Background: Mutations in NOD2, a putative intracellular receptor for bacterial peptidoglycans, are associated with a subset of Crohn's disease but the molecular mechanism linking this protein with the disease pathogenesis remains unclear. Human a defensins (HD-5 and HD-6) are antibiotic effector molecules predominantly expressed in Paneth cells of the ileum. Paneth cells also express NOD2. To address the hypothesis that the function of NOD2 may affect expression of Paneth cell defensins, we compared their expression levels with respect to NOD2 mutations in Crohn's disease. Methods: Forty five Crohn's disease patients (24 with NOD2 mutations, 21 with wild-type NOD2) and 12 controls were studied. Real time reverse transcription-polymerase chain reaction was performed with mucosal mRNA for HD-5, HD-6, lysozyme, secretory phospholipase A 2 (sPLA 2 ), tumour necrosis factor a, interleukin 8, and human hypoxanthine phosphoribosyltransferase (housekeeping gene). Immunohistochemistry with anti-HD-5 and histological Paneth cell staining were performed in 10 patients with NOD2 mutations or wild-type genotypes. Results: Ileal expression of HD-5 and HD-6, but not sPLA 2 or lysozyme, were diminished in affected ileum, and the decrease was significantly more pronounced in patients with NOD2 mutations. In the colon, HD-5, HD-6, and sPLA 2 were increased during inflammation in wild-type but not in NOD2 mutated patients. In both the colon and ileum, proinflammatory cytokines and lysozyme were unaffected by NOD2 status. Immunohistochemistry identified Paneth cells as the sole source of HD-5. Conclusion: As alpha defensins are important in the mucosal antibacterial barrier, their diminished expression may explain, in part, the bacterial induced mucosal inflammation and ileal involvement of Crohn's disease, especially in the case of NOD2 mutations.

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SummaryRecent evidence suggests that probiotic bacteria may stabilize gut barrier function via induction of anti-microbial peptides such as defensins. This study aimed to elucidate the induction mechanism of the human beta defensin-2 (hBD-2) gene by different probiotic lactobacillus strains. The expression of hBD-2 mRNA peaked at 6 h of incubation upon treatment of Caco-2 cells and increased with higher dosage of various probiotic bacteria. Deletion of nuclear factor (NF)-kB and activator protein-1 (AP-1) binding sites on the hBD-2 promoter resulted in a complete abrogation of promoter activation by probiotics. As revealed by the use of specific mitogen-activated protein kinase (MAPK) inhibitors the hBD-2 induction was dependent on the MAPK extracellular regulated kinase (ERK 1/2), p38 and c-Jun N-terminal kinase (JNK), although to varying degrees. Several Lactobacillus strains and VSL#3, a probiotic cocktail of four lactobacilli, three bifidum and one streptococcus species, induced the secretion of the hBD-2 peptide into the culture media as shown by enzyme-linked immunosorbent assay (ELISA). Thus, the present study suggests that lactobacilli and the VSL#3 bacterial mixture strengthen intestinal barrier functions through the up-regulation of hBD-2 via induction of proinflammatory pathways including NF-kB and AP-1 as well as MAPKs.

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