Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Bacteriology
Non Technical Summary
There is increasing evidence that the nutritional and health effects of food are influenced by the consumer's gut microbiota--a collection of bacteria, eukaryotes archaea and their viruses that is unique to each individual. Therefore, to better define the nutritional value of foods and our nutritional status, we need to understand how our gut microbial differences affect how we metabolize the food we consume. Dietary fiber is widely recognized as beneficial for the prevention of cardiovascular disease (CVD). However, published work and our preliminary studies suggest the benefits of fiber vary depending on the composition of the gut microbiota. Furthermore, we have identified a bacterium that lives in the gut of healthy individuals that breaks down dietary fiber and produces a compound (butyrate) that prevents CVD. Remarkably, butyrate- producing bacteria are depleted in humans with obesity, diabetes and cardiovascular disease relative to healthy individuals. In this application, we propose to use germ-free mice colonized with well-characterized human communities and subject them to defined dietary and microbial interventions to identify the specific fiber and the most effective butyrate-producing bacteria to prevent the development of CVD. This study will increase the fundamental knowledge of food-gut microbe interactions, which in turn will inform the design of prebiotic, probiotic and synbiotic (i.e., combination of diet and probiotic) strategies to prevent/treat atherosclerotic disease.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
0%
Goals / Objectives
The main goal of this project is to test the causal relationship between butyrate-producing bacteria (BPB), dietary fiber, and development of metabolic and vascular disease. We hypothesize that microbial-derived butyrate constitutes a protective mechanism against atherosclerotic plaque formation. Our specific aims are:1a. Test the contributions of five prominent butyrogenic species on metabolic and vascular disease;1b. Identify dietary substrates that enhance beneficial effects of butryrogenic bacteria.
Project Methods
Aim 1a. Test the contributions of five prominent BPB to metabolic and vascular disease.We will test the effects of 5 prominent BPB: Faecalibacterium prausnitzii (Family: Clostridiaceae; it produces butyrate from carbohydrates and acetate), Anaerostipes caccae (Family Lachnospiraceae; generates butyrate from carbohydrates, lactate, 4-hydroxybutyrate or succinate), Holdemanella biformis (Family: Erysipelotrichaceae; can generate butyrate from carbohydrates, 4-hydroxybutyrate or succinate), and Eubacterium halii (Family: Lachnospiraceae; unlike other BPB E. hallii has the capacity to also produce butyrate from lactate and acetate in a low pH environment such as the proximal small intestine). Additionally, we recently discovered taxa associated with the genus Allobacullum (Family: Erysipelotrichaceae) which is also negatively correlated with cardiometabolic traits generates butyrate in vitro (~5mM butyrate from 15 mM glucose; overnight incubation in anaerobic rich media). Six groups of 4 weeks-old GF Apoe-/- mice (n=10/group) will be colonized with the low-BD community and fed the assorted fiber diet. Two weeks after colonization, five of these groups will be inoculated with one of the five BPB listed above (single oral gavage ~108 cells). One group of mice will not receive a BPB. All mice will be on the assorted fiber diet and monitored for 16 weeks after transplantation. Colonization of taxa will be assessed by 16S rRNA gene sequencing.All animal work will be done in the Rey lab gnotobiotic facility. Cecal short-chain fatty acids including butyrate will be assessed using gas chromatography-mass spectrometry (GC/MS). 16S rRNA gene surveys of fecal DNA samples will be used to assess microbial composition and to correlate abundance of taxa with cardiovascular (e.g., atherosclerosis plaque) and metabolic phenotypes (e.g., blood glucose level) collected in the mice. We will also examine atherosclerotic lesions and plasma lipid profiles at the time of sacrifice (See below for details in experimental analyses).Aim 1b. Identify dietary substrates that enhance beneficial effects of Butyrogenic bacteria.We will use the low- and high- BD communities. These fecal samples will be resuspended anaerobically and gavaged into groups of 4-week-old GF Apoe-/- mice housed in separate gnotobiotic cages. Colonized mice will be fed the assorted fiber diet for 2 weeks to reach stability of microbial engraftment. Mice will then be switched to one of seven diets formulated to contain commonly consumed sources of fiber all supplemented at 10%: control diet (ENVIGO TD.97184 diet containing non-fermentable cellulose; n=10); inulin supplemented diet (n=10); pectin supplemented diet (n=10), resistant starch supplemented diet (n=10), short-chain fructo-oligosaccharides supplemented diet (n=10), beta-glucan supplemented diet (n=10) and glucomannan supplemented diet (n=10). Transplanted animals will be housed in a Sentry Sealed Positive Pressure system, which allows experimentation with large number of microbial communities without cross contamination. Two-weeks after the diet switch half of the mice colonized with each community will be supplemented (orally gavaged) three times (three consecutive days) with 108 cells of the BPB showing the strongest effect in aim 1a. Engraftment of the BPB will be monitored by qPCR. Cecal levels of butyrate will be examined by GC/MS at the time of sacrifice. Animals will be monitored for 16 weeks after transplantation. Microbiota composition, metabolic and vascular phenotypes will be examined as described below. Assessment of metabolic clinical traits in transplanted mice: We will assess body weight bi-weekly to determine the rate of weight gain. Fasting insulin will be measured as a surrogate for insulin sensitivity. Glucose tolerance will be measured at the end of the study through a standard oral glucose tolerance test. Additionally, we will measure fasting plasma levels of triglycerides, cholesterol, and LPS as previously.Assessment of atherosclerosis in transplanted mice: Aortic roots will be dissected and embedded in Tissue-Tek OCT compound and flash frozen on dry ice. Serial 10-μm transverse sections will be made generating ~50-100 slices spanning the entirety of the aortic root using Cryostat at the UW Translational Science Biocore. Sections will be stained with Oil Red O at even intervals to span the entire aortic root, and total plaque area and lipid area will be quantified. Quantification of the collagen content, fibrous cap thickness, and necrotic core area will be performed with Masson's trichrome staining. The composition of lesions will be examined by immunostaining with antibody to CD68 (for macrophages), CD3 (for T cells), Ly-6G (for neutrophil) and α-SMA (for smooth muscle cells). Images will be scanned using Eclipse TE300 inverted microscope at the UW Experimental Pathology laboratory and analyzed using ImageJ software. 16S rRNA gene analyses in fecal collected from transplanted mice: Genomic DNA will be extracted from feces in the Rey lab and amplicons generated from variable region 4 (V4) of the bacterial 16S rRNA gene will be sequenced using the Illuminia MiSeq platform in the UW Biotechnology Center. Sequence data will be analyzed in QIIME2 using the high-resolution sample inference method for Illumina amplicon data, DADA2, which identifies unique microbial taxa with fine-scale variation. Taxonomic assignment will be performed using a naïve Bayesian classifier trained on the Greengenes 13_8 database.