Chicken intestinal organoids: a novel in vitro system to study mucosal vaccine targeting

该项目旨在充分描述鸟类迷你模型的特征，并开发该模型的多功能潜力。最终目标是利用这些鸡的小片段来研究针对M细胞的粘膜疫苗的新策略，并增强它们在肠道上皮上的摄取。在此申请之前，罗斯林研究所申请人的实验室已经建立了从鸡肠中制备和维护这些肠道类器官的专业知识。初步数据显示，利用哺乳动物生长因子，可以从鸡小肠中制备和维持鸡类器官，与小鼠类器官相似。近年来，我们克隆了鸡上皮生长因子、R-spondin 1和Noggin，并获得了相应的蛋白。将确定生长和分化为隐窝和绒毛域所需的最佳浓度。详细的类器官的细胞和分子特征对证明鸡类器官是鸡肠道的优秀体外模型具有重要意义。从肠道不同部位制备的类器官的成分将使用罗斯林研究所目前最先进的技术进行分析。生物成像（FACS，实时成像，共聚焦和多光子显微镜），转录组学（rnas测序）和生物信息学将用于比较这些类器官的细胞组成和基因表达谱与体内肠道中的细胞组成和基因表达谱。目的2：研究M细胞在肠道上皮内对抗原的摄取。目前对鸡M细胞的免疫生物学知之甚少。因此，影响肠道上皮抗原摄取的细胞和分子机制将被研究。我们已经克隆并生产出了鸡RANKL，并将测试其与哺乳动物蛋白的同源性，以诱导鸡M细胞的能力。在M细胞存在或不存在的情况下，将使用荧光病原体、包被PAMPs的珠粒和表面修饰的纳米颗粒（如PLGA）来研究抗原摄取。MSD在佐剂和疫苗方面的专业知识将确保学生研究最先进的粘膜递送系统，以及它们如何影响不同疫苗制剂在肠道上皮中的吸收。目的是确定与诱导有益的先天反应和维持肠道完整性相关的生物标志物。目的3：研究肠道上皮中宿主-病原体的相互作用类器官也将用于研究肠道上皮在暴露于重要的禽类病原体（如艾美耳球虫和沙门氏菌）后的细胞和分子反应。类器官将暴露于艾美球虫孢子体中，并通过生物成像和转录组学监测上皮细胞内的附着、入侵和增殖。我们的数据表明，哺乳动物肠道中的沙门氏菌利用M细胞转运抗原的能力感染宿主。同时，学生还将学习鼠伤寒沙门氏菌如何与禽肠上皮相互作用。

The project seeks to fully characterise an avian minigut model and to exploit the model's multifunctional potential. The ultimate goal is to use these chicken miniguts to investigate new strategies to target mucosal vaccines to M cells and enhance their uptake across the gut epithelium. Prior to this application, the expertise to prepare and maintain these intestinal organoids from the chicken intestine has been established in the applicants' labs at The Roslin Institute.Objective 1: Characterise the optimal growth conditions and cellular composition of avian gut organoidsPreliminary data show chicken organoids, similar to mouse organoids, can be prepared and maintained from chicken small intestine using mammalian growth factors. Recently, we cloned chicken epithelial growth factor, R-spondin 1 and Noggin and the proteins were produced. The optimal concentrations required for growth and differentiation into crypt and villus domains will be established. Detailed cellular and molecular characterisation of the organoids is of great importance to show that chicken organoids are excellent in vitro models for the chicken gut. The composition of organoids prepared from different parts of the intestine will be analysed using state-of-the art technology present at the Roslin Institute. Bioimaging (FACS, live imaging, confocal and multiphoton microscopy), transcriptomics (RNAsequencing) and bioinformatics will be used to compare the cellular composition and gene expression profiles of these organoids to that in the gut in vivo.Objective 2: Study antigen uptake by M cells in the gut epitheliumLittle is known of the immunobiology of chicken M cells. Therefore the cellular and molecular mechanisms which influence antigen uptake in the gut epithelium will be studied. We have cloned and produced chicken RANKL, and in homology to the mammalian protein, its capacity to induce chicken M cells will be tested. Antigen uptake in the presence or absence of M cells will be studied using fluorescent pathogens, beads coated with PAMPs and surface modified nanoparticles such as PLGA. The expertise of MSD on adjuvants and vaccines will ensure that the student investigates the state-of-the-art mucosal delivery systems and how they influence the uptake of different vaccine preparations across the gut epithelium. The aim is to identify biomarkers that correlate with induction of beneficial innate responses and maintenance of gut integrity.Objective 3: to study host-pathogen interactions at the gut epitheliumThe organoids will also be used to study the cellular and molecular responses of the gut epithelium following exposure to important avian pathogens such as Eimeria and Salmonella. Organoids will be exposed to Eimeria sporozoites and attachment, invasion and proliferation within the epithelial cells monitored by bioimaging and transcriptomics as above. Our data show Salmonella in mammalian gut takes advantage of the M cells' capacity to transcytose antigen to infect the host. In parallel the student with also study how S. Typhimurium interacts with the avian gut epithelium.