Project description:The spatiotemporal structure of the human microbiome, proteome, and metabolome reflects and determines regional intestinal physiology and may have implications for disease. Yet, we know little about the distribution of microbes, their environment, and their biochemical activity in the gut because of reliance on stool samples and limited access to only some regions of the gut using endoscopy in fasting or sedated individuals. To address these deficiencies, we developed and evaluated a safe, ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion and maintains the viability of microbes from these locations. The collection of 240 intestinal samples from 15 healthy individuals using the device and subsequent multi-omics analyses revealed significant differences between microbes, phages, host proteins, and metabolites present in the intestines versus stool. Certain microbial taxa and gene classes were differentially enriched, and prophage induction was more prevalent in the intestines than in stool. The host proteome and bile acid profiles varied along the intestines and were highly distinct from those of stool. Correlations between gradients in bile acid concentrations and microbial abundancepredicted species that altered the bile acid pool through deconjugation. Furthermore,microbially conjugated bile acids displayed amino acid-dependent trends in concentration that were not apparent in stool. Overall, non-invasive longitudinal profilingof microbes, proteins, and bile acids along the intestinal tract under physiological conditions can help elucidate the roles of the gut microbiome and metabolome in humanphysiology and disease.
Project description:The spatiotemporal structure of the human microbiome1,2, proteome3 and metabolome4,5 reflects and determines regional intestinal physiology and may have implications for disease6. Yet, little is known about the distribution of microorganisms, their environment and their biochemical activity in the gut because of reliance on stool samples and limited access to only some regions of the gut using endoscopy in fasting or sedated individuals7. To address these deficiencies, we developed an ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion. Collection of 240 intestinal samples from 15 healthy individuals using the device and subsequent multi-omics analyses identified significant differences between bacteria, phages, host proteins and metabolites in the intestines versus stool. Certain microbial taxa were differentially enriched and prophage induction was more prevalent in the intestines than in stool. The host proteome and bile acid profiles varied along the intestines and were highly distinct from those of stool. Correlations between gradients in bile acid concentrations and microbial abundance predicted species that altered the bile acid pool through deconjugation. Furthermore, microbially conjugated bile acid concentrations exhibited amino acid-dependent trends that were not apparent in stool. Overall, non-invasive, longitudinal profiling of microorganisms, proteins and bile acids along the intestinal tract under physiological conditions can help elucidate the roles of the gut microbiome and metabolome in human physiology and disease.
Project description:Bile acids are potent antibacterial compounds and play an important role in shaping the microbial ecology of the gut. Here, we combined flow cytometry, growth rate measurements (OD600), and NMR- and mass spectrometry-based metabolomics to systematically profile the impact of bile acids on the microbiome using in vitro and in vivo models. This study confirmed that (1) unconjugated bile acids possess more potent antibacterial activity than conjugated bile acids; (2) Gram-positive bacteria are more sensitive to bile acids than Gram-negative bacteria; (3) some probiotic bacteria such as Lactobacillus and Bifidobacterium and 7α-dehydroxylating bacteria such as Clostridium scindens show bile acid resistance that is associated with activation of glycolysis. Moreover, we demonstrated that (4) as one of most hydrophobic bile acids, lithocholic acid (LCA) shows reduced toxicity to bacteria in the cecal microbiome in both in vivo and in vitro models; (5) bile acids directly and rapidly affect bacterial global metabolism including membrane damage, disrupted amino acid, nucleotide, and carbohydrate metabolism; and (6) in vivo, short-term exposure to bile acids significantly affected host metabolism via alterations of the bacterial community structure. This study systematically profiled interactions between bile acids and gut bacteria providing validation of previous observation and new insights into the interaction of bile acids with the microbiome and mechanisms related to bile acid tolerance.
Project description:Bile acids are synthesized from cholesterol and play an important role in regulating intestinal microflora. The different degrees of hydrophobicity and acidity of individual bile acids may affect their antimicrobial properties. We examined the antimicrobial effects of different bile acids on various microorganisms in vitro and confirmed whether these remain consistent in vivo. Using human bile acids, including ursodeoxycholic acid, cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid, a disc diffusion test was performed, and a rodent model was created to determine the antimicrobial effects of each bile acid. The fecal bacterial population was analyzed using a real-time polymerase chain reaction. Each bile acid showed different microbial inhibitory properties. The inhibitory activity of bile acids against microbiota which normally resides in the gastrointestinal tract and biliary system, was low; however, normal flora of other organs was significantly inhibited. Changes in microbial counts after bile acid administration in a rodent model differed in the colon and cecum. The in vivo and in vitro results show that the antimicrobial effects of bile acids against intestinal microbiota were similar. In conclusion, bile acids could be a novel treatment strategy to regulate gut microbiota.
Project description:Regulatory T cells (Tregs) were shown to protect from cholestatic liver injury. Here we investigate whether amphiregulin (AREG), a ligand for epithelial growth factor receptor (EGFR) which promotes epithelial repair, mediates innate Treg functions in cholangiopathies.
Project description:Inflammatory bowel diseases (IBD) are debilitating chronic inflammatory disorders that develop as a result of a defective immune response toward intestinal bacteria. Intestinal dysbiosis is associated with the onset of IBD and has been reported to persist even in patients in deep remission. We investigated the possibility of a dietary-induced switch to the gut microbiota composition using Winnie mice as a model of spontaneous ulcerative colitis and chow enriched with 1% Bronze tomato. We used the near isogenic tomato line strategy to investigate the effects of a diet enriched in polyphenols administered to mild but established chronic intestinal inflammation. The Bronze-enriched chow administered for two weeks was not able to produce any macroscopic effect on the IBD symptoms, although, at molecular level there was a significant induction of anti-inflammatory genes and intracellular staining of T cells revealed a mild decrease in IL17A and IFNγ production. Analysis of the microbial composition revealed that two weeks of Bronze enriched diet was sufficient to perturb the microbial composition of Winnie and control mice, suggesting that polyphenol-enriched diets may create unfavorable conditions for distinct bacterial species. In conclusion, dietary regimes enriched in polyphenols may efficiently support IBD remission affecting the intestinal dysbiosis.
Project description:Bile acids (BAs) are a group of chemically related steroids recognized as regulatory molecules whose profiles can change in different physio-pathological situations. We have developed a sensitive, fast, and reproducible ultraperformance liquid chromatography/multiple reaction monitoring/mass spectrometry method to determine the tissue and sera BA profiles in different species (human, rat, and mouse) by quantifying 31 major and minor BA species in a single 21-min run. The method has been validated according to FDA guidelines, and it generally provides good results in terms of intra- and interday precision (less than 8.6% and 16.0%, respectively), accuracy (relative error measurement between -11.9% and 8.6%), and linearity (R(2) > 0.996 and dynamic ranges between two and four orders of magnitude), with limits of quantification between 2.5 and 20 nM. The new analytical approach was applied to determine BA concentrations in human, rat, and mouse serum and in liver tissue. Our comparative study confirmed and extended previous reports, showing marked interspecies differences in circulating and hepatic BA composition. The targeted analysis revealed the presence of unexpected minoritary BAs, such as tauro-alpha-Muricholic acid in human serum, thus allowing us to obtain a thorough profiling of human samples. Its great sensitivity, low sample requirements (25 µl of serum, 5 mg of tissue), and comprehensive capacity to profile a considerable number of BAs make the present method a good choice to study BA metabolism in physiological and pathological situations, particularly in toxicological studies.
Project description:We investigated the age-dependent changes in urinary excretion of glucuronidated bile acids at the C-3 position. Bile acid 3-glucuronides accounted for 0.5% of urinary bile acids in neonates, and the proportion of bile acid 3-glucuronides plateaued at 1-3 years of age. The 3-glucuronides of secondary bile acids were first secreted at 3 months of age, the same time as the establishment of the gut bacterial flora in infants. A considerable portion of bile acid 3-glucuronides were present as non-amidated forms. Our results indicate dynamic hepatic enzyme activity in which the levels of uridine 5'-diphospho-glucuronosyltransferases (UGTs) differ by age group, with higher glucuronidation activity of UGTs towards nonamidated bile acids than amidated bile acids.
Project description:Human noroviruses (HuNoVs) cause sporadic and epidemic outbreaks of gastroenteritis in all age groups worldwide. We previously reported that stem cell-derived human intestinal enteroid (HIE) cultures support replication of multiple HuNoV strains and that some strains (e.g., GII.3) replicate only in the presence of bile. Heat- and trypsin-treatment of bile did not reduce GII.3 replication, indicating a nonproteinaceous component in bile functions as an active factor. Here we show that bile acids (BAs) are critical for GII.3 replication and replication correlates with BA hydrophobicity. Using the highly effective BA, glycochenodeoxycholic acid (GCDCA), we show BAs act during the early stage of infection, BA-dependent replication in HIEs is not mediated by detergent effects or classic farnesoid X receptor or Takeda G protein-coupled receptor 5 signaling but involves another G protein-coupled receptor, sphingosine-1-phosphate receptor 2, and BA treatment of HIEs increases particle uptake. We also demonstrate that GCDCA induces multiple cellular responses that promote GII.3 replication in HIEs, including enhancement of 1) endosomal uptake, 2) endosomal acidification and subsequent activity of endosomal/lysosomal enzyme acid sphingomyelinase (ASM), and 3) ceramide levels on the apical membrane. Inhibitors of endosomal acidification or ASM reduce GII.3 infection and exogenous addition of ceramide alone permits infection. Furthermore, inhibition of lysosomal exocytosis of ASM, which is required for ceramide production at the apical surface, decreases GII.3 infection. Together, our results support a model where GII.3 exploits rapid BA-mediated cellular endolysosomal dynamic changes and cellular ceramide to enter and replicate in jejunal HIEs.