The role of innate immune cell polarity in the canine respiratory tract following canine distemper virus infection using in vitro and ex vivo models

犬瘟热病毒(CDV)是一种高度传染性的麻疹病毒，在家畜和野生动物中可引起严重的全身疾病，累及呼吸道。先天性免疫细胞在多种病毒性呼吸道疾病的发病机制中起着关键作用。然而，有关犬瘟热的肺天然免疫的知识还很零碎。这个设想的项目是在我们以前工作的基础上进行的，可以证明先天性免疫细胞能够携带CDV促进呼吸道细胞间的传播，限制先天性免疫细胞的抗病毒信号通路可以促进犬瘟热病毒从肺释放。该项目的第一部分旨在研究CDV对体外培养的天然免疫细胞的极化效应。在这里，我们将用流式细胞术分析CDV对肺和血液来源的巨噬细胞表型的影响，以及单核细胞来源的树突状细胞的成熟状态。此外，病毒对巨噬细胞和树突状细胞的作用将通过全转录组分析和功能分析来表征，包括混合白细胞反应，以及迁移、吞噬和一氧化氮释放试验。在第二部分中，将利用犬气-液界面培养和精密肺切片研究巨噬细胞极性和树突状细胞成熟对CDV细胞进入和调节的天然免疫细胞将CDV传递到呼吸道上皮细胞能力的影响。此外，还将确定病毒诱导的细胞病变效应和超微结构变化，如纤毛病理和细胞凋亡诱导，以及感染培养物的再生能力。这项研究将对CDV感染时肺天然免疫功能紊乱及其在疾病发病机制中的影响提供机制方面的见解。阐明病原体调节天然免疫细胞可塑性的调控机制，将有助于发现麻疹病毒病的治疗靶点，防止病毒向其他宿主传播。

Canine distemper virus (CDV) is a highly contagious morbillivirus, which causes severe systemic disease with involvement of the respiratory tract in domestic and wildlife carnivores. Innate immune cells play a key role in the pathogenesis in a variety of viral respiratory diseases. However, the knowledge about pulmonary innate immunity in canine distemper is still fragmentary. The envisioned project is based on our previous work, in which it could be demonstrated that innate immune cells are able to carry CDV to facilitate cell-to-cell transmission in the respiratory tract and that restriction of antiviral signaling pathways of innate immune cells enhance virus release from the lung in canine distemper. The first part of the project aims at investigating the polarizing effect of CDV upon innate immune cells in vitro. Here the ability of CDV to influence the phenotype of pulmonary and blood-derived macrophages, and the maturation state of monocyte-derived dendritic cells will be analyzed by flow cytometry. In addition, virus-mediated effects upon macrophages and dendritic cells will be characterized by whole transcriptome analyses and functional assays, including mixed leukocyte reaction, as well as migration, phagocytosis and nitric oxide release assays. In the second part, the impact of macrophage polarity and dendritic cell maturation upon CDV cell entry and the capacity of modulated innate immune cells to transmit CDV to the airway epithelium will be investigated using canine air-liquid interface cultures and precision-cut lung slices. In addition, virus-induced cytopathic effects and ultrastructural changes, such as ciliary pathology and apoptosis induction, as well as the regenerative capacity of infected cultures will be determined. The study will give mechanistic insights in the dysfunction of pulmonary innate immunity in CDV infection and its impact on disease pathogenesis. Elucidating the regulatory mechanisms through which pathogens regulate innate immune cell plasticity will contribute to the discovery of therapeutic targets in morbillivirus diseases and prevention of virus transmission to other hosts.