Progress 12/01/15 to 11/30/19
Outputs
Target Audience:Our primary targeted audience were other researchers in this field including: nutritional scientists, microbiologists, chemists, scientists in vascular biology and medical doctors. Seminars related to this work were given at the Experimental Biology Annual Meeting (Orlando, FL), at the department of Pathology at Brown University (Providence, RI), The Hormel Institute, at University of Minnesota, (Austin, MN), at the Department of Microbiology. at the University of Chicago, (Chicago, IL), and at the Gordon Research Conference Animal-Microbe Symbioses. (West Dover, VT). Changes/Problems:We had a contamination event at our germ-free facility that caused delays in the completion of aim 3. What opportunities for training and professional development has the project provided?This proposal has provided training opportunities for 3 PhD students, 2 postdocs and 2 undergraduates. How have the results been disseminated to communities of interest?Our results were presented at more than 30 national and international seminars. Additionally, posters and short talks were presented by trainees at the American Heart Association and Beneficial Microbes (American Society for Microbiology) meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Consumption of diets rich in fruits and vegetables reduces the risk of cardiovascular disease (e.g., atherosclerosis). Some of the health benefits associated with these dietary components are derived from pigments present in plants called flavonoids. However, our bodies do not absorb most flavonoids as they are present in the plants. Flavonoids are metabolized by the trillions of microbes that inhabit our gut (i.e., gut microbiota). These products of microbial metabolism have local effects in the gut and also enter our circulation. Our work so far has demonstrated that the effects of flavonoids depend on the presence of bacteria in the gut. We have identified bacterial products made from flavonoids that are associated with protection against cardiovascular disease. We also tested whether these compounds are responsible for the beneficial effects associated with flavonoid consumption. We have also discovered that the diet that is consumed together with the flavonoids modulates their beneficial effects. We found that diets rich in fiber are needed to observe the full effects of flavonoids. Objective 1: 100% completed. To assess the extent of interpersonal variation in gut microbial conversion of a natural source of mixed anthocyanins in vitro and its relationship with microbiota composition and health status. This aim was completed during year 1 (please see Report 2016). Objective 2: 100% completed. To assess whether interpersonal variation in gut microbial metabolism of anthocyanins modulates protective effects of these flavonoids in a gnotobiotic genetic mouse model of atherosclerosis (apo E -/- mice). This aim has been the focus of the last three years. We are using a different flavonoid (quercetin, which is more common than anthocyanins in our diet) because we were having problems with the instability of anthocyanins in the mouse food. Below I describe our main results. We assessed if quercetin, one of the typical flavonol-type flavonoids, has a beneficial effect on the development of atherosclerosis in mice and its effect is dependent on dietary fiber and gut microbiota. Conventionally-raised (ConvR) Apolipoprotein E (ApoE)-deficient mice were fed a low-plant polysaccharide (LPP) diet or the LPP diet supplemented with 0.1% (w/w) quercetin for 16 weeks. Unexpectedly, quercetin did not affect plasma lipid profile or atherosclerosis lesion size. We then fed ConvR or germ-free (GF) ApoE KO mice a high-plant polysaccharide (HPP) diet or the HPP diet supplemented with quercetin. In contrast to the LPP diet, quercetin significantly reduced the atherosclerotic lesion size in ConvR mice fed the HPP diet (aortic sinus mean plaque area; 15.5±5.7×104 μm2 in ConvR mice fed the HPP diet, 9.9±1.9×104 μm2 in ConvR mice fed the HPP plus quercetin diet). Moreover, quercetin did not affect atherosclerotic lesion size in GF ApoE KO mice. Untargeted metabolomics in plasma revealed that levels of derivatives 3-(3-hydroxyphenyl)propionic acid and its sulfated form were significantly elevated in ConvR mice fed the HPP plus quercetin diet while these levels were below the level of detection in GF mice. Another downstream catabolite of quercetin, homovanillate sulfate which could be derived from breakdown of 3,4- dihydroxyphenylacetate, was also elevated in ConvR mice fed the HPP plus quercetin. These results suggested that quercetin inhibits the development of atherosclerosis in the presence of dietary plant polysaccharide and the beneficial effect on atherosclerosis is dependent on gut microbiota, which is associated with quercetin metabolites in the circulation. Additional experiments were performed to individually test the role of3-(3-hydroxyphenyl) propionic acid on atherosclerosis, however no effects on atherosclerosis was seen in the presence of this metabolite. The metabolite was delivered to the animals using subcutaneously implanted pellets. We spent a lot of efforts optimizing this experiment. However, we were never able to observe the increase in the plasma levels of this metablite. We have decided to write the results in a publication (submission Spring 2020). Objective 3: To test effects of PCA-producing and PCA-degrading bacteria in a gnotobiotic genetic mouse model of atherosclerosis (apo E -/- mice). We have completed ~50% of this aim during year 2017/2018. The goal during the no-cost extension year was to finish this aim. The idea of this aim was to test different routes of flavonoid degradation as modulators of the effects of flavonoids. In our in vitro screen of 100 human gut isolates, in which bacteria were grown anaerobically with the flavonoid, we have identified by HPLC taxa that can metabolize flavonoids to 3,4 dihydroxyphenylacetic acid (DOPAC) and other organisms that make 3,4 dihydroxybenzoic acid (PCA). We were planning to test in gnotobiotic ApoE mice whether the different routes of flavonoid degradation convey similar benefits against atherosclerosis using synthetic communities. In our preliminary studies in mice, we have found that flavonoid-degrading bacteria do not colonize well unless the high fiber diet is used. Unfortunately, our germ-free facility suffered a contamination event in 2019 and we lost temporarily access to germ-free ApoE-/- mice. We are re-deriving these animals GF and plan to complete these experiments.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Kasahara K, Rey FE. The emerging role of gut microbial metabolism on cardiovascular disease. Curr Opin Microbiol. 2019 Aug;50:64-70. doi: 10.1016/j.mib.2019.09.007. Epub 2019 Nov 3.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Rodriguez-Casta�o GP, Dorris MR, Liu X, Bolling BW, Acosta-Gonzalez A, Rey FE. Bacteroides thetaiotaomicron Starch Utilization Promotes Quercetin Degradation and Butyrate Production by Eubacterium ramulus. Frontiers Microbiology.Front Microbial. 2019 May 29;10:1145. doi: 10.3389/fmicb.2019.01145.
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Progress 12/01/17 to 11/30/18
Outputs
Target Audience:We primarily targeted other researchers in this field including nutritional scientists, microbiologists, chemists, scientists in vascular biology and medical doctors. A seminar on this work was given at Wisconsin Medical College (Milwaukee, WI), Experimental Biology Annual Meeting (San Diego, CA), American Society of Gastroenterology Annual Meeting Washington,D.C), and Universidad Nacional de Cordoba (Argentina). I talked about this work at "The Larry Meiller Show" (Wisconsin Public Radio). I participated in a program (Bacteriology) where we explained in general terms the importance of gut microbes to human health, nutrition and this wok, in which listeners called in with questions. Additionally, I discussed this work at UW Lobby day 2018 in Washington D.C. At this event, I discussed with members of congress the importance of federally funded research and discussed the implications of our findings with the Hatch grant. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This grant provided opportunities to train one undergraduate student, one graduate student (MS), one visiting graduate (PhD) student and a postdoctoral fellow in techniques related to anaerobic microbiology, gnotobiology and cardiovascular disease. It also provided opportunities for a postdoc to present at the 2018 American Heart Association General Meeting, and the 2018 Beneficial Microbes meeting and the training of a visiting graduate student from Colombia to present at the 2018 Beneficial Microbes meeting. How have the results been disseminated to communities of interest?We have presented our results at the 2018 American Heart Association General Meeting (Chicago, Il), 2018 Beneficial Microbes meeting (Madison, WI),American Society of Gastroenterology Annual Meeting (Washington, DC), 2018 Experimental Biology Annual Meeting (San Diego, CA) and at various universities including University of California-Los Angeles (Department of Genetics) and Medical College of Wisconsin (Microbiology). What do you plan to do during the next reporting period to accomplish the goals?We will continue working in Aim 3 and plan complete experiments to try to understand how different routes of flavonoid degradation modulate the beneficial effects of flavonoids on CVD, and what bacteria are the most effective.
Impacts
What was accomplished under these goals?
Consumption of diets rich in fruits and vegetables reduces the risk of cardiovascular disease (e.g., atherosclerosis). Some of the health benefits associated with these dietary components are derived from pigments present in plants called flavonoids. However, our bodies do not absorb most flavonoids as they are present in the plants. Flavonoids are metabolized by the trillions of microbes that inhabit our gut (i.e., gut microbiota). These products of microbial metabolism have local effects in the gut and also enter our circulation. Our work so far has demonstrated that the effects of flavonoids depend on the presence of bacteria in the gut. We have identified bacterial products made from flavonoids that are associated with protection against cardiovascular disease. We are testing whether these compounds are responsible for the beneficial effects associated with flavonoid consumption. We have also discovered that the diet that is consumed together with the flavonoids modulates their beneficial effects. We have found that diets rich in fiber are needed to observe the full effects of flavonoids. Objective 1: 100% completed. To assess the extent of interpersonal variation in gut microbial conversion of a natural source of mixed anthocyanins in vitro and its relation with microbiota composition and health status. This aim was completed during year 1 (please see Report 2016). No work on this aim was done during 2018. Objective 2: 80% completed. To assess whether interpersonal variation in gut microbial metabolism of anthocyanins modulates protective effects of these flavonoids in a gnotobiotic genetic mouse model of atherosclerosis (apo E -/- mice). This aim has been the focus of the last two years. We are using a different flavonoid (quercetin, which is more common than anthocyanins in our diet) because we were having problems with the instability of anthocyanins in the mouse food. Below I descibe our main findings. We assessed if quercetin, one of the typical flavonol-type flavonoids, has a beneficial effect on the development of atherosclerosis in mice and its effect is dependent on dietary fiber and gut microbiota. Conventionally-raised (ConvR) Apolipoprotein E (ApoE)-deficient mice were fed a low-plant polysaccharide (LPP) diet or the LPP diet supplemented with 0.1% (w/w) quercetin for 16 weeks. Unexpectedly, quercetin did not affect plasma lipid profile or atherosclerosis lesion size. We next fed ConvR or germ-free (GF) ApoE KO mice a high-plant polysaccharide (HPP) diet or the HPP diet supplemented with quercetin. In contrast to the LPP diet, quercetin significantly reduced the atherosclerotic lesion size in ConvR mice fed the HPP diet (aortic sinus mean plaque area; 15.5±5.7×104 μm2 in ConvR mice fed the HPP diet, 9.9±1.9×104 μm2 in ConvR mice fed the HPP plus quercetin diet). Moreover, quercetin did not affect atherosclerotic lesion size in GF ApoE KO mice. Untargeted metabolomics in plasma revealed that levels of derivatives 3-(3-hydroxyphenyl)propionic acid and its sulfated form were significantly elevated in ConvR mice fed the HPP plus quercetin diet while these levels were below the level of detection in GF mice. Another downstream catabolite of quercetin, homovanillate sulfate which could be derived from breakdown of 3,4-dihydroxyphenylacetate, was also elevated in ConvR mice fed the HPP plus quercetin. These results suggest that quercetin inhibits the development of atherosclerosis in the presence of dietary plant polysaccharide and the beneficial effect on atherosclerosis is dependent on gut microbiota, which is associated with quercetin metabolites in the circulation. We are in the process of testing complex human microbiotas with different capacity to degrade flavonoids (identified in aim 1) on the development of atherosclerosis. Objective 3: To test effects of PCA-producing and PCA-degrading bacteria in a gnotobiotic genetic mouse model of atherosclerosis (apo E -/- mice). We have completed ~50% of this aim during year 2017/2018. This aim will be the focus of the work done duing 2019 (No-cost Extension year). The idea of this aim is to test different routes of flavonoid degradation as modulators of the effects of flavonoids. In our in vitro screen of 100 human gut isolates, in which bacteria were grown anaerobically with the flavonoid, we have identified by HPLC taxa that can metabolize flavonoids to 3,4 dihydroxyphenylaceticacid (DOPAC) and other organisms that make 3,4 dihydroxybenzoic acid (PCA). We are testing in gnotobiotic mice whether the different routes of flavonoid degradation convey similar benefits to the host using synthetic communities. In our preliminary studies in mice, we have found that flavonoid-degrading bacteria do not colonioze well unless the high fiber diet is used. We are planning to use this diet for our future experiments. Conclusion: Altogether our data suggest that microbes modulate beneficial effects of flavonoids. Using diet(s) that promote robust colonization of flavonoid-metabolizing bacteria may be key to achieve highest efficacy.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Kasahara K, Krautkramer KA, Org E, Romano KA, Kerby RL, Vivas EI, Mehrabian M, Denu JM, B�ckhed F, Lusis AJ, Rey FE. 2018. Interactions between Roseburia intestinalis and diet modulate atherogenesis in a murine model. Nature Microbiology 3, 1461-71.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Dill-McFarland KA#, Tang ZZ, Kemis JH, Kerby RL, Chen G, Palloni A, Sorenson T, Rey FE, Herd P. 2019. Social relationships, social isolation, and the human gut microbiota. Scientific Reports 9 (1), 703.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Skye SM, Zhu W, Romano KA#, Guo CJ, Wang Z, Jia X, Kirsop J, Haag B, Lang J, DiDonato JA, Tang WHW, Lusis AJ, Rey FE, Fischbach MA, Hazen SL. 2018. Microbial transplantation with human gut commensals containing CutC is sufficient to transmit enhanced platelet reactivity and thrombosis potential. Circulation Research 123 (10):1164-1176.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Yang H, Wang W, Romano KA, Gu M, Sanidad KZ, Kim D, Yang J, Pei R, Martin DA, Ozay E, Wang Y, Song M, Bolling BB, Xiao H, Minter LM, Liu Z, Rey FE and Zhang G. 2018. Common antimicrobial additive increases colonic inflammation and colitis-associated colon tumorigenesis in mice. Science Translational Medicine 10(443). pii: eaan4116
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
Rodriguez-Casta�o GP, Dorris MR, Liu X, Bolling BW, Acosta-Gonzalez A, Rey FE. Bacteroides thetaiotaomicron Starch Utilization Promotes Quercetin Degradation and Butyrate Production by Eubacterium ramulus. Under review Frontiers Microbiology.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
Bresciani L, Angelino D, Vivas E, Kerby R, Garc�a-Viguera C , Del Rio D, Rey FE, Mena P. Differential catabolism of an anthocyanin-rich elderberry extract by three gut microbiota bacterial species. Submitted to Journal of Agricultural and Food Chemistry.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Romano KA, Dill-McFarland K, Kasahara K, Kerby RL, Vivas EI, Amador-Noguez D, Herd P, Rey FE. 2018. Fecal Aliquot Straw Technique (FAST) allows for easy and reproducible sub-sampling: Assessing interpersonal variation in trimethylamine-N-oxide (TMAO) accumulation. Microbiome 18;6(1):9.1
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Progress 12/01/16 to 11/30/17
Outputs
Target Audience:We primarily targeted other researchers in this field including nutritional scientists, microbiologists, chemists, scientists in vascular biology and medical doctors. A seminar on this work was given at the Food Research Institute (UW-Madison & University of Chicago joined meeting), University of California-Los Angeles, Icahn School of Medicine, Mount Sinai, International meetings in Colombia and Germany. Outreach talks were given at FermentationFest (Sauk County, WI), and for the PEOPLE program (audience was 8th graders going into high school). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?It provided opportunities for a Postdoc to present at the atherosclerosis and Vascular Biology Meeting 2017, and for training of a visiting graduate student from Colombia. How have the results been disseminated to communities of interest?We presented informal discussions at FermentationFest, organized by Wormfarm Institute (Fermentation Fest brings together farmers, chefs, artists, and performers in the beautiful working lands of Sauk County, WI for tastings, demonstrations, cooking classes, art events, performances, food carts and more). Presented at Salon 360 radio show meeting (attendees at this event include professors in various disciplines, K-12 teachers, business people, activists, and laborers (smart folk, but not necessarily science specialists; Kansas City, MO). Our work was also highlighted by Supporters of Agricultural Research (SoAR) Foundation (Retaking the Field) and the materials distributed to various lobbyists to inform on the importance of USDA funded research. Planning to present to members of Congress (Washington DC) March 14 2018 on the importance of this type of research. What do you plan to do during the next reporting period to accomplish the goals?We will continue working in Aim 3 and plan complete experiments with synthetic communities to try to understand why bacteria are necessary for the beneficial effects of flavonoids on CVD, and what bacteria are the most effective.
Impacts
What was accomplished under these goals?
This year we focused on Specific Aim 3: To test effects of flavonoid-metabolizing bacteria in a gnotobiotic genetic mouse model of atherosclerosis (APOE -/- mice). The beneficial effect of flavonoids on atherosclerosis depends on gut bacteria. We supplemented a diet high in plant polysaccharides (HPP) with 0.2% quercetin (HPP+Q; both diets were irradiated to eliminate any potential microbes from the diet). Six-old week apolipoprotein E-deficient (APOE-/-) mice germ-free and conventionally raised were fed control diet or the diet supplemented with quercetin for 16 weeks. All groups of mice (4 groups, n=10-17 mice/group), were housed in in gnotobiotic conditions for the duration of the experiment. Mice were euthanized at 22 weeks of age; at this point we collected liver/intestine and peripheral blood samples for biochemical analyses, and dissected/fixed sinus aortic tissue for histological analyses of atherosclerotic plaques. Plasma and hepatic lipid profile (total cholesterol, HDL-cholesterol, triglycerides) analyses indicate that none of these lipids were affected by Quercetin either in germ-free mice or in colonized animals. Atherosclerotic lesions were visualized and quantified in the aortic sinus using Oil Red O staining. We found that conventionally-raised mice showed a 40% reduction (p<0.01) in response to dietary supplementation with quercetin, whereas germ-free animals were not affected by treatment with the flavonoid for 16 weeks. HPLC/MS measurements of cecal contents disclosed high levels of quercetin in the cecum of germ-free animals but no flavonoid was detectable in colonized mice. Altogether these results suggest that gut microbes metabolize quercetin and this effect may be associated with improvement in vascular disease. Dietary fiber modulates the effect of Quercetin on atherosclerosis. We tested the impact of dietary fiber on the effect of quercetin. Colonized APOE-/- mice were fed 0.2% quercetin mixed with either a high-plant polysaccharide diet or a diet that lack polysaccharides (LPP) accessible to gut microbes (i.e., it contained cellulose, which cannot be degraded by the vast majority of gut bacteria). Four group of animals were exposed to one of four diets (i.e., LPP, LPP+Q, HPP, HPP+Q; 8=10 animals/group) for 16 weeks. After this time, animals were euthanized and atherosclerotic lesions were visualized and quantified in the aortic sinus using Oil Red O staining. As mentioned previously, colonized animals fed the HPP diet supplemented with quercetin exhibited a significant reduction in atherosclerosis (compared to HPP fed mice). This effect was not observed in animals fed a diet with low content of complex plant polysaccharides. Quercetin caused changes in distal gut microbiota composition in both diets. Remarkably, these effects varied dramatically between diets. Among the taxa that increased in the HPP diet were members of the Lachnospiraceae, which have the capability to generate butyrate (a microbial-derived short fatty acid, with anti-inflammatory properties). Taxa that metabolize Quercetin/Rutin require plant polysaccharides for robust colonization. Using germ-free mice colonized with synthetic communities, we tested whether the ability to colonize by microbes adept at metabolizing flavonoids depended on dietary fiber. We tested 3 quercetin degrading organisms: (i) Eubacterium Ramulus, (ii) Flavonifractor plautii, and (iii) Clostridium CG-19. These organisms can degrade quercetin/ rutin to 3,4 dihydroxyphenylaceticacid (DOPAC), acetate, and butyrate. No other bacterium from the >100 human gut isolates we have screened is capable of degrading these flavonoids. We colonized germ-free B6 mice with a core community consisting of 9 human gut isolates commonly found in the distal human gut including: Akkermansia muciniphila, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Clostridium leptum, Clostridium symbiosum, Collinsella intestinalis, Defulfovibrio piger, Escherichia coli, Ruminococcus torques. Mice were colonized into three major group (n=10/group). Each group was also colonized with one of the 3 flavonoid-degrading organisms mentioned above (Group 1 was co-colonized with Eubacterium Ramulus, Group 2 with Flavonifractor plautii, and Group 3 with Clostridium CG-19). Each of the 3 groups were divided into 2 sub-groups that received either a HPP or LPP diet. E. ramulus and F. plautii were only able to colonize in mice fed the HPP diet but not in the LPP diet whereas CG-19 colonized in both diets but its levels of colonization were 5X higher in the HPP diet relative to LPP diet. Our results suggest that the abundance of plant-polysaccharides in diet impacts the abundance of bacteria capable of degrading flavonoids, and potentially the capacity of the community to respond to their presence. Many studies aimed at studying the role of flavonoids use synthetic diets, which typically lack fermentable plant polysaccharides. This may be problematic if the effect of the particular flavonoid depends of bacterial metabolism as suggested by our data. Conclusion: Altogether our data suggest that microbes modulate beneficial effects of flavonoids. Using diet(s) that promote robust colonization of flavonoid-metabolizing bacteria (e.g, HPP) may be key to achieve highest efficacy. Butyrate-producing bacteria as potential mediators of the beneficial effects of quercetin. As mentioned above, consumption of quercetin/rutin increase abundance of members of the Lachnospiraceae, which include many organisms capable of generating butyrate. Additionally, organisms capable of degrading quercetin have the capability to make butyrate. Humans with metabolic and inflammatory diseases frequently harbor lower levels of butyrate-producing bacteria in their gut. However, it is not known whether variation in the levels of these organisms is causally linked with disease development and whether diet modifies these effects. Using germ-free APOE-/- mice colonized with synthetic microbial communities that differ in their capacity to generate butyrate we found that the prominent human-gut associated butyrate producer Roseburia intestinalis interacts with dietary plant polysaccharides to (i) effect global changes in histone post-translational modifications and gene expression in the intestine, (ii) improve intestinal barrier function, (iii) lower systemic inflammation and (iv) ameliorate atherosclerosis. Altogether, our results illustrate how modifiable diet-by-microbiota interactions impact cardiovascular disease, and suggest that strategies aimed at increasing the representation of butyrate-producing bacteria may provide protection against atherosclerosis. Furthermore, we speculate that some of the beneficial effects of flavonoids are mediated by butyrate-producing bacteria.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Romano KA, Martinez-del Campo A, Kasahara K, Chittim CL, Vivas EI, Amador-Noguez D, Balskus EP, Rey FE. 2017. Metabolic, Epigenetic, and Transgenerational Effects of Gut Bacterial Choline Consumption. Cell Host Microbe 22(3):279-290
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Romano KA, Dill-McFarland1 KA, Kasahara K, Kerby R, Vivas EI, Amador-Noguez D, Herd P, Rey FE. Fecal Aliquot Straw Technique (FAST) allows for easy and reproducible sub-sampling: Assessing interpersonal variation in trimethylamine-N-oxide (TMAO) accumulation. Submitted to Microbiome Journal October 2017
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Kasahara K, Krautkramer KA, Romano KA, Kerby RL, Vivas EI, Mehrabian M, Denu J, Backhed F, Lusis AJ, Rey FE. Interactions between butyrate-producing bacteria and diet modulate intestinal epigenetic programing and atherosclerosis. (Under review in Nature Microbiology, second round)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Yang H, Wang W, Romano KA, Gu M, Sanidad KZ, Kim D, Yang J, Pei R, Martin DA, Ozay E, Wang Y, Song M, Bolling BB, Xiao H, Minter LM, Liu Z, Rey FE* and Zhang G*. Common antimicrobial additive increases colonic inflammation and colitis-associated colon tumorigenesis in mice. (Submitted to Science Translational Medicine October 2017). Interactions/ collaboration started at NIFA meeting 2017.
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Progress 12/01/15 to 11/30/16
Outputs
Target Audience:Scientists in the field and undergraduate and graduate students in microbiology and nutrition. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This grant provided opportunities to train 2 undergraduate students, 2 graduate students and a posdoctoral fellow in techniques related to anaerbic microbiology and gnotobiology. How have the results been disseminated to communities of interest?Some results have been presented at the general meeting of the American Heart Association. What do you plan to do during the next reporting period to accomplish the goals?We are planning to pursue additional experiments to complete Objective 1, particularly by increasing the number of samples examined. Additionally, we plan to begin experiments proposed in Objectives 2. We anticipate preparing a manuscript based on data collected for Objective 1 and Objective 3 by summer 2017.
Impacts
What was accomplished under these goals?
Frequent consumption of foods rich in flavonoids is associated with reduced risk of metabolic (diabetes and obesity) and cardiovascular disease. Recent studies demonstrate that flavonoids are not absorbed as present in food, but rather heavily metabolized by gut microbes that reside the human intestine. These studies also indicate that microbial metabolites resulting from transformation of flavonoids are the ones responsible for their beneficial effects. However, there is wide variation in the type of microbes that humans harbor in their gut (particularly people with metabolic/cardiovascular disease). We hypothesized that this interpersonal microbial differences result in differential metabolism of flavonoids. We are employing well-defined environmental conditions (i.e., gnotobiotic mice, defined diets and anthocyanin sources), sequencing and biochemical approaches to minimize and monitor sources of microbial variation. Our studies to date have identified fecal samples and microbial species that can convert flavonoids into bioactive metabolites. We are currently testing whether these specific microbes enhance the beneficial effects flavonoids have on cardiovascular disease. These studies will enable the discovery of next-generation probiotics that could be given together with the flavonoids to maximize their effect. As we approach an era of personalized nutrition, our studies will help better inform how to make dietary recommendations for treatment/ prevention of CVD that are matched with the metabolic potential of the patient's gut microbes. Objective 1: Major activities completed / experiments conducted: We have tested various sources of anthocyanins (elderberry, purple carrot, purple sweet potato, red radish, chokeberry): elderberry extract most concentrated, with two main components: cyanidin 3-glucoside, cyanidin 3-sambubioside. One challenge that we faced with these "natural" sources of anthocyanins is that they also introduced multiple phenolic compounds including protocatechuic acid (PCA), which will make it hard to assess the effects of these compounds. Because of the complication observed with anthocyanins we are expanding our studies to look at other types (more stable) of flavonoids including quercetin and rutin (also prevalent in the US diet). We have also performed 16S rRNA analysis from 408 human fecal samples (same samples that are being analyzed for their capacity to metabolize flavonoids). Data collected: We assessed metabolism of flavonoids in fecal samples obtained from three healthy adults with no recent history of antibiotic use. Fecal inocula were introduced under anaerobic conditions into a diluted gut microbiota medium supplemented with the natural extract of Flavonoids (0.06% v/v) containing cyanidin 3-glucoside and cyanidin 3-sambubioside. These cultures were incubated anaerobically at 37°C and production of metabolite including PCA was monitored in samples (obtained anaerobically, acidified and filtered), as identified by its characteristic ultraviolet spectrum (absorbances at 260 and 293 nm) and by HPLC chromatographic comparison with an authentic standard used to prepare a standard curve. The HPLC analysis used a photodiode array detector coupled to a C18 reverse phase column with a buffer gradient that initiated with a 5% formic acid solution and transitioned to 5% formic acid in methanol during the 35 minute run, as routinely employed in these analyses. Microbiota composition of 313 fecal samples: 60,000 ± 19,000 sequences per sample were obtained from each sampleAll samples achieved sufficient coverage as determined by Good's coverage > 99% (Table S1), and samples were normalized to 10,000 sequences per sample for all further analyses.In total, 5,341 OTUs binned at 98% similarity were identified in the normalized dataset with 46 to 640 OTUs within individual samples. Most fecal communities were dominated by the bacterial phyla Firmicutes (69 ± 12% SD) and Bacteroidetes (22 ± 10%), though some contained high levels of Verrucomicrobia (2.8 ± 5.1%, max 43%), Proteobacteria (2.6 ± 4.9%, max 47%), or Actinobacteria (2.4 ± 2.8%, max 16%). All other phyla represented less than < 0.5% of communities on average. From these 65 individuals have cardiovascular disease and 248 do not have cardiovascular disease. We found that subjects with cardiovascular disease have a distinct microbiota composition (PERMANOVA P < 0.05). Summary statistics and discussion of results. We have found large variation in the levels of metabolites (including PCA) produced and anthocyanins consumed by fecal samples from different individuals. We are currently testing whether fecal samples from individual with cardiovascular disease metabolize flavonoids differently. Key outcomes or other accomplishments realized: The change is knowledge from this study: (i) there are large interpersonal variation in microbial metabolism of flavonoids; (ii) subjects with cardiovascular disease have a different microbiota compared to healthy subjects. Objective 2: Nothing to report this term, experiments are planned to begin next period. Objective 3: 1) Major activities completed / experiments conducted: We have screened a collection of >100 species for their capacity to metabolize flavonoids and identified species with this property. Using this information, we designed three synthetic communities of microbes that vary in their capacity to metabolize flavonoids. We colonized adult female germ-free apoE -/- mice with these defined communities. Animals were fed a defined control diet or the same diet supplemented with flavonoids (n=10/community/diet total mice=60). 2) Data collected: We have collected metabolite profiles from plasma and cecum from the mice mentioned above. Using a combination of HPLC and GC-MS, we were able to measure 15 metabolites derived from the metabolism of flavonoids. In progress: We are measuring athero lesion size for these mice and statistical analyses. 3) Summary statistics and discussion of results: We found that the composition of the microbiota determines the type and abundance of metabolites derived from the flavonoids in the plasma from these mice. We are currently analyzing how the presence of these compounds correlate with the size of the atherosclerotic lesions in these mice. 4) Key outcomes or other accomplishments realized: We have identified microbes that could serve as probiotics to maximize the effects of flavonoids.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Kazuyuki Kasahara, Robert L Kerby, Kymberleigh A Romano, Eugenio I Vivas, Margarete Mehrabian, Aldons J Lusis, Federico E Rey. 2016. Interactions Between Dietary Fiber and Gut Microbiota Modulate the Development of Atherosclerosis. Presented at the General Scientific Sessions of The American Heart Association. Nov 2016.
Abstract Published in Circulation. 2016;134:A18760
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