Prakash Nagarkatti - US grants

DESCRIPTION (provided by applicant): Smoking elicit drugs such as marijuana causes immunosuppression and increases the risk of contracting infections particularly in HIV-seropositive individuals. Delta9-tetrahydrocannabinol (THC), the major psychoactive principle marijuana has been identified as a critical immunosuppressive agent, although the mechanism by which it induces toxicity is not clear. Moreover, recreational use of marijuana during pregnancy remains a major health problem and very little is known about the consequences of prenatal exposure to THC on the immune system. Interest in cannabinoid research has increased tremendously in recent years since the discovery of cannabinoid receptors and the endogenous ligands for these receptors. The fact that cannabinoid CB2 receptors are almost exclusively expressed on immune cells raises exciting questions on the role and significance of such receptors and their endogenous ligands in immunoregulation. Recent studies from our laboratory demonstrated that cannabinoids induce apoptosis in lymphoid organs and suppress lymphocyte functions. In the current study, we will investigate the role of apoptosis in cannabinoid-induced immunomodulation following prenatal and postnatal exposure. In aim1, we will test the hypothesis that THC triggers apoptosis and immunornodulation by binding to CB1 and/or CB2 receptors on immune cells using mice deficient in CB1, CB2, or both CB1/CB2 receptors. In aim 2, we will test the hypothesis that THC-induced apoptosis is responsible for decreased T cell response to bacterial antigens by using the superantigen model and analyzing antigen-specific Vbeta3+ and Vbetal 1+ T cells. In aim 3, the mechanism by which THC induces apoptosis in T cells via the death receptor pathway and/or the mitochondrial pathway will be studied using cDNA array and mutant cell lines that are deficient in FADD, caspases and members of bcl-2 gene family. In aim 4, we will test whether prenatal exposure to THC alters positive and negative selection of T cells in the thymus and thereby affects the T cell repertoire postnatally. Lastly, in aim 5, the hypothesis that endogenous cannabinoids such as an and amide regulate the immune functions by triggering apoptosis using FAAH knockout mice will be tested. In summary, the aims listed above form the basis for understanding the mechanism of induction of apoptosis in immune cells by exogenous and endogenous cannabinoids. These studies are particularly important in understanding how substance abuse by individuals who are at a higher risk for contracting HIV infection, HIV-infected individuals, and HIV-infected pregnant mothers, will influence the course of infection.

DESCRIPTION (provided by applicant): TCDD (dioxin) is an environmental contaminant that has given rise to major human health concerns. It belongs to the family of halogenated aromatic hydrocarbons (HAH) and is considered the most toxic chemical ever created. The immune system is highly susceptible to the toxic effects of TCDD with marked effect on thymus and T cells. Recent studies from our lab demonstrated for the first time that TCDD can induce apoptosis in thymocytes and T cells in vivo involving Fas-Fas ligand (FasL) interactions. We found that Fas- and FasL-deficient mice were more resistant to TCDD-mediated immunotoxicity when compared to the wild-type mice. Because human subjects with similar mutations in Fas and FasL have been identified, our results suggested differential responsiveness to dioxin-like toxicants in the human population to immunotoxicity. Fas-FasL interactions play a critical role in T cell maturation and differentiation. Thus, any interference in these interactions can lead to autoimmunity and altered immune response to infectious agents or cancer. Thus, in the current study, we will test the central hypothesis that TCDD perturbs the development of cell repertoire by interfering with the Fas-FasL interactions in the thymus. To this end, we will pursue 5 specific aims: In aim 1, we will test whether TCDD induces apoptosis in T cells of the thymus of C57BL/6 mice through death-receptor (Fas) or the mitochondrial pathway either independently or through cross-talk. In addition, the possible role played by ER stress pathway, the third novel apoptotic pathway, will also be investigated. In aim 2, we will investigate if the interaction between T cells and stromal cells involving Fas and its ligand is responsible in induction of apoptosis in T cells using adoptive transfer of hematopoietic stem cells. In aim 3, promoter region of Fas and FasL genes will be analyzed to characterize the mechanism(s) through which TCDD induces the expression of Fas and FasL. In aim 4, we will use fetal thymic organ cultures derived from Fas-deficient and FasL-defective mice to analyze the apoptotic pathways using cDNA arrays. In aim 5, the effect of prenatal exposure to TCDD on positive and negative selection of T cells in the developing thymus will be studied using TCR transgenic mice. Together, the current study should provide novel information on how prenatal and postnatal exposure to TCDD would alter the T cell repertoire by interfering with the apoptotic pathways.

Ulcerative colitis (UC) is a chronic inflammatory condition that is immunologically mediated. Approximately 4 million people world-wide have the disease and are at increased colon cancer risk. Conventional treatment of colitis can reduce periods of active disease and help to maintain remission, but these treatments are often associated with side effects and have marginal results. For this reason, many colitis sufferers turn to complementary and alternative treatments in hopes of abating symptoms of active disease. It is estimated that about 50% of UC patients use some form of megavitamin therapy or herbal/dietary supplement. There is, however, no documentation on UC patients taking American ginseng to temper the signs and symptoms of colitis. In the current study, we present exciting data that ginseng inhibits the onset of colitis in two mouse models of colitis as well as preventing colitis-driven colon cancer. This grant aims to understand the mechanisms of this inhibition as a necessary pre-requisite to future human clinical trials. We will test the hypothesis that dietary supplementation with American ginseng inhibits UC-associated inflammation in the colon through mechanisms involving induction of apoptosis in immune cells, altered Th1/Th2 cytokine profiles, and/or changes in immune cell migration and trafficking. Because ginseng also inhibits the colitisto- cancer sequence in our model, we will examine whether it suppresses inflammation-driven DMA damage and protein nitration in colon cells. Because pRb is hyperphosphorylated and inactivated in colitis, we will further examine whether ginseng suppresses pRb hyperphosphorylation through mechanisms involving a decrease in the activation of specific kinase pathways. We will investigate whether ginseng mediates these effects through direct action on the colon cells and/or on immune cells. Finally, we propose to delineate the active ingredients of American ginseng responsible for the inhibition of colitis. Together, this study will increase our understanding of how American ginseng protects from colitis, and possibly other autoimmune conditions and will provide the rationale for expanding into clinical trials.

Multiple Sclerosis (MS) is an autoimmune disease that affects ~400,000 people in the US. It is a life-long chronic disease diagnosed primarily in young adults. There is, as yet no cure for MS. The current treatment includes use of immunosuppressive drugs that often exhibit toxic side effects.Thus, there is a pressing need for alternate and more effective treatment strategies that target the components of inflammatory cells. CAM therapies constitute an important strategy to treat and control the disease. Experimental autoimmune encephalomyelitis is an animal model of MS induced by injection of self antigens, myelin basic protein, proteolipid protein or myelin oligodendrocyte glycoprotein and adjuvants. Resveratrol (RES;3,5,4'- trihydroxystilbene), a nonflovaonoid polyphenol found in various plants including mulberries, peanuts and grapes has been shown to have beneficial effects particularly on cardiovascular diseases through its antiinflammatory properties. Our Preliminary Studies have demonstrated that RES induces apoptosis primarily in activated T cells through Fas-FasL interactions, involving aryl hydrocarbon receptor (AhR) and estrogen receptor (ER). Moreover, in vivo, RES down regulates CD44 and upregulates FoxpS. We are also excited by our findings that RES treatment decreases the inflammation and clinical symptoms of EAE. In the current study, we will test the central hypothesis that RES treatment is effective against EAE through multiple pathways incuding induction of apoptosis in myelin-specific T cells, suppression of DC functions, decreased T cell infiltration in the CMS due to CD44 down-regulation on T cells, and upregulation of T regs. We will pursue the specific aims: # 1. Study the role of Fas-FasL interactions in resveratrol (RES)-induced apoptosis in T cells and dendritic cells (DCs) involving autocrine or paracrine path ways.#2. Investigate the role of AhR in Fas-FasL upregulation and consequent apoptosis induced by RES in T cells.tf 3. Test the role of ER in the induction of apoptosis in T cells and peripheral T dysfunction following RES treatment.^ 4. Study the effect of RES on initiation and progression of EAE. These studies are aimed at providing insights into the efficacy of RES mainly in acute, remitting-relapsing and chronic forms of the disease which represent diverse pathological and immunological characteristics. Together, our studies should provide novel mechanistic clues on how RES helps in the treatment of MS and develop better CAM therapies to control the disease.

DESCRIPTION (provided by applicant): The Center for CAM Research on Inflammatory and Autoimmune Diseases aims to study the mechanistic basis of preventive and therapeutic modalities developed from plant products against inflammatory and autoimmune disorders. Autoimmune diseases are highly prevalent world-wide. These include arthritis, lupus, autoimmune hepatitis and multiple sclerosis which are mediated primarily by T cells and cause significant chronic disability. In addition, there are also a large number of inflammatory diseases that mimic autoimmune diseases, with unknown etiopathogenesis such as colitis. In the absence of effective medication which can suppress T cells and inflammatory immune response, patients often seek CAM therapy. Thus, the primary goal of the project will be to address 3 specific aims using state-of-the-art technology and an integrated approach which include 1) Studies on ligation of the AhR and ER in the use of resveratrol for the treatment of multiple sclerosis using the experimental autoimmune encephalomyelitis model, 2) Effects of American ginseng as an anti-inflammatory agent in treating colitis and colitis-driven colon cancer. 3) Elucidation of the mechanism(s) underlying cannabidiol-induced apoptosis in therapy of autoimmune hepatitis. Inasmuch as, inflammatory and autoimmune diseases are triggered by multiple factors but exhibit the common pathogenesis of immune cells destroying self-tissues/organs, the efficacy of current treatment regimen include immunosuppressive or anti-inflammatory drugs. Unfortunately, the current drugs in market cause significant toxicity to the immune and cardiovascular systems thereby limiting their use. Thus, CAM herbal therapy or novel compounds derived from plants may provide unique opportunity to treat such diseases. These studies should lead to development of translational research that extends between the Schools of Medicine, Nursing, Public Health at USC and the Dorn VA Medical Center. The project will use an Administrative Core and an Immunotoxicology Laboratory Core. The Administrative Core will provide scientific leadership including the External Scientific Advisory Committee, the Internal Advisory Committee and Steering Committee, mentorship to junior faculty, post- and predoctoral trainees as well as oversight on fiscal, personnel, and space issues. The Immunotoxicology Laboratory Core will not only serve as a source of the instrumentation resource for pursuing cutting-edge research on whole animals through single cell imaging to molecular level analysis, along with the technological support, but also perform immunotoxicity testing of the plant products. The university will provide seed funds for developmental projects in CAM and enhance recruitment of junior faculty and trainees. The establishment of this Center will provide an interactive environment for research and training in elucidating the mechanisms underlying action of CAM as well as meet the state and national goals on development of novel approaches to patient care.

Proposal Title: 2009 Research Infrastructure Improvement

Institution: South Carolina Research Authority

The South Carolina 2009 NSF EPSCoR RII proposal unites Claflin, Clemson, MUSC, SC State University, and USC and presents an integrated plan to implement a statewide vision that will give South Carolina a competitive edge in the field of biofabrication ?an emerging ?transforming? technology ? operationally defined as computer-aided, layer-by-layer deposition of biologically relevant material with the purpose of engineering functional 3D tissues and organs.

The proposed Research Plan to engineer a 3D vascular tree is divided into five ?thrust? areas. Thrust I focuses on analysis of structural-functional properties of an authentic (natural) branched vascular tree. Thrust II focuses on directed differentiation of adult stem cells into monomer units of vascular cell types, specifically to induce stem cells isolated from fat tissue to enter a smooth muscle cell lineage. Thrust III requires functional biomechanical testing of engineered, sequential segments and comparison to naturally occurring (authentic) branched vascular trees. Thrust IV is biofabrication of a branched vascular tree; that is to create linear 3-D, hollow tubular segments representing branches that can be transformed into branched ?Y? or ?T? vascular units. Thrust V focuses on accelerated tissue maturation of bioprinted, branched vascular tubes.

Intellectual Merit. A major challenge in tissue engineering is the ability to maintain viability of large masses of cells or tissue constructs upon transfer from in vitro culture conditions to in vivo hosts. Most current efforts focus on rapid fabrication of solid scaffolds, or on 2D and 3D cell biopatterning. Less emphasis has been placed on applying the principles of developmental biology and the fundamental biological process of cell and tissue self-assembly. The proposed infrastructure enhancements are essential components for engineering branched, lumenized tubules that can be scaled up (industrialized) to assemble in the future a functional, branched vascular tree, addressing perhaps the greatest obstacle to successful engineering of tissue and organ replacements.

Broader Impacts. Proposed RII activities will lead to: (1) innovative partnerships for training a new workforce in an emerging high tech, high impact industry; (2) dissemination and public education regarding advances and breakthroughs at the interface of engineering, science, and computation; (3) academic-industrial collaborations in developing and improving prototype devices for bioprinting, bioreactors, and biomechanical quantitation; (4) a vanguard position in a new S&T field that is truly global, unfettered by geographical, cultural and historic boundaries, with prime opportunities to attract underrepresented groups into a new field at the cusp of bioengineering, computation and biomedical science; and (5) opportunities for social sciences and other professional disciplines to evaluate and integrate ethical, legal, social and economic implications of ultimately implanting biofabricated replacement tissues and organs. These considerations highlight the importance of a highly science-literate, well informed press corps and public in order to understand and fairly represent multiple sides of complex issues.

DESCRIPTION (provided by applicant): Estrogens have widespread applications in health and disease. Postmenopausal women are treated with estrogen replacement therapy to relieve them of the menopausal symptoms. Between 1940-1975, an estimated 5-10 million Americans received diethylstilbestrol (DES), a synthetic estrogen, during pregnancy or were exposed to the drug in utero. Exposure to DES has been associated with an increased risk for breast cancer in DES mothers and a lifetime risk of cervicovaginal cancers in DES daughters. Exposure to DES has also been linked to a wide range of abnormalities in DES sons and daughters including immune system disorders such as increased incidence of autoimmunity, cancer and certain infections. Furthermore, a number of xenoestrogens which include environmental estrogens and phytoestrogens have been identified which bind estrogen receptors (ERs) and mediate activity similar to physiological estrogens. Thus, research on DES may serve as a template to study a growing list of endocrine disruptors. Studies from our lab demonstrated for the first time that prenatal and postnatal exposure to estradiol (E2) and DES can induce apoptosis in thymocytes, alter the expression of T cell receptor and CD molecules, and modulate positive and negative selection of T cells. In the current study, we will test the central hypothesis that prenatal exposure to DES alters the positive and/or negative selection of T cells in the thymus leading to generation of skewed T cell repertoire with enhanced reactivity towards self antigens and decreased response against non-self antigens. In aim# 1, we will investigate whether the regulation of Fas and Fas ligand genes involves estrogen responsive elements (ERE) and other transcription factors. Aim# 2 will test if prenatal exposure to DES alters positive and negative selection of T cells in the thymus and thereby skewing the immune responsiveness of mature T cells to self rather than non-self antigens. Aim# 3 will investigate the role of ERa in DES induced apoptosis and development of autoimmunity. Also, the role of T cell-stromal cell interactions in DES-induced thymocyte apoptosis will be studied. In Aim# 4, the mechanism by which prenatal DES exposure leads to increased susceptibility to autoimmune disease postnatally will be tested. Together, the studies proposed should provide novel information on the mechanism by which prenatal exposure to DES leads postnatally to increased susceptibility to autoimmunity and cancer and to development of potential approaches to prevent such immunotoxicity.

DESCRIPTION (provided by applicant): Post-traumatic stress disorder (PTSD) is a psychiatric condition with severe symptoms associated with biological dysregulation that can occur after exposure to shock. Exposure to trauma results in modulation in the hypothalamic-pituitary-adrenal (HPA) axis, which plays a critical role in the stress response that in turn interacts reciprocally with the immune system to maintain homeostasis. To date, over 1.6 million US men and women have served in the wars in Iraq (Operation Iraqi Freedom, OIF) and Afghanistan and surrounding regions (Operation Enduring Freedom, OEF). Over 35% of returned Iraq and Afghanistan veterans in Department of Veterans Affairs (VA) care have received mental health diagnoses, the most prevalent being PTSD. Inasmuch as, there is significant prevalence of PTSD in combat veterans who have served in the Iraq and Afghanistan wars, our studies have focused on addressing the pathological basis of immune dysfunction in these men and women. In the current study, we will test the central hypothesis that PTSD associates, at least in part, with dysregulation in the epigenetic mechanisms that control the differentiation of Th1/Th2/Th17/Treg cells of the adaptive immune response, thereby altering the cytokine profiles and promoting inflammatory response. The specific aims are 1) to corroborate the serum cytokine profile with severity of PTSD and to determine the role of transcription factors that regulate the differentiation of Th/T reg cells. Furthermore, whether the cytokine expression in Th/Tregs is dependent on the mitogen activated protein kinase signaling pathway will be elucidated. 2) to address whether epigenetic mechanisms play a role in Th/Treg polarization by performing high-throughput microRNAs arrays and to determine the level of hypo- or hypermethylation of Th1, Th2, Th17 and Treg cytokine/transcription factor gene promoters. The miRNA data generated will be used in silico algorithms for related target gene prediction and pathways that are dysregulated. Further studies will be performed to assess whether reversal of the cytokine expression occurs in the T cells following transfection with miRNA mimics or antagomirs of miRNAs that are downregulated or upregulated respectively. Based on the methylation data, demethylation will be attempted using inhibitors for hypermethylated gene promoters of cytokines or their transcription factors to determine whether this would lead to reversal of the Th/Treg phenotypic characteristics. Together, these studies are novel in that they will not only help understand stress-induced alterations in immune profiles in PTSD patients but will also provide useful clues on whether epigenetic markers and specific cytokines/chemokines can serve as bio-markers of PTSD. PUBLIC HEALTH RELEVANCE: Post-traumatic stress disorder (PTSD) is a psychiatric condition with severe symptoms associated with biological dysregulation that can occur after exposure to shock. Our studies are aimed at determining the immunological alterations induced in these patients and the underlying mechanisms so as to develop strategies for its prevention and therapy.

DESCRIPTION (provided by applicant): This COBRE application proposes to establish a Center for Dietary Supplements and Inflammation (CDSI) at the University of South Carolina (USC) that will pursue multidisciplinary research on how botanicals can modulate inflammation and be used to prevent and/or treat inflammatory diseases. Recently, USC has made significant strides to promote research in Complementary and Alternative Medicine (CAM) which has resulted in the award of a Program Project/Center grant to study CAM modalities to treat autoimmune diseases. Thus, the CDSI will complement the CAM program and create a unique niche at USC in inflammation research. The main goal of CDSI is to establish multi-disciplinary research that will identify the molecular mechanisms through which botanicals modulate inflammation so that they or their analogs can be used to prevent and/or treat inflammatory diseases. Americans spend~$33.9 billion/year on CAM, of which ~$20 billion is on dietary supplements. It is becoming increasingly clear that inflammation plays a critical role in the pathogenesis of not only autoimmune diseases but also a wide range of clinical disorders including cardiovascular diseases, neurodegenerative disorders, and cancer: areas under investigation in this study. Thus, understanding the mode of action of dietary supplements or their constituents on inflammation, could lead to novel treatment modalities with far ranging clinical implications. The CDSI goal will be accomplished through promotion of multi-disciplinary research pursued by 4 junior tenure-track faculty in the area of inflammatory diseases, through highly structured mentoring by eight senior faculty members, and providing them with state-of-the-art core facilities. The program will be evaluated by an External Advisory Committee consisting of nationally recognized scientists. Additionally, through institutional support, 10 new tenure-track junior faculty will be recruited and trained at USC to bolster and advance inflammation research. The long term objective of the CDSI would be to build a self-sustaining, nationally recognized multi-disciplinary Center for dietary supplements and inflammation research, by promoting innovation, faculty entrepreneurship, collaborative research projects (PPGs and Institutional Training grants), and clinical/translational research.

Administrative Core Abstract The Administrative Core will provide oversight on all aspects of the COBRE. The main focus of this Core is to enhance the research quality and productivity of the target faculty in the area of Dietary Supplements and Inflammation so that they will be successful in competing for independent, major extramural funding such as the NIH R01 awards. The specific aims are as follows: 1) To facilitate mentoring of target faculty, enhance use of research resources and monitor the transition of target faculty into independent, well-funded investigators. The Core will be responsible for organizing and integrating the scientific activities of the junior investigators and the mentors of the COBRE. It will organize the lAC and EAC meetings that will provide input to the Center Director in assessment of research progress of the target faculty and allocation of resources. The Core will foster synergistic research between the investigators within the Center and their laboratory personnel by providing an interactive environment that will be nurtured by conduct of formal discussions such as weekly seminars, journal clubs, annual retreat, research day presentations, as well as other informal interactions. It will also coordinate the use of state-of-art core facilities for research. 2) To recruit 10 new tenure-track junior faculty and provide them seed funds to initiate research on dietary supplements and inflammation. The Core will recruit an additional 10 tenure-track Assistant Professors at 5 different colleges to develop multi-disciplinary research. This will be accomplished through significant support from USC. Special consideration will be given to attracting women and underrepresented minorities. The Core will also assist in the recruitment of pre- and postdoctoral researchers, and other technical staff for target faculty. It will provide seed funds for pilot projects from junior investigators to pursue studies on dietary supplements against inflammatory diseases. 3) To oversee the financial and personnel management of all the projects and cores as well as develop long term goals. It will provide oversight in the day to day management of the Center. The Core will be responsible for communication with the university administration and other regulatory agencies. The Core will promote long term objectives of the COBRE that include: 1) advancement of innovation and entrepreneurial activity by the target faculty to enhance drug discovery, 2) helping faculty develop Startup companies, 3) Training target faculty to submit NIH SBIR/STTR grants, 4) develop NIH Program Project grants (PPGs) and 5) prepare for submission of NIH training grant for graduate students and post-doctoral fellows. 6) Facilitate clinical research and trials. Together, these activities should lead to the development of a critical mass of highly competitive scientists supported through NIH R01/Program Projects/Career Development Awards/Training Grants serving the local, regional and national unmet needs in the area of Dietary Supplements and Inflammation.

DESCRIPTION (provided by applicant): This is a renewal application aimed at investigating the epigenetic pathways through which botanicals used commonly as CAM, suppress inflammation. During the previous funding cycle, we have made outstanding progress identifying novel cellular and molecular pathways through which botanicals mediate their anti- inflammatory properties. Inflammation can trigger a wide range of diseases including autoimmune, cardiovascular, neurodegenerative, obesity, and certain types of cancer. For this reason, it is not only critical to uncover as-yet-unknown immune mechanisms and mediators of inflammation but also find novel treatment modalities. Because currently there are no medications that can effectively treat chronic inflammation and associated pain without significant side effects, our proposed studies are highly significant. Traditionally, the medicine practiced in India (Ayurveda) and China have used herbal products to treat inflammatory disorders. Also, more than 25% of the pharmaceuticals are derived from plants, which suggests that botanicals offer novel modalities against inflammation. Epigenetic modifications of chromatin and DNA have been shown recently to play a critical role in the regulation in human pathologies, including inflammation. Thus, the concept that botanicals used as CAM may mediate their effects through epigenetic regulation is highly innovative. The primary objective of the Center is to test the overarching hypothesis that botanicals currently used as CAM, regulate the epigenetic signaling pathways through interactions with specific receptors on immune cells to modulate gene expression leading to amelioration of inflammation. This will be tested using four research projects, 1) Identifying epigenetic pathways through which resveratrol (RES) triggers myeloid- derived suppressor cells (MDSCs) in the regulation of neuroinflammation. 2) Epigenetic regulation of Nrf2 signaling pathway in American ginseng (AG)-mediated suppression of inflammation in the colon and colon cancer. 3) Elucidation of the epigenetic mechanisms underlying dietary indole-mediated amelioration of inflammation in the colon specifically addressing how indoles activate AhR to promote Tregs and suppress Th17 cells. 4) Identifying the role of Sparstolonin B (a compound isolated from Sparganium stoloniferum tubers), as a TLR2 and TLR4 antagonist, thereby suppressing inflammation in the liver through epigenetic regulation. The projects are highly integrated and synergistic, all addressing epigenetic pathways so that the data generated from one project will benefit other projects. The projects will use an Administrative Core which will coordinate all activities of the Center and ensure scientific and programmatic progress. All projects will also use an Analytical Core which will perform genome-wide DNA methylation, histone methylation/acetylation, microRNA arrays, immune monitoring, bioinformatics and natural product integrity testing. Together, our CAM Center will identify epigenetic biomarkers and pathways through which botanicals suppress inflammation thereby paving the way for better treatment modality against inflammatory diseases.

Administrative Core Abstract The Administrative Core will build on the overwhelming success of Phase-1 COBRE by continuing to provide oversight on all aspects of the Center. The main focus of this Core is to continue to enhance the research quality and productivity of the Target faculty in the area of Dietary Supplements and Inflammation so that they will be successful in competing for independent, major extramural funding such as the NIH R01 grants. During COBRE Phase-1, 6 of our junior faculty ?graduated? successfully. Also, we were able to secure a P01/PPG Center involving 3 junior faculty who have graduated from the COBRE. Thus, the Phase-1 Administrative Core has been instrumental in overseeing the success of faculty and ensuring sustainability of the COBRE. The specific aims of the Administrative Core during Phase II are as follows: 1) To facilitate mentoring of target faculty, enhance use of research resources, and enable the transition of target faculty into independent, well-funded investigators. The Core will be responsible for organizing and promoting the scientific activities of the junior investigators through effective mentoring program. It will organize the Internal Advisory Committee (IAC) and External Advisory Committee (EAC) meetings that will provide input to the Center Director in assessment of research progress of the Target Faculty and Pilot Projects as well as Research Cores. 2) To recruit 5 new tenure-track junior faculty and provide them funds to initiate research on dietary supplements and inflammation. The Core will recruit an additional 5 tenure-track Assistant Professors at 4 different colleges in this area of Dietary Supplements and Inflammation to further expand multi-disciplinary research. Special consideration will be given to attracting women and underrepresented minorities which has also been a hallmark of our Phase I grant. 3) To provide oversight and sustain multi-disciplinary, collaborative research program in dietary supplements and inflammation: The Core will provide oversight in the day-to-day management of the Center and ensure that Research Cores evolve to meet the needs of current and future recruits to the COBRE. The junior investigators will be encouraged to be entrepreneurs and compete for NIH-funded SBIR/STTR grants. To sustain the COBRE research, we will apply for additional NIH P01 grants, institutional and individual pre- and post-doctoral training grants and career development awards. In summary, the overall objective of the Administrative Core is to further enhance the research infrastructure specifically in epigenetics, recruit additional junior faculty to increase the critical mass, build sustainable multi- disciplinary Center, and ensure successful transition of junior faculty into independent scientists.

Inflammation in the CNS causes a wide range of clinical disorders including Multiple Sclerosis (MS). During an MS attack, inflammation in the central nervous system (CNS) can produce partial or complete paralysis. There is, as yet, no cure for MS. In the current study, we will test the effect of resveratrol (RES; 3,5,4'-trihydroxystilbene) on experimental autoimmune encephalomyelitis (EAE), an animal model of MS primarily triggered by activation of CD4+ MBP reactive Th1 and Th17 cells. Our laboratory was the first one to demonstrate that treatment with RES can ameliorate EAE. Moreover, we made an exciting and unique discovery that RES triggers induction of a recently characterized immunoregulatory cell called myeloid derived suppressor cell (MDSC). Based on our preliminary studies, we will test the hypothesis that RES suppresses neuroinflammation during EAE through epigenetic regulation and microRNA (miR) induction leading to the generation and activation of MDSCs which suppress myelin-specific T cells. Furthermore, we will test if the MDSC induction results from the activation of aryl hydrocarbon receptor (AhR) and/or estrogen receptor (ER) by RES. Understanding the precise mechanisms underlying the effects of RES on immune response against myelin may offer effective strategies to prevent induction and progression of the disease in MS as well as neuroinflammation associated with a number of other disorders. Aim 1: We will test the central hypothesis that RES activates AhR/ER to induce granulocyte colony stimulating factor (G-CSF) and arginase 1 (Arg1), which trigger differentiation of MDSCs from hematopoietic progenitor cells and their activation respectively. We will investigate the role of AhR and ER in the induction of MDSCs. We will investigate the transcriptional regulation of G-CSF and arginase 1 (Arg1) involving dioxin responsive elements (DRE) or estrogen-responsive elements (ERE) found on the promoters of these genes. Aim 2: We will test the central hypothesis that RES triggers MDSCs through epigenetic regulation of genes involved in MDSC differentiation and functions. The role of DNA methylation as well as histone modification will be determined in RES-induced MDSC in EAE mice by assessing genome-wide methylation using MeDip- Seq and ChiP-Seq, and by determining locus-specific methylation status. The DNA methylation and histone marks in the specific gene promoters of CD11b and Gr-1 as well as arginase 1 and iNOS will be validated. Aim 3: We will test the central hypothesis that RES induces miRNA that target specific genes involved in MDSC differentiation and functions. We will identify the role of miR223 that is induced by RES in hematopoietic stem cell differentiation into MDSC. We will also test whether the downregulation of miR185 and miR340 leads to increased iNOS and arginase production respectively. Together, our studies should provide novel information on how RES suppresses neuroinflammation through the induction of MDSCs via epigenetic regulation.

AhR is a cytosolic transcription factor that can be activated by wide range of chemicals including environmental contaminants, such as 2,3,7,8-Tetrachlorodibenzodioxin (TCDD), which leads to metabolism and regulation of toxic effects. AhR can also be activated by endogenous ligands and dietary compounds. Recent groundbreaking studies demonstrated that AhR activation can also regulate T cell differentiation, specifically the differentiation of Foxp3+ regulatory T cells (T regs) and proinflammatory Th17 cells that play a role in extracellular infections and autoimmune diseases such as multiple sclerosis (MS). Interestingly, AhR ligands were shown to have contrasting effects on Treg/Th17 differentiation, the reasons for which are not clear. For example, studies, including ours, demonstrated that some AhR ligands, such as TCDD, promoted the differentiation of Tregs while dampening that of Th17 cells, while a tryptophan photoproduct, 6-formylindolo[3,2-b]carbazole (FICZ), exerted contrasting effects. We have generated exciting preliminary data indicating that AhR activation triggers dysregulation in the microRNA (miR) expression and other epigenetic pathways. Based on the above, we will test the hypothesis that AhR-ligand complex may interact with dioxin response elements (DREs) on gene promoters of microRNAs as well as induce other epigenetic pathways, which together alter the expression of the miRs, which in turn, regulate the differentiation of encephalitogenic T cells. We will test our hypothesis using an experimental model of MS called Experimental Autoimmune Encephalomyelitis (EAE). In Aim1, we will test whether TCDD and FICZ induce unique miR expression profiles in purified Tregs/Th17 cells. Based on our preliminary data, we will focus on miR31-5p and miR1192 that target the expression of FoxP3 and IL-17. Specifically, we will examine whether the interactions of AhR-ligand complex with the DREs on these miR gene promoters are responsible for the disparate responses. In Aim 2, we will test whether the contrasting effect of TCDD and FICZ is due to differential DNA methylation/hydroxymethylation of these miR gene promoters as well as due to histone modifications. In Aim 3, we will determine whether miR mimics or antagomirs as well as pharmacological intervention of DNA methylation/hydroxymethylation and histone modification would reverse the inflammatory response and clinical outcome following ligation AhR with TCDD or FICZ. The proposed studies are highly significant in that we will identify novel epigenetic pathways triggered by AhR activation leading to immune regulation. Our studies will also provide novel information on whether targeting such epigenetic pathways in vivo can prevent and treat inflammatory and autoimmune diseases.

Nontechnical Description

The Established Program to Stimulate Competitive Research (EPSCoR) holds National Conferences that bring representatives from all EPSCoR jurisdictions together to discuss strategies to promote EPSCoR goals of building STEM research and education capacity to increase their national competitiveness. This biennial event is a forum for EPSCoR project directors and participants to discuss opportunities and challenges at the local, state, national, and international levels and how EPSCoR efforts can collectively contribute to the advancement of science and engineering research and education. This meeting also showcases results from projects funded by NSF EPSCoR. The 26th National EPSCoR Conference will be held in Columbia, South Carolina from October 27-30, 2019 and is expected to have 300-400 attendees. This meeting is organized by South Carolina EPSCoR with a focus on transdisciplinary science and education.

Technical Description

The theme for the 26th National EPSCoR Conference is "Science and Partnerships across Disciplinary Boundaries". The program will include five sub-themes to provide a organizing framework for the meeting: 1) Promoting transdisciplinary research and education through NSF EPSCoR investments; 2) Broadening participation in STEM through partnerships; 3) Promoting public-private partnerships in education and research leading to economic competitiveness; 4) Big data in a transdisciplinary perspective: crossing boundaries; and 5) Evaluating transdisciplinary research and conveying its importance to a broader audience. The National Conference will showcase high-impact programs and explore the elements and strategies that make them successful over the long-term in their capacity for scientific and economic advancement. The conference program will include platform and poster sessions to encourage the active participation of EPSCoR awardees - Principal Investigators, Project Administrators, Education, Outreach, Diversity Coordinators as well as other senior personnel and early-career researchers (including students). Plenary sessions featuring national experts will highlight different aspects of the meeting theme.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Abstract: The overall objective of the COBRE Phase-2 for Dietary Supplements and Inflammation (CDSI) at the University of South Carolina (USC) is to continue to build on the success of Phase-1 and advance the research infrastructure specifically in the area of dietary supplements and inflammation by promoting multi-disciplinary research pursued by junior investigators so that they become highly competitive in obtaining NIH R01 grants and similar type of extramural funding to pursue their research. The overall objective is to investigate how botanicals can attenuate inflammation and be used to prevent and/or treat inflammatory diseases. The CDSI will test the overarching hypothesis that botanicals or their constituents regulate inflammation through epigenetic regulation of immune cell functions. In Phase-1, we have made outstanding progress in the successful transition of all of our junior faculty into extramurally-funded investigators. Additionally, we were successful in securing NIH Complementary and Alternative Medicine (CAM) Center (P01) for Epigenetic Regulation of Inflammation, in which ?graduated? faculty are leading the projects, thereby paving the path towards sustainability of CDSI. Thus, the CDSI will complement the CAM Research and create a unique niche at USC in inflammation research. The main goal of COBRE Phase-2 is to establish multi-disciplinary research that will identify the epigenetic mechanisms through which botanicals modulate inflammation so that they or their analogs can be used to prevent and/or treat inflammatory diseases. The US population spends ~$33.9 billion/year on CAM, of which ~$20 billion is on dietary supplements. It is becoming increasingly clear that inflammation plays a critical role in the pathogenesis of not only autoimmune diseases but also a wide range of clinical disorders including cardiovascular diseases, neurodegenerative disorders, obesity, aging and cancer. Thus, understanding the mode of action of botanicals or their constituents on inflammation, could lead to novel treatment modalities with far ranging clinical implications. The CDSI goal will be accomplished through promotion of multi-disciplinary research pursued by 4 junior faculty in the area of inflammatory diseases, through highly structured mentoring of each by a senior faculty and a recently graduated faculty member of the COBRE Phase-1. The junior faculty will have access to state-of-the-art research core facilities that include Flow Cytometry, Microscopy and Imaging Core, and Bioanalytical Core, to aid epigenetic studies. The program will be evaluated by an External Advisory Committees consisting of nationally recognized scientists. Additionally, through institutional support, 5 new tenure-track junior faculty will be recruited and mentored at USC to bolster and advance inflammation research. The long term objective of the CDSI would be to build a self-sustaining, nationally and internationally recognized multi-disciplinary Center for dietary supplements and inflammation research, by promoting innovation, faculty entrepreneurship, collaborative research projects such as PPGs and Institutional training grants, and clinical/translational research.

Abstract Bacterial infections during neonatal phase cause high rates of morbidity and mortality, and in developing countries are responsible for 26% of deaths. Environmental factors present during pregnancy are known to impact life-threatening infections in newborns, including Staph. aureus infections, although the mechanisms are unclear. Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental pollutant, which acts through the cytosolic aryl hydrocarbon receptor (AhR). While AhR has been well characterized for its role in regulating toxicity mediated by TCDD, recently, AhR activation was shown to regulate T cell differentiation into T regs or Th17 cells. We have generated exciting preliminary data indicating that AhR activation by TCDD suppresses T cell response to Staphylococcal enterotoxin B (SEB) and that this is mediated by epigenetic pathways including dysregulation in microRNA (miR) expression, DNA methylation, and histone modifications in activated T cells. More importantly, our studies have suggested that TCDD may exert transgenerational epigenetic effects on T cells. Based on the importance of Staph infections discussed above, we will use SEB as an antigen to test the central hypothesis that AhR activation of V?8+ T cells by TCDD, plays a crucial role in reducing pro-inflammatory Th1/Th17 cells as well as increasing anti-inflammatory Tregs and its subsets by modulating miR expression, and that this may depend on DNA methylation, histone modifications and chromatin remodeling that could be transmitted transgenerationally. Inasmuch as, SEB can activate V?8+ T cells which constitute ~30% of peripheral T cells, our studies are aimed at determining whether TCDD-induced changes persist in F0, F1, F2, and F3 generations following maternal exposure during pregnancy to TCDD or maternal/paternal exposure prior to mating (F0). In Aim 1, we will determine the transgenerational effects of TCDD on SEB-induced CD4+ T cell differentiation. We will test the effect of TCDD on the TCR clonality and diversity of the V?8+ CD4+ T cell response (Th1, 2, 17, Tregs) to SEB. In Aim 2, we will study the role of specific miRs in CD4+ T cell differentiation in F0-F3 generations. Furthermore, transfection of T cells with specific miR mimics or antagomirs will be performed to reverse T cell differentiation induced by TCDD and determine whether the effects persist across generations. In Aim 3, we will determine the role of genome-wide and locus-specific DNA methylation on CpG sites on promoters of specific miR that regulate differential expression of Th/Treg response to SEB across the generations. Aim 4 will elucidate the permissive and repressive histone modification and chromatin accessibility in TCDD-mediated transgenerational dysregulation of miR involved in CD4+ T cell differentiation. Lastly, whether these changes are imprinted through male or female germline will be assessed. The proposed studies are highly significant in that novel epigenetic pathways of TCDD-mediated immunotoxicity across generations will be identified. Also, understanding how AhR ligands mediate differential effects through epigenetic pathways would lead to development of innovative preventive and therapeutic modalities.

Abstract: This proposal requests funds of $250K with remaining funds from our institution to purchase a high-parameter, state-of-the-art flow cytometer, BD FACSymphony A5 Cell Analyzer. This flow cytometer offers novel and cutting-edge technology aimed at simultaneous detection of up to 30 parameters for the study of many inflammatory diseases that include autoimmune, cardiovascular and neurodegenerative diseases as well as obesity, aging and cancer that are important areas of research in the COBRE on Dietary Supplements and Inflammation (DSI) at the University of South Carolina (UofSC). The ability to distinguish the multiple cell types based on biomarkers, receptors and functional characteristics as well as the ability to integrate the panels into a single composite that enables the diagnosis and prognosis of disease following attempts towards prevention and treatment with plant products will be most readily performed with this high-resolution and high-throughput instrument. This instrument will be housed in the Flow Cytometry and Imaging Core of the COBRE at the University of South Carolina, School of Medicine. This core will serve the needs of the Target Faculty and Pilot Project faculty as well as their graduate and undergraduate students, postdoctoral fellows and technical staff. The existing flow cytometers such as the FC500 are antiquated and offer a maximum of 5-color capability, thereby limiting the cell subpopulations that can be identified and have no data integration capability. In addition, these flow cytometers are not supported by the vendor as they are obsolete with parts not available. Further, FACSymphony has 5 lasers, 30 parameters and 28 color detectors offering high-performance detection capability with commercial, low cost, readily-available fluorochrome conjugated antibodies, high- speed processors, optimized photomultiplier tubes for each detector as well as updated software for analysis and visualization. Importantly, the instrument can be upgraded to 10 lasers, 50 parameters with 48 colors in the future. This bench-top instrument allows for rapid analysis of low volume of sample, can detect upto 28 colors, overcomes issues associated with fluorescence compensation and is user-friendly. As detailed in the proposal, the biomedical research environment in the COBRE on DSI at the UofSC is on a trajectory for expansion with no multi-parameter flow cytometer. The absence of this capability has adversely affected the research programs of the investigators participating in this COBRE as well as the recruitment of promising scientists to the UofSC. Acquisition of the instrument and housing it in an established core facility will allow access to it by a large number of investigators and will significantly enhance the research quality and training. Dr. Narendra Singh, a Research Professor and the Flow Cytometry, Microscopy and Imaging Core Leader has >20 years experience in running flow cytometry will oversee the operation of this system.

The environmental sensor, aryl hydrocarbon receptor (AhR) serves as a ligand for pollutants as well as for plant, microbial and endogenous compounds. Following AhR ligation, the activated AhR regulates gene expression through the binding of AhR/ARNT complex to specific DNA motifs known as Dioxin Response Elements (DREs). Studies from our lab and elsewhere have shown that some AhR ligands have potent immunosuppressive properties. Inflammatory bowel disease (IBD) results from chronic inflammation in the gastrointestinal tract that affects 1.5 million people in the US. The pathogenesis of IBD involves complex interactions between gut microbiota, immune response, environmental and dietary factors, and genetic/epigenetic regulation. Recently, we made an exciting observation that the AhR ligand and plant product, I3C ameliorates colitis in mice, which was associated with anti-inflammatory effects, regulation of gut dysbiosis, and enhanced expression of ?-defensins (mBD1,2,3) by Colonic Epithelial Cells (CEC). ?-defensins constitute antimicrobial peptides (AMPs) that resist microbial colonization of epithelial surfaces in the colonic tissue. ?- defensins may also mediate anti-inflammatory effects. In fact, studies have shown defective expression of intestinal AMPs particularly defensins in IBD patients. We were excited to uncover DREs in the promoters of mouse ?-defensins (mBD1, 2, and 3). In the current study, we will test the central hypothesis that dietary indoles (I3C) attenuate colitis through AhR activation leading to increased expression of mBDs by CECs via pathways involving DREs, and/or epigenetic regulation resulting in modulation of microbiota and prevention of epithelial barrier damage. Furthermore, we propose that mBDs induced by I3C play a critical role in restoring healthy gut microbiota, preventing intestinal barrier damage and suppressing colonic inflammation through induction of Tregs. Aim 1 will test the mechanisms of mBD induction by dietary indoles. We will use reporter assay, promoter bashing and electrophoretic mobility shift assay to determine whether I3C activated AhR directly binds to the DREs to induce mBDs. We will also determine the effect of I3C on the mBD expression by using AhR cKO mice with AhR deletion in IEC, ILC3 and Tregs. In Aim 2, we will study epigenetic regulation of ?-defensins by dietary indoles. To that end, we will test whether I3C regulates mBD expression by altering histone modification and decreasing DNA methylation. We will specifically determine if I3C regulates the SATB1-mediated histone deacetylation and chromatin remodeling. In Aim 3, we will test whether administration of mBDs offers protection from colitis by regulating gut dysbiosis, preventing CEC barrier damage, enhancing Treg subsets and decreasing Th17 subpopulations to attenuate colonic inflammation. Finally, we will use mBD KO mice to test whether mBDs are required for I3C-mediated protection from colitis. The proposed studies are highly significant because they will identify novel mechanisms through which dietary indoles suppress colitis by altering the microbiota, through activation of AhR leading to increased expression of host-derived AMPs, specifically ?-defensins.

Project Summary/Abstract: Older women are more likely than men to live with disease or disability that impairs activities for independent living and threatens quality of life. The mechanisms that broadly diminish health and well-being among aging women are not fully understood, but the transition into menopause at mid-life, and the ensuing decrease in circulating estrogens, is a particularly salient milestone in the female lifespan. Numerous studies document an association between the loss of ovarian hormones at menopause and the loss of their protective effects in domains of cognition, physical ability, and immune health. Estrogens typically act upon these systems to optimize cellular metabolism supported by mitochondrial oxidative phosphorylation. Loss of estrogens at middle-age is associated with increased oxidative stress and enhanced inflammatory responses. Hormone replacement therapy is prescribed to relieve signs and symptoms of menopause, but its broad actions carry undesirable and sometimes life-threatening risks. Identifying complementary strategies that restore the proper balance of metabolic and immune activities in peri- and post-menopausal women could decelerate age-related declines in cognitive, physical, and immune health without incurring risk for other age-associated diseases. The ketogenic diet may fulfill these criteria as consuming this high fat/low carbohydrate diet fundamentally alters metabolic profiles, shifting dependence away from glucose in favor of fat-derived ketone bodies, while also attenuating inflammation. Consistent with this view our preliminary data demonstrate that a ketogenic diet improved cognitive and mitochondrial function in aging female rats. The goal of this project is to elucidate the mechanisms by which the ketogenic diet decelerates age-related decline in multiple functional domains over the female lifespan. This project will use normally aging rats to 1) determine sex-specific effects of ketogenic diet on age-related decline of brain, muscle, and immune function and 2) determine protective effects of ketogenic diet on cognition, physical function, and immune profiles in surgically estrogen-deficient, middle-aged females. These studies of nutritional ketosis in well-controlled animal models of aging and menopause will be significant because they can provide the necessary mechanistic insights to guide the translation and development of appropriate dietary interventions that physicians can recommend as primary or adjuvant therapies to older women transitioning into menopause and at risk for age-related disorders. More broadly, identifying sex-specific and age-appropriate dietary guidelines to decelerate fundamental mechanisms that drive physiological and cellular aging will broadly improve health outcomes for older women and reduce reliance on hormone replacement therapy as a first-line treatment to combat symptoms of menopause. These objectives are within the scientific scope of the University of South Carolina?s COBRE on Dietary Supplements and will accomplish rigorous scientific research on the underlying molecular and cellular mechanisms of aging leading to increased risk of disease (Alzheimer's disease, sarcopenia) in pre- and post-menopausal women.