Epithelial exosomes and TLR-mediated mucosal defense

描述(由申请人提供)：粘膜表面的上皮细胞为宿主抵御胃肠道(Gl)病原体感染提供了第一线的防御。由于Toll样受体(Toll-like Receptor，TLR)信号在Gl黏膜免疫的启动和调节中的重要性，本研究的总体目的是为了更好地了解TLR信号协调Gl上皮抗菌防御的分子机制。我们的初步研究表明，微生物刺激以TLR4依赖的方式刺激培养的Gl上皮单层顶端的外切体释放。释放的外切体穿梭于多种抗菌肽之间，并在体外显示出抗菌活性。此外，TLR4/NF-？B信号的激活会导致microRNAs(MiRNAs)的表达发生变化，miRNAs是一种在转录后水平调节基因表达的非编码的小miRNAs。选定的TLR4应答miRNAs可能以调控胞吐过程的效应分子为靶标，因此，可能参与TLR4介导的外体释放。基于这些令人兴奋的新的初步数据，我们建议检验这样一种假设，即上皮细胞外切体的释放受到TLR信号的调控，参与了miRNA介导的转录后水平的基因调控，并有助于TLR介导的Gl上皮细胞的抗菌防御。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标：i)TLR信号通路调节顶端外切体的释放以响应微生物的挑战；ii)TLR信号通过与IKK/SNAP-23相关的胞吐过程刺激上皮细胞释放外切体，参与miRNA介导的转录后调控；iii)TLR信号调节抗菌肽的外切体穿梭；以及iv)上皮外切体参与TLR介导的上皮抗菌防御。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。从这项研究中获得的信息应该为设计和实施新的治疗策略提供合理的基础。 公共卫生相关性：拟议的研究将研究上皮细胞衍生的顶端外切体和miRNA介导的转录后基因调控在TLR相关的上皮抗微生物免疫反应中的作用。总的目标是阐明TLR信号协调上皮细胞抗菌防御的分子机制，并为识别新的治疗干预靶点提供基础。

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. PUBLIC HEALTH RELEVANCE: The proposed research will study the role of epithelial cell-derived apical exosomes and miRNA-mediated posttranscriptional gene regulation in TLR-associated epithelial antimicrobial immune responses. The overall goal is to elucidate the molecular mechanisms by which TLR signaling coordinates epithelial antimicrobial defense and provide a basis for the identification of novel new targets for therapeutic intervention.