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. 

Johanna’s review published on how gut microbes can make anti-inflammatory compounds when you eat broccoli

A massive literature review led by Johanna Holman, and featuring our collaborative team of broccoli sprout and microbes researchers, was accepted for publication!

As part of her master’s of science thesis, Johanna Holman reviewed hundreds of journal articles on anti-inflammatory, health-promoting dietary compounds in broccoli and other vegetables or fruits, and how microbes in the digestive tract can transform inactive precursors from foods into those beneficial compounds. This is part of a broader research collaboration on how glucoraphanin in broccoli sprouts can be made into sulforaphane, which acts as an anti-inflammatory in humans. Humans are unable to convert glucoraphanin to sulforaphane, and a small amount of this occurs naturally thanks to enzymes in the broccoli sprouts. But, certain gut microbes can make the conversion and this has helped resolve colitis and other symptoms in mice in laboratory trials (manuscripts in preparation).

A diagram with two panels, and a cartoon mouse in the middle.  The cartoon mouse is eating broccoli, and a cartoon of the digestive tract is overlaid on the mouse's abdomen. Lines emanating from the broccoli point to the left panel, and show the compound glucoraphanin being converted into sulforaphane by the myrosinase enzyme. Lines emanating from the colon of the mouse point to the panel on the right, showing the same biochemical conversion by gut microbes.
Artwork by Johanna Holman.

If you aren’t familiar with broccoli sprouts, a lovely review on their history, current food culture, and safe production was just published by some of our colleagues: Sprout microbial safety: A reappraisal after a quarter-century.

Check out the review

Holman, J., Hurd, M., Moses, P.,  Mawe, G.,  Zhang, T., Ishaq, S.L., Li, Y. 2022. Interplay of Broccoli/Broccoli Sprout Bioactives with Gut Microbiota in Reducing Inflammation in Inflammatory Bowel Diseases. Journal of Nutritional Biochemistry, in press.

Abstract

Inflammatory Bowel Diseases (IBD) are chronic, reoccurring, and debilitating conditions characterized by inflammation in the gastrointestinal tract, some of which can lead to more systemic complications and can include autoimmune dysfunction, a change in the taxonomic and functional structure of microbial communities in the gut, and complicated burdens in a person’s daily life. Like many diseases based in chronic inflammation, research on IBD has pointed towards a multifactorial origin involving factors of the host’s lifestyle, immune system, associated microbial communities, and environmental conditions. Treatment currently exists only as palliative care, and seeks to disrupt the feedback loop of symptoms by reducing inflammation and allowing as much of a return to homeostasis as possible. Various anti-inflammatory options have been explored, and this review focuses on the use of diet as an alternative means of improving gut health. Specifically, we highlight the connection between the role of sulforaphane from cruciferous vegetables in regulating inflammation and in modifying microbial communities, and to break down the role they play in IBD.

Johanna defends her master’s thesis defense!

Johanna Holman passed her Master’s of Science these defense (we knew she would succeed)!! Johanna has worked incredibly hard over the last two years to broaden her research skills and conduct several experiments, and her defense presentation was a wonderful way to see that progression all at once. She has also earned the designation of “first Ishaq Lab grad student to defend”. The defense was attended by her thesis committee, students in the Ishaq lab, collaborators on this project, and friends and family (who brought her a flower and broccoli bouquet that can be seen in the picture below). She will officially pass after a few revisions to her thesis and a formal acceptance by the committee members, which is standard for graduate defenses.

Johanna Holman standing at a wooden podium with a white board and a projection screen behind her. Johanna is presenting her master's thesis, and the title slide of the presentation is showing on the screen.
Johanna Holman beginning her master’s defense presentation.

Johanna has been accepted to the Nutrition PhD program at UMaine, and will continue working with Dr. Li and I, as well as the full research team. Based on those preliminary results, Johanna’s doctoral work will focus on developing that new mouse model, synthesizing information from both models, and using those results to develop diet intervention trials in human patients. After her PhD, Johanna intends to conduct research at an institution here in Maine, and to continue her work connecting the biochemistry of nutrition with gut microbiology and human health.

Prevention of Inflammatory Bowel Diseases by Broccoli Sourced and Microbially Produced Bioactives.

  • Johanna Holman at her ASM Microbe 2022 poster
  • Person in a research facility holding up their arm with a mouse on it. Person is wearing a hairnet, nitrile gloves, surgical mask, and a surgical gown. They are holding their left arm up to the camera to show off a mouse with dark brown fur sitting on their arm. In the background is a metal shelf with containers of research materials.

Johanna sets a date for her master’s thesis defense!

Prevention of Inflammatory Bowel Diseases by Broccoli Sourced and Microbially Produced Bioactives.

Presented by Johanna Holman in fulfillment of her Master’s of Science in Nutrition degree at the University of Maine. Jul 25, 2022 09:30 – 10:30 AM Eastern Time (US and Canada)

Register in advance to attend this presentation over Zoom, which will also be held in person in 206 Rogers Hall at the University of Maine (no RSVP required). After registering, you will receive a confirmation email containing information about joining the meeting.

About Johanna

I met Johanna in the fall of 2019, when I was just establishing myself as a new Assistant Professor at UMaine and she was looking for an advisor for a graduate degree.  Right away, she impressed me with her background and enthusiasm for research.  I learned that Johanna began her undergraduate study as an art student before transitioning fluidly to science.  The ability to design visual aids and graphical representations of data is hugely important to science and sadly, not always a skill that scientists are trained to do, and Johanna has made some incredible art for her research.  

Once she became a science student during her undergraduate study, she worked in the laboratories of Drs. Yanyan Li, previously an Associate Professor (of nutrition) in the College of Science and Humanities, and Tao Zhang, Assistant Professor of Basic Pharmaceutical Sciences, both of Husson University in Bangor.  There, she performed nutritional biochemistry, worked with mouse models, and developed an idea of what she wanted to study in graduate school and pursue as a career.  Johanna continues to work closely with both researchers, especially now that Dr. Li has taken a position at UMaine. 

Johanna and I continued to plan her graduate work and career goals, she officially joined my lab as a Master’s Student of Nutrition at UMaine in fall 2020, and immediately got to work.  Not only did she begin preparations for the massive undertaking that is part of her project, but she began mentoring several undergraduates on and off campus, and started as a first time teaching assistant for the Chemistry department, which required navigating virtual labs.  She served as a chemistry TA for academic year 20/21 and 21/22, with up to 60 students per semester. For the last year and a half, she has been coordinating a large-scale research project with investigators at 4 different institutions and undergraduate researchers from 3 different institutions, involving hundreds of samples – while being a masters student, a graduate teaching assistant, and mentoring undergrads in the lab, and all during a pandemic!  She managed that all so well, that despite being a first-year graduate student, she was awarded a 2020-2021 University of Maine Graduate Student Employee of the Year award, and the 2022 Norris Charles Clements Graduate Student Award from the College of Natural Sciences, Forestry, and Agriculture. 

Johanna’s project focuses on whether consumption of specific broccoli sprout preparations will elicit changes in the gut microbiota, to the effect of improving the production of microbiota-specific bioactives that have local anti-inflammatory effects, and promoting intestinal homeostasis by reducing dysbiosis. Broccoli sprouts represent an effective, and accessible way to add dietary intervention to existing treatment and prevention strategies for IBD patients. This project is a continuation of previous research on bioactive compounds in broccoli, completed in the labs of Drs. Yanyan Li and Tao Zhang at Husson University in Bangor.  While some of the work may be similar, the skill set she has gained in her graduate work is entirely new.  For the 2020/2021 winter break, Johanna was managing a 40-mouse study looking at DSS-treatment and different preparations of a broccoli sprout diet for 5 weeks, which resulted in hundreds of samples collected, hundreds of data time points, and enough follow-up laboratory and analysis work to keep her occupied for an entire year.  She has learned how to culture bacteria in an anaerobic chamber, which is a notoriously fussy machine that requires regular attention, as well as to grow them under different conditions for biochemical analysis and enzyme activity.  She is currently learning additional histology skills, DNA extraction, DNA sequencing library preparation, DNA sequence analysis, and more. Recently, she has participated in a pilot study to develop an immunological model of IBD, using IL-10 knockout mice. While IL-10 mice have been used to study IBD, they have never been applied in this way to study the interaction of diet, microbes, and disease.

She has presented this work at the American Society for Microbiology annual meeting, and at the UC Davis Research Experience for Undergraduates (REU) symposium, and has several conference presentations planned for 2022. Johanna has an author on a paper in early 2022 for work she contributed to as an undergraduate, and is preparing 3 manuscripts generated from her masters work which will be submitted for peer review at a scientific journal in 2022.

Johanna has been accepted to the Nutrition PhD program at UMaine, and will continue working with Dr. Li and I, as well as the full research team. Based on those preliminary results, Johanna’s doctoral work will focus on developing that new model, synthesizing information from both models, and using those results to develop diet intervention trials in human patients. After her PhD, Johanna intends to conduct research at an institution here in Maine, and to continue her work connecting the biochemistry of nutrition with gut microbiology and human health.

Johanna receives a UMaine College of Natural Sciences, Forestry, and Agriculture Graduate Student Award for her research!

Johanna Holman received the 2022 Norris Charles Clements Graduate Student Award from the University of Maine College of Natural Sciences, Forestry, and Agriculture (NSFA), for her research and academics over the last two years for her Master’s of Science in Nutrition!! This award highlights the achievements and potential for positive impact of graduate students in agriculture, is more often awarded to doctoral students who have had more years of graduate work in which to accomplish their research, give presentations, mentor undergraduates, and otherwise develop their professional skills.

Person in a research facility holding up their arm with a mouse on it. Person is wearing a hairnet, nitrile gloves, surgical mask, and a surgical gown. They are holding their left arm up to the camera to show off a mouse with dark brown fur sitting on their arm. In the background is a metal shelf with containers of research materials.

Despite the setbacks and challenges of the pandemic, Johanna has been extremely productive and has done an extraordinary amount in just a year and a half (from start to the time of application submission) for her masters work. Johanna is currently writing up the results of her masters work into three manuscripts that we plan to submit to scientific journals for peer review this summer. She will defend her thesis at the end of the summer, just in time to start in September as a PhD student working with Dr. Yanyan Li and I!

Artwork by Johanna Holman
Artwork by Johanna Holman

From the NSFA award page: “The Norris Charles Clements Graduate Student Award was established in the University of Maine Foundation in May 1997 for the benefit of the University of Maine, Orono, with a bequest from Laurel Clements ’48 in honor of her father, Norris Charles Clements, a distinguished Maine poultry farmer who in 1953 was honored by the University of Maine as Maine’s Outstanding Farmer. Income shall be used to provide financial assistance for rewarding outstanding graduate students in agricultural sciences and to recognize the accomplishments of graduate students whose studies have the potential to make a significant contribution to Maine agriculture. Candidates should have training and be doing research in disciplines related to Maine agriculture, such as agronomy, soil science, animal and veterinary sciences, agricultural economics, entomology, plant pathology, agricultural engineering and other disciplines the dean deems contribute significantly to the well being of Maine agriculture. Students will be chosen for awards on the basis of their high academic standing, the quality of their research and their personal integrity.”

Johanna Holman is awarded UMaine Grad Student Employee of the Year!

Johanna Holman, Master’s of Nutrition student in the Ishaq Lab has been awarded the 2020-2021 University of Maine Graduate Student Employee of the Year!!!!

I met Johanna in the fall of 2019, when I was just establishing myself as a new Assistant Professor in the School of Food and Agriculture, and she was looking for an advisor for a graduate degree.  Right away, she impressed me with her background and enthusiasm for research.  I learned that Johanna began her undergraduate study as an art student before transitioning fluidly to science.  I see this is an asset – the ability to design visual aid and graphical representations of data is hugely important to science and sadly, not always a skill that scientists are trained to do. Johanna also had a number of service industry jobs, and initially in that first meeting, she was somewhat apologetic for not having been devoted to science jobs from the start.  I countered that I was pleased to see that she has worked in other industries, specifically in difficult service-related jobs.  It is often more important to have patience, dedication, and strong interpersonal skills, such as those gained by working in customer-facing jobs.  I believe that Johanna has and will continue to succeed because of her varied education and experience.

Once she became a science student during her undergraduate study, she worked in the laboratories of Drs. Yanyan Li, Associate Professor (of nutrition) in the College of Science and Humanities, and Tao Zhang, Assistant Professor of Basic Pharmaceutical Sciences, both of Husson University in Bangor.  There, she has performed nutritional biochemistry, worked with mouse models, and developed an idea of what she wanted to study in graduate school and pursue as a career. 

Johanna officially joined my lab and started as a Master’s Student of Nutrition at UMaine in fall 2020, and immediately got to work.  Not only did she begin preparations for the massive undertaking that is part of her project, but she began mentoring several undergraduates on and off campus, and started as a first time teaching assistant for the Chemistry department, which required navigating virtual labs.

Johanna’s project focuses on whether consumption of specific broccoli sprout preparations will elicit changes in the gut microbiota, to the effect of improving the production of microbiota-specific bioactives that have local anti-inflammatory effects, and promoting intestinal homeostasis by reducing dysbiosis. This project is a continuation of previous research on bioactive compounds in broccoli, completed in the labs of Drs. Yanyan Li and Tao Zhang at Husson University in Bangor.  While some of the work may be similar, the skill set is entirely new.  For the winter break, Johanna was managing a 40-mouse study for 5 weeks, which has resulted in hundreds of samples collected, hundreds of data time points, and enough follow-up laboratory and analysis work to keep her occupied for an entire year.  She has learned how to culture bacteria in an anaerobic chamber, which is a notoriously fussy machine that requires regular attention, as well as to grow them under different conditions for biochemical analysis and enzyme activity.  She will be learning DNA extraction, DNA sequencing library preparation, DNA sequence analysis, and will lead the generation of a large manuscript on the results.

It might seem too early to recommend a graduate student for this award after just one semester, but it is remarkable that a new master’s student could achieve all of this in their first semester during a pandemic.  I have informally mentored graduate and undergraduate students for years, and it is easy to spot the ones who will go far in science. Johanna has a highly successful career ahead of her, and I am honored to be one stop on that path.  This award will not only acknowledge the incredible amount of work she has accomplished, but it will support an early career researcher who has every quality to make research a hospitable and collaborate place.