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Leishmaniases are major vector-borne tropical diseases responsible for great human morbidity and mortality, caused by protozoan, trypanosomatid parasites of the genus Leishmania. In the mammalian host, parasites survive and multiply within mononuclear phagocytes, especially macrophages. However, the underlying mechanisms by which Leishmania spp. affect their host are not fully understood. Herein, proteomic alterations of primary, bone marrow-derived BALB/c macrophages are documented after 72 h of infection with Leishmania donovani insect-stage promastigotes, applying a SILAC-based, quantitative proteomics approach. The protocol was optimised by combining strong anion exchange and gel electrophoresis fractionation that displayed similar depth of analysis (combined total of 6189 mouse proteins). Our analyses revealed 86 differentially modulated proteins (35 showing increased and 51 decreased abundance) in response to Leishmania donovani infection. The proteomics results were validated by analysing the abundance of selected proteins. Intracellular Leishmania donovani infection led to changes in various host cell biological processes, including primary metabolism and catabolic process, with a significant enrichment in lysosomal organisation. Overall, our analysis establishes the first proteome of bona fide primary macrophages infected ex vivo with Leishmania donovani, revealing new mechanisms acting at the host/pathogen interface. SIGNIFICANCE: Little is known on proteome changes that occur in primary macrophages after Leishmania donovani infection. This study describes a SILAC-based quantitative proteomics approach to characterise changes of bone marrow-derived macrophages infected with L. donovani promastigotes for 72 h. With the application of SILAC and the use of SAX and GEL fractionation methods, we have tested new routes for proteome quantification of primary macrophages. The protocols developed here can be applicable to other diseases and pathologies. Moreover, this study sheds important new light on the "proteomic reprogramming" of infected macrophages in response to L. donovani promastigotes that affects primary metabolism, cellular catabolic processes, and lysosomal/vacuole organisation. Thus, our study reveals key molecules and processes that act at the host/pathogen interface that may inform on new immuno- or chemotherapeutic interventions to combat leishmaniasis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aberrant macrophage activation during intracellular infection generates immunopathologies that can cause severe human morbidity. A better understanding of immune subversion strategies and macrophage phenotypic and functional responses is necessary to design host-directed intervention strategies. Here, we uncover a fine-tuned transcriptional response that is induced in primary and lesional macrophages infected by the parasite Leishmania amazonensis and dampens NF-κB and NLRP3 inflammasome activation. Subversion is amastigote-specific and characterized by a decreased expression of activating and increased expression of de-activating components of these pro-inflammatory pathways, thus revealing a regulatory dichotomy that abrogates the anti-microbial response. Changes in transcript abundance correlate with histone H3K9/14 hypoacetylation and H3K4 hypo-trimethylation in infected primary and lesional macrophages at promoters of NF-κB-related, pro-inflammatory genes. Our results reveal a Leishmania immune subversion strategy targeting host cell epigenetic regulation to establish conditions beneficial for parasite survival and open avenues for host-directed, anti-microbial drug discovery., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background/objectives: Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis., Methodology/principal Findings: We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline., Conclusions/significance: Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential.

The interactions between the Leishmania parasite and its mammalian host cells are strongly regulated dynamic processes that take place at the molecular, cellular and organ level. Several different interaction models have been developed to take advantage of the development of imaging technologies. Here, we first review how conventional imaging techniques have been applied to fixed Leishmania-loaded tissue and cell samples. Then, we show how transgenic Leishmania expressing fluorescent or bioluminescent reporters allowed characterization of their tissue and cell host niches. Most notably, the use of whole-body imaging or intravital microscopy techniques has allowed accurate real-time monitoring of parasites in their environment. Finally, we discuss how innovative imaging technologies will allow further refinement of our understanding of the interplay between Leishmania and its hosts.

Background: Mammal macrophages (MPhi) display a wide range of functions which contribute to surveying and maintaining tissue integrity. One such function is phagocytosis, a process known to be subverted by parasites like Leishmania (L). Indeed, the intracellular development of L. amazonensis amastigote relies on the biogenesis and dynamic remodelling of a phagolysosome, termed the parasitophorous vacuole, primarily within dermal MPhi., Results: Using BALB/c mouse bone marrow-derived MPhi loaded or not with amastigotes, we analyzed the transcriptional signatures of MPhi 24 h later, when the amastigote population was growing. Total RNA from MPhi cultures were processed and hybridized onto Affymetrix Mouse430_2 GeneChips, and some transcripts were also analyzed by Real-Time quantitative PCR (RTQPCR). A total of 1,248 probe-sets showed significant differential expression. Comparable fold-change values were obtained between the Affymetrix technology and the RTQPCR method. Ingenuity Pathway Analysis software pinpointed the up-regulation of the sterol biosynthesis pathway (p-value = 1.31e-02) involving several genes (1.95 to 4.30 fold change values), and the modulation of various genes involved in polyamine synthesis and in pro/counter-inflammatory signalling., Conclusion: Our findings suggest that the amastigote growth relies on early coordinated gene expression of the MPhi lipid and polyamine pathways. Moreover, these MPhi hosting multiplying L. amazonensis amastigotes display a transcriptional profile biased towards parasite-and host tissue-protective processes.

This article provides a summary and discussion of properties of Leishmania amazonensis-loaded mouse macrophages. It illustrates how high-throughput analysis is expected to contribute to deciphering features displayed by macrophages when they are subverted as host cells for replicating Leishmania amastigotes. Firstly, we discuss features of mouse mononuclear phagocytes in steady-state conditions, including the phagocytosis of apoptotic cells. Secondly, we discuss results from ongoing investigations aimed at characterizing transcriptional signatures displayed by BALB/c mouse bone marrow-derived macrophages housing replicating L. amazonensis amastigotes. After a brief presentation on the feasibility of high-throughput microscopy relying on our robust culture system, we share some perspectives on the perpetuation of L. amazonensis in their hosts. Within this latter context, a novel question is formulated and its relevance is discussed: do the Leishmania amastigotes that persist within the mammalian dermis reach a non-replicating developmental stage? If so, is this developmental stage the only one displaying the features required for further development as promastigotes within the sand fly gut lumen?

Several lines of evidence indicate that the megakaryocyte/platelet lineage is crucial in myelofibrosis induction. The demonstration that NOD/SCID mice with functionally deficient monocytes do not develop fibrotic changes when exposed to thrombopoietin (TPO) also suggests an important role for monocyte/macrophages. However, in this animal model, the development of myelofibrosis is dependent on the level of TPO. This study was conducted to investigate whether NOD/SCID mice exposed to high TPO levels mediated by a retroviral vector would be refractory to the development of bone marrow fibrosis. We show that TPO and TGF-beta1 in plasma from NOD/SCID and SCID mice engrafted with TPO-overexpressing hemopoietic cells reach levels similar to the ones reached in immunocompetent mice, and all animals develop a myeloproliferative disease associated with a dense myelofibrosis at 8 wk posttransplantation. Monocytes in NOD/SCID mice are functionally deficient to secrete cytokines such as IL-1alpha in response to stimuli, even under TPO expression. Surprisingly, the plasma of these mice displays high levels of IL-alpha, which was demonstrated to originate from platelets. Together, these data suggest that completely functional monocytes are not required to develop myelofibrosis and that platelets are able, under TPO stimulation, to synthesize inflammatory cytokines, which may be involved in the pathogenesis of myelofibrosis and osteosclerosis.

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.

In their amastigote stage, Leishmania are obligatory intracellular parasites of mammalian macrophages, residing and multiplying within phagolysosomal compartments called parasitophorous vacuoles (PV). These organelles have properties similar to those described for the MHC class II compartments of antigen-presenting cells, sites where peptide-class II molecule complexes are formed before their expression at the cell surface. After infection with Leishmania amazonensis or L. mexicana, endocytosis and degradation of class II molecules by intracellular amastigotes have also been described, suggesting that these parasites have evolved mechanisms to escape the potentially hazardous antigen-presentation process. To determine whether these events extend to other molecules of the antigen-presentation machinery, we have now studied the fate of the MHC molecule H-2M in mouse macrophages infected with Leishmania amastigotes. At least for certain class II alleles, H-2M is an essential cofactor, which catalyses the release of the invariant chain-derived CLIP peptide from the peptide-binding groove of class II molecules and facilitates the binding of antigenic peptides. H-2M was detected in PV of mouse macrophages infected with various Leishmania species including L. amazonensis, L. mexicana, L. major and L. donovani. PV thus contain all the molecules required for the formation of peptide-class II molecule complexes and especially of complexes with parasite peptides. The present data indicate, however, that if this process occurs, it does not lead to a clear increase of SDS-stable compact (alpha)(beta) dimers of class II. In PV that contained L. amazonensis or L. mexicana, both class II and H-2M molecules often colocalized at the level where amastigotes bind to the PV membrane, suggesting that these molecules are physically associated, directly or indirectly, and possibly interact with parasite components. Furthermore, as class II molecules, H-2M molecules were internalized by amastigotes of these Leishmania species and reached parasite compartments that also contained class II molecules. Immunostaining of H-2M within parasites was increased by treatment of infected macrophages with the cysteine protease inhibitors Z-Phe-AlaCHN2 or Z-Phe-PheCHN2 or by incubation of the parasites with the same inhibitors before infection. These data thus support the idea that amastigotes of certain Leishmania species capture and degrade some of the molecules required for antigen presentation. To examine whether endocytosis of class II molecules by the parasites occurs through interactions with parasite components involving their peptide-binding groove, we made use of the fact that a large fraction of the class II molecules of H-2M(alpha) knock-out H-2(b) mice are occupied by the peptide CLIP and are unable to bind other peptides. We found that, in Leishmania-infected macrophages of these mutant mice, class II-CLIP complexes reached PV and were internalized by amastigotes. These results thus prove that endocytosis of class II molecules by amastigotes (1) is H-2M-independent and (2) does not necessarily involve the peptide-binding pocket of these molecules. Altogether, these data are compatible with an endocytic mechanism based on general properties shared by classical and non-classical class II molecules.

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.

Macrophages are apparently the only cells that in vivo allow the growth of the intracellular pathogen Leishmania. They are thus generally considered as likely candidates for the presentation of parasite Ag to CD4+ T lymphocytes known to be involved in protective and counterprotective immune responses. In the present study, we examined whether mouse macrophages infected with Leishmania were capable of stimulating T cell hybrids and a T cell clone reacting with the previously identified protective Ag LACK (Leishmania homologue of receptors for Activated C Kinase). This parasite protein is expressed in both promastigote and amastigote stages of Leishmania. We found that IFN-gamma-treated macrophages recently infected with live Leishmania promastigotes were fully competent to activate LACK-reactive T cells. However, at later times of infection, permissive macrophages infected with promastigotes were no longer able to present LACK, in spite of the presence of numerous intracellular parasites. This punctual presentation of LACK was apparently linked with the destruction, at least partial, of the intracellular parasites. In contrast, macrophages infected with live Leishmania amastigotes were always unable to stimulate the LACK-specific T cells. Amastigote-infected macrophages could, however, reactivate the T cells if LACK-delta(1), a recombinant form of LACK, was added as an exogenous protein in the culture medium. Similar results were obtained with all combinations tested involving macrophages from various origins, different activating cytokines (IFN-gamma, granulocyte-macrophage CSF, IL-4), several Leishmania species (L. amazonensis, L. major, L. donovani), and 15 different LACK-reactive T cell hybrids and clones. From these data, it is tempting to propose that the differentiation of promastigotes into amastigotes, which leads to a better survival of the parasites within macrophages, also allows them to go unnoticed by the immune system.

In their amastigote stage, Leishmania live in mammalian macrophages within parasitophorous vacuoles (PV), organelles of phagolysosomal origin that, in macrophages activated with IFN-gamma, contain major histocompatibility complex (MHC) class II molecules apparently devoid of invariant chains. We have now studied the fate of PV-associated class II molecules in mouse bone marrow-derived macrophages infected with L. amazonensis amastigotes using immunocytochemical and biochemical approaches. We have found that at least a part of these class II molecules was internalized by amastigotes and reached structures very often located in their posterior poles. This process was much more obvious if infected macrophages were incubated with protease inhibitors like antipain, chymostatin, Z-Phe-AlaCHN2 and Z-Phe-PheCHN2, or if amastigotes were pre-treated with the irreversible cysteine protease inhibitor Z-Phe-AlaCHN2 before infection, clearly indicating that amastigotes also degraded the internalized class II molecules. Study of infected macrophage cryosections by immuno-electron microscopy allowed the identification of the class II-positive structures in amastigotes as the lysosome-like organelles known as megasomes. Other PV membrane components like the prelysosomal/lysosomal glycoproteins Igp110, Igp120 and macrosialin could not be detected in megasomes of amastigotes even after treatment of macrophages with protease inhibitors, suggesting the involvement of some specific mechanism(s) for the internalization of class II molecules. Interestingly, after treatment of infected macrophages with various protease inhibitors (antipain, leupeptin, E-64, Z-Phe-AlaCHN2, Z-Phe-PheCHN2), PV membrane as well as megasomes of amastigotes become positive for invariant chains. A quantitative analysis of amastigote-associated class II molecules based on enzyme immunoassays showed that: (a) amastigotes extracted from macrophages treated with both IFN-gamma and antipain or Z-Phe-AlaCHN2 contained a much greater amount of class II than amastigotes extracted from macrophages treated with IFN-gamma alone; (b) class II molecules associated with the former were mainly intracellular and, at least some of them, were complexed with invariant chains or fragments of invariant chains; (c) amastigotes pre-incubated with Z-Phe-AlaCHN2 before infection accumulated a greater amount of intracellular class II than amastigotes pre-incubated without inhibitor, clearly indicating that the blockade of parasite cysteine proteases was sufficient to enhance the pool of these molecules within megasomes. On the whole, these data are consistent with the idea that class II molecules reaching PV are newly synthesized and still complexed with intact invariant chains or with partially degraded invariant chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Leishmania-infected M phi are potential candidates for the presentation of parasite Ag to Leishmania-specific CD4+ T lymphocytes. To assess whether infected cells could function as APC, we examined the ability of bone marrow-derived M phi infected with Leishmania amazonensis amastigotes to stimulate various CD4+, l-Ad- or l-Ed-restricted T-cell hybridomas specific for the bacteriophage lambda repressor cl protein, the human chorionic gonadotropin or OVA. A reduced capacity of infected M phi to present native Ag to most T-cell hybridomas tested was noted that was probably a result of a lower expression on their plasma membrane of stimulatory [la-peptide] complexes. Neither a reduced Ag uptake nor an altered Ag processing appeared to be at the origin of the partial inability of infected M phi to present Ag. As regards the level of plasma membrane la expression, no quantitative difference could be detected between uninfected and infected M phi. Moreover, after fixation with paraformaldehyde, the ability of plasma membrane la molecules to bind immunogenic peptides was apparently not reduced in infected M phi. So, these cells most likely expressed functional la molecules on their cell surface. Interestingly, infected M phi and M phi infected then cured by a treatment with a leishmanicidal compound were similarly impaired in their capacity to present native Ag or peptides to the hybridomas, and no recovery was noted even 24 h after the leishmanicidal treatment. Furthermore, infected M phi and M phi incubated with heat-killed amastigotes or with an amastigote homogenate exhibited similar inhibitions of Ag presentation. Taken together, these results suggest that the functional failure of infected M phi to present exogenous Ag could be because either of interferences with the events leading to the meeting of la molecules with peptides derived from these exogenous Ag or to a competition for binding to la molecules between these peptides and parasite molecules.

Leishmania-infected macrophages are potential antigen-presenting cells for CD4+ T lymphocytes, which recognize parasite antigens bound to major histocompatibility complex class II molecules (Ia). However, the intracellular sites where Ia and antigens may interact are far from clear, since parasites grow within the modified lysosomal compartment of the host cell, whereas Ia molecules seem to be targeted to endosomes. To address this question, the expression and fate of Ia molecules were studied by immunocytochemistry in Leishmania amazonensis-infected murine macrophages stimulated with gamma interferon. In uninfected macrophages, Ia molecules were localized on the plasma membrane and in perinuclear vesicles, but they underwent a dramatic redistribution after infection, since most of the intracellular staining was then associated with the periphery of the parasitophorous vacuoles (p.v.) and quite often polarized towards amastigote-binding sites. The Ii invariant chain, which is transiently associated with Ia during their intracellular transport, although well expressed in infected macrophages, apparently did not reach the p.v. Similar findings were observed with macrophages from mice either resistant or highly susceptible to Leishmania infection. In order to determine the origin of p.v.-associated Ia, the fate of plasma membrane, endosomal, and lysosomal markers, detected with specific antibodies, was determined after infection. At 48 h after infection, p.v. was found to exhibit a membrane composition typical of mature lysosomes. Overall, these data suggest that (i) Ia located in p.v. originate from secondary lysosomes involved in the biogenesis of this compartment or circulate in several endocytic organelles, including lysosomes and (ii) p.v. could play a role in antigen processing and presentation. Alternatively, the presence of high amounts of Ia in p.v. could be due to a Leishmania-induced mechanism by means of which this organism may evade the immune response.

Leishmania are protozoans of the trypanosomatidae family that cause human infections. The amastigote form of Leishmania is an obligate intracellular parasite of mononuclear phagocytes that multiplies within parasitophorous vacuoles (pv) of phagolysosomal origin. To investigate the strategies which allow Leishmania to withstand these potentially cytotoxic conditions, the distribution and activities of various lysosomal peptidases in rat macrophages infected or uninfected with Leishmania amazonensis amastigotes were studied. Specific immunoglobulins against cathepsins (cat.) B, H, L and D were used to localize these endopeptidases by immunocytochemistry. Results showed that most or even all of the secondary lysosomes in the host cell fuse with parasite-filled phagosomes, leading to translocation of the proteases in the parasitophorous vacuoles. A further study consisted in assays of five protease activities: dipeptidylpeptidases (DPP) I and II (exopeptidases), cat. B, cat. H and cat. D. Infection of macrophages was followed by a gradual increase in all these protease activities except for DPP II. These increases were apparently not related to parasite protease activities. It seems that infection by Leishmania amazonensis is followed by increased synthesis and/or reduced catabolism of host cell lysosomal proteases or alternatively by inactivation of endogenous inhibitors. Amastigote infectivity may be related, at least in part, to the development of mechanisms that allow the parasite to withstand unfavorable environmental conditions.

In mammalian hosts, Leishmania amastigotes are obligatory intracellular parasites of macrophages and multiply within parasitophorous vacuoles of phagolysosomal origin. To understand how they escape the harmful strategies developed by macrophages to kill ingested microorganisms, it is important to obtain information on the functional state of parasitophorous vacuole. For this purpose, we studied the intracellular distribution and activity of host lysosomal proteases in rat bone marrow-derived macrophages infected with Leishmania amazonensis amastigotes. Localization of cathepsins B, H, L, and D was investigated by using specific immunoglobulins. In uninfected macrophages, these enzymes were located in perinuclear granules (most of them were probably secondary lysosomes) which, after infection, disappeared progressively. In infected macrophages, cathepsins were detected mainly in the parasitophorous vacuoles, suggesting that the missing secondary lysosomes had fused with these organelles. Biochemical assays of various proteases (cathepsins B, H, and D and dipeptidyl peptidases I and II) showed that infection was accompanied by a progressive increase of all activities tested, except that of dipeptidyl peptidase II, which remained constant. No more than 1 to 10% of these activities could be attributed to amastigotes. These data indicate that (i) Leishmania infection is followed by an increased synthesis and/or a reduced catabolism of host lysosomal proteases, and (ii) amastigotes grow in a compartment rich in apparently fully active proteases. Unexpectedly, it was found that infected and uninfected macrophages degraded endocytosed proteins similarly. The lack of correlation in infected macrophages between increase of protease activities and catabolism of exogenous proteins could be linked to the huge increase in volume of the lysosomal compartment.

Leishmania amastigotes are intracellular protozoan parasites of mononuclear phagocytes which reside within parasitophorous vacuoles of phagolysosomal origin. The pH of these compartments was studied with the aim of elucidating strategies used by these microorganisms to evade the microbicidal mechanisms of their host cells. For this purpose, rat bone marrow-derived macrophages were infected with L. amazonensis amastigotes. Intracellular acidic compartments were localized by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine as a probe. This indicator, which can be detected by light microscopy by using immunocytochemical methods, mainly accumulated in perinuclear lysosomes of uninfected cells, whereas in infected cells, it was essentially localized in parasitophorous vacuoles, which thus appeared acidified. Phagolysosomal pH was estimated quantitatively in living cells loaded with the pH-sensitive endocytic tracer fluoresceinated dextran. After a 15- to 20-h exposure, the tracer was mainly detected in perinuclear lysosomes and parasitophorous vacuoles of uninfected and infected macrophages, respectively. Fluorescence intensities were determined from digitized video images of single cells after processing and automatic subtraction of background. We found statistically different mean pH values of 5.17 to 5.48 for lysosomes and 4.74 to 5.26 for parasitophorous vacuoles. As for lysosomes of monensin-treated cells, the pH gradient of parasitophorous vacuoles collapsed after monensin was added. This very likely indicates that these vacuoles maintain an acidic internal pH by an active process. These results show that L. amazonensis amastigotes are acidophilic and opportunistic organisms and suggest that these intracellular parasites have evolved means for survival under these harsh conditions and have acquired plasma membrane components compatible with the environment.

Parasites establish dynamic relationships with their hosts that allow their persistence . Leishmania spp. are unicellular parasites that have two hosts: a haematophageous insect, the sand fly, and a vertebrate. Both in animals and humans, absence of clinical lesions does not imply absence of infection. This phenomenon has major implications in epidemiology, preventive medicine, and pharmacology. Parasitic load determination in mammalian tissues or organs is currently performed by culture-based methods that are time consuming and require samples harbouring cultivable parasites. Recently, we developed a real-time PCR assay to quantify the parasitic burden of several Leishmania spp. based on detection of kinetoplastic DNA (kDNA). The efficacy of this real-time PCR assay was first evaluated in monitoring the fate of Leishmania major in C57Bl/6 mice following intradermal inoculation of the right ear pinna with low doses of infective metacyclics at 24 h, 96 h, and weekly for 7 weeks. Leishmania were established in the center of the inoculated pinna, to a lesser extent in the pinna border, and in the draining lymph node. The parasite was detected, but not quantified, in distant cutaneous sites (left ear pinna, thorax skin, tail base) and in the femoral bone marrow. Throughout the whole experiment, the parasite was detected in the glabrous tail base skin. We also investigated whether the parasites detected by our method were still viable or if we were also detecting DNA from dead parasites. Technical and biological constraints have been overcome by developing an in vitro model to monitor the persistence of Leishmania kDNA in cultured mouse macrophages after host leucocytes were exposed to L-leucine-methyl ester, a leishmanicidal molecule. The detection of kDNA by real-time PCR was correlated with the viability of the parasites in macrophages. These results show that laboratory mice (experimental host) or in vitro-grown mouse macrophages infected with Leishmania parasites can be considered useful systems in two areas. First, in pharmacology, preventive medicine and epidemiology, such a system would allow pharmacological evaluation of Leishmania-targeted drugs or vaccines to a broader extent than merely monitoring the reduction of the lesion or the prevention of lesion onset. A similar system could be developed in other animal species, particularly the dog, in order to detect asymptomatic carriage at the borders or in quarantines, or to investigate the real prevalence of Leishmania spp. The growing importance of Leishmania infantum-related disorders in humans makes such a method very interesting. A second use for this experimental system is the investigation of the prompt and renewed interactions that Leishmania parasites establish within their mammalian hosts. Establishment of a system to quantify the parasitic burden of Leishmania spp. is the perspective of this preliminary research. Recently published data show that the apparent lack of symptoms hides complex mechanisms involving very different factors such as keratinocytes, epidermal and dermal leucocytes, the extracellular matrix, and sand fly saliva. Funding: Institut Pasteur, Centre National de la Recherche Scientifique. [ABSTRACT FROM AUTHOR]