The Enterocyte-Associated Intestinal Microbiota of Breast-Fed Infants and Adults Responds Differently to a TNF-α-Mediated Pro-Inflammatory Stimulus

et al. (2013) The Enterocyte-Associated Intestinal Microbiota of Breast-Fed Infants and Adults Responds Differently to a TNF--Mediated Pro-Inflammatory Stimulus. PLoS ONE 8(11): e81762. doi:10.1371/journal.pone.0081762 The Enterocyte-Associated Intestinal Microbiota of Breast-Fed Infants and Adults Responds Differently to a TNF--Mediated Pro-Inflammatory Stimulus Manuela Centanni 0 Silvia Turroni 0 Clarissa Consolandi 0 Simone Rampelli 0 Clelia Peano 0 Marco 0 Severgnini 0 Elena Biagi 0 Giada Caredda 0 Gianluca De Bellis 0 Patrizia Brigidi 0 Marco Candela 0 Markus M. Heimesaat, Charit, Campus Benjamin Franklin, Germany 0 1 Department of Pharmacy and Biotechnology, University of Bologna , Bologna , Italy , 2 Institute of Biomedical Technologies - Italian National Research Council , Milan , Italy 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. - It is a matter of fact that the human genome does not code for sufficient information to carry out all functions necessary to maintain health. Indeed, for several aspects of our physiology, such as nutrition [1], protection from pathogens [2] and immunological wellbeing [3,4], we strictly depend on our symbiont microbial partner, the gut microbiota (GM). Being extremely dynamic and rapidly adaptable, the gut microbiome represents a plastic coding entity of the human superorganism, strategic to preserve health and homeostasis through the entire life course [5]. This GM plasticity allows our microbial counterpart to adjust the ecological services [6] in response to the specific host needs at different ages [7-10]. In particular, evolved as an integral component of the immune system, the human GM finely calibrates the immunological services at the different host ages [11], supporting the process of immune education during our infancy and maintaining a balanced immune homeostasis along the adult life. Breast-fed infants possess a peculiar GM structure which is dominated by Bifidobacterium and Enterobacteriaceae [12,13]. Within a critical time window of 8 months of life [14], the infant-type GM plays specific functions strategic for the correct maturation of the host immune system functionalities [11], modulating the T cell differentiation process [15] and leading to the acquisition of the mucosal iNKT cell tolerance [16]. These findings have been strengthened by two recent perspective surveys of GM in Danish and Swedish infants, which provided robust evidences that a low bacterial diversity in the early life is associated with an increased risk of immunological disorders later in life [17,18]. Further, the neonatal intestinal immune apparatus has been recently reported as highly responsive to microbial ligands [19], being primed to establish an intense microbe-host immunological cross-talk since birth [11]. At weaning, with the introduction of solid foods, the GM progressively acquires an adult-like profile which is dominated by Bacteroidetes and Firmicutes [20]. This phylogenetic and functional GM architecture provides different immunological functions, specifically calibrated on the needs of the adult host. Indeed, the adult-type microbiota has been reported as essential to maintain a state of alert of the adult innate and adaptive immune system [21] and, at the same time, to preserve the immunological homeostasis favoring a constitutive low-grade physiological inflammatory status [3]. Even if several steps forward have recently been made in the comprehension of the developmental trajectory of the GM-host immunological cross-talk from early infancy to adulthood, the great majority of the studies have been carried out in stool samples without providing information on the specific structure of the mucosa-associated microbiota [22]. However, bacteria interacting with the gut mucosal surface have a role of primary importance in the cross-talk with the host immune system [21]. Establishing a close interaction with the epithelial apex, mucosal microorganisms enhance the level of epithelial crosstalk at the enterocyte surface, shaping the gut immunological environment [23]. In order to shed light on the functional specificity of the GMhost immunological interaction in infants and adults and being aware of the impossibility of invasive sampling in healthy infants in the present paper we developed a non-invasive HT29 cell-based minimal model to characterize the enterocyte adherent GM fraction in human beings. In the light of the fact that stools are considered as representative of the mucosaassociated GM [24,25], our approach involved the coincubation of freshly produced and immediately processed fecal samples with monolayers of the human enterocyte line HT29 [26]. The enterocyte-associated GM fraction was subsequently characterized by a dual approach based on qPCR and the phylogenetic universal array platform HTFMicrobi.Array [27,28]. By using our ex vivo HT29 cell-based model, we investigated the phylogenetic structure of the enterocyte-associated microbiota fraction of 12 breast-fed infants and 6 young adults, whose GM composition was characterized by pyrosequencing of the 16S rDNA V4 region. In order to mimic a host inflammatory response, experiments were performed in the presence or absence of a tumor necrosis factor-alpha (TNF-)mediated inflammatory stimulus. This pro-inflammatory cytokine was selected because pivotal in the intestinal inflammatory processes [29]. According to our data, (...truncated)