Unit: Immunophysiology and Intracellular Parasitism

Director: MILON Geneviève

Leishmania spp., protozoan parasites, circulate between mammalian hosts and hematophagous sandflies. Once inoculated - by the sandflies - in the upper dermis of mammalian organisms (rodents, dogs, humans,…), invasive promastigotes (metacyclics) are phagocytosed by professional phagocytic leukocytes and they establish long-term interactions assessed by either pathogenic processes, designated as leishmaniasis, or asymptomatic parasitic processes. Whatever the level of analysis of these unique parasite-host interactions (in vivo, ex vivo, in vitro), our objectives are to decipher the mechanisms underlying asymptomatic and/or pathogenic and repair processes (both their immune and non immune components) as well as the parasite and tissue-dependent processes underlying the transmission of Leishmania spp. from mammalian host to the hematophagous insect which also acts as both a host and a vector.

In any natural habitat, there are continual encounters and interactions between microorganisms and other unicellular or multicellular organisms named "hosts". Bacteria (e.g. Listeria monocytogenes, LM), a microorganism whose dominant biotope is the soil), protozoan parasites (e.g. Leishmania spp.), when encountering such hosts establish respectively short-term (LM) or long-term interactions (Leishmania spp.), with respect to the life time of the hosts. We do enjoy spending time and efforts to design the relevant models with which to decipher the different discrete steps the invasive microorganisms - whether they are opportunistic (LM) or parasitic (Leishmania spp.) - do trigger/exploit/subvert, once delivered in their mammalian hosts. Since many years, we address many questions for better understanding the sequential discrete steps of the transient or prolonged and renewed cross-talks the microorganisms establish in their hosts especially with their multifocal immune system ; the features of these cross-talks also assess the unique steady-state structure and functions of the tissue the parasites subvert/reshape as a niche. When Leishmania is concerned, another important question is addressed, namely where and how - in the mammalian hosts - do Leishmania shape the optimal tissual microenvironment which allows the genetic program to be re-set for its transmission to the blood-pool feeder sandfly, which acts as its second host, named a vector.

The mouse mononuclear phagocytic leukocytes in vitro : host leukocytes rapidly a) invaded by Leishmania metacyclic promastigotes, b) where they differentiate as amastigotes (E. Fontan ® 31/08/03, T. Lang, M. Lebastard, E. Prina, J.-C. Antoine).

The establishment of Leishmania in mammals depends on the differentiation of metacyclic promastigotes into amastigotes mainly within macrophages. The kinetics of this process was examined using mouse bone marrow-derived macrophages infected with metacyclic promastigotes of L. amazonensis. The promastigote-specific molecules examined were down-regulated within 5 to 12h after phagocytosis whereas the amastigote-specific antigens studied were detectable from 2 to 12-24 h. The presence of amastigote characteristics, including large lysosome-like organelles called megasomes, stage-specific molecule identified in our laboratory, high cysteine protease activity and sensitivity to L-leucine-methyl ester, was followed over a 5-day period. Megasomes were observed at 48 h but probable precursors of these organelles were detected at 12 h post-infection (p.i.). An increase in the cysteine protease activity and in sensitivity to L-leucine methyl ester of the parasites was detected from 24 h. The data indicate that at 48 h p.i., parasites exhibit several amastigote features but that complete differentiation requires at least 5 days. The biogenesis of megasomes or of megasome precursors and the rise in cysteine protease activity correlate quite well with the capacity of parasites to internalize and very likely degrade host cell Major HistoCompatibility (MHC) molecules. The fact that internalization by the parasites of host cell molecules occurs very early during the differentiation process argues for a role of this process in L. amazonensis survival.

The expression "mononuclear phagocytic leukocytes" designates two related lineages : (a) the mononuclear phagocyte one and (b) the dendritic leukocyte one. During the last two years, mouse dendritic leukocytes - also named dendritic cells (DCs)-derived from mouse bone marrow progenitors have been used as host cells of either metacyclic promastigotes or recently isolated amastigotes. These phagocytic leukocytes loaded with live parasites are the relevant cells with which to monitor a pool of parasite-reactive CD4 T lymphocytes primed in mice once those have been exposed to the intradermal delivery of more or less invasive parasites : a special attention is and will be given to CD4 and CD8 T lymphocytes activated DC loaded with live parasites. The studies with dendritic leukocytes are performed in collaboration with Nathalie Winter (Unité de Génétique mycobactérienne). Of note, among the many features that have been characterized, one - the delayed/incomplete maturation of DC - is under in depth investigation.

The cutaneous tissue : a remodelable niche where Leishmania establish long-term parasitic processes and from where they are transmitted to their second host, the hematophagous sandfly (J.-C. Antoine, S. Goyard ® 30/06/03, T. Lang, M. Lebastard, L. Nicolas, E. Prina, H. Saklani ® 31/08/03,- collaboration with : E. Perret, P. Roux, S. Shorte (PFID) ; P. Douillard, S. Saeland, Schering Plough, Dardilly, G. Milon).

Secondary lymphoid organs are sites of "immunological integration", where many circulating leukocytes including T, B lymphocytes, as well as migratory non T non B leukocytes could be stopped within highly organized microenvironments. The critical portals for leukocyte entry are the high endothelial venules (HEVs) of lymph nodes, and the afferent lymphatic vascular bed for which new reagents are endly available. When we decided to design a model for deciphering the features of the intracellular parasitism driven by Leishmania at the tissual and loco-regional levels, our framework was shaped by a perception of integrative immunophysiology. Thus, models of Leishmania major delivery in C57Bl/6 mice have been established : they combine two main features of natural transmission : low dose (10, 100 to 1000 metacyclic promastigotes) and inoculation into a dermal site (the ear dermis). The evolution of the "transient" or irreversible dermal lesion could be dissociated into two distinct phases. The initial "silent" phase favoured establishment of the peak load of parasites in the dermis in the absence of lesion formation or any overt histologic change in the site. The second phase corresponds to the development of a lesion associated with an acute infiltration of neutrophils, macrophages, and eosinophils into the dermis and was coincident with the reduction of the parasite load in C57Bl/6. In BALB/c mice inoculated with either L. major or L. amazonensis, in C56Bl/6 mice inoculated with L. amazonensis, the irreversible lesions which occur are assessing very complex processes under active study focusing on the draining lymph node as well as the parasite-loaded cutaneous sites. In C57Bl/6 mice inoculated with L. majori, the onset and stable maintenance of repair processes are correlated with the presence of CD4, CD8 and dendritic leukocytes whose properties have to be further defined, especially in the healed ear where parasites are persisting at a stable number (@ 1000 parasites/ear), as well as in the distant cutaneous tissues they are able to reach. This model is processed within the framework of Leishmania transmission from the vertebrate host to the sandfly vector, a complex process also still poorly understood : it will be precious for delineating at the tissual, cellular and molecular levels the key processes which reprogram the amastigotes towards the developmental stage which is transmissible and pre-adapted to the vector. Transgenic Leishmania major expressing different genes encoding proteins tractable by fluorescence or luminescence readout assays, are constructed, as well as Leishmania major with a new suicide gene.

Keywords: Leishmania spp., Immunophysiology, Intracellular Parasitism, Parasite developmental program, Parasite transmission, Parasite invasiveness

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