Location: Diet, Genomics and Immunology Laboratory2011 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
Two experiments to confirm the ability of all-trans retinoic acid (ATRA) to directly induce an alternatively activated macrophage phenotype in vivo were conducted. ATRA reduced in vivo and in vitro phagocytosis of opsonized Staphylococcus aureus and superoxide production from lung macrophages. The results of the second transcriptomic experiment supported the first. During the course of our transcriptomic studies on the effects of ATRA on the polarization of macrophages to an alternatively phenotype, it was observed that ATRA induced high and sustained levels of the vitamin D receptor (VDR) and associated vitamin D hydroxylases, and several vitamin D target genes were up-regulated. Therefore, a study to determine potential additive and synergistic effects of ATRA and VDR was conducted by pre-treating cells with ATRA followed by exposure to VDR agonists or vitamin D precursors. A transcriptomic analysis of IL-4-treated macrophages was also conducted to determine how closely the effect resembled ATRA-treated macrophages. To assess the role of VA in mucosal immunity, the effect of VA deficiency on infectious colitis induced by C. rodentium was examined. C. rodentium infection of VA deficient mice led to increased mortality, higher colonization, and increased hyperplasia in the colon, as well as increased colonization of the spleen, indicating a breakdown in the mucosal barrier that normally prevents systemic spread of C. rodentium in nutritionally adequate mice. Previously we found that selenium (Se)- deficient mice failed to clear a H. polygyrus infection. Refeeding Se-deficient mice a Se-adequate diet rapidly restored Th2 gene expression and reduced nematode egg production in the Se-deficient mice to control levels, indicating that Th2 immunity to H. polygyrus infection was exquisitely sensitive to dietary Se levels. Diarrhea is a leading cause of mortality and morbidity in children less than five years of age in impoverished regions of the world. Our aim was to compare the intestinal microflora of healthy children to children with clinical diarrhea in a population of children from a tropical highland in Colombia, South America for the prevalence of Lactobacillus and Bifidobacterium species and associated interactions with enteric viral and bacterial pathogens. Children less than 5 years of age from two different locations were evaluated for presence of clinical diarrhea. Nucleic acids, isolated from fecal samples, were used to determine by molecular protocols the abundance of commensal bacterial species and presence of enteric pathogens compared to clinically healthy children. Overall relative abundance of commensal Bifidobacterium species was inversely correlated with incidence of diarrhea, particularly rotavirus, while certain Lactobacillus species were directly correlated with clinical diarrhea regardless of location. Our results suggested that delivery of Bifidobacterium species, or a diet rich in bifidogenic components that promote growth of Bifidobacterium species, should be beneficial for the prevention of diarrhea in at-risk children.
Smith, A.D., Botero, S., Shea-Donohue, T., Urban Jr, J.F. 2011. The pathogenicity of an enteric Citrobacter rodentium infection is enhanced by deficiencies in the antioxidants selenium and vitamin E. Infection and Immunity. 79:1471-1478.