Immunophysiology and Intracellular Parasitism  


  HEADDr. MILON Geneviève / genevieve.milon@pasteur.fr
  MEMBERSBRULE Chantal / DE LA LLAVE Emilie / DERHY Denise / Dr. LANG Thierry / LECOEUR Hervé / Dr. MELANIOU Evie / Dr. OSORIO y FORTEA José / Dr. PRINA Eric


  Annual Report

The Leishmania life traits, in the mammal hosts, assess the long-term interactions these single celled eukaryotic parasites establish and renew with i) the mammal phagocytic leukocytes left unexposed to any exogenous signal or exposed to parasite- and tissue-protective immune signals , ii) the phagocytic leukocytes or fibroblasts exposed to a balanced combination of counter- and pro-inflammatory signals, i.e. when these cells are hosting persisting Leishmania amastigotes. High content image-based readout assays have been set up to further explore in vitro, ex vivo and in vivo the unique features of the intracellular niches the cell-cycling as well as the persisting amastigote populations do remodel at the cell and tissue levels.

Designing reliableand robust readout assays for deciphering Leishmania developmental programs in the mammal phagocytic leukocyte lineages.

Each discrete step of the developmental program of Leishmania spp, in its mammal host, assesses the subversion-by intracellular amastigotes -of different leukocyte lineages displaying phagocytic properties [neutrophils, macrophages, immature dendritic cells/DCs,] or non leucocyte lineages such as fibroblasts. Once carefully sorted by a robust flow cytometry-based assay set up in our laboratory, mouse DCs hostingL.amazonensis amastigotes were shown to display transcriptional signatures of their subversion as amastigotes-shuttling cells expected to deliver unique immune signals to other non T non B leucocytes as well as to amastigotes- and tissue-protective lymphocytes. The mouse macrophages hosting L.amazonensis amastigotes not only displayed signatures of bona fide host cells where the amastigotes are multiplying but were also established as optimal host cells for high content image-based high throughput screening of small compound libraries.

Designing reliablemouse models for deciphering in real time the host tissue remodelling processes which account for Leishmania persistence.

Mice of different inbred strains receiving intradermally in the ear, transgenic bioluminescent Leishmania metacyclic promastigotes offer the relevant experimental conditions for exploring in real time simultaneously i) the parasite load fluctuations, ii) the macroscopic ear features, iii) transcriptional immune signatures captured with quantitative real time PCR. A sensitive RT PCR assay was designed and validated allowing  100 Leishmania to be detected in either the ear or the ear-draining lymph node.While longitudinally probing transcriptional signatures in both the ears and the ears-draining lymph nodes we indeed decoded (a) when the L.major or L. amazonensis amastigote-hosting leukocytes are becoming sources of signals to CD3 T lymphocytes that are retained in the draining lymph node and (b) when these activated CD3 lymphocytes are recruited in the Leishmania–loaded ears. Additional studies are in progress to highlight (a) how dermis-protective regulatory T lymphocytes are subverted to help amastigotes-loaded leucocytes to consolidate their functions as host cells of cell-cycling amastigotes, (b) when and how a balanced activation of both parasite-clearing CD3 lymphocytes and dermis-protective regulatory T lymphocytes is triggered, (c) the subsequent unique immune and dynamic signatures of the dermis where persist the amastigotes transmissible to the sand flies.

Keywords: Parasitism / Tissue remodelling / Tissue microbiology / Parasite developmental biology / Leishmania / Phagocytic leukocytes



  Publications

1. Osorio y Fortea J., Prina E., de La Llave E., Lecoeur H., Lang T., Milon G. 2007. Unveiling pathways used by Leishmania amazonensis amastigotes to subvert macrophage function. Immunol. Rev. 219 :66-74 ( PMID 17850482) .

2. Prina, E., Roux E., Mattei D., Milon G.2007. Leishmania DNA is rapidly degraded following parasite death: an analysis by microscopy and real-time PCR. Microb.Inf.9:1307-1315(PMID 17890124).

3. Lecoeur, H., P. A. Buffet, G. Morizot, S. Goyard, G. Guigon, G. Milon, T. Lang. 2007. Optimization of topical therapy for Leishmania major localized cutaneous Leishmaniasis using a reliable C57Bl/6 model. Plos.Neglected Trop.Dis:1-10(PMID 18060082).

4. Osorio y Fortéa J., Prina E, Lang T, Milon G, Davory C, Coppée JY, Regnault B.2008. AffyGCQC: a web-based interface to detect outlying genechips with extreme studentized deviate tests. J Bioinform Comput Biol. 6 :317-334 .(PMID: 18464325)

Milon, G. 2008. Leishmaniaparasites: could we consider them as living organisms per se ? Microbes Infect. 10 :1077-1081. (PMID: 18672090)





Activity Reports 2009 - Institut Pasteur
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