P050 Proinflammatory action of 5-HT in colon epithelial cell response to gut bacteria through NOX2 induction
J.-A. Kim*, S. Banskota, S. Regmi, P. Grung, B.-S. Jeong, H. W. Chang
Yeungnam University, College of Pharmacy, Gyeongsan, South Korea
Biopsy materials from inflammatory bowel disease (IBD) patients contain more invasive Escherichia coli (E. coli), which are termed adherent-invasive E. coli (AIEC), than in normal controls. Bacterial growth and virulence is known to be influenced not only by local environmental factors, but also by host signals such as amines. Serotonin (5-hydroxytryptamine, 5-HT), an enteric neurotransmitter and paracrine hormone, is increased in the gut in IBD patients, suggesting its role in the pathogenesis of IBD. The present study investigated proinflammatory role of 5-HT in inducing adhesion and invasion of commensal E. coli through colon epithelium.
The adhesion and invasion of commensal bacteria to colon epithelial cells was examined by co-culture of HT29 colon adenocarcinoma cells or CCD841 human colon epithelial cells with bacteria (E. coli O157:H7/pCM18 and E. coli K-12 BW25113/pCM18) in the presence of 5-HT. To identify signalling pathway, siRNA transfection, RT-polymerase chain reaction (PCR), and Western blot analyses were performed. Reactive oxygen species (ROS) were measured by lucigenin chemiluminescence assay.
Similar to the case of pathogenic E. coli, 5-HT stimulated adhesion of commensal E. coli to colon epithelial cells, and the adhesion was inhibited by the treatment with DPI (NADPH oxidase inhibitor), vitamin C (antioxidant), and siRNAs for NOX2 but not for NOX1. Co-treatment of colon epithelial cells with 5-HT and commensal E. coli synergistically increased not only ROS production but also gene expressions of IL-8, ICAM-1, NOX2, TLR-2, and TLR4. However, E-cadherin and claudin 2, junction proteins, were synergistically down regulated by co-treatment with 5-HT and commensal E. coli. Transfection of colon epithelial cells with TLR-2 or TLR-4 siRNA significantly suppressed commensal E. coli-induced changes in ICAM-1, E-cadherin, and NOX2 expression. Although silencing of TLR-2 or TLR-4 did not suppress 5-HT-alone-induced ROS production, it totally suppressed synergistic ROS increase in commensal E. coli and 5-HT-co-treated colon epithelial cells. In addition, knocking down of NOX2 blocked 5-HT-induced commensal E. coli adhesion to colon epithelial cells without blocking commensal E. coli-induced induction of TLR2 and TLR4.
5-HT induces colon epithelial NOX2 expression, which enhances epithelial TLR2/TLR4 induction response to commensal E. coli, thereby enhancing the E. coli adhesion and invasion into colon epithelium.