Nathalie Courret

The biogenesis and properties of the parasitophorous vacuoles that harbour Leishmania in murine macrophages

Leishmania are protozoan parasites that, as amastigotes, live in the macrophages of mammalian hosts within compartments called parasitophorous vacuoles. These organelles share features with late endosomes/lysosomes and are also involved in the trafficking of several major histocompatibility complex (MHC)-encoded molecules. Improved knowledge of the parasitophorous vacuoles may help clarify how these protozoa persist in their hosts.

Presentation of the Leishmania antigen LACK by infected macrophages is dependent upon the virulence of the phagocytosed parasites.

We have previously demonstrated that murine macrophages (Mϕ) infected with Leishmania promastigotes, in contrast to Mϕ infected with the amastigote stage of these parasites, are able to present the Leishmania antigen LACK (Leishmania homologue of receptors for activated C kinase) to specific, I‐Ad‐restricted T cell hybrids and to the T cell clone 9.1‐2. These T cells react with the LACK (158 – 173) peptide, which is immunodominant in BALB/c mice. Here, we show that the level of stimulation of the LACK‐specific T cell hybridoma OD12 by promastigote‐infected Mϕ is clearly dependent upon the differentiation state of the internalized parasites. Thus, shortly after infection with log‐phase or stationary‐phase promastigotes of L. major or of L. amazonensis, Mϕ strongly activated OD12. The activity was transient and rapidly lost. However, under the same conditions, activation of OD12 by Mϕ infected with metacyclic promastigotes of L. major or of L. amazonensis was barely detectable. At the extreme, Mϕ infected with amastigotes were incapable to stimulate OD12. Thus, the presentation of LACK by infected Mϕ correlates with the degree of virulence of the phagocytosed parasites, the less virulent being the best for the generation/expression of LACK (158 – 173)‐I‐Ad complexes. While the intracellular killing of the parasites appears to be an important condition for the presentation of LACK, it is not the only requisite. The partial or total destruction of intracellular L. amazonensis amastigotes does not allow the presentation of LACK to OD12. A preferential interaction of LACK (158 – 173) with recycling rather than newly synthesized MHC class II molecules does not explain the transient presentation of LACK by Mϕ infected with log‐phase or stationary‐phase promastigotes because brefeldin A strongly inhibited the presentation of LACK to OD12. Taken together, these results suggest that virulent stages of Leishmania, namely metacyclics and amastigotes, have evolved strategies to avoid or minimize their recognition by CD4+ T lymphocytes.

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.

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.

Kinetics of the intracellular differentiation of Leishmania amazonensis and internalization of host MHC molecules by the intermediate parasite stages

The establishment of Leishmania in mammals depends on the transformation 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 appearance 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 12h 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 appearance 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 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.

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

ABSTRACT The production of cytokines by CD4 lymph node T lymphocytes derived from BALB/c mice recently infected in the ear dermis with high (106 parasites) or low (103 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-γ) 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-γ and later on IL-4; the level of IFN-γ 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-γ and then of IL-4 also characterized L. major antigen-reactive CD4 T cells, suggesting that the early production of IFN-γ 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 106 LP, but in this case CD4 T lymphocytes did not release IFN-γ 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.