Mazet Julie M, Chiodetti Ana L, Mahale Jagdish N, Gérard Audrey
The Kennedy Institute of Rheumatology, The University of Oxford.
The Kennedy Institute of Rheumatology, The University of Oxford;
J Vis Exp. 2019 Jul 16(149). doi: 10.3791/59819.
Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.
细胞因子是细胞分泌的小蛋白，介导对有效免疫反应至关重要的细胞间通讯。细胞因子的一个特点是它们具有多效性，因为它们由多种细胞类型产生且能影响多种细胞类型。因此，不仅要了解哪些细胞产生细胞因子，还要了解它们在何种环境中产生，这对于确定更具特异性的治疗方法很重要。在此，我们描述一种在细菌感染后原位可视化细胞因子产生的方法。该技术依靠共聚焦显微镜在其天然环境中对产生细胞因子的细胞进行成像。为此，将组织切片与细胞因子染色一起对多种细胞类型的标志物进行染色。该方法的关键在于，在收获感兴趣的组织之前，直接在体内阻断细胞因子的分泌，从而能够检测在产生细胞内积累的细胞因子。这种方法有多个优点。首先，产生细胞因子的微环境得以保留，这最终可能有助于了解细胞因子产生所需的信号以及受这些细胞因子影响的细胞。此外，这种方法能指示体内细胞因子产生的位置，因为它不依赖于对产生细胞进行人工体外再刺激。然而，无法同时分析接收细胞因子的细胞中的细胞因子下游信号传导。同样，观察到的细胞因子信号仅对应于细胞因子分泌被阻断的时间窗口。虽然我们描述了在小鼠被细胞内细菌单核细胞增生李斯特菌感染后脾脏中细胞因子干扰素γ的可视化，但这种方法可能潜在地适用于大多数器官中任何细胞因子的可视化。
