CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain.

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. We investigated the role of CD11c- and CD11b-expressing leukocytes in T. gondii transport during the early step of parasitism from the mouse small intestine and during subsequent parasite localization in the brain. Following intragastric inoculation of cyst-containing parasites in mice, CD11c+ dendritic cells from the intestinal lamina propria, the Peyer patches, and the mesenteric lymph nodes were parasitized while in the blood, parasites were associated with the CD11c- CD11b+ monocytes. Using adoptive transfer experiments, we demonstrated that these parasitized cells triggered a parasitic process in the brain of naive recipient mice. Ex vivo analysis of parasitized leukocytes showed that single tachyzoites remained at the cell periphery, often surrounded by the host cell plasma membrane, but did not divide. Using either a dye that labels circulating leukocytes or an antibody known to prevent CD11b+ circulating leukocytes from leaving the microvascular bed lumen, and chimeric mice in which the hematopoietic cells expressed the green fluorescent protein, we established that T. gondii zoites hijacked CD11b+ leukocytes to reach the brain extravascular space.

Characterisation of a Leishmania mexicana knockout lacking guanosine diphosphate-mannose pyrophosphorylase.

In eukaryotes, the enzyme GDP-mannose pyrophosphorylase (GDP-MP) is essential for the formation of GDP-mannose, the donor of activated mannose for all glycosylation reactions. Unlike other eukaryotes, where deletion of GDP-mannose pyrophosphorylase is lethal, deletion of this gene in Leishmania mexicana has no effect on viability, but leads to the generation of avirulent parasites. In this study, we show that the null mutants have a perturbed morphology and cytokinesis, retarded growth and increased adherence to the substratum where they form large colonies. The null mutants attach avidly to mouse macrophages, but unlike the wild type organisms, they do not bind to the complement receptor 3 and are slow to induce phagocytosis. Once internalised, they localise to the phagolysosome, but in contrast to wild type organisms which transform into the intracellular amastigote and establish in the macrophage, they are cleared by 24 h in culture and by 5 h in vivo. The null mutants are hypersensitive to human but not mouse complement and to temperature and acidic pH. Surprisingly, in view of the lack of several known host-protective antigens, injection of the mutant parasites into BALB/c mice confers significant and long lasting protection against infection, suggesting that these temperature sensitive mutants are an attractive candidate for a live attenuated vaccine.

Leishmania spp.: on the interactions they establish with antigen-presenting cells of their mammalian hosts.

Identification of macrophages as host cells for the mammalian stage of Leishmania spp. traces back to about 40 years ago, but many questions concerning the ways these parasites establish themselves in these cells, which are endowed with potent innate microbicidal mechanisms, are still unanswered. It is known that microbicidal activities of macrophages can be enhanced or induced by effector T lymphocytes following the presentation of antigens via MHC class I or class II molecules expressed at the macrophage plasma membrane. However, Leishmania spp. have evolved mechanisms to evade or to interfere with antigen presentation processes, allowing parasites to partially resist these T cell-mediated immune responses. Recently, the presence of Leishmania amastigotes within dendritic cells has been reported suggesting that they could also be host cells for these parasites. Dendritic cells have been described as the only cells able to induce the activation of naive T lymphocytes. However, certain Leishmania species infect dendritic cells without inducing their maturation and impair the migration of these cells, which could delay the onset of the adaptive immune responses as both processes are required for naive T cell activation. This review examines how Leishmania spp. interact with these two cell types, macrophages and dendritic cells, and describes some of the strategies used by Leishmania spp. to survive in these inducible or constitutive antigen-presenting cells.

Toxoplasma gondii and mucosal immunity.

Toxoplasma gondii, an intracellular parasite infects the host through the oral route. Infection induces a cascade of immunological events that involve both the components of the innate and adaptative immune responses. Alteration of the homeostatic balance of infected intestine results in an acute inflammatory ileitis in certain strains of inbred mice. Both the infected enterocytes as well as the CD4 T cells from the lamina propria produce chemokines and cytokines that are necessary to clear the parasite whereas CD8 intraepithelial lymphocytes secrete transforming growth factor beta that reduces the inflammation. In this review, we describe the salient features of this complex network of interactions among the different components of the gut-associated lymphoid tissue cell population that are induced after oral infection with T. gondii.

Intradermal inoculations of low doses of Leishmania major and Leishmania amazonensis metacyclic promastigotes induce different immunoparasitic processes and status of protection in BALB/c mice.

In order to simulate the natural long term parasitisms which may occur in mammals infected with Leishmania, cutaneous leishmaniases due to Leishmania major or Leishmania amazonensis were induced using a model based on the inoculation of 10-1000 metacyclic promastigotes into the ear dermis of BALB/c mice. The final outcome of these parasitisms was dependent upon the number of inoculated parasites. Only some of the mice inoculated with ten parasites displayed cutaneous lesions, whereas most mice infected with 100 metacyclics and all mice infected with 1000 metacyclics developed progressive lesions. We found, using the latter experimental conditions, that the onset of the pathology was associated with: (a) parasite multiplication in the inoculation site and the draining lymph node correlating with an increase of the lymph node cell number, especially in L. major-infected mice; and (b) the detection of lymph node cells, at least in part CD4(+) T lymphocytes, able to produce high levels of interferon-gamma, interleukin (IL)-4, IL-10 and IL-13. Thereafter, mice infected by L. major harboured few parasites in the ear and had a 100-fold reduction in lymph node parasite load between 23 and 40 weeks post-inoculation. In contrast, the parasite loads of L. amazonensis-infected mice remained stable in the ear and increased in nodes during the same period of time. Only L. major-infected mice that exhibited cutaneous lesions in the primary site were resistant to the re-inoculation of 1000 metacyclic promastigotes, whereas all L. amazonensis-primary infected mice remained susceptible to a second homologous challenge. These results are the first to document that a status of resistance to re-infection, referred to concomitant immunity, is coupled to the development of primary progressive lesions in L. major-infected BALB/c mice. Such a protective status is absent in L. amazonensis-infected BALB/c mice.

The levels and patterns of cytokines produced by CD4 T lymphocytes of BALB/c mice infected with Leishmania major by inoculation into the ear dermis depend on the infectiousness and size of the inoculum.

The production of cytokines by CD4 lymph node T lymphocytes derived from BALB/c mice recently infected in the ear dermis with high (10(6) parasites) or low (10(3) parasites) doses of Leishmania major metacyclic promastigotes (MP) was examined over a 3-week period following inoculation. Results were compared with those obtained when mice were injected with less infectious parasite populations, namely, stationary-phase or log-phase promastigotes (LP). Cells were purified 16 h and 3, 8, and 19 days after inoculation, and the amounts of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) released in response to LACK (Leishmania homolog of receptors for activated C kinase) or total L. major antigens were assessed. We found that LACK-reactive T cells from mice inoculated with a high dose of parasites first produced IFN-gamma and later on IL-4; the level of IFN-gamma produced early by these cells was dependent upon the stage of the promastigotes inoculated, the highest level being reached with cells recovered from mice inoculated with the least infectious parasites, LP; sequential production of IFN-gamma and then of IL-4 also characterized L. major antigen-reactive CD4 T cells, suggesting that the early production of IFN-gamma does not impede the subsequent rise of IL-4 and finally the expansion of the parasites; after low-dose inoculation of MP, cutaneous lesions developed with kinetics similar to that of lesions induced after inoculation of 10(6) LP, but in this case CD4 T lymphocytes did not release IFN-gamma or IL-4 in the presence of LACK and neither cytokine was produced in response to L. major antigens before the onset of lesion signs. These results suggest the existence of a discreet phase in terms of CD4 T-cell reactivity for at least the first 8 days following inoculation, a time period during which parasites are able to grow moderately. In conclusion, the levels and profiles of cytokines produced by Leishmania-specific CD4 T lymphocytes clearly depend on both the stage of differentiation and number of parasites used for inoculation.

Biogenesis of Leishmania-harbouring parasitophorous vacuoles following phagocytosis of the metacyclic promastigote or amastigote stages of the parasites.

Protozoan parasites Leishmania alternate between a flagellated promastigote form and an amastigote form. In their mammalian hosts, Leishmania survive and multiply in macrophages. Both forms can be internalized by these host cells at different stages of the infectious process and eventually establish themselves within parasitophorous vacuoles exhibiting phagolysosomal properties. To determine whether the biogenesis of these organelles differs according to the parasitic stage used to initiate infection, we compared their formation kinetics after phagocytosis of either metacyclic promastigotes or amastigotes of L. amazonensis or of L. major by mouse bone-marrow-derived macrophages pre-exposed or not to IFN-gamma. After 10 minutes of contact, an accumulation of F-actin was observed around the promastigotes and amatigotes undergoing phagocytosis or those that had already been internalized. This accumulation was transient and rapidly disappeared at later times. At 30 minutes, most of the promastigotes were located in long, narrow organelles that were exactly the same shape as the parasites. The latter were elongated with their cell bodies near to the macrophage nucleus and their flagella towards the periphery. This suggests that promastigote phagocytosis mainly occurs in a polarized manner, with the cell body entering the macrophages first. Most, if not all, of the phagocytosed promastigotes were located in organelles that rapidly acquired phagolysosomal properties. At 30 minutes, lamp-1, macrosialin, cathepsins B and D were detected in 70-98% of these compartments and about 70% of them were surrounded by rab7p. These late endosome/lysosome 'markers' were recruited through fusion with late endocytic compartments. Indeed, when late endosomes/lysosomes were loaded with fluorescein dextran, 81-98% of the promastigote-harbouring compartments contained the endocytic tracer 30 minutes after infection. Electron microscopy of infected macrophages previously loaded with peroxidase confirmed that the phagosomes rapidly fused with late endocytic compartments. When the amastigote stage of L. amazonensis was used to initiate infection, the kinetics of acquisition of the different late endosome/lysosome 'markers' by the phagosomes were similar to those measured after infection with metacyclics. However, more rab7p(+)-phagosomes were observed at early time points (e.g. 90% were rab7p(+) at 30 minutes). The early endosome 'markers', EEA1 and the transferrin receptor, were hardly detected in parasite-containing compartments regardless of the parasitic stage used to infect macrophages and the time after infection. In conclusion, both metacyclic- and amastigote-containing phagosomes fuse with late endosomes/lysosomes within 30 minutes. However, with L. amazonensis, the time required for the formation of the huge parasitophorous vacuoles, which are characteristic of this species, was much shorter after infection with amastigotes than after infection with metacyclic promastigotes. This indicates that the initial fusions with late endosomes/lysosomes are followed by a stage-specific sequence of events.