Improving Disease Resistance of Livestock Through Identifying Mechanisms of Vitamin D Signaling in the Immune System

<p>NON-TECHNICAL SUMMARY:<br/> Infectious diseases are estimated to cost the livestock industries several billions of dollars in lost revenue every year. The goal of this project is to decrease the economic and food safety costs of those diseases through improving disease resistance of animals. Vitamin D deficiency is correlated with increased risk for infectious diseases in humans, the same is likely true for cattle and pigs. This project will identify host and pathogen factrs that affect vitamin D metabolism in cattle and pigs, and the influence of vitamin D compounds on immunity of cattle and swine. This information gained from this project is expected to generate strategies, therapies, and nutritional recommendations that will improve disease resistance of livestock.
<p>APPROACH: <br/> Objective 1: Identify effects of pathogen factors on vitamin D metabolism in bovine and porcine immune cells. Lipopolysaccharide (LPS) activates a vitamin D signaling pathway in in bovine macrophages and preliminary data indicates the same for swine. Several experiments to follow-up the preliminary data are planned to study the influence of pathogen associated molecules on vitamin D metabolism in vitro and in vivo (experiments 1 and 2).<br/>Experiment 1.1: Evaluate the influence of TLR signaling on vitamin D metabolism in macrophages. Monocytes from peripheral blood of pigs and cattle will be evaluated for CYP27B1 and VDR gene and protein expression in response to activation with TLR ligands (dsRNA, lipolysaccharide, lipoteichoic acid, peptidoglycan, and triacylated lipopeptides). The ability of the pig monocytes to synthesize 1,25(OH)2D3 will be accomplished
by addition of physiological concentrations of 25(OH)D3 (25-100 ng/mL) to resting and TLR-stimulated monocytes, followed by analysis of CYP24A1 gene expression (and genes identified from experiment 2). Upregulation of CYP24A1 gene expression in response to TLR-stimulation and addition of 25(OH)D3 will indicate that TLR-stimulated monocytes are produce 1,25(OH)2D3. A treatment with an inhibitor of CYP27B1, ketoconazole, also will be included to demonstrate that effects of 25(OH)D3 in TLR-stimulated monocytes are dependent on CYP27B1 enzyme activity. Alternatively, CYP27B1 expression will be inhibited with siRNA specific for the CYP27B1 transcript to determine the requirement of the CYP27B1 enzyme. The actual amount of 1,25(OH)2D3 produced by monocytes as a result of LPS and 25(OH)D3 treatments would be an ideal measure, but there are currently not reliable assays to directly measure
1,25(OH)2D3 in cell culture because of metabolites produced in culture that interfere with the assay.<br/>Experiment 1.2: Determine if the vitamin D pathway is activated in immune cells in vivo as a result of influenza infection. Lung tissue samples, alveolar macrophages, and draining lymph nodes will be collected from necropsies of H1N1-infected and control pigs. Those tissue samples will be evaluated for CYP27B1, CYP24A1, and VDR gene and protein expression using quantitative PCR and immunohistochemical assays.<p>Objective 2: Determine the influence of vitamin D signaling in bovine and porcine immune responses. Other than upregulation of nitric oxide and RANTES production in bovine macrophages, the effects of vitamin D signaling on immunity of pigs and cattle is unknown. This objective seeks to identify the effects of vitamin D signaling on innate and adaptive immunity in vitro, and to develop
a model to study the effects of vitamin D on disease resistance in vivo. <br/>Experiment 2.1: Identify genes regulated by vitamin D signaling in bovine and porcine immune cells. To identify the genes regulated in pigs and cattle LPS-stimulated and non-stimulated monocytes or epithelial cells will be treated with 0 or 10 nM 1,25(OH)2D3 for 6 and 24 h. RNA sequencing will then be performed on the mRNA isolated from the cells, and bioinformatics analysis will be performed to identify specific genes and pathways affected by 1,25(OH)2D3. RNA sequencing results from bovine and porcine immune cells will be validated with real-time PCR and appropriate functional assays.<br/>Experiment 2.2: Effects of vitamin D signaling on antigen-specific T cell responses of pigs. In cattle, humans, and mice, 1,25(OH)2D3 inhibits T cell proliferation and IFN-gamma and IL-17 production in vitro. To determine if
1,25(OH)2D3 that is produced by macrophages does the same in pigs, peripheral blood mononuclear cells (PBMCs; consist of macrophages, T cells and B cells) will be collected from H1N1-vaccinated and control and pigs. The PBMCs will be stimulated in culture with UV-killed H1N1 with and without 25(OH)D3. At the end of the culture period, the PBMCs will be labeled with fluorescent-conjugated antibodies specific for macrophages (CD14), T helper cells (CD4), and B cells (CD19), sorted via FACS according to cell type and evaluated for CYP27B1, VDR, and CYP24A1 gene expression to identify which cell populations are capable of producing and responding to 1,25(OH)2D3 in response to antigen stimulation. Effects of 25(OH)D3 on T cell proliferation and antigen-specific responses of each cell type in the PBMC cultures also will be evaluated to determine if 1,25(OH)2D3 produced in the PBMC cultures
regulates antigen-specific immune responses of pigs.<br/>Experiment 2.3: Identify a method to precisely manipulate and maintain serum 25(OH)D3 concentrations in pigs. Considerable debate exists over the serum concentrations of 25(OH)D3 needed to support proper immunity. The ability to precisely and steadily control 25(OH)D3 concentrations in a relevant animal model of human immunity and vitamin D physiology needs to be developed in order to test the hypothesis that proper immune function relies on sufficient 25(OH)D3 availability. This experiments seek to identify the dosage amounts and intervals of vitamin D3 or 25(OH)D3 administration that can precisely maintain serum 25(OH)D3 at concentrations that are considered deficient (<10 ng/mL), insufficient (25 ng/mL), sufficient (50 ng/mL), high (75 ng/mL) and upper limit (100 ng/mL) for the human population, and are typical concentrations of
swine housed outdoors (high) or from modern swine operations (deficient). Newly weaned pigs fed a vitamin D deficient diet and housed indoors at the UF swine unit will be divided into three groups (4/group) and administered the following treatments: 1) no supplemental vitamin D, 2) 10 ?g/kg BW vitamin D3/week subcutaneously, or 3) 10 ?g/kg BW 25(OH)D3/week subcutaneously. The treatments will start at weaning and continue for six weeks. Pigs will be weighed weekly and serum samples will be collected on the day of administration and again one day later and be analyzed for 25(OH)D (ELISA), 1,25(OH)2D (RIA), calcium and phosphorous (atomic absorption) concentrations. The study will be repeated as necessary to make adjustments and to demonstrate reproducibility from one experiment to the next.