Expression of NOD2 in Paneth cells: a possible link to Crohn's ileitis

Abstract

Background and aims: Genetic variation in NOD2 has been associated with susceptibility to Crohn's disease (CD) and specifically with ileal involvement. The reason for the unique association of NOD2 mutations with ileal disease is unclear. To identify a possible link, we tested expression of NOD2 in intestinal tissue of CD patients and controls.

Patients and methods: Fifty five specimens of ileum or colon from 21 CD patients, seven ulcerative colitis (UC) patients, and five controls with pathology other than CD or UC were stained for NOD2 using an immunoperoxidase method.

Results: Using a monoclonal antibody against NOD2 developed in our laboratory, we detected uniform expression of NOD2 in terminal ileum Paneth cells from controls and patients as well as in metaplastic Paneth cells in the colon. Mechanical purification showed enriched expression of NOD2 mRNA in ileal crypts. In Paneth cells, NOD2 was located in the cytosol in close proximity to the granules that contain antimicrobial peptides. We detected minimal NOD2 in the villous epithelium of the ileum or in the colonic epithelium from both CD patients and controls.

Conclusions: These results suggest a role for NOD2 in the regulation of Paneth cell mediated responses against intestinal bacteria and a plausible mechanism to explain the selective association of NOD2 mutations with ileal disease. The impaired capacity of CD associated mutations to sense luminal bacteria may result in increased susceptibility to certain gut microbes.

Figure 1

Characterisation of 2D9, a monoclonal anti-NOD2 antibody. (A, B) HEK293T cells were transfected with empty pcDNA3 vector or pcDNA3 vector to express HA tagged Nod2 (114 kD), HA tagged Nod1 (110 kD), HA tagged Ipaf (118 kD), Myc tagged Apaf-1 (138 kD), Flag tagged RICK (63 kD), or Flag tagged Pypaf-1 (120 kD). Cell lysates were immunoblotted with 2D9, a mouse monoclonal anti-NOD2 antibody (A), mouse monoclonal anti-HA antibody (16B12), mouse monoclonal anti-Flag antibody (M2), or mouse monoclonal anti-Myc antibody (9E10) (B). (C) HEK293T cells were transfected with empty pcDNA3 vector or pcDNA3 vector to express HA tagged wild-type Nod2 or mutant Nod2 lacking caspase recruitment domains (ΔCARDs) or expressing CARDs alone (CARDs). Immunoblottings with 2D9 or anti-HA antibody are shown. (D) HEK293T cells were transfected with empty pcDNA3 vector or pcDNA3 vector to express wild-type Nod2 or mutant Nod2, P268S, P268S/R702W, P268S/G908R, or P268S/L1007fs. Cell lysates were blotted with 2D9. (E) HEK293T cells were transfected with empty pcDNA3 vector or pcDNA3 vector to express wild-type Nod2. Cell lysates were immunoprecipitated with 2D9 or M2, an isotype matched control antibody. Immunorecipitates were blotted with rabbit polyclonal anti-Nod2 antibody. (F) HL60 cells were cultured with or without tumour necrosis factor α (TNF-α) for 24 hours. Cell lysates from 2×10⁸ cells were immunoprecipitated with 2D9 or M2. Immunoprecipitates were blotted with rabbit polyclonal anti-Nod2 antibody.

Figure 2

Expression of NOD2 in terminal ileum from Crohn’s disease (CD) patients and controls. (A–D) Uninvolved terminal ileum from a CD patient stained with 2D9 (A, C, D) or B-14 isotype matched control antibody (B). (E, F) Uninvolved ileum from a CD patient stained with antilysozyme antibody. (D) and (F) are high power views of Paneth cells from (A) and (E), respectively, illustrating NOD2 labelling at the periphery of secretory granules and lysozymes inside the granules. (G) Inflamed terminal ileum from a CD patient stained with 2D9, showing Paneth cell staining (indicated by arrowheads). (H, I) Medium power view of Paneth cells stained with 2D9 (arrowheads) from two different CD patients homozygous for the L1007fsinsC mutation. Magnification: 250× (A, B, E); 500× (C, G); 1000× (H, I); 2500× (D, F). The results shown in panels A–D, E, F, G, H, and I are from different patients and are representative of all patients and controls studied.

Figure 3

Expression of NOD2 mRNA in crypt and villus epithelial cells. (A) Whole villi and (B) crypts containing Paneth cells (arrows) were obtained by calcium chelation and mechanical shaking of mucosal tissue dissected from surgically resected small intestinal segments, and viewed under the 20× objective in an inverted phase microscope. (C) Quantitative real time reverse transcription-polymerase chain reaction (RT-PCR) analysis of crypt and villus epithelium shows enrichment of lysozyme and NOD2 mRNA expression in crypts. Lysozyme is approximately 40-fold enriched in crypts and NOD2 approximately 20-fold enriched in crypts from the terminal ileum compared with villi or colonic crypts. Relative expression of NOD2 and lysozyme mRNA was determined by real time RT-PCR, using intron spanning primers, and NOD2:glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lysozyme:GAPDH ratios, calculated for each sample. Results are shown as relative expression compared with the level in small intestinal crypts.

Figure 4

Expression of NOD2 in colon from Crohn’s disease (CD) patients and controls. (A, B) Uninvolved colon from a CD patient stained with 2D9 (A) or with isotype matched control antibody (B). (C) Inflamed colon from a CD patient showing no detectable 2D9 staining in the colonic epithelium. (D) Distal colon from a CD patient with Paneth cell metaplasia (indicated by arrowheads) stained with 2D9. (E) Inflamed colon from a CD patient showing focal NOD2 staining in colonic epithelium. (F) Inflamed colon from an ulcerative colitis patient showing focal NOD2 staining in colonic epithelium (indicated by arrowheads). (G–I) High power views of (D), (E), and (F), respectively. Magnification: 500× (A–F); 2500× (G–I).

Figure 5

NOD2 protein expression in lamina propria mononuclear phagocytes in Crohn’s disease (CD). (A) NOD2 protein is detected within lamina propria mononuclear cells (arrows) in CD by immunohistochemical staining using the 2D9 antibody. (B) No positive staining is noted in parallel sections incubated with no primary antibody. (C) At higher magnification, NOD2 protein is detected in the cytoplasm of lamina propria cells. (D) Immunohistochemical staining using an anti-CD68 antibody verifies the identity of lamina propria monocytes. All sections were counterstained with haematoxylin and viewed under a 40× (A, B) or 100× (C, D) objective.