Margherita T. Cantorna - US grants

interferon gamma; retinoids; genetic regulation; genetic translation; secretion; protein biosynthesis; messenger RNA; transcription factor; T lymphocyte; genetic transcription; RNase protection assay; human subject; northern blottings; immunoelectron microscopy; nutrition related tag;

The objective of this proposal is to define molecular and cellular targets of vitamin D in the immune system. There is a large body of anecdotal data to suggest that there is a link between vitamin D status and the human autoimmune disease multiple sclerosis. Experimentally, vitamin D deficiency exacerbates experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Furthermore supplementation of mice with vitamin D can suppress and prevent symptoms of EAE. Two targets of vitamin D in the immune system have been identified the interleukin (IL)-4. secreting type-2 helper T (Th2) cells and transforming growth factor (TGF)-beta1 (two cytokines shown to be protective in EAE) secreting cells. The hypotheses to be tested are: 1. Vitamin D status regulates Th cell differentiation. 2. Vitamin D treatment of EAE results because of the induction of Th2 cells. 3. Vitamin D induction of Th2 cells and protection from EAE depends on TGF-beta1 synthesis. 4. Vitamin D negatively regulates Th1 effector cells. Th cell differentiation and function will be measured as a function of increasing vitamin D both in vitro and in vivo in Th cells from T cell receptor transgenic mice. T cells from vitamin D treated mice will be isolated and tested for their ability to transfer protection from EAE. TGF-beta1 will be neutralized in vitro and in vivo to determine if vitamin D functions via the transcriptional upregulation of TGF-beta1. Similarly, TGF-beta1 supplementation will be done and compared to vitamin D treatment for the mechanisms by which they suppress EAE. Th2 cell deficient mice will be used as a source of Th1 effector cells and vitamin D will be tested as a direct regulator of Th1 function. A better understanding of how vitamin D effectively blocks EAE is necessary for proper nutritional counseling and optimal treatment of multiple sclerosis patients.

[unreadable] DESCRIPTION (provided by applicant): The objective of this project is to define the cellular and molecular targets of vitamin D as a regulator of inflammatory bowel disease (IBD). IBD are immune mediated diseases of unknown etiology affecting the gastrointestinal tract. Higher incidences of IBD occur in the northern climates of the United States and Europe; places where sunshine and vitamin D synthesis in the skin are low. Experimentally; vitamin D deficiency exacerbated symptoms of IBD in mice, which spontaneously develop enterocolitis {interleukin (IL) 10 knockout (KO)}. Supplementation with hormonally active vitamin D (1,25(OH)2D3) for as little as 2 weeks ameliorated IBD symptoms in these mice. IL10 KO mice, which cannot respond to vitamin D (vitamin D receptor; VDR/IL10 double KO) develop a fulminating form of IBD, which was transferred to T and B cell deficient mice by both CD4+ or CD8+ T cell injections. A second experimental model of IBD (CD45RBhigh induced) was more severe when the T cells were VDR KO compared to wildtype. The proposal described here will test the hypothesis that vitamin D is a physiological regulator of the CD4+ and CD8+ T cells, which cause and/or suppress IBD disease. IBD inducing T cells will be isolated and treated in vitro and in vivo with and without vitamin D and the functions of the T cells will be compared for the ability of the cells to induce disease in naive mice. The use of VDR KO mice will be included to determine whether there is a physiological role for vitamin D in the development/regulation of T cells, which induce or suppress IBD. The experiments described here are designed 1) to determine the mechanisms underlying 1,25(OH)2D3 mediated suppression of experimental IBD; 2) to determine whether IBD develops in germfree VDR/IL10 double KO mice 3) to determine which T cell functions and genes are targets of vitamin D in CD4+ T cells 4) to determine which T cell functions and genes are targets of vitamin D in CD8+ T cells and 5) to determine whether CD4+ and/or CD8+ T cells from VDR KO mice can transfer or suppress IBD. A better understanding of the mechanisms underlying vitamin D regulation of T cells and IBD may lead to improved therapies for patients with IBD. [unreadable] [unreadable] [unreadable]

SUMMARY (revised) The vitamin D hypothesis proposes that vitamin D regulates gastrointestinal homeostasis by multiple mechanisms and that changes in vitamin D status affect the composition of the microbiota, the development of the immune response, clearance of gastrointestinal infections and the ability to reinstate homeostasis following injury or infection. During the last grant cycle we determined several novel mechanisms by which vitamin D regulated T cells to control homeostasis in the gut. Our new preliminary data demonstrate that disruptions in the microbiota would inhibit vitamin D regulation of immune function and the metabolism of vitamin D by the host. These novel areas of investigation serve as the focus of the present application. The gastrointestinal immune system is a population of heterogeneous cells whose role is to maintain ignorance of the large number of antigens present in food as well as the microbiota. The microbiota in turn shapes the immune response. Germfree mice have reduced B cell IgG1, IgG2a and IgA responses, but hyper-IgE antibodies. Hyper-IgE is a symptom of dysbiosis and a failure of homeostasis. Vitamin D deficient and vitamin D receptor knockout mice have hyper-IgE and dysbiosis of the microbiota. In addition, following injury vitamin D regulates the T and B cell response in part through regulation of the microbiota. Perturbations of homeostasis, following gastrointestinal infection, of vitamin D deficient mice was severe and associated with the reduced production of antigen specific antibody responses. Not only was there an effect of vitamin D on the microbiota but the microbiota affected vitamin D metabolism. This proposal will focus on the effects of vitamin D on B cells and the microbial interactions that control the B cell response and microbial regulated tissue vitamin D metabolism. Our novel central hypothesis is that ?Vitamin D and the microbiota cooperate to regulate B cells and the vitamin D responsiveness of the host.? The three aims are: Aim 1: Determine the direct and indirect targets of vitamin D on B cells in vivo that are important for maintaining homeostasis, induction of antibody responses, and protection from GI infection. Aim 2: Determine which of the effects of vitamin D depend on the microbiota. Aim 3: Determine the role of the microbiota in the induction of 24,25(OH)2D by B cells.

DESCRIPTION (provided by applicant): There are profound effects of vitamin D on immune function. There is data to suggest that increased levels of vitamin D (either through diet, supplements or sunlight exposure) are beneficial for multiple sclerosis (MS) patients. In addition, active vitamin D (1,25(OH)2D3) treatment of experimental autoimmune encephalomyelitis (EAE) blocks the development of disease. In the first year of life infants undergo considerable fluctuation in their circulating vitamin D (25(OH)D3) levels. We hypothesize that early changes in vitamin D have profound effects on the thymus such that iNKT cells fail to develop and as a result autoimmune diseases like MS are more likely to develop. iNKT cells require adequate vitamin D in utero and the expression of the vitamin D receptor (VDR) for both development and function. iNKT cells have been implicated as inhibitors and regulatory cells in EAE and MS. The hypothesis to be tested is that vitamin D regulates iNKT cells and as a result is important for shaping the developing immune response and preventing and controlling the development of autoimmunity. The goals of this proposal are to determine the molecular mechanisms underlying vitamin D and the VDRs effects on iNKT cells. The aims are to determine the plasticity of the iNKT cell response as a function of changes in vitamin D status, determine the role of vitamin D in the thymus for iNKT cell development, determine how changes in vitamin D affect proliferation, survival and death of iNKT cells, and to determine the role of active vitamin D (1,25(OH)2D3) regulation of iNKT cells in suppression of EAE and whether changes in vitamin D status in vivo or 1,25(OH) 2D3 in vitro regulates human NKT cell expansion and function. NKT cells are novel targets in MS and a better understanding of the mechanisms by which their development and function are regulated by vitamin D and 1,25(OH)2D3 would be critical for manipulating NKT cells therapeutically. PUBLIC HEALTH RELEVANCE: There is at present a great deal of misinformation about vitamin D and vitamin D supplements as immune system modulators in the public forum. Identifying the cellular and molecular targets of vitamin D in the immune system is important so that rational decisions about the use of vitamin D supplements and/or active vitamin D compounds to manipulate the immune system and prevent or treat autoimmune diseases like multiple sclerosis.

? DESCRIPTION (provided by applicant): Nutritional status, immune function, and the gut microbiota are all factors in protection against gut infection. Citrobacter rodentium causes a gastrointestinal (Gl) infection in mice that models foodborne infections with enteropathogenic Escherichia coli in humans. The goal of this project is to determine how vitamin A deficiency worsens and intervention with the vitamin A metabolite retinoic acid (RA) affords protection against C. rodentium infection. Our preliminary data show that vitamin A deficiency (A-) resulted in a chronic C. rodentium infection that was lethal in 40% of the A- mice. In contrast, chronically infected A- mice treated with RA were able to clear the infection. Here we will determine the mechanisms by which vitamin A deficiency results in failure to clear infection and RA helps to resolve this GI infection. We hypothesize that vitamin A/RA is required for protection mediated by: 1) T cells, 2) intestinal epithelial cells, and 3) maintaining homoeostasis of the gut microbiota. Our approach includes the use of vitamin A deficient and adequate mice, and A- mice treated with RA, and the use of 3 novel animal models: mice lacking retinoid signaling in T cells or in intestinal epithelial cells specifically, and germfree mice. Mice with a tissue-specifi dominant negative RA receptor will be used to block retinoid signaling selectively in T cells (aim 1) and intestinal epithelial cells (aim 2) to probe mechanisms by which vitamin A controls GI infection. Methods to be used will include imaging of bioluminescent C. rodentium to localize the bacteria in the intestine and immunological and histological methods to characterize cell types, functions and tissue histopathology. In addition, germfree mice will be used to determine the role of the microbiota in the vitamin A-mediated protection from C. rodentium (aim 3). It is expected that resolution of C. rodentium infection (clearance) will require vitamin A signaling in both intestinal T cells and epithelium as well as maintaining homeostasis of gut microbiota. Understanding the mechanisms by which vitamin A deficiency and RA intervention regulate these processes will be critical for informing public health messages as well as for the possible use of vitamin A/RA for protection from GI infection.

PROJECT SUMMARY The primary goal of this training program entitled ?Research Training in Physiological Adaptations to Stress? is to provide a new generation of future scientists with comprehensive research training and educational experiences that emphasize a translational approach to understanding the physiological mechanisms which trigger and mediate organismal stress adaptation. Value added training in business entrepreneurship using a boot camp approach, and didactic training in team dynamics through the Penn State MBA program is also emphasized. Graduate students presently encounter little formal training in the domains of regulatory science and team dynamics, and we therefore also propose an inter-disciplinary curriculum which includes training for emerging academic and nonacademic career preparation. An understanding of the business, legal and regulatory issues which shape key milestones in the biomedical science pathway, and the ability to work in diverse teams will allow our trainees to be leaders of innovation. Here, rigorous research design and core competencies necessary for effective communication are also emphasized. Collectively, students will be well positioned for emerging career paths associated with the 21st century biomedical workforce. The training program takes advantage of existing faculty biomedical expertise within nine participating programs who have amassed an impressive record of student training while maintaining research excellence (total annual direct cost of ~$8,439,420 and an average of ~$351,643 per faculty). We propose selection of 6 students per year for a period of two years (total of 18 trainees). Additional institutional matching funds will allow for the training of 8 additional students. The institutional commitment to our training program is outstanding. A foundational course entitled ?Physiological Adaptations to Stress? has also been designed specifically for the training program, and will serve as a capstone experience to emphasize program goals using multiple levels of scientific inquiry (cells to human). Defining a new conceptual framework for hypothesis-driven research to inform mechanisms of stress adaptation within a rich academic environment with dedicated mentoring, strong research support and institutional commitment is proposed. We envision trainees will continue to participate in program activities at the conclusion of their tenure as a T32 Fellow.

SUMMARY PARENT AWARD The vitamin D hypothesis proposes that vitamin D regulates gastrointestinal homeostasis by multiple mechanisms and that changes in vitamin D status affect the composition of the microbiota, the development of the immune response, clearance of gastrointestinal infections and the ability to reinstate homeostasis following injury or infection. During the last grant cycle we determined several novel mechanisms by which vitamin D regulated T cells to control homeostasis in the gut. Our new preliminary data demonstrate that disruptions in the microbiota would inhibit vitamin D regulation of immune function and the metabolism of vitamin D by the host. These novel areas of investigation serve as the focus of the present application. The gastrointestinal immune system is a population of heterogeneous cells whose role is to maintain ignorance of the large number of antigens present in food as well as the microbiota. The microbiota in turn shapes the immune response. Germfree mice have reduced B cell IgG1, IgG2a and IgA responses, but hyper-IgE antibodies. Hyper-IgE is a symptom of dysbiosis and a failure of homeostasis. Vitamin D deficient and vitamin D receptor knockout mice have hyper-IgE and dysbiosis of the microbiota. In addition, following injury vitamin D regulates the T and B cell response in part through regulation of the microbiota. Perturbations of homeostasis, following gastrointestinal infection, of vitamin D deficient mice was severe and associated with the reduced production of antigen specific antibody responses. Not only was there an effect of vitamin D on the microbiota but the microbiota affected vitamin D metabolism. This proposal will focus on the effects of vitamin D on B cells and the microbial interactions that control the B cell response and microbial regulated tissue vitamin D metabolism. Our novel central hypothesis is that ?Vitamin D and the microbiota cooperate to regulate B cells and the vitamin D responsiveness of the host.? The three aims are: Aim 1: Determine the direct and indirect targets of vitamin D on B cells in vivo that are important for maintaining homeostasis, induction of antibody responses, and protection from GI infection. Aim 2: Determine which of the effects of vitamin D depend on the microbiota. Aim 3: Determine the role of the microbiota in the induction of 24,25(OH)2D by B cells. SUMMARY ADMINISTRATIVE SUPPLEMENT. This administrative supplement would be used to do some important experiments in gnotobiotic mice that were not possible until recently. We would like to rederive a Cyp27B1 reporter strain as germfree. Cyp27B1 is the key enzyme controlling production of 1,25(OH)2D. These germfree Cyp27B1 ko/+ and ko/ko reporter mice will then be immunized or colonized with a single microorganism (one commensal and one pathogen) to determine which immune or microbial signals induces expression of Cyp27B1 in B cells or other immune cells. At the completion of these experiments we will definitively determine under which conditions B cells or other immune cells produce Cyp27B1 in vivo. Without this additional supplement we would not be able to do these experiments. In addition, we want to have enough male and female mice so that we can evaluate sex as an independent variable. The parent grant proposed to do these experiments only in germfree wildtype mice of mixed sex. In addition to the experiments in Cyp27B1 ko/+ and ko/ko mice; we would like to double the numbers of male and female wildtype germfree mice so that we have enough male and female mice to determine the role of sex on expression of Cyp27B1 and metabolism of vitamin D.