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ARS Home » Northeast Area » Beltsville, Maryland (BHNRC) » Beltsville Human Nutrition Research Center » Diet, Genomics and Immunology Laboratory » Research » Research Project #426494

Research Project: Regulatory Mechanisms Induced by Health-Promoting Bioactive Food Components on Sex Steroid Hormone-Dependent Pathways, Cancer Cell-Stromal Cell Interactions, and the Gut Microbiome

Location: Diet, Genomics and Immunology Laboratory

2019 Annual Report

The overall goal of the project is to elucidate the molecular and cellular mechanisms that respond to selected food components to reduce the risk of chronic diseases such as cancer and obesity, with a focus on immune modulation in relation to obesity. A secondary aim is to further develop the utility of a porcine model to test the effect of probiotics and prebiotics on health maintenance through modulation of the gut microbiome and metabolome. Objective 1. Validate protective effects of bioactive food components such as glyceollins, indoles, and isothiocyanates on development of prostate cancer, and elucidate the regulation of sex steroid hormone-dependent pathways and cancer cell-stromal cell interactions as mechanisms of action by these bioactive food components. (NP107; C3, PS3B, C4, PS 4B) Objective 2. Study, in a swine model or other models as approriate, diet and gut microbiome interactions, focusing on the role of Lactobacillus, Bifidobacterium, and Bacteroides species, for the prevention of obesity and obesity-related metabolic syndrome. (NP107; C3, PS3B, C4, PS 4B)

For Objective 1, a complementary cell culture and tumor xenograft model will be used to test and identify efficacies of bioactive compounds from the diet and elucidate the mechanisms of how these bioactive food components act. The research is expected to identify mechanisms where food components alter biological processes such as proliferation, apoptosis, cell cycles, intracellular cell signaling, inflammation, metastasis, and post-transcriptional message regulation from both cell culture and whole animal studies. Genes involved in pathways mediated by the sex steroid hormones estrogen and androgen, orphan receptors, and cytokine-mediated pathways will be characterized. The conditions that modulate these pathways will include the use of plants with different phytochemical composition to delineate the role of specific compounds along with related food matrix effects. For Objective 2, a juvenile porcine animal model as a surrogate model for humans will be used to validate the effect of selected prebiotics and probiotics on the modulation of the host immune and metabolic responses to an obesogenic diet. The research will use a whole nutrigenomic approach where transcriptomics, metabolomic and metagenomic changes are integrated to identify biomarkers associated with health and disease that can be used as targets for nutritional interventions. Data generated from these studies is expected to reveal mechanisms of action of prebiotic and probiotic products added to the diet.

Progress Report
This is the final report for 8040-51530-056-00D entitled “Regulatory Mechanisms Induced by Health-Promoting Bioactive Food Components on Sex Steroid Hormone-Dependent Pathways, Cancer Cell-Stromal Cell Interactions, and the Gut Microbiome.” Objective 1 focused on the in-vitro and in-vivo effects of glyceollins, indoles, and isothiocyanates on prostate cancer development. Phenethyl isothiocyanate (PEITC), a candidate anticancer compound found in certain cruciferous vegetables, was shown to inhibit androgen-responsive LNCaP human prostate cancer cell tumor growth in our xenograft model. PEITC significantly inhibited tumor platelet/ endothelial cell adhesion molecule (PECAM-1/CD31) expression, a marker of angiogenesis. PEITC significantly attenuated the LNCaP cell plating efficiency correlating with the inhibition of integrin family proteins (ß1, a2, a6) mRNA expression. Using RNAseq, PEITC were found to induce molecular changes that may contribute to tumor growth suppression to include differentially regulated genes in inflammation and extracellular matrix pathways such as an insulin-like growth factor binding protein 3, fibronectin, thyroxine degradation enzyme, and integrin beta 6. The data suggests that PEITC may be a dietary factor that inhibits androgen-responsive prostate tumor growth indirectly by selectively targeting factors involved in the tumor microenvironment. For the indoles, we focused on cruciferous vegetable-derived indole-3-carbiol (I3C) and its dimer 3’,3’-diindolylmethane (DIM). Several novel effects with the immune system as a target were found: 1) I3C and DIM affected immune cells such as macrophages and T-cells through the activation of the arylhydrocarbon and CD84-mediated pathways, supporting a role for I3C in the modulation of the host immune system. 2) We observed that when I3C was administered at concentrations equal to its consumption as a dietary supplement, intestinal damage occurred in the animal model, providing cautionary information regarding the use of I3C as a dietary supplement. 3) We demonstrated that I3C exerts specific effects on the gut microbiome and is associated with the inhibition of prostate cancer tumor growth, providing a novel mechanism through which I3C may protect against prostate cancer. For the soy-derived glyceollin, we found glyceollins to be active in inhibiting the androgen-dependent pathway in-vitro. However, using the prostate cancer xenograft model, we found little effects of glyceollins on the androgen-dependent pathway and tumor growth. This lack of effect by glyceollins in vivo appeared to be due to the low bioavailability of glyceollins; circulating levels of glyceollins were 10-fold less than the concentration used to induce the in-vitro effects. Hence, the bioavailability of a food bioactive may play a critical role in their biological efficacies such as cancer prevention. Overall, the research from Objective 1 provided molecular and mechanistic information related to cancer prevention by diet-derived compounds. The work led to more than 25 peer-reviewed publications associated with the project. Objective 2 focused on elucidating the effect of dietary patterns supplemented with prebiotics and probiotics and how their symbiotic relationship affects the immune response, metabolomic profile and microbiome composition and function in the pig as a translational human model. We showed that Cocoa powder (CP), a rich dietary source of polyphenols, given with Lactobacillus rhamnossus LGG probiotic to healthy young pigs at a comparable dose as the one used in humans increased bacterial taxa diversity and changed the bacterial inferred metabolic profile. There was an enrichment of host endogenous Bifidobacteriaceae, Lactobacillaceae and Lachnospiraceae bacterial taxa including saccharolytic butyrate producing Roseburia genus, demonstrating the prebiotic effect of CP. We also demonstrated a modulation of colonic microbial metabolites in the biofluid and host tissues of CP treated pigs. O-methyl epicathecin glucuronide conjugates were consistently found in serum, urine and visceral adipose tissue with the presence of 5-(hydroxyphenyl)-y-valerolactone-sulfate in the brain cortex of pigs at a higher dose of CP. Furthermore, an anti-inflammatory gene expression response was detected in the intestinal mucosa and brain. A follow-up whole transcriptome analysis of peripheral blood and microbiome composition in pigs fed CP and probiotic bacteria Lactobacillus rhamnosus (LGG) after a challenge with infective A. suum parasite eggs, a well-established model of allergeric responses in humans were performed. Feeding CP to pigs activated pro-inflammatory and metabolic pathways that were reduced in pigs fed LGG, both before and after infection with A. suum. Specific anti-A. suum IgG2 antibodies were decreased in LGG + CP-fed pigs compared to pigs fed CP alone, while pigs fed LGG had a significantly reduced expression of Eosinophil peroxidase (EPX), Interleukin 13 (IL-13), Eotaxin 3 (CCL26), Toll-like receptor 2 (TLR2), TLR4, and TLR9 and Interleukin-1Beta (IL1B) in the tracheal-bronchial lymph node (TBLN) independent of CP treatment. This suggests that probiotic intervention may reduce the localized prototypical Th2-related markers of infection with A. suum in the TBLN. Although feeding CP does not appear to affect the A. suum-induced Th2-associated cytokine response, feeding LGG + CP reduced anti-A. suum antibodies. Using human food-grade Bifidobacterium lactis as an early colonizer in the pig intestine, we showed a mild modulation of the microbiome composition from an obese microbial phenotype (increased Firmicutes, Lactobacilli and Proteobacteria) to a profile with reduced Firmicutes and increased Bacteroidetes including genus from the Prevotellaceae family when pigs were fed a high-fat diet. An enhanced metabolic function diversity was seen in HF-fed pigs with probiotic relative to a non-probiotic treated group, suggesting a modulatory role of Bifidobacterium lactis when animals consumed a high fat diet. Hence, dietary probiotics constitute a purposeful attempt to modify the relation in the microbial environment in ways that may benefit the host's health. 8040-51530-056-08H: Effect of Lowering Cholesterol on Markers of Cardiovascular Disease in Pigs Fed Atherogenic and Heart-Healthy Diets (TUFTS UNIVERSITY). Final report: The impact on the host microbiome composition of two isocaloric dietary patterns with 38% energy from fat designed to represent a western diet (WD) rich in saturated fatty acids, refined carbohydrates, low in fiber and high in cholesterol versus a heart healthy diet (HHD) rich in unsaturated fatty acids, unrefined carbohydrates, fruits/vegetables and fiber with low cholesterol content were compared. Half of the pigs within each diet were also provided statin (Atorvastatin -Lipitor) using a dose comparable to the one used to treat human dyslipidemia. The diversity of the fecal microbiome at a genus level was transiently affected with a reduced Shannon Diversity index one month after WD or HHD dietary intervention with no significant change in diversity at other taxonomic levels or after 6 months of dietary intervention. Fecal bacterial communities derived from pigs fed the WD and HHD clustered and separated by diet type independent of gender, but affected by statin in the HHD dietary group only. Significant alterations in taxa associated with WD and HHD groups were detected as early as one month. Association analysis suggest a diet-dependent microbial mechanism might be involved in the early development of atherosclerosis as members from Lachnospiraceae family were highly correlated with early host dyslipidemia and with inflammatory response and presence of atheromatous lesions predominantly in the left anterior descending proximal (LAD) and LAD/Left circumflex (LCX) bifurcation six months post-intervention. These data document for the first time that Ossabaw pigs with a diet-induced dyslipidemia and early stage atherosclerosis have a distinctive bacterial profile. Taken together these results represent a new model to examine mechanistic pathways of dietary patterns and/or drug interactions and its effect on modulating microbiome in developing atherosclerosis. 8040-51530-056-16: Prebiotic Effect of Dietary Agaricus Bisporus Mushroom on Intestinal Microbiota Composition and Host Immunological Function (MUSHROOM COUNCIL UNITED STATES). Final report: A study was designed to determine the potential prebiotic effect of dietary mushrooms on the host immune response, and intestinal microbiota composition and function. Six-week-old pigs were fed a pig grower diet alone or supplemented with either three or six servings of freeze-dried white button (WB)-mushrooms for six weeks. Host immune response was evaluated in peripheral blood mononuclear cells (PBMC), and alveolar macrophages (AM) after stimulation with Salmonella typhymurium-Lipopolysaccharide (LPS). Isolated DNA from fecal and proximal colon contents were used for 16S rDNA taxonomic analysis and linear discriminant analysis effect size (LEfSe) to determine bacterial abundance and metabolic function. Pigs gained weight with no difference in body composition or intestinal permeability. Feeding mushrooms reduced LPS-induced IL-1ß gene expression in AM with no change in LPS-stimulated PBMC or the intestinal mucosa transcriptome. LEfSe indicated increases in Lachnospiraceae, Ruminococcaceae within the order Clostridiales with a shift in bacterial carbohydrate metabolism and biosynthesis of secondary metabolites in the mushroom-fed pigs. These results suggested that feeding WB mushrooms significantly reduced the LPS-induced inflammatory response in AM and positively modulated the host microbiota metabolism by increasing the abundance of Clostridiales taxa that are associated with improved intestinal health.

1. Inhibition of tumor growth by dietary compound may be associated with gut microbial interactions. Little information is available in the literature regarding the role of gut microbiome, interaction of the gut microbiome with diet-derived compounds on prevention of prostate cancer. ARS scientists at Beltsville, Maryland, in collaboration with University of Maryland and Sichuan University scientists, utilized a metagenomic approach to study the effects of a cruciferous-derived compound on the gut microbiome and its association with tumor growth in a rodent model. The study identified specific changes in the gut microbiome, i.e. the abundance of the phylum Deferribacteres, that is associated with protection against prostate tumor growth.

2. Extensive degradation and low bioavailability of orally consumed corn miRNAs. The literature regarding bioavailable and physiological effects of food-derived miRNA remain unclear. ARS scientists at Beltsville, Maryland, in collaboration with University of Maryland scientists and scientists at the NIH, utilized corn as model food to examine the fate of miRNA during digestion in a rodent model. It was demonstrated that corn miRNA is extensively degraded during the digestion process and little, if any, enters the host tissues. The study indicated that miRNA from food may not be biologically active due to their low bioavailability.

3. Maternal probiotics supplement influence a newborn’s gut microbiome. Maternal probiotics supplement influence a newborn’s gut microbiome. The impact of maternal supplementation with probiotics on the newborn remains largely unknown. ARS scientists at Beltsville, Maryland, addressed this problem using a swine model. Early dietary supplementation with probiotic Bifidobacterium lactis (Bb12) of pregnant sows and their piglets after birth induced a differential microbiome profile with increased bacterial taxa diversity. These changes may provide a metabolic advantage for the host. This study showed a potential modulation of the newborn gut microbiome through maternal feeding of probiotics.

Review Publications
Wu, Y., Wan, J., Choe, U., Pham, Q., Schoene, N., He, Q., Li, B., Yu, L., Wang, T.T. 2019. Interactions between food and gut microbiota: Impact on human health. Annual Review of Food Science & Technology. 10:389-408.
Choe, U., Yu, L., Wang, T.T. 2019. Selected lipid-based transfection reagents activate NF-kB and MAP kinases signaling pathways, induced cytokines mRNA expression in human THP-1 macrophage. Analytical Biochemistry: Methods in the Biological Sciences. 573:73-76.
Wu, Y., Li, R.W., Huang, H., Fletcher, A., Yu, L., Pham, Q., Yu, L., He, Q., Wang, T.T. 2019. Inhibition of tumor growth by indole-3-carbinol in a prostate cancer xenograft model may be associated with disrupted gut microbial interactions. Nutrients. 11(2). pii.E467.
Choe, U., Yu, L., Wang, T.T.Y. 2018. The science behind microgreens as an exciting new food for the 21st century. Journal of Agricultural and Food Chemistry. 7:66(44).
Huang, G., Xue, J., Sun, X., Wang, J., Wang, Y., Wang, T.T., Yu, L. 2018. Suppression of T lymphocyte activation by 3-chloro-1,2-propanediol mono- and di-palmitate esters in vitro. Toxicology In Vitro. 51:54-62.
Wang, T.T., Pham, Q., Kim, Y.S. 2018. Elucidating the role of CD84 and AHR in modulation of LPS-induced cytokines production by cruciferous vegetable-derived compounds indole-3-carbinol and 3,3'-diindolylmethane. International Journal of Molecular Sciences. 19(2):pii:E339.
Liu, Jie, Li, Y., Yang, P., Wan, J., Chang, Q., Wang, T.T., Lu, W., Zhang, Y., Wang, Q., Yu, L. 2017. Gypenosides reduced the risk of overweight and insulin resistance in C57BL/6J mice through modulating adipose thermogenesis and gut microbiota. Journal of Agricultural and Food Chemistry. 65(42):9237-9246.
Gao, B., Yu, L., Liu, J., Wang, T.T., Luo, Y., Yu, L., Zhang, H., Gong, L. 2017. Home-based preparation approaches altered the availability of health beneficial components from carrot and blueberry. Journal of Food Science and Nutrition. (42):9237-9246.
Choe, U., Li, Y., Gao, B., Yu, L., Wang, T.T., Sun, J., Chen, P., Liu, J., Yu, L. 2018. Chemical compositions of cold-pressed broccoli, carrot and cucumber seed flours, and their in vitro gut microbiota modulatory, anti-inflammatory and free radical scavenging properties. Journal of Agricultural and Food Chemistry. 66(35):9309-0317.
Solano Aguilar, G., Jang, S., Lakshman, S., Gupta, R., Beshah, E., Sikaroodi, M., Vinyard, B.T., Molokin, A., Gillevet, P., Urban Jr, J.F. 2018. The effect of dietary Agaricus bisporus mushroom on intestinal microbiota composition and host immunological function. Nutrients. 10(11):1721.
Walker, M., Matthan, N., Solano Aguilar, G., Jang, S., Lakshman, S., Molokin, A., Faits, T., Urban Jr, J.F., Johnson, W.E., Lamon-Fava, S., Lichtenstein, A.H. 2019. A Western dietary pattern and atorvastatin induce epicardial adipose tissue interferon signaling in the Ossabaw pig. Journal of Nutritional Biochemistry.
Walker, M., Matthan, N., Goldbaum, A., Meng, H., Lamon-Fava, S., Lakshman, S., Jang, S., Molokin, A., Solano Aguilar, G., Urban Jr, J.F., Lichtenstein, A.H. 2019. Dietary patterns influence epicardial adipose tissue fatty acid composition and inflammatory gene expression in the Ossabaw pig. Journal of Nutritional Biochemistry. 70:138-146.
Walker, M., Matthan, N., Lamon-Fava, S., Solano-Aguilar, G., Jang, S., Lakshman, S., Molokin, A., Urban Jr, J.F., Faits, T., Johnson, W.E., Lichtenstein, A.H. 2019. A Western-type dietary pattern induces an atherogenic gene expression profile in the coronary arteries of the Ossabaw pig. Current Developments in Nutrition. 3(5).