Unit: Immunophysiology and Intracellular Parasitism
Director: MILON Geneviève
Leishmania spp., protozoan parasites, circulate between mammalian hosts and pool blood-feeding sandflies. Once inoculated by the sandflies - in the upper dermis of mammalian organisms (rodents, dogs, humans, ), invasive promastigotes (qualified as metacyclics) are phagocytosed by professional phagocytic leukocytes and their progeny establishes long-term interactions assessed by either pathogenic processes, designated as leishmaniasis, or long-term 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 as well as repair processes (both their immune and non immune components) and the parasite and tissue-dependent processes underlying the transmission of Leishmania spp. from mammalian host to the blood-feeding 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/L.m ), a microorganism whose dominant biotope is the soil), protozoan parasites (e.g. Leishmania spp.), when encountering delivered to such hosts establish respectively short-term (L.m) 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 processes the invasive microorganisms - whether they are opportunistic (L.m) or parasitic (Leishmania spp.) - do trigger/exploit/ subvert, once delivered in their mammalian hosts. Since many years, we address many questions for delineating 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 tissue microenvironment which allows the genetic program to be re-set for its persistence and its further transmission to the blood-pool feeder sandfly, which acts as its second host, also designated as a vector.
The mouse mononuclear phagocytic leukocytes in vitro : host leukocytes a) rapidly and silently invaded by Leishmania metacyclic promastigotes, b) where they differentiate as amastigotes [E. Prina, J.-C. Antoine ; project facilitated by G. Milon, collaboration with N. Winter (Unité Génétique mycobactérienne).
Once delivered to mammals, the Leishmania establishment, the expansion of its progeny as intracellular amastigotes, as well as its transport from the point of entry to distant tissues depend on different steps. Some of these steps can be deciphered in vitro. The Leismania spp parasites do subvert as host cells mouse mononuclear phagocytes they actively maintain as non/un-activated host cells. 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. Using quantitative techniques (real time quantitative PCR, simultaneous analyte quantitation in small samples ) many features of these phagocytic leukocytes loaded with live parasites are sequentially characterized. Some of 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: indeed the silent entry of the parasites could assess the re-programmation of otherwise short lived cells as long lived cells able to act as shuttle cells while delivering amastigotes from the point of delivery and of primary expansion to distant tissues.
The cutaneous tissue: a niche where Leishmania establish long-term parasitic processes tractable to real time in vivo imaging (T. Lang, M. Lebastard6 31/03/04, H. Lecoeur since 1/11/04, G. Milon) collaboration with the "Plate-Forme d'Imagerie Dynamique" (M.A. Nicola, E. Perret, P. Roux, S. Shorte) and with the "Pôle de Recherche Clinique" (G. Morizot, P. Buffet).
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 now 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 the concept promoted by one of us, namely the concept of integrative immunophysiology in mammals. Indeed, the live parasites are remarkable organisms for probing the features of the steady-state conditions of each tissue as well as its remodeling property. Thus, models of Leishmania major or L.amazonensis delivery in C57Bl/6 or BALB/c mice have been established: they combine two main features of natural transmission : low dose (10, 100 to 1000 sometimes 10000 metacyclic promastigotes) and inoculation into a dermal site (the ear dermis). In C57BL/6 mice the evolution of the "transient" dermal lesion where L.major was multiplying could be dissociated into 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 histological 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. major, 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 also relevant 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 to delineate at the tissue, 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, have been constructed. Using luciferase-expressing L.major, and the ear-based model we develop, new therapeutic approaches are under active studies in collaboration with G. Morizot and P. Buffet.
Keywords: Leishmania spp., Immunophysiology, Intracellular Parasitism, Parasite developmental program, Parasite transmission, Parasite invasiveness, Parasite fitness and perpetuation