蛋白磷酸酶1负向调控TLR信号效应及相关分子机制研究

Toll-like receptors (TLRs) take critical roles in host defense against infection by detecting conserved components of invading microbial pathogens. Although TLRs have a critical role in both innate and adaptive immune response, it has become apparent that loss of negative regulation of TLR signaling is strongly associated with the pathogenesis of inflammatory and autoimmune disease. Though they have been investigated extensively, the detailed mechanisms by which TLR signaling is fine tuned remain unclear. Protein phosphatase 1 (PP1) is a major protein Serine/Threonine phosphatase and participates in many signal pathway. However, the role of PP1 in regulation of innate immune response, especially its function in TLRs pathway has not been described. In our recent work, we find that lipopolysaccharide (LPS) stimulation inhibits the enzymatic activity of PP1, but has no effect on the expression of PP1. Furthermore, PP1 has been shown to attenuate TLRs-activated proinflammatory cytokine and type I IFN production in Macrophage. These facts suggest PP1 is a potential negative regulator of TLRs. Then we use over-expression and RNA interference to study the cytokine secretion and the alteration of related signaling molecular. We will further use the technology of co-immunoprecipitation and dephosphorylation to investigate the target molecular and protein phosphorylation site of PP1. This project will support the feedback circuit of TLR-PP1-TLR. This research add new insight into the innate immune homeostasis and regulatory mechanism of immune response. It also provides a new target for prevention and treatment of infectious diseases.

Toll样受体（TLR）是免疫细胞识别病原保守成分启动固有免疫和适应性免疫的重要分子, 其过度活化将导致免疫病理反应, 因此TLR信号通路受到复杂而精细的调控。蛋白磷酸酶1(PP1)作为非常重要的丝氨酸/苏氨酸磷酸酶参与调节多种信号途径。但PP1与TLR信号调节的关系尚未明确。我们前期研究发现，LPS不影响巨噬细胞（Mφ）中PP1蛋白的表达,但明显抑制PP1的磷酸酶活性。且过表达PP1显著降低TLR配体诱导Mφ分泌前炎性因子和I型干扰素。提示PP1负相调控TLR信号效应。本课题通过过表达和干扰技术，研究PP1表达改变后TLR配体诱导Mφ分泌细胞因子及相关信号分子的变化，并通过免疫共沉淀和去磷酸化实验探讨PP1负向调控TLR信号的作用靶分子及氨基酸位点。从而提出TLR-PP1-TLR的反馈环路。该项目的完成为进一步阐明天然免疫稳态维持和精细调控机制提供理论基础，并为防治感染性疾病提供新靶点。

Toll样受体（TLR）是免疫细胞识别病原保守成分启动固有免疫和适应性免疫的重要分子，其过度活化将导致免疫病理反应，因此TLR信号通路收到复杂而精细的调控。蛋白磷酸酶1（PP1）作为非常重要的丝氨酸/苏氨酸磷酸酶参与调节多种信号途径，调节细胞周期、基因表达、蛋白合成、糖脂代谢、组织分化发育以及记忆形成等生理过程。但PP1在天然免疫反应中的作用及其分子机制并不明确。我们首次发现蛋白磷酸酶1(PP1)参与负向调节巨噬细胞TLR信号通路。TLRs配体不影响PP1蛋白在小鼠巨噬细胞系RAW264.7中的表达，但明显抑制了PP1的磷酸酶活性。同时还发现PP1抑制TLR和RLR诱导的巨噬细胞中IRF3活化。进而证明了PP1以磷酸化依赖的方式抑制TRIF-、RIG-1-、MAVS-、TBK1-、IKKε-活化的IRF3、IFN-b报告基因的表达。最后证明PP1在Ser385 和Ser396位点去磷酸化IRF3从而减弱TLR-和RLR-刺激后的巨噬细胞分泌I型干扰素。因此。PP1作为IRF3的磷酸酶成为TLR和RLR诱导的免疫反应的负反馈调节子。进一步阐明PP1在天然免疫稳态维持中发挥精细调控作用。