Influence of Crohn’s disease related polymorphisms in innate immune function on ileal microbiome

RESEARCH ARTICLE Influence of Crohn’s disease related polymorphisms in innate immune function on ileal microbiome Ellen Li ID1*, Yuanhao Zhang1, Xinyu Tian ID1, Xuefeng Wang2, Grace Gathungu3, Ashley Wolber4, Shehzad S. Shiekh4, R. Balfour Sartor4, Nicholas O. Davidson5, Matthew A. Ciorba5, Wei Zhu6, Leah M. Nelson7, Charles E. Robertson7, Daniel N. Frank7 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Medicine, Stony Brook University, Stony Brook, NY, United States of America, 2 Department of Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, United States of America, 3 Department of Pediatrics, Stony Brook University, Stony Brook, NY, United States of America, 4 Department of Medicine, University of North Carolina, Chapel Hill, NC, United States of America, 5 Department of Medicine, Washington University St. Louis, St. Louis, MO, United States of America, 6 Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY, United States of America, 7 Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, United States of America * OPEN ACCESS Citation: Li E, Zhang Y, Tian X, Wang X, Gathungu G, Wolber A, et al. (2019) Influence of Crohn’s disease related polymorphisms in innate immune function on ileal microbiome. PLoS ONE 14(2): e0213108. https://doi.org/10.1371/journal. pone.0213108 Editor: Xiaonan Han, Cincinnati Children’s Hospital Medical Center, UNITED STATES Received: November 14, 2018 Accepted: February 14, 2019 Published: February 28, 2019 Copyright: © 2019 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Clinical, genotyping, and sequencing data can be accessed through the dbGAP authorized access system (Request access to: phs000255.v2). In order to request access to any of the individual-level datasets within the controlled-access portions of the database, the Principal Investigator (PI) and the Signing Official (SO) at the investigator’s institution will need to cosign a request for data access, which will be reviewed by an NIH Data Access Committee at the appropriate NIH Institute or Center (https://dbgap. ncbi.nlm.nih.gov/aa/wga.cgi?page=login). Abstract We have previously identified NOD2 genotype and inflammatory bowel diseases (IBD) phenotype, as associated with shifts in the ileal microbiome (“dysbiosis”) in a patient cohort. Here we report an integrative analysis of an expanded number of Crohn’s disease (CD) related genetic defects in innate immune function (NOD2, ATG16L1, IRGM, CARD9, XBP1, ORMDL3) and composition of the ileal microbiome by combining the initial patient cohort (Batch 1, 2005–2010, n = 165) with a second consecutive patient cohort (Batch 2, 2010– 2012, n = 118). These combined patient cohorts were composed of three non-overlapping phenotypes: 1.) 106 ileal CD subjects undergoing initial ileocolic resection for diseased ileum, 2.) 88 IBD colitis subjects without ileal disease (predominantly ulcerative colitis but also Crohn’s colitis and indeterminate colitis, and 3.) 89 non-IBD subjects. Significant differences (FDR < 0.05) in microbiota were observed between macroscopically disease unaffected and affected regions of resected ileum in ileal CD patients. Accordingly, analysis of the effects of genetic and clinical factors were restricted to disease unaffected regions of the ileum. Beta-diversity differed across the three disease categories by PERMANOVA (p < 0.001), whereas no significant differences in alpha diversity were noted. Using negative binomial models, we confirmed significant effects of IBD phenotype, C. difficile infection, and NOD2 genotype on ileal dysbiosis in the expanded analysis. The relative abundance of the Proteobacteria phylum was positively associated with ileal CD and colitis phenotypes, but negatively associated with NOD2R genotype. Additional associations with ORMDL3 and XBP1 were detected at the phylum/subphylum level. IBD medications, such as immunomodulators and anti-TNFα agents, may have a beneficial effect on reversing dysbiosis associated with the IBD phenotype. Exploratory analysis comparing microbial composition of the disease unaffected region of the resected ileum between 27 ileal CD patients who subsequently developed endoscopic recurrence within 6–12 months versus 34 patients who did PLOS ONE | https://doi.org/10.1371/journal.pone.0213108 February 28, 2019 1 / 22 Ileal microbiome in ileal Crohn’s disease Funding: This work was supported in part by the National Institute of Health grants P30DK52574 (N. O.D.), UH2DK083994 (E.L.), and HG005964 (D.N. F.), the Barnes-Jewish Foundation (E.L. donation from Dr. Carl Lyss and the Lenauers), The BarnesJewish Hospital Foundation, the MAC Crohn’s and Colitis Foundation senior Research award Ref.370763, the Pakula IBD Innovation Fund at Washington University St. Louis and the Givin’ It All for Guts Foundation https://givinitallforguts.org/(M. C.). 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. not, suggested that microbial biomarkers in the resected specimen helped stratify patients with respect to risk of post-surgical recurrence. Introduction Inflammatory bowel disease (IBD) describes a group of disorders in which the intestines become inflamed. Two major types of IBD are ulcerative colitis (UC) and Crohn’s disease (CD). UC is limited to the colon or large intestine. Crohn’s disease, on the other hand, can involve any part of the gastrointestinal tract from the mouth to the anus. Most commonly, though, it affects the ileum or the colon or both. Abnormal host-microbial interactions and genetic susceptibility are implicated in the pathogenesis of IBD, reviewed in [1]. We previously examined the effects of the NOD2 and ATG16L1 polymorphisms on ileal microbial composition in 1) ileal CD subjects undergoing initial ileal resection, 2) IBD colitis without ileitis (predominantly ulcerative colitis) subjects and 3) subjects without inflammatory bowel disease (non-IBD) [2–4]. These previous studies identified NOD2 genotype and C. difficile infection, in addition to IBD phenotype, as associated with shifts in ileal microbiota or “dysbiosis” [2–4]. We also found that differential expression of genes involved in Paneth cell function were associated with shifts in ileal microbial composition [5]. In the current study, the effects of an expanded panel of Crohn’s disease risk alleles [6–10] that are associated with defects in innate immunity (NOD2, ATG16L1, IRGM, CARD9, XBP1, ORMDL3) on ileal microbial composition, were analyzed by combining the original (...truncated)