Nod2 is required for the regulation of commensal microbiota in the intestine

Abstract

Mutations in the Nod2 gene are among the strongest genetic risk factors in the pathogenesis of ileal Crohn's disease, but the exact contributions of Nod2 to intestinal mucosal homeostasis are not understood. Here we show that Nod2 plays an essential role in controlling commensal bacterial flora in the intestine. Analysis of intestinal bacteria from the terminal ilea of Nod2-deficient mice showed that they harbor an increased load of commensal resident bacteria. Furthermore, Nod2-deficient mice had a diminished ability to prevent intestinal colonization of pathogenic bacteria. In vitro, intestinal crypts isolated from terminal ilea of Nod2-deficient mice were unable to kill bacteria effectively, suggesting an important role of Nod2 signaling in crypt function. Interestingly, the expression of Nod2 is dependent on the presence of commensal bacteria, because mice re-derived into germ-free conditions expressed significantly less Nod2 in their terminal ilea, and complementation of commensal bacteria into germ-free mice induced Nod2 expression. Therefore, Nod2 and intestinal commensal bacterial flora maintain a balance by regulating each other through a feedback mechanism. Dysfunction of Nod2 results in a break-down of this homeostasis.

Fig. 1.

Nod2 ligand induces bacteria-killing activity in intestinal crypt secretions. (A) Crypts were isolated from the terminal ileum of C57BL/6 mice. We stimulated 2,000 crypts with MDP for 30 min in iPIPES buffer at concentrations of 10, 100, and 1000 μg/mL. Secretions were mixed with E. coli at 1 × 10³ cells for 1 h at 37 °C, and bacteria killing was measured by colony counting of serial dilutions. Reduced bacterial numbers in comparison with nonstimulated controls were plotted as percent killing. (B) Crypts were stimulated with 1 μM CCH, 1 mg/mL MDP, and 1 mg/mL MDP_DD for 30 min. Bactericidal activity of crypts secretions was determined against E. coli and Listeria monocytogenes by incubating secretions and bacteria for 1 h. (C) Crypts from MyD88-deficient mice were stimulated, and bactericidal activity against E. coli was measured as in (B). Error bars represent standard deviation. Data are representative of 2 or 3 independent experiments.

Fig. 2.

Nod2 is required to induce bactericidal activity in crypt secretions of the terminal ileum. (A) Crypts were isolated from the terminal ileum of wild-type and Nod2-deficient mice. Granules within Paneth cells were stained with 0.25% amido black. (B) We stimulated 2,000 crypts from wild-type and Nod2-deficient mice with CCH (1 μM), MDP, MDP_DD, and MDP_LL (1 mg/mL), and secretions were subjected to a bactericidal activity assay against E. coli. (C) Crypts from wild-type and Nod2-deficient mice (Left) or from wild-type and Rip2-deficient mice (Right) were stimulated with CCH as in (B), and secretions were tested for bactericidal activity against E. coli, S. typhimurium, and Listeria monocytogenes. Error bars represent standard deviation. Data are representative of 5 independent experiments.

Fig. 3.

Nod2 is required for the regulation of commensal bacteria flora in the terminal ileum. (A) Terminal ilea were isolated from littermates of Nod2^+/+, Nod2^+/−, and Nod2^−/− mice, and DNA samples were prepared. Fecal samples from the same animals were collected, and DNA was isolated. Bacteroides, Firmicutes, and Bacillus in the terminal ileum and feces were detected by PCR using primers for species-specific 16S rRNA genes. Primers for β-actin were used to show loading control of host genomic DNA. Data are representative of 3 independent littermate sets. (B) Bacteroides and Firmicutes in the terminal ileum in Nod2^+/+, Nod2^+/−, and Nod2^−/− mice were quantified by real-time PCR using primers for species-specific 16S rRNA genes. Data were normalized by real-time PCR data for the β-actin gene in host genomic DNA. The p-values were determined by Student's t test. (C) Commensal bacterial flora in the terminal ileum of wild-type and Rip2-deficient mice were detected as (A). (D) Bacteroides in the terminal ileum in Rip2-deficient mice were quantified as (B).

Fig. 4.

Nod2 suppresses colonization of pathogenic commensal bacteria in the terminal ileum. (A) H. hepaticus (5 × 10⁸/mouse) was inoculated into wild-type and Nod2-deficient mice via gastric gavage. Fresh feces samples were collected from mice at 0, 3, 7, and 14 days after inoculation, and DNA was purified. H. hepaticus colonization was quantified by real-time PCR using H. hepaticus-specific primers and 100 ng of purified DNA per reaction. Data were normalized by real-time PCR data for Eubacteria using the bacteria 16S RNA gene primer sets, which detect all bacterial strains. The p-values were determined by Student's t test. (B) Terminal ilea were isolated from wild-type and Nod2-deficient mice, and DNA samples were prepared. H. hepaticus colonization was quantified by real-time PCR using H. hepaticus-specific primers and 100 ng of purified DNA per reaction. Data were normalized by real-time PCR data for the β-actin gene in host genomic DNA. The p-values were determined by Student's t test.

Fig. 5.

Nod2 and Rip2 gene expression are dependent on commensal flora. (A) BALB/c mice were re-derived into GF conditions, and mRNA was extracted from the terminal ileum of 2-month-old BALB/c mice under SPF (n = 3) and GF (n = 3) conditions. The expression of Nod2, Rip2, MyD88, and Tirap/Mal was examined by quantitative real-time PCR. (B) The expression of Nod2 and MyD88 was examined as in (A) in the terminal ileum of 5-month-old BALB/c mice under SPF (n = 3) and GF (n = 3) conditions. (C) Two-month-old BALB/c mice raised under GF conditions were colonized with Lactobacillus plantarum and incubated for 1 month. Nod2 expression in the terminal ileum was examined by quantitative real-time PCR, together with 3-month-old BALB/c mice housed under SPF and GF conditions as controls. (D) Nod2 expression in the terminal ileum was examined as in (C) in 3-month-old GF mice and GF mice colonized with probiotic E. coli strain Nissle 1917 for 1 month. Each bar represents data from a single mouse. The p-values were determined by Student's t test.