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

项目摘要 本项目将开发新的人体胃肠道体外模型， 对微生物的天然细胞反应以及免疫耐受和激活的诱导。发展 胃肠道类器官，复杂的原代上皮细胞群的三维永久培养， 在细胞外基质中，已经彻底改变了胃肠发育，微生物学和 免疫学在过去的五年里在我们的项目中，我们组建了一个跨学科的调查团队， 拥有生物工程（Wilking，Chang），免疫学（Bimczok，Jutila）和人类微生物组的专业知识 研究（步行），以显着推进3D肠道类器官-微生物组共培养系统。我们的团队已被 最近建立了一个毫米流体肠道芯片平台，GoFlowChip，它概括了管腔和基底 在人类肠道类器官中流动。在平行的研究中，我们建立了原代人类的共培养， 单核细胞来源的DC和人胃球状体，我们已经成功地感染了H。幽门。这里我们 我们寻求利用我们两种模型的独特功能，并将它们联合收割机整合到一个单一的分析平台中， 研究从胃肠道腔采集抗原以诱导适应性粘膜免疫，或 宽容具体来说，我们试图定义和量化候选机制的贡献，包括 跨上皮树突形成和Fc受体依赖性胞吞作用使单核吞噬细胞 （MNP）以获得管腔抗原。我们假设MNP抗原获得的不同机制是 可以使用GOFlowChip平台进行定量分析，并且定植细菌和 流体动力学调节上皮抗原转运。为了验证我们的假设，我们将（1）开发和验证一个 基于芯片的类器官-DC共培养系统，具有管腔和基底外侧流动能力。(2)量化净效应 生物学复杂性对胃肠道类器官生物学和MNP相互作用的影响。(3)阐明参与的机制， 通过人MNP从胃肠道腔取样细菌抗原。这种方法将使我们能够 优化我们的集成GoFlowChip共培养系统，作为疫苗研究领域的强大新工具 或药物递送以及肠道微生物群对抗原取样的影响。拟议的研究是 概念创新，因为它整合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） 参与粘膜宿主-微生物相互作用。GoFlowChip平台具有技术创新性，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 流体技术转化为3D类器官培养物，并再现了自然发生在细胞之间的亲密相互作用。 胃肠道上皮和前哨MNP。这项研究意义重大，因为它将提供一个 令人难以置信的强大的新工具，以解决基本的，机械的问题，在人类粘膜生物学， 微生物学和人类健康和疾病背景下的免疫学。

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.