Progress 03/28/14 to 03/27/19
Outputs
Progress Report Objectives (from AD-416): Objective 1: Determine whether bioactive food components, such as vitamins A and D or lycopene, acting via vitamin A and D-receptor- mediated pathways and nuclear factor-kappaB signaling, exhibit dose- dependent inhibitory effects on macrophage-mediated remodeling of adipose tissue toward a pro-inflammatory phenotype in response to high fat diets in a swine model. [NP 107 Component 3, Problem Statement 3B]. Objective 2: Determine whether bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Compare responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling. [NP 107 Component 3, Problem Statement 3B] Approach (from AD-416): For Objective 1, in vitro and in vivo porcine models will be used to test the hypothesis that vitamin A or vitamin D or metabolites of dietary compounds that signal through retinoic acid receptor signaling pathways, such as lycopene, will promote an anti-inflammatory phenotype of adipose tissue macrophages and inhibit pro-inflammatory responses of adipose tissue macrophages to inflammatory ligands via inhibition of NF-kappaB signaling and epigenetic regulation of macrophage polarization. For Objective 2, a mouse model will be used to test the hypothesis that bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox-sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, will be used to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling. This is the final report for Project # 8040-51000-058-00D entitled ⿿Dietary Regulation of Immunity and Inflammation which has been replaced by a new Project # 8040-53000-021-00D. For additional information, see the new project report. Progress was made on two of three objectives which fall under National Program 107, and directly addresses Statement 3B: Identify Roles of Food, Food Components and Physical Activity in Promoting Health and Preventing Disease. This work resulted in 36 publications. Data and technologies developed during the current reporting period laid the foundation for studies proposed in the new project plan. We previously demonstrated that the most bioactive vitamin A (VA) metabolite, all-trans retinoic acid (ATRA), increased T helper 2- associated responses induced in pigs by infection with the parasitic nematode Ascaris suum We also showed that ATRA potentiated the mRNA expression of several interleukin 4 (IL-4, a T helper 2 cytokine)-induced chemokines associated with alternative activation (M2a) in porcine macrophages (Mfs) in vitro. During the current reporting period, several mechanisms whereby ATRA affects IL-4 signaling were examined using large-scale real time PCR and RNA-Seq analysis. Twenty-three genes associated with M2a markers in other species were found to be independently upregulated by both IL-4 and ATRA. The combination of IL-4 and ATRA synergistically induced several markers including transglutaminase 2 (one of the few cross-species markers for anti-inflammatory Mfs) and the anti-inflammatory protein, IL- 1R antagonist. These effects were recapitulated in the human Mf cell line THP-1 and were independent of ATRA⿿s effect on NF-kB activation and induction of the vitamin D (VD) receptor. Given the prevalence of allergic and parasitic diseases worldwide and the close similarities in the porcine and human immune responses, these findings have important implications for the nutritional regulation of allergic inflammation at mucosal surfaces. During the current reporting period the Porcine Translational Research Database was continually expanded (9,451 additional genes, 34 additional data fields) (http://tinyurl.com/hxxq3ur). It now provides high level, manual, annotation for 16,780 genes commonly studied in pigs, mice or humans. A fully correct and annotated genome is essential for high throughput methods of gene and protein analysis as well as performing cross species analysis of function. Our previously published work demonstrated the utility of the manual annotation process in identifying errors and improving annotation of version 10.2 of the porcine genome. We applied the same analysis techniques to 4,592 genes to version 11.1 of the porcine genome. We discovered that only half of the sequences were correctly assembled and annotated. We also discovered that a significant number of genes were still not represented in the genome. These data will be used to improve and annotate the next version of the porcine genome. Next generation sequencing technology was developed for porcine transcriptomics (RNASeq, microRNASeq). In addition to providing whole genome level comparison of factors that are induced in response to VA and vitamin D treatment of Mfs, M1 and M2 Mf polarization, and Trichuris suis and Toxoplasma gondii infection. This technology also provided the sequences for several thousand missing or novel genes including genes involved in immunomodulation and nutrient metabolism. The sequences for 1,300 of these genes were submitted to Genbank. Furthermore, a non- redundant microRNA database for pigs was created, including several hundred previously unidentified microRNA. Using alternative species as a model for human testing required a careful and thorough analysis of the genome and proteome. During the current reporting, we published 3 additional analyses focusing on comparative aspects of the pig, mouse and human immunome, the collection of genes and proteins involved in immunity and inflammation. Although we found an overwhelming similarity between humans and pigs, we also identified several areas where the human and mouse were more similar. We expanded this analysis to 3,244 genes and proteins involved in nutrition and metabolism. This analysis reveals that pigs have roughly 3-fold less unique genes than the mouse or human. When a gene is missing from one or more genomes the frequency and similarity of gene preservation between pigs and humans is overwhelmingly similar. However, in some cases these data provided mechanistic data for previously described differences in pig and human physiology. Overall, the majority of the parameters examined were more similar between pigs and humans than mice, suggesting that evaluating nutrition interventions in pigs, will provide data that can bridge the gaps between those obtained in mice as nutritional models for humans. Basic descriptions of the innate lymphoid cell (ILC) function in mice including responses to aryl hydrocarbon ligands and VA were described. The ILC was shown to be important in the early induction of immune and inflammatory processes, epithelial barrier integrity, and homeostasis. This included an observation that ILC type 2 are predominantly dependent on fatty acid (FA) metabolism during a worm infection, and increase the utilization of FA to produce interleukin 13 (IL-13) when VA is limited representing a host adaptation to maintain barrier immunity under dietary modulation. Previous work demonstrated that a deficiency in selenium (Se) worsened the outcome of gastrointestinal bacterial and parasitic infections by exacerbating the Th1 inflammatory response or ablating the Th2 response, respectively. To investigate this, studies were performed to determine if the knock out (KO) of selenoproteins in specific immune cell types mimicked the negative effect on immunity resulting from feeding a selenium deficient diet using our two infectious disease models. Deleting selenoproteins in one immune cell type (Mfs, dendritic cells) did not replicate what is observed in mice fed a Se deficient diet and infected with bacteria or parasites. Of note, it has not been possible to successfully generate KO of selenoproteins in intestinal epithelial cells by crossing TRSP floxed mice (KO of all selenoproteins) or GPX4 floxed mice (KO of only GPX4). Mice with only one functional allele did not show any adverse affects on the ability to clear a Cr infection. The knock out of selenoproteins in T-cells did delay the clearance of Cr and these results will be followed up on. Attempts to reverse the effects of Se deficiency on immune function with exogenous antioxidants including N- acetyl cysteine, vitamin E, tempo, coenzyme Q10 and ebselen, a glutathione peroxidase mimic, were not successful suggesting a unique role for the selenoproteins in immune function. In total, these results suggest that recapitulation of the effects of Se deficiency on host immunity cannot be achieved solely by knocking out selenoprotein function in specific immune cells or by compounds with antioxidant activity. Studies were conducted to characterize the effects of VA deficiency on Cr infections. Infected VA deficient mice express increased pathology, bacterial load, delayed clearance, and enlarged goblet cells with greater amounts of mucus, and higher levels of tissue associated bacteria and increased mortality. We noted that vitamin A deficient mice had enlarged goblet cells. Further analysis indicated an altered mucus production and an accumulation in goblet cells of VA deficient mice, and included alterations in the types of mucus produced by VA-deficient mice as revealed by altered lectin staining patterns. Thus, VA is important for proper mucus production that is critical for resistance to Cr infections. These studies highlight the importance of both Se and VA in gastrointestinal immunity to challenge by common classes of gut pathogens. Additional collaborative studies were conducted with ARS scientists from the Environmental Microbial and Food Safety (EMFSL), Beltsville Maryland, demonstrating that a pomegranate peel extract (PPX) reduced the colonic pathology, development of a systemic bacterial infection, and mortality in Cr infected mice. 16s rRNA gene sequencing studies demonstrated that PPX-treatment altered the microbiome of mice with PPX mice having decreased diversity and a decreased Firmicutes/Bacteroidetes ratio. Importantly, the relative abundance of Cr reached 22% in water-treated but only 5% in PPX-treated infected mice. These results suggest that the consumption of pomegranate polyphenols altered the microbiome, making it more resistant to displacement by infection with Cr, indicating that pomegranate polyphenols may mitigate the pathogenic effects of food-borne bacterial pathogens. In collaboration with scientists in the Diet, Genomics and Immunology Laboratory, we investigated the effect of indole 3-carbinol, a bioactive component of cruciferous vegetables, on a Cr infection. The consumption of I3C significantly reduced peak fecal excretion of Cr, Cr colonization of the colon, reduced colon crypt hyperplasia, and the expression of Cr- induced inflammatory markers. The expression of cytotoxic T cell markers CD8 and FasL mRNA were increased in I3C -fed infected mice. In-vitro, I3C inhibited Cr growth and adhesion to Caco-2 cells. Thus, I3C alleviates Cr- induced murine colitis through multiple mechanisms including the inhibition of Cr growth and the adhesion to colonic cells in vitro and the enhancement of cytotoxic T cell activity. In collaboration with scientists from the Animal Parasitic Diseases Laboratory, we investigated the effect of oenin, punicalagin or krill oil on Cr infections. Significant effects were found on the cytokine response to the infection with the treatments reducing the pro-inflammatory response of the infection. Accomplishments 01 Pomegranate peel extract improves the outcome of bacterial colitis. Food-borne bacterial infections remain a serious threat to human health. ARS researchers at the Beltsville Human Nutrition Center and the Beltsville Agricultural Research Center found that a pomegranate peel polyphenol extract decreased damage to colon tissue caused by bacterial infection in mice. Pomegranate peels are considered an agricultural waste product but can serve as a source for health promoting polyphenols. Pomegranate peels may serve as a source of polyphenols or as a food additive for humans or livestock and help reduce the effects of common bacterial infections of the gut.
Impacts
(N/A)
Publications
- Chen, C.T., Perry, T.L., Chitko-Mckown, C.G., Smith, A.D., Cheung, L., Beshah, E., Urban Jr, J.F., Dawson, H.D. 2019. The regulatory actions of retinoic acid on M2 polarization of porcine macrophages. Developmental and Comparative Immunology. 98(9):20-23.
- Wang, Y., Liu, F., Urban Jr, J.F., Paerewijck, O., Geldhof, P., Li, R.W. 2019. Ascaris suum infection was associated with a worm-independent reduction in microbial diversity and altered metabolic potential in the porcine gut microbiome. International Journal for Parasitology. 49(3-4) :247-256.
- Coelho, C.H., Gazzinelli-Guimaraes, P.H., Howard, J., Barnafo, E., Alani, N.A., Muratova, O., Mccormack, A., Kelnhofer, E., Urban Jr, J.F., Narum, D. , Anderson, C., Langhorne, J., Nutman, T.B., Duffy, P.E. 2019. Chronic helminth infection does not impair immune response to malaria transmission blocking vaccine Pfs230D1M-EPA/Alhydrogel® in mice. Scientific Reports. 37(8):1038-1045.
- Leroux, L., Nasr, M., Valanparamabil, R., Tam, M., Rosa, B.A., Siciliani, E., Hill, D.E., Zarlenga, D.S., Jaramillo, M., Weinstock, J.V., Geary, T.G. , Stevenson, M.M., Urban Jr, J.F., Makedonka, M., Jardim, A. 2018. Analysis of the Trichuris suis excretory/secretory proteins as a function of life cycle stage and their immunomodulatory properties. Scientific Reports. 8:15921.
- Jarjour, N.N., Schwarzkopf, E.A., Bradstreet, T.R., Shchukina, I., Lin, C., Huang, S., Lai, C., Cook, M.E., Taneja, R., Stappenbeck, T., Randolph, G. J., Artyomov, M.N., Urban Jr, J.F., Edelson, B.T. 2019. Bhlhe40 mediates tissue-specific control of macrophage proliferation in homeostasis and type 2 immunity. Nature Immunology. 20:687-700.
- Hang, L., Kumar, S., Blum, A.M., Urban Jr, J.F., Fantini, M.X., Weinstock, J.V. 2019. Heligmosomoides polygyrus bakeri infection decreases Smad7 expression in intestinal CD4+ T cells which allows TGF-beta to induce IL10- producing regulatory T cells that block colitis. Journal of Immunology. (8) 2473-2481.
- Metwali, A., Thorne, P., Winckler, S., Metwali, N., Urban Jr, J.F., Elliott, D.E., Ince, M., Guan, X., Beyatli, S., Truscott, J. 2018. Mechanisms of exacerbating lung inflammation in inflammatory bowel disease. Digestive Disease and Science.
- Li, S., Bostick, J.W., Ye, J., Qiu, J., Zhang, B., Urban Jr, J.F., Avram, D., Zhou, L. 2018. Aryl hydrocarbon receptor signaling cell intrinsically inhibits intestinal group 2 innate lymphoid cell function. Immunity. 49(5) :915-928,e5.
- Li, Y., Liu, W., Guan, X., Chen, H., Zavazava, N., Blumberg, R.S., Weiner, G.J., Urban Jr, J.F., Elliott, D.E., Ince, N.M., Truscott, J., Karakay, B. 2018. Helminth-induced production of TGFÿ and suppression of graft-versus- host disease is dependent on Interleukin 4 production by host cells. Journal of Immunology. 201(10):2910-2922.
- Beasely, H., Bennett, H.M., Coghlan, A., Cotton, J., Doyle, S.R., Gordon, D., Harsha, B., Huckvale, T., Lomax, J., Holroyd, N., Reid, A.J., Ribeiro, D., Rinaldi, G., Shafie, M., Stanley, E., Tracey, A., Berriman, M., Hallsworth-Pepin, K., Martin, J., Ozersky, P., Rosa, B.A., Tyagi, R., Zhang, X., Mitreva, M., Laetsch, D.R., Koutsovoulos, G., Kumar, S., Kaur, G., Blaxter, M., Howe, K.L., Leach, A.R., Mutowo, P., Rawlings, N., Kuo, T. , Lee, T.J., Ke, H., Tsai, I.J., Wheeler, N.J., Day, T.A., Zamanian, M., Beech, R.N., Parkinson, J., Seshadri, S.L., Kikuchi, T., Maizels, R.M., Partono, F., Babayan, S., Allen, J.E., O'Boyle, N., Wang, L., Osuna, A., Cruz-Bustos, T., Samblas, M.G., Cuellar, C., Cooper, P.J., Devaney, E., Harcus, Y., Hodgkinson, J., Bah, G., Tanya, V.N., Eberhard, M.L., Asano, K. , Rodriguez, P.F., Sato, H., Gilleard, J.S., Matthews, J.B., Cook, J., Toldeo, R., Scholz, T., Schnyder, M., Allan, F., Emery, A., Olson, P.D., Rollinson, D., Castillo, E., Kalbe, M., Eom, K.S., Horak, P., Mitreva, M., Hawdon, J.M., Urban Jr, J.F., Hill, D.E., Zarlenga, D.S., Bisset, S.A., Pfarr, K., Makepeace, B., Taylor, D.W. 2018. Comparative genomics of the major parasitic worms. Nature Genetics. 5:163-174.
- George, N.S., Cheung, L., Luthria, D.L., Santin, M., Dawson, H.D., Bhagwat, A.A., Smith, A.D. 2019. Pomegranate peel extract alters the microbiome in mice and dysbiosis caused by Citrobacter rodentium infection. Journal of Food Science and Nutrition.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): Objective 1: Determine whether bioactive food components, such as vitamins A and D or lycopene, acting via vitamin A and D-receptor- mediated pathways and nuclear factor-kappaB signaling, exhibit dose- dependent inhibitory effects on macrophage-mediated remodeling of adipose tissue toward a pro-inflammatory phenotype in response to high fat diets in a swine model. [NP 107 Component 3, Problem Statement 3B]. Objective 2: Determine whether bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Compare responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling. [NP 107 Component 3, Problem Statement 3B] Approach (from AD-416): For Objective 1, in vitro and in vivo porcine models will be used to test the hypothesis that vitamin A or vitamin D or metabolites of dietary compounds that signal through retinoic acid receptor signaling pathways, such as lycopene, will promote an anti-inflammatory phenotype of adipose tissue macrophages and inhibit pro-inflammatory responses of adipose tissue macrophages to inflammatory ligands via inhibition of NF-kappaB signaling and epigenetic regulation of macrophage polarization. For Objective 2, a mouse model will be used to test the hypothesis that bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox-sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, will be used to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling. A molecular marker of both pig and human anti-inflammatory cells is activated by metabolites of vitamin A. The most bioactive vitamin A metabolite, all-trans retinoic acid (ATRA), potentiated genes induced by the a signaling molecule called IL-4 to activate a macrophage cell (M2a) that has anti-inflammatory properties. These responses are necessary to maintain immune homeostasis and limit inflammation. We have recently demonstrated that the pig M2a and human THP-1 cells respond to both IL-4 and ATRA to produce an important enzyme called transglutaminase 2 (TGM2) that is a marker for both pig and human cells. In addition, the combination of ATRA and IL-4 synergistically increased a protein called IL1RN that binds to certain cell receptors and inhibits the action of selective proinflammatory proteins. These experiments suggest novel ways to inhibit or enhance inflammatory responses mediated by cells that regulate the appropriate control of inflammation in pigs and humans. Tools were developed to better characterize the genome of pigs. A correct and annotated genome is essential for high throughput methods of gene and protein analysis, as well as to compare genes from animals used to model humans. ARS scientists at the Beltsville Human Nutrition Center, Beltsville Maryland previously demonstrated the utility of our manual annotation process to identify errors and improve the annotation of an early version of the porcine genome. Version 11.1 of the porcine genome was recently released and we have now applied the same tools to 2,800 targeted genes in the latest version (11.1) of the pig genome and found that many errors exist in gene assembly and annotation. For example, only half of the gene sequences were correctly assembled and annotated, and a small but significant number of genes were not represented in the genome. Chief among these were genes in the interleukin 1 family which express proteins critical to controlling inflammation. We have continued assembling a non-redundant library of manually verified RNA (11,250) and protein (8,677) sequences and intend to continue this work in conjunction with our domestic and international partners. Similarities and differences were found in nutritional-related genes in mice, pigs and humans to improve modeling. ARS scientists at the Beltsville Human Nutrition Center, Beltsville, Maryland have recently published 4 comparative analyses focusing on the comparative aspects of pig, mouse and human genes and proteins involved in immunity and inflammation (the immunome). Genes between humans and pigs were generally similar, but several were unique to humans and mice. The analysis was extended to genes and proteins involved in nutrition and metabolism with a focus on questions related to the nutritional regulation of physiology of the mouse, pig and human. Are these genes 1) in specific pathways, 2) structurally and functionally similar, 3) expressed in similar cell types, and 4) regulated in a similar manner to stimuli? Pigs had roughly 4-fold less unique genes than mice or humans. When a gene is missing from one or more genomes, the frequency and similarity of gene preservation between pigs and humans is overwhelmingly similar. Notably, physiological differences such as the lack of porcine salivary lipase and amylase activities is likely related to the absence of these genes in the porcine genome. This analysis suggests that evaluating nutrition interventions in pigs will provide useful data to bridge the gaps found in using mice as nutritional models for humans. Studies were performed to determine if the knock out (KO) of selenoproteins in specific cell types mimicked the effect of feeding a selenium deficient diet using our two infectious disease models. Deleting selenoproteins in one immune cell type (macrophages, dendritic cells, or lymphocytes) did not replicate what is observed in mice fed a selenium-deficient diet and infected with bacteria. A lack of an effect was also observed in worm-infected mice with selenoproteins deleted in macrophages or dendritic cells, but preliminary results indicate that the deletion of selenoproteins in lymphocytes may have an effect on worm clearance. Of note, it has not been possible to successfully generate KO of selenoproteins in intestinal epithelial cells by crossing TRSP floxed mice (KO of all selenoproteins) or GPX4 floxed mice (KO of only GPX4). Only mice with one allele KO for TRSP or GPX4 are produced and will be tested further. Diets deficient in vitamin A (VA) produced statistically significant changes in bacterial populations in the fecal microbiome. A more in-depth analysis of the data is underway. Bacterial infection of VA deficient mice express increased pathology, bacterial load, delayed clearance, and enlarged goblet cells with greater amounts of mucus, and higher levels of tissue associated bacteria. Studies using labels for sugar motifs on glycoproteins in mucus showed alterations in the types of mucus produced by VA deficient mice. In addition, the hosts of genes associated with mucus production and goblet cell function were down- regulated by VA deficiency. This is important in understanding how diet deficiencies effect the health of the intestine. Pomegranate peel extract (POM) reduced the pathogenicity of an intestinal bacterial infection. ARS scientists from the Environmental Microbial and Food Safety (EMFSL), Invasive Insect Biocontrol and Behavior Laboratory (IIBBL) and Beltsville Human Nutrition Center, Beltsville, Maryland tested the effect of POM on infectious colitis. The results demonstrate that POM reduced colonic pathology, development of a systemic bacterial infection, mortality, colonic mucosa damage and bacterial translocation. It does not alter peak bacterial load, clearance, or the immune response to infection indicating that the health effect is on intestinal barrier function that may be related to expression of the Ang4 gene. In addition, POM treatment decreased the loss of goblet cells and mucin production, which likely ablates the severity of the infection. Dietary supplements improve glucose tolerance and reduce bacterial infection. ARS scientists at the Beltsville Human Nutrition Center, Beltsville, Maryland and colleague at the University of Maryland- Baltimore Medical School treated mice with a genetic and high-fat diet induced obesity with an immune protein called IL-25. There was decreased weight gain and improved glucose tolerance in both genetic and diet- induced obesity in mice given IL-25. In another study, the effects of feeding indole 3-carbinol mice with a bacterial infection was studied and showed to ameliorate the pathological effects of infection. This work demonstrates the utility of mouse models of metabolic and infectious disease to screen for bioactive components that can improve health. Accomplishments 01 Feeding probiotic reduced allergenic responses to worm infection. Infection in livestock such as the pig continues to be a problem for our food system, safety of food products and impacts human health. ARS scientists at the Beltsville Human Nutrition Center, Beltsville, Maryland, seek to elucidate beneficial effects of probiotics in feed to overcome infection in the pig. The effects of feeding the probiotic Bifidobacterium animalis subspecies lactis (Bb12) on parasitic nematode Ascaris suum infection in the pig were examined. Parasite-protective antibody responses in the serum and intestinal fluid were found to be significantly increased, and an inflammatory cell population in the small intestine was inhibited in infected pigs fed Bb12 without affecting the normal expulsion of the worm. The expression of genes associated worm-protective responses was enhanced along with certain anti-inflammatory genes after feeding Bb12 to worm infected pigs. Thus, feeding a probiotic can improve intestinal function during a worm infection by reducing components of a strong allergenic response without compromising normal worm expulsion and may benefit healthy livestock production. 02 Infection protective cells in the intestine are activated in response to diet and changes in gut microbiome. Tuft cells in the intestine are known to act as sentinels for infectious agents [e.g., helminths (worms) and bacterial microbiota] and express taste-signaling elements. In this work, the G protein-coupled receptor Sucnr1 was shown to be expressed specifically in tuft cells, but not in other intestinal epithelial cells. Dietary succinate and perturbations in the microbiota activate tuft cells, and subsequently type 2 immunity, via tuft cell- expressed Sucnr1. Modulating this pathway using dietary succinate or specific Sucnr1 agonists may be a strategy for fighting bacterial and parasitic infections or other type 2 immune-related metabolic disorders such as obesity. 03 Novel strategy for prevention of allergic airway inflammation identified using genetic deficient mice. Allergy and allergic asthma are significant health burdens and sound strategy to prevent these diseases are necessary. ARS scientists at the Beltsville Human Nutrition Center, Beltsville, Maryland, and colleagues at the Virginia Commonwealth University, Richmond, Virginia characterized a mouse strain with a conditional deficiency in the gene A disintegrin and metalloproteinase (ADAM) 10, and used the strain to understand the role of ADAM10 on selected immune cells during the development of allergic and anaphylactic responses. ADAM10 was found to be important in the immune response. This study provides science-based information to support that targeting ADAM10 represents a novel strategy for modulating allergic airway inflammation in the pathogenesis of allergy and allergic asthma.
Impacts
(N/A)
Publications
- Solano Aguilar, G., Shea-Donohue, T., Madden, K., Quinones, A., Beshah, E., Lakshman, S., Xie, Y., Dawson, H.D., Urban Jr, J.F. 2018. Bifidobacterium animalis subspecies lactis modulates the local immune response and glucose uptake in the small intestine of juvenile pigs infected with the parasitic nematode Ascaris suum. Gut Microbes. 19:1-15.
- Dawson, H.D., Lunney, J.K. 2018. Porcine cluster of differentiation (CD) Markers 2017 Update. Research in Veterinary Science. 118:199-246.
- Smith, A.D., Panickar, K.S., Urban Jr, J.F., Dawson, H.D. 2018. Impact of micronutrients on the immune response of animals. Annual Review of Animal Biosciences. 6:227-254.
- Damie, S.R., Martin, R.K., Cockburn, C.L., Lownik, J.C., Carlyon, J.A., Smith, A.D., Conrad, D.H. 2017. ADAM10 and Notch1 on murine dendritic cells control the development of type 2 immunity and IgE production. Allergy. 148(4):542-551.
- Lei, W., Ren, W., Ohmoto, M., Urban Jr, J.F., Matsumoto, I., Margolskee, R. F., Jiang, P. 2018. Activation of intestinal tuft cell-expressed Sucnr1 triggers type 2 immunity in the mouse small intestine. Proceedings of the National Academy of Sciences. (21):5552-5557.
- Lorentsen, K., Cho, J., Luo, X., Zuniga, A., Urban Jr, J.F., Zhou, L., Gharaibeh, R., Jobin, C., Kladde, M., Avram, D. 2018. Bcl11b is essential for licensing Th2 differentiation during helminth infection and allergic asthma. Nature Communications. 9(1)1679.
- Martin, R.K., Damle, S.R., Zellner, M.P., James, B.N., Valentine, Y.A., Elkowich, A.J., Lownik, J.C., Demeules, M.M., Khandjian, L.M., Urban Jr, J. F., Conrad, D.H. 2018. B1 cell IgE impedes mast cell-mediated enhancement of parasite expulsion through B2 IgE blockade. Cell Reports. 22(7):1824- 1834.
- Huang, Y., Mao, K., Chen, X., Sun, M., Kawabe, T., Li, W., Usher, N., Zhu, J., Urban Jr, J.F., Paul, W.E., Germain, R.N. 2018. S1P dependent inter organ trafficking of group 2 innate lymphoid cells supports host defense. Science. 359:114�119.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): Objective 1: Determine whether bioactive food components, such as vitamins A and D or lycopene, acting via vitamin A and D-receptor- mediated pathways and nuclear factor-kappaB signaling, exhibit dose- dependent inhibitory effects on macrophage-mediated remodeling of adipose tissue toward a pro-inflammatory phenotype in response to high fat diets in a swine model. [NP 107 Component 3, Problem Statement 3B]. Objective 2: Determine whether bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Compare responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling. [NP 107 Component 3, Problem Statement 3B] Approach (from AD-416): For Objective 1, in vitro and in vivo porcine models will be used to test the hypothesis that vitamin A or vitamin D or metabolites of dietary compounds that signal through retinoic acid receptor signaling pathways, such as lycopene, will promote an anti-inflammatory phenotype of adipose tissue macrophages and inhibit pro-inflammatory responses of adipose tissue macrophages to inflammatory ligands via inhibition of NF-kappaB signaling and epigenetic regulation of macrophage polarization. For Objective 2, a mouse model will be used to test the hypothesis that bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox-sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, will be used to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling. Previously we demonstrated that the most bioactive vitamin A metabolite, all-trans retinoic acid (ATRA) potentiated the mRNA expression of several IL-4 induced chemokines (chemokine (C-C motif) ligand 11 ((CCL11), CCL17, CCL22 and CCL26) associated with alternative activation (M2a) in porcine macrophages in vitro. These responses are necessary to maintain immune homeostasis and limit inflammation. We extended these findings to human macrophages. In human THP-1 cells, IL-4 exposure led to an increase in the mRNA for the M2a macrophage-associated chemokines, CCL11, CCL13, CCL17, CCL18, CCL22 and CCL26, as well as the M2a-associated surface markers, CD206, CD209 and CD274 and the M2a associated enzyme TGM2. ATRA synergistically increased IL-4�induced CCL2, CCL13, CCL18, CCL22, CCL26 and TGM2 mRNA and CCL13, CCL18 and CCL26 protein levels, while having no effect or slightly down-regulating MRC1, CD209 and CD274 mRNA expression. The effects of ATRA on mRNA levels were greater at 24 compared to 48 hrs; however, greater differences in protein levels were observed at 48 hrs for CCL13, CCL18 and CCL26. In contrast to porcine monocytes and macrophages, ATRA decreased IL-4 induced CCL17 mRNA and protein expression in THP-1 cells. Thus, in porcine monocytes and macrophages and in human macrophages, ATRA selectively increased signaling in response to IL-4. Ongoing experiments include the effects of ATRA, vitamin D and/or IL-4 on nuclear factor-kappaB signaling, inflammasome activation and TGM2 activity in pig macrophages and human THP-1 cells. We are also assisting the Food Components and Health Laboratory (FCHL) in the analysis of whole blood transcriptomic data from humans fed beta carotene or lycopene. We generated a mouse strain with KO of selenoproteins in macrophages. Much to our surprise, these mice did not exhibit a phenotype in either of our infectious disease models (Cr, Hpb), especially considering the importance of macrophages in the clearance of a Hpb infection. However, preliminary results suggest that KO of selenoproteins in BMDM may affect inflammasome activation and this will be pursued further. Mice with selenoprotein expression KO in T cells are being generated by crossing floxed TRSP mice with Lck-cre mice. We expect to see a phenotype as alterations in the immune responses in non-infectious disease models have been observed. Our studies examining the effect of VA deficiency on the fecal microbiome are progressing and significant changes have been observed with distinct groupings for VA adequate and deficient mice in a principle component analysis of the data. We have observed enlarged goblet cells in VA deficient mice that are strongly positive for staining with Periodic Acid-Schiff. We are currently determining how mucus production is altered by VA deficiency. We have identified unique changes to mucins in goblet cells and the intestinal mucus layer by staining with lectins that have specificity for different sugar moieties and plan on examining the expression of genes involved in mucin production and export. In collaboration with ARS scientists from the Environmental Microbial and Food Safety (EMFSL) and the Invasive Insect Biocontrol and Behavior Laboratory (IIBBL), we have tested the effect of pomegranate peel extracts on infectious colitis induced by Cr. The results demonstrate that the pomegranate peel extract (POM) reduces colonic pathology and development of a systemic infection by Cr in vivo. POM treatment completely prevents infection induced mortality and reduces damage to the colonic mucosa, thus preventing translocation of bacteria to the systemic compartment that is characteristic of untreated mice. Interestingly, POM treatment does not alter peak Cr load, clearance, or the cytokine response to infection indicating that the effect is on barrier function. One gene, Ang4, which is important for the control of enteric bacteria and barrier function, is increased in POM treated mice. Several other collaborations have occurred over the last year. Our expertise in the use of Cr has resulted in requests for collaboration. The Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia asked for our assistance in characterizing a mouse strain with a conditional knock out of A disintegrin and metalloproteinase (ADAM) 10, a member of a transmembrane zinc-dependent protease family that mediates ectodomain shedding and regulated intramembrane proteolysis, in dendritic cells. Given its important role in cell-cell and cell-matrix interactions, ADAM10 has been studied in many physiological and disease processes including neuron homeostasis and development, Alzheimer�s disease, and allergy. We tested the KO mouse strain in our Cr model and found that ADAM10 was important for preventing systemic dissemination of bacteria and mortality. A manuscript containing these and other results has been submitted for publication. A second collaboration with the University of Maryland-Baltimore Medical School also utilized our expertise using the Cr model. In this study bi- directional communication between the gut and the brain were demonstrated. Mice with moderate-level traumatic brain injury (TBI) had colonic paracellular flux and glial fribrillary acid protein expression after Cr infection that was significantly increased compared to shams. Importantly, brain lesions were significantly larger and neuroinflammation was increased in TBI-injured mice infected with Cr compared to CCI alone. These results demonstrate bi-directional communication between the gut and the brain and have implications for treatment of individuals suffering from TBI. Accomplishments 01 Protective immunity to nematode infections. ARS scientists at the Beltsville Human Nutrition Center, Beltsville Maryland, and colleagues at the University of Maryland, Baltimore Medical School demonstrated that proteins in mice genes (IL-25 and the receptor subunit IL-17RB) are increased during the primary and secondary infection of mice worm (Heligmosomoides polygyrus bakeri) (Hpb). Mice without the IL-25 gene had a reduced response and response against secondary infection of Hpb was severely impaired. Treating infected mice with the IL-25 protein restored protection against Hpb. This study demonstrates that IL-25 will be useful to individuals who are developing therapies for Hpb. 02 Impact of bidirectional brain/gut interactions on both gastrointestinal and brain health in mice. ARS scientists at the Beltsville Human Nutrition Center, Beltsville, Maryland, and colleagues at the University of Maryland, Baltimore Medical School examined changes in mucosal barrier properties and enteric glial cell response in the gut after experimental Traumatic brain injury (TBI) in mice, as well as effects of the enteric pathogen Citrobacter rodentium (Cr) on both gut and brain after injury. Moderate-level TBI induced changes in colon morphology, including increased mucosal depth and smooth muscle thickening, increased paracellular permeability and decreased and protein expression. Cr infection in chronically-injured mice worsened the brain lesion injury and increased inflammation. These results demonstrate bidirectional brain/gut interactions have repercussions for individuals suffering from TBI, but also indicates that alterations to the gut from disease, diet, or the microbiome may impact recovery from other traumatic brain events including stroke. This research is of value to those interested in how diet and inflammation may affect brain/ gut interactions.
Impacts
(N/A)
Publications
- Bhagwat, A.A., Young, L., Smith, A.D., Bhagwat, M. 2017. Transcriptomic analysis of swarm motility phenotype of Salmonella enterica serovar Typhimurium mutant defective in periplasmic glucan synthesis. Current Microbiology. doi:10.1007/s00284-017-1267-1.
- Shea-Donohue, T., Qin, B., Smith, A.D. 2017. Parasites, nutrition, immune responses, and biology of metabolic tissues. Parasite Immunology. 39(5). doi: 10.1111/pim.12422
- Ma, E.L., Smith, A.D., Desai, N., Cheung, L., Hanscom, M., Stoica, B.A., Loane, D.J., Shea-Donohue, T., Faden, A.I. 2017. Bidirectional brain-gut interactions and chronic pathological changes after traumatic brain injury in mice. Brain Behavior and Immunity. pii: S0889-1591(17)30207-6. doi: 10. 1016/j.bbi.2017.06.018.
- Pei, C., Zhao, C., Wang, A.J., Fan, A.X., Grinchuk, V., Smith, A.D., Sun, R., Xie, Y., Lu, N., Urban Jr, J.F., Shea-Donohue, T., Zhao, A., Yang, Z. 2016. Critical role for Interleukin-25 in host protective Th2 memory response against Heligmosomoides polygyrus bakeri. Infection and Immunity. 18:84(12):3328-3337.
- Wilhelm, C., Harrison, O., Schmitt, V., Pelletier, M., Spencer, S., Urban Jr, J.F., Ploch, M., Ramalingam, T., Siegel, R., Belkaid, Y. 2016. Critical role of fatty acid metabolism in ILC2 mediated barrier protection during malnutrition and helminth infection. Journal of Experimental Medicine. 213(8):1409-1418. doi: 10.1084/jem.20151448.
- Mclean, L.P., Smith, A.D., Cheung, L., Urban Jr, J.F., Sun, R., Grinchuk, V., Dasai, Zhao, A., Raufman, J.P., Shea-Donohue, T. 2016. Type 3 muscarinic receptors contribute to intestinal mucosal homeostasis and clearance of nippostrongylus brasiliensis through induction of Th2 cytokines. American Journal of Physiology - Gastrointestinal and Liver Physiology. 311(1):G130-141. doi: 10.1152/ajpgi.00461.2014.
- Weeks, J.C., Roberts, W.M., Robinson, K.J., Keaney, M.I., Vermeire, J.J., Urban Jr, J.F., Lockery, S.R., Hawdon, J.M. 2016. Microfluidic platform for electrophysiological recordings from host-stage hookworm Ascaris suum larvae: a new tool for anthelmenthic research. International Journal for Parasitology: Drug and Drug Resistance. 6:314-328. doi: 10.1016/j.ijpddr. 2016.08.001.
- Dawson, H.D., Chen, C.T., Gaynor, B., Shao, J.Y., Urban Jr, J.F. 2017. The porcine translational research database: A manually curated, genomics and proteomics-based research resource. Biomed Central (BMC) Genomics. doi: 10. 1186/s12864-017-4009-7.
- Hang, L., Blum, A.M., Kumar, S., Urban Jr, J.F., Mitreva, M., Geary, T.G., Jardim, A., Stevenson, M.M., Lowell, C.A., Weinstock, J.V. 2016. Downregulation of the Syk signaling pathway in intestinal dendritic cells is sufficient to induce dendritic cells that inhibit colitis. Journal of Immunology. 197(7):2948-2957. doi: 10.4049/jimmunol.1600063.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): Objective 1: Determine whether bioactive food components, such as vitamins A and D or lycopene, acting via vitamin A and D-receptor- mediated pathways and nuclear factor-kappaB signaling, exhibit dose- dependent inhibitory effects on macrophage-mediated remodeling of adipose tissue toward a pro-inflammatory phenotype in response to high fat diets in a swine model. [NP 107 Component 3, Problem Statement 3B]. Objective 2: Determine whether bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Compare responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling. [NP 107 Component 3, Problem Statement 3B] Approach (from AD-416): For Objective 1, in vitro and in vivo porcine models will be used to test the hypothesis that vitamin A or vitamin D or metabolites of dietary compounds that signal through retinoic acid receptor signaling pathways, such as lycopene, will promote an anti-inflammatory phenotype of adipose tissue macrophages and inhibit pro-inflammatory responses of adipose tissue macrophages to inflammatory ligands via inhibition of NF-kappaB signaling and epigenetic regulation of macrophage polarization. For Objective 2, a mouse model will be used to test the hypothesis that bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox-sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, will be used to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling. The central hypothesis being tested in objective 1 is that vitamin A (VA), either alone or in concert with cytokines like IL-4 or nutrients like vitamin D (VD), will foster the development of alternatively-activated macrophages (M2 macrophages) and/or inhibit the development of inflammatory macrophages (M1 macrophages) through epigenetic mechanisms. These macrophages (M1 or M2) differentially regulate the metabolic function of adipocytes and other cells; M1 macrophages are associated with insulin resistance and M2 macrophages with maintaining insulin sensitivity. To determine whether the bioactive VA metabolite, all-trans retinoic acid (ATRA) inhibits the development of M1 macrophages, primary porcine macrophages were treated with ATRA and then challenged in vitro, with an array of pro-inflammatory (lipopolysaccharide (LPS) + interferon- gamma (IFN-g), leptin, interleukin-1b (IL-1b), and nigericin (an inflammasome activator) or anti-inflammatory mediators (IL-4, IL-10 and IL-1B receptor antagonist). We found that ATRA reduced basal TNF protein, as well as TNF protein stimulated by LPS + IFN-g, IL-1b, and leptin but did not affect TNF protein stimulated by nigercin. We also found that ATRA decreased basal IL-1b protein, as well as IL-1b protein stimulated by LPS + IFN-g, but increased IL-1b protein stimulated by nigercin. These data suggest that ATRA acts to inhibit M1 macrophage polarization upstream of IL-1 b synthesis and inflammasome activation. These mechanistic data will allow us to focus our efforts. Studies to translate these findings to the human THP-1 human monocytic cell line are ongoing. One epigenetic-based, regulatory mechanism whereby ATRA may mediate M1 macrophage polarization is thorough a small class of regulatory RNA known as microRNA. The exact number of porcine microRNAs is currently unknown. To address this uncertainty, we constructed a 1,033 gene porcine microRNAome by manually screening 1,956 human, mouse or bovine sequences for homology. Using our recently constructed non-redundant porcine RNA library and microRNA sequences as templates, we determined primary porcine macrophage microRNA induction or repression in response to M1- (LPS + IFN-g) or M2- (interleukin-4 (IL-4)), VA or VD) polarizing stimuli. We found 24 microRNA that were induced by LPS/IFN-g, including the microRNA, mir-155, that has previously been showed to be LPS induced in human and mouse macrophages and regulates responses to VD in human macrophages. Predicting miRNA-binding sites in mRNA is problematic because different methods often yield different results and cross-species conservation is somewhat limited. To address this, we screened 2,131 differentially regulated genes, using 3 separate software programs, for conserved mir-155 biding sites. We found that human-porcine conservation of Mir-155 targets sites occurs at five times the rate of human-mouse conservation. We also found 82 candidate genes with binding sites that were conserved among 3 species (pig, mouse and humans) including several that are associated with insulin resistance. These gene targets will be used to determine whether ATRA and/or VD regulate M1 macrophage differentiation through mir-155-mediated responses. These epigenetics- based integrated analysis tools will be useful for modeling human inflammatory and metabolic disease in pigs because of the close evolutionary and functional features of the two species. Studies to translate these findings to human THP-1 cells are being conducted. We have obtained and are breeding multiple selenoprotein KO strains. Several KO strains, selenoprotein P, GPX1 and GPX2, were Helicobacter positive. A substantial effort was made to rid the strains of Helicobacter prior to their use in our models since Helicobacter spp. are known to influence gut immunity and inflammation, as thus, would be a confounding variable in our experiments. We successfully eradicated Helicobacter spp. from our mice and are currently evaluating the effect of KO of selenoprotein P in our two model systems. We are also currently evaluating the effect of KO of selenoprotein 15 in our two model systems. Additional experiments testing GPX1 and GPX2 mice will be initiated during the latter half of year 3. Experiments to identify sulfhydryl compounds which can reverse the effects of selenium deficiency including N-acetyl cysteine, 2-ME, lipoic acid, CoQ10 and its reduced form, and vitamin E in the Citrobacter rodentium (Cr) and/or Hpb models have not yielded any antioxidants that can substitute for the role of selenium. Due to the negative results obtained thus far and to allow additional time and resources to be devoted to use of the KO mouse models, this line of research has been terminated. Preliminary evidence, however, indicates that the GPX mimetic, ebselen, was able to reverse the effect of Se deficiency in the Cr model. This suggests that one of the four selenoprotein GPXs found in mice may be critical for controlling inflammation in response to a gastrointestinal bacterial infection. We found that VA deficiency exacerbated Cr infections leading to higher rates of mortality, increased colonic colonization and pathology, and spread to the systemic compartment. Unfortunately, these initial observations were also observed and published by another research group immediately prior to our publication effort. We have some additional unique observations on goblet cell dysfunction that we are following up on in hopes of still being able to publish our results. To further explore the role of VA in mucosal immunity, we fed pregnant mice control (1X), or VAS (5X) AIN-93G-based diet beginning on day 14 of gestation but this had no effect on the course of Cr infections. A one-time high bolus dose (20,000 U/Kg) of VA administered just prior to infection had no effect, so these studies will no longer be pursued. To determine how quickly various aspects of immunity to Cr can be recovered, we fed VAD mice, a VAS diet starting the day of, or 7 days after infection. We found that administering VAS diet at the time of infection reversed many adverse effects of VAD on Cr infection while starting 7 days post- infection was only partially effective. These observations will be part of the eventual manuscript. A study to examine the effect of VAD on the microbiome was just completed and a principle component analysis of the data indicates the VAD does alter the microbiome. Additional analyses are being performed to identify specific families of bacteria that are altered by VAD. In collaboration with ARS scientists from Environmental Microbial and Food Safety (EMFSL) and the Invasive Insect Biocontrol and Behavior Laboratory (IIBBL), we tested the effect of pomegranate peel extracts on pathogenic bacteria, both in vitro and in vivo. The results suggest that the pomegranate peel extract (POM) alters swarming properties of Cr in vitro and reduced the pathogenicity of Cr infections in vivo. To gain further insight into the mechanisms by which POM affected swarming, RNAseq data were generated for Cr grown under swarming conditions in the presence or absence of POM. Significant differences in gene expression have been identified and the analysis is continuing. A study to determine the effect of feeding POM on the microbiome was just completed and preliminary analysis indicates major POM-induced changes to the microbiome, including an increase in Akkermansia muciniphila. The abundance of this bacterial strain has been reported to inversely correlate with inflammation and metabolic disorders, associated with obesity, and is less abundant in patients with inflammatory bowel disease (IBD). These results raise the possibility that POM may have beneficial effects for IBD patients and could impact the development of metabolic syndrome. Accomplishments 01 Conducted an in-depth analysis of the porcine, murine and human inflammasomes. Herein ARS scientists at the Beltsville Human Nutrition Center, Beltsville Maryland, provide an expanded in silico analysis using an improved assembly of the porcine transcriptome that provides an in depth analysis of genes that are related to inflammasomes. Comparisons of the expansion or contraction of orthologous gene families indicated more similar rates and classes of genes in humans and pigs than in mice; however, several novel porcine or artiodactyl- specific paralogs or pseudogenes were identified. Despite these similarities, two out of four canonical inflammsome pathways that included the Absent in melanoma 2 (AIM2) and NLR family and CARD domain containing 4 (NLRC4) pathways were found to be missing in pigs. This work supports using swine to model both human immunological and inflammatory responses to infection. However, caution must be exercised as pigs differ from humans in several fundamental pathways. 02 Defined the host-adapted state of Citrobacter rodentium (Cr) by transcriptomic analysis. Citrobacter rodentium is a mouse pathogen that mimics many aspects of enteropathogenic Escherichia coli infections. Host-adapted (HA) Cr cells that are shed at the peak of infection have been reported to be hyperinfective. The exact mechanism underlying this phenomenon has remained elusive since the pathogen loses it�s HA �status� immediately upon sub-culturing in laboratory media. By sequencing the entire transcriptome from the feces of infected mouse and media grown Cr, ARS scientists at the Beltsville Human Nutrition Center, Beltsville Maryland, observed that the entire transcriptional machinery, as well as several transcriptional regulators to be differentially expressed, including fis, a global regulator that affects growth, replication, and expression of the type 3 secretion system, which is critical for inducing disease. Notably absent among the HA expressed genes were 19 fimbrial operons and non- fimbrial adhesions and several non-LEE encoded effectors. This work provides basic mechanistic observations related to Citrobacter rodentium infectivity that relate to human and animal health. 03 Demonstrated the selenoproteins regulate the activity of inflammatory cells that expel parasitic worms. The micronutrient selenium (Se) induces an alternatively activated anti-inflammatory macrophage or M2 cell that is dependent upon prostaglandin J2 (15d-PGJ2). ARS scientists at the Beltsville Human Nutrition Center, Beltsville, Maryland and colleagues at Pennsylvania State University, University Park, Pennsylvania showed that mice with an adequate level of Se in the diet expressed normal levels of M2 cells after infection with a parasitic nematode worm Nippostrongylus brasiliensis (Nb). In contrast, Nb- infected mice on a Se deficient diet had reduced M2 cells, and the worms produced more infective eggs and were more slowly expelled from the intestine. Nb Infection of genetically modified mice that did not express selenoproteins in the macrophage had fewer M2 cells and showed reduced worm clearance and higher egg production.. Notably, blocking prostaglandin production in Nb-infected mice using the nonsteroidal anti-inflammatory drug indomethacin inhibited worm expulsion, but the protective response was restored by giving 15d-PGJ2. This work is important to those in animal and human health where parasitic worms are a problem as well as understanding the importance of adequate Se in the diet.
Impacts
(N/A)
Publications
- Dawson, H.D., Smith, A.D., Chen, C.T., Urban Jr, J.F. 2016. An in-depth comparison of the porcine, murine and human inflammasomes; lessons from the porcine genome and transcriptome. Veterinary Microbiology. pii:S0378- 1135(16)30137-7. doi: 10.1016/j.vetmic.2016.05.013.
- Vannella, K.M., Ramalingam, T.R., De Queiroz, P.R., Sciurba, J., Barron, L. , Borthwick, L., Smith, A.D., Mentink-Kane, M., White, S., Thompson, R.W., Cheever, A.W., Bock, K., Moore, I., Fitz, L.J., Urban Jr, J.F., Wynn, T.A. 2016. Acidic Chitinase Limits Allergic Inflammation and Promotes Intestinal Nematode Expulsion. Nature Immunology. 17(5):538-44 doi: 101038/ ni.3417.
- Sun, R., Notari, L., Vanuytsel, T., Madden, K., Bohl, J., Ramalingam, T.R., Wynn, T.A., Urban Jr, J.F., Zhao, A., Shea-Donohue, T. 2016. Interleukin- 13 receptor a1-dependent responses in the intestine are critical to parasite clearance. Infection and Immunity. 84(4):1032-1044. doi: 10. 1128IAI.00990-009915.
- Everts, B., Tussiwand, R., Dreesen, L., Fairfax, K.C., Huang, S.C., Smith, A.M., Oneil, C.M., Lam, W.Y., Edelson, B.T., Urban Jr, J.F., Murphy, K.M., Pearce, E.J. 2016. CD103+ dendritic cells suppress Helminth-driven Type 2 immunity through constitutive expression of IL-12. Immunity. 213(1):35-51. doi: 10.1084/jem.20150235.jeq201612
- Nelson, S.M., Shay, A.E., James, J.L., Carlson, B., Urban Jr, J.F., Prabhu, K.S. 2016. Selenoprotein expression in macrophages is critical for optimal clearance of parasitic helminth Helminth Nippostrongylus brasiliensis. Journal of Biological Chemistry. 291(6):2787-2798. doi: 10. 1074/jbc.M115.684738.
- Guo, L., Huang, Y., Chen, X., Hu-Li, J., Urban Jr, J.F., Paul, W.E. 2015. Innate immunological function of TH2 cells in vivo. Nature Immunology. 16:1051-1059.
- Huang, Y., Guo, L., Qiu, J., Chen, X., Hu-Li, J., Siebenlist, U., Williamson, P.R., Paul, W.E., Urban Jr, J.F., Paul, W.E. 2015. IL-25- responsive, lineage-negative, KLRG1(hi) cells are multipotential �inflammatory� type-2 innate lymphoid cells. Nature Immunology. 16(2):161- 169. doi: 10.1038/ni.3078
- Chen, F., Wu, W., Millman, A., Craft, J.F., Chen, E., Patel, N., Boucher, J.L., Urban Jr, J.F., Kim, C.C., Gause, W.C. 2014. Neutrophils prime a long-lived effector macrophage phenotype that mediates accelerated helminth expulsion. Nature Immunology. 15(10):938-946. doi: 10.1038/ni. 2984.
- Smith, A.D., Yan, X., Chen, C.T., Dawson, H.D., Bhagwat, A.A. 2016. Understanding the host-adapted state of Citrobacter rodentium by transcriptomic analysis. Archives Of Microbiology. 198:353-362.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): Objective 1: Determine whether bioactive food components, such as vitamins A and D or lycopene, acting via vitamin A and D-receptor- mediated pathways and nuclear factor-kappaB signaling, exhibit dose- dependent inhibitory effects on macrophage-mediated remodeling of adipose tissue toward a pro-inflammatory phenotype in response to high fat diets in a swine model. [NP 107 Component 3, Problem Statement 3B]. Objective 2: Determine whether bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Compare responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling. [NP 107 Component 3, Problem Statement 3B] Approach (from AD-416): For Objective 1, in vitro and in vivo porcine models will be used to test the hypothesis that vitamin A or vitamin D or metabolites of dietary compounds that signal through retinoic acid receptor signaling pathways, such as lycopene, will promote an anti-inflammatory phenotype of adipose tissue macrophages and inhibit pro-inflammatory responses of adipose tissue macrophages to inflammatory ligands via inhibition of NF-kappaB signaling and epigenetic regulation of macrophage polarization. For Objective 2, a mouse model will be used to test the hypothesis that bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox-sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, will be used to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling. To use the pig as a translational species to determine the most fruitful nutritional interventions in the human inflammatory pathway it is necessary to understand mechanisms involved in the inflammatory process in multiple species. To this end, we conducted several in silico and in vitro analyses to determine whether central pathways involved in inflammation and alternatively activated macrophage development are conserved among the three species (mouse, pig and human). We determined shared functional domains and regulatory family members of nine related pathways involved in inflammation, the so called inflammasomes. Several key members of these pathways were sequenced in our laboratory. Based upon our analysis we determined that several pig inflammasome pathways are intermediate in similarity between the mouse and the human; however; some of them are not conserved in pigs or mice. A preliminary analysis of whole genome expression profiles of macrophages indicates that the lipopolysaccharide (LPS, a proinflammatory mediator) -induced transcriptome appears to be more conserved among humans and pigs while the LPS-induced microRNAome and targets appears similar in all 3 species. However, the pig and human IL-4-induced, alternatively activated macrophage, transcriptomes are dramatically more similar. Several novel observations were also noted including selective, high-level induction of the epigenetic regulator histone deacetylase 9 (HDAC9) and the histone demethylase, KDM6B by IL-4 and numerous histone demethylases and methyltransferases by LPS. These analyses strengthen the notion that swine are a scientifically and ethically acceptable intermediate species (rodent-human) for conducting inflammation-related research relevant to humans. The central hypothesis being tested in objective 1 is that vitamin A (VA) , either alone or in concert with cytokines like IL-4 or nutrients like vitamin D (VD), will foster the development of alternatively-activated macrophages. We determined that the bioactive vitamin A metabolite, all- trans retinoic acid (ATRA) increases vitamin D3 receptor (VDR) mRNA and protein level in macrophages and epithelial cells. We found that vitamin D (VD) significantly regulated a total of 464 genes out of 5,000 compared. Of these, 256 genes were significantly regulated only by the combination of VA and VD (160 down-regulated and 96 up-regulated). ATRA- regulated 32 VD3-regulated genes whose expression was potentiated (>50%) in the same direction by ATRA, but not significantly affected by ATRA alone. Several of these genes are associated with alternatively-activated macrophages (MRC1, IL1RN). Fifteen genes were additively and three genes were synergistically affected by the combination of VA and VD. Several of these genes (PLXNA1, TIMP1) are also associated with alternatively activated macrophages. It is clear from these data that VA increases VD�s effect on the macrophage transcriptome as 2/3 of the VD regulated genes (305/464) were additively or synergistically induced or repressed by the combination of VA and VD. We used gene expression studies to identify candidate selenoprotein genes affected by H. polygyrus bakeri (Hpb) infection in both selenium- adequate and -deficient mice, and associated with the rebound in immunity upon re-feeding an adequate diet. To gain further insights into which selenoproteins are critical for immune function we have obtained nine Sel KO mouse strains for analysis, including two conditional KOs of TR1 and GPX4 (normally embryonically lethal) and are currently breeding them to obtain sufficient numbers for testing. Preliminary results suggest that deletion of the peroxidase-like domain of selenoprotein P increases the pathology of a C. rodentium (Cr) infection. Conditional KO of TR1 and GPX4 in T-cells, macrophages and gut epithelial cells will be generated for testing with our two model systems (Hpb, Cr). Additional experiments testing thiol/antioxidant compounds for their ability to substitute for selenium, including N-acetyl cysteine, 2-ME, lipoic acid, CoQ10 and its reduced form, and vitamin E in the Cr and/or Hpb models have not yielded any antioxidants that can substitute for the role of selenium. We demonstrated that Hpb cyst formation is a STAT6 dependent process and knock-out of IL-13 produces a cyst phenotype similar to that seen in selenium deficient mice where IL-13 expression is decreased suggesting a role for IL-13 in cyst formation and are currently looking for effects of selenium on collagen deposition in these cysts. We have completed studies on the effect of VA deficiency on immunity to Cr infection and demonstrate that VA deficiency increases the colonization of the colon, increased colonic pathology that was associated with disruption of barrier function, and produces mice with enlarged goblet cells that stain heavily for mucins suggesting altered goblet cell function in VA deficient mice. A collaborative study with another ARS scientist from the Environmental Microbial and Food Safety Laboratory (EMFSL) was undertaken to compare gene expression in Cr grown under three different nutrient conditions and examine changes in bacterial gene expression. Major nutrient-dependent changes in bacterial gene expression were observed indicating that we will be able to determine dietary-induced bacterial metabolomic changes in addition to compositional changes to the microbiome. In collaboration with ARS scientists from Environmental Microbial and Food Safety (EMFSL) and the Invasive Insect Biocontrol and Behavior Laboratory (IIBBL), we tested the effect of pomegranate peel extracts on pathogenic bacteria, both in vitro and in vivo. The results suggest that the pomegranate peel extract alters swarming properties of Salmonella in vitro and reduced the pathogenicity of Cr infections in vivo. As a result of these studies a new project has been developed in EMFSL that includes collaboration with the Diet, Genomic, and Immunology Laboratory (DGIL). Accomplishments 01 Muscarinic receptors that bind acetylcholine improve resistance to bacterial pathogens in the intestine. Citrobacter rodentium is a bacterial infection of mice that mimics bacterial induced gastrointestinal disease in humans. The type 3 muscarinic receptors (M3R) is known to regulate mucosal homeostasis within the colon and mice deficient in M3R were more susceptible to Cr infection. ARS scientists in the Beltsville Human Nutrition Research Center, Beltsville, Maryland, found most of the systemic immune response in the M3R deficient mice was intact, but mucus-producing cells in the colon were reduced, making it easier for the bacteria to bind to the intestine and begin the disease process. They also found that M3R contributed to the development of inflammatory macrophage cells that play a role in eliminating bacterial infection. This study showed that M3R is important in host defense against Cr infection through effects on goblet cell mucus production and in the modulation of macrophage function. These results demonstrate that M3R plays a previously unrecognized role in gut mucosal immunity and suggests that engagement of these receptors by activating compounds in the diet could enhance resistance to certain bacterial infections. 02 Diets low in protein can influence host immune responses and the degree of competition between co-infecting parasites. Most humans are concurrently infected with multiple parasite species and live in environments transmission in co-infected hosts under these conditions. To test how diets low in protein affect immunity and co-infection outcomes, ARS scientists in the Beltsville Human Nutrition Research Center, Beltsville, Maryland, gave mice a standard or low protein diet, dosed with two species of worm parasites (alone and in combination), and then challenged with a bacterial pathogen. They found that co- infection influenced parasite survival and reproduction via the type of host immunity that was affected by the diet. In pigs infected with whipworm and nodular worm and each worm parasite induced unique immune response types with both positive and negative effects on survival of each of the parasites. This information can be used by livestock producers to develop low cost diets that are appropriate to control worm infection when conventional drug treatments cannot be used because of organic farming restrictions with fluctuating dietary resources, yet little is known about individual health and pathogen. 03 Cell �cross-talk� promotes resistance to infection and tissue repair. Worm parasites can cause considerable damage when migrating through host tissues, which requires rapid tissue repair important to prevent bleeding and release of intestinal bacteria throughout the body. Studies showed that mice lacking the ability to make antibodies or one of the general cellular receptors for these antibodies had impaired intestinal repair after invasion of the tissue by a parasitic worm. The impairment was also associated with suppressed production of a protein messenger chemokine released by muscle cells in the intestine and inflammatory macrophage cells, indicating that inflammatory cells and muscle cells communicate to promote intestinal repair. This �cross- talk� feature could be harnessed in clinical settings of impaired wound healing. Other studies in mice demonstrated cell �cross-talk� between inflammatory neutrophil cells in the lungs and macrophages promoted the killing of parasitic worm larvae migrating through the lungs. In addition, cells of the innate immune system called innate lymphoid cells-2 or ILC2 were activated by specific protein cytokine messenger molecules to facilitate expulsion of adult worms from the intestine, while ILC3 cells that are generated by a different set of cytokines can control fungal infection. These studies provide insight into some of the protein messenger molecules and related receptors for these protein that provide communication signals between cells to improve resistance to infection and promote tissue repair. How diet can affect cellular communication to improve health is under study. 04 Construction of a porcine non-redundant transcriptome and analysis pipeline. Because the porcine genome is in its first draft, there are a significant number of problems that impede its use in determining whole genome analysis of gene expression, including gene fragmentation, duplicate sequencing of genes and failure to sequence certain areas of the genome. To address this ARS scientists in the Beltsville Human Nutrition Research Center, Beltsville, Maryland, constructed a non- redundant transcriptome containing 6,545 sequences that include the vast majority of genes that have been deemed biologically important enough to study, including the vast majority of genes that are related to epigenetics, nutrition, metabolism, immunity and inflammation (approximately 32% of the pig genome). These sequences include 1,347 fragmented genes, 970 duplicated genes and 1,042 genes missing from the genome and 1,395 full-length gene sequences that have been submitted to Genbank (Bioproject: PRJNA80971). These sequences and pipeline were used to perform several transcriptomic and epigenetic�based studies (miRNA and mRNASeq expression profiling) including responses of porcine alveolar macrophages to vitamin A and vitamin D or IL-4 or LPS/IFN-g and in vivo intestinal immune responses to T. suis. Since its introduction this year, this non-redundant transcriptome and analysis pipeline has also been used by 3 other labs within ARS, an external MTA has been executed to transfer this technology to the Arkansas Children�s Hospital Research Institute and commercial and international (Denmark, Belgium) interest has been expressed in this technology. These comprehensive and integrated analysis tools will be useful for global analysis and data-mining of the porcine immune and inflammatory responses as well as modeling human disease in pigs because of the close evolutionary and functional features of the two species.
Impacts
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Publications
- Andreasen, A., Petersen, H., Kringel, H., Iburg, T., Skovgaard, K., Dawson, H.D., Urban Jr, J.F., Thamsborg, S. 2015. Immune and inflammatory responses in pigs infected with Trichuris suis and Oesophagostomum dentatum. Veterinary Parasitology. 207(3-4):249-258.
- Budischak, S.A., Sakamoto, K., Megow, L.C., Cummings, K.R., Urban Jr, J.F., Ezenwa, V. 2015. Resource limitation alters the consequences of co- infection for both hosts and parasites. International Journal for Parasitology. 45(7):455-463.
- Von Bieren, J.E., Volpe, B., Sutherland, D.B., Burgi, J., Verbeek, S., Marsland, B.J., Urban Jr, J.F., Harris, N.L. 2015. Immune antibodies and helminth products promote CXCR2-dependent repair of parasite-induced injury. PLoS Pathogens. 11(3):e1004778.
- Mahadwar, G., Chauhan, K.R., Bhagavathy, G., Murphy, C.F., Smith, A.D., Bhagwat, A.A. 2015. Swarm motility of Salmonella enterica serovar Typhimurium is inhibited by compounds from fruit peel extracts. Letters in Applied Microbiology. 60:334-340.
- Wang, A., Smith, A.D., Li, Y., Urban Jr, J.F., Thirumalai, R.R., Wynn, T., Lu, N., Shea-Donohue, T., Yang, Z., Zhao, A. 2014. Genetic deletion of IL- 25 (IL-17E) confers resistance to dextran sulfate sodium-induced colitis in mice. Cell & Bioscience. DOI: 10.1186/2045-3701-4-72.
- Merenstein, D.J., Tan, T.P., Molokin, A., Smith, K.H., Roberts, R.F., Shara, N.M., Mete, M., Sanders, M.E., Solano Aguilar, G. 2015. Safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12- supplemented yogurt in healthy adults on antibiotics: A phase I safety study. Gut Microbes. 6(1):66-77. DOI: 10.1080/19490976.2015.
- Hibberd, P.L., Kleimola, L., Florina, A.M., Botelho, C., Haverkamp, M., Andreyeva, I., Poutsiaka, D., Fraser, C., Solano Aguilar, G., Syndman, D.R. 2014. No evidence of harms of probiotic Lactobacillus rhamnosus GG ATCC 53103 in healthy elderly-a Phase I Open Label Study to assess safety, tolerability and cytokine responses. PLoS One. 1:9(12):e113456. DOI: 10. 1371/journal.pone.0113456.
- Mclean, L.P., Smith, A.D., Cheung, L., Sun, R., Grinchuk, V., Vanuytsel, T. , Desai, N., Urban Jr, J.F., Zhao, A., Raufman, J.P., Shea-Donohue, T. 2015. Type 3 Muscarinic Receptors Contribute to Clearance of Citrobacter rodentium. Inflammatory Bowel Diseases. DOI: 10.1097/MIB.0000000000000408.
- Stephensen, C.B., Dawson, H.D., Claycombe, K.J. 2015. Impact of nutrition on immune function and the inflammatory response. Journal of Nutrition. 145(5):1039S-1108S. DOI: 10.3945/jn.114.194571.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): Objective 1: Determine whether bioactive food components, such as vitamins A and D or lycopene, acting via vitamin A and D-receptor- mediated pathways and nuclear factor-kappaB signaling, exhibit dose- dependent inhibitory effects on macrophage-mediated remodeling of adipose tissue toward a pro-inflammatory phenotype in response to high fat diets in a swine model. [NP 107 Component 3, Problem Statement 3B]. Objective 2: Determine whether bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Compare responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling. [NP 107 Component 3, Problem Statement 3B] Approach (from AD-416): For Objective 1, in vitro and in vivo porcine models will be used to test the hypothesis that vitamin A or vitamin D or metabolites of dietary compounds that signal through retinoic acid receptor signaling pathways, such as lycopene, will promote an anti-inflammatory phenotype of adipose tissue macrophages and inhibit pro-inflammatory responses of adipose tissue macrophages to inflammatory ligands via inhibition of NF-kappaB signaling and epigenetic regulation of macrophage polarization. For Objective 2, a mouse model will be used to test the hypothesis that bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox-sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, will be used to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling. This is a new project plan replacing and expanding on 1235-51000-055-00D. Several porcine adipose tissue macrophage isolations were performed to optimize yield and viability. Based upon these results, the lab has tested and purchased an automated magnetic bead isolation unit. Several human and monocyte blood isolations were performed to determine the best time course for addition of macrophage polarizing ligands such as retinoic acid and interleukin-4. Progress was made in identifying candidate selenoprotein genes affected by selenium deficiency and associated with the rebound in immunity upon re-feeding adequate diet. The expression of selenoproteins GPX2 and GPX3 is increased by infection. However, GPX2 activity is further enhanced by selenium deficiency while GPX3 expression is decreased by selenium deficiency. Re-feeding adequate diet decreases expression of GPX2 but increases expression of GPX3. GPX1 activity also rapidly rebounds after re-feeding adequate diet. Thioredoxin reductase and selenophosphate synthase 2 expression is decreased by infection but to a lesser extent in selenium deficient mice and this trend starts to reverse upon re-feeding. Cyst formation is a STAT6-dependent process and knock-out of IL-13 produces a cyst phenotype similar to that seen in selenium deficient mice where IL-13 expression is decreased, suggesting a role for IL-13. Several thiol compounds were tested including N-acetyl cysteine, 2- mercaptoethanol, lipoic acid, and Co-enzyme Q10, for their ability to alter the pathogenesis of C. rodetnium infection. Both Co-Q10 and 2-ME reduced the level of colonization in the colon and these results will be pursued further. Preliminary studies on the effect of Vitamin A (VA) deficiency on immunity were completed and demonstrate that VA deficiency increases the colonization of the colon, and causes barrier function disruption, but only in the presence of pathogenic bacteria. Th1/Th17 cytokine production was blunted by VA deficiency but not chemokine production. There was severe damage to the colonic epithelial layer and disruption of crypt architecture. In addition, goblet cells were enlarged in VA deficient mice and stained more heavily for mucins, raising the possibility that export of mucins into the intestinal lumen is impaired in VA deficient mice. Accomplishments 01 Vitamin A is critical for immunity to gastrointestinal bacterial infections. Vitamin A deficiency remains a significant problem in developing countries and sub-clinical deficiencies may exist in at-risk populations such as low-income children or the elderly in the U.S. We demonstrated that vitamin A deficiency impairs the ability to mount an effective immune response to bacterial infection in mice that is very similar to E. coli infections in humans and supports epidemiological studies showing more frequent and severe diarrheal disease in vitamin A individuals. These results highlight the need of health care workers to evaluate the diets of at risk populations to insure that adequate levels of vitamin A are being consumed for optimal immune function.
Impacts
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Publications
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