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The modulation by anthrax toxins of dendritic cell activation

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Title:
The modulation by anthrax toxins of dendritic cell activation
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English
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Chou, Ping-Jen
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
LPS
Legionella
Bacillus
DCs
Infection
Dissertations, Academic -- Molecular Medicine -- Doctoral -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Bacillus anthracis produces lethal toxin (LT) and edema toxin (ET) and they suppress the function of LPS-stimulated dendritic cells (DC). Because DCs respond differently to various microbial stimuli, we compared toxin effects in bone marrow DCs stimulated with either LPS or Legionella pneumophila (Lp). DCs were enriched with GM-CSF for 9 days, purified by positive selection, and treated with toxins for 6h; cells were then stimulated with either LPS or Lp-infection for 24h. DC cytokine production and maturation marker expression varied depending upon cell stimulus and the mouse strain used. LT but not ET was more toxic for cells from BALB/c than from C57BL/6 (B6) as measured by 7-AAD uptake; however, ET suppressed CD11c expression. LT suppressed IL-12, IL-6, and TNF-α in cells from BALB/c and B6 mice but increased IL-1β in LPS-stimulated cultures. ET also suppressed IL-12 and TNF-α but increased IL-6 and IL-1β in Lp-stimulated cells from B6. Regarding maturation marker expression, LT increased MHCII and CD86 while suppressing CD40 and CD80; ET, on the other hand, generally decreased marker expression across all groups. We conclude that the modulation of cytokine production by anthrax toxins is dependent on variables including the source of the DCs, the type of stimulus and cytokine measured, and the individual toxin tested. However, LT and ET enhancement or suppression of maturation marker expression is more related to the marker studied than the cell stimulus or cell source. Anthrax toxins are not uniformly suppressive of DC function but instead can increase function under defined conditions.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2008.
Bibliography:
Includes bibliographical references.
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by Ping-Jen (Joe) Chou.
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Title from PDF of title page.
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Document formatted into pages; contains 86 pages.
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Includes vita.

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ABSTRACT: Bacillus anthracis produces lethal toxin (LT) and edema toxin (ET) and they suppress the function of LPS-stimulated dendritic cells (DC). Because DCs respond differently to various microbial stimuli, we compared toxin effects in bone marrow DCs stimulated with either LPS or Legionella pneumophila (Lp). DCs were enriched with GM-CSF for 9 days, purified by positive selection, and treated with toxins for 6h; cells were then stimulated with either LPS or Lp-infection for 24h. DC cytokine production and maturation marker expression varied depending upon cell stimulus and the mouse strain used. LT but not ET was more toxic for cells from BALB/c than from C57BL/6 (B6) as measured by 7-AAD uptake; however, ET suppressed CD11c expression. LT suppressed IL-12, IL-6, and TNF- in cells from BALB/c and B6 mice but increased IL-1§ in LPS-stimulated cultures. ET also suppressed IL-12 and TNF- but increased IL-6 and IL-1§ in Lp-stimulated cells from B6. Regarding maturation marker expression, LT increased MHCII and CD86 while suppressing CD40 and CD80; ET, on the other hand, generally decreased marker expression across all groups. We conclude that the modulation of cytokine production by anthrax toxins is dependent on variables including the source of the DCs, the type of stimulus and cytokine measured, and the individual toxin tested. However, LT and ET enhancement or suppression of maturation marker expression is more related to the marker studied than the cell stimulus or cell source. Anthrax toxins are not uniformly suppressive of DC function but instead can increase function under defined conditions.
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The Modulation by Anthrax Toxins of Dendritic Cell Activation by Ping-Jen (Joe) Chou A dissertation submitted in partial fulfillment of the requirement s for the degree of Doctor of Philosophy Department of Molecular Medicine College of Medicine University of South Florida Major Professor: Thomas W. Klein, Ph.D. Burt Anderson, Ph.D. Dmitry Gabrilovich, Ph.D. Raymond Widen, Ph.D. Date of Approval: October 17, 2008 Keywords: LPS, Legionella, Bacillus, DCs, infection, immunity, Th1 Copyright 2008, Ping-Jen (Joe) Chou

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DEDICATION This dissertation is dedicated to th e late Dr. Herman Friedman whose passion and love for science has insp ired and guided me making this study possible.

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ACKNOWLEDGEMENTS I would like to acknowledge many people for helping me during my doctoral studies. First and foremost, I w ould especially like to thank my advisor, Dr. Thomas Klein, for his quality m entorship and generous commitment. Not only had he encouraged and stimulat ed me to develop independent and analytical thinking, he assisted me tr emendously with experiment troubleshooting and scientific writing. A particular menti on must be made of the late Dr. Herman Friedman to whom I am dedicating this dissertation, whose wisdom and love in science made this dissertation possible for me The privilege of being part of the Dr. Friedman Dr. Kleins team will alwa ys be the most rewarding experience of my life. In the same lin e of thought I owe a special note of gratitude to my supervisor Cathy Newton, M.S., who has literally changed my life since 2000 when she offered me the opportunity to work in an academic research setting. She contributed a great deal to my pr ofessional as well as my personal development. I also wish to thank my teammates Izabella Perkins, Lily Lu, Jim Rogers, Marisela Agudelo, Tracy Sher wood, Liang Nong, and Kellie Larsen for their love and friendship. An additi onal thanks is given to Sumi Lee for introducing me to this wonderful team. I am also extremely grateful for having an exceptional committee and wish to thank Dr. Burt Anderson, Dr. Dmitry Gabrilovich, Dr. Ra ymond Widen, and my outside Chair Dr. Scheld for their continual support and expertise. I would also like to acknowledge the faculty, staff, and students of USF Medical College, who made this journey a fun and dynamic learning experience. I would like to extend a special thanks to Sally Bakers, BJ Seller, Helen ChenDuncan, Deborah Kingsbury, Kathy Zahn, Andrew Conniff, Dr. Larry Solomonson, Dr. Michael Barber, and Dr. Duane Eichler for taking care of the administrative matters. I am also thankful to Dr. Karoly (Char lie) Szekeres, Dr. Nick Burdash, Dr. Jonathan Harton, Dr. Kenneth Ugen, Dr. Andreas Seyfang, and Zhigang Yuan who were insightful in teaching me their know-hows. Finally I would like to thank my family for I would not be here today without their endless love and suppor t. I am grateful for t he rich family values and traditions Mom and Dad have instilled in me; and to my thicker-than-blood brothers Wayne and Jimmy, I thank you for br inging the best out of me. I also wish to express my sincere gratitude to my in-laws in Korea and my extended family in Taiwan for believing in me. Last but not least, I owe my most special thanks to my beloved wife Soo for maki ng me complete. He r love, dedication, and encouragement have fueled me to be a better person each day. And coming home to see her happy and our baby Alex smiling at us has already made me the luckiest man alive.

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i TABLE OF CONTENTS LIST OF FIGURES ...............................................................................................iv ABSTRACT ..........................................................................................................v INTRODUCTION ..................................................................................................1 Anthrax Disease ............................................................................................1 Bioterrorism. ........................................................................................1 Brief History. ........................................................................................1 Bacillus Anthracis ..........................................................................................2 Morphology and Physiology. ...............................................................2 Epidemiology and Transmission. .........................................................2 Pathogenicity. ......................................................................................2 Anthrax Toxins Effects on Immunity ..............................................................3 Entry of Anthrax Toxins. ......................................................................3 Toxin Effects on T Cell and B Cell Immunity .......................................4 Toxin Effects on Innate Immunity. .......................................................5 Toxin Effects on Dendritic Cells (DC) ............................................................6 Biology of DC. .....................................................................................6 Maturation of DC .................................................................................7 Surface Markers ..................................................................................7 Cytokines .............................................................................................8 Toxin Effects on DCs. ..........................................................................8 Microbial Stimulation .....................................................................................9 Lipopolysaccharide (LPS). ...................................................................9 Legionella pneumophila (Lp). ..............................................................9 Project Significance .....................................................................................10

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ii OBJECTIVES .....................................................................................................12 Aim One: To Determine Effect s of Anthrax Toxins on DC Maturation Following Stimul ation with Lp infection ....................................13 Aim Two: To Determine Toxin Effects on Cytokine Production in Response to LPS and Lp in DCs from BALB/c and C57BL/6 mice. ..........................................................................................................14 Aim Three: To Determine Toxi n Effects on Surface Marker Expression in Response to LPS and Lp in DCs from BALB/c and C57BL/6 Mice. ...........................................................................................15 MATERIALS AND METHODS ............................................................................16 Mice and bacteria ........................................................................................16 Isolation and Purification of Dendritic Cells .................................................16 Anthrax toxin treatment ...............................................................................17 Flow Cytometry Analysis and Cell Viability. .................................................18 Enzyme-linked Immunosorbent Assay ........................................................19 CFU Determinations ....................................................................................19 Statistical Analysis .......................................................................................20 RESULTS ...........................................................................................................21 Aim One: To Determine Effect s of Anthrax Toxins on DC Maturation Following Stimul ation with Lp infection ....................................21 CD11c Enrichment by GM-CSF and Incubation Time .......................21 Legionella Growth Unaffected by Anthrax Toxin Pretreatment ...................................................................................23 Modulation by LT and ET of DC Cytokine Production in Response to Lp Infection .................................................................25 Aim Two: To Determine Toxin Effects on Cytokine Production in Response to LPS and Lp in DCs from BALB/c and C57BL/6 mice. ..........................................................................................................31 CD11c Purification by CD11c Positive Magnetic Sorting ...................31 Cytokine Production by Purified versus Non-purified DCs .................33 LT Treatment is More Toxic than ET in vitro for DCs from BALB/c Mice ....................................................................................36 LT and ET Can either Enhance or Suppress Cytokine Production in Purified DC Cultures ..................................................41

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iii Aim Three: To Determine Toxi n Effects on Surface Markers Expression in Response to LPS and Lp in DCs from BALB/c and C57BL/6 Mice. ...........................................................................................45 LT and ET Differentially Stimulate DC Maturation Marker Expression ......................................................................................45 DISCUSSION .....................................................................................................49 Dendritic Cells Enrichment by GM-CSF and Incubation Time .....................50 Legionella Intracellular Growth Unaffected by Toxin Treatment ..................51 Functional Similarity between CD11c Selected and Non-Selected Cells ..........................................................................................................52 LT is More Toxic in vitro for BALB/c than C57BL/6 ......................................54 Differential Immune Regulation by LT and ET .............................................56 Cytokine Response ...........................................................................56 Maturation Marker Expression ...........................................................58 Speculations on the Mechani sms for Toxin Modulation ...............................58 Lethal Toxin .......................................................................................58 Edema Toxin .....................................................................................62 Summary .....................................................................................................65 REFERENCES ...................................................................................................66 APPENDICES ....................................................................................................81 Appendix A: Cytokine Profile from Various Multiplicity of Infection ..............82 Appendix B: Cytokine Kinetics from Affinity-purified DC ..............................83 Appendix C: Cytokine Effects Attenuated Using Heated Toxins ..................85 ABOUT THE AUTHOR ...........................................................................End Page

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iv LIST OF FIGURES Figure 1. Enrichment of CD 11c Cells with Incubation Time ................................22 Figure 2. Intracellular Lp Growth Unaffected by Anthrax Toxin Pretreatment ......................................................................................24 Figure 3. Lethal Toxin Suppresse s Effectively Proinflammatory Cytokines ...........................................................................................27 Figure 4. Edema Toxin Differentia lly Regulates Proinflammatory Cytokines ...........................................................................................29 Figure 5. CD11c Purification by CD11c-Positive Magnetic Sorting .....................32 Figure 6. Comparison of Cytoki ne Production by Purified and Nonpurified DC Populations Fo llowing Stimulation with Legionella or LPS ..............................................................................34 Figure 7. Magnetic Bead Sorted BALB/ c and C57BL/6 Dendritic Cells are 95% Positive for CD11c ..............................................................38 Figure 8. LPS Stimulation: LT decreases cell viability and ET decreases CD11c ..............................................................................39 Figure 9. Lp Infection: LT decreas es cell viability and ET decreases CD11c ...............................................................................................40 Figure 10. LT and ET Differentiall y Regulate Cytokine Production in Purified DC Cultures ..........................................................................43 Figure 11. LT and ET Differentially Stimulate DC Maturation Marker Expression .........................................................................................47 Figure 12 Mechanisms for Lethal Toxin Modulation ...........................................61 Figure 13 Mechanisms for Edema Toxin Modulation ..........................................64

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v The Modulation by Anthrax Toxins of Dendritic Cell Activation Ping-Jen (Joe) Chou ABSTRACT Bacillus anthracis produces lethal toxin (LT) and edema toxin (ET) and they suppress the function of LPS-stim ulated dendritic cells (DC). Because DCs respond differently to various microbial stim uli, we compared toxin effects in bone marrow DCs stimulated with either LPS or Legionella pneumophila (Lp). DCs were enriched with GM-CSF for 9 days, purif ied by positive selection, and treated with toxins for 6h; cells were then stimul ated with either LPS or Lp-infection for 24h. DC cytokine production and maturati on marker expression varied depending upon cell stimulus and the mouse strain used. LT but not ET was more toxic for cells from BALB/c than from C57BL/6 (B6) as measured by 7-AAD uptake; however, ET suppressed CD11c expressi on. LT suppressed IL-12, IL-6, and TNFin cells from BALB/c and B6 mice but increased IL-1 in LPS-stimulated cultures. ET also suppressed IL-12 and TNFbut increased IL-6 and IL-1 in Lp-stimulated cells from B6. Regarding maturation marker expression, LT increased MHCII and CD86 while suppressing CD40 and CD80; ET, on the other hand, generally decreased marker expre ssion across all groups. We conclude that the modulation of cytokine production by anthr ax toxins is dependent on

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vi variables including the source of the DCs, the type of stimulus and cytokine measured, and the individual toxin tested. However, LT and ET enhancement or suppression of maturation marker expression is more related to the marker studied than the cell stimulus or cell sour ce. Anthrax toxins are not uniformly suppressive of DC function but inst ead can increase function under defined conditions.

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1 INTRODUCTION Anthrax Disease Bioterrorism. Human anthrax is of interest and concern among the medical and scientific comm unity around the world particularly after its use as biological weapon in the U. S. immediately following the attack on the World Trade Center (61). In the fall of 2001, bioterrorism was achieved each time the news media alerted the public concerning letters tain ted with anthrax spores distributed through the U.S. postal system. Hoaxes involving envelopes with harmless powders and threatening notes made the problem even worse for authorities, and created chaos among Amer icans as they opened their mail (5). Brief History. Anthrax is blamed for several devastating plagues that killed both humans and livestock. The first reco rded incident dates back to 1500 B.C. when the Egyptians described a plague of boils affecting the Pharaohs cattle. Scientists eventually named the disease, anthrax, and it emerged in World War I as a biological weapon due to ease of laboratory production and deadliness via aerosol dissemination (5, 86). Seve ral countries are believed to have experimented with anthrax, but its use in warfare has been limited. Prior to the attack of 9-11, the average natural occurrenc e in the U.S. is 1-2 cases per year, and the last reported death is over 25 years ago (5, 158).

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2 Bacillus Anthracis Morphology and Physiology. Bacillus anthracis is the etiological agent of anthrax. It is a Gram-positive, facultat ive anaerobic, spore-forming, large bacillus about 1 1.2 m in width and 3 5 m in length (34, 93). Under conditions of environmental stress, these bacteria can na turally form spores which can persist in the soil and survive for decades. Spores can also be formed in culture and in the tissues and exudates of dead animals, but not in the blood or tissues of living animals. The endospores are ellipsoidal shaped and located centrally in the sporangium. They are highly refractile to light and resistant to staining (34, 93). Epidemiology and Transmission. Anthrax is a major disease threat to herbivorous animals like cattle, sheep, and to a lesser extent horses, hogs, and goats (140). In humans, anthrax can occu r in three distinct clinical forms: cutaneous, inhalational (or pulmonary), and gastrointestinal. Cutaneous anthrax accounts for more than 95% of human cases, and is characterized by large black skin lesions classically found on hands, forearms, or head. Inhalational anthrax often results in death due to respir atory distress (pulmonary edema) and septicemia, and is therefore regarded as the most significant fo rm in biological warfare. Gastrointestinal anthrax results from ingestion of meat derived from diseased animals. Because of strict cont rol measures, this form is not seen in the U.S (93, 140). Pathogenicity. The principal virulence factors of B. anthracis are encoded on two virulence plasmids: capsular polypeptide on plasmid pXO2, and anthrax toxins on pXO1 (34). T he bacilli are covered by antiphagocytic, polyglutamic

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3 capsule which helps to evade host imm unity and mediate the early invasive stage of infection. The capsule, t hough nontoxic, functions to protect the organism against the bactericidal componen ts of serum and phagocytes (93). Anthrax toxins are a heat-labile, heterogeneous protein complex made up of three components called protective antigen (PA), lethal factor (LF), and edema factor (EF) (25, 38, 95). And in binary complexes these proteins form the anthrax toxins: lethal toxin (LT) = PA + LF and edema toxin (ET) = PA + EF (25, 95). Anthrax Toxins Effects on Immunity Entry of Anthrax Toxins. Edema toxin (ET) and lethal toxin (LT) share a receptor-binding subunit PA but have di fferent catalytic subunits, EF and LF, respectively. PA facilitates the transloca tion of either factor across the cell membrane through receptor-mediated endocyt osis (25, 95, 164) PA binds to surface anthrax-toxin receptors that are ubiquitously expressed on cells and tissues (164). Upon binding to these receptors either tumor endothelial marker 8 (TEM8) (15, 17, 155) or capillary morphogenesis protein 2 (CMG2) (129) together with the coreceptor low-density lipoprotein receptor-related protein 6 (LRP6) (154), PA becomes the substrate of a furin-like membr ane protease. The cleaved fragments of PA self-associat e to form ring-shaped heptamers capable of binding up to three LF or EF molecu les (150). The formation of PA-EF or PALF complexes results in their entry into lipid rafts, followed by their endocytosis into acidic endosomal compartment (78) The drop to lower pH triggers PA heptamers to undergo conformational change and form a channel that assists the

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4 translocation of LF and EF into the cytosol (25). Once inside the cell, LF is a zinc-dependent metalloprotease that cl eaves most members of the MAPK kinases family and disrupts intracellular si gnaling, resulting in pro-inflammatory response suppression (25, 77, 95). EF is a calciumand calmodulin-dependent adenylate cyclase that increases the intracellular concentration of cAMP, resulting in immune cell m odulation (25, 80, 95). Though LT has been shown to be more toxic than its counter part, it is speculated that these toxins work in concert to promote disease (8, 142). Toxin Effects on T Cell and B Cell Immunity The adaptive immune response is highly specific for a particular pathogen, and improves with each successive encounter with the same pathogen. T and B lymphocytes are central to this branch of immunity because of t heir antigen-specific nature in recognizing individual pathogens. T lymphocyte activation requires the activation of MAPK signaling pathway (35), which is a common ta rget for both toxins. Disruption of this pathway by LT and ET impairs t he T cell receptor (TCR)-dependent T cell activation and proliferation ( 27, 44, 123). LT inhibits the activation of Erk1, 2, p38, and JNK MAPKs (27, 44, 123), while ET blocks all MAPKs except p38 (123). LT and ET together thus suppress cytokine production including IL-2, IL-4, IFN, TNF, and IL-5, and surface marker ex pression CD69 and CD25 (27, 44, 108, 123). Moreover, this impairment correlates with the inhibition of nuclear factor of activated T cells (NFAT) and activation protein (AP)-1 which are downstream transactivators of MAPK activation (123).

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5 Though activation of antigen-specific B lymphocytes requires the presence of activated T lymphocytes, LT can also inhibit B cell activation directly by targeting the MAPK pa thway (8). Fang et al. showed that LT inhibits B cell proliferation and antibody production in vitro and in vivo by the proteolysis of MAPKKs (45). Their data suggest an ef fective mechanism through which LT could attenuate protective humoral immune responses elicited by B. anthracis (45) Toxin Effects on Innate Immunity. Innate immunity plays a vital role in immune surveillance against pathological infectious agents (163). Phagocytic cells, such as dendritic cells, macrophages and polymorphonuclear neutrophils, are mediators of innate immunity, and they recognize, phagocytize and eliminate microbes through induction of the oxi dative burst and cytokine expression (8, 126). Macrophage activation requires the activation of MAPK pathway which induces downstream biosynthesis of pro-inflammatory cytokines (e.g., TNF, IL1 IL-6), chemoattractants (e.g., IL-8, RANTES), and enzymes (e.g., COX-2, iNOS) (22, 70, 127). LT has been shown to interfere with this process by cleaving the amino-terminal extensions of the catalytic domain from six of seven mitogen-activated protein kinase kinases (M APKK) (37, 39, 148). In addition, LT induces killing of macrophages in certai n susceptible mouse strains (90, 91), which has been associated with a region on Chromosome 11 that encodes the kinesin-like motor protein Kif1c (120, 153). ET is also a potent inhibitor of MAPK

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6 pathway by catalyzing large amounts of cAMP from ATP; cAMP can interfere with intracellular signaling at multiple levels (8, 68). There is less information on neutrophils compared to macrophages; nonetheless current studies suggest that B. anthracis uses both toxins to evade killing by these cells (8). In neutrophils, ET and LT inhibit the oxidative burst elicited by lipopolysaccharide (LPS) and muramyl dipeptide (MDP) (160). In addition, the increase in cAMP by ET inhibits neutrophils ability to phagocytize (105, 152); while LT inhibits neutrophils ability to move (chemotaxis) by impairing its actin filament assembly (40). In monocytes, accumulation of cAMP induced by ET was shown to block TNFproduction, thus impairing antimicrobial activities (56). Toxin Effects on Dendritic Cells (DC) Biology of DC. DCs are the most efficient antigen-presenting cells and are central to the integration of innate and adap tive immunity (1, 82, 115, 133). For participation in innate immunity, DCs ex press pattern recognition receptors (PRR) such as the Toll-like receptors (T LR) which allow them to detect within minutes microbial stimuli as well as ti ssue damage and necrosis (41). Since the identification of distinct subsets of DCs, much attention has been given to understanding the functions of these subpopulations in i mmune induction and regulation (62). In this study, we use bone marrow-derived dendritic cells (BMDC) isolated from the tibia and femu rs of BALB/c and C57BL/6 (B6) mice.

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7 Maturation of DC DCs upon stimulation by microbial antigens go through a maturation process from immature ce lls to mature, antigen presenting cells. Immature DCs are central to innate i mmunity and are immunological sensors that sample the environment for micr obial antigens. Upon sensing antigens, immature DCs begin to produce cytok ines such as IL-12, IL-6, TNF, and IL-1 that contribute to innate and adaptive immuni ty (see below). As they continue to mature, DCs migrate to secondary lymphoid organs where they present processed antigen to nave T cells to launch an adaptive immune response (1, 82, 133). Maturing DCs express, in addition to cytokines, high levels of adhesion and co-stimulatory molecules (e.g., CD40, CD80, CD86), as well as MHC II molecules, which interact with T helper cells (see below) (69). Increasing evidence suggests that DCs polarize the type of T cell response by expressing a selective set of T cell-polarizing mole cules. For example, DCs exposed to intracellular bacteria promote T-helper type I (Th1) responses, whereas certain parasites promote DCs to drive the development of Th2 cells (33, 65). Surface Markers Maturing DCs begin to process ingested microbial antigens into peptide fragments, which are then presented by MHC molecules to T lymphocytes (53). In addition to incr easing the expression MHC molecules, maturing DCs increase the expression of T cell polarization, co-stimulatory molecules such as CD80 (B7-1), CD86 (B 7-2), ICAM1, etc (2 6). B7 (CD80, CD86) proteins are recognized on T ce lls by CD28 and cytotoxic T lymphocyteassociated antigen 4 (CTLA-4). Interact ion of these molecules dictates the activation and expansion of a ll effector and regulatory Th cells subsets (21, 52).

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8 Another set of molecules that provi de co-stimulatory signals is CD40 ligand (CD40-L) on T cells and CD40 on DCs; ex pression of CD40 facilitates the activation of helper T cells (116). Cytokines In addition to co-stimulatory molecules, maturing DCs can also communicate via the release of cytokines. Immature DCs readily secrete IL-12 upon stimulation with microbial antigens such as lipopolysaccharide (LPS) and viral DNA and dsRNA signaled through the TL R (64, 88). In addition to activating T cells and cell mediated immunity, IL-12 is well known for contributing to local inflammation (143). Other proinflammatory and T ce ll-stimulating cytokines produced by immature DCs include IL-6, IL-1 and TNF. IL-6 stimulates acute phase proteins from liver (76) and also is important in the development of Th17 cells (107). IL-1 is important pro-inflammatory cytokine regulating fever and the acute phase response as well as activating T cells (118). TNFis a septic shock-inducing cytokine that has shown to cause apoptosis in a variety of immune cells (23, 130) as well as support the development of T helper cells (79). Toxin Effects on DCs. Several studies designed to examine the effects of anthrax toxins on immunity have studi ed their effects on DCs. LT has been reported to induce necrosis in BALB/c-der ived DCs but apoptosis in cells from B6 mice in vitro DCs from humans are reported to also respond with apoptosis following LT treatment (3, 8). Agra wal and colleagues were the first to demonstrate an effect of anthrax toxins on DC function. Using purified LT, they showed that the function of mous e splenic DCs was compromised in vitro in terms of LPS-stimulated expression of co-stimulatory molecules and

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9 proinflammatory cytokines (1). Additional work by Tournier et al. using bone marrow-derived DCs instead of splenic DCs showed that both LT and ET could suppress or enhance cytokine production d epending on the cytokine tested, and concluded the toxins cooperated to suppr ess the innate immune response (142). In both studies, the authors proposed that this disruption might impair both innate and adaptive immunity to B. anthracis infection; it was also proposed that the toxins might not only alter immunity to ant hrax but predispose a ffected individuals to other diseases due to immune modulation (135). Microbial Stimulation Lipopolysaccharide (LPS). Studies examining the effe ct of anthrax toxins on DC function have used DCs stimulated wit h either lipopolysaccharide (LPS) or B. anthracis organisms. LPS is a major component of the Gram-negative bacterial cell envelope, which elicits potent proinflammatory responses in immune cells. LPS consists of three components; Lipid A, Core polysaccharide, and O antigen. This endotoxin activa tes B cells and induces macrophages and other cells to release IL-1, IL-6, TNF, among other cytokines (6, 31, 43, 106). LPS can also induce DC maturation in vitro and in vivo resulting in increased expression of co-stimulatory molecu les and production of proinflammatory cytokines that influence the subsequent immune response (1, 63, 73, 142). Legionella pneumophila (Lp). Lp is an intracellular, Gram-negative bacterial pathogen causing Legionnaires disease (99). Adaptive cellular immunity plays an important role in host defense to Legionella (84, 102).

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10 Legionnaires disease is prevalent among immune compromised individuals, including transplant patients, patients re ceiving corticosteroids, and patients suffering from AIDS (72). Project Significance It is clear from the evidence so far that anthrax toxins can modulate immune cell function including DC function. Unfortunately, the effects of the toxins on DC immune functions are limited only to cells stimulated with either B. anthracis alone or stimulated with L PS. These initial studies examining only two methods of DC stimulation o ffer a limited insight into the immunobiology of these toxins and therefore we designed studi es to compare toxin effects in DCs stimulated with either LPS or Lp. Com parison of these two stimuli offer the opportunity to examine toxin effects on DCs stimulated in very different ways in order to more fully understand the potency of the toxins to modulate DC function under different conditions. The results obtained from these studies will have important applications in several differ ent ways. First, because DCs are pivotal in bridging innate and adaptive immuni ty, a fuller understanding of the modulation capabilities of these cells by ant hrax toxins will provide greater insight into the current use of DCs as alter native therapeutics for vaccine development (132, 159), anti-cancer ther apy (47, 100), and other im munotherapy (94). As we will show below, the toxins enhance and suppress DC function suggesting the use of these toxins as potential imm unosuppressive agents or adjuvants in immunotherapy (131). Secondly, our understanding of the pathogenesis,

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11 prevention, and treatment of anthrax is incomplete. Treatment with oral ciprofloxacin or doxycycline is effective only to kill the vegetat ive bacteria (96). These antibiotics are of limited benefits if a threshold level of anthrax toxin has already been produced following colonizati on. The use of antitoxin, such as antibodies, receptor decoys, translocation inhibitors, LF or EF inhibitors, etc., has been of major focus and extensive research recently to neutralize toxin effects (117). However, a full understanding of the immunomodulating potential of LT and ET, especially as it relates to DC stimulation under varying conditions, is imperative in the successful management of B. anthracis infected individuals.

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12 OBJECTIVES This project investigates the suppressive nature of anthrax lethal and edema toxins on activated mouse BM-DC cells. Agrawal et al. have shown previously the LT suppressive role on LPS-activated splenic DC in vitro (1), while Tourniers group in vivo studies has demonstrated ET is suppressive on IL-12 and IFNin LPS-challenged BALB/c and B6 mice (142). Our lab has previously shown that infection of DCs with Legionella induces a spectrum of cellular changes consistent with Th1 immune polariz ation (84, 85, 103). For example, Lp infection causes an increase in the producti on of polarizing cytokines such as IL12, IL-6, and TNFalong with increased MHC class II expression. In addition, in contrast to LPS stimulat ion, Lp stimulates DCs through TLR 2 and TLR 9 rather than TLR 4 (93). The cellular mechanism s mediating these responses to Lp differ in many ways to those occurring following LPS or B. anthracis stimulation; therefore, we hypothesize that modulation of DC f unction by anthrax toxin treatment will vary significant ly in cells stimulated with Lp rather than LPS as in previous studies. Accordingly, to st udy further anthrax toxins effects on DC function, we designed studies to test their effects not only following LPS treatment but also following infection wit h Lp. Our data support the conclusion that anthrax toxins are not uniformly suppressive of DC function but rather

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13 modulate function up or down depending on variables such as the function tested, the stimulus used to activate t he DCs such as either LPS or Lp, and the genetic variation in innate immune re sponse mechanisms in the host cell. Aim One: To Determine Effects of Anthrax Toxins on DC Maturation Following Stimulation with Lp infection The overall aim of this study is to ex amine the modulating effect of anthrax toxins on the maturation response of DCs following stimulation with microbial antigens. The purpose of aim one is to examine the in-vitro immune response of toxin-treated BM-DC fo llowing exposure to L. pneumophila in BALB/c mouse model. Several experimental parameters need to be considered initially, which include DC enrichment, toxin concentration, and Legionella surviv ability in toxintreated DCs. Maturation can be measur ed by quantitating the expression of various cell surface markers and cytokines released from the cell. The defining DC marker is the CD11 lym ph node homing receptor (18). GM-CSF is used to induce myeloid DC progenitor cells from mouse bone marrow to become immature BM-DCs and then these immature cells can be further matured by treatment with microbial ant igens. We will characterize DC maturation using commonly described surface markers, CD11c, CD11b, and F4/80 and cytokines such as IL-12, IL-6, TNF, and IL-1 For toxin concentration, it has been previously reported that the effective concentration required to suppress DC cytokine response was in the microgram /ml range (1). One caveat, however, other studies have shown necrosis and apoptosis of macrophages following toxin

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14 exposure (16, 74, 90, 97, 109, 151). Therefore, it is im perative that we determine the effective concentration of lethal and edema toxins require d to modulate the immune response of Lp-infected DCs without overdosing or killing them. Parallel to this reasoning, it is just as impor tant to understand how Lp survives in toxintreated DCs. Our lab has previously observed that Lp does not grow inside of DCs (99, 103); it will be of interest to determine if anthrax toxin treatment modulates this growth suppressive effect. Aim Two: To Determine Toxin Effect s on Cytokine Production in Response to LPS and Lp in DCs from BALB/c and C57BL/6 mice. Several toxin studies using animal model s or cell cultures from different mouse strains have displayed varied f unctional responses, suggesting the degree of toxin susceptibility may be dependent upon genetic background (90, 92). To study anthrax toxin specificity, two types of mice will be used: one being a toxin-susceptible strain, BALB/c, and the other one, toxin-resistant strain, B6. In addition to this variable, we will also assess DC maturation using LPS and Legionella infection. Comparison of t hese two microbial st imuli will provide a greater understanding of how anthrax toxins modulate DC maturation due to a variety of stimulating signals. DC matu ration will be assessed by the cytokine response profile of IL-12, IL-6, TNF, and IL-1 In addition, the purity and viability of the DCs from the various gr oups will be determined by the expression of CD11c and the DNA intercalating dye 7-AAD, respectively.

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15 Aim Three: To Determine Toxin Effect s on Surface Marker Expression in Response to LPS and Lp in DCs from BALB/c and C57BL/6 Mice. DC maturation can be evaluated by dete rmining the sequential expression of surface proteins or markers. Prev ious reports showed a mixed effect of anthrax toxins on marker expression. Fo r example, In their study on LT treated splenic DC, Agrawals group noted significant inhibition of co-stimulatory molecules CD80, CD86, and CD40 following LPS stimulation (1). However, another study using germinating spores showed otherwise; Tournier et al. claimed that toxins secretion from live B. anthracis did not impair maturation of DC from BALB/c and B6 mice (142). We will analyze by flow cytometry the expression of maturation markers MHC II, CD40, CD80, and CD86 from BALB/c and B6 DC stimulated with eit her LPS or Legionella as our model of infection.

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16 MATERIALS AND METHODS Mice and bacteria BALB/c and C57BL/6 (B6) we re purchased from NCI-Har lan (Fredrick, MD). They were used at 9 to 13 weeks of age. They were housed and cared for at the University of South Florida Health Sciences Center Animal Facility, which is fully accredited by the American Association for Accreditation of Laboratory Animal Care. A virulent strain of Legionella pneumophila (Lp, M124), serogroup 1 isolate from a case of Legionnaires disease at Tampa General Hospital (Tampa, FL), was cultured on buffered charcoal yeast extract plates for 48h from a passage 3 stock stored at 80C. The concentration of bacteria was determined by spectrophotometer. For infect ion of DC cultures, see below. Isolation and Purification of Dendritic Cells The femurs and tibias were removed fr om euthanatized mice. The bone marrow cells were flushed out of the leg bones with buffer RPMI 1640 and antibiotic/antimycotic solution (Sigma). The red blood cells in the suspension were lysed using ammonium-chlor ide potassium, and the cells were resuspended in RPMI 1640 supplemented with 50 M 2-ME, 10% BGS, antibiotics, 100mM L-glutamine, and 5ng/ml GM-CSF. Briefly, on day 0, 1 x

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17 10 6 /ml cells were seeded in 3ml medium in 6-well plates (Costar). On day 1, non-adherent cells were removed by gent le washing, and the wells were replaced with fresh medium. On day 3, t he wells were replenished with nutrients by replacing 1 ml of fresh medium. Afte r day 9 in culture, dendritic cells were loosely attached to culture plates and were easily harvested by gently rinsing for subsequent use. For further purific ation by magnetic cell sorting, an AutoMACS Separator Pro (Miltenyi Biotec, France) was used according to the manufacturers protocol. Up to 10 8 total GM-CSF enriched cells were resuspended in running buffer (PBS plus 0.5%, culture grade bovine serum albumin, and 0.75 mg/ml EDTA) at room temperature and t hen incubated with CD11c Microbeads N418 (Miltenyi Biotec, France) for 15min at 4-8C. Cells were re-suspended in buffer (10 7 cells/ml) and sorted with AutoMACS Separator Pro using program posse l for positive selection. Anthrax toxin treatment The recombinant toxin components PA, LF, and EF, purchased from List Biological Laboratories, Inc. (Campbell, CA), were rec onstituted in sterile, 1%BSA in PBS buffer and stored in aliq uots at -80C according to the manufacturers instruction. DCs were preincubated with PA 200ng/ml alone or with either LF 0.01 50ng/ml or EF 0.1 50ng/ml for 5h, at 37C, in complete RPMI (RPMI 1640 supplemented with 50 M 2-ME, 5% FCS, 100mM Lglutamine). The cells were washed wit h HBSS, resuspended in culture medium, and stimulated with either LPS (1 g/ml) or Legionella pneumophila for 24 hours.

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18 For Legionella infection, DCs in cultur e medium were infected for 50 min with viable bacteria at a 10-20:1 ratio (bacteria-to-cells) (Appendix A) and then washed to remove excess extracellular bac teria. Culture cells and supernatants were harvested after 18-24 hours (Appendix B) and analyzed for cytokines, viability, and surface marker expression. Flow Cytometry Analysis and Cell Viability. Surface markers were analyzed by flow cytometry. Following treatment and incubation for 24 hours, DCs were re-suspended to 1 x 10 6 cells/ml in PBS containing 2% fetal calf serum, Fc re ceptors blocked with anti-Fc receptor Fc RII/RIII antibodies for 15min on ice, stained with FITC(Fluorescein Isothiocyanate), PE(R-Phycoerythrin ), and APC(Allophycocyanin) conjugated monoclonal antibodies to CD11c, CD40, CD80 (B7.1), CD86 (B7.2), and MHC II (BD Pharmingen, San Jose, CA) for an additional 30min. To assess cell viability, labeled cells were centrifuged, resuspended and incubated for 5min on ice with 7-amino-actinomycin D (7-AAD) (BD Pharmingen, San Jose, CA), and then suspended in 1ml 2% FCS-PBS buffer. Stained cells were analyzed by flow cytometry using FACSCant o II (Becton-Dickinson, Mountain View, CA) and the program BD FACSDiva Software v5.0.1 (B ecton-Dickinson, Mountain View, CA). Some data were further analyzed using Fl owJo 7 (TreeStar, Inc., San Carlos, CA) to exclude dead (7-AAD-positive) cells.

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19 Enzyme-linked Immunosorbent Assay The proinflammatory cytoki nes IL-12p40/p70, IL-6, IL-1 and TNFwere measured in 24-h DC cultur e supernatants by ELISA. Medium-bind, 96-well Costar enzyme immunoassay (EIA) plates were coated with anti-murine IL12p40/p70 (Pharmingen, San Diego, CA) in Na HCO, pH 8.2. After 2h at 37C, the plates were blocked for 1 h at 37C. Culture supernatants and serial dilution of murine IL-12p40/p70 standard (Pharmingen) were added for 1 h, followed by biotinylated anti-murine IL-12p40/p70 for 1 h, and then Horse Radish Peroxidase for 30 min. After the substrate TMB (S igma) was added, plates were allowed to develop for 15-45 min, and stopped with H 2 SO 4 Units were calculated from the standard curve, which was performed for each plate. The plates were washed between each step with two to five changes of nanopure water. IL-6 ELISA was performed by the same prot ocol with anti-IL-6 in PBS, biotinylated anti-IL-6 antibody, and recombinant IL -6 for standards. IL-1 and TNFELISA were coated with anti-IL-1 and anti-TNFantibodies respectively in carbonate, pH 9.5. Biotinylated and recombinant antibodies were used accordingly. CFU Determinations At 0h, 24h, 48h, and 72h, DC cult ures in the 96-well plates were lysed with 0.1% saponin (Sigma). The lysates were dilu ted in Hanks balanced salt solution, plated on BCYE plates, and incubated at 37C for 72 h. CFU counts were determined on an AutoCount apparatus (Dynatech Labs, Chantilly, VA).

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20 Statistical Analysis The data approximately follow a normal dist ribution and the observations were independent to each other; therefore, the real values in ng/ml or pg/ml from any two groups were compared by one-tailed ttest (unequal variance). Based on the alpha = 0.05 level, statistical significance is noted by and between compared sample groups where p < 0.05. The values in Figure 1 are expressed as percent of control wherein the control is the Legionella only (Lp only) treated group.

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21 RESULTS Aim One: To Determine Effects of Anthrax Toxins on DC Maturation Following Stimulation with Lp infection CD11c Enrichment by GM-CSF and Incubation Time To determine the optimal time poin t to harvest quality DCs from bone marrow cell cultures, DCs collected from day 7, 8 and 9 were analyzed by flow cytometry for surface markers CD11c, CD11b, and F4/80. The bone marrow cell cultures were enriched with GM-CSF, an important colony stimulating factor necessary to drive the DC development from their progenitor cells. Figure 1 shows that as F4/80 expression gradually decreases with incubation time, CD11c increases its expression from 59% on day 7 to about 77% on day 9. As for CD11b, it remains a constant 100% expres sion regardless of collection times. As cells became more CD11c positive we also noticed t hat the population became more homogenous as indicated by t he forward-and-side sc atter dot plot. The subpopulation below t he gate gradually diminished every day after day 7.

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Day 7 Day 8 Day9 CD11c CD11b F4/8020% 38% 20% 10% 37% 33% 7% 32% 42% 41% 59% 31% 69% 23% 77% Day 7 Day 8 Day9 CD11c CD11b F4/8020% 38% 20% 10% 37% 33% 7% 32% 42% 41% 59% 31% 69% 23% 77% Figure 1. Enrichment of CD11c Cells with Incubation Time Bone marrow cells isolated from mouse femu rs and tibias were cultured for 7, 8, and 9 days in medium containing G M-CSF 10ng/ml and analyzed by flow cytometry for expression of F4/80, CD11b, and CD11c. This is a representative data of 2 experiments. 22

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23 Legionella Growth Unaffected by Anthrax Toxin Pretreatment To determine whether anthrax toxins affect L. pneumophila in DCs, we monitored the bacterial growth over a period of three days. DCs, in general, do not support Legionella growth even in pe rmissive A/J mice wherein Legionella thrives in derived thioglycolate-elicited macrophages (125). In this study, DCs were preincubated with either LT or ET fo r 6 hours, infected with Legionella at an MOI of 10; and CFU counts were determined at 24h, 48h, and 72h post infection. At time 0h, DCs from all groups demons trated efficient uptake of bacteria averaging 50to 60-thousand colonies (Figur e 2). However, regardless of toxin treatment or not, Legionella growth stead ily declined throughout the course of the 3-day period. These data suggest that anthrax toxin does not affect L. pneumophila intracellular growth.

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Colony Forming UnitsTime (Hours) Colony Forming UnitsTime (Hours) Figure 2. Intracellular Lp Growth Unaffected by Anthrax Toxin Pretreatment Dendritic cells from BALB/c mice were infected with L. pneumophila MOI 10:1, and the cells were washed and incubated for 24, 48, or 72h with or without toxin. The cells were lysed, and CFU counts were determined. Data are the mean and standard error of the mean ( SEM) of three experiments. 24

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25 Modulation by LT and ET of DC Cytokine Production in Response to Lp Infection It has been reported that LT suppresse s cytokine production by splenic DCs stimulated with LPS and that the effective toxin concentration was in the microgram/ml range (1). Ho wever, other studies involving bone marrow-derived DCs rather than spleni c and stimulated with other stimuli such as B. anthracis spores, suggested that suppression of cytokine production was not uniformly observed (142). And, in fact, LT treat ment increased certain cytokines as did treatment with ET (142), suggesting that the toxin effects may differ depending upon the source of the DC and the cell stimul us used. To extend these studies, therefore, we examin ed the effect of various toxi n concentrations in bone marrow DC cultures infected with t he intracellular pathogen, Legionella pneumophila (Lp) (Figure 3 and 4). DCs were cultur ed and treated with 200ng/ml protective antigen (PA) for 6 hours and increasing co ncentrations of either LF (0.01 50ng/ml) or EF (0.1 50ng/ml) followed by infection with Lp for 50 min. The cells were washed and resuspended in medium and cultured for an additional 18 hours. Culture supernatants were collect ed and analyzed by ELISA for IL-12, IL6, IL-1 and TNF. PA treatment alone did not significantly effect cytokine production; however, LF combined wit h PA dose-dependently decreased the production of all four cytokines (Figur e 3). EF plus PA treatment also suppressed IL-12 and TNFat 50ng/ml, but significantly increased the production of IL-6 and IL-1 even at relatively low c oncentrations (Figure 4). These toxin effects were attenuated using toxins heated to 56 C for 35 minutes (Appendix C) confirming previous reports (142) that only active toxins modulate

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26 cytokines in DCs. These results suggested LT treatment was uniformly more suppressive when added to Lp-infected DCs while ET was less suppressive and even capable of enhancing cytok ine production depending upon the concentration of the toxin.

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27 Figure 3. Lethal Toxin Suppresses Eff ectively Proinflammatory Cytokines DC cultures were pretreated for 5-6h wit h protective antigen (PA) 200ng/ml and LF at 0.01 50ng/ml. The cultures we re then infected with Lp for 50 min and supernatants were analyzed 18h later by ELISA for IL-12p40/p70, IL-6, IL-1 and TNF. The results are expressed as percentage of the Lp only control and are the means of six experiments. p < 0.05, compared to Lp control.

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% of Control % of ControlIL-12p40/p70 IL-6 IL-1 TNF* * * **5 0 1 0 1 0 0 1 ng/ml5 0 1 0 1 0 0 1 ng/ml5 0 1 0 1 0 0 1 ng/ml5 0 1 0 1 0 0 1 ng/ml % of Control % of ControlIL-12p40/p70 IL-6 IL-1 TNF* * * **5 0 1 0 1 0 0 1 ng/ml5 0 1 0 1 0 0 1 ng/ml5 0 1 0 1 0 0 1 ng/ml5 0 1 0 1 0 0 1 ng/ml Figure 3. 28

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29 Figure 4. Edema Toxin Differentially Regulates Proinflammatory Cytokines DC cultures were pretreated for 5-6h wit h protective antigen (PA) 200ng/ml and EF at 0.01 50ng/ml. The cultures we re then infected with Lp for 50 min and supernatants were analyzed 18h later by ELISA for the pro-inflammatory cytokines indicated. The results are expressed as percentage of the Lp only control and are the means of four experiments. p < 0.05, compared to Lp control.

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% of Control % of ControlIL-12p40/p70 IL-6 IL-1 TNF* * * * *5 0 1 0 1 ng/ml5 0 1 0 1 ng/ml5 0 1 0 1 ng/ml5 0 1 0 1 ng/ml % of Control % of ControlIL-12p40/p70 IL-6 IL-1 TNF* * * * *5 0 1 0 1 ng/ml5 0 1 0 1 ng/ml5 0 1 0 1 ng/ml5 0 1 0 1 ng/ml Figure 4. 30

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31 Aim Two: To Determine Toxin Effect s on Cytokine Production in Response to LPS and Lp in DCs from BALB/c and C57BL/6 mice. CD11c Purification by CD11c Positive Magnetic Sorting The reports from most mouse DC st udies consider a minimum of 95% CD11c expression the gold standard for DC purity. Our DCs enriched with GMCSF for 9 days were about 75-80% CD11c positive. To further increase this percentage, DCs were purified by labeling the cells with anti-CD11c magnetic nanoparticles for positive selection, then sorting by AutoMACS Separator Pro as described in Materials and Methods. Again the cells were analyzed by flow cytometry for surface marker CD11c, CD1 1b, and F4/80. Clearly, the forwardand-side scatter dot plot (Figure 5A) s hows the subpopulation from the smaller gate was completely removed after the selection, leaving a homogenous DC population in the larger gate. The right panel in Figure 5B shows that the cells removed were those of CD11c-negative, CD11b-positive, and F4/80-intermediate population which could likely be DC progenitor cells and macrophages.

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AB C Non-purified Purified F4/80 CD11b CD11c 9%28% 24% 1%46% 48% 47%52%5%94% Non-purified Purified SSC FSCAB C Non-purified Purified F4/80 CD11b CD11c 9%28% 24% 1%46% 48% 47%52%5%94% C Non-purified Purified F4/80 CD11b CD11c 9%28% 24% 1%46% 48% 47%52%5%94% Non-purified Purified SSC FSC Non-purified Purified SSC FSC Figure 5. CD11c Purification by CD11c-Positive Magnetic Sorting F4/80 and CD11b single positive cells were removed by magnetic sorting by AutoMACS Separator Pro. (A) A scattergram before (non-purified) and after (purified) the selection. (B) F4/80, CD11b, and CD11c subtypes in non-purified and purified populations. 32

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33 Cytokine Production by Purified versus Non-purified DCs To determine whether there is any difference in function between DCs that were about 75% CD11c-positive or non-purified and CD11c-sorted cells or purified, we compared the cytokine released from these two populations following microbial stimulation. Non-purified and purified DCs were either stimulated with LPS 1 g/ml or infected with Legionel la at MOI of 10:1. The supernatants were collected 24h post st imulation and assayed by ELISA for proinflammatory cytokines. Figure 6A shows that non-purified DCs produced more IL-12 than purified ones following LPS stimulation, yet produced about the same amount following Lp infection. As for IL-6 (Fig. 6B) and TNF(Fig. 6D) production, both populations displayed similar secretion pattern following microbial stimulation. In terms of IL-1 LPS was again more potent at inducing non-purified DCs to release a bit more cytokines than the pur ified counterpart. When comparing the cytokine profiles si de by side, the general pattern of cytokine secretion was very similar; and the minor difference seen in IL-12 and IL-1 was at best half-fold. These data suggest that the 20% difference in CD11c expression between the purified and non-purified DCs would not have contributed much difference in function agai nst microbial stimulation. However, to conform to current standard, DCs test ed in subsequent studies were purified by bead selection.

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34 Figure 6. Comparison of Cytokine Produc tion by Purified and Non-purified DC Populations Following Stimulation with Legionella or LPS Non-purified and purifi ed DCs were stimulated with either LPS 1 g/ml or Lp (10:1). Supernatants collected 24 post st imulation were analyzed by ELISA for (A) IL-12, (B) IL-6, (C) IL-1 and (D) TNF. The graphs show the mean of four experiments with SEM.

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NonPurifiedPurified IL-12p40/p70 IL-6 IL-1 TNFng/ml ng/ml pg/ml pg/ml (A) (B) (C) (D) NonPurifiedPurified IL-12p40/p70 IL-6 IL-1 TNFng/ml ng/ml pg/ml pg/ml (A) (B) (C) (D) Figure 6. 35

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36 LT Treatment is More Toxic than ET in vitro for DCs from BALB/c Mice The studies in Aim 1 were done using partially purified CD11c+ cell preparations from BALB/c mice. Howeve r, DCs from this mouse strain have been reported to be sensitive to the to xic effects of LT and because the above studies showed LT suppressed cytokine pr oduction, we measured DC viability in cells from BALB/c mice and B6 mice whic h are less susceptible to killing by LT (3, 48, 90, 142). We also felt it im portant to study toxin effects in DC preparations purified by positive selecti on with anti-CD11c magnetic microbeads. DCs from both mouse strains were isol ated, purified, and treated or not with either LT or ET, and stimulated with either LPS or Lp infection for 24 hours; the cells were then incubated with the imperm eable, DNA intercalating dye, 7-AAD, followed by flow cytometry analysis to asse ss viability. The results showed that DCs from both strains lose CD11c marker following 24 hours incubation without GM-CSF (Figure 7, 8, and 9) going from >95% CD11c positive to between 60 and 70% positive after 24 hours. Treat ment with LPS alone had no effect on viability in either strain but LT pretreated cells from BALB/c mice with LPS decreased cell viability after 24 hours (Figur e 8); viability of cells from B6 mice was less affected by the LT. Treatment with ET had no effect on viability. Interestingly, ET treatment did signific antly suppress the expression of CD11c in cells from both mouse strains (Figure 8 and 9). Results with Lp-stimulated cells showed that infection of the cultures significantly increased CD11c expression but decreased viability due to the apoptotic e ffect of Lp (4, 20, 57, 75, 101, 165) with cells from B6 mice somewhat more sensitive (Figure 9). Treatment with LT

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37 decreased cell viability even more in ce lls from BALB/c mice and decreased the expression of CD11c in cells from B6 mice ET treatment was not as toxic as LT; however, overall it decreased the expres sion of CD11c in both strains as with LPS-stimulated cells. From thes e results, it is clear that LT is more toxic than ET for DCs from BALB/c mice when the cells are stimulated with either LPS or Lp; however, it appears that ET suppresses CD11c expression in both groups. Because cells from B6 mice were more resi stant to toxicity they were included in subsequent cytokine studies. In addition, we decided to gate on the CD11c+ cells that were also 7AAD negative in subsequent activation surface marker studies to determine the toxin effects on viable, CD11c+ DCs.

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DC at 0h BALB/c C57BL/6CD 11c7-AAD DC at 0h BALB/c C57BL/6CD 11c7-AAD Figure 7. Magnetic Bead Sorted BALB/ c and C57BL/6 Dendritic Cells are 95% Positive for CD11c DCs from BALB/c and B6 mice were purif ied by magnetic cell sorting, incubated, and analyzed by flow cytometry for CD11c expression and viability by 7-AAD uptake. DCs from both mouse strain s were about 95% CD11c+ and viable at the start of incubation. The data are representative of 3-4 experiments. 38

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DC only LPS LPS + PA + LFLPS + PA + EF BALB/c C57BL/6CD 11c7-AAD DC only LPS LPS + PA + LFLPS + PA + EF BALB/c C57BL/6CD 11c7-AAD Figure 8. LPS Stimulation: LT d ecreases cell viability and ET decreases CD11c Purified DCs from BALB/c and B6 mice we re pretreated for 56h with protective antigen 200ng/ml and either LF 50ng/ml or EF 50ng/ml and then stimulated for 24h with LPS 1 g/ml. The samples were analyz ed by flow cytometry for CD11c expression and viability by 7-AAD uptake. The data are repr esentative of 3-4 experiments. 39

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DC only Lp Lp + PA + LFLp + PA + EF BALB/c C57BL/6CD 11c7-AAD DC only Lp Lp + PA + LFLp + PA + EF BALB/c C57BL/6CD 11c7-AAD Figure 9. Lp Infection: LT decreases ce ll viability and ET decreases CD11c Purified DCs from BALB/c and B6 mice we re pretreated for 56h with protective antigen 200ng/ml and either LF 50ng/ml or EF 50ng/ml and then infected with Lp 10:1 for 50min, washed and incubated for 24h. The samples were analyzed by flow cytometry for CD11c expression and viability by 7-AAD uptake. The data are representative of 3-4 experiments. 40

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41 LT and ET Can either Enhance or Suppress Cytokine Production in Purified DC Cultures Having established that LT was more toxic than ET especially in BALB/c DC cultures, we re-examined the effects on cytokine production of both toxins in affinity-purified DC cultures from both BALB/c (sensitive) and B6 (resistant) mice stimulated with either LPS or Lp. It was hypothesized that cytokine suppression by LT would be greater in cells from BALB/ c mice because of its toxic effect on these cells. DCs were purified and stimulated for 24 hours and supernatants harvested and analyzed for IL-12, IL-6, IL-1 and TNF. Figure 10A shows that for IL-12 production LT was suppressive onl y in cells from BALB/c while ET was suppressive in cells from both sources and following both stimuli. For IL-6 (Fig. 10B), LT was suppressive only in BALB/c ce lls stimulated with LP S, while in cells stimulated with Lp, the toxin was suppressive in both strains. Since in Figure 8 and 9 we showed that LT is more toxic for BALB/c cells, it is possible that suppression of IL-12 and IL-6 in BALB/c ce lls is partly due to the toxic effect of the toxin. ET treatment moderately s uppressed IL-6 following LPS stimulation but enhanced the cytokine response in cells from B6 mice stimulated with Lp. IL1 levels were surprisingly enhanced in cells from both strains following LT treatment and LPS stimulation, but were suppressed in both cell groups following Lp stimulation. ET treatm ent, on the other hand, had the reverse effect causing a suppression following LPS treatment and enhancement following Lp stimulation (Fig. 10C). TNFlevels were suppressed in all groups by both toxins (Fig. 10D). It appears from the results that LT and ET could both suppress and enhance

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42 cytokine production in DC cultures and t hat this was more dependent upon the cytokine measured and the cell stimulus than on the mous e strain source of the cells. LT suppressed cytokines such as IL-12 and IL-6 in cells from sensitive BALB/c, the toxin could also suppress IL-1 and TNFequally in cells from both mouse strains. Yet it increased IL-1 production from both strains following LPS stimulation. ET was also surprisingly qui te suppressive in cells from both strains, but it too could enhance select cytoki nes induced by the two different stimuli independent of the m ouse strain used. Clearly the modulation of cytokine production by anthrax toxins is dependent on many variables including the source of the cells, the type of stimulus and cytokine measured, and the individual toxin tested.

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43 Figure 10. LT and ET Differentially Regul ate Cytokine Production in Purified DC Cultures Affinity-purified DCs from BALB/c and C57B L/6 (B6) mice were pretreated for 56h with protective antigen (PA) 200ng/ml an d either LF 50ng/ml or EF 50ng/ml and then stimulated for 24h with either LPS 1ug/ml or Lp (10-20:1 bacteria to cell ratio). Culture supernatants were harvested and analyzed by ELISA for (A) IL12p40/p70, (B) IL-6, (C) IL-1 and (D) TNF. Data are the mean SEM cytokine concentrations in culture supernat ants from three to four independent experiments. p < 0.05, compared to DC only; p < 0.05, compared to LPS or Lp only.

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LPS Lp (A) IL-12p40/p70 (B) IL-6 (D) TNF(C) IL-1 BALB/C BALB/C C57BL/6 C57BL/6 BALB/C BALB/C C57BL/6 C57BL/6 BALB/C BALB/C C57BL/6 C57BL/6 BALB/C BALB/C C57BL/6 C57BL/6* * * ** pg/ml pg/ml ng/ml ng/ml * * * * * * * ** ** ** LPS Lp (A) IL-12p40/p70 (B) IL-6 (D) TNF(C) IL-1 BALB/C BALB/C C57BL/6 C57BL/6 BALB/C BALB/C C57BL/6 C57BL/6 BALB/C BALB/C C57BL/6 C57BL/6 BALB/C BALB/C C57BL/6 C57BL/6* * * ** pg/ml pg/ml ng/ml ng/ml * * * * * * * ** ** ** Figure 10 44

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45 Aim Three: To Determine Toxin Effect s on Surface Markers Expression in Response to LPS and Lp in DCs from BALB/c and C57BL/6 Mice. LT and ET Differentially Stimulate DC Maturation Marker Expression The above studies showed that LT and ET can modulate the production of cytokines associated with immune maturati on and polarization of the DCs. To further examine toxin effects on DC maturation, we examined their effects on DC maturation marker development follo wing stimulation with LPS and L. pneumophila in purified, CD11c+ cells from both BALB/c and B6 mice. The markers studied were those important in adaptive immunity including MHC II, CD40, CD80 (B7-1), and CD86 (B7-2). St imulated and toxin treated cells were stained with fluorescent antibodies and anal yzed by flow cytometry. Maturation marker expression was analyzed on CD11c+ cells that were also viable as judged by 7-AAD exclusion. Surprisingly, LT greatly increased the expression of MHCII across both strains and stimuli (Fig. 11A) while ET had a moderate suppressive effect. Regarding CD40 expression, both toxins suppressed the response to LPS while, on the other hand, LT increased CD40 expression in Lpstimulated cells (Fig. 11B). Suppression by both toxins was generally observed across all groups for CD80 marker expressi on (Fig. 11C); while enhancement of the CD86 marker was observed except in the case of LPS-st imulated, B6 cells, where ET significantly suppressed marker development (Fig. 11D). From these results it appears that LT and ET can either enhance or suppress maturation marker expression and that this is generally more rela ted to the marker studied than the cell stimulus or cells source LT tended to increase MHCII and CD86

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46 while suppressing CD40 and CD80; and ET on the other hand tended to decrease all of the markers.

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47 Figure 11. LT and ET Differentially Stimulate DC Maturation Marker Expression Affinity-purified DCs from BALB/c and C57B L/6 (B6) mice were pretreated for 56h with protective antigen (PA) 200ng/ml and either LF 50ng/ ml or EF 50ng/ml and then stimulated for 24h with either LPS 1ug/ml or Lp (10-20:1 bacteria to cell ratio). Cells were harvested and analyzed by flow cytometry for (A) MHC class II, (B) CD40, (C) CD80, and (D) CD86. Data are the mean SEM percent positive of the gated population from thr ee to four independent experiments. p < 0.05, compared to DC only; p < 0.05, compared to LPS or Lp only.

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MHC II0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50%BALB/cC57BL/6BALB/cC57BL/6% Gated CD 400% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%BALB/cC57BL/6BALB/cC57BL/6% Gated (A) (B) LPS Lp * * * CD 800% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%BALB/cC57BL/6BALB/cC57BL/6% Gated CD 860% 10% 20% 30% 40% 50% 60% 70% 80% 90%BALB/cC57BL/6BALB/cC57BL/6% Gated(C) (D) * * * * * * * * * * MHC II0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50%BALB/cC57BL/6BALB/cC57BL/6% Gated CD 400% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%BALB/cC57BL/6BALB/cC57BL/6% Gated (A) (B) LPS Lp * * * CD 800% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%BALB/cC57BL/6BALB/cC57BL/6% Gated CD 860% 10% 20% 30% 40% 50% 60% 70% 80% 90%BALB/cC57BL/6BALB/cC57BL/6% Gated(C) (D) * * * * * * * * * * MHC II0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50%BALB/cC57BL/6BALB/cC57BL/6% Gated CD 400% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%BALB/cC57BL/6BALB/cC57BL/6% Gated (A) (B) LPS Lp * * * CD 800% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%BALB/cC57BL/6BALB/cC57BL/6% Gated CD 860% 10% 20% 30% 40% 50% 60% 70% 80% 90%BALB/cC57BL/6BALB/cC57BL/6% Gated(C) (D) * * * * * * * * * * Figure 11 48

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49 DISCUSSION Previous reports have indicated a role of LT and ET in immune suppression thus facilitating Bacillus anthracis infection and disease (8). A number of molecular mechanisms for di srupting signaling pathways have been described for the toxins that can impai r the functions of many immune cells, including T-cells (27, 44, 108), B-ce lls (45), neutrophils (40, 105, 160), monocytes (71), dendritic cells (1, 3, 18, 24, 119, 142), and macrophages (12, 31, 42, 111). Dendritic ce lls are potent antigen presenting cells and play an important role in innate and adaptive imm unity (9, 82, 115, 133). These cells are now known to have highly diverse charac teristics when isolated from various areas in the host and the characteristics diversify even further when the cells are stimulated by various microbial antigens. To date, the effect of anthrax toxins on DC maturation to only two microbial antigens, LPS and anthrax spores, has been reported. To more fully understand toxi n effects, we wanted to study cells treated with a third type of stimulus, Legion ella, a bacterial agent known to infect and alter DC maturation (84, 85, 121). Ou r findings show that LT and ET can either enhance or suppress DC functions, and that the outcome is dependent upon several factors including the agent us ed to stimulate the cells, the DC function tested, and the genetic background of the DC donor mice.

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50 Dendritic Cells Enrichment by GM-CSF and Incubation Time Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immune-stimulating cytokine secreted by macrophages, Tcells, epithelial cells, mast cells and fibroblasts that prom otes a strong, long-lasting, systemic immune response (54). GM-CSF drives st em cells to become granulocytes and monocytes; and further induces monocyt es to maturation upon exiting the circulation and become macrophages (157). In 1992, GM-CSF was identified by Inaba et al. to proliferate DC precursors in mouse blood (60). Shortly after this discovery, the same group claimed GM-CSF, but not M-CSF, coul d generate DCs in la rge quantity from adherent cells of stroma isolated from leg bone marrow (59). In their study, DCs harvested at day 7 after GM-CSF treatment contained the intracellular antigens, M342 and 2A1, and the surface antigen 33D1, NLDC145 characteristics of dendritic cells (59). Today, CD11c is recognized as the gold standard for mouse DCs; however, at 7 days in Inabas report the cells were much less than 100% positive for this mark er (59). We therefore examined if CD11c could be increased by extending the incubation time up to 9 days and found that the CD11c expres sion increased with time in the presence of GM-CSF (10ng/ml). Similar to previous findi ngs (59), we saw a continued reduction in F4/80 antigen in our cultures as shown in Figure 1. Howe ver, along with the increase in CD11c, the cell number declined significantly beginning on day 8, and by day 10 only 25% of the cells remained compared to day 7, even when incubated at a higher (20ng/ml) GM-CSF c oncentration (data not shown). From

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51 this, it appears GM-CSF treatment for 7 to 8 days is optimal for the development of DCs in vitro, a finding supported by prev ious studies (104). In addition to bone marrow, GM-CSF is currently used to generate DCs from other tissues including spleen (83) and liver (114, 139). Legionella Intracellular Growth Un affected by Toxin Treatment There is very little information on the intracellular growth of Lp in DCs. Although it is known that macrophages from A/J mice (99, 125, 162) do not restrict the growth of Lp by mechani sms involving deficiencies in phagosomelysosome fusion (50, 149), bone marrow and splenic DCs from these mice restricted Lp growth (99). In our studies, bone marrow DCs from BALB/c mice also restricted the growth of Lp (Figure 2). Interestingly, at the same MOI of 10, the BALB/c DCs ingested 10-fold more Legi onella compared to cells from A/J mice (99); however, BALB/c (and B6) DCs were killed up to 40% as determined by 7-AAD stain (Figure 9) while A/J DC viability remained constant as measured by MTT assays (99). These studies dem onstrate, that unlike macrophages, DCs from several different mouse strains restrict the growth of Lp and survive for several days in spite of the fact th at Lp has been shown to induce apoptosis (165) in macrophages. It was also important to determine if treatment of Lp-infected DCs with toxin affected the growth of Lp in our assays because shifting the intracellular antigen load by toxin treatment coul d skew DC immune responsiveness as an indirect consequence of toxin effects. Presumably, Legionella do not express

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52 the mammalian anthrax-toxin receptors TEM8 (15, 17, 155) and CMG2 (129) and the coreceptor LRP6 (154); and therefore the bacteria should not be directly affected by the toxins. However, since anthrax toxins are generally believed to suppress immune cell function (8), we s peculated that Lp intracellular growth might be affected by toxin treatment. Howeve r, this was not the case as seen in the results from Figure 2. The decrease in Lp colony-forming units was basically the same in either the presence or absenc e of LT or ET. Thus, treatment of infected DCs with the toxins had little effect on the number of intracellular bacteria per cell culture and therefore littl e effect on the bactericidal function of the cells. Functional Similarity between CD11c Selected and Non-Selected Cells The cell surface marker that defines a dendritic cell in mouse is the CD11c marker. This marker is a type I tr ansmembrane protein wh ich belongs to a member of the leukocyte integrin family (138). Integrins are heterodimeric proteins which consist of an alpha c hain and a beta chain; CD11c is thus comprised of two subunits, gp150 (CD11c ) and gp95 (CD18) (13, 28, 29). In addition, CD18 associates with two ot her proteins, lymphocyte functionassociated antigen-1 (LFA-1), CD 11a, and macrophage-1 antigen (Mac-1), CD11b (19, 146). The mouse CD11c ant igen is present on DCs in lymphoid organs (137) and blood, on Langerhans ce lls in the epidermis (122), on DC progenitors in the bone marrow, and in-vitro generated bone marrow-derived DCs (59). In spleen and lymph node, CD11c is expressed at high levels on

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53 conventional CD11c + CD45R mPDCA-1 DCs (66, 98, 136, 156), and at moderate levels on CD11c + CD45R + mPDCA-1 + plasmacytoid DCs (pDC). CD11c is reported to be weakly expr essed on NK cells, B cells, and T cell subsets (133, 139). The two main functions of the integrins are attachm ent to the extracellular matrix (ECM) and to mediat e signal transduction from ECM to the cell (58). CD11c, in particular, binds to complem ent fragment, iC3b, provisional matrix molecules (fibrinogen), the Ig superfamily intracellular adhesion molecule (ICAM1, ICAM-2), and also recognizes va scular cell adhesion molecule (VCAM-1) (124). In addition to cell migration, it has been suggested that this homing receptor also functions in phagocytosis cytokine production by monocytes and macrophages, and induction of T-cell prol iferation by Langerhans cells (122, 124). Furthermore, Sadhu et al. have recently suggested a novel role for CD11c during leukocyte recruitment, antigen upt ake, and the survival of DC (124). Because some of our studies were done with only culture-purified DCs, we wanted to compare the cellularity and func tion of these cells versus more highly purified DCs that were affinity purifi ed for CD11c positive cells. Purification removed a small cell populati on, enriched slightly for F4/80+ cells (37 to 47%), and significantly enriched for CD11c+ cells (52 to 95%) (Figure 5). When cytokine secretion profiles to stimulation with Lp and LPS were compared (Figure 6), there was really very little di fference between the two cell populations suggesting that although the populations diffe red by cell size and the expression of CD11c, they were func tionally homogeneous in terms of cytokine production.

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54 The GM-CSF, culture-purified BM cells contain a small and la rge population that were CD11cand CD11c+, respectively, and we speculate that both populations when stimulated in culture can mature and function as DCs and that the small population contain DC precur sors. In addition, like in human DCs (133) CD11c appeared to be an inducible surface ant igen the expression of which was upregulated upon microbial stimulation and downregulated by ET treatment (Fig. 8 and 9). Therefore, the DC precursors can function like mature DCs following stimulation by microbial products. LT is More Toxic in vitro for BALB/c than C57BL/6 Several reports showed that LT is mo re toxic for BALB/c-derived BM-DCs than for cells isolated from B6 mice (3, 119, 142); and our current results support these findings. LT toxici ty occurred within 24 hours in LPS-stimulated cultures (Fig. 8); however, in Lp-stimulated cells, LT increased toxicity in BALB/c cells but had an attenuating effect in B6 cells on the enhanced toxicity induced by this intracellular pathogen (Fig. 9). On t he other hand, in cont rast to LT, ET treatment was not toxic in cultures from either mouse strain. In addition to BMDCs, LT toxicity has also been reported in mouse macrophages (90, 92) and in DC cultures derived from spleen (1) and lung (24). The mechanisms of toxicity appear to involve both caspase depende nt and independent mechanisms and also to depend on the extent of activation/ma turation of the DCs (3 119). In fact, in DCs from B6 mice, LT toxicity was attenuated in matured cells pretreated with microbial stimuli such as LPS (119). In our studies, the cells were not pretreated

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55 with LPS but rather with LT followed by LPS for 24 hours; cell damage, as measured by 7-AAD uptake, increased 8 fold in BALB/c mice while only 4 fold in B6 mice. Thus, the mouse strain sele ctive toxicity occurred prior to cell maturation by LPS. In contrast to L PS treatment, our results with Lp-stimulated cells were quite different, in that as expected, treatment with the intracellular pathogen was more harmful than LPS for DCs from both mouse strains. Although LT increased toxicity in BALB/ c cells, it surprisingly attenuated the toxicity in B6 cells. Legionella infection was more toxic in these cells and it is likely that LT treatment suppressed the intracellular life cycle of Lp and thus its apoptotic effect (4, 20, 57, 75, 101, 165). Because the LT toxic effect in BALB/c mice occurred within 24 hours, it is possi ble that this early toxicity is due to necrosis rather than apoptosis as suggested by others (3). This cytotoxic effect has also been correlated with the pres ence of Kif1c gene (87, 153) and early necrosis via Nalp1b activation (3, 119). Studies by Pezards group first report ed LT, not ET, accounts for the toxin mortality in mice (112); however, two recent studies showed a significant effect of ET in whole-animal mortality studies (46, 151). Whether ET is more toxic than LT in vivo remains unclear; however, we show ET is less toxic on cultured DCs and this is consistent with other in vitro studies (142). In addition to toxicity, we show for the first ti me that ET suppressed the L PSand Lp-induced expression of the CD11c lymph node homing receptor (Fig. 8 and 9). This finding is in contrast to an earlier report showing t hat CD11c expression was increased 24 hours following phagocytosis of B. anthracis spores (18). This study used human

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56 monocyte-derived DCs stim ulated with attenuated, to xin negative spores, and these were as potent as fully pathogenic spores in increasing CD11c expression. Thus, the effect on CD11c in this study probably had little to do with toxin production. Since CD11c expression may facilitate DC migrat ion to the lymph node and promote the development of adaptive immunity, it is possible that the CD11c suppression by ET may prevent effective DC migration and lead to progression of the infecti on following spore germination. Differential Immune Regulation by LT and ET Cytokine Response The DC cytokine profile (Figure 10) indicated that production was differentially regulated by both toxins. In addition, the effect of either LT or ET on any one cytokine varied depending on the type of stimuli and/or mouse strains. Our results with LPSand Lp-stimulated B M-DCs treated with either LT or ET, support the data of Tournier et al. and Cleret et al. as they observed a similar type of differential regulation by to xins studies using BM-DC and lung DC, respectively, stimulated with nontoxigenic (LF /EF ) mutant anthrax spores RP42 (24, 142). Furthermore, ev en though there were three related studies examining the effect of LT on LPS-activated DC in vitro (1, 3, 119), ours is the only study also investigating the effect of ET treatment on LPS-activated DCs. LT, in addition to its lethality, was generally suppressive on IL-12, IL-6, and TNFin BALB/c-derived DCs stimulated with either LPS or Lp. This was also seen in LPS-activated splenic DCs (1) and lung DCs (24) from BALB/c mice. This is

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57 consistent with the action of LT which is a metalloprotease that cleaves the Nterminus of many MAP kinases res ponsible for the production of many proinflammatory cytokines (39, 109, 111, 142, 147). However, in our hands, LT was not uniformly suppressive. For exam ple, it minimally stimulated IL-12 production in B6 cultures and robustly increased IL-1 in LPS-stimulated cultures from both mouse strains. T he mechanism of this increase is not known at this time. ET, though non-toxic in our study, is nonetheless an important factor contributing to the pathogenesis of anthrax infection and is speculated to work synergistically with LT (32, 113, 142). We showed that ET suppressed IL-12 and TNFin both BALB/cand B6-derived DC cultures, which is consistent with the results from DC stimulated wit h mutant anthrax spores (24, 142). However, ET also increased cytokine production such as IL-6 in Lp-stimulated B6 DCs and IL1 in Lp-stimulated DCs from both stra ins (Figure 10). This enhancement is similar to that seen in lung DCs stimulat ed with anthrax spores (24) and human monocytes stimulated with LPS (56). ET is an adenylate cyclase and increases intracellular cAMP in target cells thus modulating many ph ysiological processes (36, 80). It has been shown that elevated cAMP or it s analogs contributed to the increase of IL-6 in human monocytes (56) and the increase of IL-10 in DCs (67) and splenocytes (55). Our results suggest this ET effect may be extended to the production of IL-1 in Lp-infected BM-DCs.

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58 Maturation Marker Expression In addition to cytokines, we also studied the effect of LT and ET on DC maturation marker development following stimulation with LPS or Lp. Unlike with toxin effects on viability and cytokines w herein the source of the cells and the type of stimulus contributed to the outcome, with marker expression, these variables had a lesser influence. For example, LT treatment enhanced MHC class II and CD86 expression across all groups while ET was generally suppressive across all groups for all of t he markers. Our results with LPS are at variance with those obtained with splenic DC s (1) wherein it wa s reported that all markers were suppressed by LT while we saw an increase in MHC class II and CD86. Other studies with lung DCs infect ed with anthrax spores or treated with toxin showed little change in MHC class II and CD86 expression (24) while those with BM-DCs stimulated with ant hrax spores in the presence or absence of toxins showed an increase in CD86 si milar to what we saw. Speculations on the Mechanisms for Toxin Modulation Lethal Toxin Dendritic cells express toll-like receptors (TLRs) on their cell surface to detect microbial stimuli as well as tissue damage and necrosis (41). LPS activates DCs through TLR4 which leads to downstream gene activation via MAPK kinase and/or NF B signaling cascade (110) as illustrated in Figure 12. Legionella, on the other hand, activate s our DCs through TLR2 and/or TLR9 (103, 121), which ultimately leads to gene activation through MAPKK and/or

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59 NF B cascade, albeit slightly different from that of LPS-induced recruitment of signaling molecules upstream of either cascade. Hence the two activation pathways are drawn separate in the diagr am. This gene activation induced by either stimulus leads to innate imm une response including co-stimulatory molecule expression and cytokine secreti on, all of which define DC maturation and function, respectively. Lethal toxin in our study modulates ac tivated DC response in a number of ways, namely induction of cell death, suppression or enhancement of certain proinflammatory mediators and maturation markers, and yet there is no effect on IL-12 and CD80 from B6-deriv ed DCs following LPS stimul ation. In terms of suppression, LF is a metalloprotease ( 89) that cleaves all members of the MAPKK family except for MEK5 (161) and disrupts the downstream signaling (39, 111, 148). Subsequently this l eads to gene inactivation and therefore suppression of markers and cytokines (39). Consistent with our finding, LT has been shown to induce cell death particularly in DCs derived from toxin-susceptible BALB/c mice in vitro (24, 142). There are two possible mechanisms contributing to this toxicity. One, p38 MAPK has been associated with survival in macrophages (10, 141). We therefore speculate that disruption of MAPKK upstream p38 may lead to cell lysis. Two, LF targets the Nalp1b gene which has rec ently been shown to play a major role in defining toxin sensitivity in mice (16) Nalp1b protein is a key component of a very large protein complex called infla mmasome (16, 144). The mechanism by which LF affect this inflammasome is unclear at this time as depicted by the

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60 question marks in the figure. One functi on of inflammasome is to activate caspase-1 and caspase-5 activities (10, 16, 144). And caspase-1 activation has been associated with apoptosis (119, 144). The enhancement effect in our st udy appears to be more dependent on the type of microbial stimuli relative to the type of mouse strains. LT significantly increased IL-1 in LPS stimulation; yet its enhancing effect on CD40 was from that of Lp infection. This LF-induced IL-1 secretion may be associated with caspase-1 activities because they cleave pro-IL-1 proteins (30). This cleavage subsequently results in the secretion of t he cytokine. In addition, it has recently been shown that LF may have other substrates, which ar e likely to contribute to downstream gene activation (11).

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Figure 12 Mechanisms for Lethal Toxin Modulation Lp, Legionella pneumophila LPS, lipopolysaccharide. TLR, toll-like receptor. MAPK, mitogen-activated protein kinase. NF B, nuclear factor kappa B. JNK, cJun N-terminal kinase. ERK, extracellular regulated kinase. IL, interleukins. MHCII, major histocompatibility comple x class II. LF, lethal factor. 61

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62 Edema Toxin Similar to the differential regulation by LT, edema toxin can also modulate the response of activated DCs by promot ing their viability, and by suppressing and enhancing certain cytokine secretion as well as co-stimulatory marker expression. The basic activation pathway remains the same as previously described. In terms of suppression, once EF is inside the cytosol and bound to calmodulin it functions as an adenylate cyclase and increases cAMP concentration (2, 25, 81). cAMP serves as an intracellular secondary messenger and can modulate the activity of various cellular process, including neurotransmission, inflammation processes, and water homeostasis (7, 14, 128, 145). As shown in Figure 13, there are a number of players upstream MAPKK signaling pathway that can lead to gene activation, such as MAPKK kinase Raf and GTPase Rho (8, 10, 51). Src kinase is activated by TLR4 which can also leads to gene activation through accumulati on of a number of different proteins like c-Jun, IRF-1, and CREB (49). The di rect molecular target for cAMP is Protein Kinase A (PKA) (49). The suppre ssion in our study is very much likely associated with cAMP-activated PKA that t hen leads to inhibition (49, 67). PKA can inhibit Raf either directly or indirect ly through Ras (8, 10, 51). This inhibition leads to gene inactivation and ultimately suppression. PKA can also inhibit gene activation via Rho and Src kinase signaling pathways (8, 10, 51). In terms of viability, cAMP response element binding pr otein (CREB) has been associated with increase in cell viabilit y (10, 49), which may explain why we observed ET partially helps promote cell survival from Lp-induced apoptosis in

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63 Figure 9. CREB can either be acti vated via a PKA-dependent or independent pathways (134). ET enhancement effect is dependent on bot h stimuli and mouse strain in our study. ET induced IL-6, IL-1 production, and CD86 expression from B6 DCs following Lp infection. One study has shown that PKA activation can increase IL10 secretion from DC and macrophages vi a the catalytic subunit from PKA, which leads to gene activation (Figure 13) (67).

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Figure 13 Mechanisms for Edema Toxin Modulation Lp, Legionella pneumophila LPS, lipopolysaccharide. TLR, toll-like receptor. MAPK, mitogen-activated protein kinase. NF B, nuclear factor kappa B. JNK, cJun N-terminal kinase. ERK, extracellular regulated kinase. IL, interleukins. MHCII, major histocompatibility complex class II. EF, edema factor. Ras, GTPase. Raf, mitogen-activated protein kinase kinase kinase. Rho, GTPase. Src, proto-oncogene tyrosine kinase. CREB, cAMP response element binding protein. IRF-1, interferon r egulatory factor-1. c-Jun, tr ansactivator. cAMP, cyclic adenosine monophosphate. PKA, protein kinase A. 64

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65 Summary In conclusion, the modulating effect of anthrax toxins on DC maturation and function has been reported using cult ure systems of splenic, lung, and bone marrow cells; microbial stimulation by anthrax and LPS; and cells from toxin sensitive and resistant strains. Our results with BM-DCs from both strains and stimulated with LPS and Lp conf irm the relative toxic natu re of LT on cells from BALB/c mice; however, we also show that LT and ET can attenuate DC toxicity due to intracellular infection by an agent such as Lp suggesting the modulation of necrosis and apoptosis by anthrax toxins is dependent upon the relative activity of these processes within the cell. Our re sults also confirm previous reports that LT suppresses IL-12, IL-6, and TNFin LPS-stimulated DCs from BALB/c mice, however, we also show for t he first time that LT can increase IL-12 in B6 cells and IL-1 in cells from both strains. LT al so increased the expression of MHC II and CD86 which was not observed in st udies using splenic and lung DCs. Regarding the effect of ET, we showed for the first time that it suppressed the homing receptor, CD11c, in response to LPS and Lp stimulation, but increased the production of IL-6 in Lp-stimulated B6 cells as well as IL-1 in cells from both strains. Together, the data support the conclusion that anthrax toxins are not uniformly suppressive of DC function but rather modulate function up or down depending on variables such as the function tested, the stimulus used to activate the DCs, and genetic variation in innate immune response mechanisms in the host cell.

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81 APPENDICES

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APPENDIX A Appendix A: Cytokine Profile from Various Multiplicity of Infection BALB/c BM-DCs enriched with GM-CSF were infected with L. pneumophila at various concentrations (5:1, 10:1, 20:1, and so on) in 2ml media for 50min, then washed with HBSS followed by 18h overnight incubation at 37C, 0.5% CO 2 Culture supernatants were analyzed by ELISA for IL-12 and IL-6. The graphs show the representative data of 2 experiments with standard deviation. 82

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83 Appendix B: Cytokine Kineti cs from Affinity-purified DC Purified BM-DCs from BALB/c mice infect ed at MOI 20:1 (bacteria to cells) in <500 l media for 50mins were washed to remove excess bacteria and incubated for 6, 12, 18, 24, 48, 72h as indicated by the time points on x-axis. DCs only were collected 18h later. The samples were assayed for secretion of IL-12, IL-6, IL-1 and TNFby ELISA. The graphs show the representative of two preliminary experiments with standard deviation.

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Appendix B 84

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85 Appendix C: Cytokine Effects Attenuated Using Heated Toxins BALB/c DCs mice were pretreated for 5-6 h with protective antigen (PA) 200 ng/ml and either LF (50ng/ml) or EF (50ng/ml) or combination (25+25ng/ml), followed by LPS (1 g/ml) stimulation for 24h. LF and EF used on the right panel were heated to 56C for 35mins prior to their use. Cultur e supernatants were harvested and analyzed by ELISA for IL-12, IL-6, IL-1 and TNF. Data are the standard deviation of cytokine concentration s in culture super natants from two independent experiments.

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Appendix C 86

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ABOUT THE AUTHOR Ping-Jen (Joe) Chou was born in Taiwa n and grew up in Belize. He received his bachelor degree in Biology from University of South Florida, Tampa, in 2002. That fall he entered the docto ral program in the Department of Molecular Medicine (formerly the D epartment of Medica l Microbiology and Immunology) at USF, and was under the quality mentorship of Dr. Thomas Klein and the late Dr. Herman Friedman. Data from this project have been accepted for publication in DNA & Cell Biology in 2008.