骨桥蛋白对巨噬细胞中Toll样受体触发的炎症反应的调控机制研究

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns which are associated with groups of pathogens. By triggering immune response, TLRs play an important role in host defense against invading microbial pathogens. However, mechanisms underlying down-regulation of TLR-triggered inflammatory response are largely unknown. Osteopontin has been implicated in various inflammatory diseases as a glycoprotein.Our previous work has demonstrated that mRNA levels of TLR2, TLR4, TLR9, MD2, CD14 and MyD88 were significantly higher in OPN-/- mice than that in wild-type(WT) mice after treatment with P.acnes. Furthermore, the mRNA expression levels of TNF-α, IL-6, IL-12 and IFN-γ were markedly increased in the liver of OPN-/- mice compared with that of WT mice. Thereafter, we hypothesize that OPN might play as a negative regulator of TLRs-mediated inflammatory responses in macrophages. In this project, we will investigate the mechanism(s) underlying OPN-mediated signaling pathway in down-regulating TLRs response using OPN-/- mice as well as kupffer cells and peritoneal cells isolated from OPN-/- mice and WT mice. Our work will provide new insights into the function of OPN in infectious diseases、autoimmune diseases and tumor. This study will contribute to our better understandings of the regulatory mechanism(s) of TLRs signal pathways in immune recognition and modulation of immune response. It'll offer valuable clues for new approaches of immunoregulatory therapy (in infectious diseases, autoimmune disorders and human tumors).

Toll样受体（TLRs）在机体抵抗外界病原微生物感染中发挥重要的保护作用，但TLRs信号通路的负向调控机制仍不明确。骨桥蛋白为一分泌性糖蛋白，可参与机体多种炎症性反应。我们前期研究中发现骨桥蛋白缺失（OPN-/-）小鼠在痤疮丙酸杆菌刺激下其TLR2、TLR4、TLR9、CD14、MD2、MyD88及炎症性细胞因子IL-6、TNF-α、IL-12p40、IFN-γ水平与野生型小鼠相比均显著升高，据此我们提出假设：OPN可作为负向调控因子下调TLRs介导的炎症反应。本项目拟引入OPN-/-小鼠作为研究对象，利用前期建立的原代细胞分离培养平台，运用ELISA、Western Blot及实时定量PCR方法研究OPN负向调控TLRs信号通路的分子机制。本项目的研究将进一步揭示TLRs信号通路的调节机制，对深入探索免疫识别与免疫应答调控的研究具有重要的理论意义，并为寻找免疫调节治疗的新途径指明方向。

Toll样受体（Toll-like receptors，TLRs）可识别多种外侵病原微生物，激活信号转导通路并诱导炎症因子的释放，在机体免疫防御中发挥重要的保护作用。然而，TLRs信号通路的异常激活可引起炎症性疾病或自身免疫性疾病的发生。骨桥蛋白（Osteopontin， OPN）为一磷酸化糖蛋白，作为炎症因子参与多种细胞介导的免疫反应，但OPN在TLRs介导的炎症反应中的作用仍不明确。本研究将利用OPN基因敲除小鼠（OPN-/-）探讨OPN在TLRs介导的炎症反应中的作用。我们发现OPN-/-腹腔巨噬细胞与野生型细胞相比对LPS呈强反应性，释放大量的炎症性细胞因子；进一步实验揭示OPN的两种亚型（胞内OPN及分泌OPN）发挥相反的作用：胞内OPN抑制炎症因子的释放，而分泌OPN可促进炎症反应。随后，利用磷酸化特异的表达谱芯片分析发现，OPN-/-来源的巨噬细胞其Akt的磷酸化水平虽然与未刺激细胞相比上调，但其上调幅度与野生型巨噬细胞相比仍显著降低。此外我们发现，作为Akt和mTOR的下游分子，4EBP1的磷酸化水平在OPN-/-巨噬细胞中显著降低；同时，作为Akt的负向调控因子，GSK3β呈高度磷酸化状态。本研究揭示胞内OPN可通过影响GSK3β和4EBP1的磷酸化水平负向调控TLR4介导的免疫反应，丰富了OPN在天然免疫系统中的作用。