A42 SPECIES SPECIFIC BACTERIAL LIPOPOLYSACCHARIDES INDIVIDUALLY MODULATE INTESTINAL EPITHELIAL PERMEABILITY AND INFLAMMATION THROUGH TOLL-LIKE RECEPTOR 4

Patients presenting with Inflammatory bowel disease, both Crohn’s disease and ulcerative colitis, have been shown to exhibit an altered microbiome. This shift in the microbial content led us to question the importance of several of these microbes in inflammatory processes present in these diseases, and more specifically whether lipopolysaccharides from the cell wall of gram-negative bacteria differentially affect resident cell’s functions. We aimed to investigate the inflammatory contribution of five major species of gram-negative gamma-proteobacteria found to be altered in presence during IBD progression and evaluate their effect on intestinal epithelial monolayers through LPS-TLR4 activation. TLR4/MD2/CD14 co-transfected cells were seeded in 96 well plates and maintained in DMEM cell culture medium during stimulation. The cells were treated with 1ng/ml of LPS from either E.coli, K.pneumoniae, S.enterica, P.aeurginosa or S.marcescens for 24hrs at 37oC in 5% CO2. Induction of NF-κB was analysed using the QUANTIblue SEAP reporter. Permeability and stimulation assays were performed on CACO-2 human colonic epithelial carcinoma cells which were grown in either 24 well plates for cytokine analysis or to a tight monolayer on 0.4µm transwell plates. Cells were then incubated for 24hrs with or without species specific LPS and supernatants were collected for cytokine analysis or FITC-Dextran flux measured as a proxy for permeability. ZO-1 staining was also performed to classify monolayer integrity after treatment. We demonstrated that LPS from these different species had individual capacities to induce NF-κB from HEK-TLR4 cells in a TLR4 dependent manner. Additional work using human intestinal colonic epithelial cell monolayers (CACO-2) demonstrated that the cells responded to the serotype specific LPS in a distinct manner inducing TNF-α and IL-8 in significantly altered proportions. Furthermore, permeability of Caco-2 monolayers was also differentially altered by the serotype specific LPS through regulation of ZO-1. Our results suggest that specific species of bacteria may be potentiating the pathogenesis of IBD and shaping chronic inflammatory diseases through their TLR4 serotype-specific LPS responses. This may also include modulation of intestinal epithelial permeability and inflammatory response providing a potential target for microbial therapy. None

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