2010 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.
Oral and ocular exposure of pigs to a whole extract of light-roast peanuts induced Ara h 1- and Ara h 2-specific IgE antibody that is necessary for allergic disease, but an overt anaphylactic response to feeding whole peanuts or intracutaneous exposure to peanut extract was not observed. This is in contrast to simultaneous infection with the pig large roundworm, Ascaris suum, and expression of an allergic reaction to intracutaneous injection of worm antigens. The whole extract of light-roast peanuts apparently lacks allergenic properties. A whole extract of dark-roast peanuts that have been shown to be more allergenic in humans will be tested for induction of allergic disease after sensitization that includes oral feeding.
A total of 74 anti-human or anti-mouse antibodies were screened for cross reactivity to porcine proteins by flow cytometry. Monoclonal antibodies were identified that are putatively reactive with IL-17A, T-bet, TREM2, GITR ligand, IL-4 receptor, ROR gamma, and the TSLP receptor that help define the effect of vitamin A on the phenotype and function of alternatively activated macrophages and T regulatory cells.
The effect of added selenium and vitamin A in the diet on gastrointestinal immunity to bacterial and parasitic infections was evaluated. Studies comparing the effect of short- vs. long-term selenium deficiency on Citrobacter rodentium, a mouse model that simulates food borne illness from E. coli in humans, demonstrated that only long term deficiency resulted in increased pathology due to an enhanced pro-inflammatory expression of IFN-gamma, IL-17A, and IL-22 as well increased expression of both CXCL and CCL chemokines. This affect could be mimicked over a shorter period of time if the mother was made deficient as well as the pups from birth. This observation suggested that inadequate diets in during gestation and through the neonatal period of development had significant negative effects on immune function. Selenium deficiency also impaired the host resistance to a Heligmosomoides polygyrus, a worm infection that simulates intestinal food allergy, in the small intestine. Part of the host response to invasion of the parasite into the intestinal sub-mucosa is accumulation of immune cells and cyst formation around the worm. Gene expression studies of both cyst and non-cyst small intestinal tissue suggest that a number of genes associated with alternatively activated macrophage and goblet cell function were down regulated in selenium deficient mice, providing a possible mechanism to explain the delayed parasite clearance in selenium deficient mice. This mechanism could provide targets for control of allergic inflammation in the intestine.
Retinoic acid (active component in vitamin A) has been shown to affect development of both allergic responses through Th2 cells and inflammation through Th17 responses. Studies designed to determine if super-nutritional levels of vitamin A in the diet altered the normal course of infection with either C. rodentium or H. polygyrus were negative suggesting that vitamin A levels 5X above adequate levels were not affect immunity or inflammation to these pathogens.
A reduced distribution of bifidobacterium species is associated with allergy and with clinical diarrhea in children. The intestinal microbiota of normal children were compared to those with wheezing phenotype as an indicator of allergy, and with diarrhea as an indicator of enteric disease. The distribution of commensal bacterial species and the presence of potentially pathogenic bacteria, viruses, and parasites in the feces were examined by molecular techniques. The results showed a reduced distribution of bifidobacterium species in subjects with expression of allergy and diarrhea compared to normal subjects. Samples from subjects with diarrhea also showed an increase in potentially pathogenic species of bacteria, parasites, and viruses. The work suggests that feeding probiotic bifidobacterium could provide a more normal distribution of these probiotic bacteria in the intestine and promote intestinal health similar to what has been demonstrated in controlled studies feeding probiotic bifidobacterium to pigs where local expression of allergic and infectious disease was altered.
Application of digital gene expression, known as RNA-Seq, to pig alveolar macrophages to provide genomic-based biomarkers of disease related to nutrition status. Explanted pig alveolar macrophases of > 99% purity were stimulated in vitro for 24 hours with all trans retinoic acid (ATRA) and the RNA isolated for sequencing. This technique has major advantages over other techniques, such as microarray and real time PCR for high throughput genomic analysis, and provides direct quantitative comparison of transcript copy number, within sample comparison of different genes relative to one another, as well as between sample comparisons of the same gene. Analysis to date has released that ten of the top twenty expressed genes were immune related. Several genes were associated with immune and inflammatory pathways including adipokins related to adipocyte activation, alternatively activated macrophage markers related to cell activation and tissue remodeling, and asthma and allergy-related markers related to chemokin and chemokine receptor pathways of allergic inflammation. Also induced were genes associated with folate metabolism, iron metabolism, lipid metabolism, and vitamin A metabolism. Over 90% of these observations are novel and will help define the effect of vitamin A on the phenotype and function of macrophages related to disease expression and tissue repair in the lung.
Yasuda, K., Dawson, H.D., Wasmuth, E.V., Roneker, C.A., Chen, C., Urban, J.F., Welch, R.M., Miller, D.D., Lei, X.G. 2009. Supplemental dietary inulin influences expression of iron and inflammation related genes in young pigs. Journal of Nutrition. 139(11):2018-2023.
Kannan, P., Dharne, M., Smith, A.D., Karns, J.S., Bhagwat, A.A. 2009. Motility revertants of opgGH mutants of Salmonella enterica serovar Typhimurium remain defective in mice virulence. Current Microbiology. 59(6):641-645.
Liu, L., Dharne, M., Kannan, P., Smith, A.D., Meng, J., Fan, M., Boren, T., Ranallo, R., Bhagwat, A.A. 2010. Osmoregulated periplasmic glucans synthesis gene family of Shigella flexneri. Archives Of Microbiology. 192(3):167-174.
Liu, Q., Kreider, T., Bowdridge, S., Liu, Z., Song, Y., Gaydo, A.G., Urban, J.F. Jr., Gause, W.C. 2010. B cells have distinct roles in host protection against different nematode parasites. Journal of Immunology. 184(9):5213-5223.
Noland, G.S., Chowdhury, D.R., Urban Jr, J.F., Zavala, F., Kumar, N. 2010. Helminth infection impairs the immunogenicity of a Plasmodium falciparum DNA vaccine, but not irradiated sporozoites, in mice. Vaccine. 28(17):2917-2923.
Morales-Tirado, V., Sojka, D.K., Katzman, S.D., Lazarski, C.A., Finkelman, L.D., Urban Jr, J.F., Fowell, D.J. 2010. Critical requirement for the Wiskott-Aldrich syndrome protein in Th2 effector function. Blood. 115(17):3498-3507.
Flores-Mendoza, L., Sotelo-Mundo, R.R., Dawson, H.D., Mwangi, W., Hernandez, J. 2010. Characterization of porcine CD205. Developmental and Comparative Immunology. 34(7):715-721.
Sanders, M.E., Akkermans, L.M.A., Haller, D., Hammerman, C., Heimbach, J.T., Hormannsperger, G., Huys, G., Levy, D.D., Lutgendorff, F., Mack, D., Phothirath, P., Solano-Aguilar, G., Vaughan, E. 2010. Safety assessment of probiotics for human use. Gut Microbes. 1(3):164-185.
Herbert, D.R., Yang, J.Q., Hogan, S.P., Groschwitz, K., Khodoun, M., Munitz, A., Orekov, T., Perkins, C., Wang, Q., Brombacher, F., Urban Jr, J.F., Rothenberg, M.E., Finkelman, F.D. 2009. Intestinal epithelial cell secretion of RELM-beta protects against gastrointestinal worm infection. Journal of Experimental Medicine. 206(13):2947-2957.