O0033 THE PROBIOTIC STRAIN E. COLI NISSLE 1917 INDUCES HUMAN BETA DEFENSIN-2 BY A NF-k-B DEPENDENT MECHANISM IN CACO-2 CELLS

Abstract

Introduction: E. coli Nissle 1917 is an alternative treatment option to 5-ASA in remission maintenance of ulcerative colitis. The mechanism of action of this probiotic strain is still insufficiently understood. We have recently demonstrated the ability of E. coli Nissle 1917 to induce the human beta defensin-2 in CaCo-2 cells. Our aim was to investigate the E. coli Nissle mediated transcriptional regulation of the hBD-2 gene. Methods: To study the activation of the hBD-2 promoter and putative binding sites for NF-kappaB by E. coli Nissle, Luciferase reporter gene assays were applied. After transfection of CaCo-2 cells with the respective plasmids for 24 h, cells were incubated with E. coli Nissle and other strains, whose influence on hBD-2 expression had been predefined by real time PCR. To prove the direct impact of NF-kappaB, a hBD-2 promoter plasmid with 2 mutated binding sites of NF-kappaB (−186/−205 and −572/−596) was used for transfection. Results: The hBD-2 promoter was activated by E. coli Nissle as well as an uropathogenic E. coli strain, analogous to hBD-2 transcript induction in real time PCR. Remarkably, the bacterial supernatant prompted a 13 fold higher promoter activation compared to bacterial pellets. A NF-kappaB reporter plasmid was moderately activated only by E. coli Nissle. Additionally, the supernatant had the strongest capacity to activate NF-kappaB mediated transcription. After mutation of two NF-kappaB binding sites, hBD-2 promoter activation by complete bacteria, bacterial supernatant and the positive control IL-1beta; was almost completely blocked. Conclusion: The abrogation of hBD-2 promoter activation by E. coli Nissle following insertion of mutated NF-kappaB sites, indicates an important role of this transcription factor. The induction of the antimicrobial peptide hBD-2 through moderate activation of NF-kappaB, represents a novel mode of probiotic action.