Location: Diet, Genomics and Immunology Laboratory2013 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:
Previously, we showed that all-trans retinoic acid (ATRA), the most active vitamin A (VA) metabolite, increased an anti-inflammatory/anti-parasitic macrophage phenotype due to treatment with the cytokine, interleukin-4 (IL-4) and increased the expression of IL-4 in the lungs of pigs infected with the parasite, Ascaris suum. We demonstrated that macrophages, treated in vitro with IL-4 or isolated from the lungs of A. suum infected pigs, had 2-8-fold increases the mRNA for enzymes involved in ATRA generation. At the epigenetic level, ATRA increased the mRNA of CYP26A1, an enzyme involved in ATRA catabolism, through a 7-fold increase in an activator histone binding to the CYP26A1 promoter. In contrast, IL-4 pre-treatment reduced ATRA-induced CYP26A1 mRNA levels 4-fold through a 4 fold increase, above control, in the binding of a repressor histone to the promoter. These data indicate that, VA, via metabolism to ATRA, and IL-4, by favoring conditions that lead to higher levels of ATRA, may synergistically sustain an anti-inflammatory/anti-parasitic immune response. Bifidobacterium animalis subspecies lactis and Lactobacillus acidophilus are two of the most common probiotic species used in food products in North America and Europe and are found in the human diet via formulations, supplements, and dairy products. Despite the purported health benefits of probiotics, the contribution of the delivery vehicle on probiotic survival and host physiology has not been systematically assessed. To assess this, we compared the effects of a five week administration of dairy-based vehicles (2% fat-milk or 2% fat acidified-milk) versus PBS as delivery vehicles (placebo) for lyophilized B. lactis or L. acidophilus in newborn piglets. Bacteriological and molecular analysis of microbiota composition indicated increased survival of both probiotic strains administered with 2% fat milk. Probiotic treatment induced production of pro-inflammatory cytokines by peripheral blood mononuclear cells in response to LPS stimulation. Intestinal epithelial resistance on explanted intestinal jejunum from pigs fed both strains showed enhanced intestinal resistance/reduced permeability. We have demonstrated that VA deficient (VAD) mice infected with a mouse pathogen that mimics many aspects of food-borne E. coli infections in humans, Citrobacter rodentium (Cr), exhibited higher levels of bacteria in the colon than control or vitamin A supplemented (VAS) Cr-infected mice. More bacteria were also recovered from spleens of VAD mice indicating altered intestinal barrier function. This was confirmed by increased colonic pathology. Infected VAD-mice switched to a VAS diet at D0 or D7 had colonic Cr levels between those of VAD- and VAS-mice, and less bacteria was recovered from their spleens indicating partial restoration of barrier function. Growth of Cr was inhibited by D21 in mice on all diets except VAD-mice that also had decreased mRNA expression of both Th1 and Th17 cytokines. Decreased expression of two genes encoding ion transport proteins, Slc26a3 and CA4, in infected VAD mice correlated with the incidence of diarrhea in VAD mice.
Yang, Z., Grinchuk, V., Urban Jr, J.F., Bohl, J., Sun, R., Notari, L., Yan, S., Ramalingam, T., Keegan, A.D., Wynn, T.A., Shea-Donohue, T., Zhao, A. 2013. Macrophages as IL-25/IL-33-responsive cells play an important role in the induction of type 2 immunity. PLoS One. 8(3):e59441,1-11.