■禽流感病毒(AIV)感染首先影响鸡的呼吸道。上皮细胞激活宿主免疫系统,这导致诱导免疫相关基因并产生针对外部环境病原体的抗病毒分子。在这项研究中,采用鸡气管上皮细胞(TECs)体外模型研究鸡呼吸道对禽呼吸道病毒感染的免疫反应。
■使用18天龄的胚胎鸡蛋来培养原代鸡TECs。进行上皮细胞特异性基因标记的逆转录聚合酶链反应(RT-PCR)和免疫细胞化学(ICC)分析以确认其特征,形态学,原代培养鸡TECs的生长规律。此外,研究对AIV感染或多聚肌苷酸-多聚胞苷酸(聚(I:C))治疗的细胞免疫反应,TECs感染H5N1病毒或poly(I:C)。然后,免疫应答通过RT-qPCR和Western印迹进行验证.
■TECs表现出多边形形态并形成集落型细胞簇。RT-qPCR结果显示,H5N1感染诱导TECs中抗病毒基因显著表达。我们发现TECs用poly(I:C)处理并暴露于AIV感染介导的信号通路激活,导致抗病毒分子的产生(例如,促炎细胞因子和趋化因子),由于连接蛋白的丢失而受损。我们观察到核因子κB(NF-κB)和丝裂原活化蛋白激酶(MAPK)途径的激活,它们通过调节用聚(I:C)和途径抑制剂处理的TECs中促炎细胞因子和趋化因子的释放而参与炎症反应。此外,我们的发现表明,poly(I:C)处理通过影响细胞膜中的连接蛋白来损害上皮细胞屏障。
■我们的研究强调了体外TEC模型在揭示病毒感染机制和了解鸡呼吸道宿主免疫反应方面的实用性。
UNASSIGNED: Avian influenza virus (AIV) infections first affect the respiratory tract of chickens. The epithelial cells activate the host immune system, which leads to the induction of immune-related genes and the production of antiviral molecules against external environmental pathogens. In this study, we used chicken tracheal epithelial cells (TECs) in vitro model to investigate the immune response of the chicken respiratory tract against avian respiratory virus infections.
UNASSIGNED: Eighteen-day-old embryonic chicken eggs were used to culture the primary chicken TECs. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC) analysis of epithelial cell-specific gene makers were performed to confirm the characteristics, morphology, and growth pattern of primary cultured chicken TECs. Moreover, to investigate the cellular immune response to AIV infection or polyinosinic-polycytidylic acid (poly (I:C)) treatment, the TECs were infected with the H5N1 virus or poly (I:C). Then, immune responses were validated by RT-qPCR and western blotting.
UNASSIGNED: The TECs exhibited polygonal morphology and formed colony-type cell clusters. The RT-qPCR results showed that H5N1 infection induced a significant expression of antiviral genes in TECs. We found that TECs treated with poly (I:C) and exposed to AIV infection-mediated activation of signaling pathways, leading to the production of antiviral molecules (e.g., pro-inflammatory cytokines and chemokines), were damaged due to the loss of junction proteins. We observed the activation of the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, which are involved in inflammatory response by modulating the release of pro-inflammatory cytokines and chemokines in TECs treated with poly (I:C) and pathway inhibitors. Furthermore, our findings indicated that poly (I:C) treatment compromises the epithelial cell barrier by affecting junction proteins in the cell membrane.
UNASSIGNED: Our study highlights the utility of in vitro TEC models for unraveling the mechanisms of viral infection and understanding host immune responses in the chicken respiratory tract.