IRAK1单倍型介导IL-1R/TLR信号调节Th17功能异常在SLE发病中的作用及机制

The gene of interleukin-1-receptor-associated kinase 1(IRAK1),known as an gene for close relationship with many kinds of immune inflammatory diseases, has been found to be associated with the susceptibility of systemic lupus erythematosus(SLE) patients with genome-wide analyses from various global areas. It has been postulated that IRAK1 may regulate the dysfunctions of Th17 cells mediated by IL-1R/TLR signal pathways. In our previous work,we found that there is much difference of IRAK1 haplotypes between SLE patients and nomal subjects. In present project, the expression levels and functions of associated ranscription factors on periphery blood Th17 cells from patients with SLE will be detected by real-time PCR and immuno-blotting respectively based on different haplotypes and will be analysed to be associated with clinical phenotypes of SLE patients based on different IRAK1 haplotypes .From these bases on,co-expression vehicles containing different mutants of IRAK1 will be constructed and transferred into Th17 cells. Then, Effects on both the expression levels and functions of correlated transcription factor of Th17 cells will be observed in transferred cells.The anti-inflammatory effects with different mutants will be explored in C57/6-lpr mice. The aims of this project are to make a close connection between predisposing genes and immune-mediated inflammmation of SLE,and to explore the relationship between inflammation formation and immune disturbance of SLE mediated by inflammatory signaling pathways and to provide some novel evidences to establish biologic therapies on slectively turning off inflammatory signal to treat SLE.

全基因扫描技术证实白介素-1受体相关激酶1（IRAK1）是SLE发病的易感基因，而IRAK1与多种免疫炎症性疾病密切相关，并推测IRAK1可通过介导IL-1R/TLR信号途径诱导Th17细胞的功能异常。本课题选择IRAK1为切入点，以SLE患者为研究对象，在前期已明确IRAK1基因外显子单倍型在SLE存在显著差异工作基础上，分析SLE患者不同单倍型的Th17相关转录因子表达和功能的差异及其与临床关联性。构建IRAK1基因外显子区不同突变位点载体，借荧光素酶报告基因、染色体免疫共沉淀等方法，观察对Th17下游相关转录因子功能的影响及可能机制。在此基础上，选择狼疮鼠动物模型，观察给予不同表达的基因载体对狼疮炎症活动的影响。本研究将易感基因与SLE免疫性炎症形成有机结合，探讨炎症信号通路调控SLE免疫异常及其与炎症形成的关系，为开展选择性关闭炎症信号通路的生物治疗提供依据。

系统性红斑狼疮（systemic lupus erythematosus, SLE）是一种典型的慢性自身免疫性疾病，其发病机制十分复杂。GWAS研究发现，IRAK1是SLE的易感基因，在TLR/IL-1R信号通路中发挥着关键的作用。IRAK1被IRAK4激活，并在Thr209处发生磷酸化，进而IRAK1发生自身磷酸化，并与TRAF6发生相互作用，形成IRAK1-TRAF6复合物，促进NF-κB的活化及其核转位，并最终导致SLE的发病。此外，TNFAIP3及TNIP1也被GWAS确认为SLE的易感基因，在巨噬细胞中C/EBPβ可与TNFAIP3基因的启动子结合，并增加TNFAIP3 mRNA的表达。而在TNIP1基因敲除小鼠中，C/EBPβ LAP(liver-enriched activating protein)亚基表达却显著增加，同时出现狼疮样表现。我们的研究发现，SLE 患者CD4+T细胞中IRAK1表达增强，且与患者疾病严重程度呈正相关，同时异常增高的 Th17 细胞频率及 IL-17A 水平均与 IRAK1 表达 量呈正相关， 当狼疮初始CD4+T细胞中IRAK1的表达和磷酸化被抑制后，外周血Th17细 胞分化受阻、IL-17A 生成减少。进一步研究发现，SLE中IRAK1的活性明显增强，且NF-κB通路也异常活化；体外通过IRAK-Inh刺激SLE患者PBMCs 24h后，能够对NF-κB通路相关蛋白起到一定的抑制作用。动物实验证实，IRAK1在B6.lpr狼疮鼠脾脏单核细胞中异常活化，导致NF-κB通路被激活。IRAK-Inh能够逆转IRAK1及NF-κB的异常活化并对狼疮鼠肾脏损伤起到一定的治疗作用。同时，我们还发现，C/EBPβ mRNA在SLE患者外周血PBMC中表达升高,与TNIP1/TNFAIP3 mRNA表达、疾病严重程度呈正相关。而在患者PBMC中TNIP1和TNFAIP3蛋白表达下降。TNIP1干扰后THP-1细胞C/EBPβ LAP蛋白表达升高，而TNFAIP3干扰后THP-1细胞TNFAIP3蛋白、C/EBPβ LAP蛋白表达升高，证明在单核细胞中TNIP1和TNFAIP3蛋白抑制可以诱导C/EBPβ功能性表达。综上，SLE中存在IRAK1活性异常增高，且存在TNFAIP3相关分子的调控缺陷；利用IRAK-Inh抑制其活性能够对SL