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Phenotypic Changes in Dendritic Cells When Challenged with Cowpox Virus


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PHENOTYPIC CHANGES IN DENDRITIC CELLS WHEN CHALLENGED WITH COWPOX VIRUS By JUSTIN R. DeBERNARDIS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

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ACKNOWLEDGMENTS I would first like to thank my mentor (Ly le L. Moldawer, Ph.D.) for his immense contribution to this work and to my growth as a scientist. His insight and guidance over the past two years has been the driving force that helped me over the many hurdles I encountered. I would also like to thank my committee members (Richard W. Moyer, Ph.D.; and Michael Clare-Salzler, M.D.) whos e expertise in the ar eas of virology and immunology, respectively, have added to my scie ntific understanding related to this work. I must also thank the members of the Moldawer lab, Amer Abouhamze, Cindy Tannahill, Jen Lanz, and Ricardo Ungaro for their incredible help and support with all of my experiments. Their assistance was invaluable in completing this project. I would like to thank Philip Efron, M.D., for hi s expertise in analyzing all of my flow cytometry data. I would like to also thank Amy MacNeil and Michael Duke (of the Moyer lab) for the help and insight in growing and maintaining my virus stocks. Finally, I thank my parents for their unconditional love and support. I would like to thank all of my family and friends whose support for me has persisted through th e many difficult times of my educational career. ii

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TABLE OF CONTENTS page ACKNOWLEDGMENTS..................................................................................................ii LIST OF FIGURES...........................................................................................................iv ABSTRACT.......................................................................................................................vi CHAPTER 1 INTRODUCTION........................................................................................................1 Dendritic Cell Biology..................................................................................................1 Poxvirus Pathogenesis..................................................................................................3 Poxvirus and Dendritic Cell Interactions......................................................................5 2 MATERIALS AND METHODS.................................................................................7 Generation of Bone Marrow Derived DCs...................................................................7 LPS Stimulation of DCs...............................................................................................7 Cowpox Virus Infection of DC.....................................................................................8 Flow Cytometric Analysis of DCs................................................................................8 Infectivity Assessment via Flow Cytometry.................................................................9 Cytokine Measurements...............................................................................................9 Analysis of Cell Viability.............................................................................................9 Statistical Analyses.....................................................................................................10 3 RESULTS...................................................................................................................11 Rate of Infection Was Determined Using a CPV/GFP Vector....................................11 Cell Death from CPV at an MOI of 10 Occurs within 24 Hours................................11 LPS Is a Potent Stimulator of DC Maturation............................................................12 DC Maturation Proceeds Normally in TNF Receptor Type-I Null Mice and in an Exaggerated Pattern in IL-10 Null Mice................................................................19 Cowpox Virus Does Not Induce DC Maturation.......................................................20 Cowpox Virus Inhibits DC Ability to Respond to LPS following an Infection........22 4 DISCUSSION.............................................................................................................32 LIST OF REFERENCES...................................................................................................38 BIOGRAPHICAL SKETCH.............................................................................................42 iii

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LIST OF FIGURES Figure page 1 Schematic diagram depicting a DC response to external stimulus and subsequent activation of T-cells....................................................................................................2 2 Determination of infectivity rate using CPV/GFP vector........................................12 3 Analysis of cell viability after 2 h of cowpox virus infection.................................13 4 Analysis of cell viability after 6 h of cowpox virus infection..................................13 5 Analysis of cell viability after 24 hr cowpox virus infection...................................14 6 Contour plot showing the effect of LPS stimulation on CD86 and MHC Class II surface expression....................................................................................................15 7 TNFproduction in DCs from C57BL/6 wild type, sTNFR-I null, and IL-10 null mice when stimulated with LPS...............................................................................17 8 mIL-12 p70 production in DCs from C57BL/6 wild type, sTNFR-I null, and IL-10 null mice when stimulated with LPS........................................................................17 9 IL-10 production in DCs from C57BL/6, sTNFR-I null, and IL-10 null mice when stimulated with LPS.................................................................................................18 10 Contour plot showing CD86 and MHC class II expression of DCs from TNFR I null mice stimulated with LPS.................................................................................20 11 Contour plot showing DC maturation in response to cowpox virus........................21 12 Flow cytometric analysis of MHC-II expression in DCs stimulated with LPS following a 6 h cowpox infection.............................................................................23 13 Flow cytometric analysis of MHC-II expression in GFP expressing vs. nonexpressing DCs stimulated with LPS following a 6 h cowpox virus infection...24 14 Flow cytometric analysis of CD86 expression in DCs stimulated with LPS following a 6 h cowpox infection.............................................................................25 15 Flow cytometric analysis of CD86 expression in GFP expressing vs. nonexpressing DCs stimulated with LPS following a 6 h cowpox virus infection...........................26 iv

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16 TNFproduction by DCs stimulated with LPS following a 6 h cowpox infection (CPV/GFP)...............................................................................................................27 17 IL-12 p70 production by DCs stimulated with LPS following a 6 h cowpox infection....................................................................................................................28 18 IL-10 production in DCs stimulated with LPS following a 6 hr cowpox infection..29 19 Flow cytometric analysis of MHC class II expression in DCs infected with cowpox virus in the presence of anti-IL10 in culture............................................................30 20 Flow cytometric analysis of CD86 expression in DCs infected with cowpox virus in the presence of anti-IL10 in culture.........................................................................31 v

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PHENOTYPIC CHANGES IN DENDRITIC CELLS WHEN CHALLENGED WITH COWPOX VIRUS By Justin R. DeBernardis December 2004 Chair: Richard W. Moyer Major Department: Molecular Genetics and Microbiology With the increasing threat of bioterrorism, the need to understand the pathological mechanisms behind poxvirus infections has become of paramount importance. Poxviruses are one of the most feared applications of bioterrorism, partly due to the virus rapid dissemination in the target population, as well as its high morbidity and mortality. Poxvirus, unlike many other viruses, maintains the ability to modulate its hosts immune system in a manner that promotes its survival and infectivity. The objective of this study was to determine how poxvirus interacts with the hosts primary antigen recognition system, dendritic cells (DCs), a major component of the immune system that has the ability to link the innate and acquired immune responses. Immature DCs serve as sentinels in peripheral tissues where they undergo a programmed maturation to become an efficient antigen presenting cell characterized by high surface expression of MHC class II and CD86, production of high levels of IL-12, and ability to prime nave T-cells. Little is known, however, regarding the DC maturation response to vi

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poxvirus infection. Examination of DC surface markers as well as cytokine production demonstrated that DCs do not undergo a vigorous activation or maturation in response to cowpox virus infection. Furthermore, concomitant cowpox virus infection also attenuated CD86 expression when DCs were subsequently stimulated with bacterial lipopolysaccharide (LPS). In DCs infected with poxvirus, production of TNFand IL-12p70 was also significantly reduced and in some cases undetectable when compared to DCs that were stimulated with LPS alone. Interestingly, levels of IL-10 were significantly increased in the poxvirus infected DCs when compared to DCs stimulated with LPS, which had relatively low to undetectable levels. These findings suggest that DCs are not readily activated by cowpox virus infections, and more interestingly, cowpox virus infections alter the subsequent DC response to a microbial challenge. The findings confirm that poxvirus infections alter innate immune responses, and may explain the ability of poxvirus infection to evade recognition by the innate immune system. vii

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CHAPTER 1 INTRODUCTION Dendritic Cell Biology Steinman and his colleagues first characterized dendritic cells (DCs) in the 1970s as a novel splenic cell population with immunological properties. Since then, DCs have become the subject of much research interest, particularly in microbial recognition [1-3]. DCs, characterized by a stellate morphology, exist in all lymphoid tissues and have been observed in many areas of the reticuloendothelial system. When compared to other antigen presenting cells (APCs) of the immune system, DCs have a 10-100 fold greater capacity to stimulate nave T-cells than other APCs [4-7]. After encountering foreign antigen or danger signals, DCs can amplify their ability to present antigen to T-cells through various mechanisms [8,9]. Additionally, in comparison to other APCs, DCs lack the ability to eliminate antigen via antibody production or microorganism destruction [8,9]. While DCs exist as one of several phenotypically different subtypes, our studies are focusing on the bone marrow-derived CD34 + DC population [9]. These myeloid DCs are capable of accessing most tissues via the bloodstream. Immature DCs are initially directed to peripheral tissues where they act as sentinels for the detection of self and nonself. Found primarily in tissues such as the skin, lung epithelium, gut and other interfaces with the environment, immature dendritic cells continuously sample their environment through endocytosis and pinocytosis. In the absence of any stimulus, immature DCs migrate from peripheral tissues to draining lymph nodes, and are relatively non-immunogenic because they express low levels of MHC class II and co1

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2 stimulatory molecules, and exhibit little capacity to present antigen [10]. However, in the event that an immature DC encounters a foreign antigen or a danger signal in the periphery, they migrate to the draining lymph node and lose their capacity to take up antigen. The activated DC increases surface expression of MHC-peptide complexes, upregulates expression of costimulatory molecules such as CD86, and increases production of proinflammatory cytokines such as IL-12p70 and tumor necrosis factor alpha (TNF-) [11] (Figure 1). ImmatureDCSkin microorganism MaturingDC Cytokines andchemokines Lymphnode T-cells T-cells T-cells TH1response TH2response Up-regulation ofSurface Proteins(i.e.MHC II, CD86) Migration ImmatureDCSkin microorganism MaturingDC Cytokines andchemokines Lymphnode T-cells T-cells T-cells TH1response TH2response Up-regulation ofSurface Proteins(i.e.MHC II, CD86) Migration Figure 1. Schematic diagram depicting a DC response to external stimulus and subsequent activation of T-cells Note: As demonstrated here, the immature myeloid DC rests in the periphery until it encounters stimulation, such as a foreign antigen or an endogenous danger signal. Subsequently the DC undergoes a maturation process. The mature DC attains the ability to migrate to the local lymph nodes by altering its pattern of chemokine receptors, and induces either a Th1 or a Th2 response, thus linking the innate and acquired immune systems.

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3 Maturation of DCs can be initiated by a number of different endogenous and exogenous stimuli, including proinflammatory cytokines (TNF-), CD40 ligand, bacterial cell antigens, as well as some viral antigens. Recent interest has focused on a family of toll-like receptors, which currently number ten, that are involved in the recognition of both pathogen associated microbial products as well as endogenous danger signals [12]. Interestingly, the nature of the stimuli determines the type of DC and lymphocyte response (e.g., Th1 vs. Th2) [13,14]. For example, extracellular bacterial infections and their products appear to stimulate the maturation of a type I DC, characterized by their ability to produce high levels of IL-12 and to generate a Th1 type of acquired immune response. In contrast, helminthic infections appear to induce a more type II DC maturation response associated with elevated production of IL-10 and the generation of a Th2 type of acquired immune response. This property of DCs reaffirms that they can serve as the link between the innate and acquired immune systems. DC development is guided by environmental factors which may become the Achilles heel for immune responses. Therefore, it is critical to fully understand this pathway and how it progresses in response to different pathogens. Poxvirus Pathogenesis Poxviruses, along with a number of other viruses, share mechanisms capable of modulating the host immune response. Large DNA viruses like poxviruses express a wide array of immune regulatory genes, taking up approximately 50% of the total genome, dedicated to protecting the virus from the host immune system [15]. Poxviruses contain linear double-stranded DNA genomes with termini that contain covalently closed hairpin loops. Chordopoxvirinae, the family of poxviruses that infects vertebrates, has a

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4 genome that can range in size from 135,000 base pairs to 289,000 base pairs. Genes that are located toward the center of the genome are generally highly conserved among all poxviruses and tend to be involved in common viral functions such as replication and virus assembly [15,16]. Genes that are located towards the termini are typically more variable and are involved in immune response modulation specific to the individual poxvirus. Poxviruses have developed two classes of immunoregulatory proteins: those that share and those that do not share sequence homology with host proteins [16]. These immunoregulatory proteins, including cytokine receptor antagonists and caspase inhibitors, have a dramatic effect on the classical maturation process of DCs, a phenomenon that is not well understood and is consequently the focus of this study. Many poxviruses possess a protein that is capable of down regulating MHC class I, which is responsible for presenting all intracellular antigens. Down regulation of MHC class I would lead to decreased immune recognition by CD8 + CTLs of any viral agents. The mechanism of action is thought to occur at the endoplasmic reticulum, where these immune regulatory proteins target newly formed MHC class I molecules for retention and degradation via an endosomal pathway [17,18]. In addition to proteins that regulate the cell-mediated immune response, poxviruses also employ a number of mechanisms that modulate the extracellular environment. Most of theses mechanisms utilize a soluble receptor homolog that binds free inflammatory cytokines and prevents them from binding to their receptors, ultimately decreasing the inflammatory response and inhibiting CTL-mediated cytolysis. Poxviruses also have receptor homologs to bind many different cytokines including interferon (IFN) , and IL-18, and the TNF family of molecules

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5 [19-23]. Another important immune regulatory mechanism is the ability to inhibit apoptosis of the host cell. Poxviruses have a strategy that disrupts the apoptotic pathway by preventing the activation of caspase-8, which is a protein responsible for initiating apoptosis secondary to death domain activation. The most extensively studied anti-apoptotic protein is the CrmA protein of cowpox viruses [24]. CrmA acts on the different apoptotic pathways in many different manners. CrmA is a serine protease inhibitor (serpin) and exerts this action on granzyme B, which is a serine protease that is delivered to virally infected cells by NK cells and CTLs. Granzyme B is the initiator protein of perforin-dependent apoptosis [25]. CrmA also has the ability to inhibit caspase-8 and caspase-10 in order to inhibit apoptosis at other critical points in the pathway [26]. Poxvirus and Dendritic Cell Interactions Since DCs play a pivotal role in the recognition of nonself during microbial invasion, its role in the early detection of poxvirus infection would be crucial to the development of a successful immune response. There have been a number of earlier studies that have examined the interactions between DCs and poxvirus. Most of these studies have employed vaccinia virus strains and human peripheral blood monocyte derived DCs. These studies have generally shown that replicating vaccinia virus strains will infect human derived DCs and will express early virus genes, but are often nonpermissive for viral DNA replication or late protein synthesis [27]. Furthermore, the studies suggest that poxviruses fail to activate human DCs, and these DCs become resistant to subsequent maturation by endogenous activation agents [28-30]. However, those studies have not examined whether murine bone marrow derived DCs respond in a similar fashion to cowpox infections, whether the infected DCs are still responsive to an extrinsic activation agent, like bacterial lipopolysaccharide (LPS), and whether the

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6 resistance to activation by LPS is limited only to those DCs directly infected by cowpox virus, or whether the suppression of activation is secondary to a bystander effect. The specific aims of the current study were therefore three-fold: 1) to determine whether poxvirus could infect murine myeloid DCs, 2) to examine whether infection with poxvirus would lead to maturation of those cells directly infected by the virus, as well as by DCs that were not directly infected (bystander effect), and 3) to determine whether poxvirus infection altered the capacity of myeloid derived DCs to undergo maturation in response to an extrinsic inflammatory challenge (bacterial LPS). In this manner, we hope to determine whether poxvirus infections activate the innate immune system in a manner comparable to that seen with other TLR receptor based ligands, and whether poxvirus infections modulate the subsequent ability of DCs to respond to subsequent inflammatory challenges. This latter point is particularly relevant to the secondary opportunistic infections that may contribute to the high mortality associated with poxvirus infections.

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CHAPTER 2 MATERIALS AND METHODS Generation of Bone Marrow Derived DCs Female C57/BL/6 mice between eight and twelve weeks of age were obtained from The Jackson Laboratory (Bar Harbor, ME). Additional IL-10 null (C57BL/6 il10-/) and TNFR I null (C57BL/6 tnfr1-/) mice were bred locally and maintained at the Animal Care Services, University of Florida. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Florida College of Medicine prior to these studies. DCs derived from murine bone marrow were generated as described by Oberholzer et. al. [31]. Briefly, bone-marrow cells harvested from the femur and tibia of C57BL/6 female mice were depleted of red blood cells by lysis with ammonium chloride. Thereafter, approximately 10 6 cells were cultured on 24-well plates (Costar, Corning, NY) in RPMI 1640 (Cellgro, Herndon, VA) with 10% heat inactivated fetal calf serum, 0.000375% 2-mercaptoethanol (Sigma Chemical Co., St. Louis) and 1% penicillin-streptomycin-neomycin (Gibco, Grand Island, NY), pH 7.2-7.4, supplemented with 500 U/ml of recombinant murine GM-CSF (R&D Systems, Minneapolis, MN) and 1,000 U/ml of recombinant murine IL-4 (PharMi ngen, San Diego, CA), and incubated in a 5% CO 2 atmosphere at 37 C for a total of four to five da ys. The medium was replaced on day two with additional recombinant cytokines. LPS Stimulation of DCs DCs were stimulated on day four with varying doses of LPS (Escherichia coli 055:B4), ranging from 0.1 g/ml to 10 g/ml and supplemented with recombinant 7

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8 cytokines. DCs were collected for flow cytometric analysis, and supernatant was collected for cytokine analysis at various times ranging from two to 24 hours. Cowpox Virus Infection of DC DCs were incubated on day four with varying quantities of the Brighton-Red strain of cowpox virus (CPV) for two hours in serum-free media. DCs were infected with either wild-type cowpox virus or recombinant cowpox virus expressing green fluorescent protein (GFP). The recombinant cowpox virus expressing GFP under a CMV promoter was produced according to Stern et al. [32]. The number of infectious particles ranged from 10 5 to 10 7 particles/ml for 10 6 DCs, yielding approximate MOIs from 0.1 to 10. Thereafter, the plate was centrifuged (Beckman GPR centrifuge) at 1,000 rpm for five minutes and the media was completely replaced and supplemented with recombinant GM-CSF and IL-4. DCs were incubated in complete media for periods between six and 24 hours and then harvested for flow cytometric analysis, or cytokine secretion. For experiments in which maturation of DCs was subsequently examined after poxvirus infection, 10 g/ml of LPS was added after the six hour infection, for varying amounts of time. Flow Cytometric Analysis of DCs Six hours following poxvirus infection with or without stimulation by LPS, cells were harvested and centrifuged at 1,200 rpm for five minutes. The supernatant was collected and frozen at -70 C for cytokine analysis. The cell pellet was washed with 1% flow buffer (1% bovine serum albumin (BSA), 1mM EDTA (Fisher Scientific, Atlanta, GA) and 0.1% sodium azide (NaN 3 : Sigma Chemical Co., St. Louis, MO) in Hanks balanced salt solution without phenol red, calcium and magnesium (Cellgro, Herndon, VA)), and resuspended in 500 l of 1% paraformaldehyde solution (25% Formaldehyde

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9 (FischerChemical, Fair Lawn, NJ) in 1% flow buffer) to fix the cell membrane and inactivate any active viral particles. Prior to flow cytometric analysis, cells were resuspended in 4% BSA flow buffer and blocked with CD16/CD32 Fc antibodies (PharMingen, San Diego, CA) followed by staining, as described below. DCs were identified using anti-CD11c and anti-CD8 antibodies, and DC maturation was determined based on the relative surface expression of CD86 and MHC Class II (Immunotech, Miami, FL). Antibodies were either directly conjugated with PE (PharMingen, San Diego, CA), or indirectly conjugated to an anti-biotinylated antibody labeled with APC (Molecular Probes, Eugene, OR). Samples were analyzed on a FACSCalibur instrument with Lysis II Software (Becton Dickinson Systems, San Jose, CA). Infectivity Assessment via Flow Cytometry Infection efficiency of DCs was assessed using a recombinant cowpox virus/GFP (CPV/GFP) construct (provided as a gift by Dr. Pete Turner, Department of Molecular Genetics and Microbiology). DCs were infected at an MOI of 10 as previously outlined, then collected and fixed in 1% paraformaldehyde. Cells expressing GFP were quantified on a FACSCalibur instrument to determine the rate of infection. Cytokine Measurements DC culture supernatants were collected and measured for concentrations of murine TNF, IL-12p70 and IL-10 by specific ELISA using commercially available reagents (Endogen for TNF, R&D Systems for IL-10, and BD-Pharmingen, Inc. for IL-12p70). Analysis of Cell Viability DCs were infected for two hours at various MOI and then allowed to incubate for various amounts of time ranging from two to twenty four hours. DCs were then treated with 6 mg/ml yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5

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10 diphenyltetrazolium bromide) for four hours, lysed with isopropanol and read on the VERSAmax microplate reader. Statistical Analyses Data are presented as the mean S.E.M. Differences among treatment groups were determined by analysis of variance, after passing tests of normality. In those cases, where normality was not confirmed, differences among groups were compared using the Kruskal-Wallis ANOVA on ranks. Post hoc comparisons against the controls were performed using the Dunnetts multiple range test. In some cases, a pair t-test against controls was employed with Bonferronis correction for multiple comparisons. In all cases, significance was determined at the 95% confidence level.

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CHAPTER 3 RESULTS There were three specific aims to the study: 1) to determine whether poxvirus directly infected myeloid-derived murine DCs, and whether these infections were cytotoxic; 2) to determine whether infection with poxvirus induced DC maturation, and this maturation was dependent on cell infection, or resulted from a bystander effect on locally infected cells; and, 3) whether infection with poxvirus altered the ability of DCs to respond to a subsequent prototypical inflammatory stimulus, induced by LPS. Rate of Infection Was Determined Using a CPV/GFP Vector Myeloid derived DCs were generated from mixed cell populations obtained from the bone marrows of C57BL/6 mice. After culturing these cells for four days with IL-4 and GM-CSF, cells were infected with increasing doses of a recombinant CPV expressing GFP. The percentage of DCs infected with the CPV/GFP was analyzed by flow cytometry. As shown in Figure 2 (n=12), at MOIs approximating 10, approximately 46% of the CD11c + cells were expressing GFP, indicative of an active infection. Cell Death from CPV at an MOI of 10 Occurs within 24 Hours DC viability was analyzed at two, six and 24 hours after infection with wild-type CPV at a MOI of 0.1, 1, and 10 pfu/cell. Cell viability was determined by the reduction of the chromophore MTT, determined in 96 well microtiter plates. Because the assay was performed in a 96 well plate, we could not distinguish cell viability between infected 11

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12 A. DC infected w/wild-type CPVB. DC infected w/CPV/gfpPercentage of DCsexpressing gfpwere averaged across 12 samples and rate of infection was determined to be 46.5% A. DC infected w/wild-type CPVB. DC infected w/CPV/gfpPercentage of DCsexpressing gfpwere averaged across 12 samples and rate of infection was determined to be 46.5% Figure 2. Determination of infectivity rate using CPV/GFP vector Note: Immature DCs (CD11c + ) were infected with either wild-type cowpox virus or CPV/GFP at MOI of 10 for six hours, as outlined in the Materials and Methods section. DCs were harvested and stained for flow cytometry using biotinylated antibodies to CD11c + and analyzed on a FACSCalibur instrument. CD11c + cells staining positive for GFP were designated as cells positive for cowpox virus infection. cells and those incubated with poxvirus but not infected. As shown in Figures 3-5, significant reductions in cell viability were not observed until six hours after infection with an MOI of 10. There was some decrease in cell viability at 24 hours with infections at an MOI of 1. However, at the highest infection (MOI of 10 pfu/cell) at 24 hours, cell viability declined 55% suggesting that cytolysis was similar to infectivity (46%). LPS Is a Potent Stimulator of DC Maturation The second specific aim was intended to examine whether infection with cowpox virus induced DC maturation, and whether maturation was dependent upon cell infection. To establish a positive control for DC maturation, immature DCs were stimulated with

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13 MTT 2 hr. CPV infection1.31.351.41.451.51.55no virus0.1110Virus MOIMean Absorbance @ 570/690 Figure 3. Analysis of cell viability after 2 hr. of cowpox virus infection. Note: DCs were infected at MOI0.1, 1, 10 of CPV, or not infected for two hours, and the virus was removed by washing the plates. The cells were then allowed to incubate for an additional two hours. DCs were then treated with MTT for four hours, lysed with isopropanol and water, and absorbance was recorded at 570/690nm. There was no difference in cell viability after two hours following infection with varying MOIs of cowpox virus. Values represent the mean of eight replicates. MTT 6hr. CPV infection0.90.9511.051.11.151.2no virus0.1110Virus MOIMean Absorbance @ 570/690* p <0.05 vs. control MTT 6hr. CPV infection0.90.9511.051.11.151.2no virus0.1110Virus MOIMean Absorbance @ 570/690* p <0.05 vs. control Figure 4. Analysis of cell viability after 6 hr. cowpox virus infection

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14 Note: DCs were infected at MOI0.1, 1, 10 of cowpox virus, or not infected, as described in Figure 3, and allowed to incubate for six hours. DCs were then treated with MTT for four hours, lysed with isopropanol and absorbance was recorded at 570/690nm. Data were analyzed by two way ANOVA and Dunnetts multiple range test. An asterisk (*) indicates that the cell viability, as determined by MTT reduction, was reduced in the group of cells treated with an MOI of 10 cowpox virus, at the p<0.05 confidence level. Values represent the mean of eight replicates. MTT 24 hr. CPV infection00.511.52no virus0.1110Virus MOIMean Absorbance @ 570/690*** p<0.05 vs. control MTT 24 hr. CPV infection00.511.52no virus0.1110Virus MOIMean Absorbance @ 570/690*** p<0.05 vs. control Figure 5. Analysis of cell viability after 24 hr. cowpox virus infection Note: DCs were infected at MOI0.1, 1, 10 of CPV, or not infected, as described in Figure 3, and allowed to incubate for 24 hours. DCs were then treated with MTT for four hours, lysed with isopropanol and absorbance was recorded at 570/690nm. Data were analyzed by two way ANOVA and Dunnetts multiple range test. An asterisk (*) indicates that the cell viability, as determined by MTT reduction, was reduced in the group of cells treated with an MOI of 1 and 10 cowpox virus, at the p<0.05 confidence level. Values represent the mean of eight replicates. bacterial LPS, a potent TLR4 ligand and activator of innate immunity [33]. A number of criteria were used to assess the maturation status of the DC population, including the cell surface expression of MHC class II and the costimulator molecule CD86. As shown in Figure 6, cell surface expression was determined by flow cytometry, and CD11c + cells

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15 were gated for MHC class II and CD86 expression. A representative contour plot is shown where CD11c + cells were additionally stained with FITC labeled MHC class II and APC labeled CD86 antibodies. Mature DCs were identified by the proportion of cells who were high expressers of both MHC class II and CD86. The summary results are presented in Table 1. With 10 g/ml of LPS as a stimulant, DCs expressing both MHC-II and CD86 increased from 24.1% to 41.3% (p<0.05). A. UnstimulatedDCsB. 10g LPS-2hrs A. UnstimulatedDCsB. 10g LPS-2hrs Figure 6. Contour plot showing the effect of LPS stimulation on CD86 and MHC Class II surface expression. Note: GM-CSF and IL-4 generated DCs were stimulated with 10 g LPS for 24 hrs and harvested as outlined in the Materials and Methods section. DCs were then stained with conjugated antibodies to CD11c, CD86, and, MHC class II. Cell debris was gated out by omitting all signals that were outside the forward and side scatter channels. CD11c+ cells were gated, and then analyzed for surface expression of MHC-II and its costimulatory molecule, CD86, as a measure of maturation. The x-axis represents the intensity of cell staining for MHC class II, and the y-axis represents the intensity of staining for CD86. Panel A is representative analysis of unstimulated DCs while Panel B is a representative analysis of LPS stimulated DCs. It can be inferred that cells within the upper right-hand quadrant (high expression for both MHC class II and CD86) exhibit a

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16 mature phenotype, and cells that lie within the lower left-hand quadrant exhibit an immature phenotype (MHC class II and CD86 low). Table 1Summary of DC maturation from C57BL/6, TNFR I null and IL-10 null mice following stimulation with LPS. B6TNFR k/oIL-10 k/oNo LPS1ug10ugNo LPS1ug10ugNo LPS1ug10ugExp. 131.5339.6239.3424.0641.3641.3324.0838.2840.322539.6240.45Exp. 230.4154.6859.1643.2553.0957.9239.5863.3364.3541.7662.5659.8745.3652.7958.9235.2463.1164.88Mean31.94548.78549.672534.417546.71549.65537.4163.2264.615**Std. Dev.7.31940111.81611.3758711.455987.22406410.135553.0688430.1555630.374767Std. Err.3.6597015.9080025.6879375.727993.6120325.0677752.170.110.265 p<0.05 vs. no LPSB6TNFR k/oIL-10 k/oNo LPS1ug10ugNo LPS1ug10ugNo LPS1ug10ugExp. 131.5339.6239.3424.0641.3641.3324.0838.2840.322539.6240.45Exp. 230.4154.6859.1643.2553.0957.9239.5863.3364.3541.7662.5659.8745.3652.7958.9235.2463.1164.88Mean31.94548.78549.672534.417546.71549.65537.4163.2264.615**Std. Dev.7.31940111.81611.3758711.455987.22406410.135553.0688430.1555630.374767Std. Err.3.6597015.9080025.6879375.727993.6120325.0677752.170.110.265 p<0.05 vs. no LPS Note: Following 24 hr. stimulation with either nothing, 1 g LPS, or 10 g LPS, cells were harvested and stained for flow cytometry as outlined in the Materials and Methods section. Mature DCs were then identified using the same gating criteria as previously described. Percentages of mature DCs were calculated and compared among treatment groups of the three different strains of mice. Values represent the mean of either four or two replicates. An asterisk (*) indicates that the differences in percent maturation versus unstimulated controls was significant at the p<0.05 level of confidence, as determined either by ANOVA (TNFR I knockout, or paired t-test with Bonferronis correction; C57BL/6) In addition to cell surface molecule expression, the activation or maturation status of DCs was evaluated by the release of Th1 and proinflammatory cytokines, TNFand IL-12p70, as well as the anti-inflammatory cytokine, IL-10 at 24 hours after stimulation. As shown in Figures 7-9, LPS stimulated DCs to produce increased quantities of both TNFand IL-12 over the first 24 hours, but did not increase the production of IL-10. Caution must be used in interpreting these results since they represent a single replicate. Additional replicates are presented in later tables when the maturation response to LPS is directly compared to cowpox virus infected cells.

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17 010002000300040005000600070008000no LPS 1g LPS10g LPSLPS treatmentpg/mL mTNFFigure 7. TNFproduction in DCs from C57BL/6 wild type, sTNFR-I null, and IL-10 null mice when stimulated with LPS. Note: Following a 24 hr stimulation with either nothing, 1 g LPS, or 10 g LPS, cells were harvested and supernatant was collected as outlined in the Materials and Methods section. TNFconcentrations were determined by ELISA. represents DCs obtained from C57BL/6 mice; DCs obtained from TNFR-I null mice, and DCs obtained from IL-10 null mice. Values of zero indicate that TNFlevels were below detectable limits. Values represent a single replicate. No statistical analyses were performed. 050100150200250300350400no LPS1g LPS10g LPSLPS treatmentpg/mL mIL-12 p70 Figure 8. mIL-12 p70 production in DCs from C57BL/6 wild type, sTNFR-I null, and IL-10 null mice when stimulated with LPS.

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18 Note: Following 24hr. stimulation with either nothing, 1 g LPS, or 10 g LPS, cells were harvested and supernatant was collected as outlined in the Materials and Methods section. IL-12 concentrations were determined by ELISA. represents DCs obtained from C57BL/6 mice; DCs obtained from TNFR-I null mice, and DCs obtained from IL-10 null mice. Values of zero indicate that IL-12 levels were below detectable limits. Values represent a single replicate. No statistical analyses were performed. 0510152025no LPS1g LPS 10g LPSLPS treatmentpg/mL mIL-10 Figure 9. IL-10 production in DCs from C57BL/6, sTNFR-I null, and IL-10 null mice when stimulated with LPS. Note: Following 24 hr. stimulation with either nothing, 1 g LPS, or 10 g LPS, cells were harvested and supernatant was collected as outlined in the Materials and Methods section. represents DCs obtained from C57BL/6 mice; DCs obtained from TNFR-I null mice, and DCs obtained from IL-10 null mice. Values of zero indicate that IL-10 levels were below detectable limits. Values represent a single replicate. No statistical analyses were performed.

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19 DC Maturation Proceeds Normally in TNF Receptor Type-I Null Mice and in an Exaggerated Pattern in IL-10 Null Mice As an initial effort to determine the dependence of endogenous TNFand IL-10 signaling on DC maturation in response to LPS, these studies were also carried out with DCs obtained from TNFR-I and IL-10 null mice. Because these were preliminary studies and were not the primary focus of the experiment, the studies looking at cell surface expression were replicated only two times (n=4), whereas the cytokine measurements were determined only once. As shown in the contour plot in Figure 10, CD11c + cells from TNFR I null mice responded to LPS stimulation with a response similar to DCs from wild type animals with marked increases in both CD86 and MHC class II expression. Studies from the Moldawer laboratory have previously shown that lethality to endotoxicosis is dependent upon TNF signaling through the TNFR I receptor [34]. These data are further summarized in Table 1 and Figures 7-9. Surprisingly, DCs from TNFR I null mice had barely detectable TNFrelease, suggesting that TNF signaling is required for amplification of TNFproduction in response to LPS. Furthermore, IL-12p70 production in response to LPS was dramatically reduced, especially at the lower dose of LPS, suggesting that this response was also dependent to some extent on TNF signaling. Interestingly, DCs from IL-10 knockout mice showed both an exaggerated expression of cell surface molecules, and dramatically increased levels of TNF and IL-12, consistent with its role as an endogenous anti-inflammatory agent [35].

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20 A. No LPSB. 10g LPS for 24hrs A. No LPSB. 10g LPS for 24hrs Figure 10. Contour plot showing CD86 and MHC class II expression of DCs from TNFR I null mice stimulated with LPS. Note: DCs were stimulated with 10 g LPS for 24 hrs and harvested as outlined in the Materials and Methods section. DCs were then stained with fluorescent conjugated antibodies to CD11c, CD86, and, MHC class II. Cell debris was gated out by omitting all signals that were outside the forward and side scatter channels. Next, cells were gated on CD11c + expression, and the resulting positive cell population was then analyzed for surface expression of MHC class II and its costimulatory molecule, CD 86, as a measure of maturation. The x-axis represents the signal intensity for MHC class II, and the y-axis represents the signal intensity for CD86. Panel A represents DCs not stimulated with LPS while Panel B represents DCs stimulated with 10 g/ml of LPS. All cells that lie within the upper right-hand quadrant are designated as exhibiting a mature phenotype (MHC II high and CD86 high ) and all cells that lie within the lower left-hand quadrant exhibit an immature phenotype. Cowpox Virus Does Not Induce DC Maturation DCs infected with cowpox virus displayed dramatically different cell surface expression and cytokine production profiles than did DCs stimulated with LPS alone. As shown in the contour plot in Figure 11, cowpox virus infection did not induce any increase in the expression of MHC class II or CD86. With doses as high as 10 MOI,

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21 there was no increase in cell surface expression, despite a significant increase in cell death at 24 hours. B. MOI 0.1 C. MOI -1D. MOI -10 A. No CPV B. MOI 0.1 C. MOI -1D. MOI -10 A. No CPV Figure 11. Contour plot showing DC maturation in response to cowpox virus. Note: Cultured DCs were infected with cowpox virus at MOI of 0.1, 1, and 10 for two hours, washed them and allowed to incubate for an additional 6 hrs. The percentage of DCs exhibiting a mature phenotype was then compared to cultured DCs that were not infected with CPV. DCs were stained with conjugated antibodies to CD11c, CD86, and, MHC-II as outlined in the Materials and Methods section. Cell debris was gated out by omitting all signals that were outside the forward and side scatter channels. Next, cells were gated on CD11c + and the resulting cell population was then analyzed for surface expression of MHC-II and its costimulatory molecule, CD86, as a measure of maturation. he x-axis represents the signal intensity for MHC class II, and the y-axis represents the signal intensity for CD86. Panel A represents DCs not stimulated with cowpox virus while Panel B represents DCs stimulated with 0.1 MOI of cowpox virus, Panel C with 1 MOI of cowpox virus and Panel D with 10 MOI of cowpox virus. All cells that lie within the upper right-hand quadrant are designated as exhibiting a mature phenotype (MHC II high and CD86 high ) and all cells that lie within the lower left-hand quadrant exhibit an immature phenotype. As demonstrated here, cowpox infection was not associated with any increase in the percentage of cells with a mature phenotype.

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22 Cowpox Virus Inhibits DC Ability to Respond to LPS following an Infection To examine whether DCs infected with cowpox virus could respond to a subsequent inflammatory challenge like LPS, DCs infected with 10 MOI of cowpox virus were challenged after six hours with 10 g of LPS. To distinguish the direct effects of cowpox virus on infected cells, from cells incubated with virus but not infected, a recombinant cowpox virus expressing GFP was employed, and GFP positive (indicative of infection) and GFP negative DCs were gated to examine their cell surface expression. Looking at the DCs as a group in their entirety, MHC class II expression was increased in all DCs exposed to LPS (at a significance level of p=0.07, by two-way ANOVA), regardless of whether they had been pre-exposed to cowpox virus (Figure 12). By gating on the GFP expressing cells, we could subdivide the analysis into cells directly infected by cowpox virus and those exposed presumably to a bystander effect. There was no difference in the MHC class II expression induced by LPS in cells directly infected with cowpox virus and those not infected, although cells expressing GFP tended to have higher MHC class II expression than cells not expressing GFP (Figure 13).

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23 01020304050607080901002hr6hr12hrtime% surface MHC-II expression* p<0.07 vs. No LPS 01020304050607080901002hr6hr12hrtime% surface MHC-II expression* p<0.07 vs. No LPS Figure 12. Flow cytometric analysis of MHC-II expression in DCs stimulated with LPS following a 6hr. cowpox infection. Note: DCs were infected with recombinant cowpox virus expressing GFP at an MOI of 10 for two hours, and then allowed to rest for an additional six hours. The DCs were then stimulated with 10 g LPS for 2, 6, and 12 hrs. as outlined in the Materials and Methods section. DCs were harvested and stained for flow cytometry as outlined in the Materials and Methods section. GFP+ cells were recognized as cells positive for cowpox virus infection. Biotinylated antibodies to MHC II were detected using APC and analyzed on a FACSCalibur instrument. X---X indicates cowpox virus infection followed by LPS stimulation, ---LPS stimulation alone, -----cowpox virus infection alone, and ----no treatment. Data represent the mean of three replicates, and were analyzed by two way, ANOVA. The differences in MHC class two expression among the four groups was p=0.07.

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24 0102030405060708090 100 2hr6hr12hrtime% surface MHC-II expression LPSNo LPS 0102030405060708090 100 2hr6hr12hrtime% surface MHC-II expression LPSNo LPS Figure 13. Flow cytometric analysis of MHC-II expression in GFP expressing vs. nonexpressing DCs stimulated with LPS following a 6 hr. cowpox virus infection. Note: DCs were infected with cowpox virus expressing GFP at an MOI of 10 for two hours, and then allowed to rest for an additional six hours. The DCs were then stimulated with 10 g LPS for 2, 6, and 12 hrs. as outlined in the Materials and Methods section. GFP+ cells were considered to be cells positive for cowpox virus infection. Biotinylated antibodies to MHC-II were detected using APC and analyzed on a FACSCalibur instrument. ---positive for infection by cowpox virus with no LPS stimulation, ---negative for infection by cowpox virus with no LPS stimulation, ---positive for infection with CPV followed with LPS, X----X negative infection of CPV followed with LPS. Interestingly, the CD86 response was very different. When DCs were infected with cowpox virus for six hours and then stimulated with LPS for varying amounts of time, CD86 expression was significantly less than seen in cells stimulated with LPS alone (Figure 14). While CD86 expression in this treatment group was higher than the cowpox virus infection only treatment group, these results suggest that the virus infection has clearly reduced the DCs ability to express surface CD86. Interestingly, this reduction in

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25 class II expression in cowpox virus infected cells stimulated with LPS appeared to affect both the cells actually infected and those not infected, but experiencing a bystander effect. (Figure 15) Thus, it appears that the suppression in LPS induced CD86 expression by CPV may not be due to a direct cytolytic effect of the virus, but rather secondary to humoral factors released by the cells. 01020304050607080901002hr6hr12hrtime% surface CD86 expression ** p<0.05 vs. No LPS 01020304050607080901002hr6hr12hrtime% surface CD86 expression ** p<0.05 vs. No LPS Figure 14. Flow cytometric analysis of CD86 expression in DCs stimulated with LPS following a 6 hr. cowpox infection. Note: DCs were infected with recombinant cowpox virus expressing GFP at an MOI of 10 for two hours, and then allowed to rest for an additional six hours. The DCs were then stimulated with 10 g LPS for 2, 6, and 12 hrs. as outlined in the Materials and Methods section. DCs were harvested and stained for flow cytometry as outlined in the Materials and Methods section. GFP+ cells were recognized as cells positive for cowpox virus infection. Biotinylated antibodies to CD86 were detected using APC and analyzed on a FACSCalibur instrument. X---X indicates cowpox virus infection followed by LPS stimulation, ---LPS stimulation alone, -----cowpox virus infection alone, and ----no treatment. Data represent the mean of three replicates, and were analyzed by two way, ANOVA. The asterisk (*) indicates that the CD86 expression in the LPS treated cells were different than controls (p<0.05). Prior infection with cowpox virus attenuated the CD86 expression to subsequent LPS stimulation.

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26 01020304050607080902hr6hr12hrtime% surface CD86 expression LPSNo LPS 01020304050607080902hr6hr12hrtime% surface CD86 expression LPSNo LPS Figure 15. Flow cytometric analysis of CD86 expression in GFP expressing vs. nonexpressing DCs stimulated with LPS following a 6 hr. cowpox virus infection. Note: DCs were infected with cowpox virus expressing GFP at an MOI of 10 for two hours, and then allowed to rest for an additional six hours. The DCs were then stimulated with 10 g LPS for 2, 6, and 12 hrs. as outlined in the Materials and Methods section. GFP+ cells were considered to be cells positive for cowpox virus infection. Biotinylated antibodies to CD86 were detected using APC and analyzed on a FACSCalibur instrument. ---positive for infection by cowpox virus with no LPS stimulation, ---negative for infection by cowpox virus with no LPS stimulation, ---positive for infection with CPV followed with LPS, X---X negative infection of CPV followed with LPS. There was no difference in the CD86 expression between GFP+ and GFPexpressing cells stimulated with LPS or not, although CD86 expression tended to be higher in the GFP+ cells. These findings suggest that the effect of cowpox virus infection was observed regardless of whether the cells were GFP expressing. As shown in Figures 16-18, prior infection with cowpox virus also dramatically altered the pattern of cytokine production in DCs treated with LPS. For example, the production of TNFand IL-12 were significantly reduced in DCs that were infected with cowpox virus. IL-10 levels were unaffected.

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27 -1000010002000300040005000600070002hr6hr12hrtime pointpg/mL mTNF-**** p<0.05 vs. control -1000010002000300040005000600070002hr6hr12hrtime pointpg/mL mTNF-**** p<0.05 vs. control Figure 16. TNFproduction by DCs stimulated with LPS following a 6 hr. cowpox infection (CPV/GFP). Note: DCs were infected with a recombinant cowpox virus expressing GFP at an MOI of 10 for two hours, and then allowed to rest for an additional six hours. The DCs were then stimulated with 10 g LPS for two, six, and 12 hrs, as outlined in the Materials and Methods section. Additionally, some DCs were infected with cowpox virus at an MOI of 10 in the presence of 1 g/ml of anti-mIL10 antibody in culture. TNFwas determined by ELISA. DCs infected with CPV alone, DCs infected with CPV followed by LPS, DCs stimulated with LPS alone, no treatment, and DCs stimulated with CPV in the presence of anti-IL10. Values of zero indicate that TNFlevels were below detectable limits. Values represent the mean of three replicates each (with the exception of the anti-IL-10 studies which were repeated only once). An asterisk (*) indicates that the differences in TNFproduction in response to the LPS was significantly different than from no treatment controls, as determined by two way ANOVA and Dunnetts multiple range test. It is important to note that the TNFproduction by DCs infected with cowpox virus and then stimulated with LPS was not different than no treatment controls.

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28 -5000500100015002000250030003500400045002hr6hr12hrtime pointpg/mL mIL-12 p70* p<0.05 vs. control** -5000500100015002000250030003500400045002hr6hr12hrtime pointpg/mL mIL-12 p70* p<0.05 vs. control** Figure 17. IL-12 p70 production by DCs stimulated with LPS following a 6 hr. cowpox infection. Note: DCs were infected with a recombinant cowpox virus expressing GFP at an MOI of 10 for two hours, and then allowed to rest for an additional six hours. The DCs were then stimulated with 10 g LPS for two, six, and 12 hrs, as outlined in the Materials and Methods section. Additionally, some DCs were infected with cowpox virus at an MOI of 10 in the presence of 1 g/ml of anti-mIL10 antibody in culture. IL-12 was determined by ELISA. DCs infected with CPV alone, DCs infected with CPV followed by LPS, DCs stimulated with LPS alone, no treatment, and DCs stimulated with CPV in the presence of anti-IL10. Values of zero indicate that IL-12 levels were below detectable limits. Values represent the mean of three replicates each (with the exception of the anti-IL-10 studies which were repeated only once). An asterisk (*) indicates that the differences in TNFproduction in response to the LPS was significantly different than from no treatment controls, as determined by two way ANOVA and Dunnetts multiple range test. It is important to note that the IL-12 production by DCs infected with cowpox virus and then stimulated with LPS was not different than no treatment controls.

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29 -2000200400600800100012002hr6hr12hrtime pointpg/mL mIL-10********* p<0.05 vs. control -2000200400600800100012002hr6hr12hrtime pointpg/mL mIL-10********* p<0.05 vs. control Figure 18. IL-10 production in DCs stimulated with LPS following a 6 hr. cowpox infection. Note: DCs were infected with a recombinant cowpox virus expressing GFP at an MOI of 10 for two hours, and then allowed to rest for an additional six hours. The DCs were then stimulated with 10 g LPS for two, six, and 12 hrs, as outlined in the Materials and Methods section. Additionally, some DCs were infected with cowpox virus at an MOI of 10 in the presence of 1 g/ml of anti-mIL10 antibody in culture. IL-10 was determined by ELISA. DCs infected with CPV alone, DCs infected with CPV followed by LPS, DCs stimulated with LPS alone, no treatment, and DCs stimulated with CPV in the presence of anti-IL10. Values of zero indicate that IL-10 levels were below detectable limits. Values represent the mean of three replicates each (with the exception of the anti-IL-10 studies which were repeated only once). An asterisk (*) indicates that the differences in IL-10 production in response to the LPS was significantly different than from no treatment controls, as determined by two way ANOVA and Dunnetts multiple range test. Because cowpox virus infection alone was shown to increase IL-10 production, DCs were also treated with an antibody against IL-10 during cowpox virus infection. IL-10 blockade appeared to increase both MHC class II and CD86 expression in response to CPV infection (Figures 19,20), although these studies were replicated only once.

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30 01020304050607080901002hr6hr12hrtime% surface MHC-II expression Figure 19. Flow cytometric analysis of MHC class II expression in DCs infected with cowpox virus in the presence of anti-IL10 in culture. Note: DCs were infected with cowpox virus at an MOI of 10 in the presence or absence of anti-IL-10 antibody in culture for 2, 6, and 12 hrs. DCs were harvested and then stained for flow cytometry as outlined in the Materials and Methods section. GFP+ cells were recognized as cells infected with cowpox virus. Biotinylated antibodies to MHC-II were detected using APC and analyzed on a FACSCalibur instrument. ---no treatment controls, ----cowpox virus infection in the presence of anti-IL-10 antibodies, ----cowpox virus infection in standard culture medium. Values represent a single replicate. Although no statistics were performed because of the single replicate, there was a trend towards higher MHC class II expression in cells infected with cowpox virus in presence of neutralizing antibodies to IL-10.

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31 010203040506070802hr6hr12hr ti m e % surface CD86 expression Figure 20. Flow cytometric analysis of CD86 expression in DCs infected with cowpox virus in the presence of anti-IL10 in culture. Note: DCs were infected with cowpox virus at an MOI of 10 in the presence or absence of anti-IL-10 antibody in culture for 2, 6, and 12 hrs. DCs were harvested and then stained for flow cytometry as outlined in the Materials and Methods section. GFP+ cells were recognized as cells infected with cowpox virus. Biotinylated antibodies to CD86 were detected using APC and analyzed on a FACSCalibur instrument. ---no treatment controls, ----cowpox virus infection in the presence of anti-IL-10 antibodies, ----cowpox virus infection in standard culture medium. Values represent a single replicate. Although no statistics were performed because of the single replicate, there was a trend towards higher CD86 expression in cells infected with cowpox virus in presence of neutralizing antibodies to IL-10.

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CHAPTER 4 DISCUSSION The present studies examined the response of murine bone marrow derived DCs to ex vivo infection with cowpox virus. Cowpox virus is pathogenic in mice and has been used frequently in the past to study poxvirus pathogenesis (particularly pneumonia) in murine systems [36]. The DC maturation response to poxvirus was compared to the response to bacterial LPS, a prototypical inflammatory product of Gram negative bacteria. In addition, several additional studies were performed to examine the role of endogenous mediators, most notably TNFand IL-10 in modulating the DC response to poxvirus and to bacterial LPS. The results were unequivocal. Infection of murine bone-marrow derived DCs with a pathogenic strain of cowpox virus resulted in dose-dependent infection of cells that led to cell death. Cell death was not immediate, and at MOIs of 1 or 10 pfu/cell, reduced cell vitality was evident by six to 24 hours. The incidence of cell death was not complete by 24 hours, with only a 55% reduction in cell vitality at that time point. This onset and degree of DC killing by cowpox virus is comparable to the degree of human peripheral blood monocyte derived DC killing by vaccinia, as reported by others [28,29]. Despite this significant biological effect, there was no evidence that infection with cowpox virus induced maturation of the DCs. Activation and maturation of DCs is a staged process that involves not only the increased presentation of cell surface proteins involved in antigen presentation (such as MHC class II, CD80, CD83 and CD86, as well as a variety of chemokine receptors), but also the synthesis and release of a number of cytokines, 32

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33 including TNF-, IL-12, IFN-, and IL-10, which are involved in the polarization of T cells. Bacterial lipopolysaccharide is a prototypical microbial pathogen recognized by TLR4 signaling, and is a potent activator of DC maturation [37]. A number of groups have shown that it is perhaps the most potent DC maturation agent (along with CD40 ligand), resulting in increased expression of MHC class II and CD86, and stimulates the release of TNF-, IL-12, IFN-, and IL-10 [38]. The studies reported here demonstrate that the maturation of DCs in response to bacterial LPS is only modestly dependent upon TNFsignaling. Although TNFis a strong maturation agent for DCs [39,40], the present studies suggest that maturation to LPS proceeds in a near normal fashion even in TNFR I null animals. CD86 and MHC class II expression were unaffected by LPS induced maturation in DCs from TNFR I null mice, although the production of TNFand IL-12p70 were modestly reduced. In contrast, it appears that an endogenous IL-10 response plays some role in moderating the DC maturation response to LPS. DCs obtained from IL-10 null mice showed an exaggerated increase in expression of CD86 and MHC class II, and a dramatic increase in the production of cytokines, IL-12p70 and TNF-. Endogenous IL-10 production is presumed to suppress the production of Th1 cytokines and reduce the magnitude of the inflammatory response from systemic endotoxicosis [41]. In contrast to bacterial LPS, these studies demonstrated that cowpox virus infection failed to significantly induce the increased expression of either MHC class II or CD86 in these murine, myeloid derived DCs. In this regard, the studies are comparable to the work of Drillen et al. and Engelmayer et al., who examined human peripheral blood monocyte-derived DC maturation in response to ex vivo vaccinia virus infection [28,29].

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34 In both those studies, the authors demonstrated that vaccinia virus infection was modestly lethal to the human DCs within 72 hrs, and failed to alter either the expression of MHC class II, CD83 or CD86 expression, or stimulate the production of TNFor IL-6. Interestingly, Drillen et al. [28] showed that a modified vaccinia virus Ankara, unlike wildtype vaccinia, induced a dramatic immunogenic response, and was capable of moderately activating human DCs. In our studies, the effect of cowpox virus infection on DC maturation was not simply an absence of any response. Rather, a significant release of IL-10 was observed in DCs infected with cowpox virus. This was not seen to the same degree with LPS stimulation. Interestingly, although these studies were performed only once, blocking the IL-10 response with a monoclonal antibody increased CD86 and MHC class II expression on the cowpox virus infected DCs. IL-10 is an important regulatory molecule produced by DCs that is known to have a number of biological effects on both antigen presenting cells and T lymphocytes. IL-10 is a potent suppressor of DC maturation and suppresses Th1 responses [42,43]. It is possible that the poxvirus production of IL-10 could have acted in an autocrine fashion to reduce cell surface expression. Interestingly, coincubation of immature DCs expressing IL-10 with nave T cells is known to generate a population of T regulatory cells with potent immune suppressive properties [44,45]. These T regulatory cells are primarily responsible for suppressing antigenic responses to self. It is interesting to speculate that the failure of DCs to undergo a classical maturation and presentation of antigens to T cells may be one mechanism by which poxvirus can subvert the development of efficient antiviral immunity. Furthermore, the increased expression of IL-10 by these phenotypically immature DCs may lead to the expansion of the toleragenic T cells. Pathogenic poxvirus

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35 in mice, such as cowpox virus, may employ these mechanisms as a tool for immune evasion. More interesting is the observation that poxvirus infection suppressed the ability of these predominantly immature, myeloid derived DCs to undergo maturation to a subsequent prototypical maturation agent, bacterial LPS. Six hours of cowpox virus infection dramatically reduced the response of these immature myeloid DCs to a subsequent maturation signal by bacterial LPS. The increase in MHC class II expression was not affected by the prior cowpox virus infection, whereas CD86 expression was significantly reduced. This is not surprising since MHC class II molecules exist preformed in DCs and their presentation does not require new protein synthesis [46]. In addition, the expression of TNFand IL-12 were significantly reduced by the prior cowpox virus infection, whereas IL-10 expression was increased when compared to LPS treatment alone. Although these and other studies have demonstrated that cowpox virus infection can kill a significant proportion of DCs at 24 hours, and suppress the synthetic rate of endogenous proteins [47], the suppression in gene expression and protein synthesis are not necessarily complete. In this regard, the current studies extend the earlier findings of Drillen and Engelmeyer. Drillen et al. have argued that poxvirus gene expression is required to suppress DC activation [28], Engelmeyer et al. showed that immature human peripheral blood mononuclear cell DCs infected with vaccinia virus were also resistant to maturation by a cocktail of endogenous cytokines (IFN-, TNF-, IL-1) [29]. Our findings would suggest that cowpox virus infections are more insidious than merely blocking the maturation response of DCs. We would conclude that the effect of

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36 cowpox virus is not a passive effect on the innate immune response, but rather is an active process to generate a DC population that actively suppresses innate and acquired immune responses through the increased expression of IL-10. As previously stated, DCs infected with cowpox virus are resistant to maturation and express high levels of IL-10. In addition, we can now show that DCs not directly infected by cowpox virus but in co-culture with infected cells also fail to show increased CD86 expression in response to LPS stimulation. This response was detected by using a cowpox virus recombinant that expresses GFP off a CMV promoter. DCs that express GFP are infected whereas DCs that are not expressing GFP were likely not infected by the virus. Under conditions of an MOI of 10, approximately 46% of the DCs were expressing GFP, consistent with their infection. GFP negative CD11c + cells infected with poxvirus also did not increase their expression of CD86 in response to LPS stimulation, suggesting that these cells were also resistant to activation by LPS. One possible explanation is that those DCs infected with cowpox virus (GFP expressing) are secreting a humoral factor that suppresses CD86 expression in the DCs that are not infected with cowpox virus. Conclusions The present findings suggest that cowpox virus, a pathogenic agent in the mouse, can readily infect myeloid-derived dendritic cells under ex vivo conditions. This infection is cytopathic to the cells but does not induce a classical DC activation, similar to that seen with bacterial lipopolysaccharide. Rather, the infected DCs secreted high levels of the anti-inflammatory cytokine, IL-10. In response to a subsequent prototypical inflammatory challenge, bacterial LPS, prior cowpox virus infection blunts the activation and maturation response. It appears that poxvirus infections can subvert the innate

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37 immune response by DCs through a directed effort to prevent and modify the recognition by DCs. In addition, the DC response to poxvirus infection predisposes the host to a toleragenic phenotype through the increased expression of IL-10.

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BIOGRAPHICAL SKETCH Justin DeBernardis was born on July 27 th 1979, in Altoona, Pennsylvania. He grew up there with his parents, Ross and Michele, younger brother Marc, and older sister Lori. In 1991, Justin and his family moved to Jacksonville, Florida. Justin graduated from Nease High School in 1997 and immediately enrolled at the University of Florida. He graduated with honors from the University of Florida with his Bachelor of Science in microbiology and cell science in 2001. Justin began his masters in medical sciences program with a concentration in immunology at the University of Florida in 2002. In 2003, he enrolled in a concurrent degree program to obtain his masters in business management, and finally graduated from both programs in the summer of 2004. Following graduation, Justin plans to pursue a project management career in the bio-pharmaceutical industry. 42


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Permanent Link: http://ufdc.ufl.edu/UFE0007340/00001

Material Information

Title: Phenotypic Changes in Dendritic Cells When Challenged with Cowpox Virus
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0007340:00001

Permanent Link: http://ufdc.ufl.edu/UFE0007340/00001

Material Information

Title: Phenotypic Changes in Dendritic Cells When Challenged with Cowpox Virus
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0007340:00001


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PHENOTYPIC CHANGES IN DENDRITIC CELLS WHEN CHALLENGED WITH
COWPOX VIRUS

















By

JUSTIN R. DeBERNARDIS


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2004















ACKNOWLEDGMENTS

I would first like to thank my mentor (Lyle L. Moldawer, Ph.D.) for his immense

contribution to this work and to my growth as a scientist. His insight and guidance over

the past two years has been the driving force that helped me over the many hurdles I

encountered. I would also like to thank my committee members (Richard W. Moyer,

Ph.D.; and Michael Clare-Salzler, M.D.) whose expertise in the areas of virology and

immunology, respectively, have added to my scientific understanding related to this work.

I must also thank the members of the Moldawer lab, Amer Abouhamze, Cindy Tannahill,

Jen Lanz, and Ricardo Ungaro for their incredible help and support with all of my

experiments. Their assistance was invaluable in completing this project. I would like to

thank Philip Efron, M.D., for his expertise in analyzing all of my flow cytometry data. I

would like to also thank Amy MacNeil and Michael Duke (of the Moyer lab) for the help

and insight in growing and maintaining my virus stocks. Finally, I thank my parents for

their unconditional love and support. I would like to thank all of my family and friends

whose support for me has persisted through the many difficult times of my educational

career.














TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S .................................................................................................. ii

LIST OF FIGURES ......... ......................... ...... ........ ............ iv

ABSTRACT ........ .............. ............. ...... ...................... vi

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

D endritic C ell B biology ........................................................... ... ...............
Poxvirus Pathogenesis .............. ...... ...... .......... .......................... .3
Poxvirus and D endritic Cell Interactions................................... ....................... 5

2 M ATERIALS AND M ETHODS ........................................ ........................... 7

Generation of Bone Marrow Derived DCs .............. ............................................7
L P S Stim ulation of D C s ............................................................. ......................... 7
Cow pox V irus Infection of D C .......................................................... ............... 8
Flow Cytom etric A analysis of D Cs.......................................... ........... ............... 8
Infectivity A ssessm ent via Flow Cytom etry...................................... .....................9
C ytokine M easurem ents ............................................................... ....................... 9
A analysis of C ell V ability ........................ ...................... .. .. ....... .......... .......
Statistical A analyses ................................................. ....... .............. 10

3 R E S U L T S ...................................... ................................................. 1 1

Rate of Infection Was Determined Using a CPV/GFP Vector.................................11
Cell Death from CPV at an MOI of 10 Occurs within 24 Hours ...............................11
LPS Is a Potent Stimulator of DC Maturation...............................................12
DC Maturation Proceeds Normally in TNF Receptor Type-I Null Mice and in an
Exaggerated Pattern in IL-10 Null Mice......................................................19
Cowpox Virus Does Not Induce DC Maturation ................... ...........................20
Cowpox Virus Inhibits DC Ability to Respond to LPS following an Infection........22

4 D ISCU SSION ...................................................................... .......... 32

LIST OF REFEREN CES ............................................................................. 38

B IO G R A PH IC A L SK E TCH ..................................................................... ..................42
















LIST OF FIGURES


Figure page

1 Schematic diagram depicting a DC response to external stimulus and subsequent
activation of T-cells ............ .................. ............ .... .. .. .. .. ........ .. ..

2 Determination of infectivity rate using CPV/GFP vector ............. ..............12

3 Analysis of cell viability after 2 h of cowpox virus infection.............................13

4 Analysis of cell viability after 6 h of cowpox virus infection............................13

5 Analysis of cell viability after 24 hr cowpox virus infection..............................14

6 Contour plot showing the effect of LPS stimulation on CD86 and MHC Class II
surface expression. ........................................... ... .... ........ ......... 15

7 TNF-a production in DCs from C57BL/6 wild type, sTNFR-I null, and IL-10 null
mice when stimulated with LPS ...................... ........... ...... ......... 17

8 mIL-12 p70 production in DCs from C57BL/6 wild type, sTNFR-I null, and IL-10
null mice when stimulated with LPS........ .......................................... 17

9 IL-10 production in DCs from C57BL/6, sTNFR-I null, and IL-10 null mice when
stimulated with LPS. ............................................... .............. 18

10 Contour plot showing CD86 and MHC class II expression of DCs from TNFR I
null m ice stim ulated w ith LPS. ........................................ .......................... 20

11 Contour plot showing DC maturation in response to cowpox virus ......................21

12 Flow cytometric analysis of MHC-II expression in DCs stimulated with LPS
following a 6 h cowpox infection. ........................................ ........................ 23

13 Flow cytometric analysis of MHC-II expression in GFP expressing vs.
nonexpressing DCs stimulated with LPS following a 6 h cowpox virus infection...24

14 Flow cytometric analysis of CD86 expression in DCs stimulated with LPS
following a 6 h cowpox infection. ........................................ ........................ 25

15 Flow cytometric analysis of CD86 expression in GFP expressing vs. nonexpressing
DCs stimulated with LPS following a 6 h cowpox virus infection.........................26









16 TNF-a production by DCs stimulated with LPS following a 6 h cowpox infection
(C PV /G FP). ...........................................................................27

17 IL-12 p70 production by DCs stimulated with LPS following a 6 h cowpox
infection................................................ ............. ... ....... ...... ............ 28

18 IL-10 production in DCs stimulated with LPS following a 6 hr cowpox infection..29

19 Flow cytometric analysis of MHC class II expression in DCs infected with cowpox
virus in the presence of anti-IL 10 in culture. ................................... ... ..................30

20 Flow cytometric analysis of CD86 expression in DCs infected with cowpox virus in
the presence of anti-IL10 in culture. ........ ............ ................. ..... ............... 31















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

PHENOTYPIC CHANGES IN DENDRITIC CELLS WHEN CHALLENGED WITH
COWPOX VIRUS

By

Justin R. DeBernardis

December 2004

Chair: Richard W. Moyer
Major Department: Molecular Genetics and Microbiology

With the increasing threat of bioterrorism, the need to understand the pathological

mechanisms behind poxvirus infections has become of paramount importance.

Poxviruses are one of the most feared applications of bioterrorism, partly due to the

virus' rapid dissemination in the target population, as well as its high morbidity and

mortality. Poxvirus, unlike many other viruses, maintains the ability to modulate its

host's immune system in a manner that promotes its survival and infectivity. The

objective of this study was to determine how poxvirus interacts with the host's primary

antigen recognition system, dendritic cells (DCs), a major component of the immune

system that has the ability to link the innate and acquired immune responses. Immature

DCs serve as sentinels in peripheral tissues where they undergo a programmed

maturation to become an efficient antigen presenting cell characterized by high surface

expression of MHC class II and CD86, production of high levels of IL-12, and ability to

prime naive T-cells. Little is known, however, regarding the DC maturation response to









poxvirus infection. Examination of DC surface markers as well as cytokine production

demonstrated that DCs do not undergo a vigorous activation or maturation in response to

cowpox virus infection. Furthermore, concomitant cowpox virus infection also

attenuated CD86 expression when DCs were subsequently stimulated with bacterial

lipopolysaccharide (LPS). In DCs infected with poxvirus, production of TNF-ac and IL-

12p70 was also significantly reduced and in some cases undetectable when compared to

DCs that were stimulated with LPS alone. Interestingly, levels of IL-10 were

significantly increased in the poxvirus infected DCs when compared to DCs stimulated

with LPS, which had relatively low to undetectable levels. These findings suggest that

DCs are not readily activated by cowpox virus infections, and more interestingly, cowpox

virus infections alter the subsequent DC response to a microbial challenge. The findings

confirm that poxvirus infections alter innate immune responses, and may explain the

ability of poxvirus infection to evade recognition by the innate immune system.














CHAPTER 1
INTRODUCTION

Dendritic Cell Biology

Steinman and his colleagues first characterized dendritic cells (DCs) in the 1970s

as a novel splenic cell population with immunological properties. Since then, DCs have

become the subject of much research interest, particularly in microbial recognition [1-3].

DCs, characterized by a stellate morphology, exist in all lymphoid tissues and have been

observed in many areas of the reticuloendothelial system. When compared to other

antigen presenting cells (APCs) of the immune system, DCs have a 10-100 fold greater

capacity to stimulate naive T-cells than other APCs [4-7]. After encountering foreign

antigen or danger signals, DCs can amplify their ability to present antigen to T-cells

through various mechanisms [8,9]. Additionally, in comparison to other APCs, DCs lack

the ability to eliminate antigen via antibody production or microorganism destruction

[8,9]. While DCs exist as one of several phenotypically different subtypes, our studies

are focusing on the bone marrow-derived CD34+ DC population [9]. These myeloid DCs

are capable of accessing most tissues via the bloodstream. Immature DCs are initially

directed to peripheral tissues where they act as sentinels for the detection of self and

nonself. Found primarily in tissues such as the skin, lung epithelium, gut and other

interfaces with the environment, immature dendritic cells continuously sample their

environment through endocytosis and pinocytosis. In the absence of any stimulus,

immature DCs migrate from peripheral tissues to draining lymph nodes, and are

relatively non-immunogenic because they express low levels of MHC class II and co-









stimulatory molecules, and exhibit little capacity to present antigen [10]. However, in the

event that an immature DC encounters a foreign antigen or a danger signal in the

periphery, they migrate to the draining lymph node and lose their capacity to take up

antigen. The activated DC increases surface expression of MHC-peptide complexes,

upregulates expression of costimulatory molecules such as CD86, and increases

production of proinflammatory cytokines such as IL-12p70 and tumor necrosis factor

alpha (TNF-a) [11] (Figure 1).




O O
Cytokines and O
chemokines
Immature 0
DC O






_Lymph
Up-regulation of | node
Surface Proteins
(i.e. MHC II, CD86) Y




Figure 1. Schematic diagram depicting a DC response to external stimulus and
subsequent activation of T-cells

Note: As demonstrated here, the immature myeloid DC rests in the periphery until it
encounters stimulation, such as a foreign antigen or an endogenous 'danger signal'.
Subsequently the DC undergoes a maturation process. The mature DC attains the ability
to migrate to the local lymph nodes by altering its pattern of chemokine receptors, and
induces either a Thl or a Th2 response, thus linking the innate and acquired immune
systems.









Maturation of DCs can be initiated by a number of different endogenous and

exogenous stimuli, including proinflammatory cytokines (TNF-ac), CD40 ligand,

bacterial cell antigens, as well as some viral antigens. Recent interest has focused on a

family of toll-like receptors, which currently number ten, that are involved in the

recognition of both pathogen associated microbial products as well as endogenous danger

signals [12]. Interestingly, the nature of the stimuli determines the type of DC and

lymphocyte response (e.g., Thl vs. Th2) [13,14]. For example, extracellular bacterial

infections and their products appear to stimulate the maturation of a type I DC,

characterized by their ability to produce high levels of IL-12 and to generate a Thl type

of acquired immune response. In contrast, helminthic infections appear to induce a more

type II DC maturation response associated with elevated production of IL-10 and the

generation of a Th2 type of acquired immune response. This property of DCs reaffirms

that they can serve as the link between the innate and acquired immune systems. DC

development is guided by environmental factors which may become the Achilles' heel

for immune responses. Therefore, it is critical to fully understand this pathway and how

it progresses in response to different pathogens.

Poxvirus Pathogenesis

Poxviruses, along with a number of other viruses, share mechanisms capable of

modulating the host immune response. Large DNA viruses like poxviruses express a

wide array of immune regulatory genes, taking up approximately 50% of the total

genome, dedicated to protecting the virus from the host immune system [15]. Poxviruses

contain linear double-stranded DNA genomes with termini that contain covalently closed

hairpin loops. Chordopoxvirinae, the family of poxviruses that infects vertebrates, has a









genome that can range in size from 135,000 base pairs to 289,000 base pairs. Genes that

are located toward the center of the genome are generally highly conserved among all

poxviruses and tend to be involved in common viral functions such as replication and

virus assembly [15,16]. Genes that are located towards the termini are typically more

variable and are involved in immune response modulation specific to the individual

poxvirus.

Poxviruses have developed two classes of immunoregulatory proteins: those that

share and those that do not share sequence homology with host proteins [16]. These

immunoregulatory proteins, including cytokine receptor antagonists and caspase

inhibitors, have a dramatic effect on the classical maturation process of DCs, a

phenomenon that is not well understood and is consequently the focus of this study.

Many poxviruses possess a protein that is capable of down regulating MHC class I,

which is responsible for presenting all intracellular antigens. Down regulation of MHC

class I would lead to decreased immune recognition by CD8+ CTLs of any viral agents.

The mechanism of action is thought to occur at the endoplasmic reticulum, where these

immune regulatory proteins target newly formed MHC class I molecules for retention and

degradation via an endosomal pathway [17,18]. In addition to proteins that regulate the

cell-mediated immune response, poxviruses also employ a number of mechanisms that

modulate the extracellular environment. Most of theses mechanisms utilize a soluble

receptor homolog that binds free inflammatory cytokines and prevents them from binding

to their receptors, ultimately decreasing the inflammatory response and inhibiting CTL-

mediated cytolysis. Poxviruses also have receptor homologs to bind many different

cytokines including interferon (IFN) ca, 3, and y, IL-18, and the TNF family of molecules









[19-23]. Another important immune regulatory mechanism is the ability to inhibit

apoptosis of the host cell. Poxviruses have a strategy that disrupts the apoptotic pathway

by preventing the activation of caspase-8, which is a protein responsible for initiating

apoptosis secondary to death domain activation. The most extensively studied anti-

apoptotic protein is the CrmA protein of cowpox viruses [24]. CrmA acts on the different

apoptotic pathways in many different manners. CrmA is a serine protease inhibitor

(serpin) and exerts this action on granzyme B, which is a serine protease that is delivered

to virally infected cells by NK cells and CTLs. Granzyme B is the initiator protein of

perforin-dependent apoptosis [25]. CrmA also has the ability to inhibit caspase-8 and

caspase-10 in order to inhibit apoptosis at other critical points in the pathway [26].

Poxvirus and Dendritic Cell Interactions

Since DCs play a pivotal role in the recognition of nonself during microbial

invasion, its role in the early detection of poxvirus infection would be crucial to the

development of a successful immune response. There have been a number of earlier

studies that have examined the interactions between DCs and poxvirus. Most of these

studies have employed vaccinia virus strains and human peripheral blood monocyte

derived DCs. These studies have generally shown that replicating vaccinia virus strains

will infect human derived DCs and will express early virus genes, but are often

nonpermissive for viral DNA replication or late protein synthesis [27]. Furthermore, the

studies suggest that poxviruses fail to activate human DCs, and these DCs become

resistant to subsequent maturation by endogenous activation agents [28-30]. However,

those studies have not examined whether murine bone marrow derived DCs respond in a

similar fashion to cowpox infections, whether the infected DCs are still responsive to an

extrinsic activation agent, like bacterial lipopolysaccharide (LPS), and whether the









resistance to activation by LPS is limited only to those DCs directly infected by cowpox

virus, or whether the suppression of activation is secondary to a bystander effect.

The specific aims of the current study were therefore three-fold: 1) to determine

whether poxvirus could infect murine myeloid DCs, 2) to examine whether infection with

poxvirus would lead to maturation of those cells directly infected by the virus, as well as

by DCs that were not directly infected (bystander effect), and 3) to determine whether

poxvirus infection altered the capacity of myeloid derived DCs to undergo maturation in

response to an extrinsic inflammatory challenge (bacterial LPS). In this manner, we hope

to determine whether poxvirus infections activate the innate immune system in a manner

comparable to that seen with other TLR receptor based ligands, and whether poxvirus

infections modulate the subsequent ability of DCs to respond to subsequent inflammatory

challenges. This latter point is particularly relevant to the secondary opportunistic

infections that may contribute to the high mortality associated with poxvirus infections.














CHAPTER 2
MATERIALS AND METHODS

Generation of Bone Marrow Derived DCs

Female C57/BL/6 mice between eight and twelve weeks of age were obtained from

The Jackson Laboratory (Bar Harbor, ME). Additional IL-10 null (C57BL/6110--) and

TNFR I null (C57BL/6tnfr-/-) mice were bred locally and maintained at the Animal Care

Services, University of Florida. All animal protocols were approved by the Institutional

Animal Care and Use Committee of the University of Florida College of Medicine prior

to these studies. DCs derived from murine bone marrow were generated as described by

Oberholzer et. al. [31]. Briefly, bone-marrow cells harvested from the femur and tibia of

C57BL/6 female mice were depleted of red blood cells by lysis with ammonium chloride.

Thereafter, approximately 106 cells were cultured on 24-well plates (Costar, Corning,

NY) in RPMI 1640 (Cellgro, Herndon, VA) with 10% heat inactivated fetal calf serum,

0.000375% 2-mercaptoethanol (Sigma Chemical Co., St. Louis) and 1% penicillin-

streptomycin-neomycin (Gibco, Grand Island, NY), pH 7.2-7.4, supplemented with

500 U/ml of recombinant murine GM-CSF (R&D Systems, Minneapolis, MN) and

1,000 U/ml of recombinant murine IL-4 (PharMingen, San Diego, CA), and incubated in a

5% CO2 atmosphere at 370C for a total of four to five days. The medium was replaced on

day two with additional recombinant cytokines.

LPS Stimulation of DCs

DCs were stimulated on day four with varying doses of LPS (Escherichia coli

055:B4), ranging from 0.1 [tg/ml to 10 [tg/ml and supplemented with recombinant









cytokines. DCs were collected for flow cytometric analysis, and supernatant was

collected for cytokine analysis at various times ranging from two to 24 hours.

Cowpox Virus Infection of DC

DCs were incubated on day four with varying quantities of the Brighton-Red strain

of cowpox virus (CPV) for two hours in serum-free media. DCs were infected with

either wild-type cowpox virus or recombinant cowpox virus expressing green fluorescent

protein (GFP). The recombinant cowpox virus expressing GFP under a CMV promoter

was produced according to Stern et al. [32]. The number of infectious particles ranged

from 105 to 107 particles/ml for 106 DCs, yielding approximate MOIs from 0.1 to 10.

Thereafter, the plate was centrifuged (Beckman GPR centrifuge) at 1,000 rpm for five

minutes and the media was completely replaced and supplemented with recombinant

GM-CSF and IL-4. DCs were incubated in complete media for periods between six and

24 hours and then harvested for flow cytometric analysis, or cytokine secretion. For

experiments in which maturation of DCs was subsequently examined after poxvirus

infection, 10 [tg/ml of LPS was added after the six hour infection, for varying amounts of

time.

Flow Cytometric Analysis of DCs

Six hours following poxvirus infection with or without stimulation by LPS, cells

were harvested and centrifuged at 1,200 rpm for five minutes. The supernatant was

collected and frozen at -700 C for cytokine analysis. The cell pellet was washed with 1%

flow buffer (1% bovine serum albumin (BSA), ImM EDTA (Fisher Scientific, Atlanta,

GA) and 0.1% sodium azide (NaN3: Sigma Chemical Co., St. Louis, MO) in Hanks'

balanced salt solution without phenol red, calcium and magnesium (Cellgro, Herndon,

VA)), and resuspended in 500 ul of 1% paraformaldehyde solution (25% Formaldehyde









(FischerChemical, Fair Lawn, NJ) in 1% flow buffer) to fix the cell membrane and

inactivate any active viral particles. Prior to flow cytometric analysis, cells were

resuspended in 4% BSA flow buffer and blocked with CD16/CD32 Fc antibodies

(PharMingen, San Diego, CA) followed by staining, as described below. DCs were

identified using anti-CD 11 c and anti-CD8 antibodies, and DC maturation was determined

based on the relative surface expression of CD86 and MHC Class II (Immunotech,

Miami, FL). Antibodies were either directly conjugated with PE (PharMingen, San

Diego, CA), or indirectly conjugated to an anti-biotinylated antibody labeled with APC

(Molecular Probes, Eugene, OR). Samples were analyzed on a FACSCalibur instrument

with Lysis II Software (Becton Dickinson Systems, San Jose, CA).

Infectivity Assessment via Flow Cytometry

Infection efficiency of DCs was assessed using a recombinant cowpox virus/GFP

(CPV/GFP) construct (provided as a gift by Dr. Pete Turner, Department of Molecular

Genetics and Microbiology). DCs were infected at an MOI of 10 as previously outlined,

then collected and fixed in 1% paraformaldehyde. Cells expressing GFP were quantified

on a FACSCalibur instrument to determine the rate of infection.

Cytokine Measurements

DC culture supernatants were collected and measured for concentrations of murine

TNFa, IL-12p70 and IL-10 by specific ELISA using commercially available reagents

(Endogen for TNFc, R&D Systems for IL-10, and BD-Pharmingen, Inc. for IL-12p70).

Analysis of Cell Viability

DCs were infected for two hours at various MOI and then allowed to incubate for

various amounts of time ranging from two to twenty four hours. DCs were then treated

with 6 mg/ml yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5-









diphenyltetrazolium bromide) for four hours, lysed with isopropanol and read on the

VERSAmaxTM microplate reader.

Statistical Analyses

Data are presented as the mean + S.E.M. Differences among treatment groups

were determined by analysis of variance, after passing tests of normality. In those cases,

where normality was not confirmed, differences among groups were compared using the

Kruskal-Wallis ANOVA on ranks. Post hoc comparisons against the controls were

performed using the Dunnett's multiple range test. In some cases, a pair t-test against

controls was employed with Bonferroni's correction for multiple comparisons. In all

cases, significance was determined at the 95% confidence level.














CHAPTER 3
RESULTS

There were three specific aims to the study: 1) to determine whether poxvirus

directly infected myeloid-derived murine DCs, and whether these infections were

cytotoxic; 2) to determine whether infection with poxvirus induced DC maturation, and

this maturation was dependent on cell infection, or resulted from a bystander effect on

locally infected cells; and, 3) whether infection with poxvirus altered the ability of DCs

to respond to a subsequent prototypical inflammatory stimulus, induced by LPS.

Rate of Infection Was Determined Using a CPV/GFP Vector

Myeloid derived DCs were generated from mixed cell populations obtained from

the bone marrows of C57BL/6 mice. After culturing these cells for four days with IL-4

and GM-CSF, cells were infected with increasing doses of a recombinant CPV

expressing GFP. The percentage of DCs infected with the CPV/GFP was analyzed by

flow cytometry. As shown in Figure 2 (n=12), at MOIs approximating 10,

approximately 46% of the CD 1 c cells were expressing GFP, indicative of an active

infection.

Cell Death from CPV at an MOI of 10 Occurs within 24 Hours

DC viability was analyzed at two, six and 24 hours after infection with wild-type

CPV at a MOI of 0.1, 1, and 10 pfu/cell. Cell viability was determined by the reduction

of the chromophore MTT, determined in 96 well microtiter plates. Because the assay

was performed in a 96 well plate, we could not distinguish cell viability between infected









A. DC infected w/wild-type CPV B. DC infected w/CPV/gfp

a0 DCcpvempty.011 DCcpvgfp.016



''0.

1'- "'13'4_0" 1 2 16310.4
o 0




1 0 101 2 10 4 10 0 101 02 1
FL1-Height GFP

Percentage of DCs expressing gfp were averaged
across 12 samples and rate of infection was determined
to be 46.5%
Figure 2. Determination of infectivity rate using CPV/GFP vector

Note: Immature DCs (CD1 c ) were infected with either wild-type cowpox virus or
CPV/GFP at MOI of 10 for six hours, as outlined in the Materials and Methods section.
DCs were harvested and stained for flow cytometry using biotinylated antibodies to
CD lc+ and analyzed on a FACSCalibur instrument. CD1lc+ cells staining positive for
GFP were designated as cells positive for cowpox virus infection.


cells and those incubated with poxvirus but not infected. As shown in Figures 3-5,

significant reductions in cell viability were not observed until six hours after infection

with an MOI of 10. There was some decrease in cell viability at 24 hours with infections

at an MOI of 1. However, at the highest infection (MOI of 10 pfu/cell) at 24 hours, cell

viability declined 55% suggesting that cytolysis was similar to infectivity (46%).

LPS Is a Potent Stimulator of DC Maturation

The second specific aim was intended to examine whether infection with cowpox virus

induced DC maturation, and whether maturation was dependent upon cell infection. To

establish a positive control for DC maturation, immature DCs were stimulated with









MTT 2 hr. CPV infection


1.55

o 1.5
S1.45

"- 1.4
)1.35

1.3


no virus 0.1 1 10


Virus MOI

Figure 3. Analysis of cell viability after 2 hr. of cowpox virus infection.

Note: DCs were infected at MOI- 0.1, 1, 10 of CPV, or not infected for two hours, and
the virus was removed by washing the plates. The cells were then allowed to incubate for
an additional two hours. DCs were then treated with MTT for four hours, lysed with
isopropanol and water, and absorbance was recorded at 570/690nm. There was no
difference in cell viability after two hours following infection with varying MOIs of
cowpox virus. Values represent the mean of eight replicates.


MTT 6hr. CPV infection


0D
0 1.2
o 1.15
0
Cm 11
me 1.05

=00.95
D 0.9
M


no virus 0.1 1 10


* p<0.05 vs. control


Virus MOI


Figure 4. Analysis of cell viability after 6 hr. cowpox virus infection









Note: DCs were infected at MOI- 0.1, 1, 10 of cowpox virus, or not infected, as
described in Figure 3, and allowed to incubate for six hours. DCs were then treated with
MTT for four hours, lysed with isopropanol and absorbance was recorded at 570/690nm.
Data were analyzed by two way ANOVA and Dunnett's multiple range test. An asterisk
(*) indicates that the cell viability, as determined by MTT reduction, was reduced in the
group of cells treated with an MOI of 10 cowpox virus, at the p<0.05 confidence level.
Values represent the mean of eight replicates.

MTT 24 hr. CPV infection


O 1.5

> 1

@0.5

0


no virus 0.1 1 10


Virus MOI
p<0.05 vs. control

Figure 5. Analysis of cell viability after 24 hr. cowpox virus infection

Note: DCs were infected at MOI- 0.1, 1, 10 of CPV, or not infected, as described in
Figure 3, and allowed to incubate for 24 hours. DCs were then treated with MTT for four
hours, lysed with isopropanol and absorbance was recorded at 570/690nm. Data were
analyzed by two way ANOVA and Dunnett's multiple range test. An asterisk (*)
indicates that the cell viability, as determined by MTT reduction, was reduced in the
group of cells treated with an MOI of 1 and 10 cowpox virus, at the p<0.05 confidence
level. Values represent the mean of eight replicates.


bacterial LPS, a potent TLR4 ligand and activator of innate immunity [33]. A number of

criteria were used to assess the maturation status of the DC population, including the cell

surface expression of MHC class II and the costimulator molecule CD86. As shown in

Figure 6, cell surface expression was determined by flow cytometry, and CD1 c+ cells










were gated for MHC class II and CD86 expression. A representative contour plot is

shown where CD1 lc cells were additionally stained with FITC labeled MHC class II

and APC labeled CD86 antibodies. Mature DCs were identified by the proportion of

cells who were high expressers of both MHC class II and CD86. The summary results

are presented in Table 1. With 10 jtg/ml of LPS as a stimulant, DCs expressing both

MHC-II and CD86 increased from 24.1% to 41.3% (p<0.05).


A. Unstimulated DCs


B. 10pg LPS-2hrs


0



T-
Qr



U


DC1 ug LPSyesFIX 025


MHCII FTC


Figure 6. Contour plot showing the effect of LPS stimulation on CD86 and MHC Class
II surface expression.

Note: GM-CSF and IL-4 generated DCs were stimulated with 10 ag LPS for 24 hrs and
harvested as outlined in the Materials and Methods section. DCs were then stained with
conjugated antibodies to CD1 c, CD86, and, MHC class II. Cell debris was gated out by
omitting all signals that were outside the forward and side scatter channels. CD1 lc+
cells were gated, and then analyzed for surface expression of MHC-II and its
costimulatory molecule, CD86, as a measure of maturation. The x-axis represents the
intensity of cell staining for MHC class II, and the y-axis represents the intensity of
staining for CD86. Panel A is representative analysis of unstimulated DCs while Panel B
is a representative analysis of LPS stimulated DCs. It can be inferred that cells within the
upper right-hand quadrant (high expression for both MHC class II and CD86) exhibit a


0
r






, -
0



O




o
^0










mature phenotype, and cells that lie within the lower left-hand quadrant exhibit an
immature phenotype (MHC class II and CD86 low).


Table 1- Summary of DC maturation from C57BL/6, TNFR I null and IL-10 null mice
following stimulation with LPS.
B6 TNFR k/o IL-10 klo
No LPS lug 10ug No LPS lug 10ug No LPS lug 10ug
Exp. 1 31.53 39.62 39.34 24.06 41.36 41.33
24.08 38.28 40.32 25 39.62 40.45
Exp. 2 30.41 54.68 59.16 43.25 53.09 57.92 39.58 63.33 64.35
41.76 62.56 59.87 45.36 52.79 58.92 35.24 63.11 64.88
Mean 31.945 48.785 49.6725 34.4175 46.715 49.655 37.41 63.22 64.615

Std. Dev. 7.319401 11.816 11.37587 11.45598 7.224064 10.13555 3.068843 0.155563 0.374767
Std. Err. 3.659701 5.908002 5.687937 5.72799 3.612032 5.067775 2.17 0.11 0.265
p<0.05 vs. no LPS
Note: Following 24 hr. stimulation with either nothing, 1 |tg LPS, or 10 |tg LPS, cells
were harvested and stained for flow cytometry as outlined in the Materials and Methods
section. Mature DCs were then identified using the same gating criteria as previously
described. Percentages of mature DCs were calculated and compared among treatment
groups of the three different strains of mice. Values represent the mean of either four or
two replicates. An asterisk (*) indicates that the differences in percent maturation versus
unstimulated controls was significant at the p<0.05 level of confidence, as determined
either by ANOVA (TNFR I knockout, or paired t-test with Bonferroni's correction;
C57BL/6)


In addition to cell surface molecule expression, the activation or maturation status

of DCs was evaluated by the release of Thl and proinflammatory cytokines, TNF-a and

IL-12p70, as well as the anti-inflammatory cytokine, IL-10 at 24 hours after stimulation.

As shown in Figures 7-9, LPS stimulated DCs to produce increased quantities of both

TNF-a and IL-12 over the first 24 hours, but did not increase the production of IL-10.

Caution must be used in interpreting these results since they represent a single replicate.

Additional replicates are presented in later tables when the maturation response to LPS is

directly compared to cowpox virus infected cells.










8000
7000
6000
5000
4000
3000
2000


1000
0


I I


no LPS


1 g LPS


10pg LPS


LPS treatment


Figure 7. TNF-a production in DCs from C57BL/6 wild type, sTNFR-I null, and IL-10
null mice when stimulated with LPS.

Note: Following a 24 hr stimulation with either nothing, 1 ptg LPS, or 10 ptg LPS, cells
were harvested and supernatant was collected as outlined in the Materials and Methods
section. TNF-a concentrations were determined by ELISA. 1 represents DCs
obtained from C57BL/6 mice; 1 DCs obtained from TNFR-I null mice, and 1 DCs
obtained from IL-10 null mice. Values of zero indicate that TNF-a levels were below
detectable limits. Values represent a single replicate. No statistical analyses were
performed.
400
350 -


300
250
200
150
100
50
0


no LPS


1pg LPS


10pg LPS


LPS treatment



Figure 8. mIL-12 p70 production in DCs from C57BL/6 wild type, sTNFR-I null, and
IL-10 null mice when stimulated with LPS.


F-LJ


I I I I


I I









Note: Following 24hr. stimulation with either nothing, 1 |tg LPS, or 10 |tg LPS, cells
were harvested and supernatant was collected as outlined in the Materials and Methods
section. IL-12 concentrations were determined by ELISA. N represents DCs obtained
from C57BL/6 mice; E' DCs obtained from TNFR-I null mice, and E DCs obtained
from IL-10 null mice. Values of zero indicate that IL-12 levels were below detectable
limits. Values represent a single replicate. No statistical analyses were performed.




25


no LPS 1pg LPS lOpg LPS
LPS treatment


Figure 9. IL-10 production in DCs from C57BL/6, sTNFR-I null, and IL-10 null mice
when stimulated with LPS.

Note: Following 24 hr. stimulation with either nothing, 1 |tg LPS, or 10 |tg LPS, cells
were harvested and supernatant was collected as outlined in the Materials and Methods
section. S represents DCs obtained from C57BL/6 mice; = DCs obtained from
TNFR-I null mice, and E DCs obtained from IL-10 null mice. Values of zero indicate
that IL-10 levels were below detectable limits. Values represent a single replicate. No
statistical analyses were performed.









DC Maturation Proceeds Normally in TNF Receptor Type-I Null Mice and in an
Exaggerated Pattern in IL-10 Null Mice

As an initial effort to determine the dependence of endogenous TNF-a and IL-10

signaling on DC maturation in response to LPS, these studies were also carried out with

DCs obtained from TNFR-I and IL-10 null mice. Because these were preliminary studies

and were not the primary focus of the experiment, the studies looking at cell surface

expression were replicated only two times (n=4), whereas the cytokine measurements

were determined only once. As shown in the contour plot in Figure 10, CD1 lc cells

from TNFR I null mice responded to LPS stimulation with a response similar to DCs

from wild type animals with marked increases in both CD86 and MHC class II

expression. Studies from the Moldawer laboratory have previously shown that lethality to

endotoxicosis is dependent upon TNF signaling through the TNFR I receptor [34]. These

data are further summarized in Table 1 and Figures 7-9. Surprisingly, DCs from TNFR I

null mice had barely detectable TNF-a release, suggesting that TNF signaling is required

for amplification of TNF-a production in response to LPS. Furthermore, IL-12p70

production in response to LPS was dramatically reduced, especially at the lower dose of

LPS, suggesting that this response was also dependent to some extent on TNF signaling.

Interestingly, DCs from IL-10 knockout mice showed both an exaggerated expression of

cell surface molecules, and dramatically increased levels of TNFa and IL-12, consistent

with its role as an endogenous anti-inflammatory agent [35].












TNF control 1.016


MHCII FITC


A. No LPS


Figure 10. Contour plot showing CD86 and MHC class II expression of DCs from TNFR
I null mice stimulated with LPS.

Note: DCs were stimulated with 10 |tg LPS for 24 hrs and harvested as outlined in the
Materials and Methods section. DCs were then stained with fluorescent conjugated
antibodies to CD1 c, CD86, and, MHC class II. Cell debris was gated out by omitting all
signals that were outside the forward and side scatter channels. Next, cells were gated on
CD1 c+ expression, and the resulting positive cell population was then analyzed for
surface expression of MHC class II and its costimulatory molecule, CD 86, as a measure
of maturation. The x-axis represents the signal intensity for MHC class II, and the y-axis
represents the signal intensity for CD86. Panel A represents DCs not stimulated with
LPS while Panel B represents DCs stimulated with 10 [tg/ml of LPS. All cells that lie
within the upper right-hand quadrant are designated as exhibiting a mature phenotype
(MHC IIh1gh and CD86high) and all cells that lie within the lower left-hand quadrant
exhibit an immature phenotype.


Cowpox Virus Does Not Induce DC Maturation

DCs infected with cowpox virus displayed dramatically different cell surface

expression and cytokine production profiles than did DCs stimulated with LPS alone. As

shown in the contour plot in Figure 11, cowpox virus infection did not induce any

increase in the expression of MHC class II or CD86. With doses as high as 10 MOI,


B. 10j g LPS for 24hrs

TNF 10ug/ml 1.020





coe




0

100 101 102 103
MHCII FITC


0_
0o
O
07-
0

0
0O
1a











there was no increase in cell surface expression, despite a significant increase in cell

death at 24 hours.


A. No CPV
at "DCnovirus,010
20-----


/1


1 34
101 T 10 10
MHCII FITC
C. MOI -1
a DC01,0 us Ol2


B. MOI- 0.1


Quad Events % Gated %Total
IRUL A07 I -V C,71
;U.P IR 'i7,1 117 g'
S LL 7303 38.21 7 *',
LR 1324 6.93 1.32
S'


Quad Events % Gated % Total


D. MOI 10


(31.3- Evenns Ga=r3 ,T3, 1
UL 1365 4_4 1 J 6
UR C10482 531 01 10 48 o
LL '789 3612 ; 0'i
LR 1192 6 0 1 1


MI II I 3
MHCIh F[TC


UL 1363 .ff5 1.36
UR 11472 5537 11 47
LL 6990 34 10 6 999
LR 673 3.28 C 67


Figure 11. Contour plot showing DC maturation in response to cowpox virus.

Note: Cultured DCs were infected with cowpox virus at MOI of 0.1, 1, and 10 for two
hours, washed them and allowed to incubate for an additional 6 hrs. The percentage of
DCs exhibiting a mature phenotype was then compared to cultured DCs that were not
infected with CPV. DCs were stained with conjugated antibodies to CD1 Ic, CD86, and,
MHC-II as outlined in the Materials and Methods section. Cell debris was gated out by
omitting all signals that were outside the forward and side scatter channels. Next, cells
were gated on CD1 c and the resulting cell population was then analyzed for surface
expression of MHC-II and its costimulatory molecule, CD86, as a measure of maturation.
he x-axis represents the signal intensity for MHC class II, and the y-axis represents the
signal intensity for CD86. Panel A represents DCs not stimulated with cowpox virus
while Panel B represents DCs stimulated with 0.1 MOI of cowpox virus, Panel C with 1
MOI of cowpox virus and Panel D with 10 MOI of cowpox virus. All cells that lie within
the upper right-hand quadrant are designated as exhibiting a mature phenotype (MHC
IIhigh and CD86high) and all cells that lie within the lower left-hand quadrant exhibit an
immature phenotype. As demonstrated here, cowpox infection was not associated with
any increase in the percentage of cells with a mature phenotype.









Cowpox Virus Inhibits DC Ability to Respond to LPS following an Infection

To examine whether DCs infected with cowpox virus could respond to a

subsequent inflammatory challenge like LPS, DCs infected with 10 MOI of cowpox virus

were challenged after six hours with 10 tg of LPS. To distinguish the direct effects of

cowpox virus on infected cells, from cells incubated with virus but not infected, a

recombinant cowpox virus expressing GFP was employed, and GFP positive (indicative

of infection) and GFP negative DCs were gated to examine their cell surface expression.

Looking at the DCs as a group in their entirety, MHC class II expression was increased in

all DCs exposed to LPS (at a significance level of p=0.07, by two-way ANOVA),

regardless of whether they had been pre-exposed to cowpox virus (Figure 12). By

gating on the GFP expressing cells, we could subdivide the analysis into cells directly

infected by cowpox virus and those exposed presumably to a bystander effect. There was

no difference in the MHC class II expression induced by LPS in cells directly infected

with cowpox virus and those not infected, although cells expressing GFP tended to have

higher MHC class II expression than cells not expressing GFP (Figure 13).










100
90
80
70
60
50


* p<0.07 vs. No LPS


ZhIzzzczzzQQ~


Figure 12. Flow cytometric analysis of MHC-II expression in DCs stimulated with LPS
following a 6hr. cowpox infection.

Note: DCs were infected with recombinant cowpox virus expressing GFP at an MOI of
10 for two hours, and then allowed to rest for an additional six hours. The DCs were then
stimulated with 10 |tg LPS for 2, 6, and 12 hrs. as outlined in the Materials and Methods
section. DCs were harvested and stained for flow cytometry as outlined in the Materials
and Methods section. GFP+ cells were recognized as cells positive for cowpox virus
infection. Biotinylated antibodies to MHC II were detected using APC and analyzed on a
FACSCalibur instrument. X---X indicates cowpox virus infection followed by LPS
stimulation, m---- LPS stimulation alone, A------A cowpox virus infection alone, and
*-----* no treatment. Data represent the mean of three replicates, and were analyzed by
two way, ANOVA. The differences in MHC class two expression among the four groups
was p=0.07.


6hr


time


12hr


,6
t








1 UU -
.0 90
S 80 LPS
CL 70 -
6
e 60 -_ No LPS
6 50 -
40
o 30
-1 20
(n 10


2hr 6hr 12hr

time

Figure 13. Flow cytometric analysis of MHC-II expression in GFP expressing vs.
nonexpressing DCs stimulated with LPS following a 6 hr. cowpox virus
infection.

Note: DCs were infected with cowpox virus expressing GFP at an MOI of 10 for two
hours, and then allowed to rest for an additional six hours. The DCs were then stimulated
with 10 |tg LPS for 2, 6, and 12 hrs. as outlined in the Materials and Methods section.
GFP+ cells were considered to be cells positive for cowpox virus infection. Biotinylated
antibodies to MHC-II were detected using APC and analyzed on a FACSCalibur
instrument. *----* positive for infection by cowpox virus with no LPS stimulation, m----
* negative for infection by cowpox virus with no LPS stimulation, A ----A positive for
infection with CPV followed with LPS, X----X negative infection of CPV followed with
LPS.


Interestingly, the CD86 response was very different. When DCs were infected with

cowpox virus for six hours and then stimulated with LPS for varying amounts of time,

CD86 expression was significantly less than seen in cells stimulated with LPS alone

(Figure 14). While CD86 expression in this treatment group was higher than the cowpox

virus infection only treatment group, these results suggest that the virus infection has

clearly reduced the DC's ability to express surface CD86. Interestingly, this reduction in









class II expression in cowpox virus infected cells stimulated with LPS appeared to affect

both the cells actually infected and those not infected, but experiencing a bystander

effect. (Figure 15) Thus, it appears that the suppression in LPS induced CD86 expression

by CPV may not be due to a direct cytolytic effect of the virus, but rather secondary to

humoral factors released by the cells.

100
90
80
80 -

70 -
00
a) 60 i
O 50 ______
o 40 T
0 30
= 20
S10
0

2hr 6hr 12hr

p<0.05 vs. No LPS time

Figure 14. Flow cytometric analysis of CD86 expression in DCs stimulated with LPS
following a 6 hr. cowpox infection.

Note: DCs were infected with recombinant cowpox virus expressing GFP at an MOI of
10 for two hours, and then allowed to rest for an additional six hours. The DCs were then
stimulated with 10 |tg LPS for 2, 6, and 12 hrs. as outlined in the Materials and Methods
section. DCs were harvested and stained for flow cytometry as outlined in the Materials
and Methods section. GFP+ cells were recognized as cells positive for cowpox virus
infection. Biotinylated antibodies to CD86 were detected using APC and analyzed on a
FACSCalibur instrument. X---X indicates cowpox virus infection followed by LPS
stimulation, m---- LPS stimulation alone, A------A cowpox virus infection alone, and
*-----* no treatment. Data represent the mean of three replicates, and were analyzed by
two way, ANOVA. The asterisk (*) indicates that the CD86 expression in the LPS
treated cells were different than controls (p<0.05). Prior infection with cowpox virus
attenuated the CD86 expression to subsequent LPS stimulation.









= 90
0
80 -
2 70 LPS
X 60
S50 -
50 No LPS
8 40
0
s30
S20 -
10
0
2hr 6hr 12hr
time

Figure 15. Flow cytometric analysis of CD86 expression in GFP expressing vs.
nonexpressing DCs stimulated with LPS following a 6 hr. cowpox virus
infection.

Note: DCs were infected with cowpox virus expressing GFP at an MOI of 10 for two
hours, and then allowed to rest for an additional six hours. The DCs were then stimulated
with 10 |tg LPS for 2, 6, and 12 hrs. as outlined in the Materials and Methods section.
GFP+ cells were considered to be cells positive for cowpox virus infection. Biotinylated
antibodies to CD86 were detected using APC and analyzed on a FACSCalibur
instrument. *----* positive for infection by cowpox virus with no LPS stimulation, m----
* negative for infection by cowpox virus with no LPS stimulation, A ----A positive for
infection with CPV followed with LPS, X---X negative infection of CPV followed with
LPS. There was no difference in the CD86 expression between GFP+ and GFP-
expressing cells stimulated with LPS or not, although CD86 expression tended to be
higher in the GFP+ cells. These findings suggest that the effect of cowpox virus
infection was observed regardless of whether the cells were GFP expressing.


As shown in Figures 16-18, prior infection with cowpox virus also dramatically

altered the pattern of cytokine production in DCs treated with LPS. For example, the

production of TNF-a and IL-12 were significantly reduced in DCs that were infected


with cowpox virus. IL-10 levels were unaffected.










7000 *

6000

a 5000

z 4000
I-
E 3000
-J
E 2000

1000

0 III I

-1000 2hr 6hr 12hr

time point

p<0.05 vs. control

Figure 16. TNF-a production by DCs stimulated with LPS following a 6 hr. cowpox
infection (CPV/GFP).

Note: DCs were infected with a recombinant cowpox virus expressing GFP at an MOI of
10 for two hours, and then allowed to rest for an additional six hours. The DCs were then
stimulated with 10 ]g LPS for two, six, and 12 hrs, as outlined in the Materials and
Methods section. Additionally, some DCs were infected with cowpox virus at an MOI of
10 in the presence of 1 g/ml of anti-mIL10 antibody in culture. TNF-a was determined
by ELISA. F DCs infected with CPV alone, E DCs infected with CPV followed by
LPS, E[i] DCs stimulated with LPS alone, l no treatment, and N DCs stimulated
with CPV in the presence of anti-ILl0. Values of zero indicate that TNF-a levels were
below detectable limits. Values represent the mean of three replicates each (with the
exception of the anti-IL-10 studies which were repeated only once). An asterisk (*)
indicates that the differences in TNF-a production in response to the LPS was
significantly different than from no treatment controls, as determined by two way
ANOVA and Dunnett's multiple range test. It is important to note that the TNF-a
production by DCs infected with cowpox virus and then stimulated with LPS was not
different than no treatment controls.









4500 *
4000
3500
C 3000
r 2500
2000
-I 1500
S1000
500 -
0 -
-500 2hr 6hr 12hr
time point
p<0.05 vs. control

Figure 17. IL-12 p70 production by DCs stimulated with LPS following a 6 hr. cowpox
infection.

Note: DCs were infected with a recombinant cowpox virus expressing GFP at an MOI of
10 for two hours, and then allowed to rest for an additional six hours. The DCs were then
stimulated with 10 ptg LPS for two, six, and 12 hrs, as outlined in the Materials and
Methods section. Additionally, some DCs were infected with cowpox virus at an MOI of
10 in the presence of 1 ptg/ml of anti-mILl0 antibody in culture. IL-12 was determined
by ELISA. DCs infected with CPV alone, I DCs infected with CPV followed by
LPS, E[H DCs stimulated with LPS alone, E no treatment, and E DCs stimulated
with CPV in the presence of anti-IL10. Values of zero indicate that IL-12 levels were
below detectable limits. Values represent the mean of three replicates each (with the
exception of the anti-IL-10 studies which were repeated only once). An asterisk (*)
indicates that the differences in TNF-a production in response to the LPS was
significantly different than from no treatment controls, as determined by two way
ANOVA and Dunnett's multiple range test. It is important to note that the IL-12
production by DCs infected with cowpox virus and then stimulated with LPS was not
different than no treatment controls.









1200,

1000

o 800

S600 *

E 400 *

.. 200 -



-200 2hr 6hr 12hr

time point
p<0.05 vs. control

Figure 18. IL-10 production in DCs stimulated with LPS following a 6 hr. cowpox
infection.

Note: DCs were infected with a recombinant cowpox virus expressing GFP at an MOI of
10 for two hours, and then allowed to rest for an additional six hours. The DCs were then
stimulated with 10 |tg LPS for two, six, and 12 hrs, as outlined in the Materials and
Methods section. Additionally, some DCs were infected with cowpox virus at an MOI of
10 in the presence of 1 [tg/ml of anti-mILl0 antibody in culture. IL-10 was determined
by ELISA. F DCs infected with CPV alone, El DCs infected with CPV
followed by LPS, [IE DCs stimulated with LPS alone, M no treatment, and I
DCs stimulated with CPV in the presence of anti-IL10. Values of zero indicate that IL-
10 levels were below detectable limits. Values represent the mean of three replicates
each (with the exception of the anti-IL-10 studies which were repeated only once). An
asterisk (*) indicates that the differences in IL-10 production in response to the LPS was
significantly different than from no treatment controls, as determined by two way
ANOVA and Dunnett's multiple range test.


Because cowpox virus infection alone was shown to increase IL-10 production,

DCs were also treated with an antibody against IL-10 during cowpox virus infection. IL-

10 blockade appeared to increase both MHC class II and CD86 expression in response to

CPV infection (Figures 19,20), although these studies were replicated only once.









100

o 90 -
8 0 -


60 -
S 50
M 40
30
= 20
10
o 10
0
2hr 6hr 12hr

time

Figure 19. Flow cytometric analysis of MHC class II expression in DCs infected with
cowpox virus in the presence of anti-IL 10 in culture.

Note: DCs were infected with cowpox virus at an MOI of 10 in the presence or absence
of anti-IL-10 antibody in culture for 2, 6, and 12 hrs. DCs were harvested and then
stained for flow cytometry as outlined in the Materials and Methods section. GFP+ cells
were recognized as cells infected with cowpox virus. Biotinylated antibodies to MHC-II
were detected using APC and analyzed on a FACSCalibur instrument. *---- no
treatment controls, m-----m cowpox virus infection in the presence of anti-IL-10
antibodies, A-----A cowpox virus infection in standard culture medium. Values
represent a single replicate. Although no statistics were performed because of the single
replicate, there was a trend towards higher MHC class II expression in cells infected with
cowpox virus in presence of neutralizing antibodies to IL-10.









80

o 70

S60 .

X 50 -

| 40

o 30

2 20

| 10

0
2hr 6hr 12hr

time

Figure 20. Flow cytometric analysis of CD86 expression in DCs infected with cowpox
virus in the presence of anti-IL10 in culture.

Note: DCs were infected with cowpox virus at an MOI of 10 in the presence or absence
of anti-IL-10 antibody in culture for 2, 6, and 12 hrs. DCs were harvested and then
stained for flow cytometry as outlined in the Materials and Methods section. GFP+ cells
were recognized as cells infected with cowpox virus. Biotinylated antibodies to CD86
were detected using APC and analyzed on a FACSCalibur instrument. *----* no
treatment controls, m-----m cowpox virus infection in the presence of anti-IL-10
antibodies, A-----A cowpox virus infection in standard culture medium. Values
represent a single replicate. Although no statistics were performed because of the single
replicate, there was a trend towards higher CD86 expression in cells infected with
cowpox virus in presence of neutralizing antibodies to IL-10.














CHAPTER 4
DISCUSSION

The present studies examined the response of murine bone marrow derived DCs to

ex vivo infection with cowpox virus. Cowpox virus is pathogenic in mice and has been

used frequently in the past to study poxvirus pathogenesis (particularly pneumonia) in

murine systems [36]. The DC maturation response to poxvirus was compared to the

response to bacterial LPS, a prototypical inflammatory product of Gram negative

bacteria. In addition, several additional studies were performed to examine the role of

endogenous mediators, most notably TNF-a and IL-10 in modulating the DC response to

poxvirus and to bacterial LPS.

The results were unequivocal. Infection of murine bone-marrow derived DCs with

a pathogenic strain of cowpox virus resulted in dose-dependent infection of cells that led

to cell death. Cell death was not immediate, and at MOIs of 1 or 10 pfu/cell, reduced cell

vitality was evident by six to 24 hours. The incidence of cell death was not complete by

24 hours, with only a 55% reduction in cell vitality at that time point. This onset and

degree of DC killing by cowpox virus is comparable to the degree of human peripheral

blood monocyte derived DC killing by vaccinia, as reported by others [28,29]. Despite

this significant biological effect, there was no evidence that infection with cowpox virus

induced maturation of the DCs. Activation and maturation of DCs is a staged process

that involves not only the increased presentation of cell surface proteins involved in

antigen presentation (such as MHC class II, CD80, CD83 and CD86, as well as a variety

of chemokine receptors), but also the synthesis and release of a number of cytokines,









including TNF-a, IL-12, IFN-a,P and IL-10, which are involved in the polarization of T

cells. Bacterial lipopolysaccharide is a prototypical microbial pathogen recognized by

TLR4 signaling, and is a potent activator of DC maturation [37]. A number of groups

have shown that it is perhaps the most potent DC maturation agent (along with CD40

ligand), resulting in increased expression of MHC class II and CD86, and stimulates the

release of TNF-a, IL-12, IFN-a,P and IL-10 [38]. The studies reported here demonstrate

that the maturation of DCs in response to bacterial LPS is only modestly dependent upon

TNF-a signaling. Although TNF-a is a strong maturation agent for DCs [39,40], the

present studies suggest that maturation to LPS proceeds in a near normal fashion even in

TNFR I null animals. CD86 and MHC class II expression were unaffected by LPS

induced maturation in DCs from TNFR I null mice, although the production of TNF-a

and IL-12p70 were modestly reduced. In contrast, it appears that an endogenous IL-10

response plays some role in moderating the DC maturation response to LPS. DCs

obtained from IL-10 null mice showed an exaggerated increase in expression of CD86

and MHC class II, and a dramatic increase in the production of cytokines, IL-12p70 and

TNF-a. Endogenous IL-10 production is presumed to suppress the production of Thl

cytokines and reduce the magnitude of the inflammatory response from systemic

endotoxicosis [41].

In contrast to bacterial LPS, these studies demonstrated that cowpox virus infection

failed to significantly induce the increased expression of either MHC class II or CD86 in

these murine, myeloid derived DCs. In this regard, the studies are comparable to the

work of Drillen et al. and Engelmayer et al., who examined human peripheral blood

monocyte-derived DC maturation in response to ex vivo vaccinia virus infection [28,29].









In both those studies, the authors demonstrated that vaccinia virus infection was modestly

lethal to the human DCs within 72 hrs, and failed to alter either the expression of MHC

class II, CD83 or CD86 expression, or stimulate the production of TNF-a or IL-6.

Interestingly, Drillen et al. [28] showed that a modified vaccinia virus Ankara, unlike

wildtype vaccinia, induced a dramatic immunogenic response, and was capable of

moderately activating human DCs. In our studies, the effect of cowpox virus infection on

DC maturation was not simply an absence of any response. Rather, a significant release

of IL-10 was observed in DCs infected with cowpox virus. This was not seen to the same

degree with LPS stimulation. Interestingly, although these studies were performed only

once, blocking the IL-10 response with a monoclonal antibody increased CD86 and MHC

class II expression on the cowpox virus infected DCs. IL-10 is an important regulatory

molecule produced by DCs that is known to have a number of biological effects on both

antigen presenting cells and T lymphocytes. IL-10 is a potent suppressor of DC

maturation and suppresses Thl responses [42,43]. It is possible that the poxvirus

production of IL-10 could have acted in an autocrine fashion to reduce cell surface

expression. Interestingly, coincubation of immature DCs expressing IL-10 with naive T

cells is known to generate a population of T regulatory cells with potent immune

suppressive properties [44,45]. These T regulatory cells are primarily responsible for

suppressing antigenic responses to self. It is interesting to speculate that the failure of

DCs to undergo a classical maturation and presentation of antigens to T cells may be one

mechanism by which poxvirus can subvert the development of efficient antiviral

immunity. Furthermore, the increased expression of IL-10 by these phenotypically

immature DCs may lead to the expansion of the toleragenic T cells. Pathogenic poxvirus









in mice, such as cowpox virus, may employ these mechanisms as a tool for immune

evasion.

More interesting is the observation that poxvirus infection suppressed the ability of

these predominantly immature, myeloid derived DCs to undergo maturation to a

subsequent prototypical maturation agent, bacterial LPS. Six hours of cowpox virus

infection dramatically reduced the response of these immature myeloid DCs to a

subsequent maturation signal by bacterial LPS. The increase in MHC class II expression

was not affected by the prior cowpox virus infection, whereas CD86 expression was

significantly reduced. This is not surprising since MHC class II molecules exist

preformed in DCs and their presentation does not require new protein synthesis [46]. In

addition, the expression of TNF-a and IL-12 were significantly reduced by the prior

cowpox virus infection, whereas IL-10 expression was increased when compared to LPS

treatment alone. Although these and other studies have demonstrated that cowpox virus

infection can kill a significant proportion of DCs at 24 hours, and suppress the synthetic

rate of endogenous proteins [47], the suppression in gene expression and protein

synthesis are not necessarily complete. In this regard, the current studies extend the

earlier findings of Drillen and Engelmeyer. Drillen et al. have argued that poxvirus gene

expression is required to suppress DC activation [28], Engelmeyer et al. showed that

immature human peripheral blood mononuclear cell DCs infected with vaccinia virus

were also resistant to maturation by a cocktail of endogenous cytokines (IFN-a, TNF-a,

IL-p) [29].

Our findings would suggest that cowpox virus infections are more insidious than

merely blocking the maturation response of DCs. We would conclude that the effect of









cowpox virus is not a passive effect on the innate immune response, but rather is an

active process to generate a DC population that actively suppresses innate and acquired

immune responses through the increased expression of IL-10.

As previously stated, DCs infected with cowpox virus are resistant to maturation

and express high levels of IL-10. In addition, we can now show that DCs not directly

infected by cowpox virus but in co-culture with infected cells also fail to show increased

CD86 expression in response to LPS stimulation. This response was detected by using a

cowpox virus recombinant that expresses GFP off a CMV promoter. DCs that express

GFP are infected whereas DCs that are not expressing GFP were likely not infected by

the virus. Under conditions of an MOI of 10, approximately 46% of the DCs were

expressing GFP, consistent with their infection. GFP negative CD lc+ cells infected

with poxvirus also did not increase their expression of CD86 in response to LPS

stimulation, suggesting that these cells were also resistant to activation by LPS. One

possible explanation is that those DCs infected with cowpox virus (GFP expressing) are

secreting a humoral factor that suppresses CD86 expression in the DCs that are not

infected with cowpox virus.

Conclusions

The present findings suggest that cowpox virus, a pathogenic agent in the mouse,

can readily infect myeloid-derived dendritic cells under ex vivo conditions. This

infection is cytopathic to the cells but does not induce a classical DC activation, similar to

that seen with bacterial lipopolysaccharide. Rather, the infected DCs secreted high levels

of the anti-inflammatory cytokine, IL-10. In response to a subsequent prototypical

inflammatory challenge, bacterial LPS, prior cowpox virus infection blunts the activation

and maturation response. It appears that poxvirus infections can subvert the innate






37


immune response by DCs through a directed effort to prevent and modify the recognition

by DCs. In addition, the DC response to poxvirus infection predisposes the host to a

toleragenic phenotype through the increased expression of IL-10.















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BIOGRAPHICAL SKETCH

Justin DeBernardis was born on July 27th, 1979, in Altoona, Pennsylvania. He

grew up there with his parents, Ross and Michele, younger brother Marc, and older sister

Lori. In 1991, Justin and his family moved to Jacksonville, Florida. Justin graduated

from Nease High School in 1997 and immediately enrolled at the University of Florida.

He graduated with honors from the University of Florida with his Bachelor of Science in

microbiology and cell science in 2001. Justin began his master's in medical sciences

program with a concentration in immunology at the University of Florida in 2002. In

2003, he enrolled in a concurrent degree program to obtain his master's in business

management, and finally graduated from both programs in the summer of 2004.

Following graduation, Justin plans to pursue a project management career in the bio-

pharmaceutical industry.