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United States Department of Agriculture

Agricultural Research Service


Location: Diet, Genomics and Immunology Laboratory

2011 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.

4. Accomplishments
1. Initial studies looking at the effects of vitamin A (VA) deficiency on Citrobacter rodentium infection were fruitful. VA deficiency caused mortality in C. rodentium-infected mice where no mortality was observed in VA adequate mice. Higher levels of C. rodentium were found in the colon of VA-deficient mice and a high level of colonization also was observed in the spleen, indicating a breakdown in the mucosal barrier that normally prevents systemic spread of C. rodentium in nutritionally adequate mice. The colon/body weight ratio was also increased in the VA-deficient mice and correlated with increased colonic hyperplasia. This, coupled with the increased colonization of the spleen, suggested that there was substantial damage in the colonic mucosa. Future studies will include histological examination and gene expression.

2. The content of the Beltsville Human Nutrition Research Center's Porcine Immunology and Nutrition (PIN) Database was expanded greater than 2-fold. The number of entries is now 4,070 genes related to immunity and nutrition. Over 3,000 of these genes were mapped to the porcine genome. New link-outs were provided to the Innate Immune Data Base and Affymetrix microarray identifications were assigned. Several hundred new genes and corresponding real-time PCR assays were added to the database. A significant amount of time was devoted to annotation of the porcine genome. A website was maintained and revised to coordinate the efforts of the annotation committee. These efforts will aid the greater scientific community in the use of the pig in genomics-based models for humans.

3. An extract of dark-roast peanuts, which have been shown to be more allergenic in humans, was tested in pigs for induction of allergic disease after sensitization (intracutaneous, intraperitoneal, ocular, and oral). A group of pigs were exposed to peanut extract (no adjuvant) immediately after birth and another group was sensitized after weaning. Similar to our results with a light roast peanut extract, oral and ocular exposure of pigs to a whole extract of light-roast peanuts induced Arah 1- and Arah 2-specific IgE antibody that is necessary for allergic disease, but an overt anaphylactic response to feeding whole peanuts or to intracutaneous exposure to peanut extract was not observed. Significantly increased mRNA levels of the pro-inflammatory cytokine IL-2 were seen in the jejunum of pigs that were orally sensitized. Increased mRNA levels of the alternatively activated macrophage marker CD206 was observed in pigs that had been sensitized via the eye compared to those that were sensitized via oral and intracutanous routes. The research provides a test of sensitization routes using whole peanut preparations that are equivalent to exposures faced by humans.

4. Preliminary data suggest that GPX2KO mice may have delayed clearance of Citrobacter rodentium compared to wild-type control mice. A Mac knock out (KO) mouse line (obtained from Dolph Hatfield’s laboratory at the NIH), which lacks expression of selenoproteins in macrophages, does not appear to have a phenotype after infection with C. rodentium or the parasitic nematode Heligmosomoides bakeri. Another conditional mouse KO in which selenoproteins are not expressed in T-cells has shown greater promise and will be used in further studies with Citrobacter rodentium.

5. The effect of vitamin A (VA) on alternatively activated macrophages was tested in combination with Ascaris infection as a surrogate for allergic inflammation. Previously we demonstrated that pre-treatment with all-trans-retinoic acid (ATRA), a metabolite of VA with the greatest biological potency, increased allergic inflammation in the lungs and liver of pigs given an acute Ascaris infection. We have now demonstrated that ATRA increased the number and percentages of macrophages expressing markers for alternatively activated macrophages, including IL-4R, CD273, CD274, and CD62L proteins, in the lungs of pigs given a low-dose chronic infection of Ascaris. This infection model more closely resembles the natural course of human and pig Ascaris infection.

6. Gene expression studies indicated that selenium deficiency is down-regulating the localized Th2 response to the nematode parasite Heligmosomoides bakeri (Hb). The cytokines IL-4 and IL-13 are down-regulated as are a number of genes associated with alternatively activated macrophages, including YM1, YM2, Arg-1, and Relm-a. These results suggest that macrophage function is being altered by selenium deficiency. To determine how quickly functional gene expression can be recovered, deficient mice were infected with Hb and switched to a selenium-adequate diet 10 days post-infection. Egg production, a surrogate marker for worm expulsion, rapidly declined to undetectable levels over 96 hours in selenium re-fed mice while mice maintained on the deficient diet continued to shed eggs. Gene expression studies on these mice indicate the there is an up-regulation of IL-4, IL-13, and genes associated with alternatively activated macrophages in selenium re-fed mice. These results suggest that a defect in macrophage function may be responsible for delayed egg expulsion in selenium-deficient mice.

Review Publications
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.

Smith, A.D., Cheung, L., Botero, S. 2011. Long-term selenium deficiency increases the pathogenicity of a Citrobacter rodentium infection in mice. Biological Trace Element Research. Available:

Shea-Donohue, T., Notari, L., Stiltz, J., Sun, R., Madden, K.B., Urban Jr, J.F., Zhao, A. 2010. Role of enteric nerves in immune-mediated changes in protease activated receptor 2 effects on gut function. Neurogastroenterology & Motility. 10:1138-e291.

Zhao, A., Urban Jr, J.F., Sun, R., Stiltz, J., Morimoto, M., Notari, L., Madden, K., Ramalingam, T., Wynn, T., Shea-Donohue, T. 2010. Critical role of intestinal epithelial cell-derived IL-25 in enteric nematode infection-induced changes in intestinal function. Journal of Immunology. 185:6921-6929.

Yagi, R., Junttila, I., Wei, G., Urban Jr, J.F., Zhao, K., Paul, W., Zhu, J. 2010. The transcription factor GATA3 actively represses RUNX3 protein-regulated production of interferon-gamma. Immunity. 32:507-517.

Santin, M., Gomez-Munoz, M., Solano Aguilar, G., Fayer, R. 2011. Development of a new PCR protocol to detect and subtype Blastocystis spp. from humans and animals. Parasitology Research. 109(1):205-212.

Saenz, S.A., Siracusa, M.C., Perrigoue, J.G., Spencer, S.P., Urban Jr, J.F., Tocker, J.E., Budelsky, A.L., Kleinschek, M.A., Kambayashi, T., Artis, D. 2010. IL25 elicits a multipotent progenitor cell population that promotes TH2 cytokine responses. Nature. 464(7923):1362-1366.

Dawson, H.D. 2011. Comparative assessment of the pig, mouse, and human genomes: A structural and functional analysis of genes involved in immunity. In: McAnulty,P.A., Dayan, A., Hastings, K.H., Ganderup, N.-C., editors. The Minipig in Biomedical Research. Boca Raton, FL: CRC Press. p. 321-341.

Last Modified: 08/18/2017
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