Leishmania-infected macrophages sequester endogenously synthesized parasite antigens from presentation to CD4+ T cells

CD4⁺ T cell lines raised against the protective leishmanial antigens GP46 and P8 were used to study the presentation of endogenously synthesized Leishmania antigens by infected cells. Using two different sources of macrophages, the 14.07 macrophage cell line (H-2^k) which constitutively expresses major histocompatibility complex (MHC) class II molecules, and elicited peritoneal exudate cells, we found that cells infected with Leishmania amastigotes presented little, if any endogenously synthesized parasite antigens to CD4⁺ T cells. In contrast, promastigote-infected macrophages did present endogenous parasite molecules to CD4⁺ T cells, although only for a limited time, with maximal presentation occurring within 24 h of infection and decreasing to minimal antigen presentation at 72 h post-infection. These observations suggest that once within the macrophage, Leishmania amastigote antigens are sequestered from the MHC class II pathway of antigen presentation. This allows live parasites to persist in infected hosts by evading the activation of CD4⁺ T cells, a major and critical anti-leishmanial component of the host immune system. Studies with drugs that modify fusion patterns of phagosomes suggest that the mechanism of this antigen sequestration includes targeted fusion of the parasitophorous vacuole with certain endocytic compartments.     

Antibiotic Transport in Bacteria

Abstract

Survival or destruction of intramacrophage pathogen Leishmania depends in part on modulation of their host cell phagosome, capabilities of the infected macrophages to present parasite antigen to the host's immune system. Macrophages house these parasites as amastigotes in the acidic phagolysosomal compartment. Leishmania phagolysosome is the potential site for processing and presentation of its antigen as well as being the target site for chemotherapy in leishmaniasis. It is thought that the parasites are killed from macrophage activation by lymphokines secreted from either helper T1 cells or CD8⁺ T cells. Characterization of both the host and parasite molecules in the compartment in the context of biogenesis of Leishmania-phagolysosome and processing of the parasite antigen by this compartment are discussed. Trafficking of different drugs and new agents through this compartment and their role in chemotherapy and necessity of developing new drug carrier are also stressed.     

Leishmania major-specific CD8+ T cells are inducers and targets of nitric oxide produced by parasitized macrophages

Lines of Leishmania major-specific CD8⁺ T cells were derived from the lymph nodes and spleens of CBA mice, immune following resolution of a primary infection, 7 days after secondary challenge with viable L. major. Specific stimulation of these CD8⁺ T cells by bone marrow-derived macrophages infected with L. major led to the release of interferon-γ by CD8⁺ T cells and nitric oxide by macrophages. Interestingly, the nitric oxide released by bone marrow-derived macrophages down-regulated the production of interferon-γ by specifically activated CD8⁺ T cells. The proliferation and long-term maintenance of these parasite-specific CD8⁺ T cells was impaired by the nitric oxide produced by stimulating infected macrophages as a result of cytokines released by activated CD8⁺ T cells. Taken together, the results indicate that L. major-specific CD8⁺ T cells are sensitive to the toxic effect of the nitric oxide that they induce.     

Antigen presentation by Leishmania mexicana-infected macrophages: Activation of helper T cells specific for amastigote cysteine proteinases requires intracellular killing of the parasites

Leishmania mexicana amastigotes proliferate in the phagolysosomes of mammalian macrophages. The parasites abundantly synthesize lysosomal cysteine proteinases, which are encoded by the lmcpb gene family. One of these genes was overexpressed in Escherichia coli, and the purified recombinant protein was used as an antigen to induce and establish a T helper 1 (T_h1) cell line. The T cells recognize epitopes shared by the native cysteine proteinases and the recombinant protein. Infected bone marrow-derived macrophages induced to express major histocompatibility complex class II molecules by interferon (IFN)-γ do not affect parasite viability. These macrophages fail to stimulate the proliferation of the T cell line. In contrast, strong T cell stimulation is observed after the parasites are killed by treatment with L-leucine methylester, or after activation of macrophages by IFN-γ and tumor necrosis factor-α. It is concluded that infected macrophages efficiently present this lysosomal Leishmania antigen once the parasites are inactivated and degraded. This observation may be of considerable relevance for the outcome of Leishmania infections provided that it can be extended to other parasite antigens.     

Antigenicity of the Leishmania infantum histones H2B and H4 during canine viscerocutaneous leishmaniasis

In this study we show that sera from dogs naturally infected with Leishmania infantum contain antibodies that specifically react against the parasite H2B and H4 histones. The Leishmania H2B and the amino-terminal region of the histone H4, expressed as fusion proteins, when confronted with sera from canine viscerocutaneous leishmaniasis (VCL) dogs, were recognized by 63% and 47%, respectively. No reactivity was detected when sera from dogs naturally infected with pathogens other than Leishmania were used. Using a collection of synthetic peptides covering the complete sequence of both proteins, we have determined that the main linear antigenic determinants are located in the amino-terminal domains of these histones. The humoral response against histones H2B and H4 induced during canine leishmaniasis was found to be specific for Leishmania histones, since no cross-reactivity of the VCL sera with mammal histones was observed. Also, a comparative study of the prevalence of antibodies among VCL sera against the four core histones of L. infantum was performed. Although a large heterogeneity of the humoral responses against these proteins was found, histones H2A and H3 seem to be more prevalent immunogens than histones H2B and H4 during canine natural leishmaniasis. The origin of the anti-histone humoral response and its possible implications in the pathogenesis of Leishmania infection are discussed.     

L‐Arginine deprivation impairs Leishmania major‐specific T‐cell responses

The amino acid L ‐arginine plays a crucial role in the regulation of immune responses. We have recently shown that uncontrolled replication of Leishmania parasites at the site of pathology correlates with high levels of arginase activity in nonhealing leishmaniasis and that this elevated arginase activity causes local depletion of L ‐arginine. To further our understanding of the impact of L ‐arginine deprivation in experimental leishmaniasis, here we characterize in detail the effects of L ‐arginine deprivation on antigen‐specific T cells and MΦ. The results of our study show that decrease of L ‐arginine levels in the extracellular milieu affects the biological activities of Leishmania major‐specific T cells, both at the level of the magnitude and the quality of their responses. L. major‐specific CD4⁺ T cells rendered hyporesponsive by L ‐arginine deprivation can be partially rescued by addition of exogenous L ‐arginine to produce IL‐4 and IL‐10, but not to produce IFN‐γ. Furthermore, our results show that L ‐arginine deprivation also greatly impacts parasite growth in activated macrophages. In summary, our results suggest that L ‐arginine levels affect both Th cell responses and parasite replication.     

Langerhans cells transport Leishmania major from the infected skin to the draining lymph node for presentation to antigen-specific T cells

Murine epidermal Langerhans cells (LC) have been shown to internalize Leishmania major, a cause of human cutaneous leishmaniasis, and to stimulate a vigorous parasite-specific T cell response. The present study emphasizes the critical role of LC in leishmaniasis by documenting directly that LC have the ability to transport L. major from the skin to the draining lymph node (LN). This was revealed by irreversible labeling of LC with a fluorescent cell linker and in vivo tracking. In contrast, no migration to the LN was seen with L. major-infected macrophages. These findings were consistent with the results of mixed labeling immunohistology showing that early in infection the expression of parasite antigen in the LN draining the lesion was confined to dendritic cells and could not be detected in macrophages. Furthermore, dendritic cells in LN draining the site of cutaneous infection stimulated L. mayor-primed T cells in vitro and, most notably, were able to activate unprimed T cells capable of mediating parasite-specific delayed-type hypersensitivity reactivity in vivo. Taken together, the results indicate that LC capture L. major in the skin and transport it to the regional LN for initiation of the specific T cell immune response.     

Synergy between activated Leishmania major-specific CD4+ T lymphocytes and bone-marrow-derived cells in the exacerbation of murine cutaneous leishmaniasis

Mechanisms of exaberbation of murine cutaneous leishmaniasis mediated by Leishmania major-specific CD4⁺ T lymphocytes were studied. Using a limiting dilution assay for the quantification of Leishmania parasites, the infected tissues (footpad) of lethally irradiated mice were found to contain tenfold less parasites at four days of infection than the footpads of infected unirradiated animals. Injection of bone marrow cells depleted of T cells into irradiated mice at the site of infection led to an increase in parasite numbers to levels equivalent to those seen in unirradiated mice. After injection of either L. major-specific CD4⁺ T cells, previously shown to exacerbate cutaneous leishmaniasis, into the infected footpad or the intravenous (i.v.) injection of bone marrow cells depleted of T cells, the numbers of parasites in lesions of irradiated mice never reached the values found in unirradiated control mice. In contrast, the concomitant transfer of CD4⁺ T-cell populations in situ and bone marrow cells depleted of T cells intravenously led to an increase in parasite loads in irradiated mice up to levels comparable to those of the unirradiated mice. This suggested that recruitment of myelomonocytic cells at the site of the lesions plays a role in the exacerbation of murine cutaneous leishmaniasis mediated by these CD4⁺ T lymphocytes. Finally, a similar effect was observed with T cells specific for an antigen unrelated to Leishmania, provided that this antigen was added to the L. major infecting inoculum.     

T cells and immunopathogenesis of experimental African trypanosomiasis

Summary: African trypanosomes are pathogens for humans and livestock. They are single-cell, extra-cellular parasites that cause persistent infections of the blood and induce profound immunosuppression. Here, we review recent work on experimental African trypanosomiasis, especially infections with Trypanosoma congolense, in mice with regard to mechanisms of immunosuppression and immunopathology. The center of the immunopathology is the T-cell-independent production of antibodies to the variant surface glycoprotein (VSG) of trypanosomes, the anti-VSG antibody-mediated phagocytosis of trypanosomes by macrophages, and the subsequent profound dysregulation of the macrophage system. Depending on the genetics of the host and the parasite load, the malfunction of the macrophage system is enhanced by interferon-γ produced by parasite-specific, major histocompatibility complex class II-restricted, matrix-adherent CD4⁺ T cells or downregulated by interleuin-10 produced by parasite-specific, CD4⁺CD25^high Forkhead box protein 3⁺ regulatory T cells. There is a physiological conflict of the two relevant cytokines interleukin-10 and interferon-γ in regulating the immunopathology versus regulating the induction and effect of protective immune responses. On the basis of very recent work in our laboratory, we propose a hypothetical model suggesting a cross-regulation of natural killer T cells and CD4⁺CD25^high Forkhead box protein 3⁺ regulatory T cells in experimental infections with T. congolense.     

Increased Production of Interferon‐γ by Leishmania Homologue of the Mammalian Receptor for Activated C Kinase‐Reactive CD4+ T cells among Human Blood Mononuclear Cells: An Early Marker of Exposure to Leishmania?

A prospective study was undertaken to define early predictive immunological marker(s) of exposure to Leishmania in naïve subjects who have never been exposed to any Leishmania and who were also free of any cutaneous leishmaniasis lesions. These naïve subjects could have been exposed to Leishmania in a rain forest where Leishmania guyanensis and their natural vectors and mammalian host are cocirculating. The production of interferon (IFN)‐γ in response to the Leishmania homologue of the mammalian receptor for activated c kinase (LACK), a candidate for vaccine against leishmaniasis was analysed. At the end of their stay in the rain forest, LACK‐specific CD8⁺ T cells were detected in subjects whose peripheral blood mononuclear cell (PBMC) produced IFN‐γ in response to soluble Leishmania antigens (SLA) and in those whose PBMC remained unresponsive to SLA. However, LACK‐specific CD4⁺ T cells were detected only in PBMCs from individuals who became IFN‐γ responders to SLA. In subjects whose PBMC became positive to SLA, LACK‐reactive CD4⁺ T cells producing high level of IFN‐γ were detectable before the SLA‐reactive IFN‐γ producing CD4⁺ T cells, suggesting that the former readout assay could be used as an early predictive immunological marker of exposure to Leishmania in subjects who remained disease free.     

Immunohistochemical study of hepatic fibropoiesis associated with canine visceral leishmaniasis

Hepatic fibropoiesis has been confirmed in canine visceral leishmaniasis. In fibrotic disease, hepatic stellate cells (HSC) play an important role in fibropoiesis, undergoing activation by TGF‐β to acquire characteristics of myofibroblasts. These cells show extensive capacity for proliferation, motility, contractility, collagen synthesis and extracellular matrix component synthesis. The aim of this work was to identify markers of HSC activation in 10 symptomatic and 10 asymptomatic dogs naturally infected with Leishmania (Leishmania) infantum. Eight uninfected dogs were used as controls. Alpha‐actin (α‐SMA), vimentin and cytokeratin were investigated by immunohistochemistry as HSC markers. The cytokine TGF‐β in tissue was also evaluated by immunohistochemistry. All infected dogs showed higher numbers of reticular fibres than controls. Fibropoiesis found in infected dogs was always associated with the presence of parasites and chronic granulomatous hepatitis. Positive correlation was found among fibropoiesis, parasite tissue load and expression of α‐SMA. There was no correlation between fibropoiesis, vimentin and cytokeratin markers. The expression of cytokine TGF‐β was higher in infected dogs than in controls, but not significantly different between symptomatic and asymptomatic dogs. These results confirm previous work describing the intense hepatic fibropoiesis in dogs naturally infected with Leishmania infantum, but now associated them with overexpression of TGF‐β, where α‐SMA may be a superior marker for activated HSC cells in CVL.     

Cytokine gene expression in the tissues of dogs infected by Leishmania infantum

Canine leishmaniosis (CanL) caused by the protozoan parasite Leishmania infantum is a chronic systemic disease that is endemic in certain parts of the world. The domestic dog is the most important reservoir of L. infantum and is the main source of infection for other animals and for the human population. The aim of this study was to evaluate and compare the level of expression of genes encoding particular cytokines (interleukin [IL]-12, interferon [IFN]-γ, IL-2 and IL-4) in different tissues and organs of 53 adult dogs with or without clinical signs of leishmaniosis and after treatment for the disease. Asymptomatic dogs showed high expression of genes encoding IL-4 in blood leucocytes and of genes encoding IL-12 and IL-2 in lymph nodes. Blood leucocytes from symptomatic dogs had a mixed Th1 and Th2 cytokine gene expression profile, but lymph nodes from these animals had dominant IL-2 and IFN-γ gene expression, while bone marrow appeared to be unresponsive. The predominance of IL-4 gene expression in the blood of asymptomatic dogs may favour parasite replication, while the balance between Th1 and Th2 cytokine gene expression in the blood of symptomatic dogs may be important in reducing parasite replication and delaying the dissemination of Leishmania to other organs. The drugs used to treat CanL do not completely eliminate the parasite, so the high expression of the gene encoding IL-4 in blood leucocytes and the high expression of IL-12 and IL-4 mRNA in lymph nodes may reflect the persistence of residual Leishmania amastigotes. L. infantum appears able to regulate the host immune response in order to ensure its survival, but also to prevent the host from succumbing to infection. This guarantees its transmission and the completion of its life cycle.     

Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs

The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination.     

Dendritic cells in Leishmania major-immune mice harbor persistent parasites and mediate an antigen-specific T cell immune response

Upon infection with Leishmania major, a cause of human cutaneous leishmaniasis, mice of resistant strains are able to control the infection, with lesions resolving spontaneously. A long-lasting cell-mediated immunity protects them from reinfection. Nevertheless, small numbers of viable parasites persist in the lymph nodes of these mice. We have recently documented that, in addition to macrophages, epidermal Langerhans cells can ingest L. major. Furthermore, Langerhans cells have the unique ability to transport viable parasites from the infected skin to the draining lymph node for presentation to antigen-specific T cells and initiation of the cellular immune response. During migration, Langerhans cells develop into dendritic cells. In the present study, we analyzed whether dendritic cells support the persistence of parasites in immune hosts. Immunohistological studies and assays in vitro showed that in the lymph nodes of mice that have recovered from infection with L. major, both macrophages and dendritic cells harbor viable parasites. However, only dendritic cells were able to induce a vigorous T-cell immune response to L. major in vitro in the absence of exogenous antigen. Tracking experiments conducted in vivo suggested that the infected dendritic cells in the lymph nodes are derived from Langerhans cells that have emigrated from the skin. The data demonstrate that L. major-infected dendritic cells and macrophages in lymph nodes of immune animals represent long-term host cells, but only dendritic cells have the ability to present endogenous parasite antigen to T cells. Long-term infected dendritic cells may thus allow the sustained stimulation of a population of parasite-specific T cells, protecting the mice from reinfection. Our results favor the hypothesis that the persistence of antigen supports the maintenance of T cell memory and that dendritic cells are critically involved in this process.     

Evaluation of the influence of tissue parasite density on hematological and phenotypic cellular parameters of circulating leukocytes and splenocytes during ongoing canine visceral leishmaniasis

During Leishmania infection, tissue parasitism at different sites may differ and imply distinct immunopathological patterns during canine visceral leishmaniasis (CVL). For this reason, we have assessed by flow cytometry the impact of spleen and skin parasite density on the phenotypic profile of splenocytes and circulating leukocytes of 40 Brazilian dogs naturally infected by Leishmania chagasi categorized according to splenic and cutaneous parasite load. Our major statistically significant findings demonstrated that dogs with splenic high parasitism presented a significant decrease in absolute counts of CD5⁺ T lymphocytes in comparison with dogs presenting splenic medium parasitism. Moreover, a decrease in the absolute number of circulating monocytes was observed as a hallmark of high parasitism. The increased frequency of CD8⁺ T cells is associated with low splenic parasitism during CVL. Although we did not found any significant differences between the immunophenotypic analysis performed in circulating lymphocytes according to cutaneous parasite load, there were negative correlations between CD5⁺ and CD8⁺ T cells and cutaneous parasite density reemphasizes the role of T cell-mediated immune response in resistance mechanisms during ongoing CVL. These results add new insights about the pathogenesis of CVL and may help in the establishment of additional tools for future studies on drugs and vaccine approaches.     

Antigens targeted to the Leishmania phagolysosome are processed for CD4+ T cell recognition

Processing of antigen for recognition by class II-restricted CD4⁺ T cells occurs within acidic compartments of the antigen-presenting cell. The exact nature of this compartment has yet to be precisely defined, however, but may vary depending upon the cell type studied and the antigen used. The acidic compartments of macrophages are also responsible for the degradation of ingested micro-organisms and play host to others which are adapted to an intracellular existance. To determine whether the phagolysosome (PL) formed in activated macrophages after ingestion of Leishmania parasites is also a site for entry of antigen into the class II presentation pathway, we have used the approach of genetic transformation. Hence, Leishmania were transfected with the genes for the protein antigens ovalbumin (OVA) and β-galactosidase (β-gal) and after infection were able to deliver these antigens specifically into the PL. Delivery of antigen to this site resulted in the ability of infected macrophages to present these antigens to antigen-specific CD4⁺ T cells. After taking into account the absolute levels of antigen uptake by macrophages, a 4-h processing period for OVA delivered by this or a soluble route led to equivalent levels of T cell activation. Unlike macrophages pulsed with soluble OVA, those with PL-targeted OVA still retained the ability to stimulate T cells after a 24-h processing period. This enhanced lifespan of antigen in macrophages corresponded to the kinetics of degradation of the parasite, suggesting slow release of antigen into the processing pathway. β-gal presentation from the PL was tenfold less efficient under the same conditions. In addition to providing the first information on antigen processing in a protozoan PL, these studies highlight the usefulness of genetically transformed parasites for these types of studies.     

Characterization of the antigenic determinants of the Leishmania infantum histone H3 recognized by antibodies elicited during canine visceral leishmaniasis

In the present study we show that sera from dogs naturally infected with the protozoan parasite Leishmania infantum contain antibodies that specifically react with the parasite histone H3. Using synthetic peptides covering the complete sequence of the protein we located the linear antigenic determinants within the 40 amino-terminal amino acids of the molecule. In addition to the complete form of the protein (rLiH3), two regions of the Leishmania histone H3 were expressed as recombinant proteins: the rLiH3-Nt fragment containing the 39 amino-terminal amino acids and the rLiH3-Ct fragment containing the 90 carboxyl-terminal residues. Competition experiments using the protein fragment rLiH3-Nt as competitor confirmed that the antigenic determinants of histone H3 are confined to the amino-terminal domain. This domain, which is believed to be exposed on the nucleosome surface, is also the most evolutionarily divergent region of the L. infantum histone H3. Visceral leishmaniasis (VL) sera do not react with mammalian histones, an indication that the anti-histone response elicited during Leishmania infection is triggered by the parasite histone. The results of the prevalence of anti-histone H3 antibodies in canine VL sera together with the sequence-specific characteristics of the amino-terminal region of L. infantum histone H3 indicate that the recombinant protein rLiH3-Nt may be of use for diagnosis of canine VL.     

Specific lysis of Listeria monocytogenes-infected macrophages by class II-restricted L3T4+ T cells

Mice were infected with the intracellular bacterium, Listeria monocytogenes, and T cell clones from spleens, lymph nodes and peritoneal exudates were established. The capacity of L3T4⁺, Lyt2^? T-cell clones to specifically lyse L. monocytogenes-infected macrophages was analyzed. As a source of target cells, bone marrow macrophages (BMMΦ) after 9 days of culture in hydrophobic teflon bags were used. These BMMΦ were totally Ia^?; however, significant Ia-expression could be induced by interferon-γ (IFN-γ). IFN-γ-stimulated BMMΦ, after priming with live or killed L. monocytogenes organisms were effectively lysed by the vast majority of L3T4⁺ T cell clones. In the absence of either IFN-γ stimulation or antigen priming, no lysis occurred. Cytolysis was demonstrable in a conventional 4-h ⁵¹Cr-release assay and in an 18-h neutral red uptake assay and was antigen specific and class II restricted. Native T cells from L. monocytogenes-infected mice failed to lyse stimulated, L. monocytogenes-primed BMMΦ and gained their cytolytic activity after antigenic restimulation in vitro. These data demonstrate that L. monocytogenes-specific L3T4⁺ T cells could lyse MΦ presenting listerial antigens provided that Ia antigen expression had been induced. L3T4⁺ T cell clones produced IFN-γ after restimulation with antigen plus accessory cells in vitro and IFN-γ secretion could be increased by costimulation with recombinant IL 2. These T cell clones conferred significant protection upon recipient mice which was more pronounced in the liver. The possible relevance of lysis by L3T4⁺ T cells of infected MΦ to protection against and pathogenesis of intracellular bacterial infections is discussed.     

Fast high yield of pure Leishmania (Leishmania) infantum axenic amastigotes and their infectivity to mouse macrophages

Leishmania (L.) infantum (syn. Leishmania chagasi) is a dimorphic protozoan parasite that lives in promastigote and amastigote form in its sandfly vector and mammalian hosts, respectively. Here, we describe an in vitro culture system for the generation of a pure population of L. infantum axenic amastigotes after only 4 days incubation in culture medium supplemented with fetal calf serum, human urine, l-glutamine, and HEPES at 37oC (pH 5.5). Ultrastrutural analysis and infection assays in two macrophage populations (Kupffer cells (KUP) and peritoneal macrophages (PM)) infected with axenic amastigotes demonstrated that they maintained morphological and biochemical (A2 expression) features and a similar infection pattern to tissue-derived L. infantum amastigotes. The susceptibility of the macrophage lines to axenic or tissue-derived amastigotes and promastigotes was investigated. We found a completely different susceptibility profile for KUP and PM. Liver macrophages, both KUP and immigrant macrophages, are intimately involved in the response to L. infantum infection; this difference in susceptibility is probably related to their capacity to eliminate these parasites. Our in vitro system was thus able to generate axenic amastigotes that resemble tissue-derived amastigotes both in morphology and infectivity pattern; this will help in further investigation of the biological characteristics of the host–parasite relationship as well as the process of pathogenesis.