fig? .. . 5.12 . um... I :1. .§.|.i :. igunqmwvtuw -. . u ”19.23”... i .1. . 3.. :3“... “mm". .8 —\ . I 31491.51: , sign 4.» 56.53.13. 1. I . 5‘21!- !ZI? . |5 . ‘ ‘I,.: 4 ‘ .4133 i y: ”0" z {E}. 5p.“ . is“ . a \lt‘.’l!fl.l~l§" .3:.€:...nx.:.\s.‘|ul.}lla.l..flfifin lain-11...; I. 1.. :1‘ $1. ) 2‘1... tun-5.. .‘2 mm r___ LiERz” RY ‘) '3 Michigan State UniverSIty This is to certify that the dissertation entitled IN VIVO AND IN VITRO MECHANISMS FOR DISRUPTION OF THE TOLL-LIKE RECEPTOR ACTIVATED IMMUNOGLOBULIN M RESPONSE BY 2,3,7,8—TETRACHLORODIBENZO-P-DIOXIN presented by Colin M. North has been accepted towards fulfillment of the requirements for the Ph_.p. degree in Pharmacolggy & Toxicolggy Major Professor’s Signature 9. 2/ . 09 Date ‘ MSU is an Affirmative Action/Equal Opportunity Employer PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:IProj/Acc&Pres/ClRCIDateDm.indd [X i'Ii'O AX RECEPTI IN VIVO AND IN VIT R0 MECHANISMS FOR DISRUPTION OF THE TOLL-LIKE RECEPTOR ACTIVATED IMMUNOGLOBULIN M RESPONSE BY 2,3,7,8- TETRACHLORODIBENZO-P-DIOXIN By Colin M. North A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Pharmacology & Toxicology 2009 [TWO AND I RECEPTOR 2.3.7.8-tc causes profound models of T-lnd are directly sens the Primary lg_\ cells (ASC l. ca' ll. B-cell C LL ll. Paired Box thax TCDD dis activated prim regulate 0f pl; mCI'EaSe in tht ABSTRACT IN V1 V0 AND IN VIT R0 MECHANISMS FOR DISRUPTION OF THE TOLL-LIKE RECEPTOR ACTIVATED IMMUNOGLOBULIN M RESPONSE BY 2,3,7,8- TETRACHLORODIBENZO-P-DIOXIN By Colin M. North 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistant organic pollutant that causes profound suppression of the primary immunoglobulin M (IgM) response in animal models of T-independent humoral immunity. B cells, the antibody producing cell type, are directly sensitive to TCDD, but a mechanistic understanding for TCDD disruption of the primary IgM response is incomplete. B cell differentiation into antibody-secreting cells (ASC), called plasmacytic differentiation, is regulated by Activator Protein-1 (AP- 1), B-cell CLL/lymphoma (BCL-6), B lymphocyte induced maturation protein-1 (Blimp- 1), Paired Box gene 5 (PaxS), and X-box Binding Protein-1 (XBP-l). It was hypothesized that TCDD disrupts the Toll-like receptor (TLR) ligand lipopolysacchride (LPS)- activated primary IgM response by altering expression of transcription factors known to regulate of plasmacytic differentiation. In vivo LPS administration induced a significant increase in the number of ASCs, which was dose-dependently impaired by TCDD (3, 10, or 30 ug/kg). Gene and protein expression analysis showed TCDD suppressed LPS- elicited increases in CD138 and IgM components. Blimp-l and XBP-l expression were dose-dependently impaired by TCDD. Relationships between transcription factors regulating plasmacytic differentiation in purified B cells were assessed in vitro using LPS and combinations of TCDD (0.03, 0.3, 3 or 30 nM), then analyzed by flow cytometry 24, 48, and 72 h post-treatment. TCDD caused a concentration-related suppression of Blimp-1 and phosphorylated c-Jun expression. and e if DD treatment CD69. CD80. an Kinases ( TCDD alters Bt Ni phosphor} l actuated by LP: assess the time- TCDD suppress min POSi-treatm ERK and JN K 151“ response b Wiéntially as a expression, and elevated BCL-6 expression, relative to LPS + DMSO treated B cells. TCDD treatment caused a concomitant suppression of LPS-activated MHC Class II, CD69, CD80, and CD86 upregulation as early as 24 h post-treatment. Kinases directly and indirectly regulate AP-l and BCL-6. It was hypothesized that TCDD alters BCL-6 expression and AP-l phosphorylation by changing AKT, ERK, and INK phosphorylation. Multiparametric analysis of AKT, ERK, and INK phosphorylation activated by LPS, R848, or CpG DNA in the B cell lymphoma CH12.LX was used to assess the time- and concentration-dependent effects of TCDD (0.003, 0.03, or 0.3 nM). TCDD suppressed TLR-activated AKT, ERK, and INK phosphorylation at 15, 30, and 60 min post-treatment. TCDD at 30 nM impaired R848-activated phosphorylation of AKT, ERK, and INK in primary B cells. These results suggest TCDD suppresses the primary IgM response by altering transcription factor and activation marker expression, potentially as a result of TCDD interference in TLR—activated kinase phosphorylation. To Elizabeth .\'t‘ in spite of in) h To Simone. Cc do and remind To Charles an stumbled thrr completed g: To John and Cilriosity 1h, TO GOd‘ “i Dedication To Elizabeth North, who believed in me even as times were difficult, and who stuck it out in spite of my belief that I could graduate by next year if everything went right. To Simone, Cecily, and Felicity, who teach me about myself with every little thing they do and remind me that science is not the highest pursuit in life. To Charles and Chris Ialovec, who have been the light of Christ in my life as I have stumbled through graduate school. Without their help and support I could not have completed graduate school. To John and Terry North, who set the stage for my education and encouraged my curiosity throughout life. To God, without whom all our efforts are for naught. My gifts and talents are solely for your glory. iv - ' 12 Too man} mo. ms doctoral st Haitian Lu. ar me. The man} Stei‘e Simmg decades. heir canbe ashel Mi COmmitr they have m Finally. x0: than I realiz Imomplete Acknowledgments Too many individuals provided support, guidance, training, or feedback in the process of my doctoral studies to list all valued contributors individually, but Robert Crawford, Haitian Lu, and Ale Manzan have time and again gone far beyond the call of duty to help me. The many discussions and debates will stay with me for years. Steve Simmons, to whom I owe scientific debts which I will be paying forward for decades, helped time and again in my efforts to generate lentiviral vectors. I only hope I can be as helpful and good natured to others as he has been to me. My committee members, Greg D. Fink, John J. LaPres, and Robert E. Roth have provided me with valuable feedback and guidance whenever I have asked, and for that they have my gratitude. Finally, Norbert E. Kaminski has taught me far more than I ever imagined, and likely than I realize, on this journey. Without his help and teaching my education would be incomplete, and no amount of thanks can do service to his service. LIST OF TA LIST OF FlC ABBREVlA CHXPIER l O\ er OVer C ont Histt Histt lntra Resp CHAPTER Anir Cher B ce Cell Flor Gen IgM Moi lmn P la: Plas Trai Luc Len Dir: Len Pre] C lo Star TABLE OF CONTENTS LIST OF TABLES ....................................................................................................... viii ' LIST OF FIGURES ...................................................................................................... ix ABBREVIATIONS ...................................................................................................... xi CHAPTER 1. INTRODUCTION ................................................................................. l Overarching Purpose ........................................................................................ 1 Overview of Immune System ........................................................................... 2 Control of Plasmacytic Differentiation ............................................................ 7 History of Dioxin and Dioxin-like Chemicals .................................................. 16 History of TCDD and Dioxin Immunotoxicity ................................................ 20 Intracellular Signaling involved in TCDD Disruption of the Primary IgM Response ........................................................................................................... 26 CHAPTER 2. MATERIALS AND METHODS .......................................................... 31 Animals ............................................................................................................. 31 Chemicals ......................................................................................................... 3 1 B cell Isolation .................................................................................................. 32 Cell Culture ...................................................................................................... 33 Flow Cytometry Analysis ................................................................................. 34 Gene Expression Analysis ................................................................................ 38 IgM ASC Response .......................................................................................... 38 Mouse and Human IgM Sandwich ELISA ....................................................... 39 Immunoblot Analysis of HsAHR ..................................................................... 40 Plasmid DNA isolation ..................................................................................... 41 Plasmid Vector Construction ............. - ............................................................... 41 Transfection and Selection of Stably Transfected Cells ................................... 44 Luciferase Assays ............................................................................................. 45 Lentivirus Preparation ...................................................................................... 45 Direct Assessment of Lentiviral RNA .............................................................. 45 Lentiviral Transduction and Selection of Transduced Cells ............................ 46 Preparation of Conditioned Complete RPMI-SKW ......................................... 47 Cloning by Limiting Dilution ........................................................................... 47 Statistical Analysis ........................................................................................... 48 vi CHAPTER MODELS. Sim TC I Sup TC I expi Sim TC I Tim CHAPTER 3: EXPERIMENTAL RESULTS AND DISCUSSION FOR MURINE MODELS ...................................................................................................................... 49 Simultaneous In vivo Time Course and Dose Response Evaluation for TCDD-Induced Impairment of the LPS-stimulated Primary IgM Response... 49 Suppression of the In vivo LPS-activated primary IgM response by TCDD... 49 TCDD-mediated suppression of LPS-induced changes in gene and protein expression involved in B cell to ASC transition .............................................. 61 Simultaneous In vitro Time Course and Dose Response Evaluation for TCDD-Induced Impairment of the LPS-stimulated Primary IgM Response... 74 Time and Concentration Response Profiling for TCDD Disruption of Transcription Factor Expression ....................................................................... 74 Time and Concentration Response Profiling for TCDD Disruption of Cell Surface Activation Marker Expression ............................................................ 82 Simultaneous Time Course and Concentration Response Evaluation for TCDD-Induced Impairment of the TLR-stimulated In vitro Primary IgM Response ........................................................................................................... 91 TCDD Suppression of TLR-induced Antibody Secretion in CH12.LX ........... 92 TCDD alteration of TLR-activated kinase phosphorylation ............................ 94 Discussion of Results from In vivo and In vitro Murine Models for TCDD Disruption of TLR—ligand Activated Plasmacytic Differentiation ................... l 10 CHAPTER 4: EXPERIMENTAL RESULTS AND DISCUSSION FOR HUMAN MODELS ...................................................................................................................... 120 Development of a Human in vitro model for TCDD Disruption of the LPS- activated Primary IgM Response ..................................................................... 120 Initial characterization of the SKW 6.4 cell line .............................................. 121 CHAPTER 5: CONCLUDING DISCUSSION ............................................................ 149 APPENDICES .............................................................................................................. 1 59 A. Antibodies .................................................................................................... 159 B. TaqMan Primers and Probes ........................................................................ 163 BIBLIOGRAPHY ........................................................................................................ 163 vii Table l. Dir Table 3. Dir LIST OF TABLES Table 1. Direct assessment of lentiviral RNA. ............................................................... 145 Table 2. Direct assessment of vector gene expression in antibiotic-resistant cell lines . 146 viii Figure 1. The Figure 3. B Cc igure 3. Sup Figure 4. Sup Figure 5. Sup expression..... Figure 6. TC I plasmacytic d Figure 7. Gen the absence :1 Figure 8. TC l elesated 1 cells FlEllie 9. Ger fate are intl ue Flglll‘e 10. In exPassion... Figure l 1. R. Figure 12. R.- 1:1ng l3.81 alteration of] Flgme 14. Si: TCDD allera‘ FlEUre 15, T( Cells Figme 17- Re Figure 13. Sir LIST OF FIGURES Figure l. The Plasmacytic Differentiation Control Circuit .......................................... Figure 2. B cell to plasma cell transition ...................................................................... Figure 3. Suppression by TCDD of the LPS-activated IgM ASC response ................. Figure 4. Suppression by TCDD of LPS-induced spleen cell proliferation ................. Figure 5. Suppression by TCDD of LPS-induced cell surface MHC Class II expression ..................................................................................................................... Figure 6. TCDD treatment impaired expression of phenotypic indicators for plasmacytic differentiation ........................................................................................... Figure 7. Gene expression profiles for LPS-induced plasmacytic differentiation in the absence and presence of TCDD treatment .............................................................. Figure 8. TCDD treatment suppressed LPS-stimulated generation of CD19+ Blimp-- elevated 1 cells ................................................................................................................ Figure 9. Gene expression profiles for transcription factors controlling plasma cell fate are influenced by LPS and TCDD treatments ....................................................... Figure 10. In vitro TCDD treatment disrupts LPS-activated transcription factors expression ..................................................................................................................... Figure 11. Relationship of Blimp-l to BCL-6 expression at 48 h ................................ Figure 12. Relationship of Blimp-1 to BCL-6 expression at 72 h ................................ Figure 13. Simultaneous time course and concentration response for TCDD alteration of LPS-stimulated cell surface activation marker expression ...................... Figure 14. Simultaneous time course and concentration response profiling for TCDD alteration of LPS-stimulated cell surface activation marker expression .......... Figure 15. TCDD disrupts LPS-activated CD80 and CD86 expression ....................... Figure 16. TCDD Suppression of TLR-activated IgM Responses in CH12.LX B cells ............................................................................................................................... Figure 17. Relative TLR ligand efficacy for INK phosphorylation ............................. Figure 18. Simultaneous time and concentration response profiling for TCDD alteration of TLR ligand-activated AKT phosphorylation ........................................... ix 13 51 53 56 59 64 69 72 78 80 81 84 87 89 93 96 98 I , vim,“ l-mw‘i Figure 19. Simu alteration of TH Figure 30. Simu Iteration of TL Figure Il. TCTI Figure 22. EITe Figure 23. LPS Figure 34. lmr Figure 35. Rel Figure 26. Al Figure 27. GE figure 38. G SKWAG l O l Tfigure 29. c Pummb'tin.. “we 30. . Figure 31 _ cell lines... Figure 33 Figure 19. Simultaneous time and concentration response profiling for TCDD alteration of TLR ligand-activated ERK phosphorylation ........................................... 100 Figure 20. Simultaneous time and concentration response profiling for TCDD alteration of TLR ligand-activated INK phosphorylation ............................................ 102 Figure 21. TCDD suppression of kinase phosphorylation in primary B cells .............. 107 Figure 22. Effects of R848 and TCDD on INK phosphorylation in primary B cells... 109 Figure 23. LPS stimulates IgM secretion in SKW 6.4 cells ......................................... 122 Figure 24. Iminunoblot analysis of AHR expression in SKW 6.4 cells ....................... 123 Figure 25. Relative Promoter Activities in SKW 6.4 cells ........................................... 127 Figure 26. AHR expression in G418-resistant pools of SKW 6.4 cells ....................... 129 Figure 27. GF P expression in transduced SKW 6.4 cells ............................................. 132 Figure 28. GFP expression in the presence and absence of puromycin. SKW 6.4 and SKWAGlO cells lines were evaluated for GFP fluorescence by flow cytometry ........ 135 Figure 29. GFP expression loss following 1 week of culture in the absence of puromycin ..................................................................................................................... 136 Figure 30. AHR immunoblot in 293T-HAG, HepG2, and SKW 6.4 cell lines ............ 138 Figure 31. Correlation of AHR immunofluoresence with GFP fluorescence in 293T cell lines ........................................................................................................................ 140 Figure 32. AHR expression correlates with nonviability ............................................. 141 Figure 33. Summary figure for LPS-activated changes in kinase and transcription factor activity in the presence and absence of TCDD .................................................. 155 293T 293T-HAG 393T-1L1\11R 3-.\lC M488 £647 AHR AHR-GFP APC ASC BCR BSA Ca} CITY CI? CYP1 A1 CpG DNA DLC DNP‘Fieoll DRE EXISA 293T 293T-HAG 293T-HAHR 3-MC Ax488 Ax647 AHR AHR-GP P APC ASC BCR BSA C a2+ CMV CYP CYP 1 A1 CpG DNA DLC DNP-Ficoll DRE EMSA ABBREVIATIONS human embryonic kidney 293T cell line human embryonic kidney 293T cell line expressing human AHR-GFP human embryonic kidney 293T cell line expressing human AHR 3-methylcholanthrene AlexaFluor 488 AlexaFluor 647 aryl hydrocarbon receptor aryl hydrocarbon receptor with C-terminal fused GFP antigen presenting cell or allophycocyanin antibody secreting cell B cell receptor bovine serum albumin calcium cytomegalovirus cytochrome P450 cytochrome P450 1A1 CpG oligonucleotide 1826 deoxyribonucleic acid dioxin like chemical dinitrophenol—Ficoll dioxin response element electromobility shift assay xi ERK FACS GFP LPS . ‘AC S 11th min mRVA ORF PART PERK PE PBS PCR PFC ERK FACS GFP HsAHR IgH IgJ ng 1g)» IgM INK LPS MACS MAPK min mRN A ORF pAKT pERK PE PBS PCR PFC extracellular signal-regulated kinase fluorescence activated cell sorter green fluorescent protein hours Homo sapiens aryl hydrocarbon receptor immunoglobulin u heavy chain immunoglobulin I chain immunoglobulin x light chain immunoglobulin I» light chain immunoglobulin M Jun N-terminal kinase lipopolysacchride Buffer for B cell isolation mitogen—activated protein kinase minutes messenger ribonucleic acid open reading frame phosphorylated AKT (Serine 473) phosphorylated Extracellular signal Regulated Kinase 1 and 2 (Threonine 202/Tyrosine 204) phycoerythrin Phosphate Buffered Saline polymerase chain reaction plaque forming cell xii FINK PRC PuroR QRT-PC R R848 RP.\11 SKR'A SKWAG 5140 TC DD TC R pIN K PKC PuroR QRT-PCR R848 RPMI SKWA SKWAG SV4O TCDD TCR phosphorylated Jun N-terminal Kinase 1 and 2 (Threonine 183/Tyrosine 185) Protein Kinase C puromycin resistance quantitative real-time reverse transcribed polymerase chain reaction Resiquimod Roswell Park Memorial Institute seconds SKW 6.4 AHR+ SKW 6.4 AHR-GFP+ simian virus 40 2,3,7,8-tetrachlorodibenzo-p-dioxin T cell receptor xiii i i E E.“ F E i. CHAPT Overa rcl Pro! for scientiti risk assessn' the fundame practices in 1 toxicities asso tissues affeete mechanistic ur potential healtl undertaken and Unmunotoxicit} CHAPTER 1. INTRODUCTION Overarching Purpose Protection of human and animal health is considered one of the highest purposes for scientific endeavor. Making regulatory policies designed to protect health depends on risk assessment to make qualitative and quantitative evaluation of hazards. Understanding the fundamental biology underlying chemical toxicity allows the possibility of best practices in risk assessment. The optimal result of mechanism-based decision making in risk assessment is health protection, without unintentional harm due to unanticipated factors. In the case of dioxin and dioxin like chemicals (DLCs), a basic understanding of key mechanisms exists for adaptive biological responses such as drug metabolizing enzyme induction. Unfortunately, a similar level of knowledge does not exist for toxicities associated with dioxin exposure in experimental animal models. Among many tissues affected by dioxins, the immune system is highly sensitive to dioxin and DLCs. A mechanistic understanding of dioxin immunotoxicity would be usefiil in estimating potential health risks posed by exposure to environmental dioxins and DLCs. The studies undertaken and described in this dissertation aim to advance knowledge of TCDD immunotoxicity, in an effort to fill data gaps in the understanding of molecular mechanisms underlying dioxin-associated health effects. Overview The innate ii for undesiral the dynamic complement recognize or efficiency or branches. ce humeral imr undesirable The flame deg-he ‘lindenman antigen Pres macrophag e aflllgens the; Either an eff. [Hungry Fir flaili’e amng TOUT] 0f anTllj Overview of Immune System The immune response is a highly complex and regulated process, protecting the organism through innate (soluble factors such as complement and defensins), cell- mediated (recognizing and killing foreign cell types), and humoral (antibody) immunity. The innate immune system is inherently anti-pathogenic, creating a hostile environment for undesirable agents. While important for host defense, innate immunity cannot adapt to the dynamic environment in which an organism lives. The innate immune system is complemented in vertebrate mammals by an adaptive immune system, which Teams to recognize undesirable agents in the host and attack them with increasing speed and efficiency on subsequent encounters. The adaptive immune system exists as two branches, cell-mediated immunity protecting the host from conditions such as cancer, and humoral immunity that generates antibodies to identify, neutralize, and destroy undesirable agents. The humoral, or antibody, response is specific for an agent termed the antigen, a name derived from the hypothesis that an agent causes generation of an antibody (Lindenmann 1984). Several cell types interact to produce a robust humoral response: antigen presenting cells (APCs), helper T cells (TH cells), and B cells. APCs, such as macrophages and dendritic cells, sample the surrounding microenvironment and present antigens they find to T cells. Antigens recognized as foreign activate the T cell to become either an effector T cell producing cytokines, or a memory T cell maintaining antigenic memory. Finally, the B cell is the antibody producing cell type. The B cell recognizes native antigen in the environment if its B cell receptor (BCR), a non-secreted cell surface form of antibody, binds antigen with moderate affinity. B cells internalize BCR-antigen complex foll cells as an A sun‘iial and balance bet“ host and spe in either spec Indivi stem cell pop 10 n6“ locatit immune sysrc first line senu marrow into t “ithout a guff the host, T cel ‘0 li‘mphoid 0 TWOgniZed b}. 1110\‘e3 0U! l 11 1C lhe lymphgid l The Cel and secrete larg anlibgd}. mOlec Chains (as K O r} The lighl and ht complex following antigen binding, process the antigen, and present the antigen to TH cells as an APC. In turn, stimulated TH cells secrete cytokines that increase B cell survival and proliferation. The coordinated interaction of several cell types maintains a balance between specificity of response, in which the immune system does not attack the host, and speed of response, in which an immune response is rapidly initiated. Imbalances in either specificity or speed of response lead to disease in the host. Individual cell types responsible for the antibody response arise from a common stem cell population in the bone marrow. Immune cells leave the bone marrow and move to new locations around the body as. they differentiate into the various cell types of the immune system. APCs are distributed throughout the periphery of the body, acting as first line sentries to monitor the cellular environment. T cells travel from the bone marrow into the thymus where they undergo a maturation process that removes T cells without a sufficiently capable T cell receptor (TCR) or a TCR that is strongly reactive to the host. T cells that complete the maturation process and egress from the thymus migrate to lymphoid organs where they remain resident, waiting for APCs presenting an antigen recognized by the TCR. The B cell builds a functional BCR within the bone marrow, then moves out into the periphery, and similarly to the T cell, most B cells become residents of the lymphoid tissues. The cellular function for which B cells are recognized is the ability to synthesize and secrete large amounts of antibody, sometimes called immunoglobulin.‘ Individual antibody molecules have a general structure consisting of a combination of two light chains (as K or A chains) and two heavy chains (as u, y], Y2a/2ba 73, a1, a2, a, or 8 chains). The light and heavy chains are further subdivided into variable regions and constant regi-‘OIIS The produced. “T m 5 produc biological ac initial humor predominant indit idual an cells differen biological fur ‘hesis. \i'ith microscopy c providing a p While still prr Their expressi infection. Bl mostly in the aSOCIEIICCI m(_ and have a 10. threshold {0” (II. 3002)- II is SISTER} That Ca regions. The constant region of the heavy chain designates the isotype of antibody produced, with u producing IgM, 7 producing IgG, at producing IgA, 8 producing IgE, and 6 producing IgD. Antigen binding occurs in the variable region, while many of the biological activities of antibodies depend on the constant region of the antibody. The initial humoral immune response to antigen, termed the primary response, consists predominantly of IgM. Secreted IgM is distinct from other antibody isotypes in that five individual antibodies are joined as a pentarner by the immunoglobulin J-chain (IgJ). B cells differentiate into a specialized cell type, the plasma cell, in order to accomplish the biological function of copious antibody secretion, a process that is discussed later in this thesis. With the advent of fluorescence activated cell sorting (FACS) and fluorescent microscopy distinctions among types of B cells have arisen in scientific literature, providing a partial explanation for how the antibody response can be activated rapidly, while still providing specificity of response. Some B cells, termed B1 cells on the basis of their expression of Ly-l (CD5), appear to play an important role in control of bacterial infection. B1 cells have a restricted repertoire of BCR (Bendelac er al. 2001), are resident mostly in the peritoneum and pleural cavities (Hayakawa et al. 1985), bind pathogen- associated molecules such as lipopolysacchride (LPS) (Kantor and Herzenberg 1993), and have a lower stimulus requirement for activation (Fagarasan et al. 2000). The marginal zone (MZ) B cells are very similar to the B1 cells, particularly in their lower threshold for activation (Lopes-Carvalho et al. 2005), but reside in the spleen (Balazs et al. 2002). It is thought that B1 and M2 cells provide a layer to’the humoral immune system that can react more rapidly to bacterial infection, limiting the initial rate of pathogen EXP specificiti' an Pfimary hum! l‘fflffgfi e! of follicular B C- e‘mi. lQS—‘il- I presented by . BCR outcom} an actuated. i producing hi g memory. The in antibody-3e cell to reach tl examined prio. The act stimulus: T-de “limits is New Which induces . dePendent scim produce antiboc not be discussed pathogen expansion and allowing time for other B cell subsets producing higher specificity antibodies to differentiate. In LPS-activated experimental models the observed primary humoral IgM response is likely to reflect a high proportion of BI and M2 (Fairfax et al. 2007; Martin et al. 2001). The third widely recognized B cell subset is the follicular B cell, named for its histologic localization in the “follicle” of the spleen (Hsu et al. 1983). B cell selection occurs via B cell competition for binding of native antigen presented by dendritic cells at the center of the follicle. B cells expressing high affinity BCR outcompete B cells expressing lower affinity BCR for survival signals provided by i an activated, antigen-bound BCR. Through this competitive process only those B cells producing high affinity, high specificity antibody survive to provide robust immunologic memory. The time required for selective competition within the germinal center to result in antibody-secreting cells is typically greater than the time required for a BI or MZ B cell to reach the antibody-secreting stage; thus, for antibody-secreting cell responses examined prior to five days much of the response is attributed to B1 and M2 B cells. The activation of B cells has been studied in the context of 2 different types of stimulus: T-dependent stimuli such as sheep erythrocytes (sRBC), in which an intact thymus is required for a humoral response, and T—independent stimuli such as LPS, which induces a humoral response in the absence of an intact thymus (Wortis 1971). T- dependent stimuli require the coordinated interaction of the APC, TH cell, and B cell to produce antibody, and have not been utilized for studies in this dissertation and thus will not be discussed further. T-independent stimuli, such as LPS, CpG oligonucleotides (CpG), and resiquimod (R848), activate B lymphocytes via Toll—like Receptors (TLR), evolutionarily conserved pattern recognition receptors facilitating a rapid, robust response to bacterial and viral pathogens (Rehli 2002). The B cell is commonly referred to as na'r've following egress from the bone marrow. Antigen encounter subsequently activates the B cell, and like many receptor- mediated processes an activation threshold exists for the B cell (DeFranco et al. 1985). TLR activation leads to activation of key cell signaling agents such as AKT and Nuclear Factor K B (NFKB), many of which are convergent on the mitogen-activated kinases (MAPK) (Campbell 1999; Harriett et al. 2005). Intracellular signals are integrated on an individual cell basis, resulting in differentiation into either a memory B cell or antibody- secreting plasma cell (ASC). Control of The s: engagement c and intracell u transcription 1 Jun N-temiin. ILKPK. INK l transcription f; ERK also play circuit by com ubiquitinated a. releasing a brali contradiction to differentiation. ( plasmacytic diff: controlling plasn ei'Pt‘cted to alter Control of Plasmacytic Differentiation The signaling cascade feeding into plasmacytic differentiation begins with engagement of receptors such as the BCR or TLRs, causing activation of protein kinases and intracellular calcium (Ca2+) increases, both of which feed into MAPK cascades and transcription factor networks. B cell signaling converges on several kinases, including Jun N-terminal Kinase (INK), extracellular signal-regulated kinase (ERK), and p38 MAPK. IN K phosphorylates c-Iun, one component of the Activation Protein 1 (AP-1) transcription factor, increasing the transcriptional activity of AP-l (Smeal er al. 1991). ERK also plays an important role in regulation of the plasmacytic differentiation control circuit by controlling BCL-6 abundance. Following phosphorylation by ERK, BCL-6 is ubiquitinated and targeted for proteasomal degradation (N iu et al. 1998), in effect releasing a brake controlling expression of Blimp-1. Interestingly, and in seeming direct contradiction to the role of ERK-induced degradation of BCL-6 controlling plasmacytic differentiation, constitutively active MEK, thought to activate ERK, inhibits LPS-induced plasmacytic differentiation (Rui et al. 2006). Given the apparent dual nature of ERK in controlling plasmacytic differentiation, treatments affecting ERK activation can be expected to alter frequency of plasmacytic differentiation. tutsttc \ < UwEOED «‘83?on 9593: 26.335 2 T 3.8 notfiamox “3,33% 88th S T we REESE use wAEEQQSE ASBEme $33.5 3.580 robetnmxmsSQ 33353: 35 .N Emmi .19.. > / 2n. . 225 £21 2 Fax 28 wmcoammm . 22.3 :2 c 65.5 nwEouED Pi Piasmacy alteration activation. gene expre differentiati induced mat 1 lXBP-l ) a regulatory in depicted in F 6. Blimp— 1. ar 3004). so call: In this case B 1 and P216 pm“ QUiescenL bur \ initiator of p133 ."\P~] IhTOUgh a family mem bers AP-l blIIds to re ,ohpubg e! a” y of BCL-6 and Pa; Blimul expreSSiC expression profile Profound remodeling of the B cell occurs for vigorous antibody secretion. Plasmacytic differentiation requires coordinated changes in gene expression preceding alteration in cellular phenotype. On the intracellular level this is accomplished through activation, repression, and induction of transcription factors responsible for control of the gene expression program in B cells. Research into the cellular mechanism of plasmacytic differentiation establishes AP-l , B-cell CLL/lymphoma 6 (BCL-6), B lymphocyte induced maturation protein 1 (Blimp-1), Paired Box 5 (Pax5), and X-box Binding Protein 1 (XBP-l) as key transcription factors regulating B cell fate (Igarashi et al. 2007). The regulatory interactions controlling the “plasmacytic differentiation control circuit” are depicted in Figure l. The central axis of cell fate control is the interaction between BCL- 6, Blimp-1, and Pax5. Such an interaction results in a “bistable switch” (Markevich et al. 2004), so called because the switch exist in one of two stable, mutually exclusive states. In this case Blimp-l expression is stably on or stably off. In resting mature B cells BCL-6 and Pax5 prevent expression of Blimp-1, maintaining the gene expression program of a quiescent, but vigilant, B cell. Additionally, BCL-6 directly interacts with a keystone initiator of plasmacytic differentiation, AP-l, by reducing the transcriptional activity of AP-l through a specific interaction with c-Iun (V asanwala et al. 2002), one of the Inn family members that commonly participates in the AP-l complex (Karin et al. 1997). AP-l binds to response elements within in the Blimp-l promoter to drive gene expression (Ohkubo et al. 2005), and once translated into protein, Blimp-1 silences gene expression of BCL-6 and Pax5 (Shaffer et al. 2002), creating a positive feedback loop in which Blimp-1 expression increases and allows for wholesale alteration of the cellular gene expression profile. In vivo profiling of the prdmI locus, from which Blimp-I is 10 uanSCiibed' d phfinODT‘e‘ 35 3004). 1335 l on the basis 0 the B Céll line- 2007b).14355‘ Wes (Cobalet Patti expressit PatS expressic 3006). C umula ParS expressio of interest are t. IgJ. Conversely {Delogu er al. 2 Beyond XBP~lexpressi the unfolded prc endnmesugges [itinSduction thrc Recent evidence: Simpli'theresult Course OfBCR si transcribed, demonstrates Blimp-1 expression is quantitatively related to plasma cell phenotype, as cells with the highest prdmI locus activation are plasma cells (Kallies et al. 2004) Pax5 is required for generation of mature B cells (Maitra and Atchison 2000), and on the basis of functions promoting B cell maturation and repressing genes unsuitable for the B cell lineage named the “guardian of B cell identity and function” (Cobaleda et al. 2007b). Loss of Pax5 expression allows B cell dedifferentiation into either progenitor cell types (Cobaleda et al. 2007a) and T cells (Mikkola et al. 2002). Furthermore, loss of Pax5 expression promotes transition of B cells into an ASC phenotype, and restoration of Pax5 expression in Pax5"' B cells reduces expression of Blimp-l and XBP-l (Nera et al. 2006). Cumulative evidence suggests B cells maintain their cellular character because of Pax5 expression, with loss of Pax5 allowing B cells to change phenotype. Several genes of interest are targeted by Pax5 for repression, including CD138, Blimp-1, IgH, Igrc, and IgJ. Conversely, Pax5 positively regulates genes involved in forming a functional BCR (Delogu et al. 2006). Beyond regulation of Ig genes (Calame et al. 2003), Pax5 also directly regulates XBP-l expression (Reimold et al. 1996). XBP-l is best known for its role in resolution of the unfolded protein response observed in plasma cells (Yoshida et al. 2001). Emerging evidence suggests XBP-l plays additional roles in B cell biology, aiding in signal transduction through the BCR and potentially indirectly regulating Blimp-l expression. Recent evidences suggests XBP-l induction is a differentiation-dependent event, not simply the result of an unfolded protein response, and plays a role in controlling the time course of BCR signaling (Hu et al. 2009). Both the role of XBP-l in resolving the 11 unfolded We“ potential imp“n importance of .\ .‘t'BP-l expressn doomegulation nith a decrease liShen and lien and is required. indirect regulatit Different in phenotypicall; cells. Protein ant did‘erentiation cc FOE-trams to seer decrease as B 1 i m unfolded protein response and the newly identified role in BCR signaling accentuate the potential importance of XBP-l in B cell function. Further accentuating the potential importance of XBP-l in regulating plasmacytic differentiation, RNA interference in XBP-l expression reduced IgH gene expression in plasmacytoma cell lines. The downregulation of IgH expression resulting from XBP-l interference was concomitant with a decrease in the expression of the transcription factor Octamer Binding Factor 1(Shen and Hendershot 2007). Because Octamer Binding Factor 1 upregulation precedes, and is required, for prdm] locus activation (Corcoran et al. 2005) the potential for indirect regulation of Blimp-l expression by XBP-l is apparent. Differentiation from the activated B cell to antibody-secreting plasma cell occurs in phenotypically identified stages, and appears in Figure 2 beginning with resting B cells. Protein and mRNA abundance for transcription factors of the plasmacytic differentiation control circuit changes overtime as B cells remodel their transcriptional programs to secrete antibody. As conceptualized in Figure 2, levels of Pax5 function decrease as Blimp-1 expression increases. 12 at. ,z ,. . . 4i 2.... . . . J. ,0- Um. . m «51$»- 9th . .. . - . b. q C «v v :Lb Q >LO..Cn:\< «w. -cOglnwvnE \ ; :mcuim ADUZ— ch: Amuw>: {0:3 mZUU arr—n67”. mamfl—flflEmMF— 3.0.0 0 Dmum>zv< :00 Q Naipaul .993. .3 B :56 \QSSSEEN mrxmwsmk 9582 «as Emitaam 232.553: EEKSREEEQ 3 ESSKQVN .=o.:.%=e.: :8 353m. 8 :8 m .N 3sz liufiudilli RI .1..- $11.5qu l3 “.523... i -i- . Eozocstmxé llirlli lIIII Ill! 1 11 i w Beach zooé . / FOR. 50. -.Ua \ fivfifl ..wum 532:0“. c0389 =ou-m i . :..r... . ..i .m A300: JUmVV as de uol :8 m meagex :82. ”so: :82. :91 ...—.3 «Ema—n. mama—amEmmE ...—.3 m $3933, :3 m 9:33. 14 In sum intoclassie M. MAPK signali an increase in maintaining it: Increased syml cell. In summary, plasmacytic differentiation begins with receptor activation feeding into classic MAPK signaling cascades. In B cells destined for the ASC phenotype, MAPK signaling converges on the plasmacytic differentiation control circuit, resulting in an increase in Blimp-l expression. Blimp-l upregulation represses BCL-6 and Pax5, thus maintaining its own expression, and initiating changes that increase Ig synthesis. Increased synthesis of Ig allows for increasing secretion of Ig, the hallmark of the plasma cell. 15 _ an." . .' ‘~|-v gh-‘P‘I‘M‘ . 'Itma ..~.~:.;_'r_—~-L-“‘ A! Histofl' of Deca' with EXPOSUY such 35 pol.“ dil‘é’nZOdiOXi arrears in IQ Ofchemical i' remixed rCP‘ (Bowen and 3‘ from tetrachlt' 1973). Ofall I dioxin (TCDE nith dioxin an Attenti uidely publici; and for Operat. garnered large public eye. DU dietary exposur food web (Kel li consistent and Ii Industry worker Panicular. hare l History of Dioxin and Dioxin-like Chemicals Decades of research have been dedicated to understanding toxicities associated with exposure to halogenated aromatic hydrocarbons, particularly for dioxin and DLCs such as polychlorinated biphenyls, polychlorinated dibenzodifurans, and polychlorinated dibenzodioxins. The first published reports of intentional synthesis for dibenzodioxins appears in 1957 (Gilman and Dietrich 1957), but unintentional production as a byproduct of chemical industry activity occurred as early as 1848 (Weber et al. 2008). Peer- reviewed reports of health effects attributable to DLCs began to surface in the 1950's (Bowen and Moursund 1957; Meigs et al. 1954), with evidence for adverse health effects from tetrachlorinated dibenzodioxins appearing in the 1970’s (Jensen et al. 1972; May 1973). Of all the chemical structures classified as DLCs, 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) is the most studied and potent congener for inducing toxicities associated with dioxin and DLC exposure. Attention focused on health risks posed by dioxin and DLCs increased following widely publicized exposure events, including Times Beach in Missouri, Seveso in Italy, and for Operation Ranchhand participants (Weber et al. 2008). While all the above events garnered large amounts of media attention at the time, and continue to remain in the public eye, DLCs in the environment are encountered by almost all humans through dietary exposure. The highly lipophillic nature of DLCs causes bioaccumulation in the food web (Kelly et al. 2007), such that top level consumers such as humans have consistent and life-long exposure to DLC. Additionally, some individuals, chemical industry workers (Patterson et al. 1989) and incinerator workers (Kumagai et al. 2000) in particular, have higher body burdens of DLCs owing to occupational exposure. 16 \VideSI literature- but 1 models 6513in hepammegal.‘ carcimgwe“iS syndrome ( 59¢ doses of TCDl tissue and Sp?C TC DD. sho“ in 1933). [Mike I story of the dio: profiling progre the fore in the 1 major determine ltt'drocarbon. or Cnochrome P45 Ofmice. such as Comersel}; nom reSponse to 3-.\1( CYP activity that HomesDonsi t'e Str; l -0 TCDD benVCCn Widespread exposure to DLCs, including TCDD, is well established in the literature, but human health effects beyond chloracne are tenuous. Experimental animal models establish TCDD as an agent with pleitropic biologic effects, including hepatomegaly (Alsharif and Hassoun 2004), endocrine disruption (Safe 1995), carcinogenesis (Huff et al. 1991), teratogenesis (Abbott and Bimbaum 1989), wasting syndrome (Seefeld et al. 1984), and immunological defects (Luebke et al. 2006). The doses of TCDD required for eliciting biological effects listed above range from tissue to tissue and species to species, but the immune response is highly sensitive to disruption by TCDD, showing functional perturbations at concentrations as low as 4 ng/kg (Clark et al. 1983) Unlike many contemporary nuclear receptors first cloned then characterized, the story of the dioxin receptor discovery is a fascinating example of classical biochemical profiling progressing to identification of the receptor. As the effects of TCDD came to the fore in the 1970’s and 1980’s, mouse breeding studies identified a genetic locus as a major determinant in sensitivity to biOlogical effects associated with dioxin. The Aryl hydrocarbon, or Ah, locus conferred phenotypic differences for effects of DLCs on cytochrome P450 (CYP) induction by 3-methylcholanthrene (3-MC). Responsive strains of mice, such as the C57BL/6, induce CYP enzyme expression in response to 3-MC. Conversely, nonresponsive strains, such as the DBA/2, do not induce CYP enzymes in response to 3-MC (Nebert et al. 1972). TCDD is a 30,000-fold more potent inducer of CYP activity than 3-MC, and was shown to induce CYP activity in both responsive and nonresponsive strains of mice, albeit with approximately 10-fold differences in sensitivity to TCDD between the strains (Poland and Glover 1974). Further examination identified 17 t myflhmmu— lie ' gandactn‘ation ‘- lransloeator (AR ‘ that TC DD se differential set DICs (Poland \Vith tl esponses to E cellular com pr no. ammo t" protein for TC 1976;. Bestom methodology ft Poland 1988,). a apanial N-term With a p oligonucleotide characterization encoding AI IR n the CS7BL "6 m0! helix domain cont tr _ . . anscnption facto h eat shock protein that TCDD sensitivity was inherited as an autosomal dominant trait, and conferred differential sensitivity to both the biochemical and toxicologic responses to TCDD and DLCs (Poland et al. 1979). With the Ah locus implicated as being responsible for the biochemical and toxic responses to DLCs, many laboratories began using radiolabelled TCDD as a probe for the cellular component encoded by the Ah locus. Poland et al showed that TCDD binds with high affinity to a cytosolic protein, and quantified the Bmax, Kd, and specificity of the protein for TCDD, characteristics implicating the protein as a receptor (Poland et al. 1976). Bestowed the name Ah receptor (AHR), Perdew and Poland developed a methodology for enriching liver homogenates for receptor purification (Perdew and Poland 1988), a technique that allowed Bradfield et al to purify enough AHR to perform a partial N-terminal sequencing (Bradfield et al. 1991 ). With a partial protein sequence in hand, Burbach et al used degenerate oligonucleotide probes to interrogate cDNA libraries. Culminating years of biochemical characterization for AHR and its interaction with TCDD, the identification of cDNA encoding AHR resulted in the cloning and sequencing of the mouse AHRb'I allele from the C57BL/6 mouse. DNA sequence analysis identified the AHR as a basic helix-loop- helix domain containing protein and member of the Per-ARNT—Sim family of transcription factors (Burbach et al. 1992). AHR resides in the cytosol complexed with heat shock protein 90, p23, and aryl hydrocarbon receptor associated protein 9. Upon ligand activation AHR translocates to the nucleus and dimerizes with the AHR Nuclear Translocator (ARNT), forming a heterodimeric transcription factor capable of binding a 18 dioxin response element (DRE), 5’-TNGCGTG-3’, and altering gene expression (Denison and Nagy 2003). 19 History Of The t me 1970'5- E gluococortier by chemicals and humoral Vecchi and C' immunity up The pi b} DLCs oper longitudinally. a requirement . to TC DD. char. and hare since the B cell. During ti determinant in b factor in TCDD-i humOI'al respongc l ‘ . 01”mtunotoxrcitx l-» ' . Cone med DOIC‘DC'». Hillier el (11 I99:- History of TCDD and Dioxin Immunotoxicity The potential for immune response suppression by DLCs was recognized early in the 1970’s. Because TCDD causes thymic atrophy, a result associated with gluococorticoid suppression of immunity, awareness of potential for immunosuppression by chemicals came to exist. TCDD was shown to impair function of both cell-mediated and humoral responses in rodents (Vos et al. 1973; Vos et al. 1974). Later work by Vecchi and colleagues found that a single dose of TCDD caused suppression of humoral immunity up to 42 days following treatment (V ecchi et al. 1980a; Vecchi et al. 1980b). The pioneering discoveries of Joseph Vos in the area immune response disruption by DLCs opened a path for many investigators to expand on his initial findings. Viewed longitudinally, research on TCDD-mediated disruption of humoral immunity established a requirement for AHR, identified the humoral immune response cell types most sensitive to TCDD, characterized the kinetics of TCDD-mediated humoral immunity disruption, and have since delved into the molecular mechanism of TCDD immunotoxicity within the B cell. During the early 1980’s the Ah locus received increasing attention as a major determinant in biological responses to TCDD, and with time proved to be an essential factor in TCDD-mediated disruption of humoral immunity. Initial comparisons of humoral response disruption by TCDD in Ahbb and A hdd mouse stains showed sensitivity to immunotoxicity segregated with the Ah locus (Vecchi et al. 1983). Structure-activity relationship comparisons for AHR ligand-mediated effects on the primary IgM response correlated potency for AHR activation with immune suppression (Harper et al. 1995a; Harper et al. 1995b; Tucker et al. 1986). Conclusive proof for AHR involvement in 20 TCDD disruption of the primary IgM response comes from multiple lines of evidence. Sulentic et al found that an AHR expressing B cell line, CH12.LX, is highly sensitive to TCDD, showing antibody response impairment at concentrations of TCDD as low as 30 pM. The antibody response of a second cell line that does not express AHR, BCL-l, is unaffected by TCDD even at 3 nM concentrations (Sulentic et al. 1998). Using AHR knockout mice Vorderstrasse et al proved the in vivo primary IgM response to sRBC unperturbed by 10 rig/kg TCDD treatment, whereas the primary IgM response decreased 80% in TCDD-treated AHR sufficient mice (Vorderstrasse et al. 2001). As the role of AHR in TCDD-mediated immunotoxicity was established, questions remained regarding which cell types of the humoral immune response were sensitive to TCDD. To determine the sensitive cell type in the primary IgM response comparisons of in vitro cultures stimulated using sRBC, DNP-Ficoll, or LPS are useful. While the primary IgM response to sRBC requires the participation of T cells, B cells, and antigen presenting cells, the responses to DNP-Ficoll is T-independent, requiring only APCs and B cells, and the response to LPS requires only the B cell. By comparing the ability of TCDD to suppress responses to each stimulus it is possible to exclude T cells or APCs from a role in IgM response suppression. The first evidence for sensitivity of B cells to TCDD came in the form of ,parallel, concentration-dependent TCDD suppression of primary IgM responses to sRBC, DNP-Ficoll, and LPS (Holsapple et al. 1986), thus excluding T cells and APCs. Additionally, cultures of purified mouse B cells are directly sensitive to disruption of LPS-stimulated IgM secretion (Morris et al. 1993). In effort to conclusively establish the relative roles for different immune cell types in primary IgM response disruption by TCDD, Dooley et al used separation/reconstitution 21 studies examining contributions of different cell types to the primary IgM response elicited by sRBC or DNP-Ficoll. Mice were treated in vivo with TCDD, then individual cell types (T cells, B cells, and APCs) were separated and used to reconstitute in vitro cultures. After excluding TCDD effects on APCs in preliminary results, focus turned to cultures containing all three cell types with either B cells and APCs or T cells and APCs supplied by TCDD treated mice. Only cultures containing B cells from TCDD treated mice showed the profound suppression of the primary IgM response normally associated with TCDD treatment (Dooley and Holsapple 1988). Later follow-up experiments further confirmed that T cells from TCDD exposed mice do not impair the in vitro IgM response (Dooley et al. 1990). Due to the nature of the immune system and primary humoral immune response it is necessary to stimulate a response before any disruption of that response can be observed. Many events occur within the cell following stimulation, some within seconds while others will not occur for days. It is possible for a “window of sensitivity” to exist in such a scenario, during which an event may be disrupted, in this case by TCDD. Should the disrupting agent exposure occur outside the window of sensitivity, alterations in the immune response do not occur. While in vivo exposure to TCDD prior to immune response activation causes long lasting perturbations in the immune system (Holladay et al. 1991), in vitro studies show that TCDD significantly inhibits the primary IgM response only if added within 24 hours of sRBC addition (Tucker et al. 1986) or 3 hours for LPS addition (Holsapple et al. 1986). In addition, TCDD causes relatively minor alterations in circulating Ig compared to the profound impairment of the induced IgM , response (Fan et al. 1996), suggesting that in vivo TCDD does not directly inhibit the 22 activity of plasma cells, but rather the ability of an activated B cell to differentiate into a plasma cell. Considering the relatively short window of sensitivity, a single day or less following activation, it is likely that TCDD interferes in some early event in activation- induced B cell differentiation. Interestingly, a window of sensitivity for TCDD disruption of differentiation processes also exists for adipocytes, as demonstrated in the ability of TCDD to inhibit hormone induced preadipocyte differentiation into adipocytes (Hanlon et al. 2003). The window of sensitivity for TCDD disruption of the IgM response brought research focus to early post-activation events in B cells. TCDD affects many aspects of B cell activation, including protein phosphorylation and intracellular Ca2+. In purified unactivated B cells, TCDD treatment increased basal kinase activity, but did not affect Protein Kinase C (PKC) activity. The combined treatment of phorbol-l2-myristate-1 3- acetate (PMA) and TCDD caused greater protein phosphorylation than single treatments alone, which was interpreted as resulting from activation of multiple kinases (Kramer et al. 1987). Examining both global protein phosphorylation and tyrosine phosphorylation in B cells following TCDD, in vitro cultures revealed concentration-related increases in protein phosphorylation within minutes of treatment (Clark et al. 1991; Snyder et al. 1993). B cells treated with the combination of PMA and the Ca2+ ionophore ionomycin (PMA/Io) are an experimental model of BCR activation. In examining the potential for TCDD disruption of BCR signaling, Karras et al showed PMA/Io-induced proliferation decreased as much as 50% in TCDD treated B cells. The authors attributed to the decreased proliferation caused by TCDD to alteration in intracellular Ca2+ levels 23 downstream of phospholipase C activation due to the dependence on ionomycin, not PMA, concentration (Karras and Holsapple 1994). Followup studies indicated TCDD . . . . . 2+ . . . treatment interfered w1th stability of intracellular Ca concentrations in resting B cells (Karras et al. 1996). Taken together, evidence suggests TCDD affects some early event in B cell signaling, but direct evidence of TCDD effects on any single factor to which all the above effects can be attributed remains elusive. In addition to evidence for TCDD disruption of an early event in signaling, there is also support for intracellular signaling preventing TCDD disruption of the IgM response. Unlike in viva responses or in vitro stimulated responses from in vivo treated mice, inconsistent suppression of in vitro stimulated primary IgM response hampered . initial efforts to model in vivo effects until it was recognized that different serum lots supported in vitro TCDD suppression of the IgM response (Morris and Holsapple 1991). Serum was also demonstrated to modulate TCDD-induced CYP enzyme induction in both splenocytes and hepatocytes (Morris et al. 1994), suggestive of the possibility that serum factors can alter TCDD-induced AHR signaling. Human Interferon-y (IFNy) causes a concentration-related reversal for TCDD suppression of both LPS and sRBC AFC response, as well as TCDD-induced protein phosphorylation, for cultured mouse splenocytes. In a return to the window of sensitivity paradigm, only addition IFNy to culture within 18 h of activation could reverse TCDD suppression of the IgM response(Snyder et al. 1993). IFNy also prevented TCDD-induced CYP expression in cultured primary hepatocytes, an effect attributed to a soluble, heat-labile, protease- sensitive factor that was not IFNy (Jeong et al. 1993). Taking into account the. window of sensitivity and cell signaling alteration resulting from TCDD treatment, as well as results 24 demonstrating response. ex'id from alteratior cell to plasma In sum response is an 1 ~ signaling occur precede. or occ differentiation r demonstrating that a protein-mediated signal can prevent TCDD suppression of the IgM response, evidence indicates the disruption of the primary IgM response by TCDD results from alterations at, or preceding, the level of cell signals that converge on control of B cell to plasma cell differentiation. In summary, previous literature indicates TCDD disruption of the primary IgM response is an AHR-dependent phenomenon, likely the result of TCDD alteration in cell signaling occurring within 24 h of B cell activation. The kinetics of this alteration likely precede, or occur concurrently with, cell signaling inputs feeding into the plasmacytic differentiation control circuit. 25 Intracellular Signaling involved in TCDD Disruption of the Primary IgM Response As the techniques underpinning scientific investigation advanced so did efforts to understand how TCDD disrupts B cell differentiation at the intracellular level. Clues as to how TCDD impairs the antibody response can be found in the quantity of AHR in immune cells, direct regulation of Ig genes by AHR, alteration in posttranslational modification of regulatory proteins, and activity of transcription factors important in control of plasmacytic differentiation. The ED50 for TCDD inhibition of the sRBC-induced IgM response is 0.7 pig/kg (Smialowicz et al. 1994) and 7 nM for the TNP-LPS induced in vitro IgM response (Harper et al. 1995b) in the B6C3F1 mouse. One likely reason for the sensitivity of immune cells to TCDD is cell activation causes AHR and ARNT levels increase 2 to 6 fold (Crawford et al. 1997; Williams et al. 1996). In ligand-receptor interactions one primary determinant dictating tissue response to a ligand in the amount of receptor available for binding (Clark 1933), therefore, the increase in AHR associated with leukocyte activation should increase tissue responsiveness to TCDD treatment. Indeed, even in the absence of exogenous ligand addition, activation of leukocytes causes AHR translocation into the nucleus, DNA binding, and transcription of CYP1A1 , all classically associated with TCDD-induced AHR activation (Crawford et al. 1997). Development of a cell line-based model for TCDD disruption of the IgM response allowed assessment of TCDD effects in ways that were difficult or impossible to perform using primary cells. The CH12.LX B cell line is a subclone of the CH12 lymphoma that responds to sRBC or LPS treatment by differentiating into an ASC (Bishop and 26 Haughton 1986). CH12.LX cell line, due to its TCDD- and LPS-responsive phenotype, is a highly suitable in vitro model for elucidating mechanisms of TCDD-mediated disruption of the IgM response. Because both TCDD suppression of IgM secretion and AHR dependence was well established in vivo and in vitro for both primary cells and the CH12.LX cell line, one approach to elucidating mechanism is to work upstream from a known event, such as TCDD suppression of immunoglobulin p heavy chain (IgH) expression. Activity of the 3’ or enhancer of the IgH gene plays an important role in antibody production (Arulampalam et al. 1994; Dariavach et al. 1991; Grant et al. 1995; Pettersson et al. 1997; Pettersson et al. 1990). Two DRE sites within the 3’ or enhancer were identified and shown to bind AHR (Sulentic et al. 2000), both of which were later confirmed to alter transcriptional activity of the 3’ a enhancer (Sulentic et al. 2004a) and directly bind AHR in intact cells (Sulentic et al. 2004b). Together, the observations of Sulentic et al were the first published evidence for a direct regulation of Ig secretion by AHR. During the intervening time between identification of DREs within the 3’ a enhancer and evidence for direct modulation of transcriptional activity for the 3’ a enhancer by AHR, additional observations within CH12.LX cells further established potential effects of TCDD on signaling important for plasmacytic differentiation. Two key transcriptional regulators known to enhance the activity of the 3’ a enhancer are AP- ] (Matthias and Baltimore 1993) and NFKB (Kanda et a1. 2000). There are marked increases in both DNAbinding and transcriptional activity for AP-l and NFKB following LPS activation of CH12.LX cells, with TCDD treatment causing significant impairment of AP—l activity, but not NFKB. TCDD inhibits LPS-activated AP-l DNA binding in 27 ..i: L,- . ‘ .9 'k"“’i ‘ «nap. :cr--_—‘.—__ It‘d—w I." CH12.LX cells. bu binding. EMSA su present in the AP— correlated with im Since a direct. inh 3003). and the API contain Fos. a part transcriptional act treatment. Earlier 51L idem-13mg protei Will) Increasing C thVClOpmem of: lobe an imPOrta facilitates B Cell Additionally. lll inhibition incre CH12.LX cells. mOdification, \ filnher confirr TCDD ”Baum CH12.LX cells, but not BCL-l cells, implying that AHR is required to decrease AP-l binding. EMSA supershifts were able to establish Jun, but not Fos, as one of the proteins present in the AP-l complex induced by LPS in CH12.LX cells, an outcome that directly correlated with immunoblot results assessing nuclear c-Jun quantity (Suh et al. 2002b). Since a direct, inhibitory interaction between BCL-6 and c-Jun exists (V asanwala et al. 2002), and the AP-l complex induced by LPS treatment of CH12.LX cells does not contain Fos, a partial explanation for TCDD disruption of AP-l DNA binding and transcriptional activity could be maintenance of BCL-6 activity resulting from TCDD treatment. Earlier studies illustrating TCDD-induced protein phosphorylation, without identifying proteins specifically, demonstrated a limitation that became surmountable with increasing commercial availability of antibodies, quantitative PCR techniques, and development of the CH12.LX cell line model. In example, regulation of p27kip1, known to be an important regulator of differentiation in the several cell types (Lloyd et al. 1999), facilitates B cell cycle arrest to allow for differentiation (Schrantz et al. 2000). hp], BCL-6 Additionally, linking the plasmacytic differentiation control circuit with p27 inhibition increases expression of p27klpl (Shaffer et al. 2000). LPS activation of CH12.LX cells induced increases in p27klpl expression and posttranslational modification, while TCDD treatment prevented an LPS-induced alteration of p27klp]. In . . . . . . k" 1 further confirmation for a wmdow of sensrtrvrty, greatest modulation of p27 rp by TCDD treatment occurred within 24 h of activation. Given the known importance of 28 BCL—6 in regulation of plasmacytic differentiation, documented TCDD effects on the k' l . . . . . . . . common downstream target p27 1p , and the corncrdtng wmdow of sensrttvrty for regulation by TCDD, the disruption of a signal event upstream of the plasmacytic differentiation control circuit depicted in Figure 1 seems likely. With an effect on the IgH locus established in CH12.LX, efforts were applied to determining if regulatory steps preceding IgH expression were similarly disrupted. Pax5 directly controls expression of several components of IgM, including IgH (Singh and Birshtein 1993). Concentration-response profiling of TCDD effects on Pax5 expression in LPS-stimulated CH12.LX showed that TCDD prevents downregulation of Pax5 mRNA, protein, and DNA binding (Yoo et al. 2004). Further extending the observation of TCDD effects on Pax5, Schneider et al showed TCDD caused concentration-related increases in Pax5 gene expression and prevented LPS-induced downregulation of Pax5 protein in both splenocytes and CH12.LX cells (Schneider et al. 2008). In a more detailed follow up examination of both Pax5 regulation and Blimp-1 expression in splenocytes and CH12.LX, the DNA binding activity of Blimp-1 to response elements within the Pax5 promoter was markedly reduced by TCDD treatment, and correlated with TCDD suppression of LPS-induced Blimp-1 mRNA expression. Confirming previous in vitro observations of reduced AP-l transcriptional activity, TCDD caused a concentration- dependent reduction in LPS-stimulated DNA binding of specific AP-l response elements identified in the promoter of Blimp-1 (Schneider et al. 2009). ‘ Cumulatively, the knowledge of intracellular events leading to suppression of the in vitro IgM response provide clear evidence for TCDD, directly and indirectly, affecting multiple regulatory points within B cells, including AP-l DNA binding, Blimp-1 29 upregulation, Pax5 downregulation, and IgH 3’ a enhancer activity. Above the level of intracellular signaling, the immunotoxicity of TCDD can be summarized as a dose- and AHR-dependent phenomenon, for which a window of sensitivity within the first 24 hours following in vitro activation exists. Verification and extension of in vitro mechanisms using in vivo systems would represent a valuable step forward in understanding immunotoxicity caused by TCDD. 30 CHAPTER 2. MATERIALS AND METHODS Animals Female 6-8 week old C57BL6 mice were purchased from the National Cancer Institute and housed in accordance with Michigan State University Institutional Animal Care & Use Committee policy. For in vivo studies mice were dosed by single oral gavage according to individual weights 4 days prior to LPS administration, with final doses equal to 0, 3, 10, or 30 pig/kg TCDD in sesame oil. On day 0 mice received intraperitoneal injections of phosphate buffered saline vehicle or 25 pg Salmonella Iyphosa LPS to activate as primary humoral IgM response. Each time point and treatment group consisted of 6 mice. Tissue samples were collected from mice receiving sesame oil vehicle or TCDD treatment alone on the day 0 to establish baseline effects of TCDD for all outcomes measured, then tissues were collected from all treatment groups on subsequent days. Mice were euthanized by carbon dioxide asphyxiation and spleens removed for processing into single cell suspensions by mechanical disruption. Splenocytes from individual mice were divided into separate aliquots for flow cytometry, RNA and DNA isolation, protein isolation, and IgM ASC response enumeration. Chemicals TCDD was purchased from Accustandard (New Haven, CT) and prepared in sesame oil (Sigma-Aldrich). Salmonella typhosa LPS was purchased from Sigma-Aldrich and prepared immediately prior to administration. For in vitro studies stocks of LPS, R848, and CpG ODN 1826 were prepared in individual aliquots and stored at -20° C until use. LPS was reconstituted in RPMI-CH12 31 at a concentration of 10 ug/uL without serum and other culture supplements. R848 was purchased from Alexis Biochemicals (Plymouth Meeting, PA) and prepared in cell culture grade DMSO (Sigma-Aldrich) at a concentration of l ug/uL. HPLC-purified thioester modified CpG with a sequence of 5’-TCCATGACGTTCCTGACGTT-3’ was purchased from Eurofins MWG Operon (Huntsville, AL) and prepared in Tris-buffered water at a concentration of 1.2 ug/uL. B cell Isolation Purified mouse B cells were isolated using Miltyeni Biotec B cell Isolation kits (Auburn, CA) according to manufacturer’s instructions. In brief, spleens were collected from three mice and mechanically disrupted into a single cell suspension. Following passage through a 40 um nylon mesh, splenocytes were pelleted and resuspended in MACS buffer (sterile pH 7.2 phosphate buffered saline containing 0.5% bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid). A cocktail of biotinylated antibodies specific for T cells, natural killer cells, neutrophils, dendritic cells, and macrophages were incubated with the splenocytes, then cells were washed to remove excess antibody. Following resuspension, splenocytes were incubated with anti-biotin antibodies conjugated to magnetic beads, then washed to remove excess antibody. Splenocytes were resuspended in MACS buffer and passed through a single magnetic column followed by three washes to isolate B cells. During column purification non-B cells are retained in the magnetic column while B cells pass into a collection tube. Purity of isolated B cells was verified by FACS analysis of CD19 expression, and were routinely found to be 95-98% CD19+. 32 Cell Culture SKW 6.4 cells purchased from ATCC were maintained according to ATCC recommendations in Roswell Park Memorial Institute-1640 media containing 10% bovine calf serum, 4.5 g/L glucose, 2 g/L sodium bicarbonate, 1.5 mM HEPES, 1 mM sodium pyruvate, non-essential amino acids, and 50 uM 2-mercaptoethanol (Complete RPMI-SKW). Cells were passed every two to three days and maintained at a density between 1x105 and 2x106 cells/mL. CH12.LX cells, obtained from Dr. Gregory Haughton (University of North Carolina, Chapel Hill, NC), were maintained in Roswell Park Memorial Institute-1640 media containing 10% bovine calf serum, 2.5 g/L glucose, 3 g/L sodium bicarbonate, 1 mM HEPES, 1 mM soditun pyruvate, non-essential amino acids, and 50 uM 2- mercaptoethanol (Complete RPMI-CH12). Cells were passed every 2-3 days and maintained at a density between 1x104 and 1x106 cells/mL. HEK 293T (293T) cells were purchased from Open Biosystems and maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% bovine calf serum, 4.5 g/L glucose, 3 g/L sodium bicarbonate, 1 mM sodium pyruvate, and non—essential amino acids (Complete DMEM). Cells were passed every 2-3 days upon reaching 90% confluence. Primary B cells were cultured in RPMI-CH12 at an initial density of 3x106 cells/mL. 33 Flow Cytometry Analysis For in vivo studies splenocyte samples were depleted of erythrocytes by ammonium chloride lysis. Suppliers and fluorescent labels on antibodies used can be found in Appendix A. F cyIII/II (CD16/CD32) receptors were blocked to prevent non- specific labeling with F c Block followed by incubation with phycoerythrin-labeled anti- CD19 antibodies to identify B cells. For cell surface marker staining antibodies specific for MHC Class II and CD138 were added following CD19 antibody addition. Cells were washed two times to remove excess antibody, then fixed with Cytofix (BD Biosciences). For detection of intracellular proteins splenocytes were fixed following CD19 staining, then permeabilized with Perm/Wash (BD Biosciences). Excess antibody was removed with two washes before cells were resuspended in FACS staining buffer (1x Hank’s Balanced Salt Solution with 1% BSA and 0.09% sodium azide). For indirect staining of Blimp-1 and IgJ cells were incubated with the appropriate species-specific secondary antibody, then washed two time to remove excess antibody prior to analysis. 20,000 CD19+ events were collected on a BD F ACSCalibur (BD Biosciences) using CellQuest Pro for acquisition. Data were analyzed using FlowJo 7.2 (Treestar Software, Ashland, OR). For in vitro studies of activation marker and transcription factor expression purified B cells were seeded at a density of 3x106 cells/mL in 1 mL per well on 24 well plates. Cells were treated with 0, 0.03, 0.3, 3, or 30 nM TCDD in DMSO vehicle, then activated with 30 ug/mL Salmonella typhosa LPS. At 1, 8, or 24 h post-treatment 5x105 34 cells were transferred to individual wells of a 96 well U-bottom plate for staining. Antibodies used are listed in Appendix A. To identify viable populations LIVE/DEAD Near-IR dye (Invitrogen, Carlsbad, CA) was prepared according to manufacturer’s instructions and cells incubated in 250 uL labeling buffer (Hank’s Balanced Salt Solution containing diluted LIVE/DEAD Near-IR dye) for 30 minutes, then excess dye removed with a single wash of FACS buffer. FcyIII/II receptors were blocked with PC Block. For activation marker determinations a cocktail of antibodies for detection of MHC Class II, CD69, CD80, and CD86 were added to individual wells following blocking. Cells were incubated at 4° C for 30 minutes to stain, washed twice to remove excess antibody, and fixed with Cytofix. 20,000 events were collected per sample on a BD FACS Canto 11 using FACS Diva sofiware (BD Biosciences), then analyzed using FlowJo 7.2. Cells were gated to exclude doublets from analysis by plotting forward scatter pulse height compared to forward scatter pulse area. For transcription factor expression measurements B cells fixation was performed following F c Block incubation and washing. Samples were resuspended in 90% serum/10% DMSO and stored at -80° C until the day of analysis. For staining and analysis B cells were thawed at room temperature and washed twice with FACS buffer containing 0.5% saponin (Calbiochem, San Diego, CA), then incubated for 30 minutes at 4° C to allow for through penneabilization. Until the final resuspension in FACS buffer prior to analysis all buffers and washes contained 0.5% saponin to maintain penneabilization. Following penneabilization B cells were pelleted by centrifugation, resuspended in staining buffer containing antibodies specific for phosphorylated c-Jun, Blimp-1, and BCL-6 and incubated at room temperature for 30 minutes in the dark. 35 Excess antibody was removed by washing twice, then cells were resuspended in staining buffer containing anti-rabbit IgG F (ab’)2 secondary antibody for detection of BCL-6 and incubated for 30 minutes at room temperature in the dark. Following the second staining excess antibody was removed with two washes, then cells were resuspended in staining buffer containing antibody specific for c-jun. Cells were incubated for 30 minutes at room temperature in the dark, then excess antibody was removed by washing twice. 20,000 events were collected per sample on a BD FACS Canto II using FACS Diva software, then analyzed using FIowJo 7.2. Cells were gated to exclude doublets from analysis by plotting forward scatter pulse height compared to forward scatter pulse area. ' Studies measuring kinase phosphorylation utilized either CH12.LX or purified B ’ cells. CH12.LX cells were grown in log phase until reaching a density of 6x105 cells/mL, then 1 mL of cells were transferred to 12x75 mm FACS tubes and placed in a 37° C water bath to equilibrate for 2 to 3 hours. Primary B cells were diluted to a density of 1x106 cells/mL, then 1 mL of cells were transferred to 12x75 mm F ACS tubes and placed in a 37° C water bath to equilibrate for 2 to 3 hours. 100x concentrated treatments were prepared immediately prior to treatment additiOn. TCDD concentrations used were 0, 0.003, 0.03, or 0.3 nM TCDD in DMSO vehicle coupled with 30 ug/mL LPS, 12 ug/mL CpG, or 200 ng/mL R848 for activation. Following treatment addition to individual tubes, cells were vortexed briefly and returned to the water bath for the remainder of the time course. At 15, 30, and 60 minutes (min) post—treatment cells were fixed by direct addition of 32% electron microscopy grade paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for a final concentration of 10% paraforrnaldehyde, briefly 36 vortexed, then returned to the water bath for 30 minutes. At the completion of fixation cells were pelleted by centrifugation, resuspended in 500 uL of cold _>_99.9% methanol, and stored at 4° C until all time points were completed, then transferred to -80° C for storage. On the day of analysis cells were pelleted, washed twice with staining buffer, and 3.5x105 cells were transferred to U bottom 96 well plates for staining. Following centrifugation to pellet cells samples were resuspended in staining buffers containing antibodies specific for phosphorylated ERK1/2 (Threonine 202/Tyrosine 204), JNK1/2 (Threonine 183/Tyrosine 185), or AKT (Serine 473). Samples were incubated for 30 minutes at room temperature in the dark, then pelleted and washed once to remove excess antibody. 20,000 events were collected per sample on a BD FACS Canto II using FACS Diva software for acquisition, then analyzed using FlowJo 7.2. For analysis cells were gated to exclude doublets from analysis by plotting forward scatter pulse height compared to forward scatter pulse area. To calculate % change the following formula was used: ((Geometric Mean Fluorescence of stimulated samples - Geometric Mean Fluorescence of untreated samples)/(Geometric Mean Fluorescence of untreated samples) * 100 = % change For detection of Homo sapiens AHR (HsAHR) by F ACS CD16/CD32 receptors were first blocked with Fe Block, washed twice to remove excess antibody, and fixed with Cytofix for 15 min at 4° C. Cells were permeabilized using Perm/W ash, then incubated with APC-conj ugated rabbit anti-Human AHR or APC-conjugated rabbit IgG isotype control for 30 min at 4° C prior to washing twice with Perm/Wash to remove excess antibody. Cells were analyzed for AHR immunofluoresence using a BD FACSCalibur with CellQuest Pro for acquisition and FlowJo 7.2 for analysis. 37 Gene Expression Analysis RNA was isolated from splenocytes using Trizol (Sigma-Aldrich, Saint Louis, MO) according to manufacturer’s protocol. RNA pellets obtained following isopropanol precipitation and ethanol wash were resuspended in Promega SV RNA Lysis Solution and then processed according to the manufacturer’s protocol to further purify RNA (Promega, Madison, WI). cDNA was generated using Applied Biosystems High Capacity Archive kit according to manufacturer’s instructions. TaqMan primer/probe sets were used for all gene expression analysis can be found in Appendix B, and quantified by AACt method using 188 for normalization (Livak and Schmittgen 2001). Real-time PCR was performed using ABI 7900HT or Prism 7000 real-time PCR machines. IgM ASC Response Detailed methods used for enumeration of ASC can be found in (Holsapple er al. 1984). TNP-haptenated sRBC were prepared as described in (Rittenberg and Pratt 1969). In brief, single cell suspensions of splenocytes were combined with a solution of warm 0.5% agar and TNP-haptenated sheep erythrocytes. Guinea pig complement (Cedar Lane Labs, Burlington, NC) was added, samples vortexed briefly, and aliqouted to a Petri dish. Samples were overlaid with a glass coverslip and incubated at 37°C in 95% air/5% carbon dioxide overnight. Hemolytic plaques were counted under magnification and normalized to cell number, as determined using Z1 series Coulter Counter (Beckman Coulter, Fullerton, CA), to calculate IgM ASC per 106 cells. 38 Mouse and Human IgM Sandwich ELISA 2x104 CH12.LX cells/mL were plated and incubated overnight prior to treatment for mouse IgM ELISA. Following 48 h incubation cells were pelleted by centrifugation and supematants collected for assessment of secreted IgM. For quantification of mouse IgM secretion Immulon 4 HBX strips (Thermo Electron Corporation, Milford, MA) were coated overnight at 4° C with anti-mouse IgM capture antibody (Sigma-Aldrich). Plates were then washed three times with PBS containing 0.5% Tween-20 (Sigma-Aldrich) followed by four washes with distilled water. Wells were blocked with 250 uL PBS containing 3% BSA for 1.5 h at room temperature. Following blocking, plates were either washed as described above and used immediately or frozen at -20° C for future use; 100 pL of sample or purified mouse IgM standard (Sigma-Aldrich) was added to individual wells and incubated at 37° C for 1.5 h, then washed as described above. 100 uL horseradish peroxidase conjugated anti-mouse IgM was added to each well and incubated for 1.5 h at 37° C for antigen detection. Plates were washed as described above and developed using a kinetic reaction with 2,2’-azinobis(3-ethylbenz thiazoline-sulfonic acid) (Roche, Indianapolis, IN) for detection on a Synergy HT plate reader (Bio-Tek, Winooski, VT). 1x105 SKW 6.4 cells/mL were plated and incubated overnight prior to treatment additions for human IgM ELISA. Following 96 h incubation cells were pelleted by centrifugation and supematants collected for assessment of secreted IgM. For quantification of human IgM secretion Immulon 4 HBX high strips were coated 39 overnight at 4° C with 100 uL anti-human IgM x capture antibody (Sigma—Aldrich). Plates were then washed three times with PBS containing 0.5% Tween-20 followed by 4 washes with distilled water. Wells were blocked with 250 uL PBS containing 3% BSA for 1.5 h at room temperature. Following blocking, plates were washed and 100 uL of sample or purified human IgM standard (Sigma-Aldrich) was added to individual wells and incubated at 37° C for 1.5 h, then plates washed as described above. 100 uL horseradish peroxidase conjugated anti-human IgM was added to each well and incubated for 1.5 h at 37° C for antigen detection. Plates were washed as described above and developed using a kinetic reaction with 2,2’-azinobis(3-ethylbenz thiazoline-sulfonic acid) reagent for detection on a Synergy HT plate reader. Immunoblot Analysis of HsAHR For detection of HsAHR protein cells were lysed in radioimmunoprecipitation buffer (phosphate buffer saline containing 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) supplemented with Complete MINI protease inhibitor (Roche, Indianapolis, IN). Following lysis, samples were centrifuged at 12,000 g for 15 minutes at 4° C to pellet insoluble material and supematants assessed for total protein concentration by bicinchoninic acid assay (Sigma-Aldrich). Samples were heat-denatured and separated by sodium dodecyl sulfate- polyacrylarnide gel electrophoresis. Proteins were transferred overnight to nitrocellulose membranes, followed blocking with a Tris-buffered saline solution containing 4% non-fat dry milk and 1% BSA. Membranes were incubated for 2 h with antibody specific for human AHR (H-211, Santa Cruz Biotech), washed, then incubated with an HRP- conjugated goat anti-rabbit IgG antibody for l h. Supersignal Femto West (Pierce 40 Biotechnology) was used for chemiluminescent visualization of immunoreactive bands in conjunction with Kodak X-OMAT film. Plasmid DNA isolation Plasmid DNA was isolated according to manufacturer’s instructions using Qiagen Miniprep or Maxiprep columns. Plasmid Vector Construction All DNA restriction enzymes utilized were purchased from New England Biolabs (Ipswich, MA). The HsAHR insert for pcDNA3.1-TOPO (Invitrogen) was generated by PCR amplification of pSport-hAHR2, obtained from Dr. John J. LaPres, using Invitrogen Platinum T aq DNA polymerase with the forward primer NK634 (5’- CACCATGAACAGCAGCAGCGCC-3’) and reverse primer NK636 (5’- TCAAAATTGGGCTTGGAATTAC-3’). PCR conditions were 2 min at 95° C to denature the template, 30 cycles of 30 seconds (5) at 94° C, 30 s at 58° C to anneal, and 3 min at 68° C to extend the product, with a final extension of 68° C for 30 min. PCR product was verified to be a single product of ~2.5 kb by visualization on an ethidium bromide containing agarose gel. PCR product was added directly to the TOPO cloning reaction and incubated according to manufacturer’s instructions. TOPO cloning reactions were transformed into MAX Efficiency DHSa cells. Plasmids were verified by sequencing. The HsAHR insert for pIRESneo3-HSAHR was generated by PCR from pSport- hAHR2 using the primers NK657 (5’- GCGCGCGGCCGCTTACAGGAATCCACTGGATGT-3’) and NK658 (5’- 41 GCGCCGTACGCACCATGAACAGCAGCAGCGCCAAC-3’) with Phusion High Fidelity DNA polymerase (Finnzymes USA, Woburn, MA). Conditions used were an initial denaturation phase of 98° C for 1 m, followed by 30 cycles of 98° C for 7 s and 72° C for 50 s, with a final extension of 72° C for 2 min. PCR product was sequentially digested with NotI followed by BsiWI, then gel purified. A PCR product of ~2.5 kb was excised from the agarose gel, purified with Qiagen QIAQuick gel extraction columns, ligated into pIRESneo3 using NEB T4 DNA ligase, and transformed into MAX Efficiency DHSa cells. Plasmids were verified by restriction digestion and partial sequencing. To generate the C-terminal GF P fused form of HsAHR the plasmid phCMV- CGFP was purchased from Genlantis (San Diego, CA). HsAHR suitable for insertion into phCMV-CGF P was generated by PCR, introducing a 5’ BgIII site and 3’-HindIII site using the primer NK67S (5’-GCCGCCGAGATCTGGGCACCATGAACAGCAGCA-3’) in combination with either NK676 (5’-TGGGCTTGGAAGCTTAGGAATCCACTG-3’) to maintain the native stop codon or NK677 (5’- TCAAAATTAAGCTTGGAATTACAGGAATC-3’) to mutate the final amino acid of native HsAHR from valine to lysine and the stop codon to leucine, resulting in an in- frame fusion of HsAHR to GFP. For all PCR reactions Phusion High Fidelity DNA polymerase was used. PCR product was generated by first denaturing pSport-hAhR2 at 98° C for 30 s, followed by 30 cycles of 98° C for 10 s, 68° C for 10 s, and 72° C for 35 s, with a final extension at 72° C for 2 min. The plasmid was digested with BglII and HindlII, dephosphorylated using NEB Antarctic Phosphatase (New England Biolabs) according to manufacturer’s instructions, and gel purified to remove residual uncut 42 plasmid. HsAHR PCR product was also gel purified, then ligated into phCMV-CGF P using a NEB Quick Ligation kit according to manufacturer’s instructions. Ligation reactions were transformed into ultracompetent XL-2 Blue cells (Stratagene, La Jolla, CA). Colonies were screened by restriction digest, then verified by sequencing to contain the desired HsAHR sequence. For pZeoSV2-HsAHR-GFP the HsAHR-GFP ORF was liberated from phCMV- HsAHR-GFP by digestion with BglII and NotI, followed by gel purification. pZeoSV2(+) was prepared by digesting plasmid DNA with BglII and NotI, then dephosphorylating the plasmid DNA with NEB Antarctic Phosphatase, and gel purified. HsAHR-GFP was ligated into pZeoSV2(+) by using NEB T4 DNA ligase. The ligation reaction was transformed into XL-2 Blue ultracompetent cells and verified by restriction digestion followed by partial sequencing. pLEX-HSAHR and pLEX-HsAHR-GFP were generated by digesting phCMV- HsAHR and phCMV-HsAHR-GFP with BglII and NotI overnight, then gel purifying the liberated DNA fragment containing AHR and AHR-GFP ORFs. To prepare pLEX-MCS for insertion of HsAHR and HsAHR-GFP plasmid DNA was digested with BamHI and NotI overnight, dephosphorylated, and gel purified to remove residual uncut plasmid. DNA fragments were recovered from a 0.8% low melting temperature agarose gel using Promega Wizard SV Gel Clean-up columns (Madison, WI) according to manufacturer’s instructions. HsAHR and HsAHR-GFP were ligated into pLEX-MCS using NEB Quick Ligation kit, then transformed into Invitrogen Stbl2 competent E. coli (Carlsbad, CA) according to manufacturer’s instructions. Following overnight incubation at 30° C individual colonies were screened by PCR using primers NK672 (5’- 43 it I i .l.l;d4ill..3n|ul.§..n1lnlr (it? TTTCAGCGTCAGCTACACTG-3’) and NK673 (5’- TGACTGATCCCATGTAAGTCTGTAA-3’) with an initial denaturation phase of 95° C for 2 minutes followed by 25 cycles of 95° C for 30 s, 62° C for 30 s, and 72° C for 92 seconds, and a final extension phase of 72° C for 5 min. Positive colonies with PCR product size of approximately 1.5 kb, indicative of AHR presence, were grown, plasmid DNA purified, and verified by restriction digestion. Transfection and Selection of Stably Transfected Cells For pcDNA3.l-TOPO-HSAHR, pZeoSV2-HsAHR, pZeoSV2-HsAHR-GFP, phCMV-HSAHR, and phCMV-HsAHR-GFP plasmids SKW 6.4 cells were grown in log phase for 3 days prior to electroporation, starting at a density of 5x104 cells/mL. On the day of electroporation cells were pelleted by centrifugation at 90g for 10 minutes and resuspended in fresh Complete RPMI-SKW. Cells were counted by trypan blue exclusion, then 2x106 cells were transferred to individual microcentrifuge tubes for electroporation. Cells were pelleted again by spinning at 90g for 10 minutes, then resuspended in premixed room temperature electroporation buffer consisting of 90 uL Solution V with 20 uL Supplement 1 (Lonza Walkersville Inc., Walkersville, MD). 100 uL of cells were transferred to an electroporation cuvette and electroporated using an amaxa Nucleofector set to program X-001. Following electroporation room temperature Complete RPMI-SKW was added to each cuvette and cells transferred to individual 25 cm culture flasks, Wthh were placed in a vertical orlentation 1n the 1ncubator. After 24 h, recovery flasks were placed in a horizontal position and incubated an additional 24 h. 48 h post-electroporation 1.2 mg/mL G418 (pcDNA3.l-TOPO and phCMV-CGFP 44 derived vectors) or 50 ug/mL Zeocin (pZeoSV2 plasmids) was added to culture. Cells monitored for outgrowth of antibiotic resistant populations by microscopy. For G418 resistance approximately 4-5 weeks were required to achieve a large antibiotic resistant population. Luciferase Assays Luciferase activity was measured according to manufacturer’s instructions using a Promega Luciferase Assay System (Madison, WI) on a Synergy HT plate reader. Lentivirus Preparation 293T cells were plated at a density of 6.5x106 cells per 10 cm2 culture plate and incubated 8 hours to allow for full attachment. 100 pg plasmid DNA was prepared for transfection using calcium phosphate by diluting all plasmids into 0.25 M calcium chloride solution. Diluted DNA was added as single drops to an equal volume of 2x HEPES Buffered Saline while vortexing at high speed. Once all DNA had been added precipitates were allowed for form for 5 minutes prior to dropwise addition of complexed DNA to 293T cells. Following overnight incubation transfection media exchanged for fresh Complete DMEM, then cells returned to the incubator for an additional 36 h. Lentivirus containing culture media was collected and centrifuged at 3000 rpm for 30 minutes at 4° C to pellet cell debris, then supematants were aliqouted and stored at -80° C until use. Direct Assessment of Lentiviral RNA To isolate lentiviral RNA 400 uL of lentivirus containing culture supematants were combined with 1400 uL Trizol, mixed by vortexing, then incubated for 5 minutes. 45 200 ML 1-bromo-3 -chloropropane were added to the sample, then thoroughly vortexed and incubated at room temperature for 5 minutes. Samples were centrifuged at 12,000 g for 10 minutes at 4° C, then the top phase aqueous solution was transferred to a new sample tube. RNA was precipitated by adding 600 uL 100% isopropanol to each sample tube, vortexing, then incubating for I h at -20° C. Following centrifugation to pellet RNA a single wash with 70% ethanol was performed, samples were centrifuged again to pellet RNA, dried, and RNA pellets resuspended in 20 uL of nuclease-free water. RNA was quantified by NanoDrop, then reverse transcribed using a Superscript VILO cDNA Synthesis kit (Invitrogen) according to manufacturer’s instructions. cDNA generated was analyzed by QRT-PCR using custom synthesized TaqMan primers specific for HsAHR, GFP, and PuroR. For all PCR reactions a non-reverse transcribed control reaction was also performed. Lentiviral Transduction and Selection of Transduced Cells Frozen lentiviral stocks were thawed at room temperature and supplemented with 8 pg/mL polybrene (SEQUABRENE, Sigma-Aldrich). Following 5 minutes incubation to allow polybrene to coat viral particles, SKW 6.4 cells were resuspended in virus containing media. 2x106 SKW 6.4 cells in 250 uL were then added to each well of a 24 well tissue culture plate to achieve a monolayer of cells during spin infection. Cells were centrifuged at 800g for 2 h, then washed twice with cell media prior to diluting cells to a density of 1x105 cells/mL. Following 96 h incubation puromycin was added to culture at a final concentration of 1 ug/mL. Media was changed every 2-3 days until a puromycin 46 resistant populations arose and could be frozen for storage, cloned by limiting dilution, and analyzed for AHR gene and protein expression. Transduction of 293T cells was accomplished by overlaying a 50% confluent culture with lentivirus containing media supplemented with 4 ug/mL polybrene for 4 h, then aspirating media and reculturing in Complete DMEM for an additional 48 h. Media was exchanged for fresh Complete DMEM containing 3 ug/mL puromycin and cells. monitored for generation of antibiotic resistant colonies. Within 4 days puromycin resistant colonies could be observed. Polyclonal populations of 293T-HAG, expressing HsAHR fused to GFP, and 293T-HAHR, expressing the non-fused form of HsAHR, were used to validate the production of a full length HsAHR-GF P protein using immunoblot and a FACS-based method for detection of AHR. Preparation of Conditioned Complete RPMI-SKW SKW 6.4 cells were cultured at a density of 1x105 cells/mL, then following 48 h incubation cells were pelleted by centrifugation at 3000 rpm for 30 minutes. Supernatant was collected and centrifuged again prior to storage at 4° C for up to two weeks prior to use. Cloning by Limiting Dilution Stably transduced SKW 6.4 cells were diluted to a density of 3.333 cells/mL in conditioned RPMI-SKW and 100 uL of media, approximately 1/3 of a cell, was added to each well of a round bottom 96 well plate. An average of approximately 10 individual clones were obtained from each plate using this method. 47 Statistical Analysis Results were analyzed by One-way ANOVA using Dunnet’s post-hoc analysis compared to LPS + vehicle treatment for in vivo studies. Neuman-Keuls post-hoe analysis was used for determination of statistically significant differences for multiple comparisons between treatment groups of the in vivo studies. All analysis was performed using Graphpad Prism 4.05 (Graphpad Software, San Diego, CA). P-values less than or equal to 0.05 were considered significant. For multivariate analysis of FACS data the Probability Binning approach was used. Probability Binning divides a control sample into discrete bins, 600 for all analyses performed in these studies, containing equal numbers of events. Bins derived from control samples, TLR ligand + vehicle treatment in these studies, are applied to experimental samples to compute a normalized chi-squared value. Based on the distribution of events in control and experimental samples the T00 value, analogous to a t-score, is derived. The T00 value allows ranking of experimental samples based on the relative distance of an experimental sample from the control sample (Roederer et al. 2001a; Roederer et al. 2001b). A T(x) value of 0 indicates two populations are indistinguishable from each other. T()() values greater than 6 were considered significantly different. Flowjo 8.8.6 was used for all Probability Binning. 48 CHAPTER 3: EXPERIMENTAL RESULTS AND DISCUSSION FOR MURINE MODELS Simultaneous In viva Time Course and Dose Response Evaluation for TCDD-Induced Impairment of the LPS-stimulated Primary IgM Response Suppression of the in viva LPS-activated primary IgM response by TCDD Different methods of B cell activation result in distinctly different cell signaling programs and cumulative responses (Donahue and Fruman 2007). To maximize the value of in vivo and in vitro comparisons it was deemed important to utilize similar modes of B cell activation as in previous studies. Many laboratories have reported TCDD suppression of the T cell-dependent anti-sRBC IgM antibody response (Dooley and Holsapple 1988; Holsapple et al. 1986; Luster et al. 1988; Smialowicz et al. 1994; Tucker et al. 1986; Vecchi et al. 1980a; Vorderstrasse et al. 2001), but no published studies have evaluated TCDD-mediated suppression of the in viva primary IgM response in the context of the commonly used polyclonal B cell activating stimulus LPS. Moreover, no published study to date has correlated alteration in the expression of regulatory transcription factors with in viva TCDD suppression of the IgM response to any stimulus. The rationale for using LPS in this study is that a strong polyclonal B cell activator may differentiate a proportionally larger fraction of B cells in viva than antigens such as sRBC, resulting in a potentially greater sensitivity at early time points for detection of TCDD effects. To examine the in viva effects of TCDD on the LPS-activated primary IgM response a dose of 30 tag/kg TCDD was selected, which is established to 49 cause near-maximal suppression of the sRBC-activated primary IgM response in viva (V ecchi et al. 1980a), then selected half-log decreasing doses to profile the TCDD- mediated effects. LPS in viva treatment induced a significant increase in the IgM ASC response, which was significantly suppressed by all concentrations'of TCDD in a dose- dependent manner (Figure 3). This may be the first study to demonstrate that in viva exposure to the polyclonal B cell activator, LPS, results in an increase in IgM ASC reSponse, which is suppressed by TCDD. 50 ASC per 106 Splenocytes X ‘\ ‘\\ O O O 0‘ 0 0 0 0 a“ q, e e e e; '9 Q9 Q0) ‘29 Figure 3. Suppression by TCDD of the LPS-activated IgMASC response. F our days prior to LPS treatment mice were administered a single dose of TCDD (3, 10, or 30 ,ug/kg) and/0r vehicle (sesame oil) by oral gavage. On day 0 mice received single intraperitoneal injections of either PBS or 25 ug LPS. IgMASCs were enumerated on Day 3 following LPS. Results are depicted as mean ASC per 106 splenocytes i SE of the mean. a P<0. 05 compared to sesame all + PBS, b P<0.05 compared to sesame oil + LPS, C P<0.05 compared to 3 pig/kg TCDD + LPS, d P< 0.05 compared to 10 pig/kg TCDD + LPS, and 8 P<0.05 compared to 30 pig/kg TCDD + LPS. 51 LPS treatment induced a significant increase in spleen size, as reflected in the spleen weight/body weight ratio, which was significantly attenuated by TCDD treatment (Figure 4). Treatment with TCDD alone did not significantly alter spleen weight/body weight ratio, and no treatments caused significant changes in body weight. 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EM An extensive phenotyping of splenic B cells from all treatment groups was performed, in which individual mice were evaluated for gene and protein expression associated with plasmacytic differentiation to maximize the statistical strength of this study. Fluorescence-activated cell sorting (FACS) was used to profile cell surface and intracellular protein abundance. LPS-activated B cells up—regulate expression of several cell surface proteins in the normal course of an immune response, including MHC Class II to aid in antigen presentation. Surface MHC Class II expression on CDI9+ B cells increased significantly in response to LPS, peaking on Day 1 and modestly declined by Day 3. Cell surface expression of MHC Class II declined as B cells proceed toward the ASC phenotype, consistent with our observation of peak MHC Class II expression 1 day after LPS treatment, which then declined on Days 2 and 3 (Figure 5). Administration of 30 pig/kg TCDD significantly attenuated the LPS-induced MHC Class II on the surface of CD19+ B cells at all time points evaluated, while lower doses of TCDD marginally attenuated LPS-induced cell surface MHC Class II up-regulation. 55 :1 r'v. s, 'f-‘Jrgw'x n Tvo-nr Figure 5. Suppression by TCDD of LPS-induced cell surface MHC Class [1 expression. Isolated splenocytes were incubated with F cBlock, labeled with antibodies specific for CD19 and MHC Class II, thenfixed with C ytofix. Cells were subsequently analyzed by FACS. Populations were gated byfirst identifi'ing (A) CD19+ events, then (B) the individual median fluorescence (MFI) for MHC Class 11. Results from 6 mice per group are depicted as mean MFI i SE of the mean. a P<0. 05 compared to sesame oil + PBS and b P< 0. 05 compared to sesame all + LPS. 56 > Number of cells (D Surface MHC Class II Expressron (MFI) 600 400 1201 1104 A0019+ 100 101 102 to3 CD19 (PE) ix _,I,_,~,>a[; ~A—1o pig/kg TCDD + LPS Sesame Oil + Vehicle Sesame Oil + LPS 3 its/kg TCDD + LPS 10 pig/kg TCDD + LPS - 30 pig/kg TCDD 4' LPS 100 10‘ 102 1o3 MHC Class II (FITC) I-Vehicle + PBS +Vehicle + LPS b"'3 pig/kg TCDD + LPS N «30 pig/kg TCDD + LPS b Days Post-LPS i 57 5 The IgM ASC response has been widely used to enumerate antibody secreting plasma cells. Our results show that intraperitoneal administration of LPS induced a significant increase in splenic ASCs, which was suppressed in a dose-dependent manner by TCDD treatment. Using the IgM ASC response as a phenotypic anchor, splenocytes were further characterized for CD138 expression, also termed syndecan-l , by flow cytometry as a complementary marker of plasma cells. Figure 6A shows LPS induced an + increase in frequency of CD19+ CD138 cells, which was attenuated by TCDD- treatment, an indication that suppression of the IgM ASC response was not simply due to suppression of antibody secretion, but also involved a blockade in LPS-activated B cell differentiation into ASCs. Extending the observation that appearance of CD19+ CD138+ cells was impaired by TCDD, LPS-induced generation of CD19+ Iglchigh cells (Figure 6D) and CD19+ Ith‘gh cells (Figure 6E) in TCDD-treated mice was also impaired, suggesting that TCDD blocks B cell differentiation prior to the large increase in intracellular IgJ and Iglc associated with the plasma cell phenotype. Collectively, these results demonstrate TCDD-mediated suppression of the LPS-induced primary IgM response involves failure of individual cells to not only secrete IgM, but to express cell surface markers and intracellular proteins indicative of the ASC phenotype. 58 :’""1 ‘ . l u... ,, hLJL—s.‘ ,.( ' rill—£1“! mosh-'4..— Figure 6. TCDD treatment impaired expression of phenotypic indicators for plasmacytic diflerentiation. To assess B cells for C D138 expression, isolated splenocytes were incubated with F cBlock, labeled with antibodies specific for CD] 9 and CDI38, then fixed with Cytofix. For detection of total Igic or IgJ, cells were blocked, labeled with C D] 9 antibodies, and fixed with Cyto/ix. At the completion of the time course, cells were permeabilized with ED Perm/ Wash, incubated with antibodies specific for either IgJ or Iglc and analyzed by FA CS. Populations were gated byfirst identifving CD19+ events as depicted for (A) CD1 3 8 and (B)Igic, and then gated on populations that expressed elevated levels of (C) C0138, (D) IgK, or (E) IgJ. Resultsfrom 6 mice per group are depicted as mean frequency of CD1 9+ cells i SE of the meanfor respective measurements. a P<0. 05 compared to sesame oil + PBS and b P<0.05 compared to sesame oil + LPS. 59 °/o of CD19+ Cells CD19-PE 15 10' Sesame CD138 Days Post-LPS 60 % of c019+ Cells CD19-PE Figure 6 continued Sesame Oil + PBS Sesame Oil + LPS l . ' ‘ _ 3 ug/kg 10 rig/kg 30 ug/kg TCDD + LPS TCDD + LPS TCDD + LPS 1 '1. 0.5 MB! 0.6 J ...—’02- i V' ........ N I ’ ' . ' II'I'" b a lo 0' ll ' IgK-FITC ng Days Post-LPS % of CD19" Cells 20.0w 17.5' 15.0' 12.5' 10.0' 7.5' 5.0' 2.5- Figure 6 continued aVehicIe + PBS c-e-Vehicle + LPS *3 ug/kg TCDD + LPS +10 ug/kg TCDD + LPS “'30 ug/kg TCDD + LPS IgJ 0.0 Days Post-LPS 62 TCDD-mediated suppression of LPS-induced changes in gene and protein expression involved in B cell to ASC transition Differentiation of a resting B cell into an ASC is regulated by a network of reciprocally acting transcription factors, conceptualized in Figure 1. Previous in vitro studies established that TCDD impaired expression of several latter components of the plasma cell phenotype, including expression of IgJ, lglc, Igu, Pax5, and Blimp-l (Schneider et al. 2008; Schneider et al. 2009). High levels of IgJ, Iglc, Igu, and XBP-l are characteristic of antibody-secreting plasma cells, and splenocytes isolated from LPS- treated mice showed increasing levels of IgJ, Igtc, and 1gp mRNA peaking 2 to 3 days post-LPS treatment (Figure 7A, Figure 7C, and Figure 7D respectively), with the largest fold increase in the expression of IgJ. TCDD impaired LPS-induced increases of IgJ, ng, and Ig,u mRN A levels with IgJ significantly impaired relative to LPS treatment at all time points evaluated. ng and Igu have a relatively high level of constitutive expression in B cells because they form the B cell antigen receptor, whereas IgJ is required only for the production of secreted pentameric IgM and. dimeric IgA. In this study IgJ is likely to be the best marker of Ig destined for secretion, and thus an indicator of antibody- secreting plasma cells. The expression profile for total and spliced XBP-I followed a similar pattern to IgJ, Igtc, and 1gp, peaking in response to LPS on Days 2 and 3. While not statistically significant, TCDD treatment did appear to impair induction of XBP-I, similar again to the patterns observed with Iglc and 1gp mRNA levels. 63 d “373' m1.-———--.LJ .. l1 emca Figure 7. Gene expression profiles for LPS-inducedplasmacytic differentiation in the absence and presence of TC DD treatment. Total RNA from splenocytes was isolated and cDNA synthesized for gene expression analysis. Target gene mRNA levels were normalized to 18S and the fold change was calculated by the AAC, method. (A) IgJ, (B) CD138, (C) Igtc, (D) Igu, (E) total XBP-I, or (F) spliced XBP-I. Results from 6 mice per group are depicted as mean fold change i SE of the mean. a P<0. 05 compared to sesame oil + PBS and b P<0.05 compared to sesame oil + LPS. 64 IgJ Torrie? _ .3 1.1:... II]; mmwwmmw 420 mmn. + :0 959.5 m> mmcmso Eon. Days Post-LPS CD138 0 0 321.0 2 1 mmn. ... =0 mEmmwm m> mac—EU 20". Days Post-LPS 65' Figure 7 continued IgK I3 I 1 Days Post-LPS l0 d u u 3 2 1 wmn. + :0 oEmmmm m> omen—.0 Eon. l 2 . ’9.” a 1 - 5 4 3 2 1 mmn. + :0 mEmmmm m> omcmnu Eon. I3 '2 S P. I. L. S O D- I1 8 y a D ..0 0 66 Fold Change vs Sesame Oil + PBS Fold Change vs Sesame Oil + PBS ['11 Figure 7 continued XBP-1(tota l) 5.5 - 5.0 - 4.5 4 4.0 '- 3.5 - 3.0 - 2.5 - 2.0 - 1.5 '- 1.0 - 0.5 °- A N- Days Post-LPS XBP-1 (spliced) °- Days Post-LPS 67 Previous in vitro studies with LPS-activated CH12.LX, a murine B cell line, and mouse splenocytes suggested that TCDD treatment impaired down-regulation of Pax5 (Schneider et al. 2008; Yoo et al. 2004), a necessary regulatory event for plasmacytic differentiation (Lin et al. 2002; Nera et al. 2006). In agreement with prior in vitro observations, splenocytes from mice treated with LPS showed the greatest decrease in mRNA abundance of Pax5 on Day 2 of the time course. Mice treated with TCDD prior to- LPS had elevated Pax5 mRNA levels on Day 1 of the time course, with 10 and 30 rig/kg TCDD doses preventing the Pax5 down-regulation associated with LPS treatment observed most clearly on Day 2 of the time course (Figure 9A). FACS analysis of Blimp-1 protein in CD19+ B cells showed LPS treatment induced an increase in the number of B cells expressing Blimp-1, consistent with activation that precedes the ASC stage. Expression of Blimp-l protein is quantitatively associated with ASC phenotype, increasing as B cells differentiate into plasma cells (Kallies et al. 2004), a finding supported by observations in Figure 8. The peak in vivo ASC response occurs 3 to 4 days post-LPS (unpublished observation), correlating with elevated peak frequency for CD19Jr Blimp-1 cells. TCDD treatment impaired generation elevated of CD19+ Blimp-1 B cells in LPS-treated mice (Figure 8A and B). In agreement with protein expression, measurements of Blimp-1 mRNA levels in isolated splenocytes showed that LPS—treated mice possessed significantly increased Blimp-1 mRNA levels on Days 2 and 3. For example, 10 and 30 ug/kg TCDD treatment caused significant suppression of LPS-induced Blimp-1 mRNA levels on Day 3 (Figure 9B). 68 . . . - 3M _. \“l'. " tlu‘.1-\.t.-..w:_.-_.__ Figure 8. T CDD treatment suppressed LPS-stimulated generation of CD 1 9+ Blimp- [elevated cells. Isolated splenocytes were incubated with F cBlock, labeled with antibodies specific for CDI9, then fixed with Cytofix. At the completion of the time course cells were permeabilized with BD Perm/ Wash and incubated with antibodies specific for Blimp-1 . Cells were subsequently analyzed by FA CS. (A) Representative plots from individual mice 3 days post-LPS showing the In CD19+ Blimp-18 Hated cell population. (B) Time course and dose response for generation of CD19+ Blimp-1 elevated cell population. Results from 6 mice per group are depicted as mean elevated frequency of CD1 9+ Blimp-1 cells i SE of the mean. 0 P<0.05 com ared to sesame oil + P PBS and b P<0.05 compared to sesame oil + LPS. 69 A - Sesame Sesame Oilg__+ PBS Oil ,+ LPS 'zi' ..... hit/liar” “ll/lupin? _ - . 3 vg/kg 10 pg/kg 30 (Lg/kg (W TCDD + LPS TCDD + LPS TCDD + LPS thaw : 708 . 1,; ‘_ his?! B 2 75 15.0- +0 E-Vehicle + PBS 3’ 12-5' OVehicle + LPS 0 1o.o- --3 [Ag/kg TCDD + LPS E _ +10 ug/kg TCDD + LPS g 7-5' +30 ug/kg TCDD + LPS ‘5. 5-0- b .§ 5 2.5 ° o.o . . . . ..\° 0 1 2 3 Days Post-LPS 70 BCL-6, acting in concert with Pax5, is a negative regulator of Blimp-l gene expression (Shaffer et al. 2000; Tunyaplin et al. 2004). B cells constitutively express BCL-6 under resting conditions, with initiation of the immune response causing a decrease in BCL-6 expression (Ohkubo et al. 2005), in turn allowing Blimp-1 expression to rise. In vivo LPS treatment decreased the level of BCL-6 mRNA levels on Days 1 and 2, but concomitant treatment with TCDD did not appear to significantly alter BCL-6 gene expression (Figure 9C). 71 mm: + to 223.3 8 2923228 3 .ka 2 .222 mm& + he 222.3% 2 2222228 3 .Q vaw e .2222 32% MW a $2222.» 3e\2em22 8 22.232 22 $82M 2mm 3.22 b EQBSESM 6-29% G» B $2.223 «S Hack «3 22222 6.22. 22 23 2223.28 .42.: mmSEo 3Q 22 222 ME 8.2323232 .29: @323 3,322 2% Saabfi 6.23322 293.82me 2% 35332822229. E222$M3 22222.22 22222222 QQUK 2.2.5 2N .2 N 222ME l llllll Ill...l.tu.tn. a ..Q . .- 78 ll '1': c 3 7 0 'o % 3. " "’4" I— o .C a. U! o .C a. I I-R’. I l I 8 8 8 ° O O O (V) N r- (aauasaomu ueaw amawoas) uogssadea unr-o pazelflloquoqd Blimp-1 100- 1000- r .8. .. (aauasaomH ueaw omawoag) uolssejdxa L-dwna HOLITS Hours C no g H.” U V I-N IU'T‘II 888°°8 (aouasaomH ueaw omaujoag) uogssadea unr-o -2 g .3 m +2 I'Ié'lllo 333$??? (aouesaomH ueew omewoao) uopssadeg 9-199 79 Hours Hours +Untreated --LPS --LPS + DMSO LPS + LPS + DMSO 30 nM TCDD Untreated 0102 03 104 105 0102 103 1o4 105 0102 103 104 1o5 BCL-6 (PE/Cy5.5) .1 24: Figure 11. Relationship of Blimp-l to BCL—6 expression at 48 h. Purified B cells were assessed simultaneously for Blimp-1 and BCL-6 expression 48 h post-treatment by FACS. Results depicted are concatenated analysis of at least 4 biological replicates. Plots depict immunofluoresence for viable cells, with each level of contour corresponding to 5% of the population. Numbers in the corner of each quadrant depict the percentage of viable cells within the quadrant. Results are representative of 2 separate experiments. 80 U t t d LPS + LPS + n ma 6 DMSO 30 nM TCDD 105 1.85 Blimp-1 (PE) e:.v:-,..1:.'_’.. '. 47 o 102 103 104 105 o 102 BCL-6 (PE/Cy5.5) .-.: H, 5...) . .r A,;|«'_L. 103 104 105 0102 103 104 105 Figure 12. Relationship of Blimp-1 to BCL-6 expression at 72 h. Purified B cells were assessed simultaneously for Blimp-l and BCL-6 expression 72 h post-treatment by FA CS. Results depicted are concatenated analysis of at least 4 biological replicates. Plots depict immunofluoresence for viable cells, with each level of contour corresponding to 5% of the population. Numbers in the corner of each quadrant depict the percentage of viable cells within the quadrant. Results are representative of 2 separate experiments. 81 Time and Concentration Response Profiling for TCDD Disruption of Cell Surface Activation Marker Expression TCDD dose-dependently impaired LPS-induced MHC Class II upregulation on the surface of B cells following in vivo treatment (Figure 5). In experimental models of cell-mediated immunity, 15 ug/kg TCDD reduced P815-elicited increases in CD86 on the cell surface of CD45R+ B cells (Prell and Kerkvliet 1997). Based on the in vivo observations that TCDD treatment impaired both MHC Class II and CD86, it was hypothesized that TCDD impairs upregulation of activation markers following in vitro immune response activation. An examination of both the time- and concentration- dependent changes in surface expression for activation markers CD69, CD80, and CD86 following in vitro LPS stimulation of B cells was performed LPS caused a time-dependent increase in all activation markers examined during the first 24 h following treatment (Figure 13). MHC Class II expression declined in untreated B cells in vitro following isolation, while LPS treatment maintained and increased MHC Class II expression. CD69 expression peaked at 8 h post-treatment and remained sustained through the initial 24 h period. Both CD80 and CD86 increased throughout the 24 h time course. 89.3% of B cells treated with LPS + DMSO upregulated at least one cell surface activation marker expression by 24 h, compared with 14.7% of untreated B cells. Multivariate probability binning analysis of cell surface marker expression showed TCDD caused a significant impairment of LPS-stimulated activation marker expression beginning 8 h post-treatment compared to LPS + DMSO treatment (from T(x)=6.5 for LPS + 0.03 nM TCDD to T(x)=50.0 for LPS + 30 nM TCDD), and extended 82 to 24 h (from T(x)=20.5 for LPS + 0.03 nM TCDD to T(x)=64.3 for LPS + 30 nM TCDD). Most of the suppressive effect of TCDD on LPS-elicited cell surface activation marker upregulation during the first 24 h is attributable to effects on MHC Class II and CD69, as probability binning considering only CD80 and CD86 expression showed LPS + TCDD treated B cells are indistinguishable from LPS + DMSO treated B cells (T(x) less than 1.5 for all concentrations of TCDD). 83 222222222222 ~22 2252222222222 222 2.222222% 222222322 222.222.222.22 2. 22222 .22 222x2zo< womtzw :20 85 Significant upregulation of all activation markers occurred by 24 h following LPS and was decreased by TCDD treatment, but the primary IgM response develops over the course of 72 h. While previous in vivo results showed TCDD suppressed LPS-activated MHC Class II expression as early as 24 h post-treatment, purified B cells cultured in vitro may respond to LPS differently from their in vivo counterparts. The time course of activation marker examination was therefore increased to 24, 48, and 72 h post-treatment to extend and maximize the value of data obtained from experiments assessing expression F of transcription factors. LPS treatment caused a time-related increase in total surface expression of MHC Class II, CD69, CD80, and CD86 (Figure 14), with highest observed expression &, occurring 72 h post-LPS. Multivariate probability binning analysis of activation marker expression in LPS + DMSO treated B cells to all other treatment groups showed that 30 nM TCDD impaired expression of activation marker expression as early as 24 h post-LPS (T(x)=69.4), but as activation marker expression increased in LPS + DMSO treated groups over time the impairment caused by TCDD became more apparent. By 72 h TCDD significantly impaired activation marker expression from 0.03 nM (T(x)=73.5) to 30 nM TCDD (1130:4156). 86 22222222322 N\.2 2322222222222 222 222222 2222222222 2222222222 2 22222 2 222.3292 H 222222222222 22222 2222222222 2.22 22.222.222.22 22222% 2222222322 22QU 222 .2292 .22QU E 22229 0232\2 2.22.2222 MD 2222 2222222222 222.: 22222 22222222232222 2 Nu .222 .22 .wm .32222. 2 M2 WRN 22.2: 22222522 .222 «§mm2.22 222.222; QMEQ 2.2 QQUk 322 2M. 22 .2 .m .2 .22 .2 .2 22.: 2222222 2222: 2229 4222222 22:». 22 2222222 222 22.222222 22.22222 A2 222222222 2222: 2222 m 22223.2 A22222R 22.22.222.22 22222222 22.229222 22222 2222 2222222222 -mn:\2 22.222222 QQUR 2252222222. 222222222 2222222222222 222 222222 22.: 2222222222222 .3 222mg 87 C) 8 i... .. 8 .. C 8 5 2 5 co 0 :2 u :43 O U U . I E T I I I I I If I I I I I : : 888§°§° 8888§8° " g g a ... § 2 g. g a 8 v a (aouasaJonL-l ueaw omawoao) (aouasaomH ueaw omawoag) uogssadeg 9903 uogssadeg u ssem QHW "C .93 U) m 8 ‘5 3 5 (U a o o o (D 0 I U I J: C D l I I l Ir I I I I O O O O O O O O O c O O O O O O O O O ID 0 l0 0 O O O N v- 1- fi‘ 0') N ‘- (aouasaomH ueaw omawoes) (aouasaomH ueaw 31113111099) uogsseidxa ogao uogssardxg 6900 88 —-LPS + DMSO +LPS LPS + LPS + 30 nM TCDD _ - 1I_1__.V5j Untreated . 1.93 CD80 (APC) 0102 103 104 165' 0102 103 104 1‘05 0102 103 104 1105 CD86 (PE/Cy7) Figure 15. TCDD disrupts LPS-activated CD80 and CD86 expression. Purified B cells were treated with LPS (30 ,ug/mL) + DMSO vehicle or LPS + 30 nM TCDD. Cell surface expression of CD80 and CD86 was measured 72 h post-treatment. Results depicted immuno fluorescence from concatenated samples consisting of at least 4 biological replicates. Numbers in the corner of each quadrant depict the percentage of viable cells within the quadrant. Results are representative of 2 separate experiments. 89 Single parameter examination of LPS-activated expression for the costimulatory molecules CD80 and CD86 was disrupted by TCDD in a concentration-related manner (Figure 14). CD80 and CD86 stimulate T cells during the primary humoral response, and knockout of either CD80 or CD86 results in significant impairment of humoral immunity. However, even with knockout either allele can partially compensate for the loss of the counterpart (Borriello et al. 1997). LPS treatment increased expression of both markers P simultaneously, causing 24.7% of B cells to express high levels both CD80 and CD86. H' h H' h l TCDD treatment impaired the generation CD80 1g CD86 lg B cells, reducing the 1' frequency of double positive cells to 11.5%, a level similar to that observed in untreated B cells at 72 h. “L While MHC Class II and CD69 were also inhibited by TCDD treatment at 72 h, they decreased 9.0% and 12.7%, respectively, in LPS + 30 nM TCDD compared to LPS + DMSO treated B cells. In contrast, 30 nM TCDD caused at 23.5% decrease in CD80 expression relative to LPS + DMSO treated B cells at 72 h. TCDD treatment suppressed LPS-activated CD86 expression 12.6%. 90 Simultaneous Time Course and Concentration Response Evaluation for TCDD- Induced Impairment of the TLR-stimulated In vitro Primary IgM Response While results of in vivo and in vitro studies described above indicate TCDD; mediated disruption of LPS-activated Blimp-1 expression are consistent, a pivotal, direct mechanism for TCDD-mediated disruption of the primary IgM response could not be concluded. Based on the observation that TCDD disrupted Blimp-l gene and protein expression, it was hypothesized that events preceding Blimp-1 upregulation may be altered by TCDD. One pharmacologic approach for elucidating mechanisms involved in phenotype is to exploit different ligand-receptor interactions in order to understand underlying physiology. A classic example is use of a or B adrenoreceptor agonists and antagonists to illuminate the role of different adrenergic signal programs in control of sympathetic nervous system activity. LPS, used successfully both in vivo and in vitro to drive plasmacytic differentiation of B cells, is a TLR4 and CD180 ligand (Ogata et al. 2000). Other TLR ligands are also known to drive plasmacytic differentiation, in particular the small molecule R848 and CpG oligonucleotides, acting through TLR7/8 and TLR9, respectively (Genestier et al. 2007; Philbin et al. 2005). By utilizing different TLR ligands to activate plasmacytic differentiation it may be possible to identify discrete differences between responses that provide insight into TCDD-mediated disruption of the primary IgM response. 91 TCDD Suppression of TLR-induced Antibody Secretion in CH12.LX Previous studies demonstrated that TCDD causes a concentration-dependent inhibition of the LPS-activated IgM response in CH12.LX (Sulentic et al. 1998), providing a useful in vitro model for elucidating intracellular mechanisms involved in TCDD disruption of the IgM response. The TLR ligands LPS, CpG, and R848 all cause increased secretion of IgM in CH12.LX cells (Bishop and Haughton 1986; Bishop et al. 2000; Krieg et al. 1995); however, the ability of TCDD to suppress IgM secretion activated by CpG and R848 treatment was unknown. Both CpG and R848 induced increases in IgM'secretion comparable to that observed with LPS treatment. TCDD caused a concentration-dependent suppression of the TLR ligand-induced IgM response i for all stimuli examined (Figure 16). Significant suppression occurred for all stimuli at concentrations of 0.3 nM TCDD and higher. LPS- and CpG-activated IgM responses had similar profiles of suppression caused by TCDD, with 3 nM TCDD causing a 72% decrease in LPS-induced IgM secretion and 83% decrease in CpG-induced IgM secretion. 3 nM TCDD suppressed R848-induced IgM secretion more moderately compared to other TLR ligands, declining 53%. 92 240 ~I-DMSO 1,721“ i-LPS : «g, 150 ~0-R848 g: ‘C 120 3. 9o ‘1 U) 5 so 30 [s A F—l— 0.0 0.1 0.2 0.3 0.4 2 3 TCDD Concentration (HM) Figure 16. TCDD Suppression of TLR-activated IgM Responses in CH12.LX B cells. CH12.LX cells (2x104 cells/mL) were activated with LPS (3 0 ,ug/mL), CpG (1.2 ug/mL), or R848 (0.2 ,ug/mL) and treated with 0, 0. 003, 0.03, 0. 3, 0r 3 nM TCDD in DMSO vehicle (0.035%). 48 h following activation culture supernatant IgM was determined by ELISA and normalized to total cell count. Results from triplicate determinations are represented as mean IgM per 106 cells i SEM. * indicates values that are significantly diflerentfi‘om activated cells treated with vehicle at p<0. 05. Results are representative of more than four experiments. 93 TCDD alteration of TLR-activated kinase phosphorylation Previous studies in CH12.LX cells established TCDD alteration in the LPS- activated DNA binding and transcriptional activity of AP-l (Schneider et al. 2009; Sub er al. 2002a) as early as two hours following treatment. Jun was identified as one component of the AP-l DNA binding complex, potentially resulting from TCDD disruption of LPS-induced translocation of c-Jun into the nucleus. It was hypothesized that TCDD disrupts LPS-activated plasmacytic differentiation by altering the profile of kinase phosphorylation resulting from TLR activation because c-Jun is directly regulated by JNK and BCL—6 is directly regulated by ERK. AKT, or protein kinase B, is phosphorylated within minutes of BCR activation (Donahue and Fruman 2007) and is also known to be phosphorylated in B cells following LPS treatment (Bone and Williams 2001; Hebeis et al. 2005). Furthermore, AKT is a negative regulator of the transcription factor FoxO, which in turn is a positive regulator of BCL-6 expression; therefore, activated AKT can indirectly decrease BCL-6 expression by inactivating FoxO (Omori et al. 2006). Because TCDD impairs both LPS-activated and BCR-activated primary IgM responses, it was further hypothesized that TCDD impairs TLR—activated AKT phosphorylation. Preliminary studies in primary B cells and CH12.LX cells established that LPS treatment stimulated modest phosphorylation of AKT, ERK, and JN K, consistent with the Observations of others (Krutzik et al. 2005). In the absence of B cell activation it is diffic‘ut t0 discriminate between normal response variation versus inhibition, therefore alternative B cell activation stimuli were sought. Since it had already been established that CpG and R848 activated IgM responses are suppressed by TCDD in CH12.LX cells 94 (Figure 16), their relative effectiveness for JN K phosphorylation was examined. Both the TLR9 ligand CpG and the TLR7/8 ligand R848 induced JN K phosphorylation on a level equivalent to PMA/ionomycin within 15 min of treatment (Figure 17). While 1’ MA/ionomycin is not a TLR activator, it is a robust pharmacologic mimic of BCR activation. Therefore, treatments that meet or exceed the stimulation induced by PM A/ionomycin are considered robust inducers of kinase phosphorylation. 95 878.97 1116.87 1566.27 1935.45 1752.29 Figure I 7. Relative TLR ligand .eflicacy for JNKphosphorylation. CH12.LX cells were activated by addition of LPS, PAM/Ionomycin, CpG or R848. JNKphosphorylation status was frozen 15 min following activation by direct addition of concentrated paraformaldehyde to each group. Following methanol permeabilization CH12.LX cells were stained for phosphorylated JNK and analyzed by FA CS. Histograms show the population distribution for JNKphosphorylation. Geometric mean fluorescence values are shown directly below the histograms for comparison of treatments. 96 FACS analysis of IN K phosphorylation demonstrated discrete activation patterns in response to TLR activation, particularly for R848. While LPS caused a 27% increase in JNK phosphorylation, PMA/ionomycin and CpG induced greater increases in IN K phosphorylation, 78% and 120% respectively. Viewed on a percent change basis, R848 appeared to be a more modest inducer of IN K phosphorylation when compared to CpG, increasing 99% over untreated CH12.LX. However, and only observable with FACS- based determination, R848 treatment generated two populations of CH12.LX cells, a majority population that did not phosphorylate JN K and a minority population, 33% of the total, that phosphorylates JN K to a high degree. Because kinases, particularly MAPK, act as amplifiers in cell signaling cascades (Asthagiri and Lauffenburger 2001) small perturbations can profoundly alter phenotypic outcomes. Increases or decreases in the level of kinase phosphorylation, and therefore activation, can decrease both LPS-activated Blimp-1 mRNA expression and plasmacytic differentiation (Lin et al. 2006). To assess whether TCDD alters JNK, ERK, or AKT phosphorylation, CH12.LX cells were activated with single TLR ligands in combination with O, 0.003, 0.03, or 0.3 nM TCDD in DMSO vehicle. Literature reports showing TLR ligand-induced kinase phosphorylation were used to select concentrations for stimulation of AKT, ERK, and JNK (Bishop et al. 2000; Yi and Krieg 1998; Yi et al. 1996). TCDD concentrations were selected on the basis of causing, respectively, no significant effect . 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AK cw cm 8 on on c _. one 28 . 82. r as - 8:. - 89 .. 82 r 8: r 82 (ueaw amawoas) eouasalonH Z/LMNI‘d (ueaw amauxoas) aoueseJonH leMNI‘d 822 + 959.. ad 11 Ram: mfi ...T 0.222 1.... 2282:: I1 3:; as: 8 on 3. on 2N or I b p D P mu: Ea: 2:: D I (ueaw omawoas) aouaselonls zmmrd ODD... 83 258.333 2:852 252.. BEEoimoE can u 22. .- 8m u 8: .- comp .. oomv 1 0°: :52 on 3. on on or . can cos 8: 82 comp 8: 82. (ueaw amewoes) eouasaJonH z/Lxm‘d 103 Single treatment with TCDD or LPS typically altered kinase phosphorylation less than 20% relative to untreated CH12.LX cells. Serum addition to cell cultures is known to cause greater phosphorylation of ERK and JN K than TCDD (Tan et al. 2002), consistent with the modest induction observed above. As a result, many studies of TCDD-activated kinase phosphorylation have been performed under serum-starved conditions. Since the studies described above were all performed in cultures containing 10% serum, any alteration in ERK and JNK phosphorylation caused by TCDD must be robust relative to effects observed in published studies of other researchers using reduced serum conditions. While the relative effects of LPS on kinase phosphorylation are modest, LPS treatment induced a significant increase in IgM secretion at later time point, illustrative of the point that kinases act as signal amplifiers. Indeed, TCDD treatment alone, even with modest changes in kinase phosphorylation observed in this study, activated plasmacytic differentiation of B cells, albeit at very modest levels relative to classic humoral response activators (Kramer et al. 1987). The kinetic and dynamic pattern of kinase phosphorylation resulting from TLR activation differs depending on the kinase eXamined and the TLR ligand applied. LPS treatment, as observed in preliminary studies, caused modest alterations in AKT, ERK, and JNK phosphorylation that peak at 60 min following treatment (Figure 18, Figure 19, and Figure 20). CpG treatment caused the largest increases in kinase phosphorylation, increasing as much as 132% for CpG + vehicle treatment at 30 min, and declining by 60 min post-activation. Kinase phosphorylation induced by R848 peaked earlier than other TLR ligands, increasing as much as 75% by 15 minutes post-activation, and declining at 30 and 60 min post-activation. 104 Viewed on a single parameter basis, effects of TCDD on TLR ligand-activated kinase phosphorylation are modest. TCDD at 0.003 nM did not significantly suppress kinase phosphorylation, consistent with a lack of immunosuppressive effects on the primary IgM response. TCDD alone caused no more than a 10% change in AKT, ERK, and JNK phosphorylation. ANOVA analysis of single parameters shows statistically significant impairments of kinase phosphorylation in 0.03 and 0.3 nM TCDD-treated CH12.LX cells relative to TLR ligand + vehicle treated cells. One of the strengths of flow r cytometry is that it allows simultaneous collection of information on kinase phosphorylation from each individual cell, but AN OVA is not the best tool for analyzing such data. Probability binning analysis, which is designed for analysis of multiparametric flow cytometry (Roederer et al. 2001a; Roederer et al. 2001b), comparing TLR ligand + DMSO to TLR ligand + TCDD for AKT, ERK, and JNK phosphorylation suggested higher TCDD concentrations significantly decreased kinase phosphorylation. TLR- activated CH12.LX cells treated with 0.03 nM or 0.3 nM TCDD shified at least 8 standard deviations (T00 greater than 8) away from the TLR ligand + DMSO treated groups. To further extend and validate the biological relevance of TCDD disruption for kinase phosphorylation, in vitro experiments using splenocytes were performed. Based on results obtained in the CH12.LX cell line, R848 was chosen as the B cell activation stimulus because the kinase phosphorylation elicited by R848 was intermediate in magnitude when compared to LPS and CpG. At 15 min post-treatment primary B cells were assessed for AKT, ERK, and JNK phosphorylation. 105 As depicted in Figure 21, R848 caused phosphorylation of both AKT and ERK for the majority of B cells. Probability binning analysis comparing DMSO and 30 nM TCDD treated B cells showed no distinguishable difference for phosphorylation of AKT, ERK, or JN K. TCDD at 30 nM significantly suppressed R848-activated phosphorylation ngh pERKHIgh comprised 51.3% of R848- of AKT, ERK, and JNK (T00 of 11.5). pAKT treated B cells, while 38.1% of R848 + TCDD cotreated B cells were pAKTngh r pERKngh (Figure 21). The reciprocal pAKTLOW pERKLOW populations were 26.3% of R848 treated B cells and 32.0% of R848 + TCDD treated B cells. Relative to pAKT and pERK, which increased 230.4% and 383.5% respectively, 5. pJNK increased by 94.8% relative to DMSO treated B cells. While R848 induced a more modest relative increase in pJNK, TCDD caused a measurable decrease in pJN K. R848 + TCDD cotreated B cells increased pJNK by 79.5% over DMSO, a difference of 15.3% from single treatment with R848. 106 €491! .. . ‘lwi. ll {li'fflll “wit-Inna.” étmfitmmxm m :35 382%» miEEmwmiE & EaQ .33 3:33.33 «on» \o .558 of E 2.29? 33 35.53% «33 2.23.: v.23 visage wmmStmEmK M53335 ,UQd Lo\mu:mwmx§~\o§5§ .23 3235.3 33.8% £336.32» v><~i .35 .vEma. HM VA Skhmmbama 22: .totfizmom Sfi emmm a - + .3 EmSEmE 39: £39 m vtfiihzc Vzmm KM Va «EUR .Qmmm gangs: :2: “33.33 39: £8 2923.2385me ufiiozok $33mESEQhESEStS\o 22:33 33% \S hkaEmi mmiootfimw. Bottom: #25» SE m. N .QQU& + acacia Essay: 8:338 3 a is? we fix: .3: an QQE 833% 9.:on 823% £82335: 3 .383: ES: 833:»wa 323mg £39 m REES S totfifigswém. auntie 22.23%.3 Qafifi «m Eami ll.l..7ul-:Inr.\!/..... I‘) q 107 Awwqx$mm€ me 42 me no; a: 48 m2 No3. A 4...... _ I. o . _ _ . we we “we . 2m . _ . “we QQUH 2d om+ wwwm wwwm ME 42 me No; man 42 me No; Mon 42 me No; R . Sm . : ......mda 3N . . mvd _ . . . 3o 3 EB 2.5m 82m 332:: 108 (Eld) lHVd 100 % of Max 3 4 5 0 1o 10 1o pJNK (Ax647) .. Treatment pJN K (GeoMean) - Untreated 62.63 W DMSO 63.69 TCDD 58.89 R848 124.05 R848 + TCDD 114.31 Figure 22. Eflects of R848 and TCDD on JNKphosphorylation in primary B cells. Isolated splenocytes were treated by simultaneous addition of DMSO vehicle, TCDD (30 "W, R848 (0.2 ,ug/mL), or a combined treatment of R848 + TCDD. 15 min after treatment splenocytes were fixed by direct addition of concentrated formaldehyde. Following permeabilization cells were blocked, then stained for 3220, TCRfl, pAKT: PERK and pJNK. B cells were identified as a BZZOJr TCR- population, then analyzed for pAKT: PERK, and pJNK expression. Results depicted are immunofluoresence for pJNK. Geometric mean fluorescence for individual treatments is shown below the histograms. Data Is representative of more than 2 experiments. 109 g Discussion of Results from In vivo and In vitro Murine Models for TCDD Disruption of TLR-ligand Activated Plasmacytic Differentiation In vitro models are valuable tools for examining intracellular events, but without the context of the whole animal limited information is gleaned. Science is well served when in vitro and in vivo models are used as complementary tools to develop basic understanding of life processes, thus verifying in vitro observations using in vivo models helps illuminate fundamental biology at work. Along those lines, utilization of the T- independent stimulus LPS for initiating a primary IgM response in vivo complemented in vitro LPS-activation models of humoral immunity. The kinetics of both the in vivo and in vitro response are similar, peaking 3 to 4 days following activation. Unlike sRBC-elicited responses that require interactions between multiple cell types, LPS can directly stimulate B cells. Direct stimulation increases the number of activated B cells during the initial phase of the immune response, potentially increasing the ability to detect effects occurring early in B cell activation that lead to suppression of the primary IgM response. Finally, to maintain similar intracellular signaling programs leading to plasmacytic differentiation it is important to utilize comparable activation stimuli. In assimilating the characteristics of the LPS-stimulated primary IgM response, the in vivo and in vitro models are highly compatible for exploring effects of TCDD on the B cell. In examining in vitro events implicated in TCDD-mediated suppression of the primary IgM response using an in vivo T-independent humoral immune response many results were found to be consistent between models. TCDD modestly attenuated LPS- induced increases in splenocyte number, accompanied by profound suppression of the IgM response at all concentrations of TCDD evaluated. Assessment of multiple 110 phenotypic endpoints associated with AF Cs confirms that TCDD prevents LPS-induced increases in XBP-l and all components of pentameric IgM. The observations that CD138 expression, IgJ, and ng production are suppressed advance our understanding of TCDD’s mechanism of action, as previous studies have not directly shown suppression of intracellular antibody production on a single cell basis, and as such could never conclusively establish whether TCDD suppression of the primary IgM response resulted from suppression of antibody secretion and production or the differentiation of B cells into AF C. TCDD impaired LPS-stimulated up-regulation of Blimp-1, a transcription factor established to be a central regulator of B cell differentiation. Collectively, these results strongly support the hypothesis that TCDD impairs B cell to plasma cell differentiation. In agreement with moderate attenuation of LPS-stimulated increases in cell number observed in this in vivo study, TCDD treatment modestly decreased proliferation, as measured by [3H] thymidine incorporation, in isolated splenocytes activated in vitro with LPS (Morris et al. 1993). A minor decrease in proliferation is insufficient to explain the magnitude of suppression in the IgM response observed in the present study, especially since the IgM AFC response was normalized to cell number. However, the observed effect on decreased spleen cellularity was consistent with the possibility that an event upstream of antibody production is impaired by TCDD. Disruption in the production of any single lg component or protein important in antibody secretion impairs the primary IgM response. With that knowledge, in vivo studies examined expression of all immunoglobulin components. Expression of Igu and ng are more modestly inhibited than the IgJ chain, and may provide insight on why the 111 IgM response is highly sensitive to suppression by TCDD. B cells must express antigen receptors on their surface to receive survival signals (Rajewsky 1996), hence the high constitutive levels of both 1gp and ng. TCDD inhibition of B cell antigen receptor components may result in deletion of some B cells, preventing detection of some affected B cells using current techniques. Additionally, suppression of Igu and IgK expression can be expected to impair expression of all forms of immunoglobulin, whereas the IgJ chain is essential only for IgM and lgA classes of immunoglobulin. TCDD strongly attenuates IgJ chain expression and can therefore be expected to impair markedly the assembly of pentameric IgM, an outcome verified by TCDD-mediated decrease in the AFC response. It is also plausible that less striking effects of TCDD on LPS-induced ng and Igu expression are due to the high constitutive level of both IgK and Igu. Impaired expression of Ig components disrupts antibody synthesis, but B cells must also resolve endoplasmic reticulum stress resulting from increased lg protein synthesis necessary for robust secretion of antibody. Following activation B cells up- regulate expression of XBP-I mRNA, which can be spliced to an alternative mRNA - translating to full length XBP-l , a transcription factor essential for resolution of the unfolded protein response (Reimold et al. , 2001). Consistent with an interpretation that TCDD blocks plasmacytic differentiation upstream of antibody production and secretion, TCDD treatment reduced both total and spliced XBP-I expression in LPS-treated mice (Figure 5). In addition, with the recent establishment of XBP-l as a regulator of BCR signaling (Hu et al. 2009) and interacting with BOB-1 (Shen and Hendershot 2007), another key regulator of plasmacytic differentiation, it is even more apparent that TCDD- 112 mediated disruption of XBP-l expression can feed back to alter early events in plasmacytic differentiation. Importantly, recent studies of Blimp-l conditional knockout mice and identification of XBP-l as a regulator of hepatic lipid metabolism provide interesting, indirect links for two well-recognized human TCDD toxicities, chloracne and hepatosteatosis, to Blimp-1 expression. Magnusdottir and coworkers described a keratinocyte-specific knockout of Blimp-1 in mice resulting in a phenotype remarkably { similar to the most consistently observed human health outcome produced by high dose TCDD exposure, chloracne (Zugerman 1990). Blimp-1 knockout in keratinocytes resulted in a sequela termed by the investigators as “a cyst-like structure that proved to *L be filled with lipid” in the skin of adult mice (Magnusdottir et al. 2007). If TCDD inhibits Blimp-1 expression in keratinocytes similarly to B cells, then a shared mechanism for TCDD toxicity involving deregulation of Blimp-1 in different tissues becomes more plausible. Along these lines, XBP-l, a transcription factor that is indirectly controlled by Blimp-1 in B cells, is a regulator of lipid homestasis in the mouse liver (Lee et al. 2008). Human serum dioxin-like chemical levels are associated with hepatosteatosis (Lee et al. 2006), and furthermore, TCDD induces hepatosteatosis in mice (Boverhof et al. 2005). Coupling the knowledge that TCDD reduced XBP—l expression and is downstream of Blimp-1, it is tempting to speculate that TCDD-induced deregulation of Blimp-1 is a key event in some of the toxicities produced by TCDD. Separate from their function in antibody synthesis and secretion, B cells change surface protein expression during transition into ASCs. Abundance of surface CD19, MHC Class II, and B cell antigen receptor all decrease during the later phase of 113 plasmacytic differentiation, while surface expression of CD138 is up-regulated (Fairfax et al. 2007). CD138 has been commonly used as a marker to enumerate plasma cells, and is a complementary phenotypic marker not directly involved in antibody secretion. LPS and TCDD cotreated mice were impaired in their ability to generate CD19+ CD138+ cells compared to mice treated with LPS in the absence of TCDD. Cell surface activation markers such as MHC Class II, CD69, CD80, and CD86 f initially increase following stimulation during the activation phase of plasmacytic : differentiation. TCDD suppression of MHC Class II upregulation in response to LPS treatment was observed in vivo. In vitro examination of a more comprehensive panel of activation markers following LPS activation demonstrated that TCDD suppresses more ! than just MHC Class II. The significant impairment of CD80 and CD86 may be particularly important to understanding the profound suppression of primary humoral immunity caused by TCDD. The role of CD80 and CD86 is to provide costimulatory signals to T cells, which in turn stimulate B cells via cytokine release. Because the primary IgM response typically requires interaction between B and T cells, a failure of B cells to provide sufficient stimulation to T cells would result in a spiraling failure of costimulation, eventually resulting in considerable suppression of the humoral response. Evidence indicates that TCDD interferes with LPS-induced Blimp-1 expression, and taken collectively, points toward TCDD-mediated alteration of a fundamental control switch during the primary IgM response. Blimp-1 expression is required for the generation of plasma cells (Shapiro-Shelef et al. 2003), thus failure to up-regulate Blimp- 1 expression leads to decreased plasma cell appearance, consistent with TCDD-mediated suppression of both IgM AFC response and in the frequency of CD19+ CD138+ B cells. 114 Direct evidence for deregulation of Blimp-l expression in vivo comes from the ‘ + observations that TCDD treatment dose-dependently decreased the frequency of CD19 elevated Blimp-1 B cells and Blimp-1 mRNA abundance in LPS-treated mice. In vitro studies of purified B cells replicated in vivo suppression of Blimp-1 expression, and were also able to extend the effects of TCDD to an increase in BCL-6 expression. Based on Figure 3 a sustained expression of BCL-6 will, in the long term, prevent Blimp-l F expression. BCL-6 is also known to be a direct negative regulator of CD80 expression 9 (Niu et al. 2003), thus the elevated levels of BCL-6 observed 48 and 72 h following TCDD treatment in vitro provide a partial explanation for the significant impairment of ‘FT‘. ."' -".1 CD80 expression at 48 and 72 h in LPS + TCDD treated B cells. AP-l plays a fundamental role in initiating plasmacytic differentiation by activating Blimp-l transcription (Ohkubo et al. 2005). In vitro treatment of B cells with TCDD significantly impaired LPS-activated phosphorylation of c-Jun, one component of the AP-l transcription factor. AP-l DNA binding in LPS-activated CH12.LX is almost completed abrogated by 10 nM TCDD 48 h post-treatment (Schneider et al. 2009), much more than what would be expected based on the impaired expression of phosphorylated c-Jun caused by 30 nM TCDD in primary B cells. While a portion of this difference is attributable to differences in model systems such as AHR expression, BCL-6 may be another important factor. Simple abundance of AP-l components likely underestimates the effect on AP-l-mediated transcription because BCL-6 can directly bind and impair AP—l activity (Vasanwala et al. 2002). It is likely that AP-l transcriptional activity is significantly impaired given the observation that TCDD both inhibits c-Jun phosphorylation and increases BCL-6 expression. 115 Comparing the in vitro LPS + TCDD to LPS + DMSO treated cells by probability binning showed that T00 values for both transcription factor and activation marker expression correlate with the magnitude to which the IgM response was suppressed. TCDD at 0.03 and 0.3 nM caused less LPS-activated IgM response suppression than at 3 and 30 nM, which was similarly reflected in the T(x) values showing 0.03 and 0.3 nM TCDD clustering nearer to LPS + DMSO treatment than 3 and 30 nM TCDD. Application of more sophisticated statistical analysis tools to multiparametric FACS F! allowed for better discrimination of TCDD effects by correlating changes for multiple proteins within a single cell, which was previously unachievable using single parameter ! analysis. L5 TCDD treatment consistently suppressed TLR-activated IgM responses in CH12.LX cells, and a similar pattern for TCDD suppression of kinase phosphorylation was observed regardless of the activating TLR ligand. While TCDD was less effective at suppressing the R848-activated compared to LPS- and CpG-activated IgM responses, TCDD at 0.3 nM consistently suppressed kinase phosphorylation 10-20% regardless of the TLR ligand. There is likely to be additional factors beyond kinase phosphorylation that contribute to the differences between R848 and non-R848 stimuli in TCDD-mediated suppression of the IgM response. Kinases are key regulators of AP-l activation preceding plasmacytic differentiation. Immunosuppressive concentrations of TCDD decreased TLR-activated phosphorylation of the kinases AKT, ERK, and JNK in CH12.LX cells (Figure 18, Figure 19, and Figure 20). Primary B cells were also found to be sensitive to TCDD-mediated disruption of TLR-activated kinase phosphorylation (Figure 21). TCDD impairment of 116 LPS-activated ERK and JN K phosphorylation provide novel mechanistic insight into alterations in BCL-6 and phosphorylated c-Jun expression observed in vitro. The observed increase in BCL-6 abundance caused by TCDD (Figure 10) is consistent with impairment of ERK activation, as active ERK phosphorylates BCL-6 leading to ubiquitination and degradation of BCL-6. JN K derives its name from one of its best characterized activities, the phosphorylation of c-Jun. Because TCDD impairs TLR- activated JNK phosphorylation, and therefore activity, TCDD suppression of phosphorylated c-Jun abundance is a natural consequence. ERK plays an important role in differentiation processes beyond plasmacytic differentiation of B cells. Like plasmacytic differentiation, adipocytic differentiation is L. suppressed by TCDD. Treatments that induce ERK phosphorylation reversed TCDD suppression of adipocytic differentiation, leading the authors to speculate that TCDD suppression of differentiation requires a decreased activation of ERK in order to observe suppression (Hanlon et al. 2003). Results in Figure 19 demonstrated that TCDD treatment significantly impaired TLR-activated ERK phosphorylation, and coupled with the observations of Hanlon et al, suggest that TCDD impairment of ERK phosphorylation may play an important role in suppression of cellular differentiation in multiple cell types. TCDD-mediated suppression of kinase phosphorylation may provide a partial explanation for the window of sensitivity that exists for in vitro disruption of the primary IgM response. Kinases in B cells respond rapidly to immunological stimuli, but the peak signal typically decays rapidly back to background level within hours of the initial activation (Irish et al. 2006). If the initial signaling cascade caused by TLR or BCR 117 activation has cycled from peak activation back to basal levels, and activated downstream transcription factors such as AP-l and NFKB, the window of sensitivity may be closed for TCDD. The reported window of sensitivity for the LPS-activated IgM response is 3 h (Holsapple et al. 1986). ERK phosphorylation in B cells returns to background levels within 3 hours of LPS or CpG treatment (Banerjee et al. 2006). Integrating those observations, it is tempting to speculate that the window of sensitivity for TCDD disruption of the IgM response is attributable, at least in part, to alterations in kinase F phosphorylation. Observations described above, including TCDD suppression of kinase phosphorylation, disruption of transcription factor expression controlling plasmacytic I! differentiation, and impaired expression of activation markers could all individually impair humoral immunity, yet TCDD impairs normal TLR-activated expression for all. Furthermore, TCDD-activated AHR directly regulates the Igu 3’ a enhancer (Sulentic et al. 2004b), an event in the IgM response distal to Blimp-1 up-regulation during plasmacytic differentiation. Interference in both the initiation of plasmacytic differentiation, maintenance of costimulation necessary for humoral immunity, and Ig expression provides a partial explanation for the high sensitivity of B cells to TCDD. Taken together, the cumulative mechanism for TCDD-mediated suppression of the IgM response is multifaceted. Experimental results presented in this chapter provide compelling evidence that TCDD impairs the primary IgM response via a block in plasmacytic differentiation upstream of Blimp-1 expression both in vivo and in vitro, not simply at the level of antibody production. Up-regulation of several phenotypic indicators for AF Cs, including 118 CD138 and intracellular levels of Igrc and IgJ, were impaired by TCDD exposure. In vitro studies aimed to expand on results observed in vivo demonstrated both impaired activation marker expression and reduced phosphorylation of multiple kinases resulting from TCDD treatment. Collectively, these results validate and enhance the value of mechanistic in vitro studies establishing TCDD-induced deregulation of the lgu 3’ a enhancer (Sulentic et al. 2004a; Sulentic et al. 2004b), Pax5 (Schneider et al. 2008), and Blimp-1 (Schneider et al. 2009), and proceed to expand potential mechanisms for TCDD suppression of humoral immunity. 119 CHAPTER 4: EXPERIMENTAL RESULTS AND DISCUSSION FOR HUMAN MODELS Development of a Human in vitro model for TCDD Disruption of the LPS-activated Primary IgM Response Research efforts dedicated to elucidating the mechanism of TCDD immunotoxicity have used primarily rodent models, particularly the mouse. Unfortunately, the many significant advances in understanding the molecular biology underlying AHR function in the mouse have failed to produce information that can conClusively establish the health threat posed by TCDD and DLCs to human health. In vivo experimental evaluation of TCDD effects on humans is not ethical, however, availability of human tissues for in vitro experimentation presents the opportunity to validate that mechanisms of TCDD toxicity observed in mouse experimental models occur similarly for in vitro human experimental models. While sources of primary human immune cells are commercially available there are several difficulties in their research application. Human immune cells tend to be expensive to obtain and process into useful experimental material. The purification required for modeling the direct effects of TCDD on human B cells results in significant cell loss during isolation. Due to these losses it is often difficult to obtain a sufficient number of cells for techniques such as immunoblotting, Northern Blot, or electromobility shift assays in individual donors. Additionally, the human population is likely to be polymorphic for both AHR and cell signaling components necessary for a functional primary IgM response, leading to increased variability of responses, and therefore uncertainty in analysis. Transgenic DNA alterations and introduction of foreign DNA 120 into primary human cells is difficult or impossible. Finally, unlike mouse splenic B cells, human peripheral blood B cells are not stimulated by LPS treatment to differentiate into I ASC, making it impossible to make useful comparisons of LPS treated mouse cells to human cells. Initial characterization of the SKW 6.4 cell line An alternative approach to the use of primary human B cells circumventing many of the above limitations is use of immortalized human cell lines. Cell lines provide a theoretically unlimited supply of cells with homogeneous genetic makeup and responses, opening the possibility of performing analyses and manipulations that are impractical with primary cells. While many human B cell lines are available for research, few fulfill the prerequisites to make a useful model of the primary IgM response. Human B cells must, at the very least, increase secretion of IgM antibody in response to LPS treatment and express the AHR in order to compare TCDD effects on LPS activated mouse B cell responses to human B cell responses. The SKW 6.4 cell line, a subclone of the Daudi cell line identified for its low background IgM secretion under normal conditions with the capability for increased IgM secretion in response to B cell differentiation factor (Ralph et al. 1983), was found to respond to LPS treatment by increasing the secretion of IgM approximately 2-3 fold compared to vehicle treated cells (Figure 23). Immunoblot analysis of SKW 6.4 cell lysates showed that these cells to not express AHR (Figure 24) protein, further confirmed by a lack of AHR mRNA detected by QRT-PCR. 121 40- ** __,_ A30- _l E 2’20- 2 U) -10- o- . (D D. .1 Vehicle Figure 23. LPS stimulates IgM secretion in SK W 6.4 cells. [x1 05 SK W 6. 4 cells were stimulated by addition 0f100 ,ug/mL LPS. Following 96 h incubation IgM secretion was quantified by human IgM ELISA. Results depicted are mean IgM i standard error of the mean. ** P<0.01 as determined by 1 way ANOVA with Dunnet s post-hoe analysis comparing vehicle to LPS treatment. 122 Figure 24. Immunoblot analysis of AHR expression in SK W 6. 4 cells. Total cell lysates fi‘om HepGZ (10 ,ug) and SK W 6.4 (100 pg) cells were assessed for human AHR expression. Lane 1 contains protein from HepGZ cells. A clear band at approximately 96 kilodaltons, the expected size of full length human AHR, is present in HepGZ cells. Lanes 2-5 contain cell lysatesfi'om SK W 6.4 cells. T his figure includes handwritten/drawn annotation. 123 While several approaches to enforced AHR expression were possible, the stable genomic integration of constitutively expressed human AHR was the most desirable outcome based on the expected consistency of results and cost of maintenance. The first attempt at generation of AHR expressing SKW 6.4 cells, called SKWA, was performed using the plasmid pcDNA3.l-TOPO with the open reading frame (ORF) for human AHR under the control of a Cytomegalovirus (CMV) promoter, a highly active promoter in most cell lines. 48 h post-transfection with pcDNA3.l-TOPO-HsAHR G418 antibiotic, which is inactivated by the neomycin resistance gene present within pcDNA3. 1_-TOPO- HsAHR, selection was initiated. Generation of G418-resistant cells required three weeks, after which the polyclonal pool of stable transfectants was screened for AHR gene I; expression by QRT-PCR. No AHR mRN A was detected. Further analysis of the polyclonal G418-resistant SKW 6.4 cells showed high levels of the neomycin resistance gene, demonstrating that the plasmid DNA had become stably integrated into the genome and was expressed, but AHR was not. One potential cause for the failure of pcDNA3. l-TOPO-HSAHR to generate AHR expressing cells is the construction of the plasmid. To produce functional AHR an active promoter, complete cDNA sequence, and complete polyadenylation sequence must be present. The sequence beginning the CMV promoter of pcDNA3.l-TOPO-HSAHR through the end of the polyadenylation signal is approximately 4.5 kilobases. Given the entire vector is approximately 8.1 kilobases, the majority of the plasmid consists of components necessary for expression of AHR. When supercoiled plasmid DNA is transfected into any cell line it must first be linearized prior to genomic integration. The “break site” at which the plasmid linearizes occurs randomly. Accepting that the DNA 124 break site is random, and considering the size of the sequence necessary for AHR expression, it is most likely the break occurs in some portion of the sequence necessary for AHR expression. Based on the design of the plasmid, all the selection‘pressure is placed on the neomycin resistance cassette, with no advantage to expression of AHR. In such a situation, the likelihood for generating antibiotic resistant cell lines that do not express the gene of interest is greater than the likelihood of obtaining a cell line expressing the gene of interest, especially if there is a disadvantage to transgene h‘ expression. Alternatively, another possible explanation for, the lack of AHR expression in G418-resistant SKW 6.4 cells is a phenomenon known as “promoter neighboring”, in U' which a highly active promoter recruits transcriptional machinery, outcompeting neighboring promoters for gene expression (Kimberland et al. 1999). To circumvent this issue an internal ribosome entry site (IRES) allows expression of two proteins from a single mRNA transcript. Use of an IRES addressed both the randomized break site issue and promoter neighboring effects that may have caused the failure of the pcDNA3.l- TOPO-HsAHR vector. Unlike the pcDNA3.l-TOPO-HSAHR, IRES-based vectors allow antibiotic selection pressure to be placed on both AHR and neomycin resistance gene expression simultaneously, thus the cell line cannot express the neomycinresistance cassette without also producing AHR mRNA. The ORF for human AHR was cloned into pIRESneo3 vector, generating a CMV driven AHR expression vector in which AHR and neomycin resistance should be coexpressed. SKW 6.4 cells were transfected by electroporation with pIRESneoB-HSAHR and selected with G418. This method did not produce any stable transfectants in four separate trials. 125 Following the failure of pIRESneo3-HsAHR to generate a stable transformant population it was suspected that the CMV promoter may be ineffective in the SKW 6.4 cell line. The G418 resistant SKW 6.4 cells generated using the pcDNA3.l—TOPO- HsAHR plasmid used the SV40 promoter to drive expression of neomycin resistance. A SV40 driven protein expression plasmid, pZeoSV2, was obtained based on the prior knowledge that SV40 was a sufficiently active promoter to drive expression of the neomycin resistance gene. No drug resistant colonies were ever obtained using pZeoSV2- HsAHR despite multiple attempts to generate antibiotic resistant populations. To determine relative promoter activity in SKW 6.4 cells several different luciferase reporter plasmids were obtained,each containing alternative forms of CMV or a CMV/B-actin chimeric promoter coupled with different polyadenylation signals (SV40 or Bovine Growth Hormone). SKW 6.4 cells were electroporated and luciferase activity was measured at 24, 48, and 72 h post-electroporation. Figure 25 demonstrates that CMV promoters have relatively strong activity in the SKW 6.4 cell line, which can be modestly altered depending on the polyadenylation sequence used. Additionally, even with single transient transfection sustained activity of the CMV promoter is observed. 126 100000 .00.... \ 1000 __ Luciferase Actlvlty (counts per second) 100. 20 ‘2'5 3'0 3'5 4'0 42 Hours Post Transfection 50 -I- pGL4.13 SV40 Promoter + SV40 PolyA -— pGL3 Promoterless -o- pCMV SV40 PolyA -o- pCMV BGH PolyA -I- pCMV SV40 PolyA & Enhancer Figure 25. Relative Promoter Activities in SK W 6.4 cells. SK W 6.4 cells were electroporated with equal quantities of plasmid DNA and assayed for luciferase activity 24 and 48 h post-transfection. 127 Based on the observation that CMV promoters generated relatively high luciferase reporter activity and robust GFP expression from transient transfections in SKW 6.4 cells, a new expression vector was constructed utilizing a highly active form of CMV. The promoter of the pmaxGFP vector used in transient transfection of SKW 6.4 cells to assess transfection efficiency is a CMV promoter coupled with intron A, a modification of the CMV promoter that drives higher transcriptional activity than other CMV promoters lacking intron A (Chapman et al. 1991). By selecting a highly active promoter and fusing AHR to GFP it was believed that the probability of successfully generating AHR expressing SKW 6.4 cells was improved. GFP was cloned in-frarne with the C- terminus of AHR in the vector phCMV-CGFP by mutating the native stop codon of AHR to a leucine residue, with the resulting ORF generating a GFP-fused form of AHR. A nonfused form of AHR was also generated, leaving the normal AHR stop codon intact. phCMV-HSAHR and phCMV-HsAHR-GFP were individually electroporated into separate pools of SKW 6.4 cells, then following 72 h recovery, treated with G418 to select stable cell lines. No cell lines containing AHR-GFP were ever identified in three separate electroportations. Two pools of G41 8 resistant phCMV-AHR electroporated cells were identified following 6 weeks of culture and screened for AHR mRN A by QRT-PCR (Figure 26). Both pools contained cells expressing detectable levels of AHR mRNA, but following the recovery of frozen stocks AHR mRN A could not be detected. 128 Expression of AHR mRNA in wild-type and AHR-transfected SKW 6.4 cells 10000 - Ct=28.3 7500 . Ct=28.9 5000 - 3 - 2- 1- Fold Change Compared to untransfected SKW 6.4 0- ‘28. V128. ht 6‘ 6' s“ 9°“ Figure 26. AHR expression in G418-resistant pools of SK W 6.4 cells. SK W 6.4 cells were electroporated with phCMV—AHR plasmids. Following 72 h recovery cells were grown in the presence of 1.2 mg/mL G418for 6 weeks. G418 resistant cells were assessedfor A HR mRNA expression by QRT—PCR. For graphing purposes the Ct value for AHR expression in native SK W 6.4 cells was set at 40. 129 Following the failure of plasmid-based vectors to generate sustainable populations of AHR expressing cells an alternative approach to integration of DNA into host genomes was utilized, in which lentiviral transduction of SKW 6.4 cells with replication incompetent virus consisting of an expression cassette for a single mRNA containing both HsAHR and puromycin resistance (PuroR) was performed. Lentiviral genomes are randomly inserted by the enzyme Integrase into open regions of chromatin, and unlike supercoiled plasmid DNA, without a requirement for linearization by random breakage prior to integration. Recognizing that CMV sequences differ between vendor plasmids, the CMV with intron A promoter was selected based on the knowledge that the promoter had provided transient AHR expression in the phCMV-HSAHR vector. DNA for the CMV sequence containing intron A combined with either HsAHR or HsAHR-GFP was PCR amplified from phCMV-based vectors and cloned into the pLEX-MCS vector after removing the vendor supplied CMV promoter without intron A. Initial evaluations of lentivirus produced from the pLEX-CMVi-HSAHR and pLEX-CMVi-HsAHR-GFP showed very low titers of infectious virus, from 500-1000 transfection units per mL. This contrasted sharply with titers of GFP virus obtained from pLEX-Jred/T urboGF P, ranging from 3x106 to 8x106 transfection units per mL. In consultation with OpenBiosystems technical support it was learned that the inclusion of intron A provided a splice acceptor site in the mRNA produced during viral production. The w packaging sequence of HIV, the parental virus for the pLEX-MCS vector, contains a strong splice donor. During viral production the splice donor finds the splice acceptor in the intron and excises itself, resulting in removal of both the w packaging sequence and CMV promoter for AHR 130 expression. The final outcome is low production of functional lentiviral vector (John Wakefield, personal communication). Following the explanation for the low viral production the pLEX-based vectors were rederived by excising the HsAHR and HsAHR-GFP sequences from phCMV and inserting them into pLEX-MCS to generate new transfer vector plasmids lacking intron A. Newly generated lentiviruses, pLEX-HSAHR and pLEX-HsAHR-GFP, were used separately to transduce SKW 6.4 cells. Following puromycin selection, stable pools of transformants were identified and screened for GF P expression by FACS (Figure 27), and termed SKWAG to indicate “SKW 6.4 expressing AHR-GF P”. Following puromycin selection, GFP expression occurred in more than 95% of all cells. 131 v5: (mun-Z— J" .M-Q%&-Q.m .2? A5333 of M22: @2833 933 $8 m33>2o2 .QEES. Q N no .m. .M. .2 $353238 wrotoézmoroo MEALS, S 9.58%me 3 329:3. 933 $8 333.3335 NEEQEEIGANNQ .88? 23.2398 22.5.:sz o 5.3» 38355.: 39: when w 6 ACE 628 #6 Eva. 33553: E :23388 EKG NN Emmi l 4.. .IIIII... F!‘ 132 3-95-05 gist, . M: 8. o a? 2.3 86 5.8 8.0 93. A: mv<>>v~m m U<>>Mm m U<>9Mm m? :..m cum I . \ 85 $3 So 86 - M ©<>9M m To BMm @3me ( oouosorom :1 (1:19) dch'HHV 133 Pools of puromycin resistant cells were grown in the presence of different concentrations of puromycin. Increasing antibiotic concentration allows crude control over expression of the antibiotic resistance gene, and by proxy the expression of AHR. Different pools of SKWAG were given the suffix 0.5, 1, 3, 5, or 10 to indicate the concentration of puromycin in which they were selected. Following positive F“: identification of a GFP+ population SKWAGIO cells, antibiotic selection pressure was removed and cells rescreened by FACS one week later. Removal from puromycin selection resulted in a temporary increase in GP P expression, believed to be indicative of { AHR, in SKWAG] 0 cells (Figure 28). Extended maintenance in the absence of "F puromycin resulted in progressive loss in GP P fluorescence (Figure 29), potentially resulting from silencing of lentiviral DNA following removal of selection pressure. This finding led to the decision to maintain cell lines in continuous puromycin. Following several weeks in culture a polyclonal pool of SKWAG cells arose, which was in turn screened for AHR expression by QRT-PCR. Despite relatively high levels of GFP fluorescence, no expression of AHR was detectable in any SKWAG cell lines. 134 'H.-— 1 llllllll I IIIIIIII I Illlllll ll‘llllll 100 101 1O2 103 GFP Fluoresence (AHR-GFP Expression) , TUBE NAME . ”Geom. Mean , FL1-H ,. Native SKW 6.4 5.65 SKWAG 10 with Puromy... 239.93 SKWAG 10 without Puro... 981.20 Figure 28. GF P expression in the presence and absence of puromycin. SK W 6.4 and SK WAG] 0 cells lines were evaluated for GFP fluorescence byflow cytometry. SK WAG 10 cells were grown in the absence of puromycin for 48 h and compared to SK WAG 10 cells grown in the continuous presence of puromycin. Native SK W 6.4 cells are presented as an indicator of background fluorescence. 135 5.34 GFP Expression '5' 0 200 400 600 800 1000 Cell Size Figure 29. GFP expression loss following 1 week of culture in the absence ofpuromycin. SK WAG] cells were removed from puromycin selection 1 week prior to sorting by FA CS. GFP+ cells were identified based on GFP fluorescence greater than 99% of native SK W 6.4 cells. 136 A second polyclonal cell line was derived from the HsAHR-GP P lentivirus using 293T cells, called 293T-HAG. Immunoblot of cell lysates from 293T-HAG, in contrast to the experience with SKWAG, demonstrate high expression of a protein of approximately 120 kDa that reacts with AHR specific antibodies (Figure 30), confirming the production of a GFP fused form of AHR. Immunoblot analysis also demonstrated multiple smaller bands in 293T-HAG that are detected by the anti-human AHR antibody used, suggestive of either AHR degradation or alternative splice products in the polyclonal 293T-HAG pool. Several SKW 6.4 cell lines transduced or transfected with AHR expression vectors were also assessed, but none were found to express significant amounts of AHR or AHR- GFP. Development of 293T-HAG cell line provided the material to develop a F ACS- based AHR detection method, allowing for more rapid determination of AHR expression in a pooled population. Additionally, if a minority population of SKWAG cells exists expressing AHR-GFP and is at a growth disadvantage relative to SKWAG cells expressing GFP alone it may not be possible to discriminate between populations in an immunoblot, whereas this can be immediately detected using F ACS. 137 Figure 30. AHR immunoblot in 293 T-IMG HepGZ, and SKW 6.4 cell lines. Total cellular protein was separated by SDS-PA GE and probed for AHR protein by immunoblot. From left to right lane assignments are as follows: 293T-HAG HepGZ, native SK W 6. 4, SK W 6.4 transduced with pTZV-HsAHR-GFP, SK W 6. 4 transfected with phCMV-HsAHR, SK WA G1 cells, and SK W 6.4 transduced with pTZV-HSAHR. The single band in lane 2, HepGZ, corresponds with human AHR of approximately 96 kDa. 138 While 293T cells natively express AHR, increases in AHR expression detected by F ACS are detectable as increases in immunofluoresence. Figure 31 demonstrates AHR expression is relatively consistent within 293T-GFP cells. Analysis of 293T-HAG cells shows AHR immunofluoresence increases correlate with GFP fluorescence, confirming both the effectiveness of F ACS for AHR detection as well as fusion of AHR to GFP (Figure 31). Following development and validation of the FACS—based AHR detection the screening of polyclonal SKWA and SKWAG cells was initiated. Figure 32 shows AHR immunofluoresence in SKWAG cells, shifted along both the X- and Y-axes of the graph as expected for AHR-GFP expressing cells, with over 11% of the screened cells viable and AHR expressing. However, the majority of the SKWAG cells were dead, and for the SKWA cell line less than 9% of cells were viable. Dead cell populations for both the SKWA and SKWAG cell lines are AHR positive, and were slightly, though nonsignificantly, higher in AHR immunofluoresence than their viable counterparts. The loss of viability in cultures expressing AHR could be explained by potential toxicity associated with expression of HsAHR (Craig Rowlands, personal communication). 139 293T-GFP 293T-HAG GFP Fluorescence AHR Fluorescence Figure 31. Correlation of AHR immunofluoresence with GFP fluorescence in 293 T cell lines. 293 T human kidney cells were transduced with pLEX-GF P or pLEX-HsAHR-GFP lentivirus. Following establishment of a puromycin resistant cell lines intracellular AHR expression was assessed by FA CS. 140 MU wk NE mozommxozKotzfifim «EV aoaxhomeowmo 55 Amman? onEoEEe. atotfizmom $3.3m? :.BAEQSR it EIGNNQQ. .5 mkbimvamwfiwum 5b: .3833.th 89: 38 To ACE 5:333:02 Sr: 383th 2023.5?» six NM. 3:me l4l Gonna E5v 3:233 at... an..- 5.... .0»..- .mm__.____ we... £3. .04.]... a”? _ we. a? .02 J \\ /\ D j, \ t L41 -2: mo . rod / omém wtdw N . -oomm I / m J \ , oom averse AQQOZoV SENS __ ....flw.t__,_ “a..- E... E... E. . V H H H X d l 9 w -mow W E? + E 3% $0-5? + so 3% . we 3% 2E2 3 ‘ 142 With the knowledge that the lentiviral HsAHR-GFP vector produced the desired protein, but not in SKWAG cells, it was clear that previously unanticipated difficulties in the generation of stable B cell lines were present. Transfer plasmid identity had been verified by restriction digestion prior to viral production, and the successful generation of + . . . . . GFP and puromycm resrstant cell llI‘lCS demonstrated packaged lent1v1rus was competent for gene transfer. However, the actual composition of the lentiviral pool had not been examined. Several methods for measurement of lentiviral vector exist, including QRT-PCR based techniques (Sastry et al. 2002). pLEX-HSAHR and pLEX-HsAHR-GFP lentiviral vector RNA was isolated and assessed by QRT-PCR for AHR, GFP, or puromycin resistance genes. Since the lentiviral genome is a single strand of mRNA, if a single form of virus is produced then equal quantities of each gene should be detected. QRT-PCR analysis of separate lots of virus revealed that quantities of viral genes, putatively on the same single mRNA strand, were not equal. After subtracting Ct values obtained from non-reverse transcribed samples, the pTZV-HsAHR-GFP lentivirus was found to contain no AHR mRNA but did contain significant quantities of GFP mRNA. Plasmid DNA for the pTZV-HsAHR-GFP vector was verified by QRT-PCR to contain equal copy numbers of AHR, GFP, and puromycin resistance genes. After subtracting Ct values obtained from non-reverse transcribed samples, pLEX-HsAHR-GFP-20080707 contained no lentiviral RNA. Some lots of virus, pTZV-HSAHR, pLEX-HsAHR-200806l3, and pLEX-HSAHR- GFP-20080404, did contain detectable amounts of lentiviral RNA for AHR, but Ct values were not equal for PuroR. This observation is indicative of multiple isoforms of lentivirus being produced (Table 1). 143 Y "C." ,_,.. Because polyclonal isoforms of virus were also generated from an AHR expression vector produced by an offsite collaborator (pTZV-based vectors), evidence suggests that splicing issues are not restricted simply to pLEX-HSAHR and pLEX- HsAHR-GFP, but extend to other lentiviral gene transfer vectors as well. This observation provides a potential explanation for generation of SKWAG cells that do. not express AHR mRNA, as a spliced form of the virus may remove AHR entirely or remove significant portions of the gene, while leaving GFP and PuroR intact. Isoforms that spliced out PuroR would be killed during the selection process, eliminating cell lines that contained AHR or AHR-GFP alone. Confirmation of the polyclonal viral pool was obtained from puromycin resistant cell lines SKWAG and SKWA. In the pLEX-derived vectors AHR, GFP, and PuroR are all transcribed as a single strand of mRNA, thus Ct values should be equal if every copy of the genome-integrated lentiviral vector contains the same sequence. QRT-PCR analysis of gene expression for AHR, GFP, and PuroR demonstrated variations in amounts of mRNA for each gene (Table 2), suggestive of transduction with a pool of lentiviruses containing multiple spliced isoforms. 144 Virus Lot Name pTZV-HsAHR-GFP pTZV-HSAHR pLEX-HSAHR- 2008061 3 pLEX-HsAHR-GFP- 20080707 pLEX-HsAHR-GFP- 20080404 pLEX-GFP AHR Ct Value 33 .58 16.46 15.48 21.17 18.71 Undetected OF P Ct Value 22.75 16.13 Not Evaluated Not Evaluated Not Evaluated Not Evaluated PuroR Ct Value Not Evaluated Not Evaluated 17.26 21.55 19.85 14.7 Table 1. Direct assessment of lentiviral RNA. RNA was isolated from cell culture supernatants following lentiviral vector packaging. Following reverse transcription QR T- PCR was used to directly quantifil the amount of RNA present for genes included in the gene transfer vector. PuroR gene expression was'not evaluated in pT Z V-based vectors because the gene is not included in the plasmid source used for packaging lentivirus. 145 Cell Line AHR Ct Value GFP Ct Value PuroR Ct Value SKWAG 30.6 16.28 22.41 SKWA 29.35 22.1 36.09 Table 2. Direct assessment of vector gene expression in antibiotic-resistant cell lines. QRT—PCR was performed on RNA fiom antibiotic-resistant cell lines. 146 While evidence shows that the lentiviral pool contained multiple isoforms of HsAHR and HsAHR-GF P, one method to circumvent the problem of polyclonal populations is to derive a monoclonal line of SKWAG cells expressing only full length HsAHR or HsAHR-GF P. A polyclonal pool of approximately 100x106 puromycin resistant GFP+ cells previously transduced with lentiviral HsAHR-GFP vector were sorted by FACS and cloned by limiting dilution. Out of approximately 600 viable cells recovered following sorting and plating, 50 monoclonal colonies of SKWAG cells were picked fiom 96 well plates during the weeks following cloning. 30 of 50 monoclonal colonies expanded, albeit at dramatically different rates, to populations of at least 5x105 cells, sufficient to screen for AHR protein expression by FACS. The earliest screenings were performed within 2 weeks of colony expansion, while the last colonies screen were not sufficiently expanded until 2 months following cloning. Of the 30 monoclonal cell lines screened none showed AHR immunofluoresence above the background observed with normal SKW 6.4 cells. Extensive efforts to establish AHR+ SKW 6.4 cells were ineffective. Based on the relatively low viability of SKWAG and SKWA lines, difficulty in maintaining cultures with viability in excess of 90%, variable doubling times, and occasional collapse of the viable population, evidence suggests SKW 6.4 cells expressing AHR are, at the very least, at a growth disadvantage compared to cells not expressing AHR. One consequence of a growth disadvantage is that AHR mRN A may be detectable in the days or weeks following initial gene transfer, but over time will be diluted out of the heterogeneous population of cells as the non-AHR expressing cells increase number. Several approaches 147 “:..... may be fruitful in future efforts to develop AHR+ SKW 6.4 cells, including coexpression of chaperone proteins HSP90 and XAP2 in the case that HsAHR misfolding is responsible for toxicity associated with high AHR expression, or use of an inducible expression systems activated by tetracycline, ecdysone, or isopropyl B-D-l- thiogalactopyranoside. Techniques allowing for reporter indication of functional AHR expression may also provide a useful method of detection, such as incorporation of a DRE driven red fluorescent protein in the lentiviral vector. Activation with an AHR ligand such as 6-formylindolo[ 3,2-b] carbazole, which is subsequently degraded by metabolism, would drive expression of a long-lived, highly fluorescent marker which is readily detectable by FACS, facilitating sorting for the cell population containing functional AHR. 148 CHAPTER 5: CONCLUDING DISCUSSION Results presented within this dissertation can be broadly classified as technical or scientific advances for the field of toxicology. Applied together, both advances revealed insights regarding the mechanism for TCDD disruption of primary humoral immunity, resulting in reduced uncertainty pertaining to the health hazard posed by dioxins and DLCs. The most significant technical advance came from application of F ACS to perform single cell, multiparametric analyses of the transcription factor network regulating plasmacytic differentiation. While the field of toxicology has utilized FAC S to analyze changes in the composition of the immune system, only in recent years have investigators begun to examine intracellular events involved in chemical disruption of homeostasis. Analyzing expression and activation of transcription factors, and evaluating how TCDD disrupts their expression, is a novel technical advance in immunotoxicology. Single cell analysis is particularly useful for generating computational models of biological processes, as it allows for identification and characterization of subpopulations that are not apparent in bulk lysis techniques. For example, in R848-activated CH12.LX cells there are two populations, a group that phosphorylates JN K and a group that does not (Figure 17). Bulk lysis would show that JN K was phosphorylated in the population, but would erase the ability to observe that only a minority population is actually responding strongly to immune response activation. Most illustrative of the power of multiparametric flow cytometry is the difference in the amount of information gleaned from a single experiment. A single immunoblot give two basic pieces of information, molecular weight and relative protein expression. A simple 4-color FACS experiment 149 asking the most basic question of whether a protein is expressed provides at least 24 combinations of measurements within a single cell..That does not consider the wealth of information that comes from the relative expression of a given protein. A single series of experiments measuring transcription factor expression, activation marker expression, and kinase phosphorylation can be accomplished simultaneously using splenocytes from less than three mice. As efforts to reduce, refine, and replace models employing intact animals, techniques such as multiparametric FACS T can assist investigators to garner more information with less starting material. Along these lines, the experiments described in Chapter 3 provide a proof of principle that multiparametric analyses are a powerful method facilitating reduced animal use. Studies examining TCDD effects on transcription factor expression are typically performed only at times of peak abundance with a limited range of TCDD concentrations, often covering only a single order of magnitude. The experiments described in Chapter 3 not only examine TCDD-mediated effects across a wider range of concentrations, but also across time. Moreover, the multiparametric analysis of protein expression allows single cell correlations between transcription factor expression and cell surface protein abundance. The dynamic combination of evaluating multiple times, TCDD concentrations, and proteins in a systematic manner resolves the effects of TCDD on a resolution never achieved before. Many scientific advances were made in the progress of this dissertation research, most noteworthy the improved understanding of TCDD effects on the plasmacytic differentiation control circuit both in vivo and in vitro. The solid evidence for suppression of LPS-induced Blimp-1 expression by TCDD in vivo led to examination of additional 150 regulatory events preceding increases in Blimp-1. The findings that LPS-elicited changes in BCL-6 expression are disrupted by TCDD is a novel discovery, and provides another avenue of research to understand how TCDD alters the plasmacytic differentiation control circuit. Suppression by TCDD of LPS-activated c-Jun phosphorylation (Figure 10) provided supportive evidence for results showing impairment of AP-l DNA binding and activity (Schneider et al. 2009; Sub et al. 2002a), further validating the CH12.LX cell line as a useful model for investigating TCDD immunotoxicity. Viewed cumulatively, TCDD alters expression of several transcription factors, leading to failure of B cells to achieve the ASC phenotype. One persistent difficulty in application of scientific research to policy making is the uncertainty resulting from cross-species extrapolation. The validation that in vitro disruption of Blimp-1 expression occurs in vivo suggests a persistent, nonartifactual mechanism for TCDD-mediated suppression of the primary IgM response in the mouse. Because the plasmacytic differentiation control circuit is conserved between human and mouse, validating results from murine models in human models will be indispensable in understanding dioxin and DLC effects on B cells. Concomitant with disrupted plasmacytic differentiation, the observation that TCDD impaired LPS-elicited cell surface activation marker expression is another potentially important event. Impairments of MHC Class II, CD80, and CD86 will decrease B cell — T cell interaction, resulting in compromised humoral immunity against most pathogens. In part, effects of TCDD on MHC Class II, CD80, and CD86 explain why the sRBC-elicited primary IgM response is more sensitive to TCDD than the LPS- elicited primary IgM response. LPS-activated B cells do not require presence of 151 accessory cell types to differentiate into antibody-secreting cells, whereas the sRBC- elicited primary IgM response is dependent on cell—cell interactions between T cells and B cells, interactions dependent on MHC Class II, CD80, and CD86. Going forward, evaluation of surface markers essential for cell-cell communication in the immune system has the potential for helping to identify mechanisms responsible for suppression of immune responses by chemicals beyond dioxins and DLCs. F ACS-based methods for evaluation of phosphorylation of specific kinases were unknown prior to 2002 (Perez and Nolan 2002) and have only recently been applied to understanding pharmacology (Krutzik et al. 2008). Studies described in Chapter 3 are the first to apply single cell, multiparametric analysis for phosphorylation status of specific kinases in the context of toxicology research. Examining multiple kinases within the same cell illuminates the signaling network engaged by immune response stimuli, and potentially identifying nodes within the signaling network that are altered by environmental chemicals. For example, results from Figure 21 show that in primary B cells the majority of cells increase AKT and ERK phosphorylation, but there are minority populations that upregulate neither kinase, or only single kinases. Because chemicals have the potential of affecting subsets within a given population, and examining single parameters using bulk lysis techniques such as immunoblot can obscure important biological events occurring within minority subpopulations, the potential for missing important indicators of toxicity cannot be understated. Multiparametric interrogations of single cells address some of the limitations of bulk lysis methods and represent a valuable avenue of assessment with tremendous potential for aiding toxicology studies. Additional information gleaned from dissecting the kinase signaling network can be used to add a 152 new layer of refinement on computational models designed to predict disruption of plasmacytic differentiation. While literature reports exist showing that TLR ligands cause AKT phosphorylation (Francois et al. 2005; Hebeis et al. 2005; Vivarelli et al. 2004), no reported evidence exists for R848- or CpG-activated AKT phosphorylation in B cells. Most studies of TCDD effects on specific kinases have not been performed in the context of an activated immune response. Studies in Chapter 3 are the first to illustrate TCDD Pi impairs AKT, ERK, and JNK phosphorylation in B cells even when considering only single parameter analysis, and was further extended to show TCDD impairs activation of ,1 all three kinases simultaneously within single cells. Uniting findings presented in Chapter 3, evidence suggests that TCDD suppression of the primary IgM response results from a cascade of effects, potentially beginning during the activation of kinases by immunologic stimuli. Because the window of sensitivity for TCDD-mediated suppression of the IgM response occurs early in B cell activation, and evidence presented in Chapter 3 shows TCDD disrupted TLR-activated events during the first hour following treatment, future efforts to examine TCDD effects on cell signaling may be fundamental in understanding the mechanism of TCDD immunotoxicity. There are many basic questions to be addressed in the future, such as whether AHR is required for suppression of AKT, ERK, or JN K phosphorylation. Disruption of early events in the immune response is amplified and propagated into later stages of differentiation, and coupled with direct effects of AHR on the IgH 3’a enhancer, resulting in a significant impairment of the primary IgM response. 153 Based on results obtained and described within this thesis, Figure 33 depicts the putative regulation of the plasmacytic differentiation control circuit in the presence and absence of TCDD. TLR activation causes increased phosphorylation and activation of the kinases AKT, ERK, and JNK. In turn, BCL-6 and AP-l activity is changed by reducing the abundance of BCL-6 and increasing the transcriptional activity of AP-l, allowing increased expression of Blimp-1. As Blimp-1 levels increase there is a concomitant increase in phenotypic indicators of plasmacytic differentiation. 154 Figure 33. Summary figure for LPS-activated changes in kinase and transcription factor activity in the presence and absence afTCDD. Positive regulation in depicted as 9 and negative regulation as "I. Kinases are depicted as O and transcription factors are depicted as C . Line thickness and object size represent, respectively, relative activity and abundance. 155 Transcription Factor Network Following LPS Treatment mil ”unit/gs: ‘r g: /\ m i in depitlta'r 4:. I trial/(mm "J :; :t'li, relumr Plasmacytic Differentiation Transcription Factor Network Following LPS + TCDD Treatment ./® Plasmacytic Differentiation @ 156 Extending the observations gained using in vitro and in vivo mouse models to human in vitro models would be a significant advance in understanding health risks posed by DLCs. Extensive efforts to develop an in vitro human model suitable for modeling LPS-activated plasmacytic differentiation were successful in a limited scope. The SKW 6.4 cell line was established as LPS-responsive and proved amenable to both transient and persistent genetic manipulations. However, to make a suitable model for investigating DLC effects, the expression of HsAHR was essential. Transient expression of HsAHR was observed in SKW 6.4 cells using both plasmid-based and lentiviral expression vectors, but unremitting expression of HsAHR proved untenable. Sustained expression of HsAHR, likely at higher than normal physiologic levels, caused cell death in SKW 6.4 cells. Other cell lines, in particular 293T, can accommodate high level long term expression of HsAHR. At this point there are unidentified factors in the SKW 6.4 and 293T cell lines that account for the difference in sensitivity to HsAHR. The clearest benefit to the experiences gained with HsAHR and HsAHR-OF P expression vectors was an improved understanding of how to develop future expression vectors. Secondarily, confirmation of lentiviral vector splicing as a major factor affecting both viral production and protein isoforms is valuable in preventing future failures when working with lentiviral, and likely retroviral, gene transfer vectors. In summary, significant scientific progress came in the extension of in vitro observations regarding disruptionof the plasmacytic differentiation control circuit to in vivo models, establishment of several novel mechanisms of action for TCDD in affecting the both transcription factor expression and the cellular activation process, development of a more rapid method for moderate to high throughput detection of human AHR, and 157 from efforts to develop a human model suitable for assessing TCDD effects in vitro. While data gaps still exist in fundamental understanding for TCDD suppression of humoral immunity, efforts to address the unknowns have advanced the field of dioxin immunotoxicology and opened new avenues of exploration. 158 APPENDIX A. Antibodies Fluoro- Target phore Host Isotype Type Catalog # Supplier None . Santa Cruz AHR or AP C Rabbit IgG polyclonal sc-5579 Bio tech PerCP- monoclonal, . 8220 (CD45R) Cy 5. 5 Rat Inga, K RA3- 6B2 103235 Blolegend BCL-6 None Rabbit IgG polyclonal sc-368 Santa Cruz Biotech BCL-6 None Rabbit I gG polyclonal ab1190%1 1' Abcam . None Santa Cruz Blimp-1 or PE Goat IgG polyclonal sc-13206 Bio tech c-'un FITC Rabbit I G 01 clonal sc-1694 Santa Cruz J g p y Biotech monoclonal, BD CD 1 6/3 2 None Mouse IgGbe 2. 4G2 5 53 142 Biosciences monoclonal. BD CD 1 9 PE Rat IgGZa, K 2D 4 553786 Biosciences Armenian monoclonal, . CD69 PE Hamster IgG H1.2F3 104508 Brolegend Armenian monoclonal, . CD80 APC Hamster IgG 16-10A1 104714 Brolegend monoclonal, . CD86 PE/Cy7 Rat 1802a, K GL-l 105014 Blolegend goat IgG APC Donkey IgG polyclonal sc-3 860 Sim.” cm F(ab')2 Biotech I J-chain None Goat I G 01 clonal sc-34654 Santa Cruz g g p y Biotech I K li ht h ° F ITC Rat I G monoclonal c-53080 Santa Cruz g g 0 am g l S Biotech 159 1gp. chain goat IgG MHC Class II 1- AB MHC Class II 1- AP Pax5 Phosphorylated c-jun (Ser 63) Pho sphorylated AKT (Ser473) . Phosphorylated INK (Thr183/Tyr185) Phosphorylated ERK1/2 (Thr202llyr204) . Rabbit IgG Rabbit IgG Rabbit IgG Rabbit IgG TCRB lsotype lsotype FITC FITC FITC F ITC AF647 AF 647 PE AF647 AF488 AF647 PerCP PE- Cy5.5 APC PE/Cy7 APC None Goat Donkey Mouse Mouse Goat Mouse Mouse Mouse Rabbit Donkey Bovine Goat Goat Armenian Hamster Goat Goat IgG IgG F(ab')2 IgGZa, K IgGZa, K IgG lgGi IgG IgG IgG IgG IgG IgG F(ab')2 IgG ‘F(ab')2 IgG IgG IgG 160 polyclonal polyclonal monoclonal, AF6-120.1 monoclonal, 7-16. 1 7 polyclonal monoclonal, KM- l monoclonal, M89-61 monoclonal, G9 monoclonal, D 1 3. 14.4E polyclonal polyclonal polyclonal polyclonal monoclonal, H57-597 polyclonal polyclonal FI-2020 sc-3853 1 16406 558921 sc-1974 sc-822 560378 9257 4344 A31573 sc-45099 L43018 sc-3 846 109221 sc-3 867 sc-3 887 Vector Laboratories Santa Cruz Biotech Biolegend BD Biosciences Santa Cruz Biotech Santa Cruz Biotech BD Biosciences Cell Signaling Cell Signaling Invitrogen Santa Cruz Biotech Invitrogen Santa Cruz Biotech Biolegend Santa Cruz Biotech Santa Cruz Biotech lsotype lsotype lsotype lsotype lsotype lsotype lsotype AF 647 F ITC FITC FITC FITC None PE Mouse Mouse Mouse Mouse Rabbit Rabbit Rat IgG1 IgG] IgGZa, K IgM IgG IgG IgGZa, K 161 polyclonal polyclonal polyclonal polyclonal polyclonal polyclonal polyclonal Santa Cruz 8024636 Biotech sc-2855 833:3? 554647 Bioslgilznces sc-2859 Sggtfcrlilz sc-3 870 82113123;th sc-3 888 Sgtjtirlrz 554689 BD Biosciences 162 APPENDIX B. TaqMan Primers and Probes Gene Probe Catalog # Ref Seq VIC- 18S MGB 4319413E X03205.1 FAM- CD138 MGB Mm004489l 8_m1 NM_011519.2 1g p F AM- Chain MGB Mm01718955_g1 No RefSeq # Custom synthesized using Forward primer: Ig K F AM- GGAAGATTGATGGCAGTGAACGA N G 005612 Chain MGB Reverse Primer: — GCTGTCCTGATCAGTCCAACT Probe: TCAGGACGCCAI l 1 16 lg J- FAM- Chain M GB Mm00461780_ml NM_152839.2 XBP-l FAM- (total) M GB Mm00457359_m1 _ NM_013842.2 XBP-l FAM- Custom synthesized, see (Skalet et al. (spliced) MGB 2005) for details N0 Refseq # Pax5 F AM- (BS AP) MGB Mm00435501_m1 NM_008782.2 Blimp-1 FAM- (pr dml) M GB Mm00476128_m1 NM_007548.3 BCL6 FAM‘ Mm00477633 m1 NM 009744 3 MGB - - ' F AM- c-fos MGB Mm00487425_m1 NM_010234.2 . FAM- c-Jun M GB Mm00495062_sl NM_010591.2 BIBLIOGRAPHY Abbott, B. D. and Bimbaum, L. S. (1989). TCDD alters medial epithelial cell differentiation during palatogenesis. Toxicology and applied pharmacology 99, 276-286. Alsharif, N. Z. and Hassoun, E. A. (2004). 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