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. 2010 Aug 17;107(33):14739-44.
doi: 10.1073/pnas.1003363107. Epub 2010 Aug 2.

Induction and rescue of Nod2-dependent Th1-driven granulomatous inflammation of the ileum

Amlan Biswas  1 , Yuen-Joyce Liu, Liming Hao, Atsushi Mizoguchi, Nita H Salzman, Charles L Bevins, Koichi S Kobayashi
Affiliations

Induction and rescue of Nod2-dependent Th1-driven granulomatous inflammation of the ileum

Amlan Biswas et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations in the NOD2 gene are strong genetic risk factors for ileal Crohn's disease. However, the mechanism by which these mutations predispose to intestinal inflammation remains a subject of controversy. We report that Nod2-deficient mice inoculated with Helicobacter hepaticus, an opportunistic pathogenic bacterium, developed granulomatous inflammation of the ileum, characterized by an increased expression of Th1-related genes and inflammatory cytokines. The Peyer's patches and mesenteric lymph nodes were markedly enlarged with expansion of IFN-gamma-producing CD4 and CD8 T cells. Rip2-deficient mice exhibited a similar phenotype, suggesting that Nod2 function likely depends on the Rip2 kinase in this model. Transferring wild-type bone marrow cells into irradiated Nod2-deficient mice did not rescue the phenotype. However, restoring crypt antimicrobial function of Nod2-deficient mice by transgenic expression of alpha-defensin in Paneth cells rescued the Th1 inflammatory phenotype. Therefore, through the regulation of intestinal microbes, Nod2 function in nonhematopoietic cells of the small intestinal crypts is critical for protecting mice from a Th1-driven granulomatous inflammation in the ileum. The model may provide insight into Nod2 function relevant to inflammation of ileal Crohn's disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
H. hepaticus induces enlargement of Peyer’s patches, mesenteric lymphadenopathy, and granulomatous inflammation in Nod2-deficient mice. (A–E) Age- and sex-matched wild-type and Nod2-deficient mice were inoculated with H. hepaticus (5 × 108/mouse) via gastric gavage. (A) Intestines were removed from the mice at d 0 (n = 4), 7 (n = 2), or 14 (n = 10 for each genotype) postinoculation and the number of macroscopically visible Peyer’s patches in the small intestines was counted. The P values at day 14 were determined by Student t test. (B) Representative pictures of Peyer’s patches (outlined) and mesenteric lymph nodes at day 14 postinoculation. (C) Granulomatous inflammation in the ileocecal junction of wild-type (n = 41) and Nod2-deficient (n = 38) mice at day 14 postinoculation was scored in blind manner as described in Material and Methods. The P values were determined by Student t test. (D) Representative pictures of H&E stained terminal ileum tissue at d 14 postinoculation. Granulomas are indicated by arrows. (E) The expression of IL-1β, IL-6, IFN-γ, and Spp1 in the terminal ileum and descending colon was examined by qRT-PCR 14 d postinoculation. Data were normalized to the expression of the β-actin gene. Each bar represents data from a single mouse. The P values were determined by Student t test. NS: not significant.
Fig. 2.
Fig. 2.
Exaggerated Th1 immune responses in Peyer’s patches and mesenteric lymph nodes in Nod2-deficient mice (A and B). Age- and sex-matched wild-type and Nod2-deficient mice were inoculated with H. hepaticus (5 × 108/mouse) via gastric gavage. Peyer’s patch and mesenteric lymph node cells were isolated at d 14 postinoculation and stimulated with PMA and ionomycin for 6 (A) or 3 (B) h. (A) The expression of IFN-γ and IL-4 in gated CD4 or CD8 T cells was analyzed by flow cytometry. (B) The expression of IFN-γ, T-bet, and IL-12β2 receptor was examined by qRT-PCR. Data were normalized to the expression of the β-actin gene. Each bar represents replicate data from a single mouse. The P values were determined by Student t test.
Fig. 3.
Fig. 3.
Rip2-dependence of Th1 inflammatory responses in H. hepaticus challenged mice. (A–C) Age- and sex-matched wild-type and Rip2-deficient mice were inoculated with H. hepaticus (5 × 108/mouse) via gastric gavage. (A) Intestines were removed from the mice at days 0 (wild-type, n = 4; Rip2 −/−, n = 4), or 14 (wild-type, n = 8; Rip2 −/−, n = 10) postinoculation and number of macroscopically visible Peyer’s patches in the small intestine was counted. The P value was determined by Student t test. (B) Enlarged Peyer’s patches (outlined) and mesenteric lymphadenopathy in Rip2-deficient mice. Representative pictures at day 14 postinoculation are shown. (C) Granulomatous inflammation in the ileocecal junction of wild-type (n = 15) and Rip2-deficient (n = 18) mice at day 14 postinoculation was scored in a blind manner as described in Material and Methods. The P values were determined by Student t test. (D) Peyer’s patch and mesenteric lymph node cells at day 14 were stimulated with PMA and ionomycin for 3 h. The expression of IFN-γ and T-bet was examined by qRT-PCR. Each bar represents replicate data from a single mouse. The P values were determined by Student t test.
Fig. 4.
Fig. 4.
Reconstitution of wild-type hematopoietic cells into Nod2-deficient mice did not rescue Th1 inflammatory phenotype. (A–C) H. hepaticus (5 × 108/mouse) was inoculated into wild-type and Nod2-deficient mice 6 wk after reconstitution with CD45.1 C57BL/6 bone marrow cells. (A and B) Peyer’s patch and mesenteric lymph node cells were isolated at day 14 and stimulated with PMA and ionomycin for 6 (A) or 3 (B) h. (A) The expression of IFN-γ and IL-4 in gated CD4 or CD8 T cells was analyzed by flow cytometry. (B) The expression of IFN-γ, and T-bet examined by qRT-PCR. (C) The expression of IL-1β, IL-6, IFN-γ, and Spp1 in the terminal ileal tissue was examined by qRT-PCR. (B and C) Data were normalized by the expression of the β-actin gene. Each bar represents replicate data from a single mouse. The P values were determined by Student t test.
Fig. 5.
Fig. 5.
Restoration of ileal crypt function by defensin transgene rescued Th1 inflammatory phenotype in Nod2-deficient mice. (A) Generation of Nod2−/−/HD5 transgenic mice. The expression of Nod2 and HD5 in the terminal ileum of Nod2 +/−, Nod2 −/−, and Nod2 −/−/HD-5 transgenic (HD5 tg) mice was determined by RT-PCR analysis using primers specific for Nod2 and HD5. Primers for β-actins were used as an internal control. (B) Successful restoration of crypt function in bacterial killing activity by introducing defensin transgene into Nod2-deficient mice. Crypts isolated from the terminal ileum of Nod2 +/−, Nod2 −/−, and Nod2 −/−/HD5 tg littermates were stimulated with CCH for 30 min. Secretions were mixed with E. coli (1 × 103 cells) and bacterial killing was measured by counting colonies of serial dilutions. (C and D) Nod2 +/−, Nod2 −/−, and Nod2 −/−/HD5 tg littermates were inoculated with H. hepaticus (5 × 108/mouse) via gastric gavage. (C) The expression of IL-1β, IL-6, IFN-γ, and Spp1 in the terminal ilea was examined 14 d postinoculation by qRT-PCR. (D) Peyer’s patch and mesenteric lymph node cells at day 14 were stimulated with PMA and ionomycin for 3 h. The expression of IFN-γ and T-bet was examined by qRT-PCR. (C and D) Data were normalized by the expression of the β-actin gene. Each bar represents replicate data from a single mouse. The P values were determined by Student t test. (E and F) Model of ileal CD. Please see Discussion for details. (E) Normal terminal ileum without NOD2 mutations. (F) Terminal of ileum with loss-of-function NOD2 mutations or Nod2 deficiency. Altered Paneth cell function, dysbiosis, and abnormalities of Peyer’s patch (PP) and mesenteric lymph nodes (MLN) are depicted.
See this image and copyright information in PMC

References

    1. Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inflammatory bowel disease. Nature. 2007;448:427–434. - PubMed
    1. Sartor RB. Microbial influences in inflammatory bowel diseases. Gastroenterology. 2008;134:577–594. - PubMed
    1. Abraham C, Cho JH. Inflammatory bowel disease. N Engl J Med. 2009;361:2066–2078. - PMC - PubMed
    1. Hugot JP, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature. 2001;411:599–603. - PubMed
    1. Ogura Y, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature. 2001;411:603–606. - PubMed

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