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，可在家养和野生食肉动物中引起涉及呼吸道的严重全身性疾病。先天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，对犬瘟热的肺部先天免疫的认识仍然是零碎的。设想的项目是基于我们之前的工作，在我们的工作中，可以证明先天免疫细胞能够携带CDV促进呼吸道细胞间的传播，并且限制先天免疫细胞的抗病毒信号通路可以增强犬瘟热中病毒从肺部的释放。项目的第一部分旨在研究CDV在体外对先天免疫细胞的极化效应。本文将通过流式细胞术分析CDV影响肺和血源性巨噬细胞表型的能力，以及单核细胞源性树突状细胞的成熟状态。此外，病毒介导对巨噬细胞和树突状细胞的影响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分析。在第二部分，巨噬细胞极性和树突状细胞成熟对CDV细胞进入的影响以及调节的先天免疫细胞将CDV传播到气道上皮的能力将通过犬气液界面培养和精确切割的肺切片进行研究。此外，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将被确定。本研究将对CDV感染中肺先天免疫功能障碍及其对疾病发病机制的影响提供机制见解。阐明病原体对先天免疫细胞可塑性的调控机制将有助于发现麻疹病毒疾病的治疗靶点和预防病毒向其他宿主的传播。

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.