Background-Abnormal host-microbe interactions are implicated in the pathogenesis of inflammatory bowel diseases. Previous 16S rRNA sequence analysis of intestinal tissues demonstrated that a subset of Crohn's disease (CD) and ulcerative colitis (UC) samples exhibited altered intestinal associated microbial compositions characterized by depletion of Bacteroidetes and Firmicutes (particularly Clostridium taxa). We hypothesize that NOD2 and ATG16L1 risk

We tested the hypothesis that Crohn’s disease (CD)-related genetic polymorphisms involved in host innate immunity are associated with shifts in human ileum–associated microbial composition in a cross-sectional analysis of human ileal samples. Sanger sequencing of the bacterial 16S ribosomal RNA (rRNA) gene and 454 sequencing of 16S rRNA gene hypervariable regions (V1–V3 and V3–V5), were conducted on macroscopically disease-unaffected ileal biopsies collected from 52 ileal CD, 58 ulcerative colitis and 60 control patients without inflammatory bowel diseases (IBD) undergoing initial surgical resection. These subjects also were genotyped for the three major NOD2 risk alleles (Leu1007fs, R708W, G908R) and the ATG16L1 risk allele (T300A). The samples were linked to clinical metadata, including body mass index, smoking status and Clostridia difficile infection. The sequences were classified into seven phyla/subphyla categories using the Naïve Bayesian Classifier of the Ribosome Database Project. Centered log ratio transformation of six predominant categories was included as the dependent variable in the permutation based MANCOVA for the overall composition with stepwise variable selection. Polymerase chain reaction (PCR) assays were conducted to measure the relative frequencies of the Clostridium coccoides – Eubacterium rectales group and the Faecalibacterium prausnitzii spp. Empiric logit transformations of the relative frequencies of these two microbial groups were included in permutation-based ANCOVA. Regardless of sequencing method, IBD phenotype, Clostridia difficile and NOD2 genotype were selected as associated (FDR ≤0.05) with shifts in overall microbial composition. IBD phenotype and NOD2 genotype were also selected as associated with shifts in the relative frequency of the C. coccoides – E. rectales group. IBD phenotype, smoking and IBD medications were selected as associated with shifts in the relative frequency of F. prausnitzii spp. These results indicate that the effects of genetic and environmental factors on IBD are mediated at least in part by the enteric microbiota.

Background NOD2 single nucleotide polymorphisms have been associated with increased risk of ileal Crohn’s disease. This exploratory study was conducted to compare ileal mucosal gene expression in Crohn’s disease (CD) patients with and without NOD2 risk alleles. Methods Ileal samples were prospectively collected from eighteen non-smoking CD patients not treated with anti-TNFα biologics and nine non-smoking control patients without inflammatory bowel disease undergoing initial resection, and genotyped for the three major NOD2 risk alleles (Arg702Trp, Gly908Arg, Leu1007fs). Microarray analysis was performed in samples from four NOD2R (at least one risk allele) CD patients, four NOD2NR (no risk alleles) CD patients and four NOD2NR controls. Candidate genes selected by significance analysis of microarrays (SAM) were confirmed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays of all the samples. Results SAM detected upregulation of 18 genes in affected ileum in NOD2R compared to NOD2NR CD patients, including genes related to lymphocyte activation. SAM also detected altered ileal gene expression in unaffected NOD2NR ileal mucosal CD samples compared to NOD2NR control samples. QRT-PCR conducted on all the samples confirmed that increased CD3D expression in affected samples was associated with NOD2R status, and that increased MUC1, DUOX2, DMBT1 and decreased C4orf7 expression in unaffected samples was associated with CD, independent of NOD2 status. Conclusions The results support the concept that NOD2 risk alleles contribute to impaired regulation of inflammation in the ileum. Furthermore, altered ileal gene expression, independent of NOD2 status, is detected in the unaffected proximal margin of resected ileum from CD patients.

Whole human genome (Agilent) expression profiling was conducted on disease-unaffected ileal RNA collected from the proximal margin of resected ileum from 47 ileal Crohn's disease (CD), 27 ulcerative colitis (UC) and 25 control patients without inflammatory bowel diseases (IBD). Cluster analysis combined with significance analysis of microarrays (SAM) and principal component analysis (PCA)and was used to reduce the data dimension to identify geneprobe clusters associated with early pathogenic changes in ileal CD and UC. Ingenuity Pathway Analysis (IPA) was used to identify the biological pathways associated with each cluster. We reduced the dimensions of the 26,765 gene probe set to 43 gene-probe clusters. Most of these clusters could be labeled as related to different biological pathways, such as Paneth cell antimicrobial peptides, the formation of organized lymphoid structures, or nuclear receptor signaling and xenobiotic metabolism. Molecular phylogenetic 16S rRNA sequence analysis was completed on 83 DNA samples from the same samples used to generate the gene expression profiles. We conducted an exploratory study to determine if the first principle component (PC1) of these clusters could be linked to specific phyla/subphyla taxa.

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