Colonic microbiota can promote rapid local improvement of murine colitis by thioguanine independently of T lymphocytes and host metabolism.

OBJECTIVE: Mercaptopurine (MP) and pro-drug azathioprine are 'first-line' oral therapies for maintaining remission in IBD. It is believed that their pharmacodynamic action is due to a slow cumulative decrease in activated lymphocytes homing to inflamed gut. We examined the role of host metabolism, lymphocytes and microbiome for the amelioration of colitis by the related thioguanine (TG). DESIGN: C57Bl/6 mice with or without specific genes altered to elucidate mechanisms responsible for TG's actions were treated daily with oral or intrarectal TG, MP or water. Disease activity was scored daily. At sacrifice, colonic histology, cytokine message, caecal luminal and mucosal microbiomes were analysed. RESULTS: Oral and intrarectal TG but not MP rapidly ameliorated spontaneous chronic colitis in Winnie mice (point mutation in Muc2 secretory mucin). TG ameliorated dextran sodium sulfate-induced chronic colitis in wild-type (WT) mice and in mice lacking T and B lymphocytes. Remarkably, colitis improved without immunosuppressive effects in the absence of host hypoxanthine (guanine) phosphoribosyltransferase (Hprt)-mediated conversion of TG to active drug, the thioguanine nucleotides (TGN). Colonic bacteria converted TG and less so MP to TGN, consistent with intestinal bacterial conversion of TG to so reduce inflammation in the mice lacking host Hprt. TG rapidly induced autophagic flux in epithelial, macrophage and WT but not Hprt-/- fibroblast cell lines and augmented epithelial intracellular bacterial killing. CONCLUSIONS: Treatment by TG is not necessarily dependent on the adaptive immune system. TG is a more efficacious treatment than MP in Winnie spontaneous colitis. Rapid local bacterial conversion of TG correlated with decreased intestinal inflammation and immune activation.

Considering external information to improve the phylogenetic comparison of microbial communities: a new approach based on constrained Double Principal Coordinates Analysis (cDPCoA).

The use of next-generation sequencing technologies is revolutionizing microbial ecology by allowing a deep phylogenetic coverage of tens to thousands of samples simultaneously. Double Principal Coordinates Analysis (DPCoA) is a multivariate method, developed in community ecology, able to integrate a distance matrix describing differences among species (e.g. phylogenetic distances) in the analysis of a species abundance matrix. This ordination technique has been used recently to describe microbial communities taking into account phylogenetic relatedness. In this work, we extend DPCoA to integrate the information of external variables measured on communities. The constrained Double Principal Coordinates Analysis (cDPCoA) is able to enforce a priori classifications to retrieve subtle differences and (or) remove the effect of confounding factors. We describe the main principles of this new approach and demonstrate its usefulness by providing application examples based on published 16S rRNA gene data sets.

Highlighting new phylogenetic specificities of Crohn's disease microbiota.

BACKGROUND: Recent studies suggest that gastrointestinal (GI) microbes play a part in the pathogenesis of Crohn's disease (CD). METHODS: Fecal samples were collected from 16 healthy individuals and 16 CD patients (age- and sex-matched). The DNA extracted from these samples were subjected to two different methods of microbiome analysis. Specific bacterial groups were quantified by real-time polymerase chain reaction (PCR) methods using primers designed using a high-throughput in-house bioinformatics pipeline. The same DNA extracts were also used to produce fluorescently labeled cRNA amplicons to interrogate a custom-designed phylogenetic microarray for intestinal bacteria. RESULTS: Even though the intersubject variability was high, differences in the fecal microbiomes of healthy and CD patients were detected. Faecalibacterium prausnitzii and Escherichia coli were more represented in healthy and ileal CD patients, respectively. Additionally, probes specific for Ruminococcus bromii, Oscillibacter valericigenes, Bifidobacterium bifidum, and Eubacterium rectale produced stronger hybridization signals with the DNA samples from healthy subjects. Conversely, species overrepresented in CD patients were E. coli, Enterococcus faecium, and species from the Proteobacteria not normally found in the healthy human GI tract. Furthermore, we detected "healthy specific" molecular species or operational taxonomic units (OTUs) that are not closely related to any known species (Faecalibacterium, Subdoligranulum, and Oscillospora species), indicating that the phylogenetic dysbiosis is broader than at strain or species level. CONCLUSIONS: These two techniques of microbiome analysis provided a statistically robust new picture of the dysbiosis in fecal microbiota from ileal CD patients. Specifically, we identified a set of six species discriminant for CD, which provides a preliminary diagnostic tool.