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

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.

Alveolar macrophages (AMs) play key roles in lung homeostasis and the response to respiratory infections e.g., porcine reproductive and respiratory syndrome virus (PRRSV) and influenza A virus (IAV) infections. AMs have unique functions tailored by their unique microenvironment including special growth factors and epithelial cell interactions. To delineate relevant AM mechanisms and develop vaccines for lung infections the use of realistic in vitro systems with proper macrophages is essential. To study lung epithelium in vitro, air-liquid interface (ALI) cultures are increasingly used. Here, air exposed lung epithelial cells provide a better model than classical submerged cultures. However, the inclusion of AMs into ALI is problematic because of their very restricted availability and the use of blood monocytes or existing macrophage lines have limited relevance due to extensive functional differences. Previously we established a new, continuous model of mouse AM like cells (MPI cells). Very recently, we established a similar pig system (pMPI cells) providing unlimited amounts of AM-like cells. Here we will investigate relevant pig pathogens, IAV and PRRSV and a new herpesvirus-based pig vaccine vector with this system. Furthermore, we will establish a novel in vitro pig lung model incorporating our unique pig AM model into a respiratory epithelium ALI system. We will analyse the impact of the cellular interactions on epithelial and AM functions, and gene expression and compare relevant findings to those in vivo. We will study relevant elements of IAV and PRRSV pathogenesis such as virus replication, innate responses and damage to epithelial functions and will also assess PRRSV vaccines in this model. We expect to establish a new system that can save rude experimental animal usage and to provide key insights into lung functions, host-pathogen interactions and veterinary vaccine development that would not otherwise be easily achievable in vitro or in vivo.