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

微生物成分对Toll样受体（TLR）的刺激诱导炎症反应，并且过度和不受控制的炎症可导致局部组织损伤或全身性疾病。一些负调控机制控制TLR介导的炎症反应并恢复免疫平衡。本研究以结肠上皮细胞株HCT-15和HT-29为研究对象，探讨TLR信号负调控因子Toll相互作用蛋白（Tollip）、IL-1受体相关激酶（IRAK）和A20在肠道天然免疫系统中的作用。首先，通过RNA酶保护测定法检查结肠上皮细胞中TLR配体（LPS; TLR 4配体，鞭毛蛋白配体; TLR 5配体）介导的Tollip、IRAK-M和A20表达。TLR配体刺激显著诱导HCT-15和HT-29细胞中Tollip、IRAK-M和A20的基因表达。接下来，我们假设这些负调节因子可能与结肠上皮细胞TLR配体诱导的耐受性的发展有关。TLR配体诱导的耐受的发展通过HCT-15和HT-29细胞中NF-κB的报告基因测定和IL-8的产生来评估。LPS和鞭毛蛋白再刺激后，NF-κB活化和IL-8产生均显著降低。为了评估负调控因子在TLR配体诱导的耐受性发展中的确切作用，我们使用表达针对Tollip、IRAK-M和A20基因的siRNA的每种载体建立了基因敲低系统。通过特异性针对IRAK-.M基因的siRNA下调IRAK-. M表达，在用TLR配体再刺激后恢复NF-κB活化和IL-8产生。然而，靶向Tollip和A20基因的siRNA治疗不能恢复TLR配体诱导的耐受。这些发现表明IRAK-M是诱导TLR配体诱导的耐受的关键分子，并且可以调节肠道炎症条件下的先天免疫平衡。

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.