Preamble :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 spend 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 cross-talks the microorganisms establish in their hosts. 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.
1. Exploration of immunologic parameters within the framework of a model perceived as a reference model - relying on the subcutaneous delivery of a large number of L. major promastigotes poor in invasive metacyclic forms.
1.1. Listeria monocytogenes as a live recombinant bacterial vector of the leishmanial molecule LACK (H. Saklani, E. Fontan, N. Glaichenhaus Nice, J.-H. Colle ®1/10/2000, G. Milon, P. Goossens ®1/5/2000).
Let us specify that LACK is the abbreviation of Leishmania of receptors for Activated C Kinase). Listeria monocytogenes are Gram positive bacteria which can accidentally reach mammalian organisms and establish transient infectious/pathogenic processes if delivered in food where they have multiplied extensively, generally at acidic pH. Thus, once designed as bacterial vectors of an heterologous gene such as the leishmanial lack gene, these recombinant Listeria could be delivered through the "oral route". As a first approach to reach such an endpoint, intragastric delivery was achieved with L. monocytogenes grown in acid buffered culture medium. These culture conditions allow the bacteria (i) to survive the gastro-duodenum milieu, (ii) to reach large intestine and (iii) to disseminate to extraintestinal tissues. Within these extra-intestinal tissues, they are perceived as a source of immunogenic signals as assessed by the presence of LACK-reactive IFN¡-secreting CD4 T lymphocytes in Peyer's patches, mesenteric lymph nodes and in the spleen. BALB/c mice were immunized with these recombinant bacterial vectors - delivered intragastrically - and their ability to be protected from a L. major challenge was monitored. Their protected status was correlated to the rapid delivery of LACK reactive IFN¡-secreting CD4 T cells in the lymph nodes draining the footpad where stationary phase L. major promastigotes were delivered.
1.2. How is translated,in the very early phases of the parasitic process, the presence of a large number of non or poorly invasive promastigotes in the inoculum delivered subcutaneously to mice of different genetic backgrounds ?(F.Tacchini-Cottier, J. Louis - Lausanne ; K. Soteriadou - I. Pasteur, Athens, G. Milon).
The presence of non invasive parasites in stationary phase promastigotes containing inoculum does induce an early inflammatory process both within the footpad and the draining lymph node. Neutrophils are dominating over mononuclear phagocytes and were shown to contribute to the T cell differentiation in the draining lymph node. In addition, they could be critical cells in the complex regulation of iron delivery to both parasites and their host cells.
2. Leishmania donovani within the spleen of BALB/c mice : characterization of the leukocyte subsets they exploit/subvert as host cells (T. Lang, P. Ave, M. Huerre, G. Milon, J.-C. Antoine).
The purpose of this study was to characterize parasite-containing leukocytes located in spleens of BALB/c mice parasitized with Leishmania donovani intravenously (iv). In particular, it was monitored whether these leukocytes express MHC class II molecules in order to determine whether they could potentially act as cells capable of immuno-stimulating Leishmania-reactive CD4+ T lymphocytes. To this end, an immunohistological analysis of spleens taken at various time points after iv delivery of L. donovani amastigotes was undertaken. Using this approach, we observed, in the red pulp, the formation of small cellular infliltrates containing heavily infected macrophages that could be stained with the monoclonal antibodies MOMA-2 and FA/11 (CD68). All of them expressed high levels of MHC class II molecules. Parasites were also detected in the white pulp, especially in MOMA-2+, FA/11+ and MHC class II+ macrophages of the periarteriolar lymphocyte sheath and in MOMA-2+ marginal zone macrophages. Infected cells were further characterized by fluorescence microscopy after their enrichment by adherence. All parasite-loaded mononuclear leukocytes, recovered by this procedure could be stained with MOMA-2 and FA/11 and thus very probably belonged to the mononuclear phagocyte leukocytic lineage. Furthermore, all of them strongly expressed both MHC class II as well as H-2M molecules, regardless of the time points after iv. parasite delivery. Analysis of the parasitophorous vacuoles (PV) by confocal microscopy showed that these compartments were surrounded by a membrane enriched in lysosomal glycoproteins lamp-1 and lamp-2, in macrosialin (a membrane protein of prelysosomes recognized by FA/11) and in MOMA-2 antigen. About 80% of the PV also had MHC class II and H-2M molecules on their membrane. Altogether, these data indicate that in the spleens of L.donovani-infected mice, a high percentage of amastigotes are located in macrophages expressing MHC class II molecules and that they live in PV exhibiting properties similar to those of PV detected in mouse bone marrow-derived macrophages exposed to a low dose of interferon ¡ (IFN¡) and infected in vitro, with L. donovani amastigotes.
3. Proechimys oris, the natural host of Leishmania amazonensis : what can we learn from the analysis of this "natural parasitism" ? (E. Prina, K. Sebastien P. Bastien UMR CNRS 5093, P. Ave, M. Huerre, J.-C. Antoine).
As you have already appreciated and will appreciate, our objectives are to decipher each step of the process parasites do drive with the most relevant experimental models. It was the rationale for introducing Proechimys oris carefully adapted to captivity by J.-C. Ganthier et al. (Faculté de Pharmacie de Châtenay-Malabry) in the animal facility of Institut Pasteur. Using laboratory-adapted L. amazonensis and more recently a parasite isolate collected from a patient, many features of this "natural parasitism" are delineated : parasite persistence in the site of inoculation (still the footpad) assesses the stable remodelling of a very transient pathogenic process
Among the many questions which are addressed, two relate (i) to the nature of the cell lineages where amastigotes persist, (ii) to the transmissible features of these amastigote to the vector Lutzomyia longipalpis (collaboration with Petr Volf).
4. Design of original models likely more relevant to decipher at the molecular, cellular and tissual levels, the more or less sustained pathogenic processes, as well as the repair processes triggered by the metacyclic stages of Leishmania major or L. amazonensis promastigotes.
4.1. Characterization and purification of a membrane glycoprotein exclusively present in Leishmania amastigotes of the mexicana complex(E. Fontan, T. Ilg Tübingen, E. Handman Melbourne, J.-C. Antoine).
It was previously mentioned that Leishmania circulate between mammalian hosts and hematophagous vectors. Within the mammalian hosts after the delivery of the metacyclic promastigotes by the sandfly in the upper dermis non-activated mononuclear phagocytes are the first host cells where the Leishmania do rapidly enter. Within their Parasitophorous Vacuole (PV), the promastigotes differentiate as amastigotes : these developmental stages are either replicative (in non-activated/de-activated macrophages), or non replicative (in activated macrophages). Therefore, in the mammalian host, the amastigote stage is the essential developmental stage with respect to the population size. Previously, a mouse monoclonal antibody was screened through an exclusive binding property, namely the binding to the membrane of live amastigotes of Leishmania amazonensis. This mAb designated (2A3-26) is a precious reagent for purifying the amastigote molecule(s). A promising step has been reached since either micro-sequencing or protein chip technology are applicable.
4.2. Biogenesis and remodelling of the parasitophorous vacuoles (PV) in mouse bone marrow-derived mononuclear phagocytes exposed to Leishmania amazonensis metacyclic promastigotes(N. Courret, C. Fréhel INSERM U411, N. Gouhier et M. Pouchelet INSERM, T. Lang, E. Prina, J.-C. Antoine).
The establishment of Leishmania in mammals depends on the differentiation of metacyclic promastigotes into amastigotes within macrophages. The kinetics of this process was examined using mouse macrophages infected with metacyclic promastigotes of L. amazonensis. The presence of amastigote characteristics, including large lysosome-like organelles called megasomes, stage-specific antigens, 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 p.i. 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. 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 mechanism in parasite survival.
4.3. In vivo models which allow to characterize the impact of the more or less invasive state of the L. major promatigotes or the Leishmania species, on the parasite-reactivity of CD4 T lymphocytes recovered from the draining lymph node of BALB/c mice (N. Courret, J.-H. Colle ®
1/10/200, T. Lang, N. Glaichenhaus Nice, D. Sacks, NIH Bethesda, E. Prina, G. Milon et J.-C. Antoine).
Following the elegant screening of the lack gene encoding the LACK molecule common to every species of Leishmania, many studies are relying on this molecule either in vitro, ex vivo or in vivo. Within our laboratory, a still too neglected result has been documented. Briefly, the display of the 158-173 AA peptide bound to I-Ad molecule at the membrane of mononuclear phagocytes strictly depends on the poor invasive property of the promastigotes used for the so-called infection. We further study the relevance of these in vitro results within the context of in vivo analysis. BALB/c mice were infected in the ear dermis with 103 to 106 Log-Phase (LP), Stationary Phase (SP), or Metacyclic (M) L. major promastigotes. CD4 T lymphocytes recovered from the draining lymph nodes and reactivity to the LACK molecule, or to a soluble lysate of L. major were monitored over a period of 21 days post-inoculation, through their cytokine profile secretion. The level of cytokine production by CD4 T cells is clearly dependent upon both the stage of differentiation and the dose of inoculated parasites. Thus, as early as day 3 post-infection with 106 LP, SP or M promastigotes, a high level of IFNg
was produced in presence of LA. At the same time point, only very small amounts of IL-4 were detected in the three groups. After the first week, the level of IFNg fell sharply and a strong increase of IL-4 was noted. When CD4 T cells were exposed to LACK strong production of IFNg which peaked at day 3 was measured, especially after inoculation of LP parasites, but only little IL-4 could be detected irrespective of the time after infection and the parasite developmental stage injected. In contrast, after infection with 103 M promastigotes which induced cutaneous lesions with a similar kinetics as 106 LP promastigotes, the production of both cytokines in response to LA was not detected before the onset of ear lesions (day 21). No production of cytokines could be observed in response to LACK.
4.4. Sequential analysis of the discrete phases driven by Leishmania major metacyclic promastigotes once delivered in the ear dermis of C57Bl/6 mice (J.-H. Colle ® 1/10/2000, M. Lebastard, L. Nicolas, Y. Belkaid, D. Sacks NIH, Bethesda, P. Volf, Université Charles - Prague, G. Milon).
Secondary lymphoid organs are sites of immunological integration, where many circulating leukocytes including T, B lymphocytes, as well as migratory dendritic 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 a perception of integrative immunophysiology. Thus, a model of Leishmania major infection in C57Bl/6 mice has been established that combines two main features of natural transmission : low dose (100 metacyclic promastigotes) and inoculation into a dermal site (the ear dermis). The evolution of the dermal lesion could be dissociated into two distinct phases. The initial "silent" phase, lasting 4-5 week, favoured establishment of the peak load of parasites in the dermis in the absence of lesion formation or any overt histopathologic changes 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 the site. The onset of pathology was correlated with the presence of cells staining for IL12p40 and IFNg in the epidermal compartment, and an expansion of T cells capable of producing IFNg in the draining lymph node. Parasite growth was not enhanced over the first 4-5 weeks in anti-CD4-treated mice. The onset and stable maintenance of repair processes were 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).
5. A model to decipher and characterize the host and parasite dependent mechanisms underlying the transmission of amastigotes from the mammalian host to the sandfly vector(L. Nicolas, J.-H. Colle ® 1/10/2000, M. Lebastard, P. Volf Charles Unviersity - Prague, G. Milon).
So far, studies of Leishmania persistence in mice have used injections of parasites administered either intravenously in the tail vein or subcutaneously in the footpad. These routes poorly reflect the natural conditions when the sandfly delivers metacyclic promastigotes intradermally. In this study, B10D2 and BALB/c mice were inoculated within the ear dermis with 104 Leishmania major metacyclic promastigotes. The parasite load was monitored by quantitative PCR in different tissues from the dermal inoculation site to distant tissues. The two sites of multiplication and persistence of parasites were the site of L. major inoculation and the draining lymph node (DLN), with a different pattern in the two mouse inbred lines. These two organs were the only sites harbouring parasites 12 months post-inoculation, with the DLN or BALB/c mice harbouring around 107 parasites, a stable load from months 3 to 12. In these two sites, 8 and 12 months after inoculation, interleukin 4 (IL-4), IFNg and inducible nitric oxide synthase transcripts parallel the parasite load while IL-10 transcript levels remain high. In addition, at early time points, from week 2 until month 3, parasite DNA was also detected in distant tissues such as the contralateral non-inoculated ear or the tail skin, indicating that blood was at least transiently the compartment through which the parasites were delivered. In contrast, L. major DNA in liver, spleen, and femoral bone marrow remained sporadic in mice of both lines. This study is discussed within the framework of Leishmania transmission from the vertebrate host to the sandfly vector, a complex process still poorly understood. This model will be precious for delineating at the tissual, cellular and molecular levels the key processes which reprogram the amastigotes towards the developmental transmissible stage which is pre-adapted to the vector.