Source: PENNSYLVANIA STATE UNIVERSITY submitted to NRP
IDENTIFICATION OF DIETARY PHYTOCHEMICALS TO MODULATE CHRONIC GI DISEASES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
Reporting Frequency
Annual
Accession No.
1029756
Grant No.
2023-67017-39058
Cumulative Award Amt.
$638,000.00
Proposal No.
2022-09408
Multistate No.
(N/A)
Project Start Date
Apr 1, 2023
Project End Date
Mar 31, 2027
Grant Year
2023
Program Code
[A1343]- Food and Human Health
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%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7021470101025%
7021452101025%
7021129101025%
7021461101025%
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.

Progress 04/01/24 to 03/31/25

Outputs
Target Audience:Through our research efforts, presentations, and publications, we have engaged our target audience of researchers and clinicians in the ralms of nutrition, food, and gut health to provide ongoing findings of the study. Changes/Problems:This past reporting period, we encountered challenges pertaining to the initialization of our in vivo studies. The preliminary feeding study was promising but not significant, which prompted a re-evaluation of the food delivery parameters, timing of food availability for the mice, as well as timing of the euthanasia and tissue collection. This necessitated long planning discussions with the Co-PIs and their personnel and a greater involvement of our laboratory technician, which set us back 5-6 months where no in vivo work was being undertaken. We wanted to ensure we were not pursuing animal experiments that could not provide robust, reproducible data. We completed (late fall 2024/early winter 2024) mouse studies with controls that confirmed our protocol was correct, and we're primed to move forward with the rest of the feeding studies and subsequent animal studies. Secondly, the challenge around the collection of ligand data for Objective 1.2 was using a narrow lead set of compounds proved to be insufficient to appropriately model the data. We're working with collaborators external to the grant to obtain larger datasets, which will provide more varied structural information, and will assist in more comprehensive modeling of the structure-activity relationships. We anticipate having this data later in the next reporting period and will then be able to execute the remainder of Obj. 1.2. What opportunities for training and professional development has the project provided?The project in the past reporting period has provided training for a graduate student throughout the year, along with an undergraduate student researcher, as well as development opportunities for our lab technician and myself. This has included in vivo training on the planning, handling, housing, feeding, and euthanizing of mice, as well as necropsy tissue harvesting for analysis. Additional training for the undergraduate and graduate student has taken place for metabolomics and biochemometrics data analysis, in accordance with the activities for Objective 1.1. These trainings will translate into greater research producitivity, opportunities for (future) employment, and career advancement, and will further the objectives of this particular project. How have the results been disseminated to communities of interest?This last reporting period, the results have been disseminated to interested communities by way of presentations at local, regional, and national meetings. Presentations by the gradaute student in the project included a poster at the 6th International Aryl Hydrocarbon Receptor Meeting in Dusseldorf, Germany, two oral presentations at the Veterinary and Biomedical Science Departmental Graduate Data Club, and a presentation at the Huck Life Sciences Symposium (the latter two at the Pennsylvania State University). What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, we will continue to focus on all three objectives. The structural identification, bioactivity confirmation, and final analysis of AHR activition of mushroom compounds will be completed (Objective 1.1), and these potential ligands will be assessed as to their activity as direct AHR ligands (Objective 3.1). Greater ligand diversity will be sought to broaden the data available for the chemometric analysis of ligand structure-activity relationships (Objective 1.2). The feeding study (Objective 2.1) will be repeated for the two active mushroom species, and the extracts and/or their active compounds will be initiated into the in vivo IBD model (Objective 3.2). We will also embark on the investigation on the role of the gut's microbial metabolism of these dietary compounds (Objective 2.2) in in vitro and in vivo models.

Impacts
What was accomplished under these goals? For the past reporting period: For Objective 1.1, 3 culinary and medicinalfungal species (reishi(Ganoderma lucidum), oyster (Pleurotus ostreatus), and beech (Hypsizygus tessulatus)) demonstrated significant effects on AHR activity in vitro. Reishi and oyster mushroom extracts activated the AHR (and associated downstream enzyme expression), while beech mushroom extracts antagonized AHR activity. Reishi and oyster mushrooms were fractionated on a normal phase system, with relevant fractions assayed in the luciferase in vitro model, and were simultaneously profiled on LC-MS to provide untargeted metabolomics data. The two datasets were integrated to provide biochemometric modeling of the active constitutents, and putatitve active molecules were identified via computational approaches.Isolation efforts are underway for those molecules which are not commercially available. For Objective 1.2, chemical descriptors of known AHR ligands were obtained, and preliminary modeling suggested a broader scope of information is needed to provide statistical rigor to the computational modeling approaches. Thus, additional ways of obtaining the data needed in a high-throughput manner are being actively pursued. For Objective 2.1, in vivo assessment for each botanical and fungal food (pepper, mushroom (reishi and oyster), carrots, and corn) was undertaken in a mouse model. Training was completed for PD, grad students, technician, and undergraduates on safe handling, housing, feeding, euthanasia, and processing of mice and mouse tissue. Preliminary data suggested mushrooms reishi and oyster were most effective in elevating AHR activity in vivo. Optimization of the feeding study and timing of tissue collection was performed, with a survey of appropriate positive controls (e.g., broccoli cultivars) to ensure reproducibility of the data. The data is being repeated this winter, with plans to expand the in vivo studies. For Objective 3.2 (misentered as 3.1 part 2 above), undertaking training by graduate student to prepare for the in vivo mose model with identified AHR ligands.

Publications


    Progress 04/01/23 to 03/31/24

    Outputs
    Target Audience:Through our research efforts and our presentations, we have engaged our target audience of researchers and clinicians in the realms of nutrition, food, and gut health to provide some preliminary findings of the study. Changes/Problems:This past year, it was more difficult than anticipated to obtain relevant quantities of botanical foods to test in the in vitro system. There was also a long-running contamination by another lab in our cell culture facility that necessitated the removal of all samples, complete decontamination, and then restarting the in vitro work. This was a challenge, and certainly was a set back by about 2 months this year where no in vitro work could progress. The system is back up and running, and we anticipate no further problems in accomplishing our objectives. What opportunities for training and professional development has the project provided?This project has provided training for one graduate student this year, along with an undergraduate student researcher, as well as additional training and development opportunities for our technician and myself. The undergrad has learned a great deal about natural product extraction and testing, as well as in vitro cell culture techniques. All of us have gained knowledge about animal handling procedures, safety when conducting animal studies, and are close to learning how to work with animals for the in vivo portions of the project (Objectives 2 and 3). The graduate student also learned about processing and interpreting mass spectrometry data for the project, which will translate into greater knowledge as the project progresses. How have the results been disseminated to communities of interest?This year the results have been disseminated to interested communities by way of presentations at local, regional, and national meetings. Presentations by the gradaute student in the project include a poster at the American Society of Nutrition meeting, and oral presentations at the American Council for Medicinally Active Plants conference, and the Huck Life Sciences Symposium (at the Pennsylvania State University). What do you plan to do during the next reporting period to accomplish the goals?For the next year, we will continue to focus on Objectives 1 and 2. Bioactive identification from peppers, berries, and mushrooms will continue through the application of biochemometric analysis of fractions (Obj 1.1), and the molecular descriptors of known ligands will be analyzed to determine commonalities in functionality (Obj 1.2). The next reporting period will also see the first in vivo studies taking place, first a feeding study (Obj 2.1) with the most promising dietary leads, and also an investigation into the role of the microbiome in AHR activation (Obj 2.2).

    Impacts
    What was accomplished under these goals? For Objective 1.1, studies have been taken to characterize bioactive chemistry from main dietary items. Peppers, mushrooms, carrots, berries, and onions were screened for AHR activity; active cultivars have been followed up with fractionation, and repeat testing is underway. Fractions have also been profiled via mass spectrometry to provide the chemoinformatic data for the biochemometric analysis of the fractions, to better identify putative bioactives once the screening is complete. For Objective 1.2, code was written in R to start to pull down physico-chemical descriptors of AHR ligands. We have begun creating a databse of known ligands, their activity, binding affinity with AHR, and these chemical descriptors to better model the data and elucidate which molecular characteristics best align with activity. For objective 2.1, training in in vivo experimentation, animal handling, and protocol development is underway to begin the in vivo trials this year.

    Publications