Epithelial exosomes and TLR-mediated mucosal defense

描述（由申请方提供）：沿粘膜表面的上皮细胞沿着提供了宿主防御胃肠道（GI）中病原体感染的前线。由于Toll样受体（TLR）信号传导在GI粘膜免疫的起始和调节中的重要性，本申请的总体目标是更好地理解TLR信号传导协调GI上皮抗微生物防御的分子机制。我们的初步研究表明，微生物挑战以TLR 4依赖性方式刺激外泌体从培养的GI上皮单层的顶侧释放。释放的外来体穿梭于多种抗微生物肽并显示出离体抗微生物活性。TLR4/NF-？B信号传导导致微小RNA（miRNAs）表达的改变，微小RNA是在转录后水平调节基因表达的小的非编码miRNAs。选择的TLR 4响应性miRNA可以靶向调节胞吐过程的效应分子，因此可能参与TLR 4介导的外泌体释放。基于这些令人兴奋的新的初步数据，我们提出检验以下假设：外泌体从上皮细胞的释放受TLR信号传导调节，并在转录后水平参与miRNA介导的基因调节，并且它有助于TLR介导的GI上皮抗微生物防御。我们将使用体外和体内感染模型以及补充的生物化学、分子和形态学方法来测试四个相互关联的特定目的：i）TLR信号传导途径调节响应微生物挑战的上皮细胞顶端外泌体的释放; ii）TLR信号传导通过IKK/SNAP-23相关的胞吐过程刺激外泌体从上皮细胞释放，其中miRNA-23参与了外泌体释放。iii）TLR信号传导调节抗微生物肽的外泌体穿梭;和iv）上皮外泌体有助于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。从这项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。 公共卫生相关性：拟议的研究将研究上皮细胞来源的顶端外泌体和miRNA介导的转录后基因调控在TLR相关上皮抗菌免疫反应中的作用。总体目标是阐明TLR信号协调上皮抗微生物防御的分子机制，并为识别治疗干预的新靶点提供基础。

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.