Functional Characterization of Inflammatory Bowel Disease-Associated Gut Dysbiosis in Gnotobiotic Mice

Abstract

Background & aims: Gut dysbiosis is closely involved in the pathogenesis of inflammatory bowel disease (IBD). However, it remains unclear whether IBD-associated gut dysbiosis contributes to disease pathogenesis or is merely secondary to intestinal inflammation. We established a humanized gnotobiotic (hGB) mouse system to assess the functional role of gut dysbiosis associated with 2 types of IBD: Crohn's disease (CD) and ulcerative colitis (UC).

Methods: Germ-free mice were colonized by the gut microbiota isolated from patients with CD and UC, and healthy controls. Microbiome analysis, bacterial functional gene analysis, luminal metabolome analysis, and host gene expression analysis were performed in hGB mice. Moreover, the colitogenic capacity of IBD-associated microbiota was evaluated by colonizing germ-free colitis-prone interleukin 10-deficient mice with dysbiotic patients' microbiota.

Results: Although the microbial composition seen in donor patients' microbiota was not completely reproduced in hGB mice, some dysbiotic features of the CD and UC microbiota (eg, decreased diversity, alteration of bacterial metabolic functions) were recapitulated in hGB mice, suggesting that microbial community alterations, characteristic for IBD, can be reproduced in hGB mice. In addition, colonization by the IBD-associated microbiota induced a proinflammatory gene expression profile in the gut that resembles the immunologic signatures found in CD patients. Furthermore, CD microbiota triggered more severe colitis than healthy control microbiota when colonized in germ-free interleukin 10-deficient mice.

Conclusions: Dysbiosis potentially contributes to the pathogenesis of IBD by augmenting host proinflammatory immune responses. Transcript profiling: GSE73882.

Keywords: CD, Crohn's disease; CE-TOFMS, capillary electrophoresis time-of-flight mass spectrometry; Crohn's Disease; Dysbiosis; GB, gnotobiotic; GF, germ-free; IBD, inflammatory bowel disease; IFN, interferon; IL, interleukin; ILC, innate lymphoid cell; IVC, individual ventilated cage; Microbiota; NK, natural killer; OTU, operational taxonomic unit; SCFA, short-chain fatty acid; Th, T helper; UC, ulcerative colitis; Ulcerative Colitis; WT, wild type; hGB, humanized gnotobiotic; rRNA, ribosomal RNA.

Figure 1

Analysis of functional dysbiosis in IBD. Stool samples were obtained from healthy control subjects and patients with IBD (Crohn's disease and ulcerative colitis). Intestinal bacteria then were inoculated into GF WT mice (hGB mice). After 2 weeks of reconstitution, gut microbiome (16S rRNA sequencing), luminal metabolome (CE-TOF/MS), and host gene expression profile (DNA microarray) in hGB mice were analyzed. For evaluation of the colitogenic capacity of the microbiota, established hGB mice were used as stool donors. Stool samples obtained from HC- or IBD-hGB mice were transplanted into either GF WT B6 mice or GF Il10^-/-mice. After 3 weeks of reconstitution, intestinal inflammation was examined. mRNA, messenger RNA.

Figure 2

IBD-associated dysbiosis is recapitulated in humanized gnotobiotic mice. (A) Stool samples were obtained from patients with CD, UC, and HCs, and then inoculated into GF mice. After 2 weeks of reconstitution, stool samples from hGB mice were collected and bacterial 16S rRNA sequences were analyzed. Sample IDs of human donors correspond to those of donor-derived hGB mice. (B) Microbial community structures were analyzed by using the Yue and Clayton dissimilarity distance metric (θ_YC) and are shown in a nonmetric multidimensional scaling plot. (C) Shannon index (α-diversity) and number of OTUs (richness) for each group. Data are presented as means ± SD. *P < .05 by Student t test. ** P < .01.

Figure 3

Microbial functional gene pathways in hGB mice. The abundance of Kyoto Encyclopedia of Genes and Genomes metabolic gene pathways was analyzed by phylogenetic investigation of communities by reconstruction of unobserved states based on 16S rRNA sequencing data in Figure 2. Significantly altered pathway genes in 3 groups (HC-, CD-, and UC-hGB mice) were identified by linear discriminant analysis effect size. The linear discriminant analysis (LDA) score is shown.

Figure 4

Fecal metabolome profiles of GF and hGB mice. (A) A heat map showing the quantified metabolic profiles. All concentrations of quantified metabolites were transformed into Z-scores and clustered according to Euclidean distance. Gray areas in the heat map indicate that respective metabolites were not detected. (B) The principal component analysis of the metabolome data. The ellipse denotes the 95% significance limit of the model, as defined by the Hotelling t test. (C) A loading scatter plot of the principal component analysis. (D) The bar graphs showing the concentration of propionate, butyrate, 5-aminovalerate, taurine, succinate, glutamate, threonine, and urea in murine feces, respectively. Data are presented as means ± SEM. *P < .05, ** P < .01. ND, not detected by 1-way analysis of variance, followed by the Tukey post hoc test. PC, principal component.

Figure 5

Host gene expression in the colonic mucosa of hGB mice. Host gene expression induced by colonization of human microbiota (HC, CD, and UC). A heat map of selected genes, which were expressed differently in CD-hGB or UC-hGB mice compared with HC-hGB mice, is shown. The color range indicates the fold expression of genes compared with the average expression in HC-hGB mice.

Figure 6

CD-associated microbiota promotes the development of colitis in IBD-prone mice. (A and B) Stool samples were isolated from HC-hGB mice (HC-03, HC-04, and HC-05) and CD-hGB mice (CD-01, CD-02, CD-03, CD-04, and CD-05), and then inoculated into GF WT B6 mice. After 3 weeks of reconstitution, cecal and colonic tissues were harvested. (A) Histologic score of colon. Each dot indicates an individual mouse. NS, not significant by Mann–Whitney U test. (B) A representative histologic image of WT B6-HC-hGB and CD-hGB mice. Scale bar: 200 μm. (C–E) Stool samples were isolated from HC-hGB mice and CD-hGB mice and then inoculated into GF Il10^-/- mice. After 3 weeks of reconstitution, cecal and colonic tissues were harvested. (C) Colonic weights. Each dot indicates an individual mouse. **P < .01 by Mann–Whitney U test. (D) Histologic score of colon. Each dot indicates an individual mouse. **P < .01 by Mann–Whitney U test. ***P < .001. (E) A representative histologic image of colonic tissues isolated from IL10^-/--HC-hGB and CD-hGB mice. Scale bar: 50 μm.