Nutrition & Food Science
Non Technical Summary
The gut microbiota is a community of different bacteriagroups that live in our guts and have a large impact on our health. It is linked to several health conditions. Changes in microbiota composition can result in disease. Obesity is an example of a disease that can precipitate from a changed microbiota. A high fat (HF) or high calorie diet can negatively change the gut microbiota composition and reduce the number of different groups of bacteria present in the microbiota. When the microbiota is composed of many different bacterial groups it means the diversity is high and it is healthy. When the number of different bacterial groups decrease, then it means diversity is lowered and this is not associated with good health.Urtica dioica L (UT) grows widely in North America and is used in different international cultures as a vegetable. In our preliminary study, UT vegetable prevented high fat diet induced fat accumulation and insulin resistance. Furthermore, it prevented HF diet induced reduction in health and diversity of gut bacteria and enhanced the proliferation of bacterial species that are associated with health, particularly weight reduction and gut health. The vegetable also induced changes in the immune system. About 80% of our immune system is located in the gut. The bacteria affect how this immune system works to protect us against various diseases.We hypothesize that the effects of UT on the microbiota influences the absorption of dietary fat in the small intestines and therefore the amount of fat that accumulates in fat tissue and liver. We further hypothesize that the effects of UT on the composition of bacterial groups in the gut are behind its impact on fat accumulation i.e reduction in obesity. To test these hypotheses, in Aim 1, we will feed mice diets with or without UT. We will analyze the composition and diversity of gut microbiota in the small intestine and amount of fat absorbed. In Aim 2 we shall study the effect of UT on the immune system and how this is connected to shaping the bacteria composition. The primary outcome of this project is generating new knowledge on how UT benefits the host in terms of a healthier gut bacteria community, a healthier immune system and prevention of obesity.We will disseminatethis information to health scientists, nutrition scientists, the food industry and the agricultural sector with the aim of promoting UT or introducing it as a speciality agricultural crop and vegetable with significant health benefits.
Animal Health Component
Research Effort Categories
Goals / Objectives
GOAL: The goal of this project is to determine the effect of Urtica dioica (UT) vegetable on gut microbiota health, intestinal immune cell phenotype and how these relate to fat absorptionand inflammation. There are two objectives.Objective 1: Determine the effects of UT vegetable on small intestine microbiota composition and diversity and how this relates to fat absorption and accumulation. We hypothesize that UT components attenuate high fat diet induced loss in bacterial diversity, increase the proliferation of specific beneficial species and reduce the amount of dietary fat absorbed and/or assimilated.To test this hypothesis, we will feed mice isocaloric diets with or without UT vegetable and analyze the small intestinal microbiota composition by sequencing the 16S rRNA gene and by targeted nonculture qPCR. We will quantify triglycerides in fecal samples and gene and protein expression or activity of proteins involved in fat absorption, fat transport, lipogenesis, lipolysis and fatty acid oxidation in intestinal tissue, adipose and liver. After identifying bacterial species whose proliferation is enhanced or inhibited by UT vegetable, we will test the effect of these bacterial species on fat absorption using in vitro mouse enteroid cultures. We will aim at completing the work described in this objective within the first two years of the project (First 24 months) Objective 2: Determine the effects of UT vegetable on the intestinal Treg cell-IgA axis and ultimate impact on bacterial diversity and intestinal inflammation. We hypothesize that the changes observed in the microbiota composition and diversity are induced by effects of UT vegetable on the phenotype of intestinal regulatory T cells (Tregs) and on immunoglobulin A (IgA) antibody secretion and actions of IgA.To test this hypothesis, we will obtain fresh intestinal contents and intestinal tissues from mice fed diets with or without UT vegetable. We will quantify IgA coated and uncoated bacteria using flow cytometry. We will separate IgA coated and uncoated bacteria and identify taxa in each fraction by 16s sequencing. From isolated single-cell suspensions of lamina propria immune cells we will quantify IgA+ cell populations and T cells that express the biomarkers CD4, FOXP3 and CD25. CD4+CD25+FOXP3+Tregs play an essential role in intestinal homeostasis and inflammation. We will aim at completing the work described in this objective within the next 1.5 years of the project (month 25-42).Between months 43-48 we shall be working on reports, publications and conference presentations that involve this work. We anticipate to generate 4 publications that we will send to Nutrition journals.We will also seek collaborators for a further project aimed at testing the vegetable in a human clinical setting, i.e translate the findings in a clinical study.
Objective 1 will be divided into 3 parts (1.1, 1.2, 1.3) for methodsObjective 1.1. Determine the effect of UT vegetable on SI microbiome communityAnimal studyWe will use eight-week-old age matched 45 male and 45 female C57BL/6J mice (Jackson labs). Males and females will be separately randomised to 5 groups of 9. Each experimental group will have 9 males and 9 females. All diets will be formulated to be isocaloric. Group 1: Low fat (LF) diet control Group 2: Low fat (LF) diet plus UT (LFUT)Group 3: High fat (HF) diet controlGroup 4: HF diet plus UT (HFUT)Group 5: HF diet plus UT and AVNM antibiotic regimeLF=10 % fat, HF =45% fatSamples collected, variables monitored and determinedFood intake, body weight, insulin resistance, glucose metabolism and fat accumulation will be determined by standard tests.Bacterial DNA will be isolated from the duodenum and jejunum contents. Microbiota membership will be analyzed by 16S rRNA sequencing and Bioinformatics. To identify organisms that correlate with and account for the major differences between microbiota communities, we will perform a random forest analysis of the 16SrRNA data.Objective 1.2. Determine the effect of UT vegetable on tissue markers of fat absorption, lipolysis, fat oxidation and lipogenesis.Tissues collected and analyzed for objective 1.2 will be serum, colon fecal samples, cecal contents, small intestine (SI) tissue, epididymal fat, skeletal muscle and liver. We shall determine:Triglyceride (TAG) content in the fecal and cecal contents to determine the extent of intestinal fat absorption.TAG and free fatty acid content in serum, small intesrtine tissue,liver and muscle.Gene expression and protein expression of proteins involved in lipid transport, TAG formation/lipogenesis, lipolysis, fatty acid oxidation in SI tissue, liver, adipose and skeletal muscle.Objective 1.3. Determine the effect of UT extract on fat absorption in vitro assays using small intestine enteroids16S sequencing will reveal species whose proliferation is enhanced by UT and species whose proliferation is inhibited by UT. We will culture 2 species whose proliferation is most highly increased and two species whose proliferation is inhibited by UT vegetable. From these four cultures we will obtain 'conditioned media' for subsequent experiments below.Intestinal crypts will be isolated (at euthanasia of animal study) and duodenum enteroids cultured and differentiated and used to create 2D monolayers in Trans well plates with 0.4 μm pore polycarbonate membrane inserts. Confluent monolayers will be used to study absorption of lipids.Bacteria will be grown in media with or without the ethanolic/water extract of UT under anaerobic conditions for 48 hours after which filtered conditioned media which we presume contains bioactive metabolites from the bacteria and UT vegetable, will be collected and pooled. Control media will have no bacteria seeded in it.The conditioned media will be divided into 2: one part will be supplemented with 300uM final free fatty acids (FFAs/palmitic acid) bound to BSA to mimic a HF dietary conditions in vitro. Enteroids not receiving FFAs will be treated with BSA only. The conditioned media will be added to enteroid monolayers for 24 hours. In summary, the enteroids will be exposed to: control media with no bacterial metabolites and no excess FFAs, control media with no bacterial metabolites but with excess FFAs, media with bacterial metabolites but no excess FFAs, media with bacterial metabolites and excess FFAs. About 50ul of conditioned or control media will be added to 450ul of enteroid growth media (10% final concentration). This treatment will be separately done with 2 bacterial species whose abundance is identified to be most highly increased by UT in the small intestine and 2 species whose abundance is most highly decreased.After 48 hours, enteroids will be harvested and analyzed for gene and protein expression for:Enzymes that catalyze uptake of fatty acids and the re-synthesis of TAG in enterocytes.Proteins that stimulate secretion of pancreatic enzymes in response to nutrients.Proteins that influence lipoprotein formation, chylomicron formation, regulate fatty acid β oxidation and fatty acid oxidation in intestine.Enteroid monolayers will enable measurement of fats in both the apical compartment and the basolateral compartments. Lipids in the media from each compartment will be extracted by the Folch partition and amounts of FFAs and triglycerides quantified by gas chromatography and colorometric kits. TAGs in the enteroid monolayer cells will be extracted and quantified similarly.Objective 2 will be divided into 2 parts (2.1, 2.2).Objective 2.1. Determine the effect of UT on proportions and taxa of immunoglobulin+ (IgA+) bacteriaWe will obtain fresh small intestine contents and scrapings from mice fed isocaloric diets with or without UT from the same animals used in objective 1 and perform the following tests:Quantify IgA levels- secretoryIgA (sIgA) in the intestinal contents and fecal contents will be quantified using an IgA-specific mouse ELISA kit.Flow cytometric quantification of IgA-bound bacteria-small intetinal contents will be processed to isolate the bacterial pellets. Pellets will be stained with anti-mouse IgA fluorophore-conjugated antibody and then SYBR green before analysis by flow cytometryto quantify IgA coated bacteria.Small intestinal contents will be processed to separate bacteria. The supernatant (with bacteria) will be centrifuged, washed with 0.1% (v/v) BSA. Streptavidin magnetic beads will be used to coat the samples with biotinylated anti-mouse IgA. Using a magnet, IgA bound bacteria will be separated from the unbound bacteria.Samples with IgA-bound and IgA unbound bacteria will be separately processed for DNA extraction and 16S rRNA sequencing and bioinformatics to identify bacterialtaxa targeted for IgA coating and those not coated byIgA.From the 16S rRNA data, we will use a random forest analysis to identify species or taxa whose IgA binding status is changed by UT diet and account for the major differences between HF and HFUT microbiota communities.Objective 2.2. Determine the effect of UT vegetable on intestinal immune cell phenotype and intestinal inflammationWe will obtain primary intestinal tissues, small intestinal mucosal scrapings and fresh fecal samples from mice fed isocaloric diets as shown in Objective 1. The small intestinal lamina propria cells will be isolated and processed to separate the epithelial cell fraction. The cell suspension will be filltered to obtain single-cell suspensions for use in flow cytometry as shown next:Identify and quantify IgA+ cells-using fluorophore-conjugated antibodies we will determine if UT induces changes in intestinal B cell populations. Isolated single-cell suspensions of intestinal lamina propria immune cells will be sorted to quantify frequency and absolute numbers of IgA producing B (IgA+ B220+) and plasma (IgA+ B220−) cells.Identify and quantify Treg cells-we will quantify markers of natural Treg cells and adaptive/induced Treg cells. This will include Foxp3+, CD25+, CD4+ cells.Protein and gene expression of inflammatory and anti-inflammatory cytokines-we will determine the expression of IL-10, ICOS, TGF-β in the mucosal scrapings and in LP cells.Foxp3+ immunohistochemistry- small intestinal tissue will be fixed in formalin, embedded in paraffin and autostained with Foxp3+ polyclonal antibody.Slides will be counterstained with haematoxylin and evaluated to enumerate Foxp3+ cells.