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

肠道先天免疫系统中Toll样受体依赖性信号传导的负调节因子的作用

基本信息

批准号：
17590643
负责人：
ISHIHARA Shunji
金额：
$ 2.24万
依托单位：
Shimane University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

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.

微生物成分刺激类似受体的受体（TLR）会诱导炎症反应，并且超过和不受控制的注射可能会导致局部组织损伤或全身性疾病。几种负调节机制控制TLR介导的炎症反应并恢复免疫平衡。在这项研究中，我们在TLR信号相关的负调节器中投入了作用，包括Toll-1-受体蛋白（TOLLIP），IL-1-受体可靠的激酶（IRAK）和A20在肠道先天性免疫系统中。对于这项研究的几个体外实验，用于肠道上皮细胞系，HCT-15和HCT-15和HT-29。最初，通过RNase Protection Assay检查了TLR配体（LPS；用于TLR4的配体，TLR4；用于TLR5的配体）介导的Tollip，Irak-M和A20的表达。用TLR配体刺激在HCT-15和HT-29细胞中显着诱导Tollip，Irak-M和A20的基因表达。接下来，我们假设这些负调节剂可能与TLR配体诱导的结肠上皮细胞的耐受性的发展有关。通过报告基因测定法对TLR配体诱导的耐受性的发展进行了NF-κB和HT-15和HT-29细胞中IL-8的产生评估。用LPS作为鞭毛蛋白重新填充后的NF-κB激活和IL-8的生产得到显着改善。为了评估负调节剂对TLR配体诱导的耐受性发展的精确作用，我们使用表达siRNA靶向Tollip，Irak-M和A20基因的每个载体靶向的每个载体建立了基因敲低系统。用TLR配体重新刺激后，由IRAK-.M基因恢复了IRAK-.M基因的IRAK-M表达下调IL-8。但是，对Tollip和A20基因的siRNA靶向治疗并未恢复TLR配体诱导的耐受性。这些发现表明，IRAK-M是诱导TLR配体诱导的耐受性的关键分子，并可能调节肠道炎症条件下的先天免疫平衡。