Project description:The intestinal microbiota has been identified as an environmental factor that markedly impacts energy storage and body fat accumulation, yet the underlying mechanisms remain unclear. Here we show that the microbiota regulates body composition through the circadian transcription factor NFIL3. Nfil3 transcription oscillates diurnally in intestinal epithelial cells and the amplitude of the circadian oscillation is controlled by the microbiota through type 3 innate lymphoid cells (ILC3), STAT3, and the epithelial cell circadian clock. NFIL3 controls expression of a circadian lipid metabolic program and regulates lipid absorption and export in intestinal epithelial cells. These findings provide mechanistic insight into how the intestinal microbiota regulates body composition and establish NFIL3 as an essential molecular link among the microbiota, the circadian clock, and host metabolism. Overall design: 1. RNAseq analysis of epithelial cell transcripts in Nfil3fl/fl and Nfil3ΔIEC mice at 6 time points across a circadian cycle. Laser captured epithelial cells from 3 mice were pooled together for RNAseq at each time point. 2. 16s sequencing analysis of fecal bacterial composition in Nfil3fl/fl and Nfil3ΔIEC mice before and after switching from chow diet to high-fat diet.
Project description:Chronic acid suppression by proton pump inhibitor (PPI) has been hypothesized to alter the gut microbiota via a change in intestinal pH. To evaluate the changes in gut microbiota composition by long-term PPI treatment. Twenty-four week old F344 rats were fed with (n = 5) or without (n = 6) lansoprazole (PPI) for 50 weeks. Then, profiles of luminal microbiota in the terminal ileum were analyzed. Pyrosequencing for 16S rRNA gene was performed by genome sequencer FLX (454 Life Sciences/Roche) and analyzed by metagenomic bioinformatics.
Project description:A mucus layer covers and protects the intestinal epithelial cells from direct contact with microbes. This mucus layer not only prevents inflammation but also plays an essential role in microbiota colonization, indicating the complex interplay between mucus composition-microbiota and intestinal health. However, it is unknown whether the mucus layer is influenced by age or sex and whether this contributes to reported differences in intestinal diseases in males and females or with ageing. Therefore, in this study we investigated the effect of age on mucus thickness, intestinal microbiota composition and immune composition in relation to sex. The ageing induced shrinkage of the colonic mucus layer was associated with bacterial penetration and direct contact of bacteria with the epithelium in both sexes. Additionally, several genes involved in the biosynthesis of mucus were downregulated in old mice, especially in males, and this was accompanied by a decrease in abundances of various Lactobacillus species and unclassified Clostridiales type IV and XIV and increase in abundance of the potential pathobiont Bacteroides vulgatus. The changes in mucus and microbiota in old mice were associated with enhanced activation of the immune system as illustrated by a higher percentage of effector T cells in old mice. Our data contribute to a better understanding of the interplay between mucus-microbiota-and immune responses and ultimately may lead to more tailored design of strategies to modulate mucus production in targeted groups. Overall design: Young (3 months) and old (19 months) male and female C57B1 mice were sacrificed by cervical dislocation. Approximately 1 cm of proximal colon was removed for microarray analysis.
Project description:We developed a non-invasive ex vivo HT29 cell-based minimal model to fingerprint the mucosa-associated microbiota fraction in humans. HT29 cell-associated fractions were characterized by the universal phylogenetic array platform HTF-Microbi.Array, both in presence or in absence of a TNF-α-mediated pro-inflammatory stimulus. A high taxonomical level fingerprint profiling of the mucosa-associated microbiota was performed on a group of 12 breast-fed infants and 6 adults (used as controls). Relative abundance of the bacterial species was assessed by using a so-called HTF-Microbi.Array, based on a ligation detection reaction (LDR) - Univerasal array (UA) assay, capable of correctly identify up to 31 intestinal bacterial groups, covering up to 95% of the human gut microbiota
Project description:Advanced age is associated with chronic low-grade inflammation, which is usually referred to as inflammaging. Elderly are also known to have an altered gut microbiota composition. However, whether inflammaging is a cause or consequence of an altered gut microbiota composition is not clear. In this study gut microbiota from young or old conventional mice was transferred to young germ-free mice. Four weeks after gut microbiota transfer immune cell populations in spleen, Peyer’s patches, and mesenteric lymph nodes from conventionalized germ-free mice were analyzed by flow cytometry. In addition, whole-genome gene expression in the ileum was analyzed by microarray. Gut microbiota composition of donor and recipient mice was analyzed with 16S rDNA sequencing. Here we show by transferring aged microbiota to young germ-free mice that certain bacterial species within the aged microbiota promote inflammaging. This effect was associated with lower levels of Akkermansia and higher levels of TM7 bacteria and Proteobacteria in the aged microbiota after transfer. The aged microbiota promoted inflammation in the small intestine in the germ-free mice and enhanced leakage of inflammatory bacterial components into the circulation was observed. Moreover, the aged microbiota promoted increased T cell activation in the systemic compartment. In conclusion, these data indicate that the gut microbiota from old mice contributes to inflammaging after transfer to young germ-free mice. Overall design: Gut microbiota from young or old conventional mice was transferred to young germ-free mice. Four weeks after gut microbiota transfer whole-genome gene expression in the distal ileum was analyzed by microarray.
Project description:Diminished colonic health is associated with various age-related pathologies. In this study, we applied an integrative approach to reveal potential interactions between determinants of colonic health in aging C57BL/6J mice. Analysis of gut microbiota composition revealed an enrichment of various potential pathobionts, including Desulfovibrio spp., and a decline of the health-promoting Akkermansia spp. and Lactobacillus spp. during aging. Intraluminal concentrations of various metabolites varied between ages and we found evidence for an increased gut permeability at higher age. Colonic gene expression analysis suggested that during the early phase of aging (between 6 and 12 months), expression of genes involved in epithelial-to-mesenchymal transition and (re)organization of the extracellular matrix were increased. Differential expression of these genes was strongly correlated with Bifidobacterium spp. During the later phase of aging (between 12 and 28 months), gene expression profiles pointed towards a diminished antimicrobial defense and were correlated with an uncultured Gastranaerophilales spp. This study demonstrates that aging is associated with pronounced changes in gut microbiota composition and colonic gene expression. Furthermore, the strong correlations between specific bacterial genera and host gene expression may imply that orchestrated interactions take place in the vicinity of the colonic wall and potentially mediate colonic health during aging. Overall design: Male C57BL/6J mice were randomly distributed into four groups that were sacrificed at 6, 12, 24 and 28 months. Mice were housed individually and fed an ad libitum semi-synthetic (AIN-93W) control diet. Upon sacrifice at 6, 12, 24 or 28 months of age, colonic scrapings were collected and RNA was isolated for microarray analyses.
Project description:Irritable Bowel Syndrome (IBS) is a disorder of the gut-brain axis, characterized by altered gut function and frequent psychiatric co-morbidity. Although altered intestinal microbiome profiles have been documented, their relevance to the clinical expression of IBS is unknown. To evaluate a functional role of the microbiota, we colonized germ-free mice with fecal microbiota from healthy controls or IBS patients with accompanying anxiety, and monitored gut function and behavior. Mouse microbiota profiles clustered according to their human donors. Despite having taxonomically similar composition as controls, mice with IBS microbiota had distinct serum metabolomic profiles related to neuro- and immunomodulation. Mice with IBS, but not control microbiota, exhibited faster gastrointestinal transit, intestinal barrier dysfunction, innate immune activation and anxiety-like behavior. These results support the notion that the microbiota contributes to both intestinal and behavioral manifestations of IBS and rationalize the use of microbiota-directed therapies in ameliorating IBS. Overall design: We extracted total RNA of colonic tissues extracted from germ-free NIH swiss mice (10-12 weeks) colonized with healthy human gut microbiota (10 mice per fecal sample, 5 fecal samples, total=50 mice), and mice colonized with human microbiota of IBS patients (10 mice per sample, 6 fecal samples, total =60 mice). We then pooled together all the RNA samples of all the mice colonized with the same microbiota, to form a representative sample for the colonization with that specific human fecal sample. We finally ran 11 samples in total through the NanoString nCounter® Gene Expression CodeSet for mouse inflammation genes.