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. ? ?
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