Application of a Mouse Ligated Peyer's Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells
Application of a Mouse Ligated Peyer's Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells
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DOI：
10.3791/3225
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发表时间：
2011-12-01
影响因子：
1.2
通讯作者：
Ohno, Hiroshi
中科院分区：
文献类型：
--
作者：
Fukuda, Shinji;Hase, Koji;Ohno, Hiroshi
我们的肠道内部居住着大量的肠道细菌。胃肠道的粘膜表面持续暴露于它们，偶尔暴露于病原体。肠道相关淋巴组织（GALT）在诱导针对这些微生物的粘膜免疫应答中发挥关键作用（1，2）。为了启动粘膜免疫应答，粘膜抗原必须从肠腔穿过上皮屏障转运到有组织的淋巴滤泡如派尔集合淋巴结中。这种抗原转胞吞作用由特化上皮M细胞介导（3，4）。M细胞是一种非典型上皮细胞，能主动吞噬大分子和微生物。与树突状细胞（DC）和巨噬细胞将抗原靶向溶酶体进行降解不同，M细胞主要转胞吞内化的抗原。这种通过M细胞的强有力的大分子转胞吞作用将抗原递送到潜在的有组织的淋巴滤泡，并且被认为是启动抗原特异性粘膜免疫应答所必需的。然而，促进M细胞摄取这种抗原的分子机制在很大程度上是未知的。我们以前曾报道，糖蛋白2（Gp2），特异性表达于肠上皮细胞中M细胞的顶端质膜上，作为一个跨细胞受体的一个子集的肠道和致病性肠细菌，包括大肠杆菌和沙门氏菌肠血清型鼠伤寒（S。Typhimurium），通过识别FimH（细菌外膜5上I型皮利的组分）。在这里，我们提出了一种应用小鼠派尔斑肠袢试验来评估M细胞的细菌摄取的方法。该方法是先前描述的小鼠肠袢测定的改进版本6，7。改进之处如下：1.异氟烷用作麻醉剂。2.建立包括Peyer集合淋巴结在内的约1cm结扎肠袢。3.通过荧光标记试剂或通过过表达荧光蛋白如绿色荧光蛋白（GFP）来荧光标记被M细胞摄取的细菌。4.用抗Gp2抗体包埋法检测Peyer集合淋巴结滤泡相关上皮中的M细胞。5.通过共聚焦显微镜分析观察M细胞的荧光细菌转胞吞作用。小鼠Peyer’s斑肠袢试验可以提供M细胞转吞何种肠道或病原菌的答案，并有助于了解M细胞如何刺激粘膜免疫系统的分子机制。
The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes(1, 2). To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells(3, 4). M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.