Regulation of Leishmania (L.) amazonensis Protein Expression by Host T Cell Dependent Responses: Differential Expression of Oligopeptidase B,Tryparedoxin Peroxidase and HSP70 Isoforms in Amastigotes Isolated from BALB/c and BALB/c Nude Mice

Leishmaniasis is an important disease that affects 12 million people in 88 countries, with 2 million new cases every year. Leishmania amazonensis is an important agent in Brazil, leading to clinical forms varying from localized (LCL) to diffuse cutaneous leishmaniasis (DCL). One interesting issue rarely analyzed is how host immune response affects Leishmania phenotype and virulence. Aiming to study the effect of host immune system on Leishmania proteins we compared proteomes of amastigotes isolated from BALB/c and BALB/c nude mice. The athymic nude mice may resemble patients with diffuse cutaneous leishmaniasis, considered T-cell hyposensitive or anergic to Leishmania´s antigens. This work is the first to compare modifications in amastigotes’ proteomes driven by host immune response. Among the 44 differentially expressed spots, there were proteins related to oxidative/nitrosative stress and proteases. Some correspond to known Leishmania virulence factors such as OPB and tryparedoxin peroxidase. Specific isoforms of these two proteins were increased in parasites from nude mice, suggesting that T cells probably restrain their posttranslational modifications in BALB/c mice. On the other hand, an isoform of HSP70 was increased in amastigotes from BALB/c mice. We believe our study may allow identification of potential virulence factors and ways of regulating their expression.     

Leishmania (Leishmania) amazonensis: experimental cutaneous leishmaniasis associated with systemic amyloidosis in mice

We infected Swiss and C57BL/6 female mice in the left hind footpad with 10⁴Leishmania (L.) amazonensis promastigotes in stationary phase. The macroscopic examination showed a nodular non-ulcerated lesion at the site of inoculation and hepatic and spleenic enlargement. Histopathologically, the primary lesion showed an extensive liquefactive necrosis and inflammatory infiltrate, mainly consisting of macrophages filled with amastigotes, and rare lymphocytes. The inflammatory reaction in liver, spleen and kidney showed amyloid deposits. Additionally, C57BL/6 had accentuated amyloidosis in both ovarian cortical and medullar region and inflammatory infiltrates in the pancreas and adrenal gland.     

Leishmania (Leishmania) infantum/chagasi: Histopathological aspects of the skin in naturally infected dogs in two endemic areas

In the New World, visceral leishmaniasis (VL), which is a progressive disease and frequently fatal, is caused by Leishmania (Leishmania) infantum/chagasi. It is endemic in many regions of Brazil and occasionally occurs in non-endemic regions when dogs from an endemic area are introduced. The aim of the present study is to compare different skin infection patterns of dogs from two leishmaniasis endemic areas. A histological analysis of dogs from Campo Grande, Mato Grosso do Sul state, a region where epidemic episodes are currently taking place, showed dermic inflammatory infiltrates, composed of numerous vacuolated parasitized macrophages, few lymphocytes, plasma cells and many degranulated mast cells. In the other region of the study, São Luís, Maranhão state, the skin of dogs presented a remarkable inflammatory reaction composed mainly of plasma cells, lymphocytes and very few parasites. We concluded that there is a difference in the skin lesion patterns of dogs with leishmaniasis that is directly related to the endemic area where the animals live.     

Leishmania tarentolae: Utility as an in vitro model for screening of antileishmanial agents

Primary screens for antileishmanial compounds use Leishmania species pathogenic to humans that must be handled under biosafety conditions that cannot be adopted or guaranteed everywhere. Leishmania tarentolae, a parasite isolated from the gecko Tarentolae annularis, has not been considered pathogenic to humans. Promastigotes of L. tarentolae have been previously used as a eukaryotic expression system for the production of recombinant proteins and in the amplification of genes involved in resistance to antileishmanial drugs. To validate the use of this Leishmania species in the screening of antileishmanial drugs, the sensitivity of axenic and intracellular amastigotes of L. tarentolae was compared to the sensitivity showed by Leishmania species causative of human leishmaniasis. The ability of L. tarentolae to grow as axenic amastigotes is first described while its ability to infect several mammalian cells has been confirmed. L. tarentolae amastigotes offer a suitable model for the in vitro screening of compounds for antileishmanial activity.     

Partial protective responses induced by a recombinant cysteine proteinase from Leishmania (Leishmania) amazonensis in a murine model of cutaneous leishmaniasis

A 500 bp fragment encoding an isoform of cysteine proteinase from Leishmania (Leishmania) amazonensis was subcloned and expressed in the pHis vector, resulting in a recombinant protein of 24 kDa, rLacys24. In Western blots of L. (L.) amazonensis extracts, antibodies directed to rLacys24 recognized a cysteine proteinase isoform of 30 kDa. Analysis by fluorescence-activated cell sorter showed a significantly higher expression of CD8⁺ lymphocytes in animals immunized with rLacys24 plus CFA, whereas a low expression of CD4⁺ lymphocytes was observed in these animals. The cytotoxicity of lymphocytes isolated from mice immunized with rLacys24 plus CFA on L. (L.) amazonensis-infected macrophages was significantly higher than that observed in the presence of lymphocytes from control animals. Immunization of BALB/c mice with rLacys24 plus CFA resulted in a low but significant decrease of foot lesions after challenge with L. (L.) amazonensis compared to those exhibited by control mice.     

Anti-leishmanial Effects of Trinitroglycerin in BALB/C Mice Infected with Leishmania major via Nitric Oxide Pathway

This study investigated whether trinitroglycerine (TNG) as nitric oxide (NO) releasing agent had anti-leishmanial effects and mediated pathology in BALB/c mice infected with Leishmania major. Cutaneous leishmaniasis (CL), a zoonotic infection caused by leishmania protozoa is still one of the health problems in the world and in Iran. NO is involved in host immune responses against intracellular L. major, and leishmania killing by macrophages is mediated by this substance. Moreover, application of CL treatment with NO-donors has been recently indicated. In our study, TNG was used for its ability to increase NO and to modify CL infection in mice, in order to evaluate NO effects on lesion size and formation, parasite proliferation inside macrophages, amastigote visceralization in target organs, and NO induction in plasma and organ suspensions. Data obtained in this study indicated that TNG increased plasma and liver-NO, reduced lesion sizes, removed amastigotes from lesions, livers, spleens, and lymph nodes, declined proliferation of amastigotes, hepatomegaly, and increased survival rate. However, TNG reduced spleen-NO and had no significant effects on spelenomegaly. The results show that TNG therapy reduced leishmaniasis and pathology in association with raised NO levels. TNG had some antiparasitic activity by reduction of positive smears from lesions, livers, spleens, and lymph nodes, which could emphasize the role of TNG to inhibit visceralization of L. major in target organs.     

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.     

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world''s most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly¹. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited ²;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance ³. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.In vitro promastigotes ⁴ and axenic amastigotes assays⁵ are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).     

The Route of Leishmania tropica Infection Determines Disease Outcome and Protection against Leishmania major in BALB/c Mice

Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.     

Leishmania mexicana: Immunology and histopathology in C3H mice

C3H mice were infected subcutaneously with 10⁵ promastigotes of Leishmania mexicana and subsequent lesions were examined at 3, 5, and 8 months. All animals developed persistent nonulcerating nodules of variable size which did not metastasize. The nodules contained amastigotes with a mononuclear infiltrate of histiocytes, lymphocytes, and plasma cells, but without formation of tuberculoid-type granulomas. Neutrophils and eosinophils were also encountered in some cases. Specific antileishmanial antibodies and delayed-type hypersensitivity to leishmanial antigen were present at 3, 5, and 8 months postinfection. L. mexicana infection in C3H mice differs from classic self-healing cutaneous leishmaniasis by the pesistence of nonhealing, nonulcerating, nonmetastasizing lesions, despite evidence of cellular and humoral immunity.     

Induction of autophagy correlates with increased parasite load of Leishmania amazonensis in BALB/c but not C57BL/6 macrophages

We investigated the role of autophagy in infection of macrophages by Leishmania amazonensis. Induction of autophagy by IFN-γ or starvation increased intracellular parasite load and the percentages of infected macrophages from BALB/c but not from C57BL/6 mice. In contrast, starvation did not affect the replication of either Leishmania major or Trypanosoma cruzi in BALB/c macrophages. In BALB/c macrophages, starvation resulted in increased monodansylcadaverine staining and in the appearance of double-membrane and myelin-like vesicles characteristic of autophagosomes. Increased parasite load was associated with a reduction in NO levels and was attenuated by wortmannin, an inhibitor of autophagy. In infected macrophages from BALB/c, but not from C57BL/6 mice, starvation increased the number of lipid bodies and the amounts of PGE₂ produced. Exogenous PGE₂ increased parasite load in macrophages from BALB/c, but not C57BL/6 mice. The cyclooxygenase inhibitor indomethacin prevented the increase of parasite load in starved BALB/c macrophages, and actually induced parasite killing. These results suggest that autophagy regulates the outcome of L. amazonensis infection in macrophages in a host strain specific manner.     

Influence of long-term treatment with pravastatin on the survival, evolution of cutaneous lesion and weight of animals infected by Leishmania amazonensis

The high toxicity of current drugs for treatment of leishmaniasis is a major hindrance for controlling the disease. Pravastatin is a well-known drug with anti-inflammatory and immunomodulatory properties that may modulate host defense mechanisms against Leishmania. We evaluated the influence of prolonged pravastatin treatment on the survival of Leishmania amazonensis-infected animals (BALB/c, C57BL6 mice and Syrian hamsters), including weekly measurement of cutaneous lesions (footpad thickness) and weight. Pravastatin improved survival of Leishmania-infected BALB/c mice but not of infected C57BL6 mice or hamsters. On the 50th week of follow-up, 71% of pravastatin-treated Leishmania-infected BALB/c mice were alive against 29% of control group (p < 0.01). Low footpad thickness was found on BALB/c pravastatin treated mice from the 14th week (p < 0.05), and 20th week onward for C57BL6 treated mice. Pravastatin treatment decreased weight loss in Leishmania-infected C57BL6 mice and Syrian hamsters, but not infected BALB/c mice. Our results points to beneficial effects of pravastatin on the evolution of the disease in the murine leishmaniasis model.     

Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

Neutrophils reduce the parasite burden in Leishmania (Leishmania) amazonensis-infected macrophages

Background

Studies on the role of neutrophils in Leishmania infection were mainly performed with L. (L) major, whereas less information is available for L. (L) amazonensis. Previous results from our laboratory showed a large infiltrate of neutrophils in the site of infection in a mouse strain resistant to L. (L.) amazonensis (C3H/HePas). In contrast, the susceptible strain (BALB/c) displayed a predominance of macrophages harboring a high number of amastigotes and very few neutrophils. These findings led us to investigate the interaction of inflammatory neutrophils with L. (L.) amazonensis-infected macrophages in vitro.

Methodology/Principal Findings

Mouse peritoneal macrophages infected with L. (L.) amazonensis were co-cultured with inflammatory neutrophils, and after four days, the infection was quantified microscopically. Data are representative of three experiments with similar results. The main findings were 1) intracellular parasites were efficiently destroyed in the co-cultures; 2) the leishmanicidal effect was similar when cells were obtained from mouse strains resistant (C3H/HePas) or susceptible (BALB/c) to L. (L.) amazonensis; 3) parasite destruction did not require contact between infected macrophages and neutrophils; 4) tumor necrosis factor alpha (TNF-α), neutrophil elastase and platelet activating factor (PAF) were involved with the leishmanicidal activity, and 5) destruction of the parasites did not depend on generation of oxygen or nitrogen radicals, indicating that parasite clearance did not involve the classical pathway of macrophage activation by TNF-α, as reported for other Leishmania species.

Conclusions/Significance

The present results provide evidence that neutrophils in concert with macrophages play a previously unrecognized leishmanicidal effect on L. (L.) amazonensis. We believe these findings may help to understand the mechanisms involved in innate immunity in cutaneous infection by this Leishmania species.     

Experimental Murine Fascioliasis Derives Early Immune Suppression with Increased Levels of TGF-�� and IL-4

In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19⁺ B cells was observed as early as week 1 post-infection while CD4⁺/CD8⁺ T cells were down-regulated. Accumulation of Mac1⁺ cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-α mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1β expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-β were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-β and IL-4 during the early stages of infection.     

Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania) amazonensis,but Not by Leishmania (Viannia) guyanensis

Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.     

Leishmania major: Disruption of signal peptidase type I and its consequences on survival, growth and infectivity

Leishmania major (L. major) signal peptidase type I (SPase I) is an endopeptidase encoded by a single-copy gene. In all organisms, SPase I is responsible for removing the signal peptide from secretory pre-proteins and releasing mature proteins to cellular or extra-cellular space. In this study, the role of SPase I in L. major is investigated by gene deletion using homologous recombination (HR). The null mutant of SPase I was not possible to create, suggesting that SPase I is an essential gene for parasite survival.The obtained heterozygote mutant by disrupting one allele of SPase I in L. major showed significantly reduced level of infectivity in bone marrow-derived macrophages. In addition, the heterozygote mutants are unable to cause cutaneous lesion in susceptible BALB/c mice. This is the first report showing that SPase I may have an important role in Leishmania infectivity, e.g. in differentiation and survival of amastigotes. Apparently, the SPase I expression is not essential for in vitro growth of the parasite.