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.

蜕膜出血/破裂会导致局部凝血酶产生强烈，并且与以下两种情况相关：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受体（PAR）起作用，诱导强烈的蜕膜细胞因子和蛋白水解反应。胎盘早剥和 CAM 之间的联系表明免疫反应受损。托尔利克受体（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。我们现在证明蜕膜细胞表达 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 的早剥相关 PTD 均伴有蜕膜 TLR 表达减少。我们假设蜕膜出血导致局部凝血酶的强烈生成，这自相矛盾通过下调诱导局部无菌炎症反应并促进上行生殖道感染 TLR 表达和功能。我们提出四个具体目标来评估之间的相互作用所有蜕膜细胞均表达凝血酶和 TLR。 1) 免疫组织化学、免疫荧光和显微切割与定量 RT-PCR 相结合将用于量化改变之间的关联 TLR 表达和与早剥相关的 PTD（有或没有相关 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 的表达。这些研究将使用激动剂、拮抗剂和小干扰细胞培养物中的 RNA (siRNA)。 3) 确定凝血酶对TLR-配体相互作用的功能影响。与对照相比，培养的蜕膜细胞将用凝血酶处理，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 和免疫分析以及通过蛋白质印迹评估 NFkB 成分。 4) 最后，与项目 I 和 III 协调在这个PO1中，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易感性。这些研究将为基本原理提供独特的见解与 PTD 相关的早剥的机制，并剖析先天免疫功能障碍在这一重大公共卫生问题。