Developing a complex in vitro airway model to study respiratory viral pathogenesis, lung macrophage function and herpesviral vaccine vectors in pigs

猪的呼吸道感染会影响大量动物，并可能导致严重的健康和福利状况。它们也给养猪户带来了沉重的经济负担。病毒和细菌都可能在受感染猪的肺部引起急性和慢性炎症。在肺中，吸入的病原体面临几个层次的宿主保护。排列在气道内的呼吸道上皮细胞彼此紧密结合，代表了防止吸入的微生物深入组织传播的感染的物理屏障。特殊的免疫细胞，肺泡巨噬细胞（AM），巡逻和保护气道。巨噬细胞是人体抵御病原体的第一道防线，它们吞噬和吞噬微生物并激活其他免疫细胞，导致组织炎症。巨噬细胞在不同的器官中是不同的，它们的组织特异性确保它们在不同的器官中对病原体做出最佳反应。在气道中，呼吸道上皮细胞和AM的特殊功能是由它们的细胞相互作用和组织特异性分泌因子定制的。深入了解这些肺部病原体如何与猪呼吸道免疫和非免疫细胞相互作用的机制，对于开发控制策略和更有效的疫苗是必要的。目前大多数发病机制研究使用活动物进行，但这使得分子机制的解剖变得困难，并且使用猪引起了伦理问题。因此，需要一种合适的体外模型，再现呼吸道上皮细胞和免疫细胞与病原体的复杂相互作用。这方面的主要障碍是猪AM的可用性有限。这些细胞可以从淘汰的猪中分离出来，但它们在组织培养中不能繁殖，供体与供体之间的变异性和细胞的频繁真菌污染进一步阻碍了它们的使用。以前，我们建立了一个新的，不断增长的小鼠AM模型（MPI细胞），最近创建了一个类似的猪巨噬细胞系统。这些细胞持续生长，并提供无限量的猪AM样巨噬细胞。为了在体外研究上皮气道功能，可以使用肺上皮细胞的气液界面（ALI）培养物。在这里，我们将我们新创建的猪AM样细胞整合到现有的呼吸上皮ALI系统，以提供更真实的猪气道模型。我们将研究由于上皮细胞和pMPI细胞中的细胞相互作用而引起的基因表达变化。此外，我们将使用这种新的共培养系统来研究两种重要的猪病原体，猪流感病毒和猪繁殖与呼吸综合征病毒的复制，以及对病毒的免疫反应。我们还将探索该系统用于评估猪疫苗的潜力。BoHV-4是一种新型的猪疱疹病毒疫苗载体。这种病毒优先在气道的上皮细胞和巨噬细胞中复制。我们将在我们的新pMPI-ALI系统中研究BoHV-4疫苗株引起的免疫应答。总之，我们希望建立一个新的系统，将减少动物实验，并将揭示肺功能和宿主-病原体相互作用的关键机制。我们希望我们的系统能够促进新的兽医疫苗的开发，并有助于评估其有效性。

Respiratory infections in pigs can affect a large number of animals and may cause serious health and welfare conditions. They are also responsible for a significant economic burden on pig farmers. Both viruses and bacteria may cause acute and chronic inflammation in the lungs of the infected pigs. In the lungs, inhaled pathogens face several levels of host protection. The respiratory epithelial cells lining the airways are bound tightly to each other representing a physical barrier to infection which prevents the spread of the inhaled microbes deep into the tissues. Special immune cells, lung alveolar macrophages (AMs), patrol and protect the airways. Macrophages represent the first line of defence against pathogens in the body, they engulf and inactivate microbes and activate other immune cells resulting in tissue inflammation. Macrophages are different in various organs and their tissue specific properties ensure that they respond to pathogens optimally in different organs. In the airways, the special functions of respiratory epithelial cells and AMs are tailored by their cellular interactions and tissue specific secreted factors. A thorough understanding of the mechanisms of how these lung pathogens interact with pig respiratory immune and non-immune cells is necessary to underpin the development of control strategies and more effective vaccines. Most pathogenesis studies are currently performed using live animals, but this makes the dissection of molecular mechanisms difficult and the use of pigs raises ethical concerns. There is therefore a need for a suitable in vitro model reproducing the complex interactions of the respiratory epithelial and immune cells with pathogens. The main obstacle to this was the restricted availability of pig AMs. These cells can be isolated from culled pigs, but they do not multiply in tissue culture and donor to donor variabilities and frequent fungal contamination of the cells further hampers their use. Previously we established a novel, continuously growing mouse AM model (MPI cells) and more recently created a similar swine macrophage system. These cells grow continuously and provide unlimited amounts of pig AM-like macrophages. To study epithelial airway functions in vitro, air-liquid interface (ALI) cultures of lung epithelial cells can be used. Here we will integrate our newly created pig AM-like cells into an existing respiratory epithelium ALI system to provide a more realistic model of the pig airway. We will study gene expression changes due to cellular interactions in both the epithelial and pMPI cells. Furthermore, we will use this new co-culture system to study the replication of two important swine pathogens, swine flu virus and porcine reproductive and respiratory syndrome virus, as well as immune responses to the viruses. We will also explore the potential of this system to evaluate pig vaccines. BoHV-4 is a new, herpesvirus-based vaccine vector in pigs. This virus replicates preferentially in the epithelial cells and macrophages of the airways. We will study the immune responses elicited by BoHV-4 vaccine strains in our new pMPI-ALI system. In summary, we expect to establish a new system that will reduce animal experiments and will reveal key mechanisms of lung function and host-pathogen interactions. We expect our system to facilitate the development of new veterinary vaccines and to contribute to the evaluation of their effectiveness as well.