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微型鸡苗来研究新的策略，将粘膜疫苗靶向M细胞，并增强它们在肠道上皮细胞的摄取。在此申请之前，申请者在罗斯林研究所的实验室已经建立了从鸡肠中制备和维护这些肠道有机化合物的专业知识。目标1：表征禽肠道有机化合物的最佳生长条件和细胞组成初步数据显示，可以使用哺乳动物生长因子从鸡的小肠中制备和维护类似于老鼠有机化合物的鸡肠道有机化合物。最近，我们克隆了鸡上皮生长因子、R-响应蛋白1和Noggin，并生产了相应的蛋白。将确定生长和分化为隐窝和绒毛域所需的最佳浓度。对有机化合物进行详细的细胞和分子表征对于证明鸡类有机化合物是鸡肠道的良好体外模型具有重要意义。从肠道不同部分制备的有机化合物的组成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)、转录学(RNA测序)和生物信息学将用于比较这些有机物的细胞组成和基因表达谱与活体肠道中的有机物。目的2：研究M细胞在肠道上皮细胞摄取抗原的情况鸡M细胞的免疫生物学知之甚少。因此，将对影响肠道上皮细胞抗原摄取的细胞和分子机制进行研究。我们已经克隆并生产了鸡RANKL，并在与哺乳动物蛋白同源性的情况下，将测试其诱导鸡M细胞的能力。在有或没有M细胞的情况下，将使用荧光病原体、包裹有PAMPs的珠子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调查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细胞的吸收。目的3：研究肠道上皮宿主与病原体的相互作用。有机类化合物还将用于研究艾美耳球虫和沙门氏菌等重要禽类病原体暴露后肠道上皮细胞和分子的反应。类有机物将暴露在艾美耳球虫子孢子上，并通过上述生物成像和转录学监测上皮细胞内的附着、入侵和增殖。我们的数据显示，哺乳动物肠道中的沙门氏菌利用M细胞跨细胞抗原的能力感染宿主。同时，这位学生还研究了鼠伤寒沙门氏菌如何与禽类肠道上皮相互作用。

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.