Location: Diet, Genomics and Immunology Laboratory2012 Annual Report
1a. Objectives (from AD-416):
This project proposes rodent models to provide molecular, genetic, and functional information to address the effects of nutrient requirements on mucosal immune responses to infectious pathogens, and pig models to provide physiologically relevant comparisons to human allergy and responses to probiotic bacteria. Common features are the use of targeted gene expression probes to elucidate innate and acquired immunity to both probiotic and pathogenic bacteria that activate Th1 responses, and allergies and worm infections that activate Th2 responses. The goal is to reveal interactions between dietary micronutrients and food components that modulate immune responses to food allergens, micro and macrobiotic organisms, and their products. Objective 1: To elucidate the role of vitamin A (VA) on the phenotype and function of alternatively activated macrophages and T regulatory cells, and identify macrophage-mediated modulation of localized nutrient delivery/partitioning in porcine models of allergy. Objective 2: To elucidate the mechanisms used by probiotic bacteria to improve respiratory and intestinal mucosal responses to allergens, and correlate intestinal microflora composition of pigs and humans with biomarkers of allergic and intestinal disease. Objective 3: To elucidate the mechanisms by which micronutrients affect gut physiology and immune competence in response to food-borne illness due to viruses, bacteria, and gastrointestinal parasites.
1b. Approach (from AD-416):
Studies will evaluate if pigs can be sensitized to peanut (PN) allergens by different routes of mucosal exposure without cholera toxin and orally challenge with over-the-counter unsalted dry-roasted PN; if Vitamin A (VA) via all-trans retinoic acid (ATRA) can exacerbate allergic disease via stimulation of Th2 dependent pathways at low doses of antigen; if alternatively activated macrophages (AAM) express retinal and retinol dehydrogenases leading to increased ATRA generation in vitro and in vivo; and if CD209 is a receptor for PN and parasite antigens that mediates functional polarization of AAM accompanied by generation of ATRA. Additional work will test if probiotic bacteria protect against allergy, and if changes in intestinal microflora in children affect the incidence of allergy and intestinal disorders such as chronic diarrhea. Finally, it will be determined if selenium (Se) deficiency impairs AAM function in a helminth-parasite infection model in mice, if chronic Se deficiency or genetic deficiencies in selenoprotein expression in immune cells or intestinal tissue alter immunity and pathology associated with Citrobacter rodentium; and if vitamin A status will alter gastrointestinal immunity to C. rodentium and Heligmosomoides polygyrus in mice.
3. Progress Report:
Alveolar macrophages (AM) normally make up greater than 90% of the cells in the airspaces of the lung and are the first line of defense against airborne particles and pathogens that contribute to lung health and disease. Information on the biology and function of these cells comes largely from studies in mice with little comparable information available for pigs and humans. To address this, we studied basal gene expression in porcine AM by Illumina-based next generation sequencing technology. More than 200 new, full, or partial length porcine genes were cloned from this project including many key immune regulatory molecules and splice variants. Real time PCR results confirm the deep sequencing data with a correlation of 0.999. Correlations of gene expression among animal samples were greater than 0.90 including both biological and technical duplicates. Results from pathways and bio-function analysis were consistent with the biological roles of AM defined in the mouse. DGIL scientists continue to enhance a database on genes and proteins related to those prominently studied in humans and mice. The database now contains 4,545 entries. New datafields were added to accommodate the unit’s new focus on epigenomics including descriptions of the promoter region of porcine genes and porcine micro RNAs. This will enhance the ability to look for regulatory regions that may be conserved among different species. As part of an international consortium, they annotated over 1,400 genes to provide the first comprehensive description of the portion of the pig genome involved in the immune response. These efforts, to be published in Nature, revealed an overwhelming similarity of pigs to humans, further validating their use as a biomedical model. To establish safety and tolerability of probiotic interventions in humans fecal samples and whole blood have been collected from patients fed probiotic bacteria, Lactobacillus rhamnossus (LGG) or Bifidobacterium lactis. Measurement of probiotic colonization have been tracked by relative quantification of probiotic bacteria in fecal samples using specific single copy gene assays. The complete change in transcriptome of RNA from whole blood from probiotic treatment are being evaluated by Illumina-based transcriptomic analysis. Mice that lack expression of selenoproteins in macrophages did not differ from normal mice infected with Citrobacter rodentium bacteria (Cr) or the parasitic nematode Heligmosomoides bakeri (Hb). This suggested that a selenium dependent defect in macrophages may not be responsible for the altered immune response to Hb as originally hypothesized. Similarly, glutathione peroxidase (GPX) isozyme 1 or 2 knockout (KO) mice had unaltered immune responses during primary and secondary Hb infection infections. In contrast, preliminary data indicated that GPX2KO mice had a higher colonic Cr burden than WT mice. Additional studies will be conducted with GPX1KO and GPX2KO to define the role of these selenoproteins in resistance to gastrointestinal bacterial infections.
Wu, S., Li, R.W., Li, W., Urban Jr, J.F., Dawson, H.D., Beshah, E. 2012. Worm burden-dependent disruption of the porcine colon microbiota by Trichuris suis infection. PLoS One. 7(4):e35470.