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. 2016 Feb;65(2):225-37.
doi: 10.1136/gutjnl-2015-309333. Epub 2015 Apr 17.

Dysbiotic gut microbiota causes transmissible Crohn's disease-like ileitis independent of failure in antimicrobial defence

Monika Schaubeck  1 , Thomas Clavel  2 , Jelena Calasan  1 , Ilias Lagkouvardos  2 , Sven Bastiaan Haange  3 , Nico Jehmlich  3 , Marijana Basic  4 , Aline Dupont  5 , Mathias Hornef  5 , Martin von Bergen  6 , André Bleich  7 , Dirk Haller  8
Affiliations

Dysbiotic gut microbiota causes transmissible Crohn's disease-like ileitis independent of failure in antimicrobial defence

Monika Schaubeck et al. Gut. 2016 Feb.

Abstract

Objectives: Dysbiosis of the intestinal microbiota is associated with Crohn's disease (CD). Functional evidence for a causal role of bacteria in the development of chronic small intestinal inflammation is lacking. Similar to human pathology, TNF(deltaARE) mice develop a tumour necrosis factor (TNF)-driven CD-like transmural inflammation with predominant ileal involvement.

Design: Heterozygous TNF(deltaARE) mice and wildtype (WT) littermates were housed under conventional (CONV), specific pathogen-free (SPF) and germ-free (GF) conditions. Microbial communities were analysed by high-throughput 16S ribosomal RNA gene sequencing. Metaproteomes were measured using LC-MS. Temporal and spatial resolution of disease development was followed after antibiotic treatment and transfer of microbial communities into GF mice. Granulocyte infiltration and Paneth cell function was assessed by immunofluorescence and gene expression analysis.

Results: GF-TNF(deltaARE) mice were free of inflammation in the gut and antibiotic treatment of CONV-TNF(deltaARE) mice attenuated ileitis but not colitis, demonstrating that disease severity and location are microbiota-dependent. SPF-TNF(deltaARE) mice developed distinct ileitis-phenotypes associated with gradual loss of antimicrobial defence. 16S analysis and metaproteomics revealed specific compositional and functional alterations of bacterial communities in inflamed mice. Transplantation of disease-associated but not healthy microbiota transmitted CD-like ileitis to GF-TNF(deltaARE) recipients and triggered loss of lysozyme and cryptdin-2 expression. Monoassociation of GF-TNF(deltaARE) mice with the human CD-related Escherichia coli LF82 did not induce ileitis.

Conclusions: We provide clear experimental evidence for the causal role of gut bacterial dysbiosis in the development of chronic ileal inflammation with subsequent failure of Paneth cell function.

Keywords: CROHN'S DISEASE; IBD BASIC RESEARCH; INTESTINAL MICROBIOLOGY; SMALL BOWEL DISEASE; TNF.

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Figures

Figure 1
Figure 1
Changes in the gut microbial ecosystem are associated with inflammation in TNFdeltaARE mice. (A) Ileitis scores in 18-week-old TNFdeltaARE (ARE) and WT littermates in CONV or GF housing. Ileitis-score groups are colour-coded according to inflammation severity: score=0 (blue); score <4 (grey); score >4 (red). Male mice are displayed as triangles, female mice as circles. (B) Representative H&E-stained sections of the distal ileum. (C) NMDS plot showing shifts of centroids (indicated by arrows) and variance of faecal bacterial profiles (circled areas) of CONV-TNFdeltaARE and WT mice at 4 weeks, 8 weeks and 12 weeks of age. 16S ribosomal RNA gene amplicons of the V3/V4 region (407 bp) in faeces were sequenced on a MiSeq platform. (D) Ileitis scores in TNFdeltaARE mice treated for 4 weeks with vancomycin and metronidazole (VM), starting at 8 weeks of age. Recurrence of ileitis after VM therapy was followed for 6 weeks (n=5–6/group). (E) Ileal expression of Tnf as fold-increase to CONV-WT mice (normalised to Gapdh). (F) Ileal bacterial density of cultivable anaerobes (as log 10 of colony forming units (cfu) per gram ileal content). (G) Intestinal bacterial diversity (Shannon effective counts). (H) Changes in caecal bacterial composition (relative abundance of total sequences) at the phylum level. *p<0.05; **p<0.01; ***p<0.001 (Two-way-ANOVA followed by Holm-Sidak test). CONV, conventional; GF, germ-free; ARE, AU-rich elements; TNF, tumour necrosis factor; NMDS, non-parametrical multiple dimensional scaling.
Figure 2
Figure 2
Ileitis in the specific pathogen-free (SPF) environment shows different grades of severity. (A) Ileitis scores in 18-week-old TNFdeltaARE (AU-rich elements, ARE) and WT littermates in the SPF facility. The three ileitis score groups are colour-coded as ‘non-responders’ (NRs) in blue (score=0), ‘low-responders’ in grey (score <4) and ‘responders’ in red (score >4). Male mice are displayed as triangles, female mice as circles. (B) Upper panel: Representative H&E-stained sections of the distal ileum. Lower panel: Representative terminal ileum sections stained against Ly-6G (red). (C) Ileal expression (normalised to Gapdh) of Tnf as fold-increase to SPF-WT-mice (dotted line) in NRs, low-responders (lowRs) and responders (Rs). (D) Quantification of Ly6G-positive granulocytes per mm2 in tissue sections of the terminal ileum.
Figure 3
Figure 3
Disease severity is associated with loss of PC function. (A) Paneth cell staining of lysozyme (green; upper panel) combined with UEA-1 (red). A representative crypt is shown in the magnification. Lower panel: Paneth cell staining of cryptin 2 (red). A representative crypt is shown in the magnification. (B) Quantification of lysozyme, UEA-1 and cryptdin-2 positive cells per crypt base in ARE-mice compared with WT mice (dotted line). **p<0.01; ***p<0.001; Two-way ANOVA followed by Holm-Sidak test.
Figure 4
Figure 4
Bacterial profiles and functional dysbiosis mirror ileitis severity. (A) Shannon effective species counts in SPF-WT and SPF-TNFdeltaARE mice. (B) Non-parametrical multiple dimensional scaling analysis showing separation of caecal bacterial communities according to genotype and ileitis severity. Responders (R; red) were clearly separated from TNFdeltaARE with low ileitis scores (low-responder, lowR; grey) and from non-responders (NR; blue). Microbiota donor-mice used in colonisation experiments are shown with halos. (C) Correlation of bacterial taxa with ileitis scores. (D) Metaproteome analysis of the colonic microbiota. Partial least squared (PLS) analysis differentiated responder (red) and NR (blue) TNFdeltaARE-mice. (E) Significant changes in abundance of protein functions belonging to the clusters of orthologous groups (COGs) main role metabolism between R and NR samples. The heatmap represents differences in the abundance of COGs. Protein group data were log10 transformed and normalised by median of bacteria protein data. Protein functions that are unique to either R or NR are shown in the grey squares. COG subroles are: C, Energy production and conversion; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism. ***p<0.001; Two-way ANOVA followed by Holm-Sidak test.
Figure 4
Figure 4
Bacterial profiles and functional dysbiosis mirror ileitis severity. (A) Shannon effective species counts in SPF-WT and SPF-TNFdeltaARE mice. (B) Non-parametrical multiple dimensional scaling analysis showing separation of caecal bacterial communities according to genotype and ileitis severity. Responders (R; red) were clearly separated from TNFdeltaARE with low ileitis scores (low-responder, lowR; grey) and from non-responders (NR; blue). Microbiota donor-mice used in colonisation experiments are shown with halos. (C) Correlation of bacterial taxa with ileitis scores. (D) Metaproteome analysis of the colonic microbiota. Partial least squared (PLS) analysis differentiated responder (red) and NR (blue) TNFdeltaARE-mice. (E) Significant changes in abundance of protein functions belonging to the clusters of orthologous groups (COGs) main role metabolism between R and NR samples. The heatmap represents differences in the abundance of COGs. Protein group data were log10 transformed and normalised by median of bacteria protein data. Protein functions that are unique to either R or NR are shown in the grey squares. COG subroles are: C, Energy production and conversion; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism. ***p<0.001; Two-way ANOVA followed by Holm-Sidak test.
Figure 5
Figure 5
Ileitis is transmissible via dysbiotic gut microbiota. (A) GF-WT and GF-TNFdeltaARE (ARE) mice were colonised at the age of 8 weeks with the complex caecal microbiota from SPF responder (+R) or non-responder (+NR) donor-TNFdeltaARE mice (orange halos in figure 4; each n=3). Age-matched and time-matched GF-WT and GF-TNFdeltaARE-mice served as controls. Ileitis was scored after 4 weeks of colonisation. SPF-donor mice and respective recipients are shown with identical symbols. (B) Increase in mesenteric lymph node (MLN) weight. (C) Ileal expression (normalised to Gapdh) of Tnf as fold-increase to SPF-WT mice. (D) NMDS plot showing clear separation between R (red) and NR (blue) recipients according to respective donor (R1–3 or NR1–3; donor and recipient are displayed using identical symbols). ARE and WT are shown as filled and open symbols, respectively. (E) OTUs associated with R or NR colonisation. Each column represents one recipient ARE mouse. Donors and percentages of total sequences (coloured scale) are indicated below the table. ***p<0.001; Two-way ANOVA followed by Holm-Sidak test. ARE, AU-rich elements; GF, germ-free; NMDS, non-parametrical multiple dimensional scaling.
Figure 6
Figure 6
Transfer of dysbiotic microbiota induces a gradual disease onset. (A) GF-WT and GF-TNFdeltaARE (ARE) mice were colonised at the age of 8 weeks with the complex caecal microbiota from inflamed SPF responder mice (open symbols). After 1 week, 2 weeks and 4 weeks of colonisation, ileitis was assessed. (B) Upper panel: Representative H&E stained terminal ileum sections of ARE-mice colonised with responder microbiota for 1 week, 2 weeks and 4 weeks (wk). Age matched GF-AREs (left) served as controls. Lower panel: Representative terminal ileum sections stained against Ly-6G (red). (C) Ileal expression (normalised to Gapdh) of Tnf as fold-increase to SPF-WT mice. Tnf expression of GF-ARE-mice is displayed as dotted line. (D) Quantification of Ly6G-positive granulocytes per mm2 in tissue sections of the terminal ileum after 1 week, 2 weeks and 4 weeks of colonisation with R microbiota. ARE, AU-rich elements; GF, germ-free.
Figure 7
Figure 7
Loss of antimicrobial peptides is secondary to the development of inflammation. (A) Paneth cell staining of lysozyme, UEA-1 and Cryptdin 2, as labelled in the figure (upper panel, 10× magnification; others, 120×). (B) Quantification of lysozyme-positive, UEA-1-positive and cryptdin 2-positive cells per crypt base in AU-rich elements (ARE) mice compared with WT mice (dotted line). *p<0.05; ***p<0.001 (One-way-ANOVA followed by Tukey's test).
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