奶牛乳腺感染时乳腺上皮细胞外泌体对PMN功能的影响及作用途径

奶牛乳腺上皮细胞是细菌感染与乳腺免疫的一个最初的重要交汇处。在这个初始交汇处，乳腺上皮细胞启动了机体对病原菌最早的免疫识别和免疫应答，并发挥了协调后续固有免疫细胞应答的重要作用。外泌体作为一种非接触介导的新型细胞间通讯模式，具有调节多形核中性粒细胞（PMN）功能的作用。我们推测乳腺感染时上皮细胞产生的外泌体可能以远距扩散的方式调节PMN生物功能，进而影响乳腺炎症的发生发展过程。因此，本项目拟分离脂多糖处理乳腺上皮细胞后产生的外泌体，研究其对奶牛PMN活化功能、跨上皮迁移功能、呼吸爆发功能、胞外诱捕网形成和吞噬功能的影响，利用细胞培养条件下稳定同位素标记技术和高通量测序技术筛选外泌体中的差异蛋白和miRNA，采用多种分子生物学手段分析差异蛋白和miRNA调控PMN生物功能的作用途径，并进行体内外验证，为全面认识乳腺感染时乳腺上皮细胞与PMN之间的相互作用，以及合理防控奶牛乳房炎提供科学依据。

The prompt detection of pathogens by the innate immune system is of prime importance, and because bovine mammary epithelial cells (bMECs) are in the front line, they could act a significant part as sentinels. Therefore, bMECs may be an initial convergence of pathogens infection and host immunity, in which the bMECs not only plays roles in local defense against pathogens infection, but also plays diverse and multifaceted regulatory roles in systemic immune response. Exosomes have been postulated to play a vital role in cell-cell communication. The tumour- and immune cell-derived exosomes have been shown to be immunomodulatory and stimulate neutrophils. Whether the release of exosomes from bMECs has an effect on neutrophil functions during mammary gland infection has not been investigated to any extent. We hypothesize that bMECs-derived exosomes could modulate neutrophil functions during mammary gland infection. Consequently, we will examine the role of the lipopolysaccharide (LPS)-induced bMECs-derived exosomes in mediating neutrophil activition, migration, reactive oxygen species (ROS) production, neutrophil extracellular trap (NET) formation, and phagocytosis. Second, we will characterize the proteome and miRNA expression profiles of bMECs-derived exosomes in response to LPS using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry for protein profiling and high-throughput sequencing for miRNA profiling. Third, we will determine if transcriptome and proteome expression profiles of bMECs-derived exosomes affect neutrophil functions, and the molecular pathways of exosome-regulated neutrophil functions will be investigated. This work will provide a better understanding of epithelial-neutrophil interactions in the mammary gland of dairy cows. Also, it is our hopes that these findings will enable us to introduce new concepts for the development of effective mastitis treatments and/or preventatives that bolster mammary immunity.