SIGIRR对H292细胞Toll样受体4、5、9致炎作用的影响及机制研究

Effects of SIGIRR on Toll-like receptor 4,5,9 inflammatory signaling pathway in H292 cells and its mechanism

目的观察SIGIRR(single Ig IL-1R-related molecule)对人气道上皮细胞株H292中Toll样受体(TLR)4、5、9致炎作用的影响,并初步了解其作用机制。方法运用脂质体转染的方法,将SIGIRR-EGFP融和蛋白真核表达载体稳定转染H292细胞,通过免疫细胞化学技术观察H292细胞中SIGIRR的过表达情况;经LPS、Flagellin、CpG DNA 2006处理后,ELISA检测重组质粒(SIGIRR-EGFP)、空质粒(pEGFP-N1)转染以及未转染的H292细胞分泌致炎因子TNF-α和IL-6水平;通过免疫共沉淀的方法了解SIGIRR与MyD88之间的关系。结果激光共聚焦显微镜显示重组融合蛋白SIGIRR-EGFP与H292内源SIGIRR共定位于细胞膜上;经LPS、Flagellin、CpGDNA 2006刺激后,重组质粒转染的H292细胞分泌的IL-6和TNF-α水平显著低于空质粒转染和未转染的H292细胞(P<0.01);免疫共沉淀提示H292细胞受到刺激后,SIGIRR与MyD88有较强相互作用,可与TLR4、5、9竞争结合MyD88分子。结论SIGIRR对H292细胞TLR4、5、9致炎通路的负性调节作用,可能是通过减少MyD88分子与TLR4、5、9的结合,从而削弱了炎症信号的细胞内转导。

Objective To characterize the modulatory effects of SIGIRR on Toll-like receptor (TLR) 4,5,9-mediated immune response in H292 cells and define its mechanisms. Methods SIGIRR mRNA levels in transfected H292 cells or wild-type were investigated by RT-PCR. The proinflammatory cytokines IL-6 and TNF-α were measured in the supernatants of H292 overexpressing the SIGIRR molecule,following treatment with various concentrations of Flagellin,LPS,and CpG DNA 2006,respectively. Moreover,interaction between SIGIRR and the TLRs adaptor myeloid differentiation factor 88 (MyD88) was observed by co-immunoprecipitation corresponding to Flagellin-,LPS-and CpG DNA 2006-stimulation,respectively. Results SIGIRR labeled with EGFP or TRITC was apparently colocalized. The protein levels for IL-6 and TNF-α were significantly decreased in SIGIRR-overexpressed H292 as compared to wild type. After stimulation,increased binding of MyD88 molecules to SIGIRR was found in overexpressed H292 compared to untransfected cells. Conclusion These results indicate that SIGIRR can inhibit TLR4,5,9-mediated immune responses in H292,probably by sequestrating MyD88 and thus obstructing the downstream signal transduction pathways normally instigated by TLR4,5,9 receptor binding and activation.