Research Project: Dietary Regulation of Immunity and Inflammation

Location: Diet, Genomics and Immunology Laboratory

2019 Annual Report

Objectives
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
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.

Progress Report
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
1. 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.

Review 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. https://doi.org/10.1016/j.dci.2019.03.020.
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. https://doi.org/10.1016/j.ijpara.2018.10.007.
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. https://doi.org/10.1016/j.vaccine.2019.01.027.
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. https://doi.org/10.1038/s41598-018-34174-4.
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. https://doi.org/10.1038/s41590-019-0382-5.
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. https://doi.org/10.4049/jimmunol.1801392.
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. https://doi.org/10.1007/s10620-018-5196-z.
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. https://doi.org/10.1016/j.immuni.2018.09.015.
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 TGFß and suppression of graft-versus-host disease is dependent on Interleukin 4 production by host cells. Journal of Immunology. 201(10):2910-2922. https://doi.org/10.4049/jimmunol.1700638
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. https://doi.org/10.1038/s41588-018-0262-1.
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. https://doi.org/10.1002/fsn3.1106.