Sorting of Leishmania-bearing dendritic cells reveals subtle parasite-induced modulation of host-cell gene expression.
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.
Institut Pasteur, Unité d'Immunophysiologie et Parasitisme Intracellulaire, Département de Parasitologie et Mycologie, Paris Cedex 15, France. herve.lecoeur@pasteur.fr
Hervé
H
Lecoeur
Emilie
E
de La Llave
José
J
Osorio Y Fortéa
Sophie
S
Goyard
Hélène
H
Kiefer-Biasizzo
Anne-Marie
AM
Balazuc
Geneviève
G
Milon
Eric
E
Prina
Thierry
T
Lang
D000818:Animals; D030801:Animals, Genetically Modified; D003713:Dendritic Cells / Q000469:parasitology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D020869:Gene Expression Profiling; D006790:Host-Parasite Interactions; D007894:Leishmania mexicana / Q000276:immunology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D015800:Protozoan Proteins / Q000096:biosynthesis; D013194:Staining and Labeling / Q000379:methods; D019076:Transgenes; D015854:Up-Regulation; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D015800:Protozoan Proteins; D000097573:Red Fluorescent Protein