Regulation of Human TLR3 & TLR9 Expression and Function

描述（由申请人提供）：细胞因子是两种基本且不同的子宫功能的关键调节因子：宿主防御和生殖。共同的细胞因子对不同子宫功能的调节导致交叉交流，对人类健康和疾病产生重大影响。细胞因子表达的变化可以满足宿主防御的需要，但改变胎儿耐受性，导致不孕或妊娠丢失。此外，雌激素和孕激素可以通过多种方式促进妊娠启动和维持，包括调节细胞因子表达，从而改变宿主防御。为了研究子宫细胞因子表达的调节，我们将注意力集中在子宫内膜上皮细胞上，子宫内膜上皮细胞是子宫细胞因子表达的主要来源，也是第一个接触植入胚胎或上升精子或微生物的子宫细胞类型。在非子宫组织中，Toll样受体（TLR）蛋白家族成员对微生物分子（和特异性宿主分子）的识别可刺激细胞因子的表达。然而，子宫内膜TLR的表达和功能仍然未被探索。初步数据表明两个总体假设：（1）TLR刺激子宫内膜细胞因子的表达和（2）TLR刺激的细胞因子表达受雌二醇和孕酮调节。为了验证这些假设，拟议的研究将集中在子宫内膜的TLR 3和TLR 9的表达和功能，这两种蛋白分别识别与病毒和细菌相关的核酸基序。首先，通过对整个子宫内膜进行免疫组织化学分析，并对取自月经周期各期的分离的上皮细胞和基质细胞进行实时定量RT-PCR（qRT-PCR）分析，确定表达TLR 3和TLR 9的子宫内膜细胞类型。还将通过qRT-PCR分析在原代子宫内膜细胞以及上皮细胞系（RL 95 -2和石川）中确定雌二醇和孕酮对体外TLR 3和TLR 9表达的影响。子宫内膜细胞和细胞系中的TLR 3和TLR 9功能将通过使用ELISA和细胞计数珠阵列（CBA）分析测量用TLR 3和TLR 9配体处理后细胞因子表达的变化来评估。最后，将使用通路抑制剂和蛋白质印迹分析来研究子宫内膜TLR在刺激刺激细胞因子表达中利用的细胞内信号以及雌二醇和孕酮对它们的调节。因此，拟议的研究将有助于了解调节人类子宫内膜细胞因子表达的机制，这可能会导致感染和生殖疾病的药物治疗的设计。

DESCRIPTION (provided by applicant): Cytokines are key regulators of two fundamental and distinct uterine functions: host defense and reproduction. The regulation of separate uterine functions by common cytokines results in cross-communication with significant impact on human health and disease. A shift in cytokine expression can serve the needs of host defense, but alter fetal tolerance contributing to infertility or pregnancy loss. Additionally, estrogen and progesterone can promote pregnancy initiation and maintenance by multiple means including modulation of cytokine expression resulting in altered host defense. To investigate the regulation of uterine cytokine expression, we have focused our attention on the endometrial epithelial cell, a prominent source of uterine cytokine expression and the first uterine cell type to contact an implanting embryo or ascending sperm or microbe. In non-uterine tissues, recognition of microbial molecules (and specific host molecules) by members of the Toll-like receptor (TLR) protein family can stimulate expression of cytokines. However, endometrial TLR expression and function remains unexplored. Preliminary data suggest two overall hypotheses: (1) TLRs stimulate expression of endometrial cytokines and (2) TLR-stimulated cytokine expression is modulated by estradiol and progesterone. In order to test these hypotheses, the proposed studies will focus on the endometrial expression and function of TLR3 and TLR9, which recognize nucleic acid motifs associated with viruses and bacteria, respectively. Initially, the endometrial cell types expressing TLR3 and TLR9 will be determined by immunohistochemical analysis of whole endometrium and real-time quantitative RT-PCR (qRT-PCR) analysis of separated epithelial and stromal cells taken from each phase of the menstrual cycle. The effects of estradiol and progesterone on expression of TLR3 and TLR9 expression in vitro will also be determined in primary endometrial cells as well as epithelial cell-lines (RL95-2 and Ishikawa) by qRT-PCR analysis. TLR3 and TLR9 function in endometrial cells and cell-lines will be assessed by measuring changes in cytokine expression after treatment with TLR3 and TLR9 ligands using ELISA and cytometric bead array (CBA) analyses. Finally, the intracellular signals utilized by endometrial TLRs in stimulating stimulate cytokine expression and their modulation by estradiol and progesterone will be investigated using pathway inhibitors and western blot analysis. Thus, the proposed studies will contribute to an understanding of mechanisms regulating human endometrial cytokine expression, which could lead to the design of pharmacologic therapies for both infectious and reproductive disorders.