The enterocyte-associated intestinal microbiota of breast-fed infants and adults responds differently to a TNF-?-mediated pro-inflammatory stimulus.
ABSTRACT: Co-evolved as an integral component of our immune system, the gut microbiota provides specific immunological services at different ages, supporting the immune education during our infancy and sustaining a well-balanced immunological homeostasis during the course of our life. In order to figure out whether this involves differences in the microbial groups primarily interacting with the host immune system, we developed a non-invasive HT29 cell-based minimal model to fingerprint the enterocyte-associated microbiota fraction in infants and adults. After depicting the fecal microbial community of 12 breast-fed infants and 6 adults by 16S rDNA amplicon pools 454 pyrosequencing, their respective HT29 cell-associated gut microbiota fractions were characterized by the universal phylogenetic array platform HTF-Microbi.Array, both in the presence and absence of a tumor necrosis factor-alpha (TNF-?)-mediated pro-inflammatory stimulus. Our data revealed remarkable differences between the enterocyte-associated microbiota fractions in breast-fed infants and adults, being dominated by Bifidobacterium and Enterobacteriaceae the first and Bacteroides-Prevotella and Clostridium clusters IV and XIVa the second. While in adults TNF-? resulted in a profound impairment of the structure of the enterocyte-associated microbiota fraction, in infants it remained unaffected. Differently from the adult-type gut microbial community, the infant-type microbiota is structured to cope with inflammation, being co-evolved to prime the early immune response by means of transient inflammatory signals from gut microorganisms.
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:Gut microbiota play an important role in human immunological processes, potentially affecting allergic diseases such as eczema. The diversity and structure of gut microbiota in infants with eczema have been previously documented. This study aims to evaluate by comparative metagenomics differences in genetic content in gut microbiota of infants with eczema and their matched controls. Stools were collected at the age of one month old from twelve infants from an at risk birth cohort in a case control manner. Clinical follow up for atopic outcomes were carried out at the age of 12 and 24 months. Microbial genomic DNA were extracted from stool samples and used for shotgun sequencing. Comparative metagenomic analysis showed that immune-regulatory TCAAGCTTGA motifs were significantly enriched in the six healthy controls (C) communities compared to the six eczema subjects (E), with many encoded by Bifidobacterium (38% of the total motifs in the C communities). Draft genomes of five Bifidobacterium species populations (B. longum, B. bifidum, B. breve, B. dentium, and B. pseudocatenulatum) were recovered from metagenomic datasets. The B. longum BFN-121-2 genome encoded more TCAAGCTTGA motifs (4.2 copies per one million genome sequence) than other Bifidobacterium genomes. Additionally, the communities in the stool of controls (C) were also significantly enriched in functions associated with tetrapyrrole biosynthesis compared to those of eczema (E). Our results show distinct immune-modulatory genomic properties of gut microbiota in infants associated with eczema and provide new insights into potential role of gut microbiota in affecting human immune homeostasis.
Project description:Tens of trillions of microorganisms constitute the gut microbiota of the human body. The microbiota plays a critical role in maintaining host immunity and metabolism. Analyses of the gut microbial composition in Korea are limited to a few studies consisting of small sample sizes. To investigate the gut microbial community in a large sample of healthy Koreans, we analyzed the 16S ribosomal RNA of 4 representative bacterial genera Lactobacillus, Bifidobacterium, Bacteroides, and Clostridium.A total of 378 DNA samples extracted from 164 infants and 214 adults were analyzed using quantitative real-time polymerase chain reaction.Analysis of 16S ribosomal RNA of 4 representative bacterial genera Lactobacillus, Bifidobacterium, Bacteroides, and Clostridium showed that the gut microbiota in infants had higher relative abundances of Bifidobacterium and Lactobacillus than that in adults, which was dominated by Bacteroides and Clostridium.To the best of our knowledge, this was the first study evaluating the distinct characteristics of the microbial community of Korean infants and adults. The differences between the 2 populations suggest that external factors such as age, diet, and the environment are important contributing factors to the change in gut microbial composition during development.
Project description:By means of a recently developed non-invasive ex vivo minimal model based on the interaction of the human enterocyte-like HT29 cell line and fecal slurries, we explored the enterocyte-associated microbiome of 21 Hadza hunter-gatherers and nine urban living Italians. Though reductionist, this model allows inferring the microbiota structural and functional arrangement as it interacts with enterocytes. Microbial suspensions obtained from Hadza or Italian stools were first evaluated for structural integrity by high resolution-scanning electron microscopy and co-incubated with HT29 cell monolayers. The enterocyte adherent microbiota fraction was then characterized by 16S rRNA gene sequencing and predictive functional profiling using PICRUSt. Compared to Italians, the Hadza enterocyte-associated microbiome was characterized by a greater amount of adhesive microorganisms with pathogenic potential, such as Proteobacteria, Erysipelotrichaceae, Enterococcus, Clostridium and Sarcina. These compositional characteristics were reflected in a functional enrichment in membrane transport, signal transduction, signaling molecules and interaction. Our results depict a new interesting mutualistic configuration of the enterocyte-associated microbiome in Hadza, stressing the importance of microbe-host interaction at the mucosal surface along the course of human evolution.
Project description:Very few studies have analyzed how the composition of mother's microbiota affects the development of infant's gut and oral microbiota during the first months of life. Here, microbiota present in the mothers' gut, vagina, breast milk, oral cavity, and mammary areola were compared with the gut and oral microbiota of their infants over the first six months following birth. Samples were collected from the aforementioned body sites from seven mothers and nine infants at three different time points over a 6-month period. Each sample was analyzed with 16S rRNA gene sequencing. The gut microbiota of the infants harbored distinct microbial communities that had low similarity with the various maternal microbiota communities. In contrast, the oral microbiota of the infants exhibited high similarity with the microbiota of the mothers' breast milk, mammary areola and mouth. These results demonstrate that constant contact between microbial communities increases their similarity. A majority of the operational taxonomic units in infant gut and oral microbiota were also shared with the mothers' gut and oral communities, respectively. The disparity between the similarity and the proportion of the OTUs shared between infants' and mothers' gut microbiota might be related to lower diversity and therefore competition in infants' gut microbiota.
Project description:Celiac disease (CD) is a unique autoimmune disorder in which the genetic factors (DQ2/DQ8) and the environmental trigger (gluten) are known and necessary but not sufficient for its development. Other environmental components contributing to CD are poorly understood. Studies suggest that aspects of gluten intake might influence the risk of CD occurrence and timing of its onset, i.e., the amount and quality of ingested gluten, together with the pattern of infant feeding and the age at which gluten is introduced in the diet. In this study, we hypothesize that the intestinal microbiota as a whole rather than specific infections dictates the switch from tolerance to immune response in genetically susceptible individuals. Using a sample of infants genetically at risk of CD, we characterized the longitudinal changes in the microbial communities that colonize infants from birth to 24 months and the impact of two patterns of gluten introduction (early vs. late) on the gut microbiota and metabolome, and the switch from gluten tolerance to immune response, including onset of CD autoimmunity. We show that infants genetically susceptible to CD who are exposed to gluten early mount an immune response against gluten and develop CD autoimmunity more frequently than at-risk infants in which gluten exposure is delayed until 12 months of age. The data, while derived from a relatively small number of subjects, suggest differences between the developing microbiota of infants with genetic predisposition for CD and the microbiota from infants with a non-selected genetic background, with an overall lack of bacteria of the phylum Bacteriodetes along with a high abundance of Firmicutes and microbiota that do not resemble that of adults even at 2 years of age. Furthermore, metabolomics analysis reveals potential biomarkers for the prediction of CD. This study constitutes a definite proof-of-principle that these combined genomic and metabolomic approaches will be key to deciphering the role of the gut microbiota on CD onset.
Project description:<h4>Background and aims</h4>It is known that postnatal functional maturation of the small intestine is facilitated by microbial colonization of the gut. Preterm infants exhibit defects in gut maturation, weak innate immunity against intestinal infection and increased susceptibility to inflammatory disorders, all of which may be related to the inappropriate microbial colonization of their immature intestines. The earliest microbes to colonize the preterm infant gut encounter a naïve, immature intestine. Thus this earliest microbiota potentially has the greatest opportunity to fundamentally influence intestinal development and immune function. The aim of this study was to characterize the effect of early microbial colonization on global gene expression in the distal small intestine during postnatal gut development.<h4>Methods</h4>Gnotobiotic mouse models with experimental colonization by early (prior to two weeks of life) intestinal microbiota from preterm human infants were utilized. Microarray analysis was used to assess global gene expression in the intestinal epithelium.<h4>Results and conclusion</h4>Multiple intestinal genes involved in metabolism, cell cycle regulation, cell-cell or cell-extracellular matrix communication, and immune function are developmental- and intestinal microbiota- regulated. Using a humanized gnotobiotic mouse model, we demonstrate that certain early preterm infant microbiota from prior to 2 weeks of life specifically induce increased NF-?B activation and a phenotype of increased inflammation whereas other preterm microbiota specifically induce decreased NF-?B activation. These fundamental differences correlate with altered clinical outcomes and suggest the existence of optimal early microbial communities to improve health outcomes.
Project description:Establishment of the human gut microbiota begins at birth. This early-life microbiota development can impact host physiology during infancy and even across an entire life span. However, the functional stability and population structure of the gut microbiota during initial colonization remain poorly understood. Metaproteomics is an emerging technology for the large-scale characterization of metabolic functions in complex microbial communities (gut microbiota).We applied a metagenome-informed metaproteomic approach to study the temporal and inter-individual differences of metabolic functions during microbial colonization of preterm human infants' gut. By analyzing 30 individual fecal samples, we identified up to 12,568 protein groups for each of four infants, including both human and microbial proteins. With genome-resolved matched metagenomics, proteins were confidently identified at the species/strain level. The maximum percentage of the proteome detected for the abundant organisms was ~45%. A time-dependent increase in the relative abundance of microbial versus human proteins suggested increasing microbial colonization during the first few weeks of early life. We observed remarkable variations and temporal shifts in the relative protein abundances of each organism in these preterm gut communities. Given the dissimilarity of the communities, only 81 microbial EggNOG orthologous groups and 57 human proteins were observed across all samples. These conserved microbial proteins were involved in carbohydrate, energy, amino acid and nucleotide metabolism while conserved human proteins were related to immune response and mucosal maturation. We identified seven proteome clusters for the communities and showed infant gut proteome profiles were unstable across time and not individual-specific. Applying a gut-specific metabolic module (GMM) analysis, we found that gut communities varied primarily in the contribution of nutrient (carbohydrates, lipids, and amino acids) utilization and short-chain fatty acid production.Overall, this study reports species-specific proteome profiles and metabolic functions of human gut microbiota during early colonization. In particular, our work contributes to reveal microbiota-associated shifts and variations in the metabolism of three major nutrient sources and short-chain fatty acid during colonization of preterm infant gut.
Project description:BACKGROUND:Acquisition of the intestinal microbiota in early life corresponds with the development of the mucosal immune system. Recent work on caesarean-delivered infants revealed that early microbial composition is influenced by birthing method and environment. Furthermore, we have confirmed that early-life environment strongly influences both the adult gut microbiota and development of the gut immune system. Here, we address the impact of limiting microbial exposure after initial colonization on the development of adult gut immunity. METHODOLOGY/PRINCIPAL FINDINGS:Piglets were born in indoor or outdoor rearing units, allowing natural colonization in the immediate period after birth, prior to transfer to high-health status isolators. Strikingly, gut closure and morphological development were strongly affected by isolator-rearing, independent of indoor or outdoor origins of piglets. Isolator-reared animals showed extensive vacuolation and disorganization of the gut epithelium, inferring that normal gut closure requires maturation factors present in maternal milk. Although morphological maturation and gut closure were delayed in isolator-reared animals, these hard-wired events occurred later in development. Type I IFN, IL-22, IL-23 and Th17 pathways were increased in indoor-isolator compared to outdoor-isolator animals during early life, indicating greater immune activation in pigs originating from indoor environments reflecting differences in the early microbiota. This difference was less apparent later in development due to enhanced immune activation and convergence of the microbiota in all isolator-reared animals. This correlated with elevation of Type I IFN pathways in both groups, although T cell pathways were still more affected in indoor-reared animals. CONCLUSIONS/SIGNIFICANCE:Environmental factors, in particular microbial exposure, influence expression of a large number of immune-related genes. However, the homeostatic effects of microbial colonization in outdoor environments require sustained microbial exposure throughout development. Gut development in high-hygiene environments negatively impacts on normal succession of the gut microbiota and promotes innate immune activation which may impair immune homeostasis.
Project description:Multiple factors help shape the infant intestinal microbiota early in life. Environmental conditions such as the presence of bioactive molecules from breast milk dictate gut microbial growth and survival. Infants also receive distinct, personalized, bacterial exposures leading to differential colonization. Microbial exposures and gut environmental conditions differ between infants in different locations, as does the typical microbial community structure in an infant's gut. Here we evaluate potential influences on the infant gut microbiota through a longitudinal study on cohorts of breast-fed infants from the neighboring countries of Armenia and Georgia, an area of the world for which the infant microbiome has not been previously investigated. Marker gene sequencing of 16S ribosomal genes revealed that the gut microbial communities of infants from these countries were dominated by bifidobacteria, were different from each other, and were marginally influenced by their mother's secretor status. Species-level differences in the bifidobacterial communities of each country and birth method were also observed. These community differences suggest that environmental variation between individuals in different locations may influence the gut microbiota of infants.