Integration of mononuclear phagocytes into the human gastrointestinal GOFlowChip for investigation of luminal antigen sampling

项目总结 该项目将开发新的人体胃肠道(GI)体外模型以了解 对微生物的自然细胞反应和诱导免疫耐受和激活。最新进展 胃肠道器官，复杂原代上皮细胞群的3D永久培养嵌入 在细胞外基质中，已经彻底改变了胃肠道发育、微生物学和 近5年来的免疫学知识。对于我们的项目，我们组建了一个跨学科的调查团队 在生物工程(Wilking，Chang)、免疫学(Bimczok，Jutila)和人类微生物组方面拥有专业知识 研究(WALK)显著推进三维肠道有机物-微生物组共培养系统。我们队有 最近建立了一个毫秒级的芯片上肠管平台，GoFlowChip，它概括了流明和基础 在人体肠道器官中流动。在平行的研究中，我们建立了原始人的共培养 我们已经成功地感染了幽门螺杆菌的单核细胞来源的树突状细胞和人的胃球。在这里，我们 寻求利用我们两个模型的独特功能，并将它们结合到一个分析平台中，以 胃肠腔抗原采样诱导黏膜适应性免疫的研究 宽容。具体地说，我们寻求界定和量化候选机制的贡献，包括 单核吞噬细胞的跨上皮树突状细胞形成和Fc受体依赖的跨细胞转运 (MNPs)以获得管腔抗原。我们假设MNP抗原获得的不同机制是 复制并可以使用GOFlowChip平台进行定量分析，并且定殖菌和 流体动力学调节上皮抗原的运输。为了检验我们的假设，我们将(1)开发并验证一个 基于芯片的有机物-DC共培养系统，具有管腔和基侧流动能力。(2)量化净效应 GI类有机物生物学和MNP相互作用的生物复杂性。(3)阐明以下机制： 用人MNPs从胃肠腔内提取细菌抗原。这种方法将使我们能够 优化我们集成的GoFlowChip共培养系统，为疫苗研究领域提供强有力的新工具 或药物输送，以及肠道微生物区系对抗原采样的影响。拟议的研究是 概念上的创新，因为它集成了所有三种必要的细胞类型(上皮细胞、微生物细胞和免疫细胞) 参与了粘膜宿主与微生物的相互作用。GoFlowChip平台在技术上具有创新性，因为 它复制了一个复杂的利用氧气的上皮细胞和一个微生物定植的管腔，是第一个结合 转化为3-D有机物培养物，并再现了自然发生在 胃肠上皮细胞和前哨MNPs。这项拟议的研究意义重大，因为它将提供一种 令人难以置信的强大的新工具来解决人类粘膜生物学中的基本机械问题， 微生物学和免疫学在人类健康和疾病的背景下。

PROJECT SUMMARY This project will develop novel in vitro models of the human gastrointestinal (GI) tract for understanding natural cellular responses to microbes and the induction of immune tolerance and activation. The development of gastrointestinal organoids, 3-D permanent cultures of complex primary epithelial cell populations embedded in an extracellular matrix, has revolutionized research in gastrointestinal development, microbiology and immunology in the past 5 years. For our project, we have assembled a trans-disciplinary team of investigators with expertise in bioengineering (Wilking, Chang), immunology (Bimczok, Jutila), and human microbiome research (Walk) to significantly advance 3-D gut organoid-microbiome co-culture systems. Our team has recently established a millifluidic gut-on-a chip-platform, the GoFlowChip, that recapitulates luminal and basal flow in human intestinal organoids. In parallel investigations, we have established co-cultures of primary human monocyte-derived DCs and human gastric spheroids that we have successfully infected with H. pylori. Here, we seek to leverage the unique capabilities of our two models and combine them into a single analytical platform to study antigen sampling from the gastrointestinal lumen for the induction of adaptive mucosal immunity or tolerance. Specifically, we seek to define and quantify the contributions of candidate mechanisms including transepithelial dendrite formation and Fc-receptor-dependent transcytosis that enable mononuclear phagocytes (MNPs) to acquire luminal antigens. We hypothesize that distinct mechanisms of MNP antigen acquisition are reproduced and can be quantitatively analyzed using the GOFlowChip platform and that colonizing bacteria and fluid dynamics regulate epithelial antigen transport. To test our hypotheses, we will (1) Develop and validate a chip-based organoid-DC co-culture system with luminal and basolateral flow capacity. (2) Quantify the net effect of biologic complexity on GI organoid biology and MNP interaction. (3) Elucidate the mechanisms involved in bacterial antigen sampling from the gastrointestinal lumen by human MNPs. This approach will enable us to optimize our integrated GoFlowChip co-culture system as a powerful new tool for the field for studies on vaccine or drug delivery and on the impact of intestinal microbiota on antigen sampling. The proposed research is conceptually innovative, because it integrates all three necessary cell types (epithelial, microbial, and immune) involved in mucosal host-microbe interactions. The GoFlowChip platform is technologically innovative, because it replicates a complex, oxygen-utilizing epithelium and a microbially colonized lumen, is the first to incorporate fluidics into 3-D organoid cultures, and reproduces the intimate interactions that naturally occur between the gastrointestinal epithelium and sentinel MNPs. The proposed research is significant, because it will provide an incredibly powerful new tool to address fundamental, mechanistic questions in human mucosal biology, microbiology, and immunology in the context of human health and disease.