Nancy I. Kerkvliet - US grants

Polychlorinated dibenzo-p-dioxin (PCDD) and structurally related halogenated aromatic hydrocarbons, including the dibenzofurans and biphenyls, are widely distributed, highly toxic environmental contaminants that produce a similar and characteristic pattern of toxic and biochemical responses in animals. Structure/activity studies suggest that the toxic isomers of these chemicals act through a common receptor (the Ah receptor) which regulates the expression of a multitude of gene batteries. In all species studied, the toxic congeners produce thymic involution and suppression of the immune response, which also is mediated, at least in part, by the Ah receptor. However despite considerable investigation, the precise nature of the immune dysfunction and the target cells affected by these chemicals have not been determined. The cell membrane antigens and receptors displayed by lymphocyte/monocyte subsets phenotypically define the cells, and, with several markers, are associated with specific functions. Therefore, modulation of the expression of these cell-surface molecules by exposure to dioxin could be expected to have significant effects on cell behavior. Since the Ah receptor regulates the expression of multiple, and, as yet, not fully defined gene batteries, it is possible that activation of the Ah gene produces altered surface antigen or receptor expression which, in turn, alters the functional activity of the cell. The specific aims of this proposal are: 1) to characterize lymphocyte/monocyte subpopulations in control and dioxin-treated mice in terms of cell-surface marker expression using multiparameter flow cytometry, 2) to characterize relative changes in lymphocyte/monocyte subpopulations following antigen challenge in control and dioxin-exposed mice by multiparameter flow cytometry, and 3) to determine if in vitro exposure of lymphocyte/monocyte subsets to dioxin produces similar alterations in surface antigen expression observed after in vivo exposure that may then be utilized for the study of dioxin-induced effects on the human immune system. Quantitative and qualitative surface marker expression of Ia, IgM, IgD, C3, Thy-1, Lyt-1, Lyt-2, L3T4 and I-J will be analyzed in response to exposure to 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin, a major immunosuppressive contaminant of technical grade pentachlorophenol. Other dioxin isomers will be examined if positive results are found with HpCCD.

Polychlorinated dibenzo-p-dioxin (PCDD) and structurally related halogenated aromatic hydrocarbons, including the dibenzofurans and biphenyls, are widely distributed, highly toxic environmental contaminants that produce a similar and characteristic pattern of toxic and biochemical responses in animals. Structure/activity studies suggest that the toxic isomers of these chemicals act through a common receptor (the Ah receptor) which regulates the expression of a multitude of gene batteries. In all species studied, the toxic congeners produce thymic involution and suppression of the immune response, which also is mediated, at least in part, by the Ah receptor. However despite considerable investigation, the precise nature of the immune dysfunction and the target cells affected by these chemicals have not been determined. The cell membrane antigens and receptors displayed by lymphocyte/monocyte subsets phenotypically define the cells, and, with several markers, are associated with specific functions. Therefore, modulation of the expression of these cell-surface molecules by exposure to dioxin could be expected to have significant effects on cell behavior. Since the Ah receptor regulates the expression of multiple, and, as yet, not fully defined gene batteries, it is possible that activation of the Ah gene produces altered surface antigen or receptor expression which, in turn, alters the functional activity of the cell. The specific aims of this proposal are: 1) to characterize lymphocyte/monocyte subpopulations in control and dioxin-treated mice in terms of cell-surface marker expression using multiparameter flow cytometry, 2) to characterize relative changes in lymphocyte/monocyte subpopulations following antigen challenge in control and dioxin-exposed mice by multiparameter flow cytometry, and 3) to determine if in vitro exposure of lymphocyte/monocyte subsets to dioxin produces similar alterations in surface antigen expression observed after in vivo exposure that may then be utilized for the study of dioxin-induced effects on the human immune system. Quantitative and qualitative surface marker expression of Ia, IgM, IgD, C3, Thy-1, Lyt-1, Lyt-2, L3T4 and I-J will be analyzed in response to exposure to 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin, a major immunosuppressive contaminant of technical grade pentachlorophenol. Other dioxin isomers will be examined if positive results are found with HpCCD.

Immune-endocrine interactions have recently been identified as an important, and perhaps central, mechanism for regulation of the immune and endocrine systems. Numerous endocrine hormones have been shown to regulate immune responses. Conversely, a number of protein products produced by cells of the immune system (cytokines and lymphokines) have been shown to regulate endocrine hormone activity. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure alters the activity of several immunoregulatory hormones, including glucocorticoids, estrogen, thyroxine, and prolactin. Preliminary data from our laboratory have shown that TCDD exposure results in an enhanced inflammatory response following antigen challenge. We postulate that enhanced inflammatory cytokine production accompanies the enhanced inflammatory response. Two of the major inflammatory cytokines [IL-1 and tumor necrosis factor (TNF)], have been shown to alter endocrine activity. When produced in pathological amounts, they also produce a cachectic response that resembles TCDD toxicity. Other cytokines produced during inflammation [e.g., PGE2, histamine] are immunosuppressive. The specific aims of this grant are to define the roles of specific inflammatory cytokines and hormones in the suppression of the cytotoxic T lymphocyte (CTL) response by TCDD and to assess the interaction of hormones and cytokines with TCDD at the level of the macrophage (Mphi) and T cell. Specific aim 1 will determine if prevention of corticosterone (CS) elevation or blockade of CS receptors in TCDD-treated animals alters the suppressive effects of TCDD on the CTL response. Specific aim 2 will determine if the production of inflammatory cytokines (TNF, IL-1, PGE2) is altered in TCDD-exposed animals. Cytokine alterations will be measured at the level of transcription in macrophages (TNF and IL-1 mRNA) as well as systemically in TCDD-treated animals. Specific aim 3 will determine if neutralization of TNF or PGE2 activity alters the elevation of circulating CS or the suppression of CTL activity in TCDD-exposed animals. Specific aim 4 will determine the direct effect of TCDD on Mphi cytokine production and the effects of coexposure to TCDD and CS on Mphi cytokine production and CTL maturation in vitro. Based on the results of these studies, the extension of the model to assess other inflammatory cytokines (e.g., IL-6, histamine) and other immunoregulatory hormones (e.g., thyroxine, androgens) in TCDD-induced suppression of CTL activity will be initiated.

The Immune system has been identified as one of the most sensitive targets for the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and structurally related halogenated aromatic hydrocarbons (HAH). The Environmental Protection Agency recently completed a reassessment of the health risks associated with environmental contamination by these chemicals. Based on a draft of the risk characterization document, TCDD affects the immune and reproductive systems at exposure levels lower than the carcinogenic dose. Thus, for the first time, data from animal studies on the immunotoxicity and reproductive toxicity of TCDD may be used as the basis for setting acceptable levels in the environment. It is therefore of great importance that we improve our understanding of the mechanisms that underlie these toxicities in order to improve our ability to extrapolate from animals to humans and to most accurately predict human health effects. Previous studies in our laboratory have shown that' exposure of C57B1/6 mice to HAH induces an Ah-receptor dependent, dose- dependent suppression of cytotoxic T lymphocyte (CTL) activity following the injection of allogeneic P815 tumor cells. Our overall goals are to understand the cellular and molecular basis by which HAH suppress allograft immunity as a model for understanding the overall impact of these widespread environmental toxicants on immune function. In studies carried out during the current grant period we have shown that suppression of CTL activity by HAH is correlated with a profound decrease in splenic production of interferon-gamma (IFN-gamma). Interleukin (IL)- 2 production is also modulated by HAH exposure, but the effects are characterized by an early elevation followed by a later suppression of IL-2 levels. In contrast, the production of IL-6 and IL-4 was not suppressed by HAH exposure even though the alloantibody response is suppressed by HAH exposure in parallel with the suppression of the CTL response. The studies proposed in this grant application are based on- the hypothesis that the alterations in cytokine production underlie the suppression of allograft immunity in HAH-treated mice. Using 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) as the prototype AhR ligand, five specific aims will be addressed: l) to define the roles of suppressed IFN-g and altered IL-2 production in the suppression of CTL activity and the alloantibody response to P8l5 tumor cells in TCDD-treated mice; 2) to characterize the cells responsible for the production of IFN-gamma and IL-2 in the allograft response; 3) to characterize by phenotype and function a TCDD-induced population of Mac-l+ cells in the spleen; 4) to further characterize the effects of TCDD exposure on the early cytokine response to allografted P815 tumor cells, focusing on IL-12, TGF-beta, IL-1 and TNF as important regulators of T cell activation; and 3) based on preliminary data that demonstrate a reduced expression of B7-1 and B7- 2 on splenic Mac-1+ and B220+ cells from TCDD treated mice, to investigate the role of altered expression of these and other costimulatory molecules on antigen presenting cells (APC) as the defective link in T cell activation in this model.

environmental toxicology; community; environmental health; health education; behavioral /social science research tag;

The studies proposed in this renewal application represent a continuation of ongoing studies to understand the role of CD4+T cell activation in the immunotoxic effects of 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD). Data obtained during the current grant period are consistent with the hypothesis that TCDD suppresses the allogeneic CTL and allantibody responses through a defect in T helper cell activity. Furthermore, current data indicate that TCDD exposure suppresses CD4+ T helper cell function by influencing the activation signals they receive from antigen-presenting cells (APC). Specifically, we have shown that TCDD exposure reduces the expression of B7 molecules on the surface on macrophages and B cells, which provide critical co-stimulatory signals necessary for full T cell activation. Consistent with the consequences of incomplete co-stimulation, we have evidence to suggest that activated CD4+ cells die instead of proliferating and differentiating into effector cells subsequent to TCDD exposure. The hypothesis to be tested in this renewal application is that T cell unresponsiveness to primary antigenic stimulation in TCDD-treated mice is due to T cell deletion resulting from defective co-stimulation by APC. We further hypothesize that toxicity is initiated in the APC by increased oxidative stress resulting from TCDD exposure. The alteration in intracellular redox balance results in partially activated APCs that are unable to provide the necessary co- stimulatory signals to T cells. The lack of generation of sufficient T cell help is the cause of suppressed immune responsiveness in TCDD-treated mice. This hypothesis will be tested by characterizing: (1) the fate of antigen-activated T cells in TCDD-treated mice; and (2) the influence of TCDD exposure on the redox state and co-stimulatory functions of APC. The proposed studies include an evaluation of oxidant production by APC, antioxidant status of APC based on measurements of glutathione and thioredoxin, expression in nitric oxide synthases, and activation of NF- kappaB. In addition, studies will determine in antioxidant treatment prevents TCDD-induced T cell apoptosis and immunosuppression. A new focus of the proposed studies is on dendritic cells, which represent the most potent APC for the activation of naive T cells. Comparative effects of TCDD on the intracellular redox status and antigen-presenting functions of macrophages and B cells will be examined as these cells play important accessory roles in sustaining the responsiveness of activated T cells. These studies will help to elucidate the mechanisms of immune suppression mediated by TCDD, an environment contaminant of concern to human health, as well as the prototypic ligand of the Ah receptor. These studies will provide insight into the natural role of this widely expressed receptor.

DESCRIPTION (Taken from the Applicant's Abstract) This project seeks to improve the academic performance of high school students by developing their knowledge and skills in science, math, and humanities using context-based environmental health science scenarios. The project is a collaboration among the Environmental Health Sciences Center, the Marine/Freshwater Biomedical Sciences Center, and the Science and Math Investigative Learning Experiences (SMILE) Program at Oregon State University; the Oregon Department of Education; and Oregon school districts with high dropout rates and high numbers of minority and disadvantaged students. The project uses the Hydroville Challenge Problems, an existing set of environmental health science scenarios, developed under NIEHS grant ESA-96-001, to improve the critical-thinking skills of high school students and to engage them in developing solutions to real-life environmental health science problems. The proposed project includes the following activities: * Adapting the Hydroville Challenge Problems into integrated classroom modules, enhancing them with additional scientific and humanities content (including risk assessment and risk perception), and aligning them with state and national education standards; * Conducting Summer Institutes to train teams of teachers in environmental health science, team teaching, and implementation of the Challenge Problem modules; * Evaluating the impact of environmental health science as an integrative context for learning using the Hydroville Challenge Problems; and * Disseminating the Challenge Problem modules nationwide. Approximately 150 science, math, and humanities teachers will be trained to use the Hydroville Challenge Problem modules in each year of the project. Teams of teachers will then incorporate the modules within the existing high school curricula. As a result of this project, it is hoped that students will demonstrate improved academic performance, decision-making skills, and attitudes toward science and school, as well as increased knowledge of environmental health issues.

Description: The overall goal of the OSUEHS Centers COEP is to increase the public's ability to understand and to make informed decisions on issues relevant to the role of environmental factors in human health and disease. The COEP has eighteen current or past partnerships and collaborations with other OSU programs. The Center's COEP works actively with the Science and Math Investigative Learning Experiences (SMILE) program. These programs jointly received an NIEHS dissemination grant in 1996 to bring environmental health science education to teachers, students and community in rural Oregon. The program has reached an increasing number of students, teachers and community members since 1996. The majority of the population affected by this program consists of Native Americans (25 percent) and Hispanics (46 percent). Each year this program focuses on a different environmental health science theme, and as a portion of the program each summer teachers come to OSU to be trained in an environmental health science subject area. Of the four themes presented yearly since 1996, two were directly related to a research focus of the Center (1996-Chemicals in the Home Environment and 1998-Water Quality and Human Health). The "Lunch with a Scientist" portion of this program also involved Center investigators. Family Science Nights are also offered as a portion of this program through the COEP each fall. The final component of this program is the High School Challenge Weekend. Center members have served as environmental experts in this program by having students work in cooperative groups to develop solutions to environmental problems. Through the OSUEHS Center/SMILE Program, educational programs have been provided to over 1225 elementary, middle, and high school students, 125 teachers and 2000 community members. Rural communities (775 individuals) have received informal science education through family science nights. Center members are actively involved in programs such as Adventures in Learning, American Women in Science, and Science Education Partnerships. Teacher training components are currently being integrated into the program. Teacher workshops are being offered in the curriculum, Exploring Environmental Issues: Focus on Risk. Sixty-five teachers in 1999 have been trained in this curriculum, with future workshops planned in 2000. Teacher training in the ToxRAP program also will be a major focus of the Center through support from the Society of Toxicology. The OSUEHS Center sponsors a yearly course, Your Health and Chemical Risks. This course and environmental health science articles in the monthly newspaper reaches over 3,500 community members each month.

DESCRIPTION (provided by applicant): Autoimmune and allergic diseases afflict millions of people worldwide. Novel immunosuppressive strategies to prevent and treat these diseases are urgently needed. Following several years of studying the potent immunosuppressive effects of TCDD, we recently discovered that activation of the transcription factor, Aryl Hydrocarbon Receptor (AHR), by TCDD, in CD4+ T lymphocytes leads to the induction of a unique subpopulation of highly immunosuppressive regulatory T cells (Treg). These AHR-induced CD4+CD25+ Treg were more potent than natural CD4+CD25+Foxp3+ Treg in vitro. The induction of AHR-Treg appears to underlie the potent suppression the graft-vs host (GVH) response in TCDD-treated mice. Furthermore, treatment of NOD mice with TCDD prevented the development of diabetes, in parallel with increased frequency of CD4+CD25+ T cells in the pancreatic lymph nodes. These data support the hypothesis that the AHR signaling pathway is a novel pathway for the induction of Treg that may be amenable to manipulation for therapeutic benefit. The goal of the proposed studies is to understand the signaling pathways that are influenced by AHR activation in responding CD4+ T cells that lead to the induction of AHR-Treg, and to use the pattern of changes in gene expression to identify other AHR ligands that might also induce AHR-Treg. Specific Aim 1 will define the changes in gene expression in CD4+ T cells that are induced by TCDD-mediated AHR activation in vitro under a variety of well-defined conditions of T cell differentiation. The second specific aim will validate the functional significance of key genes that are identified in Specific Aim 1 to support the development of a custom gene expression array for identifying other AHR ligands that induce Tregs. Specific Aim 3 will identify novel ligands for the AHR using a unique 3-tiered approach to ligand screening that includes prioritization based on lack of effects on T cell proliferation. In Specific Aim 4, we will screen novel AHR ligands for their ability to induce TCDD-like changes in gene expression in vitro during CD4+ T cell differentiation and test positive chemicals in vivo for their ability to induce AHR-Treg during an acute GVH response. These studies are timely and important in the context of understanding the pathways for the development of adaptive Tregs as the AHR may represent a useful target for therapeutic intervention in the prevention and treatment of a wide variety of human diseases. At the same time, they will greatly enhance our understanding of the molecular mechanisms underlying TCDD-induced immune suppression and may lead to useful biomarker(s) of an "immunotoxicity pathway" for dioxin-like chemicals. PUBLIC HEALTH RELEVANCE: This research will characterize a newly discovered pathway for inducing regulatory T cells and will identify new ligands for the receptor that activates this pathway. Such ligands hold promise as new therapies for treatment of immune-mediated diseases such as allergic asthma, multiple sclerosis, type 1 diabetes, lupus and psoriasis.

DESCRIPTION (provided by applicant): Autoimmune diseases afflict millions of people worldwide and are the fourth leading cause of disability in women in the US (USDHHS, 2000). Thousands more seek treatment to prevent rejection of tissue and organ transplants. Unfortunately, current immunosuppressive treatments are often inadequate and associated with many undesirable side effects. Immunoregulatory T cells (Tregs) potently suppress immune responses and offer a promising therapeutic alternative. The discovery of a novel approach to induce Tregs is important and timely. The Ah receptor (AhR) is a ligand-activated transcription factor that induces the differentiation of CD4+Tregs. Our laboratory made the original discovery in studies to define the mechanism underlying the potent immunosuppressive effects of the prototypic AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Since then, several laboratories have validated and extended the scope of Treg induction by TCDD. Studies have also shown that treatment of mice with TCDD suppresses the development of a variety of autoimmune diseases, including our own studies using the non-obese diabetic (NOD) mouse model of Type 1 diabetes, in association with increased frequency of Tregs. Together these findings support a strong case for the AhR as a therapeutic target for treatment of immune-mediated diseases via the induction of Tregs. The goal of our current grant is the discovery of alternative AhR ligands that are capable of inducing AhR-dependent Tregs. We have successfully screened small molecule compound libraries and identified a lead compound (AHRL1) that is structurally distinct from TCDD but activates AhR at nanomolar concentrations. AHRL1 induces AhR-dependent Tregs in vivo that are phenotypically identical to Tregs induced by TCDD and is immunosuppressive in a graft-vs-host response. The hypothesis to be tested in this renewal application is that activation of the transcription factor AhR in CD4+ T cells by certain AhR ligands regulates the expression of specific genes that induce their differentiation into AHR-Tregs, resulting in effective suppression of autoimmune disease. The ultimate goal of our studies is to understand the mechanisms by which AHR activation induces Tregs and identify promising compound(s) that function via AHR for treatment of immune-mediated diseases. In Specific Aim 1, we hypothesize that AHR ligands other than TCDD are effective in suppressing chronic autoimmune disease via the induction of AHR-dependent Tregs. We will determine the influence of treatment with AHRL1 on the development of diabetes in NOD mice and the association between disease-free survival and increased numbers of Foxp3+CD25+CD4+ Tregs. We will also track the AHR-Treg population over time in mice treated with AHR ligands (AHRL1 and TCDD) to identify changes in gene expression that correlate with disease outcome. In Specific Aim 2, we hypothesize that activation of AHR in T cells alters signaling pathways leading to their differentiation into Tregs. We will determine the functions of AHR required to induce Tregs by utilizing T cells derived from two distinct mouse lines that express (i) DNA-binding or (ii) nuclear localization-defective AHR. We will also determine the functional significance of specific genes that we identified to be upregulated by TCDD and AHRL1 in AHR-Tregs. The results of these studies will significantly expand our current understanding of the molecular pathways of AHR activation in T cells that lead to Treg induction and will provide an unprecedented foundation for pursuing AHR ligands as more effective, less toxic drugs for the treatment of autoimmune diseases.