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

微生物刺激Toll样受体(TLRs)可诱导炎症反应，过度或失控的炎症可导致局部组织损伤或全身性疾病。几种负性调节机制控制TLR介导的炎症反应，恢复免疫平衡。在这项研究中，我们研究了TLR信号相关的负调控因子，包括Toll相互作用蛋白(Tollip)、IL-1受体相关激酶(IRAK)和A20在肠道固有免疫系统中的作用。首先，用核糖核酸酶保护法检测TLR配体(脂多糖；TLR4、鞭毛蛋白；TLR5)介导的Tollip、IRAK-M和A20在结肠上皮细胞中的表达。TLR配体刺激可显著诱导HCT-15和HT-29细胞Tollip、IRAK-M和A20基因表达。接下来，我们假设这些负调控因子可能与TLR配体诱导的结肠上皮细胞耐受的形成有关。通过转录因子κB的报告基因检测和IL-8的产生来评估TLRL诱导的耐受性的发展。再刺激后，NF-κB的活性和IL-8的产生以及鞭毛蛋白的表达均显著降低。为了评估负调控因子在TLR配体诱导的耐受形成中的确切作用，我们建立了针对Tollip、IRAK-M和A20基因的每个表达载体的siRNA的基因敲除系统。通过针对IRAK-M基因的小干扰RNA下调IRAK-M的表达，可恢复NF-κB的激活和IL-8的产生。然而，靶向Tollip和A20基因的siRNA治疗并不能恢复TLR配体诱导的耐受。这些发现表明，IRAK-M是诱导TLR配体诱导耐受的关键分子，并可能在肠道炎症条件下调节先天免疫平衡。

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.