Host Genes Related to Paneth Cells and Xenobiotic Metabolism Are Associated with Shifts in Human Ileum-Associated Microbial Composition

et al. (2012) Host Genes Related to Paneth Cells and Xenobiotic Metabolism Are Associated with Shifts in Human Ileum-Associated Microbial Composition. PLoS ONE 7(6): e30044. doi:10.1371/journal.pone.0030044 Host Genes Related to Paneth Cells and Xenobiotic Metabolism Are Associated with Shifts in Human Ileum- Associated Microbial Composition Tianyi Zhang 0 Robert A. DeSimone 0 Xiangmin Jiao 0 F. James Rohlf 0 Wei Zhu 0 Qing Qing Gong 0 Steven R. Hunt 0 Themistocles Dassopoulos 0 Rodney D. Newberry 0 Erica Sodergren 0 George Weinstock 0 Charles E. Robertson 0 Daniel N. Frank 0 Ellen Li 0 Markus M. Heimesaat, Charite, Campus Benjamin Franklin, Germany 0 1 Department of Applied Mathematics and Statistics, Stony Brook University , Stony Brook , New York, United States of America, 2 Department of Medicine, Stony Brook University , Stony Brook , New York, United States of America, 3 Department of Ecology and Evolution, Stony Brook University , Stony Brook , New York, United States of America, 4 Department of Medicine, Washington University-St. Louis School of Medicine , Saint Louis , Missouri, United States of America, 5 Department of Surgery, Washington University-St. Louis School of Medicine , Saint Louis , Missouri, United States of America, 6 The Genome Institute, Washington University-St. Louis School of Medicine , Saint Louis , Missouri, United States of America, 7 Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado, United States of America, 8 Department of Medicine, University of Colorado , Aurora, Colorado , United States of America The aim of this study was to integrate human clinical, genotype, mRNA microarray and 16 S rRNA sequence data collected on 84 subjects with ileal Crohn's disease, ulcerative colitis or control patients without inflammatory bowel diseases in order to interrogate how host-microbial interactions are perturbed in inflammatory bowel diseases (IBD). Ex-vivo ileal mucosal biopsies were collected from the disease unaffected proximal margin of the ileum resected from patients who were undergoing initial intestinal surgery. Both RNA and DNA were extracted from the mucosal biopsy samples. Patients were genotyped for the three major NOD2 variants (Leufs1007, R702W, and G908R) and the ATG16L1T300A variant. Whole human genome mRNA expression profiles were generated using Agilent microarrays. Microbial composition profiles were determined by 454 pyrosequencing of the V3-V5 hypervariable region of the bacterial 16 S rRNA gene. The results of permutation based multivariate analysis of variance and covariance (MANCOVA) support the hypothesis that host mucosal Paneth cell and xenobiotic metabolism genes play an important role in host microbial interactions. - Funding: This work was supported partially by National Institutes of Health (NIH) UH2DK083994, the Crohns and Colitis Foundation of America, the Simons Foundation and by the Leona M. and Harry B. Helmsley charitable trust through the Sinai-Helmsley Alliance for Research Excellence (SHARE) Network and NIH R21HG005964. The authors acknowledge use of the Washington University Digestive Diseases Research Core Center Tissue Procurement Facility (P30 DK52574). No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Inflammatory bowel diseases are complex genetic disorders resulting from the interplay of genetic and environmental factors [13]. Crohns diseases (CD) and ulcerative colitis (UC) represent the two major inflammatory bowel diseases (IBD) phenotypes and are distinguished by different patterns of disease location. The inflammation in CD patients may be located anywhere along the gastrointestinal tract, but in the majority (80%) of CD patients, the terminal ileum is involved. In UC, the inflammation is confined to the colon. Because there is evidence that isolated Crohns colitis are associated with genetic factors that are distinct from ileal CD, and the overlap between genetic factors associated with UC and isolated Crohns colitis, we have focused our attention on the ileal CD subphenotype as a relatively homogenous category that is distinct from isolated colitis (CD or UC) and non-IBD controls [46]. Single nucleotide polymorphisms in the NOD2 gene and the ATG16L1 gene have been linked to alterations in innate host immunity, particularly Paneth cell function and with ileal CD phenotype [714]. We previously reported that increased CD3D mRNA expression in disease affected ileum resected from 18 ileal CD patients was associated with NOD2 genotype [15]. We also observed alterations in mRNA gene expression in the disease unaffected proximal margin of resected ileum from 19 ileal CD patients compared to 9 control non-IBD patients, regardless of NOD2 genotype [15]. The microarray dataset has recently been further expanded to include 47 ileal CD, 27 UC and 25 non-IBD control subjects (total = 99). Culture-independent microbiological technologies coupled with high-throughput DNAsequencing have uncovered alterations in human intestine-associated microbial compositions (dysbiosis) in IBD patients compared with controls [1625]. Ileal CD phenotype has been also associated with shifts in intestinal and fecal microbial composition, particularly reduced relative frequency of Faecalibacterium prausnitzii [19], [20], [23]. In addition to disease phenotype, exploratory analyses have also associated NOD2 genotype to intestinal associated microbial composition [22]. We have recently completed 16 S rRNA sequence analysis on an independent set of disease-unaffected ileal biopsies collected of 52 ileal CD, 58 colitis and 60 control patients without IBD undergoing initial surgical resection [24]. Of the 170 subjects with microbial composition data and 99 subjects with mRNA expression profiles, 84 subjects had paired microarray and microbial datasets. We report here the results of permutation based MANCOVA of these paired mRNA expression and microbial profiles in 34 ileal CD, 27 UC and 23 nonIBD control patients. Patient Characteristics As shown in Table 1, 35% of ileal CD patients harbored at least one NOD2 risk allele (NOD2R) compared to 13% of nonIBD control patients, consistent with previous studies [13]. Only one ileal CD patient was homozygous for the ATG16L1 nonrisk allele. The patients were predominantly Caucasian. The median age of surgery was lower in ileal CD patients than nonIBD control patients. Thirty percent of colitis patients had a concomitant C. difficile infection, consistent with the increased incidence of this infection noted previously in IBD patients [26], [27]. Thirty to fifty percent of IBD patients and none of the nonIBD control patients were treated with 5-aminosalicylic acid (5ASA), steroids, immunomodulators or an anti-TNFa (...truncated)