR4) pathway and poly I:C mimics viral infections through TLR3. These compounds were chosen to ensure the immunostimulatory effects were not TLR dependent. Observed stimulant effect s were found to be significant with p values <0.05 by paired t test compared to nonstimulated controls.
121 Appendix C (C ontinued) Curcumin Prevents Increased Maturati on Marker Expression and Cytokine Secretion Mature dendritic cells express elevat ed levels of co-stimulatory and antigen presenting molecules such as CD86, CD83 and HLA-DR on their surface. If the antigen pres enting machinery of DCs ar e impaired, they can not effectively engage the T cells to initiate a response. Stimulated curcumin-treated DCs do not significantly increase thei r surface expression of CD86 and CD83 above the control ( Fig. 1 A). HLA-DR surface expression is not significantly inhibited by 20 M curcumin. We can surmi se that curcumin affects the antigen presenting machinery by reducing co-stimu latory molecule expression but not the antigen presenting molecules. Mature m onocyte-derived DCs secrete IL-12, IL10, and other inflammatory cytokines. St imulated curcumin-treated DCs produce significantly lower levels of IL-12, IL-10, and TNF when compared to the controls ( Fig. 1 B) creating a Th2 permissive environment. IL-6 was also significantly reduced by curcumin ( data not shown). T hough the reduction of TNF was significant, they were not reduced to the levels of the controls. These findings correlate with those fr om the study by Kim et al.  which shows curcumin prevents immuno-stimulator y function of murine bone marrow-derived cells. They along with others show curc umin is a potent inhibitor of NFB and AP-1 activation as well as MAPK signaling [20,21] This provides a reasonable explanation for the observed reduction of IL -12 and IL-10 levels in this study.
122 Appendix C (C ontinued) TNF expression is controlled by other transcription factors such as lipopolysaccharideinduced TNF factor (LITAF)  or interferon regulatoryfactor 3 (IRF3)  that may not be affected by curcumin, allowing the transcription of some TNF independent of the NFB pathway. Curcumin Prevents DC-Induced CD4+ T Cell Proliferati on in the Mixed Lymphocyte Reaction The mixed lymphocyte reaction (MLR) is used as the basic test of DC function since it measures their ability to stimulate proliferatio n of an allogeneic T cell population. Studies show curcumin can inhibit MLR  Immature DCs will weakly stimulate prolif eration, while the mature DCs will induce a significantly more robust response ( Fig. 2 A). Increased expression of co-stimulatory markers is essential for T cell interaction and pro liferation. Curcumin-treated DCs, both stimulated and non-stimulated, show muted T cell proliferation ( Fig. 2 A). These observations and those in Fig. 1 A imply there are factors at play other than the expression levels of co-stimulatory and antigen presenting molecules. The regulation of the DC cytoskeleton is im portant in DCT Cell interactions  Little is known about the effect of cu rcumin on cytoskeletal rearrangement. One study reveals curcumin significantly alte rs the actin cytoskeleton in prostate cancer cells  Based on this premise, curcumin-induced alterations in DC cytoskeleton could account for the observations in Fig. 2 A. Analysis of the CD4 T
123 Appendix C (C ontinued) Cell cytokines produced in the MLR re vealed a Th2biased response evidenced by the increase in the IL-4:IFN ratio between untreated and curcumin treated cells ( Fig. 2 B). Curcumin Reduces Endocytosis Capture and presentation of antigen is an important feature of DC biology. This provides the link between innate and adaptive immunity. Immature DCs are highly endocytic, a feature which is lost when cells become mature. We find curcumin reduces endocytosis in non-stimulated DCs ( Fig. 2 C and D). There is a significant decrease in dextran uptake by non-stimulated cells treated with curcumin similar to stimulated cells, but not in stimulated cells. There are conflicting reports on the effects of curc umin on antigen capture; a few studies show increased endocytosis, while others show suppression  Our findings indicate curcumin interferes with antigen handlin g in human DCs. Curcumin Prevents Homotypic Cluste ring and Surface Adhesion Marker Expression DCs aggregate in clusters in response to stimuli as a visual sign of maturation  Cluster formation correlates with increased CD86, CD54, and CD80 expression. Here we show curc umin impairs homotypic DC cluster
124 Appendix C (C ontinued) formation in response to both LPS and poly I:C in a concentration-dependent manner ( Fig. 3 A). Adhesion molecules such as IC AM-1 (CD54) are important in cellular interactions and in generating T cell response. Murine antigen presenting cells (APCs) deficient in ICAM-1 have impaired ability to induce T cell responses [32,33] CD11c, a member of the in tegrin family of proteins, is also important for cell attachment and found in high levels on DCs. Curcumin significantly reduces expression of both mark ers on the DC surface ( Fig. 3 B and C). The reduced CD11c could be the result of curcumin-induced AP-1 inhibition  Curcumin Prevents DC Migration and Chemokine Secretion Mature DCs travel to the lymph nodes w here they present processed antigen to T cells. Migration towards chemoattrac tants is a featur e of mature DCs  They also secrete chemokines to attract res ponder cells to the site of injury or inflammation. Monocyte-derived DCs migrate in response to CCL19 or macrophage-inflammatory protein-3beta (MIP -3b) and CCL21 or exodus-2, which are expressed in the lymph nodes. Both chemokines bind to the CCR7 receptor on the DC surface. CCR7 expression is not affected by curcumin (data not shown). Curcumin prevents migrat ion towards CCL19 and CCL21 in a chemotaxis assay ( Fig. 4 A) and also reduces the levels of chemokines fractalkine (CX3CL1) and interf eron producing factor (IP-10) ( Fig. 4 B and C). Both fractalkine and IP-10 attract inflammatory cells to sites of inflammation. Poly
125 Appendix C (C ontinued) I:C stimulated cells did not migrate in re sponse to the chemokines, even in the absence of curcumin (data not shown). By preventing DC migration, curcumin reduces the probability of the DC encountering T cells to initiate a specific immune response. Reduced chemokine se cretion will stem the flow of inflammatory cell traffic to sites of inflammation.
126 Appendix C (C ontinued) CONCLUSIONS AND PERSPECTIVES Curcumin acts in several ways as an immune suppressor of human peripheral CD14+ monocytederrived DCs. It renders them non-responsive to the immuno-stimulants LPS and poly I:C by r educing expression of co-stimulatory and antigen presentation molecules expression and dampening the Th1-type response while promoting a Th2 permissive environment. It also reduces migration and adhesion molecule ex pression and reduces DC-induced proliferation of all ogeneic CD4+ T cells. The inhibiti on of transcription factors NFB and AP-1 and other cell signaling pathways by curcumin provide a plausible explanation for most of observations; however curc umin may be targeting other essential cellular pathways as well. Bas ed on our observations and reports from other studies, we speculate curcumin may be disrupting t he antigen handling and presenting machinery of DCs in additi on to transcription factor and signaling pathway inhibition  Elucidation of the mechani sm of action of curcumin immunosuppression could lead to clinical applications of this novel antiinflammatory agent.
127 Appendix C (C ontinued) ACKNOWLEDGEMENTS The authors would like to thank Ka roly Szekeres at the USF Flow cytometry core facility and Nancy Burke at the Moffitt analytic microscopy core facility. Thanks to Dr. Maureen Groer and Jason Beckstead at the USF College of Nursing and to Gary Bentley for his help in preparing this manuscript. This work is supported by the Joy McCann Cu lverhouse endowment to the University of South Florida, Division of Allergy and Immunology, VA Career Scientist Award and the Mabel and Ellsworth Simmons Professorship to SSM.
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134 Appendix C (C ontinued) FIGURE LEGENDS Fig. 1. Curcumin reduces maturation ma rker expression (A) and cytokine Production (B) in human monocyte-deriv ed dendritic cells. Surface marker expression is measured as mean fluorescenc e intensity (MFI) by flow cytometry. Cytokine levels were measured from cult ure supernatant usi ng a multiplex assay kit and expressed as log concentration. *Significance by one-tailed t-test of planned comparisons p < 0.05. Fig. 2. Curcumin reduces DC-induced prolifer ation of allogenieic CD4+ T cells (A) and promotes a Th2-permissive env ironment with an increased IL4:IFN ratio (B) in mixed lymphocyte reaction. Curcumin also reduces the endocytic capability of DCs. Endocytosis of fluorescently labeled dextran was measured by change in mean fluorescence intensity (MFI) between cells incubated at 37 C and 4 C (C) and imaged by confocal microscopy of a representative donor (D). *Significance by one-tailed t-test of pl anned comparisons p < 0.05. Fig. 3. Curcumin prevents homotypic clusteri ng of DCs in response to stimuli (A) and reduces surface adhesion molecule s CD54 (B) and CD11c (C). Surface marker expression is measured as mean fluorescence intensity (MFI) by flow cytometry. *Significance by one-tailed ttest of planned comparisons p < 0.05.
135 Appendix C (C ontinued) Fig. 4. Curcumin prevents DC migr ation towards CCL19 and CCL21 (A) and chemokine secretion in cu lture (B, C). Chemotaxis assay performed in 96-well microchemotaxis plate with 5 lm pore size polycarbonate filters. Chemokine levels were measured from culture s upernatant using a multiplex assay kit. *Significance by one-tailed t-test of planned comparisons p < 0.05.
136 Appendix C (C ontinued) FIGURES Figure 1. No StimLPSPoly I:C 0 1 2 3 4 5 *Log IL-10 No StimLPSPoly I:C 0 1 2 3 4 5 *Log TNF No StimLPSPoly I:C 0 1 2 3 4 5 *Log IL-12p70B A No StimLPSPoly I:C 2.5 3.5 4.5 5.5 *Log MFI No StimLPSPoly I:C 25 35 45 55 65 *Log MFI No StimLPSPoly I:C 2.5 3.5 4.5 5.5 *Log MFIDMSO Cur 20 M Cur 30 M CD83 CD86 HLA-DR
137 Appendix (Continued) Figure 2. B C 0 D No StimLPSPoly I:C 0 10000 20000 30000 Change in MFI* *DMSO Cur 20 M C DMSO Cur 20 M Cur 30 M No StimLPSPoly I:C 0 10 20 30 40 50 * *% CD4+ TC Proliferation A IL4:IFN No StimLPSPoly I:C 0 1 2 3 4Log Concentration No Stim Cur 20M LPS
138 Appendix C (C ontinued) Figure 3. A B CDMSO Cur 20 M Cur 30 M LPS Poly I:C No Stim Cur 0M Cur 20M Cur 30M No StimLPSPoly I:C 2.5 3.5 4.5 5.5 6.5 *Log MFI No StimLPSPoly I:C 2.5 3.5 4.5 5.5 6.5Log MFI *Surface CD11c Surface CD54
139 Appendix C (C ontinued) Figure 4. DMSOCur 20 MLPSCur 20 M+LPS 0 10 20 30 40 Medium CCL21 CCL19 *Percent MigrationA B C Experimental Conditions Fractalkine No StimLPS 0.0 0.5 1.0 1.5Log Concnetration IP-10 No StimLPS 0 1 2 3 4 5 DMSO Cur 20 M *Log Concentration
ABOUT THE AUTHOR Shawna Ann Shirley was born in St. Andrew, Jamaica in 1980. She earned a Bachelors degree in Biochemistr y with a minor in C hemistry from the University of the West Indies, Mona campus (Honors) in 2001. She then became a teacher of Mathematics and Science at Ardenne High School in St. Andrew Jamaica. After two years in the pr ofession she decided to pursue higher education in research. She moved to Tampa, Florida in 2003 to join the Multidisciplinary Biomedical Sciences Ph.D program at the Un iversity of South Florida, College of Medicine. She ear ned a Master of Science in Medical Sciences in 2007 and went on to complete the Ph.D. degree in Medical Sciences with a specialty cognate in Molecular Medi cine. Shawna is married to Karl Gilman, a teacher who is currently pursing a career as a mental health counselor.
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Shirley, Shawna A.
The role of curcumin in human dendritic cell maturation and function
h [electronic resource] /
by Shawna A. Shirley.
[Tampa, Fla] :
b University of South Florida,
Title from PDF of title page.
Document formatted into pages; contains 139 pages.
Dissertation (Ph.D.)--University of South Florida, 2008.
Includes bibliographical references.
Text (Electronic dissertation) in PDF format.
ABSTRACT: Curcumin is the yellow pigment found in the Indian spice curry. It has anti-inflammatory, ant-oxidant, anti-cancer, anti-viral, anti-bacterial and wound healing properties. It is widely used in industry for its flavor as a spice and as a coloring agent because of its brilliant yellow color. It is also used as a dye for textiles and as an additive to cosmetics. Dendritic cells (DCs) are the sentinels of the immune system and functions as the bridge between the innate and adaptive immune response. The effect of curcumin on DCs is poorly understood. A study shows curcumin prevents the immuno-stimulatory function of bone marrow-derived murine DCs, but no study examines the effects on human DCs. This study investigates the effects of curcumin on immature human DC maturation and function in response to immune stimulants lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly I:C).Human CD14+ monocytes isolated from the peripheral blood of donors are cultured with GM-CSF and IL-4 supplemented media to generate immature DCs. The cultures are treated with curcumin, stimulated with the above mentioned stimulants then functional assays performed. These assays include homotypic cluster formation, surface marker expression, cytokine production, chemotaxis, endocytosis, DC-induced allogeneic CD4+ T cell proliferation after mixed lymphocyte reaction, gene expression analysis and immuno-fluorescence labeling and imaging. Curcumin-induced changes in gene expression indicate the actin cytoskeleton signaling pathway is a target. Immuno-fluorescence labeling and imaging of f-actin was carried out. Curcumin reduces DC maturation in response to the stimulants used in the study. Expression of surface markers, cytokines and chemokines is reduced as well as DC-induced stimulation of allogeneic CD4+ cells after MLR.Curcumin prevents chemotaxis without affecting chemokine receptor expression and significantly reduces endocytosis in non-stimulated cells. Curcumin-treated DCs do not induce a Th1 or Th2 population in allogeneic MLR but induces a CD25+Foxp3+ regulatory cell population. Immuno-fluorescence imaging shows curcumin causes the cell to become more rounded. These data imply that curcumin inhibits f-actin polymerization and thereby prevents DC maturation and function in response to stimulation. This outlines a novel role for curcumin as an immune suppressant and shows its therapeutic potential as an anti-inflammatory agent.
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Co-advisor: Shyam S. Mohapatra, Ph.D.
Co-advisor: Richard Heller, Ph.D.
x Molecular Medicine
t USF Electronic Theses and Dissertations.