Paper published on “Steamed broccoli sprouts alleviate DSS-induced inflammation and retain gut microbial biogeography in mice”!

The Ishaq and Li labs at UMaine are delighted to announce that our paper on “Steamed broccoli sprouts alleviate DSS-induced inflammation and retain gut microbial biogeography in mice” has been published in mSystems!! The complete author list, Abstract, and Ackowledgements/Funders portions of the paper can be found at the end of this post.

This paper is part of a larger Broccoli project, in which we are evaluating the use of broccoli sprouts in the diet to enlist gut microbes to produce anti-inflammatories. You can read about the whole project here, with links to other resources.

The Premise

Broccoli sprouts are very high in a compound called glucoraphanin. When glucoraphanin comes in contact with the myrosinase enzyme, also found in the sprouts, it is transformed into a compound that acts an an anti-inflammatory in people!

If you eat raw sprouts, this conversion happens when you cut or chew the sprouts, and that anti-inflammatory will get absorbed in your stomach. If you steam or cook the sprouts, you can inactivate the enzyme and leave the glucoraphanin compound alone. Some of your gut microbes are able to use the compound, and produce the anti-inflammatory right in your gut! We are trying to understand how and when this works, so we can use it to reduce symptoms of Inflammatory Bowel Disease.

The Mouse work

In the winter of 2020-2021, we ran a 40-day study with 40 mice housed at UMaine. The mice were divided into 4 groups: “control” which ate the mouse chow, “control+DSS” which ate the mouse chow and had colitis induced by adding DSS (a salt laxative) to their drinking water, “broccoli” which ate the mouse chow with steamed broccoli sprouts mixed in, and “broccoli+DSS” which ate the mouse chow/steamed broccoli sprouts diet and had colitis induced by adding DSS (salt laxative) to their drinking water. This work was led by Johanna Holman, who was a master’s student at the time; Lousia Colicci, who was an undergrad at Husson University at the time and is applying to medical schools now; Dorein Baudewyns, who was an undergrad at Husson University at the time and is completing a graduate program in Psychology at UMaine; and Joe Balkan, who was completing his senior year of high school at the time and has since begin an undergrad degree in Biology at Tufts University where he is preparing for medical school.

The mice were weighed regularly and fecal samples assessed for blood (signs of colitis). At the end of the study, the mice were euthanized so we could study the bacteria in parts of the intestines that we can’t access in humans. We used as few mice as possible, and got as much information from this study as possible, to do as much good as we can with their sacrifice.

The Health Benefits

As we’d hoped, the broccoli+DSS mice that were eating the broccoli sprouts that were given colitis did much better than the control+DSS group who ate mouse chow during their colitis. The broccoli+DSS mice were able to keep gaining weight as they grew, had better consistency of their stool, and had lower amounts of proteins and other metabolities in their blood which indicate inflammation (lower cytokines and lipocalin). Those graphs are shown in the paper.

The Gut Microbes

We found a lot of interesting things with the microbial communities that were living in different parts of the intestines, but the most exciting was that broccoli sprouts in the diet helped microbial communities stay alive in their original gut locations even during colitis! Certain microbes like to live in particular places in our intestines based on where different ingredients in our diet get processed, or the local environment (like how acidic the intestinal neighborhood is), and this is called biogeography.

In the graph below, our control group mice (eating chow) or the broccoli group (eating chow plus sprouts), we see that microbial communites in the small intestines clustered away from the microbial communities in the large intestines.

The DSS salt laxative, and ulcerative colitis, wreak havoc on gut microbes because they cause physical damage to the lining of the intestine, which where many microbes that can be useful to us live on or near. When we induced colitis in mice that were eating mouse chow (control+DSS group), the damage to the intestines caused a loss to some of the microbes living in different places. The remaining microbes that could survive these tough conditions were basically the same ones regardless of where we we looked in the intestines.

But, if mice had colitis and were eating broccoli sprouts (broccoli+DSS), the microbes were able to survive in their original locations and preserved biogeography! This is important because where microbes live in the gut may determine if the beneficial things they make can help resolve IBD symptoms in specific locations in the gut.

Image by Johanna Holman, graph from the paper.

The Spatial Location of GLR-digesting-genes

Bejamin and Timothy Hunt are undergraduates in Biology who have been working on bioinformatics in the Ishaq Lab since December 2022 after completing Sue’s DNA Sequencing Data Analysis Class. They joined the DSS project to provide in-depth analysis on some of the sequences which matched bacteria that are known to convert GLR into the anti-inflammatory SFN, as well as analyze data comparing numbers of genes known to be involved in the process.

A cartoon of the intestines with bacteria of interest in the jejunum, ceculm and colon,
Cropped figure from the paper, made by Benjamin and Timothy.
Benjamin Hunt

The study of the bioproduction of SFN and its mucosal and luminal activity benefited from the biogeographical analysis of this study. It was interesting to note the extreme dominance of a Bacteroides species in the broccoli treatments. B. thetaiotaomicron was indicated based on BLASTN analysis and an evaluation of matching species but was not directly suggested by the dada-Silva taxonomy assignment. The indication of B. thetaiotaomicron suggested analyzing the presence of the operon BT2159-BT2156, which was generally minimally present (<100) but at relatively high counts (>100,000) in some samples. Significantly, the operon was found at locations where no Bacteroides were identified. We continue to reflect on the similarities and differences in the biogeography of bacterial abundance and operon presence highlighted in the different treatments of this study.

Benjamin and Timothy Hunt

The Next Steps

As part of this project, we cultured hundreds of bacteria from the intestines of mice to try and isolate some of the ones that turn glucroraphanin into sulforaphane. We have a large team of students and researchers participating on the culturing work, some of whom are pictured here. We’ll be providing plenty of updates on that project as we continue to process the bacteria this fall!

The Paper

Steamed broccoli sprouts alleviate DSS-induced inflammation and retain gut microbial biogeography in mice.

Johanna M. Holman1, Louisa Colucci2, Dorien Baudewyns3, Joe Balkan4, Timothy Hunt5, Benjamin Hunt5, Marissa Kinney1, Lola Holcomb6, Allesandra Stratigakis7, Grace Chen8, Peter L. Moses9,10, Gary M. Mawe9, Tao Zhang7, Yanyan Li1*, Suzanne L. Ishaq1*

1 School of Food and Agriculture, University of Maine, Orono, Maine, USA 04469 2 Department of Biology, Husson University, Bangor, Maine, USA 04401 3 Department of Psychology, University of Maine, Orono, USA 04469 4 Department of Chemical and Biological Engineering, Tufts University, Medford, Massachusetts, USA 02155 5 Department of Biology, University of Maine, Orono, Maine, USA 04469 6 Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine, USA 04469 7 School of Pharmacy and Pharmaceutical Sciences, SUNY Binghamton University, Johnson City, New York, USA 13790 8Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA 48109 9Departments of Neurological Sciences and of Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont, USA 0540110 Finch Therapeutics, Somerville, Massachusetts, USA 02143

Abstract: Inflammatory Bowel Diseases (IBD) are devastating conditions of the gastrointestinal tract with limited treatments, and dietary intervention may be effective, and affordable, for managing symptoms. Glucosinolate compounds are highly concentrated in broccoli sprouts, especially glucoraphanin, and can be metabolized by certain mammalian gut bacteria into anti-inflammatory isothiocyanates, such as sulforaphane. Gut microbiota exhibit biogeographic patterns, but it is unknown if colitis alters these or whether the location of glucoraphanin-metabolizing bacteria affects anti-inflammatory benefits. We fed specific pathogen free C57BL/6 mice either a control diet or a 10% steamed broccoli sprout diet, and gave a three-cycle regimen of 2.5% dextran sodium sulfate (DSS) in drinking water over a 34-day experiment to simulate chronic, relapsing ulcerative colitis. We monitored body weight, fecal characteristics, lipocalin, serum cytokines, and bacterial communities from the luminal- and mucosa-associated populations in the jejunum, cecum, and colon. Mice fed the broccoli sprout diet with DSS treatment performed better than mice fed the control diet with DSS, including significantly more weight gain, lower Disease Activity Indexes, lower plasma lipocalin and proinflammatory cytokines, and higher bacterial richness in all gut locations. Bacterial communities were assorted by gut location, but were more homogenous across locations in the control diet + DSS mice. Importantly, our results showed that broccoli sprout feeding abrogated the effects of DSS on gut microbiota, as bacterial richness and biogeography were similar between mice receiving broccoli sprouts with and without DSS. Collectively, this supports the protective effect of steamed broccoli sprouts against dysbiosis and colitis induced by DSS.

Importance: Evaluating bacterial communities across different locations in the gut provides a greater insight than fecal samples alone, and provides an additional metric by which to evaluate beneficial host-microbe interactions. Here, we show that 10% steamed broccoli sprouts in the diet protects mice from the negative effects of dextran sodium sulfate induced colitis, that colitis erases biogeographical patterns of bacterial communities in the gut, and that the cecum is not likely to be a significant contributor to colonic bacteria of interest in the DSS mouse model of ulcerative colitis. Mice fed the broccoli sprout diet during colitis performed better than mice fed the control diet while receiving DSS. The identification of accessible dietary components and concentrations that help maintain and correct the gut microbiome may provide universal and equitable approaches to IBD prevention and recovery, and broccoli sprouts represent a promising strategy.

Acknowledgements: All authors have read and approved the final manuscript. The authors thank Jess Majors, University of Maine, for her kind and detailed care of the mice during the trial, and for Ellie Pelletier for her informal review of the manuscript. This project was supported by the USDA National Institute of Food and Agriculture through the Maine Agricultural & Forest Experiment Station: Hatch Project Numbers ME022102 and ME022329 (Ishaq) and ME022303 (Li) which supported Johanna Holman; the USDA-NIFA-AFRI Foundational Program [Li and Chen; USDA/NIFA 2018-67017-27520/2018-67017-36797]; and the National Institute of Health [Li and Ishaq; NIH/NIDDK 1R15DK133826-01] which supported Marissa Kinney, Timothy Hunt, and Benjamin Hunt. Lola Holcomb was supported by US National Science Foundation One Health and the Environment (OG&E): Convergence of Social and Biological Sciences NRT program grant DGE-1922560, and through the UMaine Graduate School of Biomedical Sciences and Engineering. 

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