Microbial Cell Components Induced Tolerance to Flagellin-Stimulated Inflammation through Toll-Like Receptor Pathways in Intestinal Epithelial Cells

摘要

In the intestine, bacterial components activate innate responses that protect the host. We hypothesize that bacterial components reduce Interleukin-8 (IL-8) production in intestinal epithelial cells stimulated by flagellin via the Toll-like receptor (TLR) signaling pathway. Caco-2 cells were pretreated with various doses of lipopolysaccharide (LPS), lipoteichoic acid (LTA), or low-dose flagellin (LDFL) for 24 hours. Cells were then treated with flagellin (FL) 500 ng/mL (HDFL) for another 48 hours. IL-8 production was measured in the cell culture medium by ELISA. Eighty-four genes in the TLR pathway were evaluated by RT Profiler PCR Array. Pathway Studio 8.0 software was used for altered pathway analysis. HDFL induced IL-8 production by 19 fold (p<0.01). Pre-treatment with LDFL at 20, 10 or 1 ng /ml reduced HDFL-induced IL-8 production by 61%, 52% and 40%, respectively (p<0.05). LPS at 50 µg/ml decreased HDFL–induced IL-8 production by 38% (p<0.05). HDFL up-regulated CXCL10, IL-1B, IL-8, IRAK2, NF-κB1 and I-κB (all p< 0.05). Pathway Studio analysis showed that HDFL induced cell processes including inflammation, cell death and apoptosis. Pre-treatment with LDFL at 10 ng/mL down-regulated FADD, FOS, MAP4K4, MyD88, TLR2, TLR3 and TNFERSF1A compared to HDFL (all p<0.05). These down-regulated genes are integral for numerous cell functions including inflammatory response, cell death, apoptosis and infection. These results demonstrate that LPS and LDFL provoke tolerance to HDFL-induced IL-8 production. This tolerance effect was accompanied by a complex interaction of multiple genes related to inflammatory as well as other responses in the TLR pathway rather than a single gene alteration.