Life Science School, Ningxia University, Yinchuan 750021, China.
Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in Western China, Ningxia University, Yinchuan 750021, China.
Int J Mol Sci. 2023 May 11;24(10):8588. doi: 10.3390/ijms24108588.
(, Mp) is an intracellular pathogen that causes pneumonia, tracheobronchitis, pharyngitis, and asthma in humans and can infect and survive in the host cells leading to excessive immune responses. Extracellular vesicles (EVs) from host cells carry components of pathogens to recipient cells and play a role in intercellular communication during infection. However, there is limited knowledge on whether EVs derived from -infected macrophages play as intercellular messengers and functional mechanisms. In this study, we establish a cell model of -infected macrophages that continuously secrete EVs to further asses their role as intercellular messengers and their functional mechanisms. Based on this model, we determined a method for isolating the pure EVs from -infected macrophages, which employs a sequence of operations, including differential centrifugation, filtering, and ultracentrifugation. We identified EVs and their purity using multiple methods, including electron microscopy, nanoparticle tracking analysis, Western blot, bacteria culture, and nucleic acid detection. EVs from -infected macrophages are pure, with a 30-200 nm diameter. These EVs can be taken up by uninfected macrophages and induce the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 through the nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPK) signals pathway. Moreover, the expression of inflammatory cytokines induced by EVs relies on TLR2-NF-κB/JNK signal pathways. These findings will help us better understand a persistent inflammatory response and cell-to-cell immune modulation in the context of infection.
（, Mp）是一种细胞内病原体，可导致人类肺炎、气管支气管炎、咽炎和哮喘，并且能够感染和在宿主细胞中存活，导致过度的免疫反应。宿主细胞的细胞外囊泡 (EVs) 携带病原体的成分到受体细胞，并在感染过程中发挥细胞间通讯的作用。然而，关于源自感染巨噬细胞的 EVs 是否作为细胞间信使以及其功能机制，目前知之甚少。在这项研究中，我们建立了一个持续分泌 EVs 的感染巨噬细胞的细胞模型，以进一步评估它们作为细胞间信使的作用及其功能机制。基于该模型，我们确定了一种从感染巨噬细胞中分离纯 EVs 的方法，该方法采用了一系列操作，包括差速离心、过滤和超速离心。我们使用多种方法鉴定 EVs 及其纯度，包括电子显微镜、纳米颗粒跟踪分析、Western blot、细菌培养和核酸检测。来自感染巨噬细胞的 EVs 是纯的，直径为 30-200nm。这些 EVs 可以被未感染的巨噬细胞摄取，并通过核因子 (NF)-κB 和丝裂原活化蛋白激酶 (MAPK) 信号通路诱导肿瘤坏死因子 (TNF)-α、白细胞介素 (IL)-1β、IL-6 和 IL-8 的产生。此外，EVs 诱导的炎症细胞因子的表达依赖于 TLR2-NF-κB/JNK 信号通路。这些发现将帮助我们更好地理解在感染过程中持续的炎症反应和细胞间免疫调节。
