Epithelial exosomes and TLR-mediated mucosal defense

描述（由申请人提供）：沿着粘膜表面的上皮细胞在胃肠道（Gl）中提供宿主防御病原体感染的前线。由于toll样受体（TLR）信号在Gl粘膜免疫的启动和调节中的重要性，本应用的总体目标是更好地了解TLR信号协调Gl上皮抗微生物防御的分子机制。我们的初步研究表明，微生物挑战刺激外泌体以tlr4依赖的方式从培养的Gl上皮单层的顶端释放。释放的外泌体穿梭各种抗菌肽并在体外显示抗菌活性。此外，TLR4/NF-？B信号传导导致microrna （mirna）表达的改变，microrna是在转录后水平调节基因表达的小的非编码mirna。选定的tlr4响应mirna可能针对调节胞吐过程的效应分子，因此可能参与tlr4介导的外泌体释放。基于这些令人兴奋的新颖初步数据，我们提出验证TLR信号调控外泌体从上皮细胞释放的假设，并参与miRNA介导的转录后水平基因调控，并有助于TLR介导的Gl上皮抗微生物防御。我们将使用体外和体内感染模型以及互补的生化、分子和形态学方法来测试四个相互关联的特定目的：i) TLR信号通路调节上皮细胞在微生物挑战下的顶端外泌体释放；ii) TLR信号通过IKK/SNAP-23相关的胞吐过程刺激上皮细胞外泌体释放，并参与mirna介导的转录后调控；iii) TLR信号调节抗菌肽的外泌体穿梭；iv)上皮外泌体参与tlr介导的上皮抗微生物防御。该提案在概念上是创新的，因为它测试了关于tlr介导的粘膜抗菌防御的新概念。从本研究中获得的信息应该为新的治疗策略的设计和实施提供合理的基础。

DESCRIPTION (provided by applicant): Epithelial cells along the mucosal surface provide the front line of host defense against pathogen infection in the gastrointestinal (Gl) tract. Because of the importance of Toll-like receptor (TLR) signaling in the initiation and regulation of Gl mucosal immunity, the overall objective of this application is to better understand the molecular mechanisms by which TLR signaling coordinates Gl epithelial antimicrobial defense. Our preliminary studies demonstrate that microbial challenge stimulates exosome release from the apical side of cultured Gl epithelial monolayers in a TLR4-dependent manner. Released exosomes shuttle a variety of antimicrobial peptides and display antimicrobial activity ex vivo. Moreover, activation of TLR4/NF-?B signaling causes alterations in expression of microRNAs (miRNAs), small non-coding miRNAs that regulate gene expression at the posttranscriptional level. Selected TLR4-responsive miRNAs may target effector molecules that regulate the exocytotic process and, thus, are potentially involved in TLR4-mediated exosome release. Based on these exciting novel preliminary data, we propose to test the hypothesis that the release of exosomes from epithelial cells is regulated by TLR signaling with the involvement of miRNA- mediated gene regulation at the posttranscriptional level, and that it contributes to TLR-mediated Gl epithelial antimicrobial defense. We will use in vitro and in vivo infection models and complementary biochemical, molecular, and morphologic approaches to test four interrelated Specific Aims: i) The TLR signaling pathway regulates release of apical exosomes from epithelial cells in response to microbial challenge; ii) TLR signaling stimulates exosome release from epithelial cells through the IKK/SNAP-23- associated exocytotic process with the involvement of miRNA-mediated posttranscriptional regulation; iii) TLR signaling regulates exosomal shuttling of antimicrobial peptides; and iv) epithelial exosomes contribute to TLR-mediated epithelial antimicrobial defense. The proposal is conceptually innovative as it tests new concepts regarding TLR-mediated mucosal antimicrobial defense. The information obtained from this study should provide a rational basis for the design and implementation of new therapeutic strategies.