Tag: broccoli

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

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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”!

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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. 

Upcoming presentations at the American Society for Nutrition conference

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The Ishaq Lab will be presenting at a few research conferences this summer, with a few more in the works for the fall.

Broccoli sprouts in a tray

American Society for Nutrition meeting, July 22-25, 2023, Boston, Massachusetts

Steamed broccoli sprouts alleviate gut inflammation and retain gut microbiota against DSS-induced dysbiosis.

Poster, abstract P20-022-23, July 23

Authors: Johanna M. Holman*1, Lola Holcomb2, Louisa Colucci3, Dorien Baudewyns4, Joe Balkan5, Grace Chen6, Peter L. Moses7,8, Gary M. Mawe7, Tao Zhang9, Yanyan Li1, Suzanne L. Ishaq1

Affiliations: 1 School of Food and Agriculture, University of Maine, 2 Graduate School of Biomedical Sciences and Engineering, University of Maine, 3 Department of Biology, Husson University, 5 Department of Chemical and Biological Engineering, Tufts University, 6 Department of Internal Medicine, University of Michigan Medical School, 7 Departments of Neurological Sciences and of Medicine, Larner College of Medicine, University of Vermont, 8 Finch Therapeutics, 9 School of Pharmacy and Pharmaceutical Sciences, SUNY Binghamton University.

Objectives: Inflammatory Bowel Diseases (IBD) are devastating conditions of the gastrointestinal tract with limited treatments, and dietary intervention may be effective, affordable, and safe for managing symptoms. Ongoing research has identified inactive compounds in broccoli sprouts, like glucoraphanin, and that mammalian gut microbiota play a role in metabolizing it to the anti-inflammatory sulforaphane. The objectives were to identify biogeographic location of participating microbiota and correlate that to health outcomes. 

Methods: 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 40-day experiment to simulate chronic, relapsing ulcerative colitis. We monitored body weight, fecal characteristics, fecal lipocalin, and sequenced bacterial communities from the contents and mucosa in the jejunum, cecum, and colon. 

Results: Mice fed the broccoli sprout diet while receiving DSS performed better than mice fed the control diet while receiving DSS for all disease parameters, including significantly more weight gain (2-way ANOVA, p < 0.05), lower Disease Activity Index scores (2-way ANOVA, p < 0.001), and higher bacterial richness in all gut locations (linear regression model, p < 0.01 for all locations measured). Bacterial communities were assorted by gut location except in the mice receiving the control diet and DSS treatment (Beta-diversity, ANOVA, p < 0.05 for each). Importantly, our results suggested that broccoli sprout feeding completely abrogated the effects of DSS on gut microbiota, as bacterial communities were similar between mice receiving broccoli sprouts with and without DSS. 

Conclusions: Spatially resolved microbial communities provide greater insight when investigating host-microbe interactions. Here, we show that a 10% broccoli sprout 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. 

Funding Sources: This work was funded by the NIH, USDA, NSF NRT, and UMaine GSBSE.

 

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

Poster, abstract P20-021-23, July 23

Authors: Lola Holcomb*1, Johanna Holman2, Molly Hurd3, Brigitte Lavoie3, Louisa Colucci4, Gary M. Mawe3, Peter L. Moses3,5, Emma Perry6, Allesandra Stratigakis7, Tao Zhang7, Grace Chen8, Suzanne L. Ishaq1, Yanyan Li1

1 Graduate School of Biomedical Sciences and Engineering, University of Maine, 2 School of Food and Agriculture, University of Maine, 3 Larner College of Medicine, University of Vermont, 4 Department of Biology, Husson University, 5 Finch Therapeutics, 6 Electron Microscopy Laboratory, University of Maine, 7 School of Pharmacy and Pharmaceutical Sciences, SUNY Binghamton University, 8 Department of Internal Medicine, University of Michigan Medical School.

Objectives: Inflammatory Bowel Diseases (IBD) are chronic conditions characterized by inflammation of the gastrointestinal (GI) tract that burden daily life, result in complications, and disrupt the gut microbiome. Many studies on diet and IBD in mice use an ulcerative colitis model, despite the availability of an immune-modulated Crohn’s Disease model. The objective of this study was to establish IL-10 deficient mice as a model for studying the role of dietary broccoli and broccoli bioactives in reducing inflammation, modifying the immune response, and supporting GI tract microbial systems. 

Methods: Interleukin-10-knockout (IL-10-ko) mice on a C57BL/6 background, beginning at age 4 or 7 weeks, were fed either a control diet or one containing 10% raw broccoli sprouts. Diets began 7 days prior to inoculation with Helicobacter hepaticus, which triggers Crohn’s-like symptoms in these immune-impaired mice, and ran for 2 additional weeks. 

Results: Broccoli sprouts decreased (p < 0.05), fecal lipocalin (LCN2), a biomarker for intestinal inflammation, and fecal blood, diarrhea, and overall Disease Activity Index. Sprouts increased gut microbiota richness, especially in younger mice (p < 0.004), and recruited different communities in the gut (B-diversity, ANOVA, p < 0.001), especially in the colon (B-diversity, ANOVA, p = 0.03). The control group had greater prevalence and abundance of otherwise commensal bacteria which trigger inflammation in the IL-10-ko mice. Helicobacter was within the top-5 most prevalent core genera for the control group, but was not within the top-5 for the broccoli group. Disease parameters and microbiota changes were more significant in younger mice receiving broccoli.

Conclusions : A diet containing 10% raw broccoli sprouts may be protective against negative disease characteristics of Helicobacter-induced enterocolitis in IL-10-ko mice, and younger age is the most significant factor (relative to diet and anatomical location) in driving gut bacterial community richness and similarity. The broccoli diet contributes to prevalence and abundance of bacterial genera that potentially metabolize dietary compounds to anti-inflammatory metabolites in the gut, are bacteriostatic against pathogens, and may ease disease severity.

Funding Sources: This work was funded by the NIH, USDA, NSF NRT, and UMaine GSBSE.

Establishing Growth Curve Assays for Bacterial Glucosinolate Metabolism: A Study Protocol

Poster, abstract P22-030-23, July 23

Marissa Kinney*1, Ryan Wijayanayake1, Johanna Holman1, Timothy Hunt2, Benjamin Hunt 2, Tao Zhang3, Grace Chen4, Yanyan Li1 , Suzanne L. Ishaq1

1School of Food and Agriculture, University of Maine, Orono, Maine, USA 04469; 2 School of Biology and Ecology, University of Maine, Orono, Maine, USA 04469; 3 School of Pharmacy and Pharmaceutical Sciences, SUNY Binghamton University, Johnson City, New York, USA 13790; 4 Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA 48109 

Objective: Inflammatory bowel diseases (IBD) cause dysfunction of the gastrointestinal (GI) tract and can result in hospitalization, suffering and disruption to overall health. Recent work has demonstrated the anti-inflammatory capacity of a broccoli sprout-diet in artificially-induced GI inflammation in pathogen free C57BL/6 mice. Microbiota samples obtained from the GI tract of these mice will be used to study the presence and activity of broccoli glucosinolate hydrolysis to create microbial-sourced bioactives, to further understand the relationship between broccoli-diets and inflammation reduction. It is imperative to validate or replicate qPCR protocols which have been established for glucosinolate metabolism in Bacteroides thetaiotaomicron (B. theta), in other bacterial species. Additionally, this project will focus on developing new growth curve assays for glucosinolate metabolism, as these methods are lacking in published literature. 

Methods: Pathogen free C57BL/6 mice were given dextran sodium sulfate (DSS) into their drinking water to create a disease profile similar in development and morphology to human ulcerative colitis (UC), a type of IBD. DSS and a steamed broccoli sprout diet were administered. Samples were taken from the digesta, jejunum, cecum, and colon of mice fed broccoli diets. About 806 bacterial isolates will be grown up/cultured anaerobically on minimal/selective media containing glucosinolate-related compounds (glucoraphanin, sinigrin) to determine hydrolysis activity via spectroscopy to measure optical density of growth in competent isolates. Successful isolates will be further analyzed with LC/MS to confirm production of bioactive products, and with qPCR using the B. theta positive control genome to help identify gene targets (α-1,6-mannanase, glycosyl hydrolase, nicotinamide-dependent oxidoreductase, and transcriptional regulator protein) for glucosinolate conversion in isolates. 

Results  N/A

Conclusions: In the initial study from which these samples are sourced, mice fed a broccoli diet had less inflammation than those fed a control diet and DSS, and had higher bacterial diversity in their gut. We expect that bacteria isolated from the GI of broccoli-fed mice will contain more glucosinolate-metabolizing genes.

Funding: NIH (Li and Ishaq) and USDA (Li). 

Broadening Perspectives by Situating Nutrition Education in Broader Social Contexts: A Study Protocol

Poster, abstract number 1490287, July 23

Authors: Ashley Toney*1, Patricia Wolf*2, Sue Ishaq*3

Affiliations: 1Dept. of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner Research Institute, 2 Department of Nutrition Science, Purdue University, 3 School of Food and Agriculture, University of Maine

Objectives: The Microbes and Social Equity (MSE) Working Group connects microbiology with social equity research, education, policy, and practice to understand the interplay of microorganisms, individuals, societies, and ecosystems. Given the complexity and nuance of evidence-based nutrition delivery, MSE sought to provide a conceptual structure. The goals are 1) convene diverse researchers, educators, learners, and practitioners, 2) publish and present evidence-based information within socio-cultural contexts, and 3) teach audiences to define ways for integrating equity into their work.

Methods: MSE hosted a 14-week speaker series in 2021, 2022, and 2023, with researchers from various disciplines (e.g., nutrition, gut microbes, food security). The series lead into a 5-day symposium of speakers and guided discussions to generate co-written documents identifying research needs and resources. We use targeted and non-targeted event promotion to attract audiences, and reach/impact is evaluated through registration, attendance, social media views, or attendee feedback. The series provides learning sessions that build concepts over time, guiding attendees past the conceptual roadblock of being new to interdisciplinary research and grappling with grand challenges. This extended period to learn stimulates participation in activity-based collaboration during symposia, even from new members or students. 2023 symposium attendees will be surveyed on their impressions on the event, how it impacted their perspective on experimental design, whether attendees started with the series and followed up with the symposium, and whether having the preface of the series aided in being able to create actionable outputs in a guided co-working session.

Results: n/a

Conclusions: Benefits include nuanced knowledge/perspective sharing, establishing and nurturing interdisciplinary collaboration, and sparking conversations on critical topics in policy, sustainability, host and microbial metabolism, etc. In the last 3 years, MSE published 4 papers together, with ~ 50 independent papers from members. Event recordings are used as curriculum in nutrition, microbiology, and pre-med biology.

Funding Sources: NIH/NIDDK, Allen Foundation (Ishaq); National Dairy Council (Toney)

Li and Ishaq research on broccoli sprouts and gut microbes featured in the UMaine Annual Research Report!

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Every year, The University of Maine releases a summary report of the research efforts of its faculty, staff, and students. I was pleased to discover that the work that Yanyan Li and I have been doing on broccoli sprouts and gut microbes was featured in the report! A screenshot of the story is below, but you can read the story and the entire 2022 UMaine research report here.

Screenshot of an article on broccoli research from the UMaine Annual Research Report. The text of the article can be found in that report.

The Ishaq Lab welcomes a new grad student, Marissa Kinney!

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The Ishaq Lab is pleased to welcome Marissa Kinney as a Master’s of Science student in Microbiology, beginning in January 2023! She’ll be joining ‘Team Broccoli‘ to investigate the 806 bacteria we isolated from the digestive tracts of mice eating a broccoli sprout diet, in a previous experiment on broccoli sprouts, microbes, and resolving colitis.

Marissa is a recent graduate of the UMaine Microbiology bachelor’s program, where she was part of an interdisciplinary research group and was the first author on a scientific publication this year: Suppression of Methicillin-Resistant Staphylococcus aureus and Reduction of Other Bacteria by Black Soldier Fly Larvae Reared on Potato Substrate.

Marissa Kinney

Marissa Kinney 

Master of Science student, Microbiology and Animal and Veterinary Sciences

Blurb: Marissa is a Masters student who loves learning and bench microbiology. She completed her undergraduate at the University of Maine in 2021, earning a BS in Microbiology and a BS in Cellular/Molecular Biology. She devoted a large portion of her time in undergrad to research in the laboratories of Dr. Julie Gosse and Dr. Edward Bernard. Since graduating, she worked in the field of public health at UMaine’s Margaret Chase Smith Policy Center, collecting and processing data about violent and drug-related deaths in Maine. While her role at the Center was one she loved dearly, she feels a big pull towards laboratory work and academic research. She recently joined the Ishaq lab and is excited by the new opportunities this position brings. 

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

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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.

News Center Maine interviews Yanyan and Sue about broccoli sprouts!

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Yanyan Li and I sat down yesterday with Carly D’Eon, a reporter with News Center Maine, to talk about our ongoing research into broccoli sprouts, gut microbes, and Inflammatory Bowel Disease!

The story and the video can be found here.

Li and Ishaq labs made the UMaine News!

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UMaine News recently highlighted some of the collaborative work that Yanyan Li and I have been doing at UMaine! You can read more about our projects here, here, and here.

This work features researchers from other universities, including:

•Dr. Tao Zhang, PI, SUNY Binghamton

•Dr. Grace Chen, PI, MSU

•Dr. Gary Mawe, PI, UVM

•Molly Hurd, PhD student, UVM

•Bridgette Lavoie, RA, UVM

•Dr. Peter Moses, PI, Finch Therapeutics

•Johanna Holman, MS student, Umaine

•Lola Holcomb, PhD student, UMaine

•Louisa Colucci, undergrad at the time, Husson

Joe Balkan, Undergrad, Tufts

•Dr. Emma Perry, UMaine Microscopy Facility

In addition to the two new funding sources listed in the article, we have been supported by:

•Maine Agricultural and Forestry  Experimental Station

•ME0-22102 (Ishaq) and ME0-22303 (Li)

•USDA-NIFA-AFRI Foundational Program [Grant No. 2018-67017-27520]

•UMaine Institute of Medicine

•Maine Center for Genetics in the Environment

• UMaine GSBSE

Li and Ishaq labs receive NIH R15 award to study broccoli bioactives, gut microbes, and inflammation!

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The Li and Ishaq labs at UMaine, along with collaborators from multiple institutions, have been awarded R15 funding from the National Institute Of Diabetes And Digestive And Kidney Diseases of the National Institutes of Health!

This award will complement other projects/awards led by our team, which has been investigating inflammatory bowel diseases, anti-inflammatories, gut microbes, and nutrition, separately for decades and collaboratively for over two years.

  • Dr. Yanyan Li, PhD (lead PI), Assistant Professor at the University of Maine with expertise in nutrition and food science, particularly dietary bioactives and colitis;
  • myself (co-PI), with expertise in host-associated microbiology, especially GI tract;
  • Dr. Grace Chen, MD, PhD (co-I), Associate Professor at the University of Michigan, expertise in mouse models for gut microbiome and colonic host immune responses;
  • Dr. Tao Zhang, PhD (consultant), Assistant professor at Binghamton University, with expertise in metabolism, kinetics, and bioanalysis of natural products;
  • Dr. Gary Mawe, PhD (consultant), Professor at the University of Vermont, with expertise in translational research on GI tract regulation, inflammation, and IBD;
  • Dr. Peter Moses, MD (consultant), Professor Emeritus at the University of Vermont College of Medicine and Senior Researcher at GSK, with expertise in IBD and functional gastrointestinal disorders.

R15 Research Enhancement Awards are designated for projects which involve a large number of student researchers. Between the Li and Ishaq labs, there are three current graduate students, and two former undergrads who have contributed to this research, and we anticipate bringing in 1-2 additional graduate students and almost a dozen undergrads in the next year! That will include undergrads in Honors, Top Scholars, and Capstone programs at UMaine. We’ve also been assisted by the work of students, postdocs, technicians, and investigators through our collaborators, and we are ecstatic about the opportunity to continue to grow our team across institutions. And, this project will generate research that will feed back into education at UMaine through the courses that we teach, such as my microbiomes and DNA sequence analysis courses.

“Harnessing gut microbiota to reduce inflammation using broccoli-sprout diets.”

Project Summary:

Inflammatory bowel disease (IBD) is a poorly understood gastrointestinal (GI) condition characterized by inflammation. The prevailing theory is that combined genetic and environmental factors disrupt the host immune system’s interaction with gut microbiota. Our central hypothesis is that consumption of specific broccoli sprout preparations elicits changes in the gut microbiota that not only improve the production of anti-inflammatory bioactives, but also promote intestinal homeostasis. Our labs have shown there is an anatomical pattern along the GI tract where broccoli sprout-derived bioactive levels are high which correspond to diet-induced changes in gut microbial communities. We showed that gut microbiota contribute to the transformation of inactive precursors to bioactives, and that specific broccoli sprout preparations alter their capacity for biotransformation, and the susceptibility of mice to colitis. However, a significant knowledge gap remains regarding the mechanisms by which dietary bioactives modify disease risk and the role of gut microbiota. Our immediate goal is to identify the mechanisms by which broccoli sprout diets affect susceptibility to IBD in mice. Our long-term goal is to develop a dietary preparation of
broccoli sprouts which has therapeutic effects against IBD in humans. Our innovative approach uses different preparations of broccoli sprouts to help differentiate gut microbiota versus plant-derived
enzymatic activities. We employ a combination of “omics” approaches to spatially-map the microbial community and metabolite profile changes along the GI tract, to better assess changes induced by broccoli sprout diets. We complement “omics” approaches with culturing, and validate our study design using two complementary models for strategic research.

A cartoon of three gastrointestinal tracts showing the locations of inflammation in ulcerative colitis, crohn's disease, or healthy tissue. At the bottom are cross-sections showing thickening of the intestinal wall in patients with Crohn's, and ulcers in patients with colitis.
Created by Johanna Holman.

Aim 1 tests the hypothesis of an anatomical pattern where the GI tract microbiota transform broccoli compounds into bioactives, and helps us determine whether this microbial biotransformation is sensitive to dose of broccoli compounds. We will use our established DSS-mouse-model of ulcerative colitis to investigate the effects of different broccoli sprout preparations and concentrations on the microbiota along the GI tract; on the resulting concentration of bioactives in gut tissues; and on the development of colitis in mice.

A cartoon schematic of the experimental design of the project. Four mice are at the top, two have "DSS" written above them, one of which is also holding a broccoli sprout. One of the mice without DSS written on it is holding a broccoli sprout. Below the mice is a cartoon of the digestive tract with arrows emanating from it to indicate samples of microbes will be taken from different locations. The microbe images have arrows pointing to culturing equipment, and also to a biochemical pathway showing the compound glucoraphanin being converted to sulforaphane.
Created by Sue Ishaq, made with Biorender

Aim 2 tests the benefits of using an immunosuppressed mouse model in the dietary prevention study to provide a stronger translational strategy for the use of broccoli sprouts for IBD prevention. When exposed to a specific bacterial pathogen, the immunosuppressed mice develop chronic enterocolitis resembling Crohn’s disease. This diet-based approach provides critical information for developing accessible and equitable strategies for improving health of IBD patients.

A cartoon schematic of the experimental design of the project. Two mice are at the top, with the label "IL-10" crossed out above them. One mouse is also holding a broccoli sprout. Below the mice is a cartoon of the digestive tract with arrows emanating from it to indicate samples of microbes and tissue will be taken from different locations. The words weight and plasma indicate those will also be collected. The plasma and tissue samples will be used for mass-spectroscopy and histology, and the microbes will be used for DNA sequencing.
Created by Sue Ishaq, made with Biorender

Li and Ishaq labs receive Allen Foundation funding to research broccoli bioactives!

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The Allen Foundation awarded Dr. Yanyan Li, Assistant Professor of Food Science and Human Nutrition, and myself funding for a pilot project in people on broccoli sprouts, the gut microbiome, anti inflammatory compounds, and health! Dr. Li and I, as well as a team of other researchers, have been collaborating over the last three years to understand how certain gut microbes create an anti-inflammatory compound using a compound in broccoli sprouts, and how we can use this action to calm colitis. Over the next 18 months, we will be recruiting a small group of people to participate in a diet trial. This will form the first part of the PhD work for Johanna Holman, who recently defended her master’s of science at UMaine.

Project Summary:

There is increasing evidence that diet and the gut microbiota have significant impact on human health and thus impact susceptibility to disease such as inflammatory bowel disease. Indeed, a Westernized diet has been associated with higher risk for developing inflammatory bowel disease, primarily as ulcerative colitis and Crohn’s disease, while a diet rich in fruits and vegetables tends to reduce risk. Our preliminary data suggests that a specific whole-food preparation of broccoli sprouts protects against the development of colitis in a chemically-induced mouse model as well as in a transgenic mouse model of Crohn’s disease. Furthermore, the gut microbiome contributes to the generation of the active anti-inflammatory component, sulforaphane, from broccoli sprouts, and the microbiome, in turn, is altered by exposure to broccoli sprouts or its metabolites. Thus, our long-term goal is to understand the interactions between anti-inflammatory bioactives of broccoli sprouts and the gut microbiome. The current proposal aims to increase our understanding of the nutrigenomics of the human microbiome and a broccoli sprout diet in healthy subjects. Our goal is to determine the feasibility of incorporating a specific broccoli sprout preparation into whole-food diets to increase levels of anti-inflammatory bioactives from broccoli sprouts in healthy humans. These directly address the foundation’s priority of “bringing the promise of nutrigenomics or nutritional genomics to realization”. Results from this study will help determine the feasibility and potential efficacy of a whole food approach in promoting intestinal homeostasis and mitigating risk of developing inflammatory bowel disease.

Diagram of the chemical conversion of glucoraphanin to sulforaphane. In panel A, the process is shown using the plant enzyme myrosinase, and in panel B, the process is shown using bacterial myrosinase-like enzymes. In the middle of the diagram, there is a cartoon mouse eating broccoli. Panel A points to the broccoli, where that action occurs, and panel B points to the gut, where microbial conversion occurs,
Figure from Holman et . in review, artwork by Johanna Holman. Glucoraphanin hydrolysis. A. GLR hydrolysis in the presence of myrosinase upon damage to the broccoli plant. Epithiospecifier protein preferentially converts GLR to SFN-nitrile. B. GLR hydrolysis has been demonstrated by gut bacteria in the colon of mammals.  Low pH environments favor conversion to SFN-nitrile.