Thrombin Effects on Decidual TLR Expression

蜕膜出血/脱落导致强烈的局部凝血酶生成，并与两者相关 早产胎膜早破（PPROM）和绒毛膜炎（CAM）。先前的研究表明 凝血酶通过蛋白酶激活受体（PAR）起作用，诱导强烈的蜕膜细胞因子， 蛋白水解反应预防和CAM之间的联系表明免疫反应受损。toll样 受体（TLR）启动宿主先天性免疫应答。通过促进先天免疫反应， TLR提供了抵抗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TLR及其中间信号蛋白。重要的是，我们证明， 凝血酶下调蜕膜细胞TLR表达和信号传导。最后，我们证明， 伴有或不伴有CAM的妊娠相关PTD均伴有蜕膜TLR表达降低。 我们假设蜕膜出血导致强烈的局部凝血酶生成， 诱导局部无菌性炎症反应，并通过下调 TLR表达和功能。我们提出了四个具体目标，以评估之间的相互作用 凝血酶和TLR的表达。1)免疫组织化学、免疫荧光和 显微切割结合定量RT-PCR将被用于定量改变的 TLR表达和预防相关的PTD伴或不伴相关CAM。2)确定 凝血酶下调蜕膜细胞表达的TLR水平和功能的机制。研究 将剖析PAR在TLR及其下游信号中间体表达中的作用， 以及细胞因子和NFkB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（siRNA）。3)确定凝血酶对TLR-配体相互作用的功能影响。 培养的蜕膜细胞将用凝血酶与对照处理，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和免疫测定以及 通过蛋白质印迹法评估NFkB组分。4)最后，与2010年的项目一和项目三协调， 本PO 1，我们将利用小鼠模型来研究凝血酶对TLR表达和功能的影响， 以及对细菌感染的敏感性。这些研究将提供独特的见解的基本 与PTD相关的先天性免疫功能障碍的作用， 这一重大公共卫生问题。

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.