Interactions of intestinal epithelial cells with bacterial extracellular products

The enteric microflora represents one of the densest microbial populations in the biological world; as a consequence, the intestinal immune system is constantly exposed to high concentrations of antigenic materials. One of the major frontline defences in the innate immune system is the intestinal epithelial layer, which presents both a physical barrier and an immune sensor to the antigens of the lumen. The latter function is performed by the expression of pattern recognition receptors, which recognise a wide variety of bacterial antigens, and the production of inflammatory cytokines, which stimulate, or inhibit, inflammation. The overall aim of the present study was to investigate the immunomodulatory potential of extracellular products, from non-pathogenic bacteria, with intestinal epithelial cells. Two in vitro human intestinal epithelial cell lines HT29-19A and Caco-2 were shown to exhibit different expression levels of Toll-like receptors (TLRs) and the inflammatory cytokines, interleukin (IL)-8 and IL-10. These differences were reflected in their sensitivity (monitored by IL-8 release) to known TLR agonists, isolated from pathogenic bacteria. Caco-2 cells were also shown to form physiologically active tight junctions, with the formation and maintenance of domes. Both cell lines exhibited sensitivity to the cytotoxic extracellular products of the enteropathogen Clostridium difficile. Extracellular products, in crude cell-free supernatants and bacterial sonicates, from the commensal Gram-negative bacterium Escherichia coli C25, significantly increased IL-8 release in both cell lines. Lipopolysaccharides and membrane vesicles were shown to contribute to the proinflammatory effects of C25-derived extracellular products. These extracellular products were also shown to regulate bacterial internalisation in both cell lines. Crude cell free supernatants and bacterial sonicates from two lactobacilli strains Lactobacillus acidophilus 5e2 and Lactobacillus helveticus sp. Rosyjski were also found to be biologically active, stimulating IL-8 release and TLR expression modification in both intestinal epithelial cell lines. In addition, ultrapure EPSs, isolated from these lactobacilli strains, were also found to possess immunomodulatory potential. HT29-19A cells, pre-treated with EPSs, were found to be ‘primed’ to bacterial agonists, peptidoglycan and flagellin, with a significantly potentiated release in IL-8 observed. Finally, EPSs were also found to modify bacterial adherence and internalisation in both cell lines. In conclusion, data presented in this investigation has shown that the use of the intestinal epithelial cell lines, HT29-19A and Caco-2, presents a reasonable model for investigating the interaction of bacterial extracellular products with the intestinal epithelium. Additionally, it has demonstrated that extracellular products, isolated from non pathogenic, enteric-associated bacteria, possess immunomodulatory potential in vitro. If these effects were also to occur in vivo, then they could potentially contribute to intestinal homeostasis and the innate ‘priming’ of the epithelial layer to pathogens and their products.