Unit: Immunophysiology and Intracellular Parasitism
Director: MILON Geneviève
Leishmania spp - protozoan parasitic microorganisms - are now known to 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 - the latter still being neglected by too many scientists. 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. Two other protozoan parasites Plasmodium falciparum and Toxplasma gondii are studied within the frame of collaborations that are briefly displayed at the end of this report.
In any natural habitat, there are continual encounters and interactions between microorganisms and other unicellular or multicellular organisms named "hosts". Protozoan parasites (Leishmania, Plasmodium falciparum, Toxoplasma gondii), when reaching/delivered to their hosts establish long-term interactions, with respect to the life time of the hosts. We do appreciate and enjoy to address relevant questions and to design the relevant models with which to decipher the different discrete processes the invasive parasite developmental stage - does trigger/exploit/subvert, once delivered in its mammalian hosts. Since many years, we have re-addressed questions for delineating the sequential discrete steps of the transient or prolonged and renewed cross-talks Leishmania 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 insect, which acts as its second host and vector.
The mouse mononuclear phagocytic leukocytes in vitro : leukocytes a) rapidly and silently invaded by Leishmania metacyclic promastigotes, b) where the promastigotes differentiate as amastigotes [H. Lecoeur, T. Lang, J. Osorio depuis le 1/12/05, J.C.Antoine ® février 2005, G. Milon, E. Prina].
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 processes. Some of these processes can be deciphered in vitro. The Leismania spp parasites do subvert as host cell 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 three 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, Affymetrix based transcriptome profiles, simultaneous analyte quantitation in small samples ), many features of these phagocytic leukocytes loaded with live parasites are sequentially characterized. 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 many innovative approaches the real time in vivo imaging included [C. Deblanc février ® juin 2005 , H. Lecoeur, G. Milon, T. Lang ; collaboration with the "Plate-Forme Imagerie Dynamique" (M.-A. Nicola, S. Shorte), with "le Pôle de Recherche Biomédicale" (G. Morizot, P. Buffet), and J. Blackwell (Cambridge, United Kingdom)].
When we decided to design a model for deciphering the features of the intracellular parasitism driven by Leishmania at the tissue and loco-regional levels, our framework was shaped by the concept promoted by one of us, namely the concept of integrative immunophysiology as it would be tractable in laboratory mice. Indeed, the live parasites are remarkable unique organisms for probing the features of the steady-state conditions of each tissue as well as its reactivity for remodelling. 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, never more than 10.000 metacyclic promastigotes) and inoculation into a dermal site (the central part of the ear dermis). At the site of Leishmania inoculation, the metacyclic promastigotes once taken up by phagocytic leukocytes differentiate as amastigotes ; the latter are constantly renewing their intracellular niche - of note the features of the parasitophorous vacuoles they shape and reshape assess the different signals the parasites and their host cells sensed and processed. These dynamic processes could be dissociated into distinct phases. The initial "silent" phase favoured establishment and unrestricted production of the parasite progeny 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 is 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 sustained entry of CD4, CD8 lymphocytes as well as the sutstained renewal of 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 the parasites 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 so poorly understood : it will be precious to delineate at the tissue, cellular and molecular levels the key processes that reprogram the amastigotes towards the developmental stage which is transmissible and pre-adapted to the insect host and 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, new therapeutic approaches for the treatment of cutaneous lesions are under active screening in collaboration with G. Morizot and P. Buffet. In addition the real time imaging allows to screen novel vaccine preparations (collaboration with J. Blackwell, Cambridge, United Kingdom).
The parasitism at the tissue level : Toxoplasma gondii hijack mobile leukocytes to reach the brain extravascular space [collaboration with S. Darche and D. Buzoni (Département de Parasitologie, Institut Pasteur) and with N. Courret, P. Sonigo, I. Tardieux (Département Maladies Infectieuses, Institut Cochin, Paris), G. Milon].
The protozoan parasite Toxoplasma gondii enters hosts through the intestinal mucosa and colonizes distant tissues such as the brain where its progeny persists for a lifetime. It was possible to challenge the hypothesis one of us GM formulated ten years ago : are mobile CD11c- and CD11b- expressing leukocytes subverted as shuttle cells by T. gondii ? Following intragastric inoculation of cyst-containing parasites in mice, CD11c+ dendritic cells from the intestinal lamina propria, the Peyer's patches and the mesenteric lymph nodes were indeed parasitized while in the blood, parasites were associated with the CD11c- CD11b+ monocytes. Using many different approaches and among them adoptive transfer experiments, it was possible to demonstrate that T.gondii - carrying mobile leukocytes - do reaching the brain extravascular space, a niche where there is further parasite developmental processes since the parasite develop as bradyzoite - containing cysts.
The parasitism at the tissue level : the fate of Plasmodium falciparum harbouring human red blood cells in the isolated perfused human spleens [collaboration with O. Puijalon, P. David (Département de Parasitologie, Institut Pasteur), with V.Brousse, I. Safeukui, P. Buffet (Pôle de recherche biomédicale, Institut Pasteur Paris), with B.Steiniger (Marburg, Germany), and collaboration within an Alliance funded by Pr F. Shi (National University of Singapore), an Alliance that allowed Scientists to co-operate within the frame of three Entities : MIT /NUS/IP, G. Milon]
Pierre Buffet identified a surgical intervention that allows to get access to healthy human spleens, thus offering the opportunity to set-up and validate a functional isolated-perfused human spleen system. A quantitative read-out for the clearing function of the isolated-perfused human spleens indicated that the organ retains clearing and processing functions and thus that the "black box" can now be informatively opened for hypothesis-driven investigations at the tissue level. Some physiological features of red blood cell lineage - especially once the red blood cells have exited the bone marrow - are re-addressed namely their mechanical properties/deformability, features known to be regularly monitored once the red blood cells are moving through the slow microcirculation of the red pulp and while crossing the endothelial cells of the venous sinus. The potential unique features of the red blood cell - trafficking within the perifollicular zone of the human human spleen - will be tractable to further analysis. The human isolated-perfused spleens offer unique situation for exploring the trafficking of P. falciparum-harbouring human red blood cells, the spleen red pulp being a tissue compartment where P. falciparum-infected red blood cells could be innately selected or re-programmed.
Keywords: Leishmania spp., Immunophysiology, Intracellular Parasitism, Parasite developmental program, Parasite transmission, Parasite invasiveness, Parasite fitness and perpetuation, Parasitism features at the tissue level