Institut Pasteur, Unité d'Immunophysiologie et Parasitisme Intracellulaire, Département de Parasitologie et Mycologie, Paris Cedex 15, France.
Microbes Infect. 2010 Jan;12(1):46-54. doi: 10.1016/j.micinf.2009.09.014. Epub 2009 Sep 26.
Once in the mouse skin, Leishmania (L) amazonensis amastigotes are hosted by professional mononuclear phagocytes such as dendritic cells (DCs). When monitored after parasite inoculation, the frequency of amastigote-hosting DCs is very low (<1%) in both the skin and skin-draining lymph nodes. Therefore, we designed and validated an efficient procedure to purify live amastigotes-hosting DCs with the objective to facilitate quantitative and qualitative analysis of such rare cells. To this end, a L. amazonensis transgenic parasite expressing DsRed2 fluorescent protein was generated and added to mouse bone marrow-derived DC cultures. Then, a high speed sorting procedure, performed in BSL-2 containment, was setup to pick out only DCs hosting live amastigotes. This study reveals, for the first time, a unique transcript pattern from sorted live amastigotes-hosting DCs that would have been undetectable in unsorted samples. It was indeed possible to highlight a significant and coordinated up-regulation of L-arginine transporter and arginase2 transcripts in Leishmania-hosting DCs compared to un-parasitized DCs. These results indicate that arginine catabolism for polyamine generation is dominating over L-arginine catabolism for NO generation. In conclusion, this approach provides a powerful method for further characterisation, of amastigote-hosting DCs in the skin and the skin-draining lymph nodes.
一旦进入小鼠皮肤，莱姆曼原虫（L）亚马逊亚种的无鞭毛体就会被树突状细胞（DC）等专业单核吞噬细胞所寄生。在寄生虫接种后进行监测时，皮肤和引流淋巴结中的寄生 DC 的频率非常低（<1%）。因此，我们设计并验证了一种有效的方法来纯化携带活无鞭毛体的 DC，目的是便于对这些稀有细胞进行定量和定性分析。为此，生成了一种表达 DsRed2 荧光蛋白的莱姆曼原虫转基因寄生虫，并将其添加到小鼠骨髓来源的 DC 培养物中。然后，建立了一个在 BSL-2 条件下进行的高速分选程序，以仅分选携带活无鞭毛体的 DC。这项研究首次揭示了从分选的携带活无鞭毛体的 DC 中获得的独特转录模式，而在未分选的样本中这些模式是无法检测到的。与未寄生虫化的 DC 相比，在携带寄生虫的 DC 中确实可以观察到 L-精氨酸转运体和精氨酸酶 2 转录本的显著上调和协调。这些结果表明，多胺生成的精氨酸分解代谢占主导地位，而 NO 生成的 L-精氨酸分解代谢则处于次要地位。总之，该方法为进一步表征皮肤和引流淋巴结中的无鞭毛体寄生 DC 提供了一种强大的方法。
