BLACK RASPBERRIES FOR SUPPRESSION OF COLITIS INDUCED BY HUMAN-TO-MOUSE FECAL MICROBIOME TRANSFER FROM ULCERATIVE COLITIS PATIENTS.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1019994
• Project Start Date: Aug 20, 2019
• Project End Date: Jun 30, 2024
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UTAH STATE UNIVERSITY
(N/A)
LOGAN,UT 84322

Performing Department
Animal Dairy & Veterinary Sciences

Non Technical Summary
An estimated 1.4 million people suffer from inflammatory bowel disease (IBD) in the U.S. [1], including patients diagnosed with ulcerative colitis and Crohn's disease, resulting in an overall health care cost of more than $1.7 billion. IBD is associated with gut microbiome dysbiosys - a scenario in which the composition of the bacteria population has shifted toward taxa with potentially harmful health effects. Dietary interventions may ameliorate these negative effects by shifting the composition of the microbiome in favor of health-promoting bacteria species. We aim to employ dietary supplementation with black raspberries, which have been shown to suppress colon inflammation and inflammation-associated colon tumorigenesis in mice. The goal of this work is to determine whether dietary supplementation with black raspberries shifts the composition of the human microbiome from ulcerative colitis patients, and consequently, alters inflammation and biomarkers of inflammation in colon tissues. To execute this work, we will use fecal microbiome transfer to colonize mice with human bacteria from colitis patients or healthy controls. If our hypothesis is correct, we expect to gain further evidence supporting dietary intervention with black raspberries to suppress colitis episodes and improve recovery in UC patients.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	3840	1010	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
3840 - Laboratory animals;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

ulcerative colitis

inflammatory bowel disease

black raspberry

western diet

inflammation

fecal microbiome transfer

gut microbiome

Goals / Objectives
Objective 1: Determine impact of repeated fecal microbiome transfer (FMT) from healthy or ulcerative colitis (UC) human donors on colitis symptoms, microbiome composition and biomarkers of inflammation in recipient mice fed either a healthy diet (AIN), a Western type diet (TWD) or the TWD with 10% BRB. Objective 2: Determine how mice that received FMT from UC or healthy patients respond to acute colitis stress by using the DSS model of chemically-induced colitis in mice fed either a healthy diet (AIN), a Western type diet (TWD) or the TWD with 10% BRB.TimelineRecruit human donors and sequence microbiomes - year 1 Perform FMT study (in cohorts) - years 2-3 Analyze recipient mouse microbiomes- year 3 Biomarkers of inflammation (immunohistochemistry, histopathology) - years 3-4 Differential gene expression by Fluidigm - years 3-5 Data analysis - years 3 -5 Preparation of manuscripts years 4-5

Project Methods
This study will employ a fecal microbiome transfer from human donors to recipient mice and the dextran-sodium sulfate model acute colitis. Previously, we partnered with Dr. Kathleen Boynten, a gastroenterologist at the University of Utah, who has an approved IRB protocol (at the University of Utah) for collection of fecal biospecimens from ulcerative colitis patients and healthy controls, which have been made available to use for use in human-to-mouse fecal transfer experiments at USU. As we are using only biospecimens from these human subjects, a new IRB approval for USU is not required. We plan to pool human microbiome samples from either healthy or UC patients to avoid confounding due to individual donor variability. Thus, we will first screen human donor material by 16s rRNA sequencing and identify those individuals (minimum 4 for each group) that represent the most divergent microbiome profiles for healthy or colitis subjects as determined by weighted or unweighted unifrac beta diversity analyses. Recipient mice will be fed either a healthy diet (AIN93G) or a Western type diet (TWD) and given either no supplement or 10% black raspberries (by diet weight). Composition of the fecal microbiome for human donors and recipient mice will be determined by standard 16S sequencing. Alpha and beta diversity will be determined and LEfSe will be employed to identify discriminating taxa. Histological and gene biomarkers of colitis will be examined in mouse colon tissues.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:International medical community, scientific community Changes/Problems:The overall project plan has not changed, though the timeline has been adjusted to reflect impacts of covid-19 on research activities the past year. What opportunities for training and professional development has the project provided?Training of undergraduate and graduate personnel in laboratory methods, including nucleic acid extraction, PCR, RNA sequencing, data analysis. Training of undergraduate personnel in literature searching, assessment of scientific papers, and scientific writing. How have the results been disseminated to communities of interest?Nothing to report at this time. What do you plan to do during the next reporting period to accomplish the goals?We have revised our timeline, with the expectation that obtaining fecal samples from human donors can proceed this year with increasing availability of the COVID vaccine and improved protection protocols. For 2021, we plan to obtain human donor fecal samples and sequence microbiomes - year 1 For the remaining project, the revised timeline is as follows: Perform FMT study (in cohorts) - 2022 Analyze recipient mouse microbiomes- 2022 Biomarkers of inflammation (immunohistochemistry, histopathology) - 2023 Differential gene expression by Fluidigm - 2023 Data analysis - years 2021-2023 Preparation of manuscripts years 2023-2024 Preparation of literature review paper (new) 2020-2021

Impacts
What was accomplished under these goals? Our plan of work was modified this year due to the impacts of COVID-19. Our initial plan was to obtain fecal samples from ulcerative colitis patients from a collaborator at the U. of Utah; however, due to restrictions related to COVID-19, we could not obtain these samples during the pandemic. Thus, we retooled our plans to focus on activities that could be performed in the lab and remotely. The first priority was to refine and optimize a new protocol for isolation of quality DNA from poor quality stool samples, typical of patients with ulcerative colitis. We used stool samples from mice that had severe colitis to validate this protocol. The main output from this work is a new, validated protocol that is much improved with respect to recovery of DNA and processing through all of the steps necessary for bar-coding and then 16s rRNA sequencing. The second priority was to continue to engage our undergraduate researchers, even though they had to work remotely for much of the year due to COVID-19 restrictions. Students and laboratory staff assisted the PI in surveying and summarizing relevant primary literature on the topic of bioactives, the gut microbiome and gut health as the first steps in writing a new review paper for the journal Nutrients (planned submission in 2021).

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Benninghoff, A., Hintze, K. J., Monsanto, S., Rodriguez, D. M., Hunter, A., Phatak, S., Pestka, J. J., Van Wettere, A., & Ward, R. E. (2020, February 20). Consumption of the total Western diet promotes colitis and inflammation-associated colorectal cancer in mice. To appear in Nutrients, 12(2), 544

Progress 08/20/19 to 09/30/19

Outputs
Target Audience:Research team and collaborators Changes/Problems:None at this time. What opportunities for training and professional development has the project provided?Staff and student researchers participated in standard laboratory safety training (including use of biological hazardous materials) and the use of laboratory animals in research, as well as professional development training in science communication/presentations and research data management. How have the results been disseminated to communities of interest?Not yet applicable. What do you plan to do during the next reporting period to accomplish the goals?Our plan of work is unchanged. For the next year, we will focus on recruiting human donors and ulcerative colitis patients and performing initial microbiome profiling of donor samples.

Impacts
What was accomplished under these goals? In the first six months of this project, we focused on building our collaboration to identify physicians who could assist in recruiting human subjects. Also, we focused on validating the methods for isolating high quality DNA from fecal samples using new kits and protocols, as the prior kit we employed was discontinued.

Publications