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

开发复杂的体外气道模型来研究猪呼吸道病毒发病机制、肺巨噬细胞功能和疱疹病毒疫苗载体

• 批准号: NC/X002446/1
• 负责人: Gyorgy Fejer
• 金额: $ 25.79万
• 依托单位: Plymouth University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FX002446%2F1
• 关键词: Developing; complex; vitro; airway; model

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.

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。他们还负责养猪者的巨大经济负担。病毒和细菌都可能引起感染猪肺的急性和慢性炎症。在肺部，吸入的病原体面临几个级别的宿主保护。衬里的呼吸道上皮细胞彼此紧密结合，代表了感染的物理障碍，可防止吸入的微生物的传播。特殊的免疫细胞，肺肺泡巨噬细胞（AMS），巡逻和保护气道。巨噬细胞代表了针对体内病原体的第一道防线，它们吞噬并灭活微生物并激活其他免疫细胞，导致组织炎症。巨噬细胞在各种器官及其特异性特性中有所不同，可确保它们对不同器官最佳的病原体反应。在气道中，呼吸性上皮细胞和AM的特殊功能是根据其细胞相互作用和组织特异性分泌因子量身定制的。对这些肺病原体如何与猪呼吸免疫和非免疫细胞相互作用的机制有透彻的理解，这是对控制策略和更有效疫苗的发展的基础。目前，大多数发病机理研究都是使用活动物进行的，但这使得分子机制的解剖很困难，并且使用猪会引起道德问题。因此，需要一个适当的体外模型，重现呼吸道上皮和免疫细胞与病原体的复杂相互作用。这样做的主要障碍是猪AMS的可用性受到限制。这些细胞可以从被剔除的猪中分离出来，但它们不会在组织培养物中繁殖，供体变异性变异性和频繁的细胞真菌污染进一步妨碍其使用。以前，我们建立了一种新型的，不断生长的小鼠AM模型（MPI细胞），并最近创建了类似的猪巨噬细胞系统。这些细胞不断生长，并提供无限量的猪Am样巨噬细胞。为了研究上皮气道在体外的功能，可以使用肺上皮细胞的空气界面（ALI）培养物。在这里，我们将将新创建的猪AM样细胞整合到现有的呼吸性上皮ALI系统中，以提供更现实的猪气道模型。我们将研究上皮细胞和PMPI细胞中细胞相互作用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，猪流感病毒和猪生殖和呼吸综合征病毒的复制，以及对病毒的免疫反应。我们还将探讨该系统评估猪疫苗的潜力。 BOHV-4是猪中的一种新的，基于疱疹病毒的疫苗载体。该病毒在气道的上皮细胞和巨噬细胞中优先复制。我们将研究新的PMPI-ALI系统中BOHV-4疫苗菌株引起的免疫反应。总而言之，我们希望建立一个新系统，该系统将减少动物实验，并揭示肺功能和宿主 - 病原体相互作用的关键机制。我们希望我们的系统有助于开发新的兽医疫苗，并为评估其有效性做出贡献。