Thrombin Effects on Decidual TLR Expression

蜕膜出血/早剥导致局部凝血酶的强烈生成，并与 早产胎膜早破(PPROM)和绒毛膜羊膜炎(CAM)。先前的研究表明 凝血酶通过蛋白酶激活的受体(PARs)作用，诱导强烈的蜕膜细胞因子和 蛋白分解反应。胎盘早剥和CAM之间的联系表明免疫反应受损。Tolllike 受体(TLR)启动宿主的先天免疫反应。通过促进先天免疫反应， TLRs提供了抵御一系列微生物病原体的第一道防线。我们现在证明 蜕膜细胞表达TLRs及其中间信号蛋白。重要的是，我们证明了 凝血酶下调蜕膜细胞TLR的表达和信号。最后，我们证明了 伴或不伴CAM的早剥相关性PTD伴随着蜕膜TLR表达的降低。 我们推测蜕膜出血会导致强烈的局部凝血酶生成，这是自相矛盾的 诱导局部无菌性炎症反应，并通过下调表达促进上升生殖道感染 TLR的表达和功能。我们提出了四个具体目标来评估 所有蜕膜细胞均表达凝血酶和TLRs。1)免疫组织化学、免疫荧光和 显微解剖结合定量RT-PCR将被用来量化改变之间的联系 伴有和不伴有相关CAM的TLR表达和早剥相关PTD。2)确定 凝血酶下调蜕膜细胞表达TLR水平和功能的机制(S)。研究 将剖析PARs在TLR及其下游信号中间产物AS表达中的作用 以及细胞因子和NFkB的表达。这些研究将使用激动剂、拮抗剂和小干扰。 细胞培养中的RNA(SiRNAs)。3)测定凝血酶对TLR-配体相互作用的功能影响。 培养的蜕膜细胞将用凝血酶处理与对照组，然后暴露于TLR-2，3和4配体。 这些研究的终点将通过微阵列分析、RT-PCR和免疫分析以及 免疫印迹法鉴定NFkB组分。4)最后，与#年的项目一和项目三协调 因此，我们将利用一个小鼠模型来研究凝血酶对TLR表达和功能的影响。 以及对细菌感染的易感性。这些研究将提供对基本知识的独特见解 早剥相关PTD的发病机制及先天免疫功能障碍的研究 这一重大的公共卫生问题。

Decidual hemorrhage/abruption results in intense local thrombin generation and is associated with both preterm premature rupture of the membrane (PPROM) and chorioamnionitis (CAM). Prior studies indicate that thrombin, acting via protease activated receptors (PARs), induces an intense decidual cytokine and proteolytic response. The link between abruption and CAM suggests an impaired immune response. Tolllike receptors (TLRs) initiate the host innate immune response. By promoting the innate immune response, TLRs provide the first line of defense against an array of microbial pathogens. We now demonstrate that decidual cells express TLRs and their intermediate signaling proteins. Importantly, we demonstrate that thrombin down-regulates decidual cell TLR expression and signaling. Finally, we demonstrate that abruption-associated PTD with or without CAM are accompanied by decreased decidual TLR expression. We postulate that decidual hemorrhage leads to intense local thrombin generation which paradoxically induces a local aseptic inflammatory reaction and promotes ascending genital tract infections by downregulating TLR expression and function. We propose four Specific Aims to assess the interactions between thrombin and TLRs expressed by all decidual cells. 1) Immunohistochemistry, immunofluorescence and microdissection coupled with quantitative RT-PCR will be utilized to quantify the association between altered TLR expression and abruption-associated PTD with and without related CAM. 2) To determine the mechanism(s) by which thrombin down-regulates decidual cell-expressed TLR levels and function. Studies will dissect out the role of PARs in the expression of TLRs and their downstream signaling intermediates as well as cytokine and NFkB expression. These studies will use agonists, antagonists and small interference RNA (siRNAs) in cell culture. 3) To determine the functional effects of thrombin on TLR-ligand interactions. Cultured decidual cells will be treated with thrombin vs. control and then exposed to TLR-2, 3 and 4 ligands. Endpoints of these studies will be assessed by microarray analysis, RT-PCR and immunoassays as well as assessment of the NFkB components by western blotting. 4) Lastly, in coordination with Projects I and III of this PO1, we will utilize a murine model to study the effects of thrombin on TLR expression and function as well as the susceptibility to bacterial infection. These studies will provide unique insights into the fundamental mechanisms underlying abruption associated PTD and dissect out the role of innate immune dysfunction in this major public health problem.