Progress 02/15/15 to 02/14/18
Outputs
Target Audience:The new information generated by this porject has been published in scientific journals and thus reaches the entire scientific community. The practical application of our data has been futured in news articles and websites and thus has reached the general public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student was trained under this grant and performed most of the work. How have the results been disseminated to communities of interest?Yes, the work was published in the Journal of Functional Foods. After publication this project was the focus of several news articles, incuding, clean eating magazine, ScienceDaily, Newsweek, and Biotechniques. This work was also featured in the SOAR foundation document; http://supportagresearch.org/retakingthefield/ What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
All of the goals of this project were completed and were published in the Journal of Functional Foods. Summary: Consumption of broccoli mediates numerous chemo-protective benefits through the intake of phytochemicals, some of which modulate aryl hydrocarbon receptor (AHR) activity. Whether AHR activation is a critical aspect of the therapeutic potential of dietary broccoli is not known. Here we administered isocaloric diets, with or without supplementation of whole broccoli (15% w/w), to congenic mice expressing the high-affinity Ahrb/b or low-affinity Ahrd/d alleles, for 24 days and examined the effects on AHR activity, intestinal microbial community structure, inflammatory status, and response to chemically-induced colitis. Cecal microbial community structure and metabolic potential were segregated according to host dietary and AHR status. Dietary broccoli associated with heightened intestinal AHR activity, decreased microbial abundance of the family Erysipelotrichaceae, and attenuation of colitis. In summary, broccoli consumption elicited an enhanced response in ligand-sensitive Ahrb/b mice, demonstrating that in part the beneficial aspects of dietary broccoli upon intestinal health are associated with heightened AHR activity.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Hubbard, T.D., Murray, I.A., Nichols, R.G., Cassel, K., Podolsky, M., Kuzu, G., Tian, Y., Smith, P., Kennett, M.J., Patterson, A.D., and Perdew, G.H. (2017) Dietary broccoli impacts microbial community structure and attenuates chemically induced colitis in mice in an Ah receptor dependent manner. J. Functional Foods. 37, 685-698.
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Progress 02/15/16 to 02/14/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A graduate student has been trained to perform large scale mouse feeding studies and has learned how to analyze large datasets from 16S RNA sequencing. He has also learned how to work with gnootbiotic mice. How have the results been disseminated to communities of interest?We have published one article in scientific reports about the role of the Ah receptor in gut homeostasis. What do you plan to do during the next reporting period to accomplish the goals?We purposed to examine changes in the metatranscriptome of the gut microbiome after broccoli consumption. This method is not a routine procedure, during the past year we have developed the methodology necessary to isolate bacterial mRNA from cecal contents essentially free of ribosomal RNA, which makes up the majority of the total RNA isolated. We are now positioned to perform RNA seq on cecal samples. This will complete the studies proposed. We are in the process of writing up our results from these studies into one large manuscript.
Impacts
What was accomplished under these goals?
This is the second year of this study to examine the ability of dietary broccoli to alter the microbiome in the gastrointestinal (GI)tract of mice. Curiferous vegetables contain chemicals that degrade in the gastrointestinal tract to Aryl hydrocarbon receptor (AHR) ligands. Activation of the Ah receptor has been shown to have a positive effect on the immune system in the GI tract as far as maintaining appropriate homeostasis with the gut microbiome. We will determine the overall effects of broccoli consumption on the gut microbiome and how much of these effects are due to AHR activation. These studies will lead to better dietary recommendation to maintain health and even treat gastrointestinal disease. In our first series of studies we wanted to establish the role of Ah receptor expression in the gut and how it may influence the gut microbiome. Utilizing C57BL6/J Ahr−/+ and Ahr−/− co-housed littermates followed by 18 days of genotypic segregation, we examined the influence of AHR expression upon intestinal microbe composition/functionality and host physiology. 16S sequencing/quantitative PCR (qPCR) revealed significant changes in phyla abundance, particularly Verrucomicrobia together with segmented filamentous bacteria, and an increase in species diversity in Ahr−/− mice following genotypic segregation. Metagenomics/metabolomics indicate microbial composition is associated with functional shifts in bacterial metabolism. Analysis identified Ahr−/−-dependent increases in ileal gene expression, indicating increased inflammatory tone. Transfer of Ahr−/− microbiota to wild-type germ-free mice recapitulated the increase Verrucomicrobia and inflammatory tone, indicating Ahr−/−-microbial dependence. This work has been published. These data suggest a role for the AHR in influencing the community structure of the intestinal microbiota and provided important insights into methodology and key aspects of the microbiome to focus on in our broccoli studies. We have now mostly completed the studies outlined in objectives one and two. The results indicate that indeed the presence of the Ah receptor has a major influence on the ability of broccoli to alter the gut microbiome. Long term activation of the Ah receptor in the colon did not lead to an increase in inflammatory signaling.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Murray, I.A., Nichols, R.G., Zhang, L., Albert, I., Patterson, A.D., and Perdew, G.H. (2016) Expression of the aryl hydrocarbon receptor is necessary to maintain intestinal host-microbe homeostasis. Scientific Reports 6, 33969.
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Progress 02/15/15 to 02/14/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A graduate student has learned new techniques such as how to work with bacterial 16S large data sets and the development of mouse diets. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will complete the studies in objective 1 and plan on submitting a manuscript for publication.
Impacts
What was accomplished under these goals?
This is the first year of a study to examine the ability of dietary broccoli to altered the microbiome in the gastrointestinal (GI) tract of mice. Curiferous vegetables contain chemicals that degrade in the gastrointestinal tract to Aryl hydrocarbon receptor (AHR) ligands. Activation of the Ah receptor has been shown to have a positive effect on the immune system in the GI tract as far as maintaining appropriate homeostasis with the gut microbiome. We will determine the overall effects of broccoli consumption on the gut microbiome and how much of these effects is due to AHR activation. These studies will lead to better dietary recommendation to maintain health and even treat gastrointestinal disease. During the first five months of this funding period we had to wait to obtain a large batch of broccoli from a local grower. During this time we have completed a series of studies examining the host-microbiome interaction in Ahr+/- or Ahr-/- mice. A manuscript detailing these studies is being developed and will serve as a basis for examining many of the important endpoints in our studies outlined in this proposal. We purchased a large lot of broccoli from a local grower, have freeze dried a large amount of it, and ground it down into a powder. Next we performed a preliminary experiment to test 10 and 15% percent of broccoli powder into a semi-purified diet. We found that incorporation of 15% broccoli resulted in a robust induction of the Ah receptor marker gene Cyp1a1 in the intestinal tract. In our experimental design we wanted to induce Cyp1a1 activity in Ahb allele (high affinity receptor) mice, yet obtain a limited response in our control low affinity Ahd mice. Both strains are congenic for the C57Bl6/J background. This will allow us to assess what changes observed in the host and the microbiome from broccoli consumption is due to activation of the AHR. Mice were fed control or broccoli containing diet for 7 days. The results in figure 1 reveal that in Ahb mice a 55-fold level of induction of Cyp1a1 in the ileum was observed (data not shown). In contrast, only a very low level of broccoli-mediated Cyp1a1 induction was observed in the Ahd (D2N) mice. Next the total number of cecal bacteria was determined by measurement of the amount of DNA for 16S RNA using global bacterial primer and real-time PCR and no difference between control and broccoli diet were obtained (data not shown). The relative concentration of several specific bacteria in the cecal contents was assessed and the number of Firmacutes decreased, while no difference was observed in the relative concentration of Bacteroidetes (data not shown). Generally, it is believed that a decrease in Firmacutes is associated with a healthy microbiome. With the successful completion of this preliminary experiment we are now performing the large-scale mouse feeding studies outlined in objective 1.
Publications
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