Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
(N/A)
Non Technical Summary
Inflammatory bowel diseases (IBD) are an increasingly important global health problem; over 850,000 people in the United States are effected annually, and the worldwide incidence of IBD has increased since 1990. IBD are characterized by chronic inflammation and ulceration of the colon, abdominal pain, weight loss, bloody diarrhea, and an increased risk of a number of other diseases, including colorectal cancer and type 2 diabetes. While dietary, drug, and biological therapies have been useful in the management of IBD, the only 'curative' treatment currently available is surgical resectioning of the colon. In addition to not being truly curative, surgical options have significant side-effects, including increased risk of infection. Chronic inflammation has been shown to underlie IBD, as well as other chronic diseases, including liver disease and diabetes. And the aryl hydrocarbon receptor (AHR) is a crucial regulator in the gastrointestinal tract, promoting balance in the gut while lowering inflammation and protecting against numerous diseases.Botanicals contain hundreds of different phytochemicals, and have been shown to have beneficial health effects. And consumption of these foods (fruits, vegetables, nuts, fungi, etc.) has been linked to improved gastrointestinal health and lower inflammation. A diverse array of foods, including broccoli, corn, mushrooms, carrots, berries, etc., have demonstrated activity in activating the AHR in preliminary studies. However, the chemical agents of these foods that are responsible for this activity remain unknown. Using our innovative chemical profiling and data modeling approaches, we will be able to tease apart the differing chemical signatures and identify which molecules are able to activate the AHR. We will be investigating interactions at the interface of food, nutrition, the gut microbiome, and their downstream effects on chronic human disease. Using both cellular and mouse models, we will be able to ascertain their effects on the AHR as well as subsequent effects on the gastrointestinal system, including their ability to mitigate inflammatory bowel diseases.This project will provide a greater understanding of the mechanisms that underlie dietary protection against inflammatory gastrointestinal diseases. It will lead to a greater understanding of the botanical phytochemicals and their AHR activity, and the data can be used to guide dietary recommendations that modulate AHR activity to improve human gastrointestinal health and combat chronic GI diseases. Moreover, these endeavors will allow us to provide predictive models and to enhance the ability to discriminate between species or cultivars and even predict the cultivar and bioactivity of unknown botanical product. These data will also benefit botanical users (supplement manufacturers and consumers) by providing a more nuanced understanding of the relationship between phytochemistry and bioactivity of vegetable crops and botanicals. While the present study is focused on a limited selection of model crops, the innovative approaches are applicable to any nutritional or medicinal plant.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
0%
Goals / Objectives
This project is focused on the examination of diverse, phytochemical-rich foods (mushrooms, peppers, carrots, and berries) to discover their effects on modulating AHR activation, as well as elucidating the chemical agents responsible for this activity. Furthermore, we will ascretain the phytochemicals'interaction with the gut microbiome, and probe their downstream effects on gastrointestinal inflammation and disease. We will achieve this via the following objectives:Objective 1:To efficiently identify AHR-active ligands from complex dietary matrices (e.g., peppers, mushrooms, carrots, berries) and analyze structural features of active compounds that are effective in modulating AHR activity.1.1.Identify putative AHR ligands from botanical foods for in vitro AHR activation.1.2. Use chemo-informatics to identify characteristics of AHR active molecules that optimize functionality.Objective 2:To categorize AHR ligand metabolism by gut microbiome and their in vivo effect on gut homeostasis and cell development.2.1.In vivo assessment of AHR activation of each botanical food.2.2.Comparative metabolomics analysis to identify microbiome-generated metabolites and determine interactions with the gut microbiome.Objective 3:To assess protective mechanisms of dietary AHR ligands against intestinal injury and inflammation in an inflammatory bowel syndrome (IBS) model.3.1.Identification and binding activity of microbial AHR-active compoundsusing a photoaffinity ligand binding assay.3.1.Examine effect of AHR ligands on modulating chemically induced intestinal injury in an in vivo mouse model.
Project Methods
Objective 1 Efforts:AHR activation by food phytochemicals using an in vitro luciferase assay.To assess potential AHR agonist or antagonist activity, samples will be analyzed via a sensitive cell-based luciferase gene reporter assay to obtain quantitative AHR activation data. Using HepG2 40/6 and Hepa 1.1 reporter cells, we will obtain quantitative determinations of the AHR activity of samples. Furthermore, this assay is agnostic, and thus will be used to evaluate which samples agonize or antagonize AHR activity.Biochemometric modeling for determination of active constituents from foods.Samples will be profiled via an untargeted metabolomics approach to provide accurate mass (MS1) and fragmentation (MS2 and MS3) profiles. A de novo biochemometric approach will integrate the metabolomic datasets and bioactivity data. This will allow the determination of which metabolite signatures most correlate and co-vary with the biological data via a supervised multivariate statistical approach, partial least squares regression (PLS). Putative AHR ligands will be identified after being highlighted as significant from a consensus of biomarker identification approaches.Understanding structural characteristics of AHR active molecules to optimize functionality.The structure of known AHR ligands will be encoded with ChemDes, a molecular descriptor, compiling ca. 3,600 2D and 3D molecular parameters. We will build quantitative structure-activity relationship (QSAR) models using three machine learning methods, which will establish associative models between the structural characteristics of putative ligands and their experimental binding properties. The models will be utilized to identify structural analogues to the identified compounds with potential AHR activity.Objective 1 evaluation.AHR active structures will be determined by assessment in our innovative biochemometric modeling approach with multiple biomarker identification metrics, which increases the likelihood of strong correlations being discovered, and lowers the false discovery rateof the analysis. All putative AHR active structures identified through the biochemometric methods will be verified using the in vitro cell assay system.Objective 2 efforts:Analysis of the effect of the microbiome on AHR activity via in vivo studies.A semi-purified diet will be fed to 10-week-old C57BL6/J mice for two weeks followed by groups of 6 female/6 male mice being fed each diet for one week. Food intake, water consumption and the weight of mice will be examined over the week. Feces will be collected before and after start of each diet. Mice will be sacrificed, and tissues, cecal contents, and feces collected. The ability of each food to modulate AHR activity in the host will be assessed through the isolation of the duodenum, jejunum, ileum, cecum, proximal and distal colon, and liver. Each GI segment will have enterocytes and gene expression of AHR target genes (e.g., Cyp1a1, Cyp1b1, Ahrr) will be determined using qRT-PCR. To understand whether the activity seen is derived from the food itself or whether AHR structures are generated via microbiota metabolism in the gut, 6 male/6 female conventional and germ-free C57BL6/J mice will be fed irradiated semi-purified diet for one week followed by diets containing foods for one week. Liver and scrapes from the segments of the GI tract will be obtained and analyzed.Hydrophobic and hydrophilic components will be extracted from cecal and fecal will be analyzed by metabolomic analyses. The two groupings analyzed via unsupervised multivariate analysis to identify whether there is a discernable shift in the metabolome due to microbially-produced metabolites.Metabolomics analysis to identify microbiome-generated metabolites.Botanical foods that result in decreased AHR activity in germ-free mice (relative to conventional mice) will be selected for identification of microbial metabolites. Mouse cecal contents and feces will be first extracted and fractionated via flash chromatography if necessary. AHR activity will be assessed for each fraction using the cell-based luciferase assay and profiled for metabolomic analysis. This model will be used to identify potential AHR active ligands from complex mixtures through molecular network analysis using the GNPS framework. Identified features will be targeted for isolation. We will first broadly classify the phytochemical metabolites and then annotate the structures based on interpretation of the MSn data. For unknown molecules, isolation and structural elucidation will be undertaken. Identified AHR ligands will be obtained commercially or synthesized.Objective 2 evaluation.The statistical assessment of the metabolomics from the conventional and germ-free mice willdifferentiate between the two mouse groups, but also provide insight into the additional metabolites which govern the separation. The molecular networking will serve toidentify analogs present in cecal and fecal samples to be able to also identify putative AHR ligands from microbial metabolism.Objective 3 efforts:Assess whether AHR-active compounds bind to the AHR ligand binding pocket.Using the photoaffinity ligand, 2-azido-3[125I]iodo-7,8-dibromodibenzo-p-dioxin, along with cytosol from a transgenic liver specific humanized AHR mouse liver (rich source of human AHR) or wild-type mouse liver, we will assess whether the identified putative AHR ligands bind to the ligand binding pocket.Examine effect of AHR ligands on modulating chemically induced intestinal injury.Identified AHR ligands from the diet and/or from microbial metabolism will be administered as a daily gavage to groups of six wild type C57BL6/J (Ahr +/+) and knockout (Ahr -/-) mice. Each compound will be gavaged at three different concentrations, based on our previous studies. After 7 days, intestinal injury will be induced by intraperitoneal injection of trinitrobenzene sulfonic acid (TNBS). Food intake, water consumption and animal weight will be examined over 4 days, after which the animals will be sacrificed and blood and colon samples harvested. Intestinal samples will be assessed histologically by light microscopy, and we will determine serum levels of cytokines IL-6, IL-17, TNF-α, MCP-1, IFN-γ, and PGE2 using commercially available ELISA kits. Quantitative RT-PCR analysis using cDNA from the mouse tissue specimens will quantify expression levels for murine Cyp1a1, IFN-γ, IL-17A, IL-22, TNF-α, all normalized to β-actin. This experiment will be performed in both males and females.Objecte 3 evaluation.The histological scoring of intestinal sections from the mice will provide evidence to the effects of AHR ligands in chronic inflammation.Statistical analysis of inflammatory cytokines and expression levels will be analyzed by two-way ANOVA with sex and treatment being co-variables.