Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response.

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 106 cells per well on 24-well plates in order to obtain 2 x 105 macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy.

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3). Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Avaliação da infectividade parasitária a Lutzomyia longipalpis por xenodiagnóstico em cães tratados para leishmaniose visceral naturalmente adquirida / Evaluation of parasite infectivity for Lutzomyia longipalpis by xenodiagnosis in dogs treated for natural visceral leishmaniasis

O efeito de um protocolo quimioterápico multidrogas contra a leishmaniose visceral (LV) canina, sobre a capacidade de transmissão de Leishmania infantum ao vetor, foi analisado por meio de xenodiagnóstico. Trinta e cinco cães naturalmente infectados foram avaliados antes e durante o tratamento com a combinação de metronidazol, cetoconazol e alopurinol a cada três meses por até um ano. Em cada avaliação, os cães foram individualmente submetidos ao xenodiagnóstico e quantificação da carga parasitária por PCR quantitativa. O tratamento foi eficaz em bloquear a transmissibilidade parasitária do cão para o flebotomíneo (p= 0,011) nos cães avaliados. Houve significante correlação entre recuperação clínica e infectividade: cães com melhora clínica mais evidente apresentaram menores chances de transferir L. infantum ao Lutzomyia longipalpis via xenodiagnóstico (r=0,528, p= 0,002). Esses resultados demonstram que o tratamento canino com o protocolo proposto pode representar uma alternativa ao sacrifício de cães no Brasil como medida de controle da doença, uma vez que as drogas utilizadas não são aplicadas ao tratamento da LV humana em áreas endêmicas.(AU)

The outcome of a multidrug chemotherapeutic protocol against canine visceral leishmaniasis (VL) has been evaluated for its effect on dogs' capacity of transferring Leishmania infantum to sand flies by xenodiagnosis. Thirty-five naturally infected dogs were examined before and during treatment with a combination of metronidazole, ketoconazole, and allopurinol, at every three months up to one year. For each evaluation, treated dogs were individually submitted to xenodiagnosis and quantitative PCR to quantify parasite load in sand flies. The treatment was effective in blocking parasite transmission from host to sand flies (p=0.011) in the assessed dogs. There was a significant correlation between clinical improvement and sand fly infectivity: dogs that achieved better clinical conditions showed a lower chance of L. infantum transference to vector by xenodiagnosis (r=0.528, p=0.002). These results demonstrate that the treatment of dogs with the proposed protocol may represent an alternative to dog culling in Brazil for disease control, since these drugs are not used for treating human VL in endemic areas.(AU)

Chemotherapeutic potential of 17-AAG against cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis.

BACKGROUND: Leishmaniasis remains a worldwide public health problem. The limited therapeutic options, drug toxicity and reports of resistance, reinforce the need for the development of new treatment options. Previously, we showed that 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), a Heat Shock Protein 90 (HSP90)-specific inhibitor, reduces L. (L.) amazonensis infection in vitro. Herein, we expand the current knowledge on the leishmanicidal activity of 17-AAG against cutaneous leishmaniasis, employing an experimental model of infection with L. (V.) braziliensis. METHODOLOGY/PRINCIPAL FINDINGS: Exposure of axenic L. (V.) braziliensis promastigotes to 17-AAG resulted in direct dose-dependent parasite killing. These results were extended to L. (V.) braziliensis-infected macrophages, an effect that was dissociated from the production of nitric oxide (NO), superoxide (O(-2)) or inflammatory mediators such as TNF-α, IL-6 and MCP-1. The leishmanicidal effect was then demonstrated in vivo, employing BALB/c mice infected with L. braziliensis. In this model, 17-AAG treatment resulted in smaller skin lesions and parasite counts were also significantly reduced. Lastly, 17-AAG showed a similar effect to amphotericin B regarding the ability to reduce parasite viability. CONCLUSION/SIGNIFICANCE: 17-AAG effectively inhibited the growth of L. braziliensis, both in vitro and in vivo. Given the chronicity of L. (V.) braziliensis infection and its association with mucocutaneous leishmaniasis, 17-AAG can be envisaged as a new chemotherapeutic alternative for cutaneous Leishmaniasis.

Extracellular vesicles from Leishmania-infected macrophages confer an anti-infection cytokine-production profile to naïve macrophages.

BACKGROUND: Extracellular vesicles (EVs) are structures with phospholipid bilayer membranes and 100-1000 nm diameters. These vesicles are released from cells upon activation of surface receptors and/or apoptosis. The production of EVs by dendritic cells, mast cells, macrophages, and B and T lymphocytes has been extensively reported in the literature. EVs may express MHC class II and other membrane surface molecules and carry antigens. The aim of this study was to investigate the role of EVs from Leishmania-infected macrophages as immune modulatory particles. METHODOLOGY/PRINCIPAL FINDINGS: In this work it was shown that BALB/c mouse bone marrow-derived macrophages, either infected in vitro with Leishmania amazonensis or left uninfected, release comparable amounts of 50-300 nm-diameter extracellular vesicles (EVs). The EVs were characterized by flow cytometry and electron microscopy. The incubation of naïve macrophages with these EVs for 48 hours led to a statistically significant increase in the production of the cytokines IL-12, IL-1ß, and TNF-α. CONCLUSIONS/SIGNIFICANCE: EVs derived from macrophages infected with L. amazonensis induce other macrophages, which in vivo could be bystander cells, to produce the proinflammatory cytokines IL-12, IL-1ß and TNF-α. This could contribute both to modulate the immune system in favor of a Th1 immune response and to the elimination of the Leishmania, leading, therefore, to the control the infection.

A comparison of two distinct murine macrophage gene expression profiles in response to Leishmania amazonensis infection.

BACKGROUND: The experimental murine model of leishmaniasis has been widely used to characterize the immune response against Leishmania. CBA mice develop severe lesions, while C57BL/6 present small chronic lesions under L. amazonensis infection. Employing a transcriptomic approach combined with biological network analysis, the gene expression profiles of C57BL/6 and CBA macrophages, before and after L. amazonensis infection in vitro, were compared. These strains were selected due to their different degrees of susceptibility to this parasite. RESULTS: The genes expressed by C57BL/6 and CBA macrophages, before and after infection, differ greatly, both with respect to absolute number as well as cell function. Uninfected C57BL/6 macrophages express genes involved in the deactivation pathway of macrophages at lower levels, while genes related to the activation of the host immune inflammatory response, including apoptosis and phagocytosis, have elevated expression levels. Several genes that participate in the apoptosis process were also observed to be up-regulated in C57BL/6 macrophages infected with L. amazonensis, which is very likely related to the capacity of these cells to control parasite infection. By contrast, genes involved in lipid metabolism were found to be up-regulated in CBA macrophages in response to infection, which supports the notion that L. amazonensis probably modulates parasitophorous vacuoles in order to survive and multiply in host cells. CONCLUSION: The transcriptomic profiles of C57BL/6 macrophages, before and after infection, were shown to be involved in the macrophage pathway of activation, which may aid in the control of L. amazonensis infection, in contrast to the profiles of CBA cells.

Immune and inflammatory responses to Leishmania amazonensis isolated from different clinical forms of human leishmaniasis in CBA mice.

Leishmania amazonensis causes different diseases depending on the host and parasitic virulence factors. In this study, CBA mice were infected with L. amazonensis isolates from patients with localized (Ba125), diffuse cutaneous (Ba276) or visceral leishmaniasis (Ba109). Mice infected with Ba125 and Ba276 progressed rapidly and lesions displayed an infiltrate rich in parasitized macrophages and were necrotic and ulcerated. Ba109 induced smaller lesions and a mixed inflammatory infiltrate without necrosis or ulceration. Ba109 induced an insidious disease with lower parasite load in CBA mice, similar to human disease. Levels of IFN-γ, IL-4 and IL-10 did not differ among the groups. Because all groups were unable to control the infection, expression of IL-4 associated with low production of IFN-γ in the early phase of infection may account for susceptibility, but others factors may contribute to the differences observed in inflammatory responses and infection progression. Evaluation of some parasitic virulence factors revealed that Ba276 exhibits higher ecto-ADPase and 5'-nucleotidase activities compared to the Ba109 and Ba125 strains. Both Ba276 and Ba125 had higher arginase activity in comparison to Ba109. Finally, these data suggest that the differences in enzyme activities among parasites can account for differences in host inflammatory responses and infection progression.

Phagolysosomes induits par Leishmania amazonensis et Coxiella burnetii: modèle pour l´étude de fusion entre grandes vacuoles de pahgocytose / Phagolysosomes induced by Leishmania amazonensis and Coxiella burnetii: a model for the study of merger between large vacuoles of pahgocytose

Actuellement, les connaissances sur l'interaction entre grandes vacuoles de phagocytose sont tres limitées. Il est important de signaler, les travaux pionniers sur la fusion entre les vacuoles contenant différents types des particu1es chez les Acantamcebas et les études sur l'interaction entre les phagosomes contenant le Staphylococcus aureus et les endosomes. L'objectif du travail ici présenté était d'étudier l'interaction entre grandes vacuoles de phagocytose en utilisant les cellules intactes de mammifere, les macrophages ou les cellules CHO ("chinese hamster ovary"). Nous avons utilisé comme vacuoles réceptrices deux types de phagolysosomes, la vacuole parasitophore induite par le parasite Leishmania amazonensis et celle induite par la bactérie Coxiella burnetii (vacuole de Coxiella). Ces grands phagolysosomes ont été choisis parce qu'elles sont facilement repérable au microscope optique et partagent entre elles des caractéristiques similaires. La vacuole parasitophore et la vacuole de Coxiella sont addifiées, contiennent des enzymes hydrolytiques, et il est connu que, les deux vacuoles se fusionnent avec les compartiments tardifs d'endocytose. Dans um premier temps, les vacuoles contenant les particu1es inertes, comme celles dérivées de la levure, les billes de latex ainsi que, les globules rouges fixés ou opsonisés, ont été utilisées comme les vacuoles donatrices. Nous avons démontré que les particu1es dérivées de la levure, le zymosan ou la levure tuée, étaient sélectivement transférées aux vacuoles parasitophores, puisque les billes de latex ou les globules rouges également phagocytés par les macrophages, étaient exc1us de ces vacuoles. D'abord, nous avons établi une méthode en pulse-chasse pour étudier le transfert de particules zymosan aux vacuoles parasitophores dans les macrophages infectés par L. amazonensis. Nous avons démontré que le transfert était vectoriel et quantal. Les études pharmacologiques ont montré que l'alcalinisation, par des bases faibles ou l'ionophore monensine, augmentait le transfert. Nous avons également démontré que la toxine cholérique augmentait le transfert probablement par des mécanismes, au moins en partie, dépendants de sa sous-unité B et indépendants de l' AMPc intracellulaire. Nous avons alors montré que la sous-unité B purifiée ou recombinante stimulait le transfert et que d'autres molécu1es qui augmentent l'AMPc intracellulaire, comme les inhibiteurs de phosphodiesterases, la forskoline ou le Br-AMPc réduisaient le transfert. Deuxiemement, nous avons comparé la capadté de fusion entre les vacuoles induites par L. amazonensis ou par C. burnetii dans les cellules CHO, et les vacuoles contenant différents particu1es inertes...