Sorting of Leishmania-bearing dendritic cells reveals subtle parasite-induced modulation of host-cell gene expression

Hervé Lecoeur (1) , Emilie de La Llave (1) , José Osorio y Fortéa (1) , Sophie Goyard (1) , Helene Kiefer-Biasizzo (2) , Anne-Marie Balazuc (2) , Geneviève Milon (1) , Eric Prina (1) , Thierry Lang (1)

1 Immunophysiologie et Parasitisme Intracellulaire
2 Cytométrie (Plate-forme)

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.

Mots clés: DsRed2; Leishmania; Mouse dendritic cells; Flow-sorting; BSL-2

Domaines: Sciences du Vivant [q-bio]

Hervé Lecoeur, Emilie de La Llave, José Osorio y Fortéa, Sophie Goyard, Helene Kiefer-Biasizzo, et al.. Sorting of Leishmania-bearing dendritic cells reveals subtle parasite-induced modulation of host-cell gene expression. Microbes and Infection, 2010, 12 (1), pp.46-54. ⟨10.1016/j.micinf.2009.09.014⟩. ⟨pasteur-02794034⟩