Federico E. Rey, Ph.D. - US grants

? DESCRIPTION (provided by applicant): The collections of microbes (i.e., microbiota) that inhabit the human intestine have profound effects on development, physiology and health. Alterations in the gut microbiota contribute to metabolic disorders including obesity and diabetes. Gut microbes affect our physiology at least in part by metabolizing bile acids (BAs). BAs are key nodes of metabolic communication between gut microbes and the host; they are synthesized in the host liver, have antimicrobial effects, facilitate the absorption of lipids, andact as hormones to modulate glucose homeostasis, lipid metabolism, energy expenditure, and intestinal motility. Gut microbes in turn metabolize BAs and regulate their synthesis and their influence on host physiology. However, the genes that modulate the composition of the gut microbiota and abundance of individual species of BAs remain largely unknown. We propose to combine the power of the Diversity Outbred (DO) mouse panel, a newly developed resource that contains as much genetic variation as the human population, with biochemical analyses of BAs and gut microbiota profiling to identify genes and pathways that modulate gut microbial composition and abundance of BAs, and are associated with disease susceptibility. The phenotypic diversity and high-resolution genetic mapping of the DO mice will direct our use of select gnotobiotic hosts of different genetic backgrounds to experimentally validate the contributions of these genes and pathways on gut microbial composition, abundance of BAs and disease susceptibility. The proposed studies are based on three central hypotheses: (i) host genetic variation alters microbiota composition; (ii) differences in microbiota composition result in changes in BA composition and BA-dependent host signaling; and (iii) altered BA signaling contributes to the development of metabolic disease. Our preliminary studies on a small cohort of DO mice have revealed an extraordinary level of phenotypic diversity of diabetes-related traits, fecal BAs and gut microbiota composition in response to a prolonged feeding of a western-style high-fat/high-sucrose diet. Our collective expertise in gnotobiotics and gut microbiome (Rey), nutrition, obesity and diabetes (Attie, Keller), metabolomics (Wang) and statistical genetics (Broman) will enable the discovery of novel genetically-driven host-microbe interactions that modulate the development of diet-induced metabolic disease.

Project Description Gut microbiota have been associated with many different disorders, including cardiovascular disease. One common mechanism involves the production, from dietary components, of metabolites that enter the circulation and affect physiologic functions such as inflammation. We propose to perform a comprehensive screen of gut microbiota-derived metabolites that contribute to cardio-metabolic disorders. Using a panel of genetically diverse inbred strains of mice, we will identify microbes and microbiota-derived metabolites that associate with atherosclerosis, followed by validation in human cohorts and mechanistic studies in germ-free mice. The work will be done in three laboratories with complimentary skills: A. Lusis (genetics), F. Rey (microbiology), and Z. Wang (metabolomics). All of the investigators have worked together for several years. The proposal represents an extension of a screen we previously performed using a panel of 100 inbred strains of mice for atherosclerosis (900 mice total). In that screen, we observed over a 200-fold range of lesion development. We now propose to analyze the microbiomes (Aim 1) and plasma metabolomes (Aim 2) of the mice and to relate these to atherosclerosis traits. We will then prioritize the significant associations by studying these in an atherosclerosis case-control human population (Aim 3). Finally, we will study the mechanisms by which the metabolites affect disease using germ-free mouse models (Aim 4). In preliminary studies, the levels of trimethylamine-N-oxide, another microbe-derived molecule shown to contribute to human atherosclerosis, were significantly correlated with lesion development. And, using a subset of the panel, we identified two microbes (A. muciniphila and R. intestinalis) associated with cardiometabolic traits and showed that these exhibited the predicted effects when used to colonize mice. These preliminary studies provide strong validation for the overall approach. We anticipate identifying several novel metabolites associated with atherosclerosis and related traits, and exploring the underlying mechanisms. This should pave the way for novel therapies that target the microbiome.

Project Summary Human and mouse studies have identified changes in the gut microbiome associated with progression of atherosclerosis. While gut microbes provide many benefits to the host (e.g. provide metabolic capabilities not represented in our human genome), they also can be detrimental. Coexistence with our gut microbes is largely enabled by the intestinal barrier?composed of a luminal mucus layer, epithelial cells and an inner functional immunological barrier? which limits the entry of toxins and microbial pro-inflammatory molecules. Previous studies have shown that diet and microbial metabolism modulate intestinal barrier function. Recent work from our team and others has linked changes in the gut microbiome with alterations in intestinal barrier function and cardiometabolic disease. However, the role of intestinal barrier function on atherosclerosis development and the microbial, dietary and host factors that control this process remain largely unexplored. Defining these will open new avenues for disease prevention and treatment, as both diet and the gut microbiome can be modified. We have identified both microbial and host targets associated with intestinal homeostasis, inflammation, and atherosclerosis. Briefly, we examined a panel of over 100 different genetically diverse inbred strains of mice (known as the Hybrid Mouse Diversity Panel, HMDP) for both atherosclerosis susceptibility and gut microbiota composition. In this screen, we identified several bacterial taxa associated with atherosclerosis protection and experimentally validated one predicted protective microbe, Roseburia intestinalis, whose effect depends on the availability of dietary substrates (i.e., fiber) that promote its growth and butyrate production. Moreover, we showed that the beneficial effects of R. intestinalis are associated with improved intestinal barrier function and lower plasma levels of LPS. Furthermore, these effects are mimicked by delivering butyrate to the distal gut. Our HMDP studies also revealed a poorly understood protein expressed primarily in intestinal dendritic cells and macrophages, ADAM-like Decysin-1 (Adamdec1), as a protein responsive to microbiome composition and contributing to intestinal homeostasis, glucose homeostasis and systemic inflammation. In this application, we propose to follow-up on these exciting observations to gain novel mechanistic insights into how modulation of intestinal homeostasis via diet-butyrate-producing bacteria interactions and Adamdec1 affect progression of atherosclerosis. We provide a strong validation for the overall approach, and the work will be done in two laboratories with complementary skills: Dr. Rey (microbiology, gnotobiotic mouse models) and Dr. Lusis (genetics, atherosclerosis). The investigators have worked together for several years. We anticipate discovery of novel mechanisms by which gut bacteria modulate development of atherosclerosis, which should pave the way for novel cardiovascular disease therapies that target the gut microbiome.

PROJECT SUMMARY/ABSTRACT It is becoming increasingly evident that the composition and metabolites produced by the human gut microbiome influence the progression of cardiovascular diseases. While we are continuing to discover important associations between the gut microbiome and human physiology and diseases, we lack the tools and methodology to precisely manipulate gut microbiota to benefit human health. We propose to develop computational models and optimization frameworks to predict community dynamics and functions and design interventions to shift the gut microbiome to desired states. We will design novel bacterial therapeutics that operate autonomously in the mammalian gastrointestinal tract to steer the microbiome towards healthy states. These next-generation bacterial therapeutics will sense important gut microbiome metabolites, process information, and deliver species- specific antimicrobial proteins to reshape the dynamics and functions of this ecosystem. The performance of these bacterial therapeutics will be characterized in vitro using synthetic human gut microbiome communities and in gnotobiotic mouse models of cardiovascular disease. Model-guided microbiome engineering has the potential to transform human medicine and is becoming increasingly important as scientists continue to discover connections between the microbiome and human health and disease.

The age-related processes that contribute to Alzheimer's disease (AD) development, particularly in the prodromal period, are incompletely understood. Age-related reduction in gut microbiome alpha-diversity is apparent in the majority of older adults, and is suspected of contributing to brain changes, including the development of neurodegenerative disease. Our team published the first comprehensive report describing differences in the gut microbiome observed in AD dementia, including reduced diversity in gut microbiota and altered composition in people with AD dementia compared to age-matched controls. Furthermore, we found that differentially abundant genera were associated with cerebrospinal fluid biomarkers of AD, even among individuals who were cognitively unimpaired. Several studies in mouse models of AD indicate that gut microbiota play a role in the development of AD neuropathology, however to date, the mechanisms underlying these effects are virtually unknown. Recently it has also become clear that the innate immune response in AD plays a critical role in mediating the pathology associated with AD; however the interplay between systemic changes and the innate immune response in AD are not well understood, nor is it known how these factors impact the progression of AD pathology. Our overarching goal is to determine the extent to which alterations in the composition of gut microbiome exacerbate and/or accelerate the development of AD pathology. This proposal is based on the central hypothesis that age-associated gut dysbiosis and inflammation weaken gut barrier function, which in turn leads to the systemic dissemination of microbial components, driving an immune response and system wide changes that worsen AD pathology. To test this hypothesis we propose to study well-characterized participants enrolled in the Wisconsin Alzheimer's Disease Research Center as well as conventional and gnotobiotic APPPS1 mice, to address the following specific aims: 1. Determine the longitudinal relationship between gut microbiome (metagenome), gut inflammation and permeability, and the development of AD pathology in human participants, and 2. Determine the effects of modifying gut permeability on AD pathology in mice. We expect that alterations in gut microbiome composition and gut permeability exacerbate AD pathology in humans, and that impairment of intestinal barrier function and increased gut permeability alters brain homeostasis and exacerbates AD progression in mouse models of AD. Our research group has been working to determine the role of gut microbiome in the development of AD pathology for the past 5 years, and we are perfectly poised to address the proposed aims. We will leverage our expertise in clinical AD, neuroimmunology, and gut microbiology/gnotobiotic mouse models to successfully carry out the proposed project. Completion of the proposed experiments is expected to lead to the development of novel therapeutic strategies for AD and related dementias.

1 PROJECT SUMMARY 2 Cardiovascular disease (CVD) remains the leading cause of death in the United States and worldwide even with 3 advances in the understanding of risk factors for CVD, the development of prevention interventions to reduce 4 the risks, and the improvements in therapy for patients with CVD. There has been an increasing attention in the 5 contribution of the gut microbiota to age-associated CVD, as both human and mouse studies associate changes 6 in the gut microbiome with disease progression. Aging is the dominant risk factor for atherosclerotic CVD, and 7 the process of aging such as inflammation and cellular senescence can be influenced by the gut microbiome. 8 Atherosclerosis is a chronic, low grade inflammatory response that attracts cells of the innate and adaptive 9 immune systems into the atherosclerotic plaque. Both aging and atherosclerosis have been linked with 10 alterations in mucosal-associated invariant T (MAIT) cells ? a subset of innate like T cells that localize 11 preferentially to the gastrointestinal tract and recognize microbial-derived vitamin B metabolite antigens. Germ- 12 free (GF) mice are deficient in MAIT cells, and colonization of feces from conventionally-raised mice into GF 13 mice restores the MAIT frequencies in tissues, suggesting that gut microbes are necessary for the complete 14 development of MAIT cells. Our collaborator Dr. Clement has recently found that elderly subjects and patients 15 with metabolic syndrome exhibit a decreased frequency and functional defects of MAIT and that this 16 phenomenon is more pronounced in patients with CVD. Recent developments of tools and rodent models have 17 provided insight into the mode of action and characterization of MAIT cells in diseases, but their roles in aging 18 and atherosclerosis remains to be elucidated. Moreover, the contribution of human gut microbiota to the induction 19 of MAIT cells are still unknown. Using a gnotobiotic mouse model with human fecal samples, our preliminary 20 data showed that mice colonized with feces from a high MAIT subject had significantly higher abundance of 21 MAIT cells compared to mice with feces from a low MAIT subject. We now propose to extend this study to 22 characterize the frequency and function of MAIT cells during atherosclerosis development (Aim 1a); to elucidate 23 the role of MAIT cells on atherosclerosis using MR1 deficient mice (Aim 1b); and to test the contribution of distinct 24 human gut microbiota to MAIT cell induction (Aim 2a) and atherosclerosis (Aim 2b). Combining novel 25 experimental tools in studying MAIT cell, such as antigen-loaded MR1 tetramers, a magnetic bead-based 26 enrichment method of murine MAIT cells, and MAIT cell-deficient MR1-/- mice, with innovative experimental 27 approaches to investigate the role of the gut microbiota on atherosclerosis (i.e., gnotobiotic atherosclerosis- 28 prone mice colonized with human gut microbiota), we anticipate to discover novel mechanisms by which the 29 MAIT ? gut microbiota interactions modulate development of atherosclerosis, will pave the way for novel 30 microbiome-center therapies to prevent this and potentially other aging-associated diseases.