IKIP的表达调控及其在TLR介导的免疫反应中的作用

Tight regulation of TLRs signaling plays an important role in regulating inflammatory responses and maintaining immune homeostasis, yet the molecular mechanisms responsible for its negative regulation are not completely understood. It has been reported IKIP interacts with IKKβ by yeast two-hybrid screens. Given that IKKβ plays a central role in TLRs-triggered production of proinflammatory cytokines, the role of IKIP in the regulation of TLR-triggered immune response remains unknow. We found that knockdown of IKIP increased proinflammatory cytokine production in macrophages induced by LPS and PGN, whereas overexpression of IKIP had an opposite effect. We further demonstrated IKIP specificly binded with IKKβ but not with IKKα and NEMO. We hypothesize IKIP competes with NEMO for IKKβ and inhibits IKK activity and regulates TLR-triggered production of proinflammatory cytokines. By using RT-PCR, reporter gene assay, co-immunoprecipitation, CHIP and other molecular techniques, we demonstrate this hypothesis. We aim to clarify the molecular mechanisms of IKIP on regulating TLR-triggered inflammatory responses, add new insight into the regulation of TLRs signaling pathway and provide the theoretical basis to prevent bacterial infections and other diseases by using gene transfection.

严密调控TLRs通路在调节炎症反应以及维持机体免疫稳态中起着重要作用，然而其负向调节的分子机制研究并不完善。有作者通过酵母双杂交实验预测IkB激酶结合蛋白（IKIP）可以和IKKβ相互作用。考虑到IKKβ作为TLR介导炎性细胞因子表达的关键分子，IKIP是否在TLR通路中发挥的作用仍然未知。我们前期结果显示干扰掉IKIP后LPS以及PGN诱导的炎性细胞因子表达明显增加；高表达IKIP则抑制其表达。进一步实验证明IKIP特异性与IKKβ相互作用。我们假设IKIP通过与NEMO竞争结合IKKβ，抑制IκB激酶（IKK）复合物活性，进而调控TLRs介导的炎性因子的产生。本课题利用RT-PCR、报告基因、免疫共沉淀以及CHIP等试验方法证明这一假说。本研究旨在阐明IKIP负向调控TLRs介导的炎症反应的分子机制，为负向调控TLRs信号通路提供新认识，为寻找基因转染防治细菌感染等疾病提供理论基础。

核转录因子κB（Nuclear factor κB，NF-κB），是细胞内重要的核转录因子，其诱导表达的基因广泛参与各种生命活动，在免疫应答中起着非常重要的调节作用。NF-κB过度活化可以直接引起组织损伤、自身免疫性疾病以及炎症相关的癌症。因此，NF-κB信号通路的严密调控对维持机体免疫稳态起重要作用。目前，NF-κB活化的负向调节机制尚不完善。我们发现IKIP（IKK-interacting protein，IKIP）能够通过其N端的1-99个氨基酸与NEMO竞争性结合IKKα/IKKβ，抑制IκB激酶（IκB kinase, IKK）复合体的形成，从而阻碍IKKα/β的磷酸化，进而负向调节下游NF-κB信号通路的活化，抑制炎性细胞因子的表达。当LPS、poly(I:C)、TNF-α和IL-1β等刺激剂激活信号通路时，巨噬细胞特异性Ikip缺陷小鼠的IKKα/β、IκB和p65的磷酸化更强，TNF-α和IL-6的表达更多。脂多糖（Lipopolysaccharide，LPS）诱导的脓毒性休克模型和硫酸葡聚糖钠（Dextran Sulfate Sodium，DSS）诱导的结肠炎模型中，Ikip缺陷小鼠更加易感。综上所述，本课题研究揭示了IKIP在NF-κB信号中的负向调节作用及其机制，丰富了NF-κB的负向调节机制，并为IKK的激活调控提供了新的分子机制，可能有助于炎症性疾病新治疗策略的发展。

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