Role of negative regulators for Toll-like receptor-dependent signaling in gut innate immune system

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Stimulation of Toll-like receptors (TLRs) by microbial components induces inflammatory responses, and excess and uncontrolled inflammation may lead to local tissue damage or systemic diseases. Several negative regulatory mechanisms control TLR-mediated inflammatory responses and restore the immune balance. In this study, we invested role of TLR signaling-related negative regulators including Toll-interacting protein (Tollip), IL-1-receptor-assoiated kinase (IRAK) and A20 in gut innate immune system.For several in vitro experiments of this study, colonic epithelial cell lines, HCT-15 and HT-29 were used. Initially, TLR ligands (LPS; ligand for TLR4, flagellin; ligand for TLR5)-mediated expression of Tollip, IRAK-M and A20 in colonic epithelial cells were examined by RNase protection assay. Stimulation with TLR ligands significantly induced gene expression of Tollip, IRAK-M and A20 in HCT-15 and HT-29 cells. Next, we hypothesized that these negative regulators may be associated with development of TLR ligand-induced tolerance in colonic epithelial cells. Development of TLR ligand-induced tolerance was assessed by reporter gene assay for NF-κB and production of IL-8 in HCT-15 and HT-29 cells. NF-κB activation and production of IL-8 after restimulation with LPS as well-as flagellin were significantly decreased. To evaluate precise role of negative regulators on the development of TLR ligand-induced tolerance, we established gene knock-down systems using each vector expressing siRNA targeting for Tollip, IRAK-M and A20 gene. Down-regulation of IRAK-M expression by siRNA specific for IRAK-.M gene reinstated NF-κB activation and production of IL-8 after re-stimulation with TLR ligands. However, treatment of siRNA targeting for Tollip and A20 gene did not reinstate TLR ligand-induced tolerance. These findings suggest that IRAK-M is a key molecule to induce TLR ligand-induced tolerance and may regulate innate immune balance in gut inflammatory conditions.