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. 2013 Feb;123(2):700-11.
doi: 10.1172/JCI62236. Epub 2013 Jan 2.

NOD2-mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer

Aurélie Couturier-Maillard  1 , Thomas Secher, Ateequr Rehman, Sylvain Normand, Adèle De Arcangelis, Robert Haesler, Ludovic Huot, Teddy Grandjean, Aude Bressenot, Anne Delanoye-Crespin, Olivier Gaillot, Stefan Schreiber, Yves Lemoine, Bernhard Ryffel, David Hot, Gabriel Nùñez, Grace Chen, Philip Rosenstiel, Mathias Chamaillard
Affiliations

NOD2-mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer

Aurélie Couturier-Maillard et al. J Clin Invest. 2013 Feb.

Abstract

Instability in the composition of gut bacterial communities (dysbiosis) has been linked to common human intestinal disorders, such as Crohn's disease and colorectal cancer. Here, we show that dysbiosis caused by Nod2 deficiency gives rise to a reversible, communicable risk of colitis and colitis-associated carcinogenesis in mice. Loss of either Nod2 or RIP2 resulted in a proinflammatory microenvironment that enhanced epithelial dysplasia following chemically induced injury. The condition could be improved by treatment with antibiotics or an anti-interleukin-6 receptor-neutralizing antibody. Genotype-dependent disease risk was communicable via maternally transmitted microbiota in both Nod2-deficient and WT hosts. Furthermore, reciprocal microbiota transplantation reduced disease risk in Nod2-deficient mice and led to long-term changes in intestinal microbial communities. Conversely, disease risk was enhanced in WT hosts that were recolonized with dysbiotic fecal microbiota from Nod2-deficient mice. Thus, we demonstrated that licensing of dysbiotic microbiota is a critical component of disease risk. Our results demonstrate that NOD2 has an unexpected role in shaping a protective assembly of gut bacterial communities and suggest that manipulation of dysbiosis is a potential therapeutic approach in the treatment of human intestinal disorders.

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Figures

Figure 1
Figure 1. The absence of NOD2/RIP2 confers a transmissible risk for colitis, even to immunocompetent hosts.
(A) Five days after AOM administration (at 8 mg/kg), age- and gender-matched SH-WT (n = 5), SCH-WTNod2–/– (n = 5), CH-WTNod2–/– (n = 5), SCH-WTRIP2–/– (n = 5), and CH-WTRIP2–/– (n = 4) mice received ad libitum 2% (w/v) DSS (molecular mass, 35–40 kDA; TdB Consultancy) for 7 days followed by 2 days of regular drinking water. Mutant and WT animals were cohoused at a 3:2 or 1:1 ratio on the same diet for a 4-week period. WT cohoused mice were either left with or separated from mutant mice before being challenged by 1 cycle of DSS-AOM. The cross indicates necropsy. Changes in body weight of (B) CH-WTNod2–/–, (C) CH-WTRIP2–/–, and (D) SCH-WTNod2–/– and SCH-WTRIP2–/– mice were evaluated daily and compared to that in SH-WT animals. (E) Histological scoring was determined at day 9. (F) Representative H&E and PAS staining of paraffin-embedded longitudinal sections from colons of SH-WT, SCH-WTRIP2–/–, and CH-WTRIP2–/– mice at day 9. Scale bar: 100 μm. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2
Figure 2. The risk for colitis in RIP2-deficient mice is maternally transmissible.
WT animals were reared with either a RIP2-deficient or -sufficient mother since birth (n = 4). Five-week-old cross-fostered mice were then exposed to 1 cycle of DSS-AOM, as described earlier. Changes in (A) body weight and (B) colon length were evaluated daily. (C) Representative H&E and PAS staining of paraffin-embedded transversal sections from colons of CF-WT and CF-RIP2–/– mice at day 9. CF refers to cross-fostered animals. Scale bar: 100 μm. *P < 0.05; ***P < 0.001.
Figure 3
Figure 3. Enhanced expression of claudin 5 in response to dysbiosis coupled to NOD2 deficiency.
WT animals were either single housed (n = 5) or cohoused at a 1:1 ratio with Nod2-deficient mice (n = 5) on the same diet for a 4-week period. qRT-PCR analysis was performed on extracted RNAs from the colons of (A) CH-WT, (B) SCH-WT, and (C) CF-WT animals as well as SH-WT animals. Mean ± SEM. *P < 0.05; **P < 0.01. SCH, CH, and SH refer to separated cohoused, cohoused, and single-housed animals, respectively.
Figure 4
Figure 4. NOD2-driven risk for colitis-associated colorectal cancer is communicable to WT host.
Five days after AOM administration (at 8 mg/kg), SH-WT, CH-WTNod2–/–, CH-WTRIP2–/–, and SH-Nod2–/– mice were subjected to 4 rounds consisting of 2% DSS for 5 days followed by 7-day access to regular drinking water. (A) Macroscopic and (B) endoscopic quantification of colonic tumor burden in SH-WT (n = 5), CH-WTNod2–/– (n = 5), and CH-WTRIP2–/– (n = 4) animals was performed at day 56 and 55, respectively. (C) Representative H&E and PAS staining of paraffin-embedded transversal sections from colons of SH-WT, CH-WTNod2–/– and CH-WTRIP2–/– mice at day 56. Scale bar: 100 μm (D) Macroscopic quantification of colonic tumor burden in SH-WT (n = 10) and SH-Nod2–/– (n = 10) animals was performed at day 56. (E) Representative photographs of dissected colons of SH-WT and SH-Nod2–/– mice at day 56. (F) Macroscopic quantification of colonic tumor growth in SH-WT and SH-Nod2–/– animals was performed at day 56. (G) BrdU incorporation in vivo (original magnification, ×200). Mean ± SEM. *P < 0.05; **P < 0.01.
Figure 5
Figure 5. Broad-spectrum antibiotic treatment improves intestinal inflammation and tumorigenesis in Nod2-deficient mice.
Five days after AOM administration (at 12 mg/kg), Nod2+/+ (n = 4), Nod2–/– (n = 3), antibiotic-treated (Atb-treated) WT (n = 4), and Atb-treated Nod2–/– (n = 3) mice were subjected to 2% DSS for 7 days followed by 9-day access to regular drinking water. (A) Changes in body weight were monitored daily. (B) Colonic-associated Bacteroides load was determined by specific q-PCR analysis at day 16. (C–E) Ly6G, IA/IE, CD11c, and CD11b staining of the lamina propria cell population in Nod2–/– mice treated or not with antibiotics as indicated was quantified by FACS analysis. (F) Colon weight/length ratio quantification on Nod2+/+ (n = 10), Nod2–/– (n = 9), antibiotic-treated Nod2+/+ (n = 10) and antibiotic-treated Nod2–/– (n = 10) mice was performed at day 58. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 6
Figure 6. Neutralizing interleukin-6 reduces tumor progression in Nod2- and RIP2-deficient mice.
Five days after AOM administration (at 8 mg/kg) SH-WT (n = 5), SH-Nod2–/– (n = 5), and SH-RIP2–/– (n = 4) mice were exposed to 4 cycles of DSS, which consisted of 5 days separated by a 7-day period of regular water. An interleukin-6 receptor antibody (αIL-6R) (1 mg per mouse i.p.) was given once a week from day 31 to 56 or not to AOM-exposed mice. (A) Endoscopic and (B) macroscopic evaluation of mice was performed at day 55 and 56, respectively. (C) Colonic transcript levels of Reg3b, Reg3g, Il6, Ptgs2, and Spp1 in untreated mice (n = 4) and anti–interleukin-6 receptor–treated (n = 4) animals are shown as determined by q-PCR evaluation at day 56. *P < 0.05.
Figure 7
Figure 7. Interleukin-6–mediated control of disease progression is linked to NOD2-driven dysbiosis in an experimental model of colitis-associated colorectal cancer.
High-throughput 16S rRNA pyrosequencing was performed on tumoral (T) and chronically inflamed (NT) colonic specimens isolated from WT and Nod2–/– animals (n = 4). P values were determined by the Mann-Whitney U test. (A) PCoA plot generated on nonabundance-based UniFrac distance matrices, (B) CCA based on observed genera. (C) Percentage of phyla and (D) top 10 genera, with abundance identified by bray-curtis model of SIMPER analysis. F, Firmicutes; B, Bacteroidetes; P, Proteobacteria. (E and F) Nonparametric quantification of microbiota diversity within (E) tumoral and (F) nontumoral tissues. Mean ± SEM. *P < 0.05.
Figure 8
Figure 8. Dysbiotic microbial ecology of Nod2-deficient mice intrinsically instigates risk for colitis that is corrected by fecal transplantation.
Four weeks before DSS-AOM challenge, GF-Nod2–/– (n = 5) or GF-Nod2+/+ (n = 5) mice were reconstituted with fecal microbiota from either Nod2+/+ or Nod2–/– animals that were not exposed to DSS. (A and D) Changes in body weight were monitored daily. (B and E) Representative H&E and PAS staining of paraffin-embedded transversal sections at day 10. Scale bar: 100 μm. (C and F) Histological scoring was evaluated at day 10. (G and H) High-throughput 16S rRNA pyrosequencing was performed on fecal specimens isolated from all DSS-treated gnotobiotic animals (n = 5). (G) PCoA generated on nonabundance-based UniFrac distance matrices. (H) Nonparametric quantification of microbiota diversity. P values were determined by the Mann-Whitney U test. Percentage of phyla abundance on top 10 genera was identified by bray-curtis model of SIMPER analysis. Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.
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