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Major tea catechin inhibits dendritic cell maturation in response to microbial stimulation

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Title:
Major tea catechin inhibits dendritic cell maturation in response to microbial stimulation
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English
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Rogers, James L
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Dendritic cells
EGCG
TNFa
Toll-like Receptors
MHC
CD86
CD40
IL-12
Dissertations, Academic -- Molecular Medicine -- Doctoral -- USF   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Dendritic cells (DCs) are a migratory group of bone-marrow-derived leukocytes specialized for uptake, transport, processing and presentation of antigens to T cells. Exposure of DCs to bacterial pathogens can induce DC maturation characterized by cytokine production, up-regulation of co-stimulatory molecules and an increased ability to activate T cells. DCs have the ability to restrict growth of L. pneumophila (Lp), an intracellular Gram-negative bacillus that causes a severe form of pneumonia known as Legionnaires' disease, in murine ER-derived organelles (121) but replicate in human DCs (145). Even in human cells, however, lysis of the DCs does not occur for at least 24 hours which may allow DCs time to participate in the transition from innate to adaptive immunity (145).^ The primary polyphenol in green tea extract is the catechin (-)-epigallocatechin-3-gallate (EGCG) which accounts for most of the numerous reported biological effects of green tea catechins, including anti-bacterial, anti-tumor, and neuroprotective effects. Primary murine bone marrow derived DCs from BALB/c mice were treated in vitro with Lp, or stimulated for comparison with Escherichia coli lipopolysaccharide (LPS). CD11c, considered an important marker of mouse DCs, and surface expression of co-stimulatory molecules CD40, CD80, CD86, as well as class I/ II MHC molecules was determined by flow cytometry. Treatment of the cells with EGCG inhibited the microbial antigen induced up-regulation of CD11c, CD40, CD80, CD86 and MHC I/ II molecules. EGCG also inhibited, in a dose dependent manner, induced production of the Th1 helper cell activating cytokine, IL-12, and the chemokines RANTES, MIP1a, and MCP-1. However, EGCG upregulated TNFa production.^ In addition, EGCG inhibited both Lp and LPS induced expression of both TLR2 and TLR4 as well as LPS-induced NF-kB activation; all of which are important mediators of DC maturation. The modulation of phenotype and function of DCs by EGCG has implications for host interaction with microbial pathogens like Lp, which involve TLR interaction.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2007.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
Statement of Responsibility:
by James L. Rogers.
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Title from PDF of title page.
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Document formatted into pages; contains 90 pages.
General Note:
Includes vita.

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usfldc doi - E14-SFE0002176
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Major tea catechin inhibits dendritic cell maturation in response to microbial stimulation
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ABSTRACT: Dendritic cells (DCs) are a migratory group of bone-marrow-derived leukocytes specialized for uptake, transport, processing and presentation of antigens to T cells. Exposure of DCs to bacterial pathogens can induce DC maturation characterized by cytokine production, up-regulation of co-stimulatory molecules and an increased ability to activate T cells. DCs have the ability to restrict growth of L. pneumophila (Lp), an intracellular Gram-negative bacillus that causes a severe form of pneumonia known as Legionnaires' disease, in murine ER-derived organelles (121) but replicate in human DCs (145). Even in human cells, however, lysis of the DCs does not occur for at least 24 hours which may allow DCs time to participate in the transition from innate to adaptive immunity (145).^ The primary polyphenol in green tea extract is the catechin (-)-epigallocatechin-3-gallate (EGCG) which accounts for most of the numerous reported biological effects of green tea catechins, including anti-bacterial, anti-tumor, and neuroprotective effects. Primary murine bone marrow derived DCs from BALB/c mice were treated in vitro with Lp, or stimulated for comparison with Escherichia coli lipopolysaccharide (LPS). CD11c, considered an important marker of mouse DCs, and surface expression of co-stimulatory molecules CD40, CD80, CD86, as well as class I/ II MHC molecules was determined by flow cytometry. Treatment of the cells with EGCG inhibited the microbial antigen induced up-regulation of CD11c, CD40, CD80, CD86 and MHC I/ II molecules. EGCG also inhibited, in a dose dependent manner, induced production of the Th1 helper cell activating cytokine, IL-12, and the chemokines RANTES, MIP1a, and MCP-1. However, EGCG upregulated TNFa production.^ In addition, EGCG inhibited both Lp and LPS induced expression of both TLR2 and TLR4 as well as LPS-induced NF-kB activation; all of which are important mediators of DC maturation. The modulation of phenotype and function of DCs by EGCG has implications for host interaction with microbial pathogens like Lp, which involve TLR interaction.
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Major Tea Catechin Inhibits Dendritic Cell Maturati on in Response to Microbial Stimulation by James L. Rogers A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Medicine College of Medicine University of South Florida Major Professor: Thomas W. Klein, Ph.D. Nicholas Burdash, Ph.D. Peter Medveczky, M.D. Alberto Van Olphen, D.V.M.,Ph.D. Date of Approval: September 28, 2007 Keywords: Dendritic cells, EGCG, IL-12, TNF, CD86, CD40, MHC, Toll-like receptors Copyright 2007, James L. Rogers

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DEDICATION This dissertation is dedicated to my mother whose l oving support has made my studies possible.

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AKNOWLEDGEMENTS A PhD is not something that one gets alone, and the re are many professors and highly skilled technical staff as well as classmate s at USF Medical College who made this dissertation possible for me. However, pa rticular mention must be made of my major professor Dr. Thomas Klein and co-advisor Dr. Herman Friedman not only for their hard work in reviewing my work and s etting goals but most of all for their inspiration and wisdom. In this same line of thought particular thanks is given to Izabella Perkins in the lab and my committee mem bers Dr. Nicholas Burdash, Alberto Van Olphen and Peter Medveczky as well as A mal Hakki and Ilona Friedman who were all instrumental in preparation o f several of my papers. Additional thanks is given to Dr. Ray Widen for his assistance in the actual running of numerous FACS experiments. Other thanks is given to the rest of Dr. Klein’s and Dr. Freidman’s team including but not limited to Ca therine Newton, M.S., fellow classmates and other professors at the USF College of Medicine such as Dr. Burt Anderson, Dr. Susan Pross, and Dr. Ken Ugen. Additi onal thanks is given to many of the medical college staff such as Kathryn Zhan a nd the department chairman, Larry Solomonson, for their support in administrati ve matters.

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i TABLE OF CONTENTS LIST OF TABLES.............................................................................................................iv LIST OF FIGURES.............................................................................................................v LIST OF ABBREVIATIONS..........................................................................................viii ABSTRACT....................................................................................................................... ..x INTRODUCTION...............................................................................................................1 EGCG.......................................................................................................................1 Sources and Structure..................................................................................1 Antibacterial Activity of EGCG..................................................................2 Effects on Cytokine Production...................................................................2 Dendritic Cells.........................................................................................................4 Functions in Immunity.................................................................................4 DC Maturation and the Immune Response..................................................6 Phenotypic Changes Associated with DC Maturation.............................................7 Introduction..................................................................................................7 MHC Molecules...........................................................................................8 Co-Stimulatory Molecules...........................................................................8 Functional Changes Associated with DC Maturation..................................9 Cytokine Induction and Asso ciated Biological Functions...........................9 Chemokines................................................................................................10 Chemokine Receptors................................................................................13 Microbial Factors and Dendritic Cell Maturation..................................................15 Lipopolysacharide (LPS)...........................................................................15 Peptidoglycan/Murymyldipeptide (MDP).................................................15 L. pneumophila (Lp)..................................................................................16 Toll-Like Receptors...............................................................................................17 TLR2..........................................................................................................18 TLR4..........................................................................................................19 TLR5..........................................................................................................20 TLR9..........................................................................................................20 Molecular Mechanisms of Action of EGCG.........................................................20 TLR Signaling Effects...............................................................................20 MAPKs......................................................................................................20 NF B..........................................................................................................21 Antioxidant Properties of EGCG...........................................................................22 ROS and Redox Environment....................................................................23

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ii PROJECT SIGNIFICANCE..............................................................................................25 OBJECTIVES....................................................................................................................2 6 Aim 1: Determine the Effects of EGCG Treatment on Co-Stimulatory Marker Production in Response to Microbial Stimulation................................26 Aim 2: Determine the Effects of EGCG on DC Cytokine and Chemokine Production in Reponse to Microbial Stimulation..............................................27 Aim 3: Determine the Molecular Signaling Mechanisms Involved in Effects of EGCG on DC Maturation.................................................................28 MATERIAL AND METHODS.........................................................................................29 Catechins and Stimulants.......................................................................................29 Animals..................................................................................................................29 Preparation of DCs.................................................................................................29 Bacteria..................................................................................................................30 Infection.................................................................................................................30 Treatment...............................................................................................................30 Cell Viability..........................................................................................................31 Flow Cytometry (FACS)........................................................................................32 ELISA....................................................................................................................32 Bioplex Cytokine Assay........................................................................................34 P65/RelA Dna-Binding Activity............................................................................34 Statistics.................................................................................................................35 RESULTS........................................................................................................................ ..36 Aim 1: Determine the Effects of EGCG Treatment on Co-Stimulatory Marker Production in Response to Microbial Stimulation................................36 Lp Infection Induces CD11c, Co-Stimulatory Molecule and MHC Surface Molecule Expression..............................................................36 EGCG Inhibits CD11c, Co-Stimulatory Molecule and MHC Surface Molecule Expression Induced by Lp Infection.......................38 LPS Induces CD11c, Co-Stimul atory Molecules and MHC Surface Molecules That are Inhibited by EGCG Treatment.............................40 EGCG Treatment of DCs Al one Does Not Affect CD11c, Costimulatory Molecule or MHC Surface Expression........................41 Inhibitory Effects Not Due to Cytotoxity of EGCG..................................43 EGCG Treated DCs Exhibit th e Morphology of Immature DCs...............44 Aim 2: Determine Effects of EG CG on DC Cytokine and Chemokine Production in Reponse to Microbial Stimulation..............................................44 EGCG Up-Regulates TNF Production by DCs Stimulated with LPS, MDP or Infected with Lp............................................................44 EGCG Inhibits IL-12 Producti on by DCs Stimulated with MDP or LPS or Infected with Lp.......................................................................47 Inhibition of IL-12 by EGCG Does Not Depend on TNF .......................51 EGCG Inhibits RANTES, MCP1 and MIP1 Production by DC Stimulated with LPS............................................................................53

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iii EGCG Inhibits RANTES, MCP1 and MIP1 Production by DCs Infected with Lp...................................................................................55 Aim 3: Determine Molecular Signaling Mechanisms Involved in Effects of EGCG on DC Maturation.............................................................................58 Lp and LPS are Potent Indu cers of TLR2 and/or TLR4 Surface Molecule Expression............................................................................58 EGCG Inhibits Upregulati on of TLR2/TLR4 Surface Expression Induced by Lp and LPS........................................................................60 EGCG Inhibits NF B Activation by LPS..................................................61 DISCUSSION....................................................................................................................6 3 REFERENCES CITED......................................................................................................70 APPENDICES...................................................................................................................85 Appendix A. Permission Letters............................................................................86 ABOUT THE AUTHOR.......................................................................................End Page

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iv LIST OF TABLES Table 1: MHC I/II and Costimulatory molecule CD40, C86 surface molecule expression by DCs infected with Lp (10:1) and treat ed with various concentrations of EGCG and analyzed by flow cytomet ry.. .............................. 39

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v LIST OF FIGURES Figure 1. Diagram of the natural polyphenol classif ication and the chemical structure of green tea catechins. ................. ................................................... .... 1 Figure 2. DCs Direct an Immune Response.. ......... ................................................... .......... 6 Figure 3. Compared with the RPMI-1640 (untreated co ntrol), Astragalus mongholicus polysaccharides (ASP) or LPS treated DC show characteristic morphology of mature DC (needle-like protrusions).. ................ 8 Figure 4. Pathogens Induce Different Patterns of Ch emokine Expression .. .................... 12 Figure 5. Chemokine Receptor Expression on Dendriti c Cells.. ....................................... 1 4 Figure 6. Flow cytometric dot plot of CD11b and CD1 1c surface molecule expression by DCs. ................................ ................................................... ....... 36 Figure 7. Lp infection up-regulates CD40 and CD86 e xpression by DCs. Flow cytometric dot plots of CD11c and co-stimulatory mo lecule expression.. ...................................... ................................................... ............. 37 Figure 8. Lp infection up-regulates MHC class I/II epxression by DCs. Flow cytometric dot plots of CD11c and MHC I/II surface molecule expression ........................................ ................................................... ............. 38 Figure 9. EGCG inhibits Lp upregulation of MHC surf ace molecule expression by DCs infected with Lp and treated with various conce ntration of EGCG and analyzed by flow cytommetry.. ................. ............................................... 38 Figure 10. EGCG inhibits Lp upregulation of co-stim ulatory molecule CD40 and CD86 expression by DCs infected with Lp and treated with various concentrations of EGCG and analyzed by flow cytomet ry.. ........................... 39 Figure 11. EGCG inhibits CD40 and MHCII surface mol ecule expression by DCs stimulated with LPS and treated with 50 g of EGCG and analyzed by flow cytometry. ................................... ................................................... ......... 40 Figure 12. EGCG inhibits MHCI and CD86 surface mole cule expression by DCs stimulated with LPS and treated with 50 g of EGCG and analyzed by flow cytometry. ................................... ................................................... ......... 41

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vi Figure 13. Effects of EGCG on MHC class I/II molcul e expression by DCs as analyzed by flow cytometry. Numbers reflect percent ages rounded to next greater whole integer. ....................... ................................................... .... 42 Figure 14. Effects of EGCG on co-stimulatory molecu le expression by BMDCs as analyzed by flow cytometry.. ...................... ................................................... 42 Figure 15. BM derived DCs were exposed to various c oncentrations (0, 50, 100 g/ml) of EGCG for 24 h. Cell viability was analyzed with XTT assay. ....... 43 Figure 16. Effects of increasing concentrations of EGCG on TNF production in cultures of BM derived dendritic cells stimulated w ith LPS.. ......................... 45 Figure 17. Effects of increasing concentrations of EGCG on TNF production in cultures of BM derived dendritic cells stimulated with MDP. ....................... 46 Figure 18. Effects of EGCG on TNF production by dendritic cells infected 24 hr with Lp. .......................................... ................................................... .............. 47 Figure 19. Effects of ECGG on IL-12 p40/p70 product ion by BM derived dendritic cells stimulated by LPS. ................ ................................................... 48 Figure 20. Effects of increasing concentrations of EGCG on IL-12 p40/p70 production in cultures of BM-derived dendritic cell s stimulated with MDP. .............................................. ................................................... .............. 49 Figure 21. Effects of EGCG on IL-12 p40/p70 product ion by dendritic cells infected 24 hr with Lp. ........................... ................................................... ...... 50 Figure 22. Effects of EGCG (50 g/ml) on TNF production in cultures of DCs stimulated with LPS (10 ng/ml) with or without anti TNF neutralization antibody ........................... ................................................... ...... 51 Figure 23. Effects of EGCG (50 g/ml) on IL12 production in cultures of DCs stimulated with LPS (10 ng/ml) with or without anti TNF neutralization antibody (20 g/ml). ............................................ ..................... 52 Figure 24. Effects of EGCG on RANTES production by DCs stimulated by LPS (100 ng/ml). ...................................... ................................................... ............ 53 Figure 25. Effects of EGCG on MCP-1 production by D Cs stimulated by LPS (100 ng/ml). ...................................... ................................................... ............ 54 Figure 26. Effects of EGCG on MIP1production by DCs stimulated by LPS (100 ng/ml). ...................................... ................................................... ............ 55

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vii Figure 27. Effects of EGCG on RANTES production by DCs after infection by Lp ....... 56 Figure 28. Effects of EGCG on MCP1 production by DC s infected with Lp ................... 57 Figure 29. Effects of EGCG on MIP1 production by DCs infected with Lp ............... ... 58 Figure 30. Lp infection up-regulates TLR2/TLR4 surf ace expression on DCs infected with Lp.. ................................ ................................................... .......... 59 Figure 31. EGCG inhibits induced TLR2 on DCs infect ed with Lp or stimulated with LPS and treated with various concentrations of EGCG analyzed by flow cytometry. ................................ ................................................... ....... 60 Figure 32. EGCG inhibits induced TLR4 on DCs infect ed with Lp and treated with various concentrations of EGCG analyzed by flo w cytometry. .............. 61 Figure 33. EGCG inhibits DNA binding activity of p6 5/Rel A subunit from DCs stimulated with LPS. .............................. ................................................... ...... 62 Figure 34. Schematic diagram of proposed effects of EGCG on DCs. ............................. 69

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viii LIST OF ABBREVIATIONS ACK: Ammonium chloride potassium bicarbonate APCs: Antigen presenting cells APC : allophycoerythrin Ag: antigen BMDCs: bone marrow derived dendritic cells DCs: dendritic cells EGCG: (-)-Epigallocatechin-3-Gallate ERK: extracellular signal-regulated kinase FBS: fetal bovin serum FITC: fluorescein isothiocyante FSC: forward scatter GM-CSF: granulocyte-macrophage colony stimulating facto r HBSS: Hank’s balanced salt solution iDC: immature DC Jnk c-Jun N-terminal kinase mDC: mature DC MHC II: class II MHC 2-Me: 2-mercaptoethanol IFN: interferon gamma IL-12: interleukin-12 Lp Legionella pneumophila LPS: lipopolysaccharide MAPK mitogen-activated protein kinases MIP-1alpha/CCL3: macrophage inflammatory protein-1alpha MCP-1/CCL2: monocyte chemoattractant protein-1 MDP: muramyldipeptide Ml: milliliter MIP: macrophage inflammatory protein NF-B: nuclear factor kappa B NK: natural killer cell PBS: phosphate buffered salin PE: phycoerythrin PGN: peptidoglycan PI: propidium iodide RANTES: regulated on activation normal T cell expressed and secreted ROS reactive oxygen species RPMI1640: medium supplemented with 10% serum SSC: side scatter

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ix TLR Toll-like receptor TNF: tumor necrosis factor alpha g: microgram

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x MAJOR TEA CATECHIN INHIBITS DENDRITIC CELL MATURATI ON IN RESPONSE TO MICROBIAL STIMULATION JAMES L. ROGERS ABSTRACT Dendritic cells (DCs) are a migratory group of bone -marrow-derived leukocytes specialized for uptake, transport, processing and p resentation of antigens to T cells. Exposure of DCs to bacterial pathogens can induce D C maturation characterized by cytokine production, up-regulation of co-stimulator y molecules and an increased ability to activate T cells. DCs have the ability to restri ct growth of L. pneumophila (Lp), an intracellular Gram-negative bacillus that causes a severe form of pneumonia known as Legionnaires’ disease, in murine ER-derived organel les (121) but replicate in human DCs (145). Even in human cells, however, lysis of the DCs does not occur for at least 24 hours which may allow DCs time to participate in th e transition from innate to adaptive immunity (145). The primary polyphenol in green tea extract is the catechin (-)epigallocatechin-3-gallate (EGCG) which accounts fo r most of the numerous reported biological effects of green tea catechins, includin g anti-bacterial, anti-tumor, and neuroprotective effects. Primary murine bone marrow derived DCs from BALB/c mice were treated in vitro with Lp, or stimulated for comparison with Escherichia coli lipopolysaccharide (LPS). CD11c, considered an imp ortant marker of mouse DCs, and surface expression of co-stimulatory molecules CD40 CD80, CD86, as well as class I/ II MHC molecules was determined by flow cytometry. Tre atment of the cells with EGCG

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xi inhibited the microbial antigen induced up-regulati on of CD11c, CD40, CD80, CD86 and MHC I/ II molecules. EGCG also inhibited, in a dos e dependent manner, induced production of the Th1 helper cell activating cytoki ne, IL-12, and the chemokines RANTES, MIP1, and MCP-1. However, EGCG upregulated TNF production. In addition, EGCG inhibited both Lp and LPS induced ex pression of both TLR2 and TLR4 as well as LPS-induced NF-B activation; all of which are important mediators of DC maturation. The modulation of phenotype and functio n of DCs by EGCG has implications for host interaction with microbial pa thogens like Lp, which involve TLR interaction.

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1 INTRODUCTION EGCG Sources and Structure Polyphenols are natural substances found in abundan ce in fruits, vegetables and plant-derived beverages such as tea and consist of an aromatic ring that is condensed to a heterocylic ring and attached to a second aromatic ring (90). Flavonoids are the largest group of polyphenols, which include the subcasses o f flavones, isoflavones, flavanols, flavans and flavonols. Catechins are a further subc ategory of flavanols (166)(Figure 1). Figure 1. Diagram of the natural polyphenol classif ication and the chemical structure of green tea catechins. Reproduced with permission of Elsevier L imited.

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2 (-)-epigallocatechin-3-gallate (EGCG) is one of sev eral catechins found in many natural products, particularly both green and white tea. The other major catechins are (-)epicatecin (EC), (-)-epigallocatechin (EGC), and () epicatechin-3-gallate (ECG) (166). EGCG is the major catechin in green tea, and it als o accounts for most of the reported biological effects of green tea, especially its rep orted anti-tumor effects (115). These biological effects of EGCG may relate to the presen ce of the trihydroxyl group on the B ring and the gallate moiety at the 3’ position in t he C ring (120). Antibacterial Activity of EGCG EGCG reportedly also has potent antimicrobial activ ity. For example, a report published in 2001, from our own laboratory, showed that the growth of Lp in permissive macrophages could be selectively inhibited by small amounts of EGCG. These antimicrobial effects were not due to direct effect s on the bacteria, since EGCG could not alter Lp growth in medium regardless of the concentration us ed (106). Instead, antimicrobial effects were mediated by indirect eff ects of EGCG on the macrophages themselves which were activated to induce the obser ved antimicrobial activity. This activation was also mediated, at least in part, by induction of TNF and IFN production from the macrophages, since treatment of the macrop hage cultures with anti-TNF and anti-IFN g monoclonal antibodies markedly abolished the antib acterial effects of EGCG (106). Effects on Cytokine Production Cytokines are soluble proteins secreted by cells of the immune system. They have pleiotropic effects in that they act on many cell t ypes to modulate the host’s immune

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3 response (150). Various studies have shown that EGC G has immunomodulatory effects upon pro-inflammatory cytokines. For example, EGCG inhibits LPS-induced TNF a production by peritoneal macrophages from BALB/c mi ce (179). In the murine macrophage cell line, RAW264.7, EGCG decreases LPS induced TNF production in a dose-dependent fashion as well as LPS-induced TNF mRNA expression. The mechanism of action was reported to be due, in part to the down regulation of NF-kB, an oxidative stress –sensitive nuclear transcription f actor, since EGCG also inhibited LPS induced nuclear NF-kB-binding activity (179). EGCG combined with EC also reportedly inhibits TNF production by BALB/3T3 cells treated with the tumo r promoter, okadaic acid (152). However, in cultured human peripheral blood mononuc lear cells, EGCG stimulates production of TNF (143). Moreover, Matsunaga showed that EGCG selectively upregulated production of TNF by macrophages induced by bacterial infection (106). Other studies from Matsunaga show that EGCG attenuates nicotineinduced inhibition of TNF production in Lp infected macrophages (105) as wel l as attenuates suppression by cigarette smoke condensat e of TNF in response to infection with Lp (104). The effects of EGCG on IL-12, another pro-inflammat ory cytokine, has also been investigated. For example, Ahn and company reported that EGCG inhibits IL-12 production by BMDCs stimulated with LPS (3). Howeve r, in the MH-S murine alveolar macrophage cell line, EGCG selectively upregulates production of IL-12 (106). EGCG also attenuates nicotine inhibition of IL-12 produc tion in Lp infected macrophages (105). Topical application of EGCG before UVB exposure als o reportedly upregulates UVB-

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4 induced production of IL-12 in skin as well as in d raining lymph nodes from C3H/HeN mice (75). EGCG has been reported to have immunomodulating eff ects on various other cytokines. In the MH-S murine alveolar macrophage c ell line, EGCG selectively down regulates IL-10 production by macrophages induced b y bacterial infection and upregulates macrophage gamma interferon (IFN-) mRNA by EGCG but does not alter IL-6 production (106). Topical application of EGCG before UVB exposure reportedly decreases UVB-induced production of IL-10 in skin a s well as in draining lymph nodes in C3H/HeN mice (75). However, EGCG attenuates nicotin e inhibition of IL-6 production in Lp infected macrophages (105) as well as attenua tes suppression by cigarette smoke condensate of IL-6 in response to infection with L p (104). Using normal human keratinocytes stimulated with TNF, EGCG has also been reported to inhibit production of VEGF and IL-8 (160). In cultured human periphera l blood mononuclear cells, EGCG stimulates production of IL-1/ (143). The results of all of these studies establish that EGCG has inhibitory effects on pro-inflammatory cytokines such as TNF and IL-12. However, the effects of EGCG upon such pro-inflammatory cytokines, as well as ot her cytokines, varies depending upon both the host cell studied as well as the stimulus used in the study. Dendritic Cells Functions in Immunity DCs are potent APCs because of their unique charac teristic features such as very high MHC class II expression, costimulatory molecul es B7-1/2, and the ability to capture antigen at an immature stage and efficiently presen t to T cells at a mature stage (13, 22).

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5 Although T and B cells of the adaptive immune syste m express antigen receptors of enormous diversity, activation of these cells depen ds on their induction by co-stimulatory molecules and secretion of cytokines and chemokines by APCs such as DCs (126). As DCs mature, they migrate to the T cell areas of lym phoid organs, where they translate tissue-derived information into language that T hel per (Th) cells can understand. DCs do this by providing Th cells with an antigen-specific “signal 1,” a costimulatory signal 2, and a signal 3 which determines the polarization of nave Th cells into Th1 or Th2 cells. Thus, DCs provide a critical link between innate an d adaptive immunity (129). DCs are also often said to “direct” the type of imm une response delivered in response to the detected pathogen. LPS, dsRNA and o ligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) promote matu ration of DCs that direct nave T cells to a Th1 subtype. By contrast, phosph orycholine-containing glycoproteins derived from nematode parasites, cholera toxin or y east hyphae activate DCs that selectively induce Th2 cells (109)(Figure 2)

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6 Figure 2. DCs Direct an Immune Response. Reproduced with permission of Elsevier Limited. DC Maturation and the Immune Response The ability of DCs to “direct” an immune response is linked to their maturation state. In the mature state, DCs represent a potent APC for helper (CD4+) T cell activation. Interaction with activated CD4+ T cells may also result in the delivery of additional stimuli that render the DC “hyper-mature .” These DCs can subsequently induce activation of cytotoxic (CD8+) T cells (88). In addition, it is becoming increasingly clear that DCs, in an immature state, play a central role in peripherally expressed self and non-threatening foreign antigens For example, immature DCs within peripheral tissues capture cells dying by apoptosis and migrate to the draining lymph node where they present self-peptide-MHC complexes, in the absence of costimulation

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7 signals, to the circulating nave autoreactive T ce lls. This results in their inactivation either by anergy or deletion (151). There is also evidence that DCs can control periph eral tolerance through induction and maintenance of regulatory T cells. Fo r example, fusion proteins targeted to DCs lead to antigen-specific tolerance induction wh en DCs are left immature (17), and CD4+ T cells repetitively stimulated with allogenei c immature DC differentiate into IL10 producing regulatory cells, which inhibit the pr oliferation of alloreactive T cells (69). Injection of immature DCs pulsed with influenza mat rix peptide into healthy human volunteers also leads to the appearance of MP-speci fic IL-10 producing CD8+ T cells and silencing of MP-specific CD8+ T cell effector funct ion in freshly isolated T cells (33). It is important to keep in mind that the induction of T cell responses versus tolerance is a complex process which depends on much more then whe ther DCs are “mature” or “immature.” The outcome of an immune response depen ds on the phenotypic and functional change which occurs as DCs mature. Phenotypic Changes Associated with DC Maturation Introduction During the process of DC maturation, DCs lose the ability to phagocytosize, but they also produce large amounts of cytokines and ch emokines. Simultaneously, MHC class II molecules are translocated to the membrane and costimulatory molecules such as CD86 and CD40 are up-regulated. Mature DCs demonstr ate a characteristic morphology with enlarged size and numerous cytoplasmic process es ((148)(Figure 3).

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8 Figure 3. Compared with the RPMI-1640 (untreated co ntrol), Astragalus mongholicus polysaccharides (ASP) or LPS treated DC show charac teristic morphology of mature DC (needle-like protrusions). Reproduced with permission of Elsevi er Limited. MHC Molecules Whereas, in immature DCs, class II molecules are r apidly internalized and have a short half-life, maturation stimuli lead to a burst of MHC class II synthesis and translocation of the MHCII peptide complexes to the cell surface where they remain stable for days and are available for recognition b y CD4+ T cells (12). To generate CD8+ cytotoxic killer cells, DCs present antigenic pepti des on MHC class I molecules (12). Although most cells use their MHC class I molecules to present peptides derived from endogenously synthesized proteins, DCs have the cap acity to deliver exogenous antigens through the MHC class I pathway, a phenomenon know n as cross-presentation (55). Increased MHC class II expression has been shown to occur in several autoimmune diseases, including multiple sclerosis and rheumato id arthritis (49). Co-Stimulatory Molecules During DC maturation, several co-stimulatory molecu les are also expressed, with especially high levels of CD86. The MHC-peptide com plexes are found in clusters at the DC surface together with CD86 (161). It is believed that these high levels of antigenpresenting and co-stimulatory molecules, in a clust ered distribution, initiate the formation

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9 of the immunologic synapse, bringing together essen tial elements, such as the T cell receptor (TCR) and CD28, that are required for T ce ll activation (89). Low levels of the costimulatory molecules CD80 and CD86 expression on APCs leads to T cell anergy. This reportedly occurs because CTLA-4, which inhibi ts T cell responses, has a higher affinity for CD80 and CD86 than CD28, which promote s T cell responses (119). DCs from CD40-/mice do not make IL-12 or elicit CD4+ and CD8+ T cell responses, even though they are able to present pep tide Ag (44). DCs lacking cell surface expression of CD40, due to inhibited RelB function, reportedly also suppress ongoing immune responses by inducing IL-10-secreting Tregs (102). Moreover, CD40/CD40L interactions release immature DCs from suppression by CD4+CD25+ T cells, further suggesting that CD40 ligation is necessary and suff icient to abrogate tolerance and inhibit the action of Tregs (147). There is also evidence t hat suggests that costimulatory molecules on APCs may selectively influence T helpe r cell differentiation: antibodies against CD80 or CD86 selectively inhibit the develo pment of Th1 and Th2 responses, respectively (157). Functional Changes Associated with DC Maturation Cytokine Induction and Associated Biological Functi ons In the DC maturation process, cytokine genes are ex pressed with distinct kinetics in mice. Following appropriate stimulation, TNF is released rapidly (peaking at 3 h), whereas IL-6, IL-10, IL-12 and IL-23 are produced b etween 6 and 18 h after stimulation (87). The nature of the immune response is also dep endent upon the types of cytokines secreted by maturing DCs. A prime example of this i s the Th1/Th2 dichotomy. Nave Th cells differentiate into Th1 or Th2 cells depending on the cytokine microenvironments

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10 after activation through their antigen-specific rec eptors. In particular, IL-12 is a proinflammatory cytokine with immunoregulatory functio n that bridges innate resistance and antigen-specific adaptive immunity (159) and, when produced by DCs, induces Th1 differentiation and, hence, cellular immunity. This cytokine acts in concert with natural killer (NK) cell-derived IFN to further promote Th1 responses (159). Secretion of cytokines by DCs is also important for induction or reversal of tolerance. For example, attenuation by DCs of T regulatory cells depends, a t least in part, on DC secretion of IL-6 (127). In BMDCs, there is one report associating a possib le anti-inflammatory role with TNF. In particular, BMDCs produced less IL-12p40 when preincubated with TNF and then stimulated with LPS (1 ng/ml) (184). In genera l, however, TNF is recognized as a proinflammatory cytokine as well as associated with antigen-specific, cell-mediated immune responses (57). TNF also promotes DC migration from tissues into lymph nodes, can induce chemokines that are important in the recruitment of APCs, and upregulates antigen presentation(84). Chemokines Chemokines are potent chemoattractants that can be divided into four highly conserved but distinct families: CXC, CC, C, and CX 3C, based on the position of the first two cysteines in the amino terminus as well as the remaining cysteines in the carboxy portion of the molecule. Maturing DCs are also an a bundant and strategic source of chemokines, which are produced in a precise time-or dered fashion. Following stimulation with LPS, DCs show an initial burst of MIP-1 (CCL3), MIP-1 (CCL4) and IL-8 (CXCL8) production, which cease within a few hours. RANTES (CCL5) and MCP-1 are

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11 also induced, but in a more steady manner. At later time points DCs produce mainly lymphoid chemokines, such as CCL17 (TARC), CCL18 (D C-CD1), CCL19 (MIP-3) and CCL22 (MDC), that attract T and B lymphocytes ( 108, 144). Chemokines are produced by DCs in response to micro bial antigens through TLRs. For example, TLR4 is activated by LPS from Gr am-negative bacteria. Activation of different TLRs induces expression of different s ets of chemokines that recruit distinct subsets of leukocytes (Figure 4). Many different ch emokines are produced through TLR activation in DCs including IL-8 (also known as CXC L8), MIP-1 (CCL3), MIP-1 (CCL4), RANTES (CCL5) and IP-10 (CXCL10). MIP-1, MIP-1 and RANTES are reported to be induced by agonists of both TLR2 and TLR4 whereas IP-10 is preferentially induced by TLR4 agonists and IL-8 pr eferentially induced by TLR2 specific agonists. These studies suggest that patho gens can determine the nature of the immune response through differential activation of TLRs and the subsequent patterns of chemokines expression (97).

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12 Figure 4. Pathogens Induce Different Patterns of Ch emokine Expression Reproduced with permission of Elsevier Limited. Thus, in similarity to production of cytokines, the early production of chemokines is essential in shaping the immune response that fo llows in the tissue. For example, the production of IL-8 will induce the recruitment of n eutrophils, and MIP-1 and MIP-1 will induce the influx of NK cells, macrophages and immature dendritic cells (97). The stimulation of select TLRs by the pathogen and the subsequent production of a specific subset of chemokines may be the first point at whic h the immune system is tailored to a specific pathogen (97). As with cytokines, the types of chemokines produce d by DCs have been associated with Th1/Th2 immune response. In particu lar, fractalkine and IP-10 have been associated with a Th1 phenotype, whereas MDC and TA RC with a Th2 phenotype (30, 32, 43, 62, 66, 92, 108, 187). MIP-1 also reportedly upregulates Th1-type cytokine responses (74) and downregulates Th2 (96), while IP -10 selectively up-regulates antigen-

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13 driven IFNsynthesis suggesting an important role in maintain ing bias toward a Th1 response (45). Some of these effects of chemokines on T helper biasing may be direct or indirect through the action of cytokines. For exam ple, MIP-1-driven Th1 differentiation was not abrogated by anti-IFNsuggesting that the effects of MIP-1 are either direct or operating through undertermined cofactors. In contr ast, anti-IL-4 abrogated the ability of MCP-1 to drive Th2 differentiation suggesting that MCP-1 enhanced T cell-mediated IL4 production which in turn supported the Th2 phenot ype (73). Chemokines can also directly influence the polariz ing potential of DCs. For example, CCL19 reportedly programmed DCs for the in duction of Th1 rather than Th2 responses. Migrating DCs isolated form mice genetic ally deficient in CCL19 and CCL21 also presented an only partially mature phenotype, highlighting the importance of these chemokines for full DC maturation in vivo (100). Chemokine Receptors The type of chemokine receptor expressed is associa ted with the maturation state of the DC. Immature DCs respond to MIP-3, RANTES, and MIP-1 via chemokine receptors CCR1, 5 and 6, whereas mature DCs respond to MIP-3/ELC and SLC via CCR7. Down-regulation of receptors for the inflamma tory chemokines and up-regulation of receptors on mature DCs for chemokine that are e xpressed in secondary lymphoid organs allow DCs to leave the sites of inflammation and migrate to regional lymph nodes (10, 21, 35) ((Figure 5).

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14 Figure 5. Chemokine Receptor Expression on Dendriti c Cells. Reproduced with permission of Nature Publishing Group. Each immature DC population also displays a unique spectrum of chemokine responsiveness. For example, Langerhans cells migra te selectively to MIP-3 (via CCR6), blood, CD11C+ DC, to MCP chemokines (via CCR2), monocyte derivedDCs respond to MIP-1 alpha/beta (via CCR1 and CCR5), wh ile blood CD11c+DC precursors do not respond to any of these chemokines (21, 108) A number of chemokine receptors are also found on T h1 and Th2 cells. CCR5 and CXCR3 have been associated with the Th1 phenoty pe, while CCR3, CCR4, and CCR8 have been associated with the Th2 phenotype (1 24). Mice which are defective for CCR2, the receptor for MCP-1, reportedly have signi ficant defects in production of Th1type cytokines as well as delayed type hypersensiti vity responses (18). Interestingly, the expression of chemokine receptors may change depend ing on the activation status of the T cell. For example, CCR8 is only strongly expresse d in activated Th2 cells (185).

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15 Microbial Factors and Dendritic Cell Maturation Lipopolysacharide (LPS) Lipopolysaccharide (LPS), a major component of the Gram-negative bacterial envelope, elicits immediate proinflammatory respons es in the host (47). LPS is captured by LPS-binding protein (LBP) and subsequently trans ferred to CD14 (53). However, because CD14 lacks intracellular signaling domains, the complex interacts with TLR4 providing the necessary intracellular signaling cap acity (111). LPS can induce DC maturation in vitro and in vivo resulting in increased expression of costimulator y molecules and production of proinflammatory cytokin es that influence the subsequent immune response (110, 136, 139, 164). Peptidoglycan/Murymyldipeptide (MDP) Myramyldipeptide (N-acetyl-muramyl-L-alanyl-D-isog luatamine; MDP) is the smallest structural unit responsible for the immun oadjuvant activity of the peptidoglycan (PGN) in bacterial cell walls (170). (Audibert). Al though Gram-negative bacterial cell walls also contain PGN, its concentration is far gr eater in the walls of Gram-positive bacteria (Traub). MDP has been shown to exert diverse biological eff ects on immunocompetent cells in vitro (24). It enhances phagocytic and mic rocidal activities of monocytes and macrophages (29, 138). It can also augument the exp ression of immunostimulatory molecules such as MHC class II and CD40 on monocyte s and B cells (28, 56).

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16 L. pneumophila (Lp) Lp is a Gram-negative intracellular pathogen that o ften causes serious and lifethreatening pneumonia in humans known as Legionnair es’ disease with an estimated 17,000 to 50,000 patients hospitalized annually in the United States (101) (183). Unlike macrophages, DCs have the ability to restrict Lp gr owth which has been suggested as a factor allowing DCs ample time to present antigens for a cell-mediated immune response (121). In contrast to murine DCs, human DCs support Lp replication; however, lysis of the DC does not occur for at least 24 hours allowin g DC-mediated transition from innate to adaptive immunity (145). Alterations in maturati on parameters such as co-stimulatory and MHC molecules induced by Lp are essential for e ffective antigen presentation by DCs and enhanced cellular immunity against Lp. An alteration in chemokine production caused by Lp infection is another maturation parameter important in host immunity. Lp infection of cultured mouse peritoneal macrophages reportedly increases the lev els of cellular mRNAs for the neutrophil-attracting CXC chemokines, such as kerat inocyte-derived chemokine and macrophage inflammatory protein 2 (116, 176). Lp in fection also reportedly induces the gene expression of monocyte chemotactic protein 3 ( CCL7) by mouse alveolar macrophage MH-S cells (112). Neutrophil accumulatio n in Lp infected mouse lungs is reportedly mediated by CXC chemokines such as kerat inocyte-drived chemokine, macrophage inflammatory protein 2 and lipopolysacch aride-induced CXC cehmokine (CXCL6) (116, 156). Moreover, DC-mediated immune re sponse to Lp reportedly is attributed at least in part to the DC-derived expre ssion of the membrane-bound Th1 attractant fractalkine, which may promote both the chemotaxis of T cells toward Lp-

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17 capturing DCs and the adhesion between them, leadin g to clonal expansion and a Th1polarized differentiation of T cells recognizing Lp antigens (80). Toll-Like Receptors DCs have been shown to express TLRs 2, 3, 4, 5, 6 a nd 9. The activation of TLRs on DCs induces DC maturation which is characterized by the production of proinflammatory cytokines, upregulation of co-stimu latory molecules and altered expression of chemokine receptors (58, 97). TLR act ivation ultimately leads to the activation of NF-B which is essential for the induction of chemokine s and cytokines (97). TLR activation on DCs downregulates the expr ession of CCR1, CCR5 and CCR6, and upregulates the expression of CCR7. Because TLR stimulation occurs when a DC is likely to have internalized microbial pathogens, th is switch in chemokine receptor expression ensures that DCs loaded with antigens le ave the tissue and are attracted into the lymphoid organs. This modulation of chemokine-r eceptor expression and subsequent pattern of DC migration are crucial for the inducti on of an adaptive immune response (97). Structurally, TLRs are members of the type I transm embrane receptor family, first described in Drosophila and share homology to components of the IL-1 sign aling pathway (14). TLR signaling is initated by dimeriza tion of TLRs, which can form homodimers (such as TLR4) or heterodimers (such as TLR2 and TLR1) (6). TLRs and other members of the IL-1 receptor family share a h omologus intracellular domain, designated as the toll/IL-1R-like region (TIR), and have been reported to share common intermediate signaling molecules such as myeloid di fferentiation factor 88 (MyD88), IL1 receptor-associated kinase (IRAK), and tumor necr osis factor (TNF) receptor-

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18 associated factor 6 (TRAF6), for activation of NFB, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p3 8 kinase pathways (20, 118, 155). In addition to the enormous diversity of the adapti ve system, there also exists considerable diversity of recognition within innate immunity through the TLR superfamily which recognizes conserved structures c alled pathogen-associated molecular patterns (PAMPs) such as LPS. TLR4, for example, re cognizes bacterial LPS whereas TLR2 recognizes acylated outer membrane lipoprotein s of Gram-positive bacteria. The various TLRs also have a diversity of function thro ugh the selective use of intracellular adaptor molecules (125, 173, 174). For example, th e adaptor MAL is vital for TLR1 through 9 with the exception of TLR3 for the activa tion of NF-B (41, 172). TLR3 uses instead the adaptor molecule TRIF to induce NF-B and IFNa synthesis through IFNregulatory factor (IRF) 3 and 7, a signaling pathwa y that is crucial for anti-viral immunity (77, 158). This pathway is sometimes referred to as the MyD88-independent pathway. TLR4 can also activate the IRF3 signalling pathway in a process that requires the adaptors TRIF and TRAM. There are also other pathwa ys that contribute to TLR function, such as those involving Jun N-terminal ki nase (JNK) and the mitogen-activated protein kinases (MAPKs) (36, 59). TLR2 TLR2 is capable of recognizing a much broader rang e of pathogen components compared to TLR4. For example, TLR2 can recognize c omponents derived from both Gram-positive and Gram-negative bacteria and mycoba cteria such as peptidoglycan (PGN), lipoteichoic acid (LTA), bacterial lipoprote ins, lipopeptides, and lipoarabinomannan (40, 146, 181). TLR2 signalling c an induce activation of NF-B and

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19 MAPK cascades in a MyD88-dependent manner (155). Mu rine DCs deficient in TLR2 do not undergo maturation upon stimulation with PGN (1 13). TLR2 has been shown to be an important molecule res ponsible for resistance to intracellular growth of Lp in bone marrow-derived m acrophages. In particular, intracellular growth was enhanced within TLR2(-/-) compared to wild type and TLR4(-/-) macrophages. There was, however, no difference in t he bacterial growth with dendritic cells from WT or TLR-deficient mice (5). TLR4 TLR4 is a critical receptor and signal transducer for LPS, a prominent PAMP of Gram-negative bacteria, in coordination with CD14 a nd MD2 molecules (133, 135, 154, 162). LPS ligation induces NF-B activation (118) and TLR4-deficient mice are hyporesponsive to LPS (12) and derived DCs do not u ndergo maturation upon stimulation with TLR4 ligands such as LPS and lipid A (113). LPS-induced TLR4 activates two downstream pathways ; the MyD88-dependent pathway that leads to the production of proinflamma tory cytokines with quick activation of NF-kB and MAPK, and the MyD88-independent pathwa y, associated with activation of IRFs, subsequent induction of IFN, and maturatio n of DCs, with delayed activation of NF-kB and MAPK (70). Although cytokine production is severly restricted in MyD88deficient mice, some responses to LPS, including th e induction of interferon-inducible genes and the maturation of DCs are still observed (70, 76, 77).

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20 TLR5 Both humans and mice detect Lp flagellin to mount an immune response. In humans, its recognition by TLR5 correlates with res istance to Legionnaires’ disease (54). When injected into mice, Lp flagellin triggers a ro bust inflammatory response (137). TLR9 Recent results from our own laboratory suggest tha t TLR9 is also important in sensing Lp in DCs from both BALB/c and A/J mice. As evidence for the importance of TLR9, chloroquine treatment suppressed IL-12p40 pro duction in response to Lp infection, and the TLR9 inhibitor ODN2088 suppresse d Lp-induced IL-12 production in DCs from both strains (122). Molecular Mechanisms of Action of EGCG TLR Signaling Effects As mentioned above, microbial antigens trigger the activation of two downstream signaling components of TLRs including MyD88 and TR IF leading to activation of NF-B. EGCG has been shown to inhibit both of these sig naling pathways. For example, EGCG reportedly inhibits IKK and TBK1 in the MyD88 and TRIF-dependent signaling pathways, respectively (182) MAPKs The MAPKs are central to receptor signal transducti on in the activation of many immune cell genes. They are activated upon phospho rylation, which then allows them to phosphorylate and activate other intracellular fact ors. The major subgroups of MAPKs comprise ERK, JNK, and p38. Whereas ERKs are predom inantly activated by mitogenic signals, JNK and p38 are primarily activated by env ironmental stresses such as UV

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21 radiation, inflammatory cytokines, heat shcok and D NA-damaging agents (23, 72, 85). Activation of the p38 pathway is involved in IL-12 p40 promotor activity and cytokine release in DCs (4, 95, 165). However, there are som e data indicating that activation of the ERK pathway acts to suppress IL-12 secretion as wel l as DC maturation (169, 177). EGCG has previously been shown to inhibit the ultr aviolet-B-induced activation of p38-MAPK in a human keratinocyte cell line (27), while others have shown that EGCG activates ERK1/2, JNK and p38 in HeLa cells (2 5). In vascular smooth muscle cells, EGCG inhibited the platelet-derived growth f actor--induced activation ERK1/2 in a dose-dependent manner (2). In addition, EGCG sele ctively inhibited IL-1-induced activation of JNK, but not ERK1/2 or p38 MAPK, in h uman osteoarthritis chondrocytes (149). EGCG inhibited LPS-induced IL-12p40 producti on in murine macrophages by inhibiting p38 MAPK while enhancing p44/p42 ERK, le ading to the inhibition of I degradation and NF-B activation (61). In DCs, EGCG inhibited LPS-induc ed MAPKs, ERK1/2, p38 and JNK (3) Thus, it appears that MAPK activating or inhibitory effects of EGCG may be stimulus and/or cell type-dependent. NF-B NF-B is the common downstream signaling component for all TLRs and plays a critical role in immune and inflammatory responses. Most genes of inflammatory mediators such as TNF and IL-12 are regulated by NF-B because they have a B site in their 5’ flanking region (46). NF-B is sequestered in the cytoplasm of most cell type s by virtue of its association with the IB family of inhibitor proteins, which includes IB and IB. The IBs bind to the Rel homology domain, which contains the dimerization, nuclear transfer, and DNA binding functions of the NF-B/Rel protein (11). At least two

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22 of the IBs (IB and IB) undergo rapid phosphorylation at two conserved Nterminal residues in response to cell stimulation by proinfl ammatory cytokines or bacterial LPS. This phosphorylation targets them for rapid polyubi quitination followed by degradation through the 26S proteasome pathway, thereby liberat ing NF-B, which is then free to translocate to the nucleus and bind to DNA (34) EGCG is known to inhibit NF-B activation induced by many pro-inflammatory stimuli. In DCs, EGCG has previously been shown to inhibit LPS-induced NF-B p65 translocation (3). Interestingly, EGCG-mediated inh ibition of NF-B constitutive expression was reportedly found to occur at much hi gher doses of EGCG in normal human keratinocytes compared to human epidermal car cinoma cells suggesting that cancer cells were more sensitive to the effects of this compound (1). Antioxidant Properties of EGCG EGCG is a potent antioxidant, and this catechin has been associated with most of the biological effects of tea catechins, including redu ced risk of cancer, diabetes and cardiovascular disease (86). The ability of green t ea polyphenols such as EGCG to act as oxygen radical scavengers and chelate transitional metals such as iron and copper may also be of major significance for treatment of neur odegenerative diseases such as multiple sclerosis, Parkinsons disease and Alzheimer’s disea se (99). EGCG also reportedly elevates the activity of two major oxygen-radical s pecies metabolizing enzymes, superoxide dismutase and catalase in mice striatum which may also be significant for its reported neuroprotective effects (91).

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23 ROS and Redox Environment NF-B can be activated through the generation of exogen ous and endogenous reactive oxygen species (67, 71, 134) which include s mechanisms of involving TLR4 activation and function (9). In addition, LPS-induc ed NFB activation and consequent TLR4-induced TLR2 expression in endothelial cells i s reportedly mediated by NADPH oxidase (39). The involvement of ROS is postulated to regulate the activity of the upstream kinases that converge onto the NF-B signaling activation pathway (51). DC maturation has also been reported to be regulated b y the redox environment. For example, DCs grown under tightly regulated O2 in the absence of exogenous reducing agents, e.g., 2-Me, induces DC maturation (48). Tea preparations have been shown to trap reactive oxygen species, such as superoxide radical, singlet oxygen, hydroxyl radica l, peroxyl radical, nitric oxide, nitrogen dioxide, and peroxynitrite. Among tea cate chins, EGCG is most effective in reacting with most reactive oxygen species (178). H2O2-induced erythrocyte membrane damage has been reported to be inhibited by EGCG tr eatment (141), and EGCG inhibits deoxycholate induced oxidative stress as well as ac tivation of NF-B in HCT-116 cells derived from a colon carcinoma (9). EGCG in hydroph ilic ointment before UVB exposures also reportedly resulted in significant p revention of induced depletion of antioxidant enzymes such as glutathione peroxidase and catalase in mouse skin (163). In tumor cells, a differential oxidative stress enviro nment and induction of apoptosis by tea polyphenols compared to the normal cells have been reported (175, 180). Under certain conditions, catechins may undergo au tooxidation and behave like prooxidants (178). It has been reported that higher concentrations of tea polyphyenols in

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24 cell culture systems produce H2O2, which may be an important factor responsible for cellular toxicity (68, 94, 142, 175, 180).

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25 PROJECT SIGNIFICANCE A vast amount of literature exists linking EGCG to many different beneficial biological effects. Within this literature, many st udies also support an anti-inflammatory role of EGCG, although results depend upon the type of immune cell studied and stimulus used. Dendritic cells are critical to link ing innate to adaptive immunity by initially detecting PAMPs on invading pathogens and activating nave T cells. DCs are often said to “direct” an immune response, and they are important in directing a inflammatory response. The type of immune response which DCs direct depends upon their maturation state, and more specifically, upon a range of parameters such as cytokine production and costimulatory surface molecule expre ssion which change as DCs mature in response to microbial stimulation. Enhanced infl ammation is known to be a critical step in the cascade of events leading to the develo pment of many chronic diseases such as Alzheimer’s disease and multiple sclerosis, and it is widely believed that newer therapies are needed in the management of these diseases. Rec ent evidence also suggests the involvement of TLRs in these chronic inflammatory d iseases. The studies are significant because DC maturation parameters such as cytokine/c hemokine production and TLR expression are important in inflammation, and the t ype of immune response directed by DCs.

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26 OBJECTIVES These studies examine effects of EGCG upon importan t parameters of DC maturation in response to microbial products such a s LPS and Lp. In this respect, an objective of the following studies is to investigat e effects of EGCG on phenotypic maturation parameters of DCs such as costimulatory and MHC molecule surface expression. A second goal is to examine effects of EGCG on functional characteristics of DC maturation such as cytokine and chemokine produc tion. A third goal of the following studies is to examine mechanistic effects of EGCG o n DC maturation and in particular, its effects on TLR signaling pathways. EGCG is one of the most widely consumed natural products in the form of tea, particularly g reen tea. In addition, there is a vast reservoir of literature attributing many beneficial biological effects to this natural compound, particularly its anti-cancer effects. How ever, EGCG has also been reported to have anti-inflammatory properties and DCs play a ce ntral role in inflammatory and immune responses. The hypothesis to be tested is that EGCG exerts its antiinflammatory effect in part by suppressing the acti vation and maturation of DCs. Aim 1: Determine the effects of EGCG treatment on c ostimulatory and MHC molecule expression in response to microbial stimul ation. Various phenotypic changes occur upon maturation o f DCs. Among changes which occur are upregulation of costimulatory molec ule expression, particularly CD80 (B7-1) and CD86 (B7-2). DCs also upregulate MHC cla ss I/II molecule expression upon maturation. Whereas immature DCs express chemokine receptors 1-6, mature DCs express CCR7-8 and CXCR4. These phenotypic changes or the lack thereof have been implicated in the type of immune response which DCs direct. For example, antibodies

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27 against CD80 reportedly inhibit Th1 responses where as antibodies against CD86 reportedly inhibit Th2 responses (157). Low levels of CD80 and CD86 on DCs are also known to lead to T cell anergy because CTLA-4 repor tedly has a higher affinity for low expression of CD40 and CD86 compared to CD28 (119). In this aim, we will measure costimulatory and MHC surface molecule expression on mouse bone marrow-derived DCs by flow cytometry following microbial stimulation ( i.e., LPS treatment and Lp infection) with or without EGCG treatment. Aim 2: Determine the effects of EGCG on DC cytokine and chemokine production in reponse to microbial stimulation. Various functional changes also occur upon maturati on of DCs. Among these are shifts in endocytic and or phagocytic ability from one of high capacity to one of low capacity. Other changes associated with DC maturati on are cytokine and chemokine production important in determining what type of im mune response DCs will direct. For example, DC production of IL-12 drives differentiat ion of CD4 T cells to Th1 effector cells, while IL-4 production drives nave T cells t o become Th2 effectors. Among chemokines reported as important for a Th1 response are CX3CL1 (fractalkine), CXCL10 (IP10) and MIP-1. Chemokines implicated as being important for a Th 2 response are CCL17 (TARC) and CCL22 (MDC). In parti cular, MIP-1 reportedly induces Th0 cells to differentiate into Th1 effecto rs whereas MCP-1 induces Th0 cells into Th2 effects (73). In this aim, we will examine effects of microbial stimulation (e.g., LPS, Lp) either with or without EGCG treatment on c ytokine (IL-12, TNF) and inflammatory chemokine (MIP-1, MCP-1, RANTES) production by DCs using ELISA technology.

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28 Aim 3: Determine the molecular signaling mechanisms involved in effects of EGCG on DC maturation. TLRs are an evolutionary conserved family of cell surface proteins that recognize PAMPs. These PAMPs can include such microbial produ cts as LPS from Gram-negative bacteria as well as teichoic acid from Gram-positiv e bacteria. Once engaged, TLRs interact with a host of signaling proteins which cu lminates in activation of different sets of genes including cytokine and co-stimulatory mark er genes. In this respect, the TLR molecular signaling pathway is crucial to the abili ty of DCs to direct an immune response. A major transcription factor induced by T LRs is NFkB; the activation of this factor has also been shown to be modulatated by EGC G. Therefore, in this aim we will examine the modulation of TLRs and NKkB in microbia l stimulated and EGCG-treated cells. In particular, we will stimulate DCs with Lp or LPS and study TLR expression by flow cytommetry. We will use ELISA to determine NFB protein levels following stimulation and treatment with EGCG.

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29 MATERIAL AND METHODS Catechins and Stimulants EGCG was obtained from Sigma Chemical Co. (St. Lou is, MO) and stored as 5 mg/ml stock solutions. LPS from E. coli was also obtained from Sigma. The vehicle for all solutions was sterile pyrogen-free water. Animals BALB/c mice from NCI (Frederic, MD) were utilized. They were 8-10 weeks of age at the start of an experiment and kept in groups of 4 in plastic mouse cages with barrier filters and fed Purina mouse chow and water ad libitum They were housed and cared for in the University of South Florida animal facility, which is fully accredited by the American Association of Laboratory Animal Care. Preparation of DCs DCs were prepared as described previously (63) with several modifications. Briefly, bone marrow cells were flushed from the fe murs and tibias of the mice and the red cells lysed with ACK lysing buffer to deplete r ed blood cells. Pooled BM cells were plated in six-well culture plates (106 cells/ml; 3 ml/well) and cultured overnight in RPM I 1640 medium (Sigma, Saint Louis, Mo) supplemented w ith 10 % heat-inactivated fetal bovine serum, 2 mM L-glutamine, 0.1% 2-mercaptoetha nol, 1% antibiotic/ antimycotic solution (Sigma), and 10 ng/ml recombinant GM-CSF ( BD Pharmingen, San Diego, CA). Non-adherent cells were removed and the adherent ce lls were incubated with fresh GMCSF-containing medium for an additional 7-9 days, d uring which time the BMDCs became non-adherent and were harvested. The cells w ere typically about 97 % positive for CD11b and 60-70 % positive for CD11c, as measur ed by flow-cytometry analysis.

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30 Bacteria A virulent strain of Lp (M124), serogroup 1, was ob tained from a case of Legionellosis from Tampa General Hospital (Tampa, F L) and was grown on buffered charcoal-yeast extract agar (BCYE, Difco, Detroit, MI) for 48 hr. The bacterial suspensions were prepared in pyrogen-free saline, a nd the concentration of bacteria determined by spectrophotometry. Infection DCs were infected with Lp at a ratio of 10 bacteri a per cell for 30 min., washed to remove non-phagocytized bacteria and incubated in R PMI 1640 medium containing 10 % FCS with no antibiotics. In certain experiments, D Cs were infected with Lp at a ratio of 20 bacteria per cell for 40 min., washed to remove non-phagocytized bacteria and incubated in RPMI 1640 medium containing 10 % bovin e calf serum with no antibiotics. The cultures were then incubated for 48 hr at 37oC under 5 % CO2 humidified atmosphere. Treatment BMDCs, either infected or non-infected, were added at a concentration of 2 x 105 cells/ml to 24-well plastic plates for bioplex cyto kine analysis or 1 x 106 cells/ml to polypropylene tubes for flow cytommetry analysis an d various concentrations of EGCG (0, 10, 50 g/ml) were then added to each well. For ELISA, DCs, either infected or noninfected, were added at a concentration of 2 x 105 cells/ml to 24-well plastic plates (CoStar, Cambridge, MA) and various concentrations of EGCG (0, 10, 50 and 100 g) were then added to each well. For DNA binding assay s, DCs were added, at a concentration of 2x105 cells/ml (total volume of 5 ml for LPS stimulation ), or at a

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31 concentration of 1x106 cells/ml (total volume of 1 ml for Lp infection), to polypropylene tubes with 50 g/ml of EGCG. For stimulation of non-infected cells E. coli LPS (10 ng/ml or 100 ng/ml or1 g/ml) was added to each wel l/tube with the various concentrations of EGCG. In some experiments, DC cul tures treated with LPS or infected with bacteria and treated or not either EGCG were i ncubated with purified rat antimouse/rate TNF monoclonal antibody (Cat No. 554640, Pharmingen, S an Diego, Calif.). Cell Viability The XTT assay was used to assess the effects of EGC G on cell viability (In Vitro Toxicology Assay Kit XTT Based, TOX-2, Sigma, Saint Louis, MO). This assay is based on the ability of mitochondrial dehydrogenases of v iable cells to cleave the tetrazolium ring of XTT (2,3-bis[2-methoxy-4-nitro-5-sulfopheny l]-2H-tetrazolium-5-carboxyanilide inner salt) yielding orange formazan crystals which are soluble in aqueous solutions. DCs were harvested as outlined above and dispensed in t riplicates at a density of 1x106 cells/ml into a 96-well flat bottom tissue culture plate. Plates were incubated with EGCG at various concentrations (0, 10, 50, and 100 m g/ml) in 5% CO2 at 370C for 24h. Because EGCG produced an orange color at higher doses, the culture medium was replaced on day 2 with fresh culture medium (200 m l) before adding 20 l XTT (20% of the medium volume) and incubated at 370C for another 4h. The plates were read on an Emax microphage reader (Molecular Devices, Menlo Park, C A), using a wavelength of 450 nm and a reference wavelength of 650 nm. Control wells contained cells alone. Cell survival was calculated as a percentage of MTT inhibition by the following formula: survival (%) = (mean experimental absorbance/mean control absorb ance) X 100%.

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32 Flow Cytometry DCs were harvested as outlined above and analyzed f or expression of various cell surface molecules by tri-color immunofluorescent st aining with fluorescein isothiocyanate (FITC)-conjugated rat anti-major his tocompatibility complex (MHC) class II (I-Ab) and class I (H-2K), phycoerythrin (PE)-conjugated rat anti-CD86, CD4 0 and CD80 and allophycocyanin (APC)-conjugated rat antiCD11c (all from PharMingen, San Diego, CA), as well as FITC anti-mouse-TLR2 and PE anti-mouse-TLR-4 (all from eBioscience, San Diego, CA). Cells in PBS containi ng 2% heat-inactivated bovine growth serum were blocked with anti-FCR antibody (C D16/ CD32) for 15 min. Staining was performed for 30 min on ice with the various co njugated antibodies. Cells were fixed with 1% paraformaldehyde and the fluorescent-labele d cells were analyzed by flow cytommetry (Becton Dickinson, Mountain View, CA). T he instrument is equipped with lasers tuned to 488 nm and to 635 nm. In all analys es, dead cells were gated out and cells of the phagocytic lineage were identified by forwar d and orthogonal light-scattering signals. ELISA The amount of IL-12 p40/p70 and TNF a in the culture supernatants of DC cultures, 24 hours after treatment, was determined by sandwich ELISA using matched antibody pairs and protein standard for ELISA (BD P harmagen) for IL-12 and Duoset ELISA development system (R&D Systems, Minneapolis, MN) for TNF a For this purpose, medium-bind, 96-well Costar enzyme immunoa ssay (EIA) plates were coated with specific monoclonal anti-cytokine antibody for IL-12 p40/p70 or TNF a overnight at 4oC. Plates were blocked for 1 h at 37oC with PBS plus 3% BSA (IL-12 p40/p70) or 1%

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33 lipid free BSA (TNF a ) and 0.05% Tween 20. Culture supernatants or seria l dilutions of murine cytokine standard were added for 1 h, follow ed by biotinylated anti-murine IL-12 p40/p70 or TNF a and then followed by streptavidin-alkaline phosph atase (1:1,000; BD Pharmagen) for 30 min. After the substrate was adde d, plates were allowed to develop. The plates were washed between additions with three to five changes of nanopure water. The plates were read at 450 nm on an Emax microphag e reader (Molecular Devices, Menlo Park, CA). Units were calculated form the cyt okine standard curve, which was performed for each plate. The amount of MCP-1, CCL5/RANTES and CCL3/MIP-1 in the culture supernatants of DC cultures, 24 hours after treatme nt, was determined by sandwich enzyme-linked immunosorbent assay ELISA using match ed antibody pairs and protein standard for ELISA (BD Pharmagen) for MCP-1 and Duo set ELISA development system (R&D Systems, Minneapolis, MN) for RANTES an d MIP-1. For this purpose, medium-bind, 96-well Costar enzyme immunoassay (EIA ) plates were coated with specific monoclonal anti-cytokine antibody for MCP1, RANTES or MIP-1 overnight at 4oC for MCP-1 and at room temperature for RANTES and MIP-1. Plates were blocked for 1 h at 37oC with PBS plus 0.5% BSA (MCP-1) or 1% BSA (RANTES & MIP-1) and 0.05% Tween 20 in the case of MCP-1. Culture su pernatants or serial dilutions of murine cytokine standard were added for 1 h, follow ed by biotinylated anti-murine MCP1, RANTES or MIP-1, and then followed by streptavidin-alkaline phosph atase (1:200; R&D Systems) for 30 min. After the substrate was ad ded, plates were allowed to develop. The plates were washed between additions w ith three to five changes of nanopure water. The plates were read at 450 nm on a n Emax microphage reader

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34 (Molecular Devices, Menlo Park, CA). Units were cal culated form the cytokine standard curve, which was performed for each plate. Bioplex Cytokine Assay Briefly, 50 l of the culture supernatant or cytoki ne standard was plated in a 96 well filter plate coated with a multiplex of beads coupled to a ntibodies against the above mentioned cytokines and incubated for 30 min on a platform sh aker at 300 rpm at RT. After a series of washes to remove the unbound proteins, a mixture of biotinylated detection antibodies, each specific for a different epitopes, was added t o the reaction resulting in the formation of antibodies assimilated around the target protein s. Streptavidin-phycoerythrin (streptavidin-PE) was then added to bind to the bio tinylated detection antibodies on the bead surface. The data from the reaction were then collected and analyzed by using the Bio-Plex suspension array system (or Luminex 100 sy stem) from Bio-Rad Laboratories (Hercules, CA). P65/RelA Dna-binding activity DNA-binding activity of the p65/RelA subunit of NFB was determined using Trans Am™ NFB colorimetric kit (Active Motif). An equal amount of cellular extracts was added to incubation wells precoated wi th the DNA-binding consensus sequence. The presence of translocated p65/RelA sub unit was then assessed by using the Trans Am™ kit according to manufacturer instruction s. Plates were read at 450 nm, and results were expressed as OD.

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35 Statistics The results were expressed as means SD of indicat ed number of experiments. Statistical significance was determined using Stude nt’s t test for unpaired observations. A value of p < 0.05 was considered significant.

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36 RESULTS Aim 1: Determine the effects of EGCG treatment on c o-stimulatory and MHC molecule expression in response to microbial stimul ation. Lp Infection Induces CD11c, Co-stimulatory Molecule and MHC Surface Molecule Expression To characterize effects of EGCG on phenotypic matur ation of BMDCs after infection with Lp, we investigated the expression o f maturation markers MHC class I and II, CD40, CD86 and CD80 on gated populations of DCs from BALB/c mice. For this purpose, donor cells were differentiated into DCs w ith GM-CSF. DCs were greater than 97% positive for the myeloid cell-surface antigen, CD11b, and typically between 60-70% positive for CD11c as determined by flow cytometry (Figure 6). On days 7-8 of culture, DCs were infected with Lp at an MOI of 10 for 30 mi nutes and various concentrations of EGCG (10, 30 and 50 g/ml) were added to either the Lp infected or non-infected groups. Figure 6. Flow cytometric dot plot of CD11b and CD1 1c surface molecule expression by DCs.

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37 DCs infected with Lp in the absence of EGCG were ac tivated, as indicated by a increase in percentage of cells expressing both CD1 1c and the co-stimulatory molecules CD40 (71% versus 13%), and CD86 (68% versus 20%), i ndicating maturation of DCs (Figure 7). Figure 7. Lp infection up-regulates CD40 and CD86 e xpression by DCs. Flow cytometric dot plots of CD11c and co-stimulatory molecule expression. Numbe rs in quadrants reflect percentages rounded to next greater whole integer. Results are 1 of 5 i ndependent experiments with similar results. Lp was also a potent inducer of both MHC class I an d class II surface molecule expression. Cells which were double positive for M HC and CD11c increased from 14% to 32% for MHCII and from 48% to 80% for MHCI (Fig ure 8).

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38 Figure 8. Lp infection up-regulates MHC class I/II epxression by DCs. Flow cytometric dot plots of CD11c and MHC I/II surface molecule expression. Num bers in quadrants reflect percentages rounded to next greater whole integer. Results are 1 of 5 independent experiments with similar results. EGCG Inhibits CD11c, Co-stimulatory Molecule and MH C Surface Molecule Expression Induced by Lp Infection Incubation of DCs with various concentrations of E GCG (10, 30 and 50 g/ml) reduced in a dose dependent manner the upregulating effect of Lp on the percentage of cells expressing MHC I and II molecules (Figure 9). DC onlyDC + LpDC + Lp+ EGCG(10)DC + Lp+ EGCG(30)DC + Lp + EGCG(50 )MHCII MHCICD11c 14 1 47 38 32 55 12 1 16 61 123 12 47 338 1350 3 35 48 12 1129 80 <1 19 1 5616 217 3630 1816 2832 1823 Figure 9. EGCG inhibits Lp upregulation of MHC surf ace molecule expression by DCs infected with Lp and treated with various concentration of EGCG a nd analyzed by flow cytommetry. Flow cytometric dot plots of CD11c and MHC surface molec ule expression. Number in quadrants reflect percentages rounded to next greater whole integer. Results shown are 1 of 5 independent experiments with similar results.

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39 In a similar manner, incubation of DCs with variou s concentrations of EGCG (10, 30 and 50 g/ml) reduced the upregulating effect on co-stimulatory molecules CD40 and CD86 (Figure 10). DC onlyDC + LpDC + Lp+ EGCG(10)DC + Lp+ EGCG(30) DC + Lp+ EGCG(50 )CD40 CD86CD11c 113 4146 117199 2222650 173359 1147 338 138 2041 5 8 68 18 321 3344 618 3541 520 34 41 Figure 10. EGCG inhibits Lp upregulation of co-stim ulatory molecule CD40 and CD86 expression by DCs infected with Lp and treated with various conce ntrations of EGCG and analyzed by flow cytometry. Flow cytometric dot plots of CD11c and c o-stimulatory surface molecule expression. Numbers in quadrants reflect percentages rounded to next greater whole interger. Results shown are 1 of 5 independent experiments with similar results ___________________________________________________ _____________________ Percentage of CD11+cells EGCG g/ml __________________________________________________ ________ MHCII MHCI CD40 CD86 ___________________________________________________ ____________________ DC only 10 3 47 6 9 5 17 7 DC + Lp 28 12 68 15 20 3 52 19 EGCG10 13* 4 52* 10 18 8 30* 11 EGCG30 8* 3 44* 12 8* 3 17* 6 ECGC50 12* 1 42* 14 11* 4 23* 12 Table 1: MHC I/II and Costimulatory molecule CD40, C86 surface molecule expression by DCs infected with Lp (10:1) and treated with various co ncentrations of EGCG and analyzed by flow cytometry. Results expressed as mean SEM from 5 in dependent experiments. The asterisks indicate statistically significant differences of P<.05 from values of Lp infected cells.

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40 As shown in Table 1, the standard error mean (SEM) from 5 independent experiments was significantly lower for EGCG groups then values of Lp infection alone for each of the key maturation markers MHC I/II, CD 40 and CD86 LPS Induces CD11c, Co-stimulatory Molecules and MHC Surface Molecules that are inhibited by EGCG Treatment Microbial products such as LPS can also activate im mature DCs and induce DC maturation, characterized by up-regulation of co-st imulatory molecules and increased ability to activate T cells (12). EGCG treatment su ppressed LPS-induced MHC and costimulatory molecule DC surface expression similar to the effect followint Lp treatment. In particular, LPS increased the percentage of DCs double positive for CD11c and CD40/CD86 molecule surface expression whereas in th e presence of 50 g /ml EGCG surface expression was not increased following LPS treatment (Figure 11-12). DC only1317 19 51CD40 DC+LPS2326 3714 DC+LPS+EGCG(50)2313559MHCII 2725 3811 5438 71 22 13 5610CD11c Figure 11. EGCG inhibits CD40 and MHCII surface mol ecule expression by DCs stimulated with LPS and treated with 50 g of EGCG and analyzed by flow cytometry. Numbers i n quadrants reflect percentages rounded up to next greater whole intege r. Results shown are from 4 independent experiments with similar results.

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41 Similarly, EGCG inhibited the percentage of cells double positive for CD11c and MHCI and II surface molecule expression by DCs indu ced by LPS ( Figure 12 ). DC only DC+LPS DC+LPS+EGCG(50)MHCI CD861365139 111 2266 2476<1<111 57 1219 207226648 1730CD11c Figure 12. EGCG inhibits MHCI and CD86 surface mole cule expression by DCs stimulated with LPS and treated with 50 g of EGCG and analyzed by flow cytometry. Numbers i n quadrants reflect percentages rounded up to next greater whole intege r. Results shown are from 4 independent experiments with similar results. EGCG treatment of DCs alone does not affect CD11c, costimulatory molecule or MHC surface expression. To determine if the inhibitory effect of EGCG obser ved above on MHC and costimulatory molecule expression was one of drug tox icity rather than an inhibition of the microbial stimulation response, we tested the effec t of EGCG, without microbial

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42 stimulation, on the DC response. As is shown in (F igure 13) and in (Figure 14), EGCG had virtually no effect on surface marker expressio n of either MHC or co-stimulatory molecules. Thus, the effects of EGCG appeared to in volve the EGCG prevention of microbial-induced upregulation of these maturation markers as opposed to a toxic effect of EGCG on the cell. Figure 13. Effects of EGCG on MHC class I/II molcul e expression by DCs as analyzed by flow cytometry. Numbers reflect percentages rounded to n ext greater whole integer. Results shown are 1 of 3 independent experiments with similar results. Figure 14. Effects of EGCG on co-stimulatory molecu le expression by BMDCs as analyzed by flow cytometry. Numbers in quadrants reflect percentages rounded to next greater whole integer. Results shown are 1 of 3 independent experiments with simil ar results.

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43 Inhibitory Effects not Due to Cytotoxity of EGCG As a direct test of drug toxicity, cells were treat ed with varying concentrations of EGCG and viability measured by an XTT assay (Figure 15). The results show that EGCG did not reduce vaiblity at 50 m g/ml and only slightly reduced it at 100 m g/ml. Moreover, no measurable effect on DC viability occu red over a period of 48 hr following infection with Lp (data not shown). Figure 15. BM derived DCs were exposed to various c oncentrations (0, 50, 100 g/ml) of EGCG for 24 h. Cell viability was analyzed with XTT assay. P ercent (%) viability was determined by measuring the OD at 450 nm and a reference wavelength of 650 nm in a microplate reader. The results are expressed as an average of 3 independent experiment s performed in triplicate. The asterisks indicate statistically significant differences of P<0.05 fro m values obtained with non-EGCG treated DCs.

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44 EGCG treated DCs Exhibit the Morphology of Immature DCs In all cultures, cells infected with Lp or stimulat ed with LPS and which had the greatest co-stimulatory/ MHC/ CD11c molecule surfac e expression tended to be larger and more granular, indicative of a more mature DC p henotype. Conversely, EGCG treated infected/stimulated cells, which showed sup pression of co-stimulatory/MHC/ CD11c molecule surface expression, tended to be sma ller and less granular, indicative of a less mature DC phenotype comparable to the non-in fected/ EGCG treated control group as shown by flow cytommetry (Data not shown). Aim 2: Determine effects of EGCG on DC cytokine and chemokine production in reponse to microbial stimulation. EGCG Up-regulates TNF Production by DCs Stimulated with LPS, MDP or Infected with Lp. Murine derived DCs stimulated with LPS (10 ng/ml) p roduced detectable levels of TNF in the culture supernatants 24 hr after stimulatio n. The DC cultures treated with increasing amounts of EGCG showed marked enhancemen t, after 24 hours, of TNF when treated with a concentration of 50 g/ml (13). In contrast, a higher concentration (100 g/ml) markedly inhibited TNF production in the LPS stimulated cultures after 24 hours (Figure 16).

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45 Figure 16. Effects of increasing concentrations of EGCG on TNF production in cultures of BM derived dendritic cells stimulated with LPS. Resul ts expressed as mean value in ng/ml SEM from 5 independent experiments. The asterisks indicate sta tistically significant differences of P<0.05 from values of the non-EGCG treated LPS stimulated cells The effects of EGCG were examined further to determ ine effects on responses to other microbial stimulators. For this purpose, DC cultures were treated with MDP (10 g/ml) and the results showed DCs stimulated with MD P and treated with the 50 g/ml concentration of EGCG had approximately a 3 fold in crease in TNF production. Furthermore, a 100 g/ml concentration also resulted in a significant i ncrease, but less than that induced by the lower concentration (Figur e 17).

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46 Figure 17. Effects of increasing concentrations of EGCG on TNF production in cultures of BM derived dendritic cells stimulated with MDP. Resul ts expressed as mean value in pg/ml SEM from 5 independent experiments. The asterisks indicate s tatistically significant differences of P<0.05 from values from non-ECGG treated MDP-stimulated cells. Next, we examined the effect of EGCG on cytokine pr oduction by DCs after infection with Lp. The effects of EGCG on the patt ern of production of TNF in DCs infected with Lp was similar to that observed follo wing stimulation with LPS or MDP. In particular, the 50 g/ml EGCG concentration enhanced production of TNF to approximately 2.5 ng/ml, a level several fold highe r than observed in Lp infected DCs treated with 100 g/ml of EGCG (Figure 18).

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47 Figure 18. Effects of EGCG on TNF production by dendritic cells infected 24 hr with Lp. TNF levels in culture supernatants determined by ELISA and results expressed as mean value in ng/ml SEM from 3 independent experiments. The asterisk i ndicates statistically significant differences (p<0.05) from values obtained with non-EGCG treated Lp infected DCs. EGCG inhibits IL-12 production by DCs stimulated wi th MDP or LPS or infected with Lp. EGCG also had marked effects on production of IL-1 2 p40/p70 in the stimulated DC cultures. LPS treated cells without EGCG evinced marked production of this cytokine after 24 hours. However, addition of EGCG to the cultures inhibited IL-12 p40/p70. The 10 g/ml concentration of EGCG had a slight inhibitory effect. Moreover,

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48 the 50 g/ml and 100 g/ml concentrations markedly depressed IL-12 p40/p7 0 production (Figure 19). Figure 19. Effects of ECGG on IL-12 p40/p70 product ion by BM derived dendritic cells stimulated by LPS. Results expressed as mean value in ng/ml SEM from 5 independent experiments 24 hrs after stimulation of cells. The asterisk indicates statistically significant differences (p<0.05) fro m values obtained with nontreated EGCG LPS-stimulat ed cells. Similar suppressive effects were observed by EGCG t reatment of MDP stimulated DCs. The 10 g/ml concentration reduced by 50% IL-12 production, while the 50 and 100 g/ml concentrations essentially abolished the respo nse (Figure 20).

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49 Figure 20. Effects of increasing concentrations of EGCG on IL-12 p40/p70 production in cultures of BM-derived dendritic cells stimulated with MDP. Re sults expressed as mean value in ng/ml SEM from 5 independent experiments. The asterisks indi cate statistically significant differences (p<0.05) from the values of the non-EGCG treated MDP-stimula ted cells. Similarly, DCs infected with Lp and treated with EGCG showed a marked reduction (50 g/ml) or essentially abolished (100 g/ml) the response (Figure 21).

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50 Figure 21. Effects of EGCG on IL-12 p40/p70 product ion by dendritic cells infected 24 hr with Lp. Results expressed as mean value in ng/ml SEM from 3 independent experiments. The asterisks indicate statistically significant differences of P <0.05 from values obtained with non-EGCG treated Lp infected DCs. As shown previously in cell viability studies (see Figure 15), treatment of DCs with EGCG at 10 and 50 g/ml did not decrease cell viability, which indicat es that increased TNF and decreased IL-12 production levels were not due to EGCG toxicity at these concentration levels. However, a significant (p<.05 ) decrease in cell viability (75% of control) was observed when DCs were treated with th e higher concentration of 100 g/ml which may explain why TNF production levels did not continue to increase at 100

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51 g/ml. This suggests that some of the decrease of IL -12 production at 100 m g/ml may be due to cytotoxic effects of EGCG on the DCs. Inhibition of IL-12 by EGCG does not depend on TNF To determine whether inhibition of EGCG inhibited IL-12 production depended on induced TNF production, DCs were stimulated with LPS either al one or in the presence of neutralizing antibody to TNF and production of IL-12 was determined. As shown in Figure 22 TNF production by LPS stimulated DCs was decreased abo ut 3 fold with neutralization antibody. Figure 22. Effects of EGCG (50 g/ml) on TNF production in cultures of DCs stimulated with LPS (10 ng/ml) with or without antiTNF neutralization antibody (20 g/ml).

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52 However, as shown in Figure 23, anti-TNF had no effect on IL-12 production by DCs at 2 and 4 hours and minimallt decreased the effect at 24 hours in contrast to EGCG treatment which markedly diminished LPS induced IL12 production. Figure 23. Effects of EGCG (50 g/ml) on IL12 production in cultures of DCs stimula ted with LPS (10 ng/ml) with or without antiTNF neutralization antibody (20 g/ml).

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53 EGCG inhibits RANTES, MCP-1 and MIP1production by DC stimulated with LPS. DC maturation is often accompanied by production o f chemokines that assist DCs in attracting T cells for efficient antigen present ation (108). EGCG inhibited LPS-induced RANTES (Figure 24), MCP-1 (Figure 25), and MIP1(Figure 26). For the most part, significant differences were observed only at the 5 0 m g/ml concentration. Figure 24. Effects of EGCG on RANTES production by DCs stimulated by LPS (100 ng/ml). Results are expressed as mean value in ng/ml SEM from 3 i ndependent experiments 24 hours after stimulation of cells. The asterisk indicates statis tically significant differences of P<0.05 from valu es obtained non-treated EGCG LPS-stimulated cells.

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54 Figure 25. Effects of EGCG on MCP-1 production by D Cs stimulated by LPS (100 ng/ml). Results are expressed as mean value in pg/ml SEM from 3 i ndependent experiments 24 hours after stimulation of cells. The asterisk indicates statis tically significant differences (p<0.05) from value s obtained in non-treated EGCG, LPS-stimulated cells.

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55 Figure 26. Effects of EGCG on MIP1production by DCs stimulated by LPS (100 ng/ml). R esults are expressed as mean value in pg/ml SEM from 3 i ndependent experiments 24 hours after stimulation of cells. The asterisk indicates statis tically significant differences (p<0.05) from value s obtained in non-treated EGCG, LPS-stimulated cells. EGCG inhibits RANTES, MCP1 and MIP1 production by DCs infected with Lp. EGCG also attenuated Lp-induced RANTES (Figure 27) MCP1 (Figure 28) and MIP1 (Figure 29) chemokine production, which was signif icant at higher doses of EGCG.

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56 Figure 27. Effects of EGCG on RANTES production by DCs after infection by Lp (20:1). Results are expressed as mean value in pg/ml SEM from 3 indep endent experiments 24 hours after stimulation of cells. The asterisk indicates statistically sign ificant differences (p<0.05) from values obtained i n non-treated EGCG, Lp infected cells.

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57 Figure 28. Effects of EGCG on MCP1 production by DC s infected with Lp (20:1). Results are expressed as mean value in pg/ml SEM from 3 indep endent experiments 24 hours after stimulation of cells. The asterisk indicates statistically sign ificant differences (p<0.05) from values obtained i n non-treated EGCG, Lp infected cells.

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58 Figure 29. Effects of EGCG on MIP1 production by DCs infected with Lp (20:1). Results are expressed as mean value in pg/ml SEM from 3 indep endent experiments 24 hours after stimulation of cells. The asterisk indicates statistically sign ificant differences (p<0.05) from values obtained i n non-treated EGCG, Lp infected cells. Aim 3: Determine molecular signaling mechanisms inv olved in effects of EGCG on DC maturation. Lp and LPS are potent inducers of TLR2 and/or TLR4 surface molecule expression. Lp was a potent stimulator of TLR2 surface molecule expression in DCs. In particular, Lp increased the percentage of cells do uble positive for CD11c and TLR2 to 64% from 19% (Figure 30). Lp also upregulated surfa ce molecule expression of the TLR4 from16% to 34% (Figure 30).

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59 Figure 30. Lp infection up-regulates TLR2/TLR4 surf ace expression on DCs infected with Lp. DCs were infected at 10 bacteria per cell and cultured at 1x106 cells/ml. (A) Flow cytometric dot plots of CD11c and TLR 2/4 surface molecule expression. Numb ers in quadrants reflect percentages rounded to next greater whole integer. Results shown are 1 of 3 independent experiments with similar results. (B) Bar graphs of percentage of CD11c+ and TLR2/4 s urface molecule expression. Data represent mean SD from three independent experiments. Aster isks indicate statistically significant differences (p<0.05) from non-Lp infected cells. LPS was also a very potent inducer of TLR2 surface molecule expression by DCs. In particular, LPS increased the percentage of cell s double positive for CD11c and TLR2 from 28% to 76% (Figure 31). In contrast, LPS actua lly downregulated TLR4 surface expression (data not shown) which is in accord with previous reports that LPS stimulation of DCs leads to TLR4 internalization an d degradation (60).

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60 Figure 31. EGCG inhibits induced TLR2 on DCs infect ed with Lp or stimulated with LPS and treated with various concentrations of EGCG analyze d by flow cytometry. Numbers in quadrants reflect percentages rounded to next greater whole i nteger. Results shown are 1 of 3 independent experiments with similar results. EGCG Inhibits Upregulation of TLR2/TLR4 Surface Exp ression Induced by Lp and LPS. This upregulation of TLR2 by both Lp and LPS was d ramatically inhibited by increasing doses of EGCG (Figure 31). EGCG treatme nt in a dose dependent manner also inhibited TLR4 up-regulation caused by Lp infe ction (Figure 32).

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61 Figure 32. EGCG inhibits induced TLR4 on DCs infect ed with Lp and treated with various concentrations of EGCG analyzed by flow cytometry. Numbers in quadrants reflect percentages rounded to next greater whole integer. Results show n are 1 of 3 independent experiments with similar results. EGCG Inhibits NFB Activation by LPS Most genes of inflammatory mediators such as TNF and IL-12 are regulated by NFB because they have a B site in their 5’ flanking region (46). Inhibition of NFB has also been reported to suppress induction of TLR 4 and TLR2 mRNA expression in mouse DCs stimulated with LPS (8). To determine wh ether EGCG inhibition of inflammatory mediators and TLR up-regulation involv ed inhibition of NFB translocation, DCs exposed to LPS were simultaneous ly treated with EGCG. As shown in (Figure 33), LPS stimulation resulted in enhanced a ctivation of NFB whereas this stimulation was significantly inhibited by EGCG (50 g/ml).

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62 Figure 33. EGCG inhibits DNA binding activity of p6 5/Rel A subunit from DCs stimulated with LPS. Cellular extracts (16 g) obtained from DCs treated with 10 ng/ml of LPS without EGCG treatment showed increased binding of p65/Rel A subunit to NF B binding sequence when compared to EGCG (50 g/ml; 45 minute incubation) treated DCs.

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63 DISCUSSION The mechanisms underlying maturation and immunogeni city of DCs are starting to be elucidated. Immature DCs capture antigens and during maturation, MHC peptide complexes begin to form within the MHC class II com partments, followed by transport in non-lysosomal vesicles to the cell surface (132). MHC class I is also upregulated upon maturation (161). Several co-stimulatory molecules such as CD40 and CD86, are also expressed. The MHC-peptide complexes are found in c lusters at the DC surface together with CD86 (161). It is believed that these high le vels of antigen-presenting and costimulatory molecules, in a clustered distribution, initiate the formation of the immunologic synapse, bringing together essential el ements like the TCR and CD28 required for T cell activation (89). Maturing DCs c hange in many other ways, including changes in chemokine receptor expression which cont ributes to their migration to the T cell areas of lymphoid tissue (30). In this study, we examined various parameters of D C maturation in response to several microbial products and the effects of EGCG on these parameters. For example, we observed that EGCG inhibits Lp induced surface e xpression of co-stimulatory molecules by BALB/c mouse DCs. Up-regulation of th ese proteins is a central feature of DC maturation and is associated with their enhanced ability to activate resting T cells. We additionally showed that EGCG inhibited Lp induc ed up-regulation of both class MHC I and II molecules. DCs process exogenous antig ens intracellularly and present them to CD4 T cells via MHC class II molecules (168 ). Although most cells use their MHC class I molecules to present peptides derived f rom endogenously synthesized

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64 proteins, DCs have the capacity to deliver exogenou s antigens to the MHC class I pathway, a phenomenon known as cross-presentation ( 55). Up-regulation of CD11c surface expression on BMDCs by bacterial products has been reported. For example, both mycoplasma lipopro tein FL-1 and LPS have previously been reported to up-regulate CD11c on the surfaces of C57BL/6-derived mouse BMDCs (82). Our results also show an increase in CD11c in response to microbial stimulation by Lp or LPS and in addition we observed an increase i n double positive DCs which expressed both CD11c and the various MHC/costimulat ory molecules. Treatment with EGCG, however, suppressed the expression of all of these developmental markers following stimulation by microbial products. The inhibitory effects of EGCG on maturation of DC s by infection is further substantiated by our results showing that EGCG inhi bits IL-12 p40 production in DCs after Lp infection (140). IL-12p40 is a subunit of IL-12p70 whose expression is inducible and correlated with production of bioacti ve p70 by DCs (8). IL-12 production is widely regarded as an essential indicator of a full y activated DC phenotype (98). EGCG, as well as other catechins have also reportedly sup pressed IL-12 p40 production by murine peritoneal macrophages and the macrophage ce ll line, J774.1(61). In other studies with EGCG, the compound upregulated important inna te immune stimulating cytokines such as IFN and TNF. (106). In our studies, we also show that EGCG up regulates TNF production by DCs after stimulation by LPS, MDP an d Lp (140). Other studies have reported dependence of IL-12 on TNF, as well as possibly other cytokines. For example, IL-12 production by m urine macrophages in response to Mycobacterium bovis Bacillus Calmette-Gurin reportedly depends on IFN and TNF

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65 production (42). Moreover, administration of antiTNF monoclonal antibody diminished the lung levels of IL-12 and IFN induced by Cryptococcus neoformans infection in CBA/J mice (57). In order to determine dependence of IL-12 production by DCs on TNF in our system, we treated LPS stimulated DCs with TNF neutralization antibody. We show that neutralization of TNF did not significantly affect IL-12 production levels. The differences between our resu lts and those of other thus likely depends upon differences in DC biology compared to other cell types studied such as macrophages. Zakharova recently reported that addition of TNF reduced IL-12p40 production in DCs, suggesting a possible anti-inflammatory rol e for TNF (184). Our studies do not indicate a role of TNF in reduction of IL-12p40 because neutralization of TNF either with or without EGCG treatment did not affect IL-12 p40 production levels by DCs. The differences between our results and those of Zakhar ova may thus relate to differences in cell culture conditions such as levels of LPS stimu lation (1 ng/ml used by Zakharova versus 10 ng/ml in our studies), cell number and/or culture medium used. Moreover, Zakharova preincubated DCs with TNF followed by LPS stimulation whereas we did not add exogenous TNF. In addition, the majority of Zakharova studies we re done with macrophages. Maturing DCs are also an abundant and strategic sou rce of chemokines which are produced in a precise time-ordered fashion. Followi ng stimulation with LPS, DCs have an initial burst of MIP1 (CCL3), MIP1 (CCL4) and IL-8 (CXCL8) production, which cease within a few hours. RANTES (CCL5) and MCP1 a re also induced, but in a more steady manner. At later time points, DCs produce ma inly lymphoid chemokines, such as

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66 CCL17 (TARC), CCL18 (DC-CD1), CCL19 (MIP-3) and CCL22 (MDC), that attract T and B lymphocytes (107, 144). As shown in this stud y, LPS induced up-regulation of the early inflammatory chemokines RANTES, MCP1 and MIP1. This up-regulation was significantly inhibited by EGCG, particularly at hi gher concentrations of EGCG. Several other important pharmaceutical agents have been shown to suppress DC maturation and activation such as 1 Alpha, 25-dihyd roxyvitamin D3 (15, 128), resveratrol (3), aspirin (50), and glucocorticoids (130). On a molecular level, these agents typically block DC maturation by inhibiting relB, a subunit o f the NFB pathway (98). As shown in this study, EGCG inhibited both LPS an d Lp up-regulation of TLR2 and TLR4 by DCs. EGCG also inhibited activation of the p65/RelA NFB subunit in DCs treated with LPS. TLRs are critical for inducti on of downstream effecter functions in monocytes (7), and control expression of co-stimula tory molecules, as well as induction of cytokine and chemokine production by DCs (65, 15 3). TLR4 is a signal transducer for LPS, whereas TLR2 is a common transducer for a dive rse array of bacterial products (93) such as PGN from Gram-positive bacteria (93). Lp i s a Gram-negative pathogen and due to its LPS would be expected to activate TLR4 which is a receptor for Gram negative LPS, whereas TLR2 is a receptor for other bacterial products (93). However, related studies suggest that TLR2, rather than TLR4 plays a prominent role in Lp infection since purified Lp LPS as well as Lp, either viable or formalin-killed are able to activate DCs from TLR4-deficient C3H/HeJ mice but fail to activa te DCs from TLR2-knockout mice(19). In our study, we found that infection with viable Lp resulted in marked upregulation of TLR2 on DCs, and this may be related to TLR4, since microbial stimulation

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67 leads to NFB activation, and the promoter of TLR2 contains NFB sites known to upregulate TLR2 gene transcription(117). Inhibition of ERK or NFB has also been reported to suppress induction of TLR4 and TLR2 mRN A expression in mouse DCs stimulated with LPS (8). Contrary to our results, the expression of maturati on surface markers CD40, CD86 and MHC class II, was strikingly lower than wa s previously reported in DCs from A/J mice infected with live Lp compared to non-infe cted cells (81). The differences between these results and ours may be related to th e different strains of mice used. A/J mice are relatively more susceptible to Lp infectio n whereas BALB/c mice used in this study are relatively resistant. The differing resul ts also suggest that co-stimulatory and MHC class II up-regulation on BALB/c DCs may accoun t for increased resistance to infection with Lp in this mouse strain. Although not examined in the A/J model, TLR upregulation in BALB/c mice may serve as an additiona l important factor in differences between the two strains in susceptibility to Lp infection. In addition to the importance of both TLR2 and TLR4 in sepsis (103, 167), emerging data support contribution of these TLRs in diseases like atherosclerosis (123). For example, mice deficient in MyD88, a TLR-signall ing adaptor protein, are less prone to atherosclerosis (16, 114) and patients with a D2 99G polymorphisms of TLR4 have reduced risk of atheroscelorsis (79). The associati on between TLR4 function and atherosclerosis is consistent with findings showing that TLR4 mRNA and protein are more abundant in plaques in atherosclerotic lesions than in unaffected vessels (171). TLR2 also reportedly potentiates microglial interac tion with A42, a key pathogenic factor in Alzheimer Disease (AD), via the induction of the G-protein-coupled receptor

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68 mFPR2 (26). TLR signaling may also contribute to di lated cardiomyopathy, a common heart failure in young patients, by elevating dendr itic cell function (38). TLRs might also be responsible for the development of diabetes (83, 186) and experimental autoimmune encephalomyelitis (78). TLRs also play a crucial pa rt in the induction and progress of chronic inflammatory disorders such as asthma, a T helper 2 mediated chronic airway disorder (31, 37), and rheumatoid arthritis, a TH1related inflammatory joint disease (64, 131). Thus, the inhibitory effects of EGCG on TLR up-regu lation as shown in this study may have therapeutic applications. However, both TL R2 and TLR4 are likely regulated differently in human cells by EGCG. This may be par ticularly the case with TLR2 since the proximal promoter regions of mouse and human TL R2 genes does not reveal a significant level of homology (52). Assessment of the physiological relevance of the findings presented here must also take into account maximum achievable EGCG concentrations attainable in vivo. In summary, our results show that microbial product s from LPS, MDP and Lp infection of DCs can significantly impact key DC ma turation markers. These maturation markers include important co-stimulatory and MHC mo lecules as well as proinflammatory cytokines such as IL-12 and TNF. In addition, EGCG has significant inhibitory effects on DC production of the pro-infl ammatory chemokines, RANTES, MIP1 and MCP1. These studies show that DCs are suscepti ble to immune modulation following Lp infection which is likely important in transition from innate to adaptive immunity. In addition, these studies show that the polyphenol EGCG is a potent antiinflammatory small molecular weight molecule which may have potential therapeutic

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69 uses against diseases implicated in inflammation an d up-regulation of TLRs. The molecular mechanisms for the action of EGCG likely involve inhibition of ROS and TLR signaling transduction pathways which lead to downs tream activation of NFB (Figure 34). TLR4 LPS TLR2Lp EGCG Gene promoter MAPKs/IKKs p50 p65 Activated NFB I B p50 p65 P I B P proteolysis TLR9ROS EGCG MD88 MD88TRIF MD88 IRAK-4 TLR2 Proinflammatorycytokines/chemokines EGCG Figure 34. Schematic diagram of proposed effects of EGCG on DCs. Bacterial products such as LPS and Lp interact with TLRs thereby activating TLR si gnalling transduction and/or ROS which activates MAPKs/IKKs leading to activation of NF B. NF B activates many pro-inflammatory genes for pro-inflammatory cytokines/chemokines. TLRs are upregulated themselves in response to NF B which serves to further heighten the immune respons e. There is also cross-talk between TLRs as in the case of where LPS activates NF B which then activates the promoter for TLR2 thereb y upregulating TLR2 in response to LPS stimulation. EGC G inhibits ROS and/or MAPKS and NF B which downregulates many pro-inflammatory cytokines /chemokines as well as TLRs such as TLR2.

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70 REFERENCES CITED 1. Ahmad, N., S. Gupta, and H. Mukhtar. 2000. Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kappaB in cancer cells versus normal cells. Arch Biochem Biop hys 376: 338-346. 2. Ahn, H. Y., K. R. Hadizadeh, C. Seul, Y. P. Yun, H. Vetter, and A. Sachinidis. 1999. Epigallocathechin-3 gallate selectively inhi bits the PDGF-BBinduced intracellular signaling transduction pathwa y in vascular smooth muscle cells and inhibits transformation of sis-transfecte d NIH 3T3 fibroblasts and human glioblastoma cells (A172). Mol Biol Cell 10: 1093-1104. 3. Ahn, S. C., G. Y. Kim, J. H. Kim, S. W. Baik, M. K. Han, H. J. Lee, D. O. Moon, C. M. Lee, J. H. Kang, B. H. Kim, Y. H. Oh, a nd Y. M. Park. 2004. Epigallocatechin-3-gallate, constituent of green te a, suppresses the LPS-induced phenotypic and functional maturation of murine dend ritic cells through inhibition of mitogen-activated protein kinases and NF-kappaB. Biochem Biophys Res Commun 313: 148-155. 4. Aicher, A., G. L. Shu, D. Magaletti, T. Mulvania, A Pezzutto, A. Craxton, and E. A. Clark. 1999. Differential role for p38 mitogen-activated protein kinase in regulating CD40-induced gene expression in dendr itic cells and B cells. J Immunol 163: 5786-5795. 5. Akamine, M., F. Higa, N. Arakaki, K. Kawakami, K. T akeda, S. Akira, and A. Saito. 2005. Differential roles of Toll-like receptors 2 and 4 in in vitro responses of macrophages to Legionella pneumophila. Infect Immun 73: 352-361. 6. Akira, S., and K. Takeda. 2004. Toll-like receptor signalling. Nat Rev Immun ol 4: 499-511. 7. Akira, S., K. Takeda, and T. Kaisho. 2001. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2: 675-680. 8. An, H., Y. Yu, M. Zhang, H. Xu, R. Qi, X. Yan, S. L iu, W. Wang, Z. Guo, J. Guo, Z. Qin, and X. Cao. 2002. Involvement of ERK, p38 and NF-kappaB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic ce lls. Immunology 106: 38-45. 9. Asehnoune, K., D. Strassheim, S. Mitra, J. Y. Kim, and E. Abraham. 2004. Involvement of reactive oxygen species in Toll-like receptor 4-dependent activation of NF-kappa B. J Immunol 172: 2522-2529. 10. Bachmann, M. F., M. Kopf, and B. J. Marsland. 2006. Chemokines: more than just road signs. Nat Rev Immunol 6: 159-164. 11. Baldwin, A. S., Jr. 1996. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol 14: 649-683. 12. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, B. Pulendran, and K. Palucka. 2000. Immunobiology of dendritic cells. Annu Rev Immunol 18: 767-811. 13. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245-252. 14. Belvin, M. P., and K. V. Anderson. 1996. A conserved signaling pathway: the Drosophila toll-dorsal pathway. Annu Rev Cell Dev B iol 12: 393-416.

PAGE 85

71 15. Berer, A., J. Stockl, O. Majdic, T. Wagner, M. Koll ars, K. Lechner, K. Geissler, and L. Oehler. 2000. 1,25-Dihydroxyvitamin D(3) inhibits dendriti c cell differentiation and maturation in vitro. Exp H ematol 28: 575-583. 16. Bjorkbacka, H., V. V. Kunjathoor, K. J. Moore, S. K oehn, C. M. Ordija, M. A. Lee, T. Means, K. Halmen, A. D. Luster, D. T. Go lenbock, and M. W. Freeman. 2004. Reduced atherosclerosis in MyD88-null mice l inks elevated serum cholesterol levels to activation of innate im munity signaling pathways. Nat Med 10: 416-421. 17. Bonifaz, L. C., D. P. Bonnyay, A. Charalambous, D. I. Darguste, S. Fujii, H. Soares, M. K. Brimnes, B. Moltedo, T. M. Moran, and R. M. Steinman. 2004. In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination. J Exp Med 199: 815-824. 18. Boring, L., J. Gosling, S. W. Chensue, S. L. Kunkel R. V. Farese, Jr., H. E. Broxmeyer, and I. F. Charo. 1997. Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in C-C chemokine re ceptor 2 knockout mice. J Clin Invest 100: 2552-2561. 19. Braedel-Ruoff, S., M. Faigle, N. Hilf, B. Neumeiste r, and H. Schild. 2005. Legionella pneumophila mediated activation of dendr itic cells involves CD14 and TLR2. J Endotoxin Res 11: 89-96. 20. Cario, E., I. M. Rosenberg, S. L. Brandwein, P. L. Beck, H. C. Reinecker, and D. K. Podolsky. 2000. Lipopolysaccharide activates distinct signal ing pathways in intestinal epithelial cell lines expres sing Toll-like receptors. J Immunol 164: 966-972. 21. Caux, C., S. Ait-Yahia, K. Chemin, O. de Bouteiller M. C. Dieu-Nosjean, B. Homey, C. Massacrier, B. Vanbervliet, A. Zlotnik, a nd A. Vicari. 2000. Dendritic cell biology and regulation of dendritic cell trafficking by chemokines. Springer Semin Immunopathol 22: 345-369. 22. Cella, M., F. Sallusto, and A. Lanzavecchia. 1997. Origin, maturation and antigen presenting function of dendritic cells. Cur r Opin Immunol 9: 10-16. 23. Chang, L., and M. Karin. 2001. Mammalian MAP kinase signalling cascades. Nature 410: 37-40. 24. Chedid, L. A., M. A. Parant, F. M. Audibert, G. J. Riveau, F. J. Parant, E. Lederer, J. P. Choay, and P. L. Lefrancier. 1982. Biological activity of a new synthetic muramyl peptide adjuvant devoid of pyroge nicity. Infect Immun 35: 417-424. 25. Chen, C., R. Yu, E. D. Owuor, and A. N. Kong. 2000. Activation of antioxidant-response element (ARE), mitogen-activat ed protein kinases (MAPKs) and caspases by major green tea polyphenol componen ts during cell survival and death. Arch Pharm Res 23: 605-612. 26. Chen, K., P. Iribarren, J. Hu, J. Chen, W. Gong, E. H. Cho, S. Lockett, N. M. Dunlop, and J. M. Wang. 2006. Activation of Toll-like receptor 2 on microg lia promotes cell uptake of Alzheimer disease-associate d amyloid beta peptide. J Biol Chem 281: 3651-3659.

PAGE 86

72 27. Chen, W., Z. Dong, S. Valcic, B. N. Timmermann, and G. T. Bowden. 1999. Inhibition of ultraviolet B--induced c-fos gene exp ression and p38 mitogenactivated protein kinase activation by (-)-epigallo catechin gallate in a human keratinocyte cell line. Mol Carcinog 24: 79-84. 28. Cohen, L. Y., G. M. Bahr, E. C. Darcissac, and M. A Parant. 1996. Modulation of expression of class II MHC and CD40 m olecules in murine B cells by various muramyl dipeptides. Cell Immunol 169: 75-84. 29. Cummings, N. P., M. J. Pabst, and R. B. Johnston, J r. 1980. Activation of macrophages for enhanced release of superoxide anio n and greater killing of Candida albicans by injection of muramyl dipeptide. J Exp Med 152: 1659-1669. 30. Cyster, J. G. 1999. Chemokines and the homing of dendritic cells to the T cell areas of lymphoid organs. J Exp Med 189: 447-450. 31. Dabbagh, K., M. E. Dahl, P. Stepick-Biek, and D. B. Lewis. 2002. Toll-like receptor 4 is required for optimal development of T h2 immune responses: role of dendritic cells. J Immunol 168: 4524-4530. 32. Delgado, M., E. Gonzalez-Rey, and D. Ganea. 2004. VIP/PACAP preferentially attract Th2 effectors through differential regulati on of chemokine production by dendritic cells. Faseb J 18: 1453-1455. 33. Dhodapkar, M. V., R. M. Steinman, J. Krasovsky, C. Munz, and N. Bhardwaj. 2001. Antigen-specific inhibition of effector T ce ll function in humans after injection of immature dendritic cells. J Exp Med 193: 233-238. 34. DiDonato, J., F. Mercurio, C. Rosette, J. Wu-Li, H. Suyang, S. Ghosh, and M. Karin. 1996. Mapping of the inducible IkappaB phosphoryla tion sites that signal its ubiquitination and degradation. Mol Cell Biol 16: 1295-1304. 35. Dieu, M. C., B. Vanbervliet, A. Vicari, J. M. Brido n, E. Oldham, S. AitYahia, F. Briere, A. Zlotnik, S. Lebecque, and C. C aux. 1998. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med 188: 373-386. 36. Dumitru, C. D., J. D. Ceci, C. Tsatsanis, D. Kontoy iannis, K. Stamatakis, J. H. Lin, C. Patriotis, N. A. Jenkins, N. G. Copeland G. Kollias, and P. N. Tsichlis. 2000. TNF-alpha induction by LPS is regulated post transcriptionally via a Tpl2/ERK-dependent pathway. Cell 103: 1071-1083. 37. Eisenbarth, S. C., D. A. Piggott, J. W. Huleatt, I. Visintin, C. A. Herrick, and K. Bottomly. 2002. Lipopolysaccharide-enhanced, toll-like recep tor 4-dependent T helper cell type 2 responses to inhaled antigen. J Exp Med 196: 1645-1651. 38. Eriksson, U., R. Ricci, L. Hunziker, M. O. Kurrer, G. Y. Oudit, T. H. Watts, I. Sonderegger, K. Bachmaier, M. Kopf, and J. M. Pe nninger. 2003. Dendritic cell-induced autoimmune heart failure requires coop eration between adaptive and innate immunity. Nat Med 9: 1484-1490. 39. Fan, J., R. S. Frey, and A. B. Malik. 2003. TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADP H oxidase. J Clin Invest 112: 1234-1243. 40. Fan, J., Y. Li, Y. Vodovotz, T. R. Billiar, and M. A. Wilson. 2006. Hemorrhagic shock-activated neutrophils augment TLR 4 signaling-induced TLR2 upregulation in alveolar macrophages: role in hemor rhage-primed lung inflammation. Am J Physiol Lung Cell Mol Physiol 290: L738-L746.

PAGE 87

73 41. Fitzgerald, K. A., E. M. Palsson-McDermott, A. G. B owie, C. A. Jefferies, A. S. Mansell, G. Brady, E. Brint, A. Dunne, P. Gray, M. T. Harte, D. McMurray, D. E. Smith, J. E. Sims, T. A. Bird, and L. A. O'Neill. 2001. Mal (MyD88-adapter-like) is required for Toll-like rece ptor-4 signal transduction. Nature 413: 78-83. 42. Flesch, I. E., J. H. Hess, S. Huang, M. Aguet, J. R othe, H. Bluethmann, and S. H. Kaufmann. 1995. Early interleukin 12 production by macrophag es in response to mycobacterial infection depends on inte rferon gamma and tumor necrosis factor alpha. J Exp Med 181: 1615-1621. 43. Fraticelli, P., M. Sironi, G. Bianchi, D. D'Ambrosi o, C. Albanesi, A. Stoppacciaro, M. Chieppa, P. Allavena, L. Ruco, G. Girolomoni, F. Sinigaglia, A. Vecchi, and A. Mantovani. 2001. Fractalkine (CX3CL1) as an amplification circuit of polarized Th1 responses. J Clin Invest 107: 1173-1181. 44. Fujii, S., K. Liu, C. Smith, A. J. Bonito, and R. M Steinman. 2004. The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen prese ntation and CD80/86 costimulation. J Exp Med 199: 1607-1618. 45. Gangur, V., F. E. Simons, and K. T. Hayglass. 1998. Human IP-10 selectively promotes dominance of polyclonally activated and en vironmental antigen-driven IFN-gamma over IL-4 responses. Faseb J 12: 705-713. 46. Ghosh, S., M. J. May, and E. B. Kopp. 1998. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune respon ses. Annu Rev Immunol 16: 225-260. 47. Glauser, M. P., G. Zanetti, J. D. Baumgartner, and J. Cohen. 1991. Septic shock: pathogenesis. Lancet 338: 732-736. 48. Goth, S. R., R. A. Chu, and I. N. Pessah. 2006. Oxygen tension regulates the in vitro maturation of GM-CSF expanded murine bone mar row dendritic cells by modulating class II MHC expression. J Immunol Metho ds 308: 179-191. 49. Greenwood, J., L. Steinman, and S. S. Zamvil. 2006. Statin therapy and autoimmune disease: from protein prenylation to imm unomodulation. Nat Rev Immunol 6: 358-370. 50. Hackstein, H., A. E. Morelli, A. T. Larregina, R. W Ganster, G. D. Papworth, A. J. Logar, S. C. Watkins, L. D. Falo, a nd A. W. Thomson. 2001. Aspirin inhibits in vitro maturation and in vivo im munostimulatory function of murine myeloid dendritic cells. J Immunol 166: 7053-7062. 51. Haddad, J. J. 2004. Oxygen sensing and oxidant/redox-related pat hways. Biochem Biophys Res Commun 316: 969-977. 52. Haehnel, V., L. Schwarzfischer, M. J. Fenton, and M Rehli. 2002. Transcriptional regulation of the human toll-like r eceptor 2 gene in monocytes and macrophages. J Immunol 168: 5629-5637. 53. Hailman, E., H. S. Lichenstein, M. M. Wurfel, D. S. Miller, D. A. Johnson, M. Kelley, L. A. Busse, M. M. Zukowski, and S. D. Wrig ht. 1994. Lipopolysaccharide (LPS)-binding protein accelerate s the binding of LPS to CD14. J Exp Med 179: 269-277.

PAGE 88

74 54. Hawn, T. R., A. Verbon, K. D. Lettinga, L. P. Zhao, S. S. Li, R. J. Laws, S. J. Skerrett, B. Beutler, L. Schroeder, A. Nachman, A. Ozinsky, K. D. Smith, and A. Aderem. 2003. A common dominant TLR5 stop codon polymorphi sm abolishes flagellin signaling and is associated wit h susceptibility to legionnaires' disease. J Exp Med 198: 1563-1572. 55. Heath, W. R., G. T. Belz, G. M. Behrens, C. M. Smit h, S. P. Forehan, I. A. Parish, G. M. Davey, N. S. Wilson, F. R. Carbone, a nd J. A. Villadangos. 2004. Cross-presentation, dendritic cell subsets, a nd the generation of immunity to cellular antigens. Immunol Rev 199: 9-26. 56. Heinzelmann, M., M. A. Mercer-Jones, S. A. Gardner, M. A. Wilson, and H. C. Polk. 1997. Bacterial cell wall products increase monocy te HLA-DR and ICAM-1 without affecting lymphocyte CD18 expression Cell Immunol 176: 127134. 57. Herring, A. C., J. Lee, R. A. McDonald, G. B. Toews and G. B. Huffnagle. 2002. Induction of interleukin-12 and gamma interfe ron requires tumor necrosis factor alpha for protective T1-cell-mediated immuni ty to pulmonary Cryptococcus neoformans infection. Infect Immun 70: 2959-2964. 58. Hertz, C. J., S. M. Kiertscher, P. J. Godowski, D. A. Bouis, M. V. Norgard, M. D. Roth, and R. L. Modlin. 2001. Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2. J Immunol 166: 2444-2450. 59. Huang, Q., J. Yang, Y. Lin, C. Walker, J. Cheng, Z. G. Liu, and B. Su. 2004. Differential regulation of interleukin 1 receptor a nd Toll-like receptor signaling by MEKK3. Nat Immunol 5: 98-103. 60. Husebye, H., O. Halaas, H. Stenmark, G. Tunheim, O. Sandanger, B. Bogen, A. Brech, E. Latz, and T. Espevik. 2006. Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive i mmunity. Embo J 25: 683-692. 61. Ichikawa, D., A. Matsui, M. Imai, Y. Sonoda, and T. Kasahara. 2004. Effect of various catechins on the IL-12p40 production by murine peritoneal macrophages and a macrophage cell line, J774.1. Bio l Pharm Bull 27: 1353-1358. 62. Imai, T., M. Nagira, S. Takagi, M. Kakizaki, M. Nis himura, J. Wang, P. W. Gray, K. Matsushima, and O. Yoshie. 1999. Selective recruitment of CCR4bearing Th2 cells toward antigen-presenting cells b y the CC chemokines thymus and activation-regulated chemokine and macrophage-d erived chemokine. Int Immunol 11: 81-88. 63. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, and R. M. Steinman. 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures sup plemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 176: 1693-1702. 64. Iwahashi, M., M. Yamamura, T. Aita, A. Okamoto, A. Ueno, N. Ogawa, S. Akashi, K. Miyake, P. J. Godowski, and H. Makino. 2004. Expression of Tolllike receptor 2 on CD16+ blood monocytes and synovi al tissue macrophages in rheumatoid arthritis. Arthritis Rheum 50: 1457-1467. 65. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu Rev Immunol 20: 197-216.

PAGE 89

75 66. Jankovic, D., Z. Liu, and W. C. Gause. 2001. Th1and Th2-cell commitment during infectious disease: asymmetry in divergent p athways. Trends Immunol 22: 450-457. 67. Janssen-Heininger, Y. M., I. Macara, and B. T. Moss man. 1999. Cooperativity between oxidants and tumor necrosis factor in the a ctivation of nuclear factor (NF)-kappaB: requirement of Ras/mitogen-activated p rotein kinases in the activation of NF-kappaB by oxidants. Am J Respir Ce ll Mol Biol 20: 942-952. 68. Johnson, M. K., and G. Loo. 2000. Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA. Muta t Res 459: 211-218. 69. Jonuleit, H., E. Schmitt, G. Schuler, J. Knop, and A. H. Enk. 2000. Induction of interleukin 10-producing, nonproliferating CD4(+ ) T cells with regulatory properties by repetitive stimulation with allogenei c immature human dendritic cells. J Exp Med 192: 1213-1222. 70. Kaisho, T., O. Takeuchi, T. Kawai, K. Hoshino, and S. Akira. 2001. Endotoxin-induced maturation of MyD88-deficient den dritic cells. J Immunol 166: 5688-5694. 71. Kamata, H., T. Manabe, S. Oka, K. Kamata, and H. Hi rata. 2002. Hydrogen peroxide activates IkappaB kinases through phosphor ylation of serine residues in the activation loops. FEBS Lett 519: 231-237. 72. Karin, M. 1995. The regulation of AP-1 activity by mitogen-a ctivated protein kinases. J Biol Chem 270: 16483-16486. 73. Karpus, W. J., and K. J. Kennedy. 1997. MIP-1alpha and MCP-1 differentially regulate acute and relapsing autoimmune encephalomy elitis as well as Th1/Th2 lymphocyte differentiation. J Leukoc Biol 62: 681-687. 74. Karpus, W. J., N. W. Lukacs, K. J. Kennedy, W. S. S mith, S. D. Hurst, and T. A. Barrett. 1997. Differential CC chemokine-induced enhancemen t of T helper cell cytokine production. J Immunol 158: 4129-4136. 75. Katiyar, S. K., A. Challa, T. S. McCormick, K. D. C ooper, and H. Mukhtar. 1999. Prevention of UVB-induced immunosuppression i n mice by the green tea polyphenol (-)-epigallocatechin-3-gallate may be as sociated with alterations in IL10 and IL-12 production. Carcinogenesis 20: 2117-2124. 76. Kawai, T., O. Adachi, T. Ogawa, K. Takeda, and S. A kira. 1999. Unresponsiveness of MyD88-deficient mice to endotox in. Immunity 11: 115-122. 77. Kawai, T., O. Takeuchi, T. Fujita, J. Inoue, P. F. Muhlradt, S. Sato, K. Hoshino, and S. Akira. 2001. Lipopolysaccharide stimulates the MyD88independent pathway and results in activation of IF N-regulatory factor 3 and the expression of a subset of lipopolysaccharide-induci ble genes. J Immunol 167: 5887-5894. 78. Kerfoot, S. M., E. M. Long, M. J. Hickey, G. Andone gui, B. M. Lapointe, R. C. Zanardo, C. Bonder, W. G. James, S. M. Robbins, and P. Kubes. 2004. TLR4 contributes to disease-inducing mechanisms res ulting in central nervous system autoimmune disease. J Immunol 173: 7070-7077. 79. Kiechl, S., E. Lorenz, M. Reindl, C. J. Wiedermann, F. Oberhollenzer, E. Bonora, J. Willeit, and D. A. Schwartz. 2002. Toll-like receptor 4 polymorphisms and atherogenesis. N Engl J Med 347: 185-192.

PAGE 90

76 80. Kikuchi, T., S. Andarini, H. Xin, K. Gomi, Y. Tokue Y. Saijo, T. Honjo, A. Watanabe, and T. Nukiwa. 2005. Involvement of fractalkine/CX3CL1 expression by dendritic cells in the enhancement of host immunity against Legionella pneumophila. Infect Immun 73: 5350-5357. 81. Kikuchi, T., T. Kobayashi, K. Gomi, T. Suzuki, Y. T okue, A. Watanabe, and T. Nukiwa. 2004. Dendritic cells pulsed with live and dead Le gionella pneumophila elicit distinct immune responses. J Imm unol 172: 1727-1734. 82. Kiura, K., H. Kataoka, T. Nakata, T. Into, M. Yasud a, S. Akira, N. Inoue, and K. Shibata. 2006. The synthetic analogue of mycoplasmal lipopr otein FSL-1 induces dendritic cell maturation through Toll-like receptor 2. FEMS Immunol Med Microbiol 46: 78-84. 83. Kolek, M. J., J. F. Carlquist, J. B. Muhlestein, B. M. Whiting, B. D. Horne, T. L. Bair, and J. L. Anderson. 2004. Toll-like receptor 4 gene Asp299Gly polymorphism is associated with reductions in vascu lar inflammation, angiographic coronary artery disease, and clinical diabetes. Am Heart J 148: 10341040. 84. Krakauer, T., J. Vilcek, and J.J. Oppenheim. 1999. Proinflammatory cytokines: TNF, and IL-1 families, chemokines, TGF, and others., p. 775-811. In W.E.Paul (ed.), Fundamental immunology. Lippincott -Raven, Philadelphia. 85. Kyriakis, J. M., and J. Avruch. 1996. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays 18: 567-577. 86. Lambert, J. D., and C. S. Yang. 2003. Mechanisms of cancer prevention by tea constituents. J Nutr 133: 3262S-3267S. 87. Langenkamp, A., M. Messi, A. Lanzavecchia, and F. S allusto. 2000. Kinetics of dendritic cell activation: impact on priming of TH1, TH2 and nonpolarized T cells. Nat Immunol 1: 311-316. 88. Lanzavecchia, A. 1998. From antigen presentation to T-cell activati on. Res Immunol 149: 626. 89. Lechler, R., W. F. Ng, and R. M. Steinman. 2001. Dendritic cells in transplantation--friend or foe? Immunity 14: 357-368. 90. Lee, H. G., H. Kim, W. K. Oh, K. A. Yu, Y. K. Choe, J. S. Ahn, D. S. Kim, S. H. Kim, C. A. Dinarello, K. Kim, and D. Y. Yoon. 2004. Tetramethoxy hydroxyflavone p7F downregulates inflammatory media tors via the inhibition of nuclear factor kappaB. Ann N Y Acad Sci 1030: 555-568. 91. Levites, Y., O. Weinreb, G. Maor, M. B. Youdim, and S. Mandel. 2001. Green tea polyphenol (-)-epigallocatechin-3-gallate preve nts N-methyl-4-phenyl-1,2,3,6tetrahydropyridine-induced dopaminergic neurodegene ration. J Neurochem 78: 1073-1082. 92. Lieberam, I., and I. Forster. 1999. The murine beta-chemokine TARC is expressed by subsets of dendritic cells and attract s primed CD4+ T cells. Eur J Immunol 29: 2684-2694. 93. Lien, E., T. J. Sellati, A. Yoshimura, T. H. Flo, G Rawadi, R. W. Finberg, J. D. Carroll, T. Espevik, R. R. Ingalls, J. D. Radolf and D. T. Golenbock. 1999. Toll-like receptor 2 functions as a pattern r ecognition receptor for diverse bacterial products. J Biol Chem 274: 33419-33425.

PAGE 91

77 94. Long, L. H., M. V. Clement, and B. Halliwell. 2000. Artifacts in cell culture: rapid generation of hydrogen peroxide on addition o f (-)-epigallocatechin, (-)epigallocatechin gallate, (+)-catechin, and quercet in to commonly used cell culture media. Biochem Biophys Res Commun 273: 50-53. 95. Lu, H. T., D. D. Yang, M. Wysk, E. Gatti, I. Mellma n, R. J. Davis, and R. A. Flavell. 1999. Defective IL-12 production in mitogen-activa ted protein (MAP) kinase kinase 3 (Mkk3)-deficient mice. Embo J 18: 1845-1857. 96. Lukacs, N. W., S. W. Chensue, W. J. Karpus, P. Linc oln, C. Keefer, R. M. Strieter, and S. L. Kunkel. 1997. C-C chemokines differentially alter interleukin-4 production from lymphocytes. Am J Pat hol 150: 1861-1868. 97. Luster, A. D. 2002. The role of chemokines in linking innate and adaptive immunity. Curr Opin Immunol 14: 129-135. 98. Mahnke, K., and A. H. Enk. 2005. Dendritic cells: key cells for the induction of regulatory T cells? Curr Top Microbiol Immunol 293: 133-150. 99. Mandel, S., O. Weinreb, T. Amit, and M. B. Youdim. 2004. Cell signaling pathways in the neuroprotective actions of the gree n tea polyphenol (-)epigallocatechin-3-gallate: implications for neurod egenerative diseases. J Neurochem 88: 1555-1569. 100. Marsland, B. J., P. Battig, M. Bauer, C. Ruedl, U. Lassing, R. R. Beerli, K. Dietmeier, L. Ivanova, T. Pfister, L. Vogt, H. Naka no, C. Nembrini, P. Saudan, M. Kopf, and M. F. Bachmann. 2005. CCL19 and CCL21 induce a potent proinflammatory differentiation program in l icensed dendritic cells. Immunity 22: 493-505. 101. Marston, B. J., H. B. Lipman, and R. F. Breiman. 1994. Surveillance for Legionnaires' disease. Risk factors for morbidity a nd mortality. Arch Intern Med 154: 2417-2422. 102. Martin, E., B. O'Sullivan, P. Low, and R. Thomas. 2003. Antigen-specific suppression of a primed immune response by dendriti c cells mediated by regulatory T cells secreting interleukin-10. Immuni ty 18: 155-167. 103. Martin, M. A. 1991. Epidemiology and clinical impact of gram-neg ative sepsis. Infect Dis Clin North Am 5: 739-752. 104. Matsunaga, K., T. W. Klein, H. Friedman, and Y. Yam amoto. 2002. Epigallocatechin gallate, a potential immunomodulat ory agent of tea components, diminishes cigarette smoke condensate-induced suppr ession of anti-Legionella pneumophila activity and cytokine responses of alve olar macrophages. Clin Diagn Lab Immunol 9: 864-871. 105. Matsunaga, K., T. W. Klein, H. Friedman, and Y. Yam amoto. 2002. In vitro therapeutic effect of epigallocatechin gallate on n icotine-induced impairment of resistance to Legionella pneumophila infection of e stablished MH-S alveolar macrophages. J Infect Dis 185: 229-236. 106. Matsunaga, K., T. W. Klein, H. Friedman, and Y. Yam amoto. 2001. Legionella pneumophila replication in macrophages i nhibited by selective immunomodulatory effects on cytokine formation by e pigallocatechin gallate, a major form of tea catechins. Infect Immun 69: 3947-3953. 107. McColl. 2002. McColl.

PAGE 92

78 108. McColl, S. R. 2002. Chemokines and dendritic cells: a crucial al liance. Immunol Cell Biol 80: 489-496. 109. McGuirk, P., and K. H. Mills. 2002. Pathogen-specific regulatory T cells provoke a shift in the Th1/Th2 paradigm in immunity to infectious diseases. Trends Immunol 23: 450-455. 110. McWilliam, A. S., S. Napoli, A. M. Marsh, F. L. Pem per, D. J. Nelson, C. L. Pimm, P. A. Stumbles, T. N. Wells, and P. G. Holt. 1996. Dendritic cells are recruited into the airway epithelium during the inf lammatory response to a broad spectrum of stimuli. J Exp Med 184: 2429-2432. 111. Medzhitov, R., and C. Janeway, Jr. 2000. Innate immunity. N Engl J Med 343: 338-344. 112. Menten, P., A. Wuyts, and J. Van Damme. 2001. Monocyte chemotactic protein-3. Eur Cytokine Netw 12: 554-560. 113. Michelsen, K. S., A. Aicher, M. Mohaupt, T. Hartung S. Dimmeler, C. J. Kirschning, and R. R. Schumann. 2001. The role of toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells (DCS). Peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2. J Biol Chem 276: 25680-25686. 114. Michelsen, K. S., M. H. Wong, P. K. Shah, W. Zhang, J. Yano, T. M. Doherty, S. Akira, T. B. Rajavashisth, and M. Ardit i. 2004. Lack of Toll-like receptor 4 or myeloid differentiation factor 88 red uces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotei n E. Proc Natl Acad Sci U S A 101: 10679-10684. 115. Morre, D. J., D. M. Morre, H. Sun, R. Cooper, J. Ch ang, and E. M. Janle. 2003. Tea catechin synergies in inhibition of cance r cell proliferation and of a cancer specific cell surface oxidase (ECTO-NOX). Ph armacol Toxicol 92: 234241. 116. Murphy, P. M., M. Baggiolini, I. F. Charo, C. A. He bert, R. Horuk, K. Matsushima, L. H. Miller, J. J. Oppenheim, and C. A Power. 2000. International union of pharmacology. XXII. Nomencla ture for chemokine receptors. Pharmacol Rev 52: 145-176. 117. Musikacharoen, T., T. Matsuguchi, T. Kikuchi, and Y Yoshikai. 2001. NFkappa B and STAT5 play important roles in the regul ation of mouse Toll-like receptor 2 gene expression. J Immunol 166: 4516-4524. 118. Muzio, M., G. Natoli, S. Saccani, M. Levrero, and A Mantovani. 1998. The human toll signaling pathway: divergence of nuclear factor kappaB and JNK/SAPK activation upstream of tumor necrosis fact or receptor-associated factor 6 (TRAF6). J Exp Med 187: 2097-2101. 119. Nagler-Anderson, C. 2000. Tolerance and immunity in the intestinal imm une system. Crit Rev Immunol 20: 103-120. 120. Nanjo, F., K. Goto, R. Seto, M. Suzuki, M. Sakai, a nd Y. Hara. 1996. Scavenging effects of tea catechins and their deriv atives on 1,1-diphenyl-2picrylhydrazyl radical. Free Radic Biol Med 21: 895-902. 121. Neild, A. L., and C. R. Roy. 2003. Legionella reveal dendritic cell functions t hat facilitate selection of antigens for MHC class II p resentation. Immunity 18: 813823.

PAGE 93

79 122. Newton, C. A., I. Perkins, R. H. Widen, H. Friedman and T. W. Klein. 2007. Role of Toll-like receptor 9 in Legionella pneumoph ila-induced interleukin-12 p40 production in bone marrow-derived dendritic cel ls and macrophages from permissive and nonpermissive mice. Infect Immun 75: 146-151. 123. Niessner, A., S. Steiner, W. S. Speidl, J. Pleiner, D. Seidinger, G. Maurer, J. J. Goronzy, C. M. Weyand, C. W. Kopp, K. Huber, M. Wolzt, and J. Wojta. 2006. Simvastatin suppresses endotoxin-induced upre gulation of toll-like receptors 4 and 2 in vivo. Atherosclerosis 189: 408-413. 124. O'Garra, A., L. M. McEvoy, and A. Zlotnik. 1998. T-cell subsets: chemokine receptors guide the way. Curr Biol 8: R646-649. 125. Oshiumi, H., M. Matsumoto, K. Funami, T. Akazawa, a nd T. Seya. 2003. TICAM-1, an adaptor molecule that participates in T oll-like receptor 3-mediated interferon-beta induction. Nat Immunol 4: 161-167. 126. Pasare, C., and R. Medzhitov. 2004. Toll-like receptors: linking innate and adaptive immunity. Microbes Infect 6: 1382-1387. 127. Pasare, C., and R. Medzhitov. 2003. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 299: 10331036. 128. Penna, G., and L. Adorini. 2000. 1 Alpha,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and surviv al of dendritic cells leading to impaired alloreactive T cell activation. J Immunol 164: 2405-2411. 129. Perruccio, K., S. Bozza, C. Montagnoli, S. Bellocch io, F. Aversa, M. Martelli, F. Bistoni, A. Velardi, and L. Romani. 2004. Prospects for dendritic cell vaccination against fungal infections in hematopoie tic transplantation. Blood Cells Mol Dis 33: 248-255. 130. Piemonti, L., P. Monti, P. Allavena, M. Sironi, L. Soldini, B. E. Leone, C. Socci, and V. Di Carlo. 1999. Glucocorticoids affect human dendritic cell differentiation and maturation. J Immunol 162: 6473-6481. 131. Pierer, M., J. Rethage, R. Seibl, R. Lauener, F. Br entano, U. Wagner, H. Hantzschel, B. A. Michel, R. E. Gay, S. Gay, and D. Kyburz. 2004. Chemokine secretion of rheumatoid arthritis synovia l fibroblasts stimulated by Toll-like receptor 2 ligands. J Immunol 172: 1256-1265. 132. Pierre, P., S. J. Turley, E. Gatti, M. Hull, J. Mel tzer, A. Mirza, K. Inaba, R. M. Steinman, and I. Mellman. 1997. Developmental regulation of MHC class II transport in mouse dendritic cells. Nature 388: 787-792. 133. Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. Van Huffel, X. Du, D. Birdwell, E. Alejos, M. Silva, C. Galanos, M. Freud enberg, P. RicciardiCastagnoli, B. Layton, and B. Beutler. 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 ge ne. Science 282: 20852088. 134. Pullar, J. M., C. C. Winterbourn, and M. C. Vissers 2002. The effect of hypochlorous acid on the expression of adhesion mol ecules and activation of NFkappaB in cultured human endothelial cells. Antioxi d Redox Signal 4: 5-15. 135. Qureshi, S. T., L. Lariviere, G. Leveque, S. Clermo nt, K. J. Moore, P. Gros, and D. Malo. 1999. Endotoxin-tolerant mice have mutations in To ll-like receptor 4 (Tlr4). J Exp Med 189: 615-625.

PAGE 94

80 136. Rescigno, M., F. Granucci, S. Citterio, M. Foti, an d P. Ricciardi-Castagnoli. 1999. Coordinated events during bacteria-induced DC maturation. Immunol Today 20: 200-203. 137. Ricci, M. L., A. Torosantucci, M. Scaturro, P. Chia ni, L. Baldassarri, and M. C. Pastoris. 2005. Induction of protective immunity by Legionel la pneumophila flagellum in an A/J mouse model. Vaccine 23: 4811-4820. 138. Riveau, G. J., B. G. Brunel-Riveau, F. M. Audibert, and L. A. Chedid. 1991. Influence of a muramyl dipeptide on human blood leu kocyte functions and their membrane antigens. Cell Immunol 134: 147-156. 139. Roake, J. A., A. S. Rao, P. J. Morris, C. P. Larsen D. F. Hankins, and J. M. Austyn. 1995. Systemic lipopolysaccharide recruits dendrit ic cell progenitors to nonlymphoid tissues. Transplantation 59: 1319-1324. 140. Rogers, J., I. Perkins, A. van Olphen, N. Burdash, T. W. Klein, and H. Friedman. 2005. Epigallocatechin gallate modulates cytokine production by bone marrow-derived dendritic cells stimulated with lipo polysaccharide or muramyldipeptide, or infected with Legionella pneum ophila. Exp Biol Med (Maywood) 230: 645-651. 141. Saffari, Y., and S. M. Sadrzadeh. 2004. Green tea metabolite EGCG protects membranes against oxidative damage in vitro. Life S ci 74: 1513-1518. 142. Sakagami, H., H. Arakawa, M. Maeda, K. Satoh, T. Ka dofuku, K. Fukuchi, and K. Gomi. 2001. Production of hydrogen peroxide and methioni ne sulfoxide by epigallocatechin gallate and antioxidants. Antic ancer Res 21: 2633-2641. 143. Sakagami, H., M. Takeda, K. Sugaya, T. Omata, H. Ta kahashi, M. Yamamura, Y. Hara, and T. Shimamura. 1995. Stimulation by epigallocatechin gallate of interleukin-1 productio n by human peripheral blood mononuclear cells. Anticancer Res 15: 971-974. 144. Sallusto, F., C. R. Mackay, and A. Lanzavecchia. 2000. The role of chemokine receptors in primary, effector, and memory immune r esponses. Annu Rev Immunol 18: 593-620. 145. Sauer, J. D., J. G. Shannon, D. Howe, S. F. Hayes, M. S. Swanson, and R. A. Heinzen. 2005. Specificity of Legionella pneumophila and Co xiella burnetii vacuoles and versatility of Legionella pneumophila revealed by coinfection. Infect Immun 73: 4494-4504. 146. Schwandner, R., R. Dziarski, H. Wesche, M. Rothe, a nd C. J. Kirschning. 1999. Peptidoglycanand lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J Biol Chem 274: 17406-17409. 147. Serra, P., A. Amrani, J. Yamanouchi, B. Han, S. Thi essen, T. Utsugi, J. Verdaguer, and P. Santamaria. 2003. CD40 ligation releases immature dendritic cells from the control of regulatory CD4+ CD25+ T cells. Immunity 19: 877-889. 148. Shao, P., L. H. Zhao, C. Zhi, and J. P. Pan. 2006. Regulation on maturation and function of dendritic cells by Astragalus mongholic us polysaccharides. Int Immunopharmacol 6: 1161-1166.

PAGE 95

81 149. Singh, R., S. Ahmed, C. J. Malemud, V. M. Goldberg, and T. M. Haqqi. 2003. Epigallocatechin-3-gallate selectively inhibi ts interleukin-1beta-induced activation of mitogen activated protein kinase subg roup c-Jun N-terminal kinase in human osteoarthritis chondrocytes. J Orthop Res 21: 102-109. 150. Slifka, M. K., and J. L. Whitton. 2000. Clinical implications of dysregulated cytokine production. J Mol Med 78: 74-80. 151. Steinman, R. M., D. Hawiger, and M. C. Nussenzweig. 2003. Tolerogenic dendritic cells. Annu Rev Immunol 21: 685-711. 152. Suganuma, M., S. Okabe, N. Sueoka, E. Sueoka, S. Ma tsuyama, K. Imai, K. Nakachi, and H. Fujiki. 1999. Green tea and cancer chemoprevention. Mutat Res 428: 339-344. 153. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu Rev Immunol 21: 335-376. 154. Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Ta kada, T. Ogawa, K. Takeda, and S. Akira. 1999. Differential roles of TLR2 and TLR4 in recog nition of gram-negative and gram-positive bacterial cell w all components. Immunity 11: 443-451. 155. Takeuchi, O., A. Kaufmann, K. Grote, T. Kawai, K. H oshino, M. Morr, P. F. Muhlradt, and S. Akira. 2000. Cutting edge: preferentially the R-stereoiso mer of the mycoplasmal lipopeptide macrophage-activatin g lipopeptide-2 activates immune cells through a toll-like receptor 2and My D88-dependent signaling pathway. J Immunol 164: 554-557. 156. Tateda, K., T. A. Moore, M. W. Newstead, W. C. Tsai X. Zeng, J. C. Deng, G. Chen, R. Reddy, K. Yamaguchi, and T. J. Standifo rd. 2001. Chemokinedependent neutrophil recruitment in a murine model of Legionella pneumonia: potential role of neutrophils as immunoregulatory c ells. Infect Immun 69: 20172024. 157. Thompson, C. B. 1995. Distinct roles for the costimulatory ligands B7-1 and B72 in T helper cell differentiation? Cell 81: 979-982. 158. Toshchakov, V., B. W. Jones, P. Y. Perera, K. Thoma s, M. J. Cody, S. Zhang, B. R. Williams, J. Major, T. A. Hamilton, M. J. Fen ton, and S. N. Vogel. 2002. TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/betadependent gene expression in macrophages. Nat Immun ol 3: 392-398. 159. Trinchieri, G. 1995. Interleukin-12: a proinflammatory cytokine w ith immunoregulatory functions that bridge innate resis tance and antigen-specific adaptive immunity. Annu Rev Immunol 13: 251-276. 160. Trompezinski, S., A. Denis, D. Schmitt, and J. Viac 2003. Comparative effects of polyphenols from green tea (EGCG) and soybean (g enistein) on VEGF and IL8 release from normal human keratinocytes stimulate d with the proinflammatory cytokine TNFalpha. Arch Dermatol Res 295: 112-116. 161. Turley, S. J., K. Inaba, W. S. Garrett, M. Ebersold J. Unternaehrer, R. M. Steinman, and I. Mellman. 2000. Transport of peptide-MHC class II complexes in developing dendritic cells. Science 288: 522-527. 162. Vasselon, T., and P. A. Detmers. 2002. Toll receptors: a central element in innate immune responses. Infect Immun 70: 1033-1041.

PAGE 96

82 163. Vayalil, P. K., C. A. Elmets, and S. K. Katiyar. 2003. Treatment of green tea polyphenols in hydrophilic cream prevents UVB-induc ed oxidation of lipids and proteins, depletion of antioxidant enzymes and phos phorylation of MAPK proteins in SKH-1 hairless mouse skin. Carcinogenes is 24: 927-936. 164. Verhasselt, V., C. Buelens, F. Willems, D. De Groot e, N. Haeffner-Cavaillon, and M. Goldman. 1997. Bacterial lipopolysaccharide stimulates the production of cytokines and the expression of costimulatory mo lecules by human peripheral blood dendritic cells: evidence for a soluble CD14dependent pathway. J Immunol 158: 2919-2925. 165. Vidalain, P. O., O. Azocar, C. Servet-Delprat, C. R abourdin-Combe, D. Gerlier, and S. Manie. 2000. CD40 signaling in human dendritic cells is i nitiated within membrane rafts. Embo J 19: 3304-3313. 166. Weinreb, O., S. Mandel, T. Amit, and M. B. Youdim. 2004. Neurological mechanisms of green tea polyphenols in Alzheimer's and Parkinson's diseases. J Nutr Biochem 15: 506-516. 167. Weinstein, M. P., M. L. Towns, S. M. Quartey, S. Mi rrett, L. G. Reimer, G. Parmigiani, and L. B. Reller. 1997. The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacterem ia and fungemia in adults. Clin Infect Dis 24: 584-602. 168. Wilson, N. S., and J. A. Villadangos. 2005. Regulation of antigen presentation and cross-presentation in the dendritic cell networ k: facts, hypothesis, and immunological implications. Adv Immunol 86: 241-305. 169. Wittmann, M., P. Kienlin, S. Mommert, A. Kapp, and T. Werfel. 2002. Suppression of IL-12 production by soluble CD40 lig and: evidence for involvement of the p44/42 mitogen-activated protein kinase pathway. J Immunol 168: 3793-3800. 170. Wolfert, M. A., T. F. Murray, G. J. Boons, and J. N Moore. 2002. The origin of the synergistic effect of muramyl dipeptide with endotoxin and peptidoglycan. J Biol Chem 277: 39179-39186. 171. Xu, X. H., P. K. Shah, E. Faure, O. Equils, L. Thom as, M. C. Fishbein, D. Luthringer, X. P. Xu, T. B. Rajavashisth, J. Yano, S. Kaul, and M. Arditi. 2001. Toll-like receptor-4 is expressed by macropha ges in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL. Circulation 104: 3103-3108. 172. Yamamoto, M., S. Sato, H. Hemmi, H. Sanjo, S. Uemat su, T. Kaisho, K. Hoshino, O. Takeuchi, M. Kobayashi, T. Fujita, K. T akeda, and S. Akira. 2002. Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4. Nature 420: 324-329. 173. Yamamoto, M., S. Sato, H. Hemmi, S. Uematsu, K. Hos hino, T. Kaisho, O. Takeuchi, K. Takeda, and S. Akira. 2003. TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent sig naling pathway. Nat Immunol 4: 1144-1150.

PAGE 97

83 174. Yamamoto, M., S. Sato, K. Mori, K. Hoshino, O. Take uchi, K. Takeda, and S. Akira. 2002. Cutting edge: a novel Toll/IL-1 receptor dom ain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling. J Immunol 169: 6668-6672. 175. Yamamoto, T., S. Hsu, J. Lewis, J. Wataha, D. Dicki nson, B. Singh, W. B. Bollag, P. Lockwood, E. Ueta, T. Osaki, and G. Schu ster. 2003. Green tea polyphenol causes differential oxidative environmen ts in tumor versus normal epithelial cells. J Pharmacol Exp Ther 307: 230-236. 176. Yamamoto, Y., T. W. Klein, and H. Friedman. 1996. Induction of cytokine granulocyte-macrophage colony-stimulating factor an d chemokine macrophage inflammatory protein 2 mRNAs in macrophages by Legi onella pneumophila or Salmonella typhimurium attachment requires differen t ligand-receptor systems. Infect Immun 64: 3062-3068. 177. Yanagawa, Y., N. Iijima, K. Iwabuchi, and K. Onoe. 2002. Activation of extracellular signal-related kinase by TNF-alpha co ntrols the maturation and function of murine dendritic cells. J Leukoc Biol 71: 125-132. 178. Yang, C. S., P. Maliakal, and X. Meng. 2002. Inhibition of carcinogenesis by tea. Annu Rev Pharmacol Toxicol 42: 25-54. 179. Yang, F., W. J. de Villiers, C. J. McClain, and G. W. Varilek. 1998. Green tea polyphenols block endotoxin-induced tumor necrosis factor-production and lethality in a murine model. J Nutr 128: 2334-2340. 180. Yang, G. Y., J. Liao, C. Li, J. Chung, E. J. Yurkow C. T. Ho, and C. S. Yang. 2000. Effect of black and green tea polyphenols on c-jun phosphorylation and H(2)O(2) production in transformed and non-tran sformed human bronchial cell lines: possible mechanisms of cell growth inhi bition and apoptosis induction. Carcinogenesis 21: 2035-2039. 181. Yoshimura, A., E. Lien, R. R. Ingalls, E. Tuomanen, R. Dziarski, and D. Golenbock. 1999. Cutting edge: recognition of Gram-positive b acterial cell wall components by the innate immune system occurs via T oll-like receptor 2. J Immunol 163: 1-5. 182. Youn, H. S., J. Y. Lee, S. I. Saitoh, K. Miyake, K. W. Kang, Y. J. Choi, and D. H. Hwang. 2006. Suppression of MyD88and TRIF-dependent sig naling pathways of Toll-like receptor by (-)-epigallocatec hin-3-gallate, a polyphenol component of green tea. Biochem Pharmacol 72: 850-859. 183. Yu, V. L., J. F. Plouffe, M. C. Pastoris, J. E. Sto ut, M. Schousboe, A. Widmer, J. Summersgill, T. File, C. M. Heath, D. L. Paterson, and A. Chereshsky. 2002. Distribution of Legionella species and serog roups isolated by culture in patients with sporadic community-acquire d legionellosis: an international collaborative survey. J Infect Dis 186: 127-128. 184. Zakharova, M., and H. K. Ziegler. 2005. Paradoxical anti-inflammatory actions of TNF-alpha: inhibition of IL-12 and IL-23 via TNF receptor 1 in macrophages and dendritic cells. J Immunol 175: 5024-5033. 185. Zingoni, A., H. Soto, J. A. Hedrick, A. Stoppacciar o, C. T. Storlazzi, F. Sinigaglia, D. D'Ambrosio, A. O'Garra, D. Robinson, M. Rocchi, A. Santoni, A. Zlotnik, and M. Napolitano. 1998. The chemokine receptor CCR8 is preferentially expressed in Th2 but not Th1 cells. J Immunol 161: 547-551.

PAGE 98

84 186. Zipris, D., E. Lien, J. X. Xie, D. L. Greiner, J. P Mordes, and A. A. Rossini. 2005. TLR activation synergizes with Kilham rat vir us infection to induce diabetes in BBDR rats. J Immunol 174: 131-142. 187. Zlotnik, A., and O. Yoshie. 2000. Chemokines: a new classification system and their role in immunity. Immunity 12: 121-127.

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85 APPENDICES

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ABOUT THE AUTHOR James L. Rogers received his bachelor’s degree at U nion College in Schenectady, N.Y. in 1987, his J.D. degree from Suffolk University in Bo ston, MA and his M.S. in biology from New York University in 1999. After several yea rs of law practice in the biotechnology field as a patent attorney, he entere d the Ph.D. program in the Department of Medical Microbiology and Immunology (now the Dep artment of Molecular Medicine) in May, 2003.