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

背景。牛呼吸道疾病（BRD）是牛的一种多因素疾病，涉及不同病毒和细菌病原体之间的复杂相互作用，给世界范围内的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，而溶血性曼海姆病是BRD中最重要的细菌之一。目前迫切需要开发更有效的疫苗和抗菌剂，以对抗导致BRD的病原体。为了实现这一目标，我们需要对病原体和宿主之间发生的分子相互作用有更深入的了解。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，目前可用来做到这一点的方法还没有很好地发展，因此，大量的研究实验必须在牛身上进行。因此，迫切需要开发基于实验室的方法，可用于研究病毒和细菌与牛呼吸道的相互作用。其中一种方法涉及在气液界面（ALI）培养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刚宰杀的牛的肺中提取；该方法不涉及专门为此目的杀死牛。目的和目标。该项目的总体目标是开发一个三维牛气道上皮细胞（BAEC）模型，这将使我们能够研究牛呼吸道内细菌和病毒的复杂相互作用。项目分为四个阶段。在第一阶段，将使用一系列显微镜和其他技术，在42天的时间内对ALI中生长的分化baec进行鉴定和优化。将确定细胞处于最健康状态的最佳“窗口”，以便在随后的实验中使用。在第二阶段，将使用一系列显微镜和其他技术研究BRSV和BHV-1以及溶血分枝杆菌与在ALI中维持的baec的相互作用。我们还将开展合并感染研究，其中我们将调查病毒感染对随后溶血支原体感染的影响。在第三阶段，上皮细胞对这些病原体“感染”的免疫反应将被研究。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），这对保护上皮细胞免受感染很重要。我们将通过测量细胞因子的分泌来研究这种先天免疫反应。在第4阶段，将通过单独用一种病毒感染牛、单独用溶血支原体感染牛以及随后用该病毒感染溶血支原体感染牛来验证实验室研究的结果。这些感染的影响将通过评估细菌定植和呼吸道内的细胞因子反应来评估。应用和好处。该模型的发展和表征，以及它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，这种方法也可以推广到其他动物身上。这样，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。

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.