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

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.

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。

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。外泌体是一种非接触介导的新型细胞间通讯模式，在多种生物过程中发挥了重要调节作用。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影响乳腺炎症的发生发展过程。本项目采用超速离心方法分别提取了正常奶牛乳腺上皮细胞外泌体和脂多糖（LPS）刺激后奶牛乳腺上皮细胞外泌体，并对提取到的外泌体进行透射电镜观察及外泌体表面标志分子 CD9、CD63、TSG101 免疫印迹分析；确认外泌体分离成功后将外泌体与 PMN 共孵育后，分析其对 PMN 跨乳腺上皮迁移功能和胞外诱捕网（NETs）形成功能的影响；对外泌体中 miRNA和蛋白质进行了高通量测序，初步筛选了可能调控 PMN 功能的乳腺上皮细胞外泌体 miRNA 和蛋白质。构建了差异 miRNA 在乳腺上皮细胞的过表达和抑制细胞模型，验证了差异 miRNA 对炎症信号通路、炎症细胞因子的影响。结果发现，LPS 处理乳腺上皮细胞后外泌体可识别牛 PMN，并能提高 PMN 跨乳腺上皮迁移功能，但不影响 PMN 胞外诱捕网形成功能；LPS处理乳腺上皮细胞后外泌体分拣miR-193b-5p量显著降低，miR-1296_R-2、miR-126-5p、miR-141-5p量显著升高，钙稳态内质网蛋白、抑制素-2、肿瘤易感基因 101等15个蛋白含量显著升高，溶质载体家族 12 成员 2、酸性磷酸酶-1等5个蛋白含量显著降低；过表达或抑制乳腺上皮细胞内miR-126-5p、miR-193b-5p可以调控乳腺上皮细胞p-p65、IκBα 表达和磷酸化水平，进而调控炎症细胞因子基因转录。综上所述，奶牛乳腺感染时乳腺上皮细胞外泌体可以调控奶牛中性粒细胞功能及炎症信号通路，该研究为全面认识乳腺上皮细胞外泌体在乳腺感染时的作用提供了研究依据。