Tag: broccoli sprouts

Paper published on “Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease”

Sue IshaqLeave a comment

The Ishaq and Li labs at UMaine are delighted to announce that our paper on “Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease.” has been published in mSystems!! ASM was kind enough to write a press release about study, found here.

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 as a way to resolve symptoms of Inflammatory Bowel Disease.

The Premise

Broccoli sprouts are very high in a compound called glucoraphanin, which is in-active for humans. When glucoraphanin comes in contact with the myrosinase enzyme, also found in the sprouts, it is transformed into sulforaphane, which drives away insect pests but acts as an anti-inflammatory in people!

If you eat raw sprouts, most of 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 glucoraphanin, and produce the anti-inflammatory sulforaphane 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.

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.
A cartoon of a woman eating broccoli, with the digestive tract shown on her shirt, and smiling microbes in the background.

The mice in this trial are used to mimic Crohn’s Disease, which is one of the main ways that Inflammatory Bowel Diseases may be classified. Crohn’s Disease is complictaed, and involves an over-active immune response to gut microbes. This is replicated in mice that are bred to lack the genes in the DNA to make interleukin-10 (IL-10). IL-10 is an immune factor that can be used to calm the immune system and tolerate microbes which are not causing harm. Without IL-10, these mice over-react to the presence of bacteria, even those which are not causing harm, and this creates symptoms similar to Crohn’s in people.

We used two age groups of mice, and in each group, half ate a mouse chow (control) diet and half ate the mouse chow with 10% of the chow replaced by raw broccoli sprouts. Crohn’s often develops in childhood and adolescence, so our two age groups of mice reflect the juvenile stage (4-5 weeks old) and the adolescence stage (5-6 weeks old) of symptom onset. After wo weeks of symptoms, we sacrificed the mice and collected as much information as we could.

Figure 1 from the paper mentioned in this post. It shows an experimental design.

The Team

The mice, their care during the experiment, and sample collection for this project was graciously provided by University of Vermont researchers Gary Mawe and Brigitte Lavoie, and then-grad-student-now-medical-student Molly Hurd, in 2021. The SUNY Bingamton team, Tao Zhang and Allesandra Stratigakis, processed metabolite and cytokine samples and analyzed those data. The UMaine team (pictured below and led by Sue Ishaq and Yanyan Li) processed and analyzed data from different locations of gut tissue for histolgy and sequencing of bacterial communities, as well as analyzing those data, and took the lead on writing the paper.

Portrait of Lola Holcomb, wearing a block sweater on a beach at sunset
Lola standing in front of her poster which displays the results of her research on gut microbes.
Louisa Colucci. Young woman with dark hair tied in a bun is sitting in a kayak with her paddle raised. She is wearing a black top and a red life-vest over it, in a light blue, single-person kayak. The kayak is in the water, most likely a lake, with the forested shoreline in the background.

The Health Benefits were most obvious in the younger mice

The mice that were eating the broccoli sprouts in their chow and did much better than the control group who ate only mouse chow when symptoms of Crohn’s Disease were induced — and we found something really interesting… The diet worked really well in the younger mice and reduced their symtpoms of inflammation and illness for almost every metric we studied. The older, adolecent mice got some benefit from eating the raw broccoli sprouts, but not nearly as much as the younger mice! Those graphs are shown in the paper.

The Gut Microbes were most changed in the younger mice

Bacterial richness (the number of different types of bacteria present) was increased, but only in younger mice consuming a 10% raw sprout diet, which is useful because pediatric Crohn’s patients usually have fewer types of bacteria present in their gut.

Younger mice consuming broccoli sprouts also had more types of bacteria that are known to convert glucoraphanin into sulforophane, and they had more of the genes needed to do it. Crohn’s patients usually have fewer of these types of bacteria, which are also known to provide other health benefits.

The Next Steps

We are currently working on replicating and expanding this project to include more age groups, so we can understand how different diet preparations of broccoli sprouts impact immune systems and gut microbiota at different developmental periods of life. We are also really interested in understanding how sex in mice, and gender in humans, plays a role in how immune systems and microbial communities develop during a critical phase of life. We have some initial data to suggest that male and female mice respond to different diets and at differnt ages, but we aren’t sure why yet.

We hope to expand our work with people to study how these diets work in the real world, and how we can tailor diet and cooking preparations of sprouts to best meet the needs of people of different ages, health statuses, and tastes.

The paper

Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease. 

Lola Holcomb1$, Johanna M. Holman2$, Molly Hurd3, Brigitte Lavoie3, Louisa Colucci4, Benjamin Hunt5, Timothy Hunt5, Marissa Kinney2, Jahnavi Pathak1, Gary M. Mawe3,Peter L. Moses3,6, Emma Perry7, Allesandra Stratigakis8, Tao Zhang8, Grace Chen9, Suzanne L. Ishaq1*, Yanyan Li1*

1 Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine, USA 04469. 2 School of Food and Agriculture, University of Maine, Orono, Maine, USA 04469. 3 Larner College of Medicine, University of Vermont, Burlington, Vermont, USA 05401. 4 Department of Biology, Husson University, Bangor, Maine, USA 04401. 5 Department of Biology, University of Maine, Orono, Maine, USA 04469. 6 Finch Therapeutics, Somerville, Massachusetts, USA 02143. 7 Electron Microscopy Laboratory, University of Maine, Orono, Maine, USA 04469. 8 School of Pharmacy and Pharmaceutical Sciences, SUNY Binghamton University, Johnson City, New York, USA 13790. 9 Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA 48109

$ these authors contributed equally.

Keywords: Crohn’s Disease, cruciferous vegetables, sulforaphane, glucoraphanin, gut microbiota, dietary bioactives, 16S rDNA, interleukin-10 knockout 

Abstract

Crohn’s Disease (CD) is a presentation of Inflammatory Bowel Disease (IBD) that manifests in childhood and adolescence, and involves chronic and severe enterocolitis, immune and gut microbial dysregulation, and other complications. Diet and gut-microbiota-produced metabolites are sources of anti-inflammatories which could ameliorate symptoms. However, questions remain on how IBD influences biogeographic patterns of microbial location and function in the gut, how early life transitional gut communities are affected by IBD and diet interventions, and how disruption to biogeography alters disease mediation by diet components or microbial metabolites. Many studies on diet and IBD use a chemically induced ulcerative colitis model, despite the availability of an immune-modulated CD model. Interleukin-10-knockout (IL-10-KO) mice on a C57BL/6 background, beginning at age 4 or 7 weeks, were fed a control diet or one containing 10% (w/w) raw broccoli sprouts, which was high in the sprout-sourced anti-inflammatory sulforaphane. Diets began 7 days prior to, and for 2 weeks after inoculation with Helicobacter hepaticus, which triggers Crohn’s-like symptoms in these immune-impaired mice. The broccoli sprout diet increased sulforaphane in plasma; decreased weight stagnation, fecal blood, and diarrhea associated; and increased microbiota richness in the gut, especially in younger mice. Sprout diets resulted in some anatomically specific bacteria in younger mice, and reduced the prevalence and abundance of pathobiont bacteria which trigger inflammation in the IL-10-KO mouse, e.g., Escherichia coli and Helicobacter. Overall, the IL-10-KO mouse model is responsive to a raw broccoli sprout diet and represents an opportunity for more diet-host-microbiome research.

Importance

To our knowledge, IL-10-KO mice have not previously been used to investigate the interactions of host, microbiota, and broccoli, broccoli sprouts, or broccoli bioactives in resolving symptoms of CD. We showed that a diet containing 10% raw broccoli sprouts increased the plasma concentration of the anti-inflammatory compound sulforaphane, and protected mice to varying degrees against disease symptoms, including weight loss or stagnation, fecal blood, and diarrhea. Younger mice responded more strongly to the diet, further reducing symptoms, as well as increased gut bacterial richness, increased bacterial community similarity to each other, and more location-specific communities than older mice on the diet intervention. Crohn’s Disease disrupts the lives of patients, and requires people to alter dietary and lifestyle habits to manage symptoms. The current medical treatment is expensive with significant side effects, and a dietary intervention represents an affordable, accessible, and simple strategy to reduce the burden of symptoms.

Acknowledgements: 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); 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. Johanna Holman was supported by ME0-22303 (Li), and 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 the UMaine Graduate School of Biomedical Science and Engineering.

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

Sue IshaqLeave a comment

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

A cartoon of a bacteria eating broccoli and pooping out sulforaphane
Designed by Johanna Holman
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.

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.

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 Holman, graduate researcher in the Ishaq Lab
Louisa Colucci. Young woman with dark hair tied in a bun is sitting in a kayak with her paddle raised. She is wearing a black top and a red life-vest over it, in a light blue, single-person kayak. The kayak is in the water, most likely a lake, with the forested shoreline in the background.
Louisa Colucci.
A person holding a mouse in a research facility. The person is wearing a hairnet, nitrile gloves, a surgical mask, and a surgical gown. The mouse with dark brown fur is sitting on the top of the person's hand. In the background, there are metal shelves with bins and containers of research supplies.
Dorien Baudewyns, undergraduate researcher at Husson University
Joe Balkan, performing culturing of bacteria at the anaerobic chamber. Joe is wearing a face mask, and is holding up a culture plate. There are stacks of petri dishes inside the chamber.
Joe Balkan, undergraduate researcher at Tufts University, performing culturing of bacteria at the anaerobic chamber.
Close-up image of a small, dark brown mouse perched on the arm of a graduate researcher. The person is wearing a face mask and surgical gown.

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!

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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.