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United States Department of Agriculture

Agricultural Research Service


Location: Diet, Genomics and Immunology Laboratory

2013 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.

4. Accomplishments
1. Dietary probiotics enhance immunological response to antigenic challenge and maintain intestinal permeability function in young piglets. Promotion of health by enhancement of immunological response and intestinal function may be accomplished by administration of certain Probiotics, defined as “live microorganisms, which when administered in adequate amounts confer a health benefit on the host”. It was demonstrated that continuous dietary administration of probiotic strains Bifidobacterium lactis (Bl-04) and Lactobacillus acidophilus NCFM to young pigs increased Bifidobacterium spp. and Lactobacillus spp. abundance in intestinal lumen, enhanced anti-inflammatory cytokine response to antigenic challenge and maintained intestinal epithelial permeability without inducing morphometric changes in intestinal mucosa. The data generated from these in vivo studies demonstrates the probiotic properties of these strains.

2. Dietary supplementation with Lactobacillus rhamnossus (LGG) ATCC 55103 strain induced transitory changes in the gene expression profiles in the blood of elderly subjects. Blood RNA isolated from subjects was used for transcriptomic evaluation through RNA sequencing to identify differentially expressed genes (DEGs) in whole blood cells prior to consuming LGG (day 0), at the end of LGG consumption (day 28), and one month after feeding of LGG stopped. Analysis revealed a total of 44 DEGs (39 down-regulated, 5 up-regulated). These changes were not evident one month after LGG was stopped. To investigate the functional relationships between genes which are differentially expressed at day 28 and predict possible outcomes, we used DEGs to conduct pathway analysis to identify networks of functional genes. This analysis revealed activation of pathways involved in cell to cell signaling, hematological system development and function, and immune cell trafficking. Overall, LGG consumption transiently modified whole blood cell transcriptomic expression in healthy elderly subjects without inducing any adverse effect or abnormal clinical signs.

3. Restoration of adequate dietary selenium quickly activates immunity. Infection with parasitic worms is a worldwide problem in humans and livestock. We previously demonstrated that host protective expulsion from the intestine of a model worm parasite, Heligmosomoides bakeri, was delayed in mice fed a diet deficient in selenium (Se). We now demonstrate that the tissue surrounding the parasite had a decreased local immune response that inhibited host protection and made the worm more metabolically active in the face of a suboptimal Se-dependent local immune response. Notably, the process of worm expulsion was restored within two to four days after feeding a Se-adequate diet to Se-deficient mice. Expulsion was associated with an increased local immunity and activation of genes in the small intestine for enhanced oxidative enzymes and specific antibody production that reduced worm metabolic activity. These results demonstrate the resilience of the host immune system and the requirement for adequate dietary selenium for optimal immune function. This is important not only for maintaining adequate nutrition in humans and livestock to enhance immune activity against worm infection, but also for modulation of allergic diseases that accompany worm infection.

4. Macrophages activated by parasite infection regulate immunity and inflammation in the intestine. Several of our recent published studies demonstrate that the activation of intestinal macrophages by parasitic worm infection regulate immunity and inflammation. Intestinal macrophages are activated during a parasitic infection by protein molecules called cytokines that are released by intestinal epithelial cells. The study showed that the cytokine pair IL-25/IL-33 cause the macrophage to release its own set of cytokines that contribute to the expulsion of a worm infection. In addition, the activation of these cells and others called regulatory dendritic cells by the worm can down-regulate the expression of experimental colitis equivalent to the inflammatory bowel disorders that are increasingly common in humans. Some of this phenomenon relates to other protein molecules called chemokines that are signals for the migration of inflammatory cells into intestinal tissues. This work, in total, demonstrates the importance of the macrophage as a key cell controlling inflammation and immunity in intestinal tissues, and provides information on key regulatory cytokines and chemokines that can be targets to control inflammation by processes that include dietary interventions.

Review Publications
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.

Blum, A.M., Hang, L., Setiawan, T., Urban Jr, J.F., Stoyanoff, K., Leung, J., Weinstock, J.V. 2012. Heligmosomoides induces tolerogenic dendritic cells that block colitis and prevent antigen-specific gut Tcell responses. Journal of Immunology. Sept 1:189(5):2512-2520.

Groenen, M.A., Archibald, A.L., Uenishi, H., Tuggle, C., Takeuchi, Y., Rothschild, M.F., Rogel-Gaillard, C., Park, C., Milan, D., Hendrik-Jan, M., Li, S., Larkin, D., Kim, H., Franz, L.A., Caccamo, M., Hyeonju, A., Aken, B.L., Anselmo, A., Anthon, C., Auvil, L., Bouabid, B., Beattie, C.W., Bendixen, C., Berman, D.J., Blecha, F., Blomberg, J., Bolund, L., Bosse, M., Botti, S., Zhan, B., Bystrom, M., Capitanu, B., Silva, D.C., Chardon, P., Chen, C.T., Cheng, R., Choi, S., Chow, W., Clark, R.C., Clee, C., Crooijmans, R.P., Dawson, H.D., Dehais, P., De Sapio, F., Dibbits, B., Drou, N., Du, Z., Eversole, K., Fadista, J., Fairley, S., Faraut, T., Faulkner, G.J., Fowler, K.E., Fredholm, M., Fritz, E., Gilbert, J.G., Giuffra, E., Gorodkin, J., Griffin, D., Harrow, J.L., Hayward, A., Howe, K., Zhi-Liang, H., Humphray, S.J., Hunt, T., Hornshoj, H., Jeon, J., Jern, P., Jones, M., Jurka, J., Kanamori, H., Kapetanovic, R., Jaebum, K., Kim, J., Kim, K., Kim, T., Larson, G., Lee, K., Lee, K., Leggett, R., Lewin, H.A., Li, Y., Liu, W., Loveland, J.E., Lu, Y., Lunney, J.K., Ma, J., Madsen, O., Mann, K., Mathews, L., Mclaren, S., Morozumi, T., Murtaug, M.P., Narayan, J., Nguyen, D., Ni, P., Oh, S., Onteru, S., Rohrer, G.A., et al. 2012. Analysis of pig genomes provide insight into porcine demography and evolution. Nature. 491(7424):393-8.

Smith, A.D., Cheung, L., Shea-Donohue, T., Urban Jr, J.F. 2013. Selenium status alters the immune response and expulsion of adult Heligmosomodies bakeri in mice. Infection and Immunity. 81(7):2546-2553.

Dawson, H.D., Loveland, J., Pascal, G., Gilbert, J., Uenishi, H., Mann, K., Sang, Y., Zhang, J., Carvalho-Silva, D., Hunt, T., Hardy, M., Hu, Z., Zhao, S., Anselmo, A., Sinkai, H., Chen, C.T., Badaoui, B., Berman, D.J., Amid, C., Kay, M., Lloyd, D., Snow, C., Morozumi, T., Cheng, R., Bystrom, M., Bourneuf, E., Kapetanovic, R., Schwartz, J.C., Kataria, R., Astley, M., Fritz, E., Steward, C., Thomas, M., Wilming, L., Giuffra, E., Archibald, A., Bed'Hom, B., Beraldi, D., Ait-Ali, T., Blecha, F., Botti, S., Freeman, T., Hume, D.A., Lunney, J.K., Murtaugh, M.P., Reecy, J.M., Harrow, J., Rogel-Gaillard, C., Tuggle, C.K. 2013. Structural and Functional Annotation of the Porcine Immunome. Biomed Central (BMC) Genomics. 14:332-370.

Zhao, A., Yang, Z., Sun, R., Grinchuk, V., Netzel-Arnett, S., Anglin, I.E., Driesbaugh, K.H., Notari, L., Bohl, J.A., Madden, K.B., Urban Jr, J.F., Antalis, T.M., Shea-Donahue 2013. SerpinB2 is critical to Th2 immunity against enteric nematode infection. Journal of Immunology. 190(11):5779-87.

Solano Aguilar, G., Fernandez Duarte, K., Ets, H.K., Molokin, A., Vinyard, B.T., Urban Jr, J.F., Guitierrez, M. 2013. Characterization of Fecal Microbiota of Children With Diarrhea in 2 Locations in Colombia. Journal of Pediatric Gastroenterology and Nutrition. 56:503-511.

Last Modified: 05/24/2017
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