A three-dimensional air-liquid interface airway epithelial cell model to study pathogen interactions within the bovine respiratory tract

三维气液界面气道上皮细胞模型，用于研究牛呼吸道内病原体的相互作用

• 批准号: NC/L000822/1
• 负责人: Robert Davies
• 金额: $ 49.7万
• 依托单位: University of Glasgow
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FL000822%2F1
• 关键词: three; dimensional; air; liquid; interface

Background. Bovine respiratory disease (BRD) is a multifactorial condition of cattle that involves complex interactions between different viral and bacterial pathogens and causes significant economic losses to the cattle industry worldwide. Bovine respiratory syncytial virus (BRSV) and bovine herpes virus-1 (BHV-1) are two of the most important viruses and Mannheimia haemolytica is one of the most important bacterial species involved in BRD. There is an urgent need to develop more effective vaccines and antimicrobials against the pathogens responsible for BRD. To achieve this we require a greater understanding of the molecular interactions that take place between pathogens and host. In particular, we need to more fully understand the early events that take place when viruses and bacteria colonise the host respiratory tract. Unfortunately, the methods that are currently available to do this are not well developed and, consequently, a large number of research experiments have to be carried out in cattle. Consequently, there is an urgent need to develop laboratory-based methods that can be used to study the interactions of viruses and bacteria with the bovine respiratory tract. One such method involves the three-dimensional (3-D) culture of differentiated primary airway epithelial cells grown at an air-liquid interface (ALI). Crucially, these cells can be extracted from the lungs of freshly killed cattle at abattoirs; the approach does not involve killing cattle specifically for this purpose.Aims and objectives. The overall aim of the project is to develop a 3-D bovine airway epithelial cell (BAEC) model at an ALI that will allow us to investigate the complex interactions of bacteria and viruses within the bovine respiratory tract. The project comprises four phases. In phase 1, differentiated BAECs grown at an ALI will be characterized and optimized by examining the cells over a period of 42 days using a range of microscopic and other techniques. The optimum "window" during which the cells are at their healthiest for use in subsequent experiments will be identified. In phase 2, the interactions of BRSV and BHV-1, as well as M. haemolytica, with BAECs maintained at an ALI will be studied using a range of microscopy and other techniques. We will also carry out co-infection studies in which we will investigate the effect of viral infection on subsequent infection with M. haemolytica. In phase 3, the immune response of the epithelial cells to "infection" with these pathogens will be studied. Respiratory epithelial cells secrete special immune chemicals (cytokines) when they are infected with pathogens that are important in protecting the epithelial surface from infection. We will study this innate immune response by measuring cytokine secretion. In phase 4, the findings of the laboratory studies will be validated by infecting cattle with a virus alone, with M. haemolytica alone, and with the virus followed by M. haemolytica. The effects of these infections will be assessed by evaluating colonisation of bacteria and the cytokine response within the respiratory tract.Applications and benefits. The development and characterization of this model, and the demonstration that it can be used to study bacterial and viral infections of the bovine respiratory tract, will have a number of important applications and benefits to the study of respiratory disease in cattle. It will provide an invaluable tool for studying the interactions of bacteria and viruses within the bovine respiratory tract in the laboratory without the need to use cattle. Importantly, this approach could also be extended to other animals. In this way, the development of this model will save countless animals from being used in such experiments. In addition, this and future work will further our understanding of bacterial and viral infections within the bovine respiratory tract and this will contribute to the development of improved vaccines and antimicrobials.

背景牛呼吸道疾病（BRD）是一种多因素的牛病，涉及不同病毒和细菌病原体之间的复杂相互作用，并给全球养牛业造成重大经济损失。牛呼吸道合胞病毒（BRSV）和牛疱疹病毒-1（BHV-1）是两种最重要的病毒，而溶血性曼氏菌（Mannheimia haemolytica）是参与BRD的最重要的细菌之一。目前迫切需要开发更有效的疫苗和抗微生物剂来对抗导致BRD的病原体。为了实现这一目标，我们需要更好地了解病原体和宿主之间发生的分子相互作用。特别是，我们需要更全面地了解病毒和细菌在宿主呼吸道定植时发生的早期事件。不幸的是，目前可用于这样做的方法还没有很好地开发，因此，必须在牛中进行大量的研究实验。因此，迫切需要开发可用于研究病毒和细菌与牛呼吸道相互作用的实验室方法。一种这样的方法涉及在空气-液体界面（ALI）处生长的分化的原代气道上皮细胞的三维（3-D）培养。至关重要的是，这些细胞可以从屠宰场新鲜宰杀的牛的肺中提取;该方法不需要专门为此目的宰杀牛。该项目的总体目标是在ALI中开发一个3-D牛气道上皮细胞（BAEC）模型，使我们能够研究牛呼吸道内细菌和病毒的复杂相互作用。该项目包括四个阶段。在第1阶段，将通过使用一系列显微镜和其他技术在42天内检查细胞来表征和优化在ALI下生长的分化的BAEC。将确定细胞处于最健康状态的最佳“窗口”，以用于随后的实验。在第2阶段，BRSV与BHV-1的相互作用以及M.将使用一系列显微镜和其他技术研究溶血性，BAEC维持在ALI。我们还将进行合并感染研究，研究病毒感染对随后感染M的影响。溶血性在第3阶段，将研究上皮细胞对这些病原体“感染”的免疫反应。当呼吸道上皮细胞被病原体感染时，它们会分泌特殊的免疫化学物质（细胞因子），这些化学物质在保护上皮表面免受感染方面非常重要。我们将通过测量细胞因子分泌来研究这种先天免疫反应。在第4阶段，实验室研究的结果将通过单独用病毒感染牛来验证，其中M。溶血性弧菌，以及病毒后接M.溶血性这些感染的影响将通过评估呼吸道内的细菌定植和细胞因子反应来评估。应用和益处。该模型的开发和表征，以及其可用于研究牛呼吸道细菌和病毒感染的证明，将对牛呼吸道疾病的研究具有许多重要的应用和益处。它将为在实验室中研究牛呼吸道内细菌和病毒的相互作用提供宝贵的工具，而无需使用牛。重要的是，这种方法也可以扩展到其他动物。通过这种方式，这种模型的发展将使无数动物免于被用于此类实验。此外，这项工作和未来的工作将进一步加深我们对牛呼吸道内细菌和病毒感染的了解，这将有助于改进疫苗和抗菌剂的开发。