Thrombin Effects on Decidual TLR Expression

凝血酶对蜕膜 TLR 表达的影响

基本信息

批准号：
7714226
负责人：
CHARLES JOSEPH LOCKWOOD
金额：
$ 20.61万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-10 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7714226
关键词：
Abruptio Placentae Affect Agonist Allogenic Bacterial Infections Birth Cell Culture Techniques Cells Complement Coupled Decidua Decidual Cell Dendritic Cells Disseminated Intravascular Coagulation Endometrial Endothelial Cells Event Gene Expression Generations Genes Genital system Gestational Age Health Heat shock proteins Hemorrhage Host Defense Human IRAK2 gene IRAK4 gene Immune Immune System Diseases Immune response Immune system Immunoassay Immunofluorescence Immunologic Immunohistochemistry Infection Infection of amniotic sac and membranes Inflammation Inflammatory Inflammatory Response Interleukin-8 Ligands Link Lipopolysaccharides Matrix Metalloproteinases Mediating Messenger RNA Microarray Analysis Microbe Microdissection Modeling Molecular Molecular Profiling Morbidity - disease rate Mus Mutant Strains Mice NF-kappa B Natural Immunity Natural Killer Cells Pathway interactions Patients Pattern Peptidoglycan Perinatal Placenta Poly I-C Predisposition Pregnancy Premature Birth Premature Rupture Fetal Membranes Production Proteinase-Activated Receptors Public Health RNA Interference Reaction Receptor Gene Receptor Signaling Recurrence Regulation Reverse Transcriptase Polymerase Chain Reaction Role Signal Transduction Signaling Protein Stream Stromal Cells TLR2 gene TLR4 gene Testing Thrombin Time Tissues Toll-like receptors Western Blotting chemokine cytokine in vivo insight interest laser capture microdissection macrophage microbial mortality neutrophil pathogen pregnant preterm premature rupture of membranes protein expression receptor receptor expression receptor function research study response stress protein

项目摘要

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.

判决出血/破坏会导致强烈的局部凝血酶产生，并且两者都相关膜（PPROM）和绒毛膜炎（CAM）的早产过早破裂。先前的研究表明通过蛋白酶活化受体（PAR）作用的凝血酶会诱导强烈的decIDual细胞因子和蛋白水解反应。突发与CAM之间的联系表明免疫反应受损。收费像受体（TLR）启动宿主先天免疫反应。通过促进先天免疫反应， TLR提供了针对一系列微生物病原体的第一道防线。我们现在证明了 dec骨细胞表达TLR及其中间信号蛋白。重要的是，我们证明了凝血酶下调降低了dec骨细胞TLR的表达和信号传导。最后，我们证明了有或没有CAM的突发相关的PTD伴随着降低的TLR表达。我们假设决定性出血会导致强烈的局部凝血酶产生，从而自相矛盾诱导局部无菌炎症反应，并通过下调促进上升生殖道感染 TLR表达和功能。我们提出了四个特定目标，以评估所有decIDual细胞表达的凝血酶和TLR。 1）免疫组织化学，免疫荧光和与定量RT-PCR相结合的显微解剖将用于量化变化之间的关联 TLR表达和与有和没有相关CAM的突发相关的PTD。 2）确定凝血酶下调的机制可将决定性细胞表达的TLR水平和功能下调。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效应。培养的判例性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评估NFKB组件。 4）最后，与项目I和III的协调这个PO1，我们将利用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。这些研究将为基本的独特见解与PTD相关的机制，并剖析了先天免疫功能障碍在这个主要的公共卫生问题。