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Microbes and Infection xx (2014) 1e9 www.elsevier.com/locate/micinf
Original article
Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages Camila I. Morato a, Ildefonso A. da Silva Jr. a, Arissa F. Borges a, Miriam L. Dorta a, Milton A.P. Oliveira a, Sonia Jancar b, Carlos H. Serezani c, Fatima Ribeiro-Dias a,*
Q4
a
Tropical Pathology and Public Health Institute, Federal University of Goias, Goi^ania, Goias, Brazil b Institute of Biomedical Sciences, University of S~ao Paulo, S~ao Paulo, Brazil c Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA Received 1 March 2014; accepted 27 August 2014
Abstract Although Leishmania (Viannia) braziliensis is the most prevalent species that cause American tegumentary leishmaniasis (ATL), the immune response against this parasite has been poorly investigated. Upon activation, macrophages produce a series of pro-inflammatory molecules, including the lipid mediator leukotriene B4 (LTB4). LTB4 has been shown to enhance several macrophage functions, but its role in human macrophages is less known. Here, we investigated the role of LTB4 on human monocyte-derived macrophages infected with human isolate of L. (V.) braziliensis (IMG3). It was found that human macrophages produce LTB4 upon infection with Leishmania, which by autocrine or paracrine activation of its high affinity receptor BLT1, potentiates macrophage leishmanicidal activity. This LTB4 effect is mediated by increased secretion of reactive oxygen species (ROS). Moreover, Leishmania infection decreased the expression of BLT1, leading to the speculation that this could represent a parasite escape mechanism to establish a chronic inflammatory infection. Therefore, our data suggest that LTB4 could be used in therapeutic strategies to control Leishmania infection. © 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur.
Keywords: Leishmania (V.) braziliensis; LTB4; Human; Macrophage; ROS
1. Introduction American tegumentary leishmaniasis (ATL) is a disease caused by a protozoa parasite of the genus Leishmania, characterized by a broad clinical spectrum ranging from localized or disseminated cutaneous lesions and mucosal lesions affecting oral, nasal and pharynx mucosa. Several species can cause ATL, but in South America, the most prevalent species is Leishmania (Viannia) braziliensis [1,2]. Although
* Corresponding author. Setor de Imunologia, Instituto de Patologia Tropical e Sau´de Pu´blica, Universidade Federal de Goias, Rua 235 S/N, Setor Universitario, Goi^ania 74605-050, GO, Brazil. Tel.: þ55 62 3209 6116; fax: þ55 62 3209 6363. E-mail addresses: [email protected], [email protected] (F. RibeiroDias).
the immune response during ATL has been extensively investigated [3,4], the mechanisms underlying the pathogenesis of the disease remain to be understood. It is well established that macrophages bear the parasites, but the biology of human macrophages in leishmaniasis is still incompletely understood. In particular, in the case of L. (V.) braziliensis, it was shown that human macrophages are able to control the infection via cytokine/chemokine production [5]. Lipid mediators such as leukotrienes (LTs), and LTB4 in particular, are essential in the control of infections by several pathogens [6e13]. Leukotrienes are eicosanoids derived from the 5-lipoxygenase metabolism of arachidonic acid (AA) to form LTB4 and the cysteinyl LTs, LTC4, LTD4 and LTE4. Both classes of LTs act on their respective G protein-coupled receptors, BLT1 or BLT2 and CysLT1 or CysLT2 [14]. The role of LTs in leishmaniasis has been investigated in in vivo and
http://dx.doi.org/10.1016/j.micinf.2014.08.015 1286-4579/© 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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in vitro murine models of infection caused by Leishmania donovani or Leishmania (L.) amazonensis [9,15]. We have demonstrated that LTB4 production is associated with patterns of resistance and susceptibility in a murine model of L. (L.) amazonensis infection, suggesting that LTB4 can be an important endogenous mediator involved in the control of ATL [9]. Recently, Lefevre et al. [16] demonstrated that LTB4 is produced by human monocyte-derived macrophages infected with Leishmania infantum, which causes visceral leishmaniasis. Studies concerning the production and actions of LTs either in patients with ATL or in human macrophages infected with L. (V.) braziliensis are lacking. Here, we sought to investigate the role of LTB4 in the control of L. (V.) braziliensis infection in human macrophages. To this purpose we first investigated the infection pattern of human monocytederived macrophages infected with a human isolate of L. (V.) braziliensis (IMG3). Then we found that LTB4 is produced by the infected macrophages, which also express the receptor BLT1. Studies using selective receptor antagonists and inhibitors of LT synthesis showed that LTB4 is a critical mediator involved in human leishmanicidal activity by enhancing the generation of ROS.
2.3. Human macrophage differentiation and culture All protocols were approved by the Ethics Committee of the Hospital das Clínicas/UFG (n. 190/2011). Venous blood samples (~10 ml) were obtained from healthy blood donors (n ¼ 77) and collected in vacuum tubes (Greiner bio-one, Vacuette) containing EDTA solution. Blood was centrifuged at 200 g for 5 min, and the plasma was discarded. The pellet was diluted (v/v) in phosphate-buffered saline (PBS) containing 0.01 mM EDTA, pH 7.3 and layered on Percoll gradient (GE Healthcare). Mononuclear cells (MNC) were suspended in RPMI 1640 medium supplemented with 15% FCS, 11 mM sodium bicarbonate, 2 mM L-glutamine, 100 U/ ml penicillin and 100 mg/ml streptomycin (complete RPMI medium). Cells (2 105) were distributed into 24-well plates (TPP, Techno Plastic Products) on 13-mm round glass coverslips (Deckglaser). Complete RPMI medium was replaced every 48 h for 5 days of culture (36 C, 5% CO2), allowing non-adherent cells to be removed and adherent monocytes to differentiate into macrophages. 2.4. Infection of monocyte-derived macrophages and quantification of parasite burden
2. Materials and methods 2.1. Reagents Cell culture reagents were from SigmaeAldrich (St. Louis, USA), unless otherwise stated. Fetal calf serum (FCS) was purchased from Cripion Biotechnology to be used with parasites, and from Gibco Life Technology for using with the cells. The FLAP inhibitor MK0591 was from Merck KgaA; the BLT1 antagonist, CP-105, 696 was from Pfizer. The LTB4 and LTB4 enzyme immunoassay (EIA) kits were from Cayman Chemical Co., and the antibodies against BLT1 were purchased from Enzo Life Science. The peroxidase-conjugated secondary antibody (anti-rabbit-HRP conjugate) and chemiluminescence system were from Santa Cruz Biotechnology, and the Trizol reagent was from Invitrogen. The Revert Aid TM First Strand cDNA Synthesis kit was from Thermo Scientific. 2.2. Parasite culture Throughout this study we employed the human clinical isolate MHOM/BR/2003/IMG L. (V.) braziliensis (IMG3) that was well characterized by us [17]. The isolate was obtained from a patient with localized cutaneous leishmaniasis and stored in the Leishbank (Central West's Bank of Leishmania sp., Federal University of Goias (UFG), Brazil). Parasites were cultured in Grace's Insect Medium supplemented with 20% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin, at 26 C. Passages of parasites were performed every three days, always starting the culture with 5 105 parasites/ml, up to a maximum of six times. To infect macrophages, promastigotes were used on the 6th day of growth (stationary phase).
Concentrations of 1 108 promastigotes/ml were suspended in complete RPMI medium and added to macrophages (2 106/well). Considering that monocytes represent ~5e20% of MNC, the MOI varied between ~50:1 to 200:1 (parasites: macrophage). After parasite addition plates were not centrifuged to synchronize the phagocytosis. Cultures were incubated for 4 h (36 C, 5% CO2), washed three times with warmed complete RPMI medium to remove extracellular parasites, and the coverslips were then removed to stain the cells and analyze the parasite burden at the first 4 h. In another wells, complete RPMI medium was added again, cultures were re-incubated and coverslips harvested at different time points (24 h, 48 h, 72 h). Cells were fixed with methanol, stained with the commercial Instant Prov kit (Newprov) and analyzed under light microscopy (1000; Nikon) to determine the parasite burden. The percentage of infected cells (300 cells counted in random fields) and the mean number of intracellular amastigotes per infected cell (at least 50 infected cells were counted) were obtained by blinded counts. The parasite burden was calculated as: Parasites/ 100 macrophages ¼ percentage of infected cells mean number of parasites per infected cell. All experiments were done in duplicate. 2.5. Pharmacological treatment of monocyte-derived macrophages Inhibition of leukotriene biosynthesis or actions was achieved by treating macrophages with the 5-lipoxygenase-activating protein (FLAP) inhibitor MK0591 or the leukotriene B4 receptor 1 antagonist (BLT1) CP-105, 696, respectively. The compounds (103 M) were dissolved in DMSO (vehicle) and stored at 80 C until use. For the experiments, compounds
Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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were diluted in complete RPMI medium and added to the macrophages in different concentrations, 30 min before infection. The vehicle control was added at the same final concentration used in the experimental compounds. The compounds were added back or not after 4 h of incubation (when cultures were washed to eliminate extracellular parasites), as indicated in the figure legends. After 4 h or 48 h of incubation, coverslips were removed, and the parasite burden was evaluated as described above. In another set of experiments, macrophages were treated or not with LTB4, dissolved in ethanol (3 104 M) and further diluted in PBS. We have done three different treatment protocols: 1) macrophages were stimulated with LTB4 plus parasites; 2) LTB4 was added 4 h after the infection; and 3) LTB4 was added 24 h after the infection. In all circumstances, coverslips were harvested 48 h after the initial 4 h of infection. Vehicle control (ethanol) was added in all time points tested. None of the compounds or vehicles (MK0591, CP-105,696, DMSO and ethanol) was toxic to cells or parasites (data not shown). A MTT assay was used to evaluate cytotoxicity [18]. To check the activity of MK0591 (106 M), macrophages were incubated in absence or presence of MK0591 for 30 min before parasite addition. After 30 min, LTB4 was measured as described below (at 2.6), showing that MK0591 was inhibiting LTB4 production. To check the ability of CP-105,696 to block BLT1 receptors, macrophages were incubated in the presence or absence of CP-105,696 (106 M) before parasite and LTB4 addition. Results showed that CP-105,696 blocked the macrophage leishmanicidal activity induced by LTB4 (Supplementary Fig. 1). 2.6. Measurement of LTB4 As the amount of adherent cells in each MNC preparation is variable and the number of macrophages is low, to measure LTB4 production after 4 days of culture (2 107 MNC/well, 6-well plates) adherent cells were scrapped from the plates, counted and then plated again (1 106/well). After 24 h, macrophages were incubated or not with MK059, followed by promastigote parasites (MOI: 10 parasites:1 macrophage) and after 0.5 h or 4 h, the supernatants were collected. LTB4 was determined by enzyme immunoassay (EIA) kits according to manufacturer's instructions. The results were expressed in pg/ mL, and the detection limit was 13 pg/mL. 2.7. Quantification of BLT1 expression Human macrophages were scrapped from the plates (2 107 MNC/well, 6-well plates), counted and then plated again (107 cells/well, 6-well plates). They were infected as described above (MOI: 10:1), and BLT1 expression was evaluated by real-time polymerase chain reaction (qPCR, 6 h) and Western blotting (24 h), as previously described [19]. After 6 h of macrophageeparasite interaction, macrophages were washed 3 with warmed PBS (37 C) and RNA was extracted using Trizol. cDNA synthesis was performed with the Revert Aid TM First Strand cDNA Synthesis kit, according
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to the manufacturer's instructions. Human BLT1 specific primers (sense primer 50 - TATGTCTGCGGAGTCAGCATGTACGC-30 and antisense 50 CCTGTAGCCGACG CCCTATGTCCG-30 ) and the housekeeping gene 50 -GAPDH (GAGTCAACGGAT TTGGTCGT-30 sense primer, antisense 50 -TTGATTTTG GAGGGATCTCG-30 ) were used. qPCR was performed using the Stratagene cycler Mx3005PTM QPCR Systems according to the manufacturer's instructions. Gene expression was analyzed by 2-DDCT method and expressed as the percentage of uninfected macrophages. For immunoblotting experiments, infected macrophages were washed 3 with warmed PBS and lysed, and protein content was measured using a BCA kit (Pierce). Proteins (20 mg) were separated in 10% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After blocking, membranes were incubated with anti-BLT1 followed by peroxidase-conjugated secondary antibody (anti-rabbit-HRP conjugate) for 2 h at room temperature. Visualization was performed with chemiluminescence system. Densitometric analysis was performed, and the intensity of the protein band was divided by that of the bactin. This ratio was expressed relative to that of the uninfected macrophages (100%). 2.8. Measurement of ROS To measure ROS production, we employed the NBT (nitro blue tetrazolium) assay, adapted from Muniz-Junqueira & Paula-Coelho [20]. Briefly, macrophages were placed in glass coverslips and infected with parasites. After 1 h, macrophages were incubated with Tris-Hanks containing 0.05% NBT for 20 min at 36 C, 5% CO2. Coverslips were washed, fixed with methanol, and stained with a solution of 1.4% safranin and 28.6% glycerol in distilled water. The percentage of ROSpositive macrophages was evaluated microscopically. In one protocol, the percentage of macrophages that were strongly positive (dark blue staining) among ROS-positive cells were counted. 2.9. Inhibition of ROS production To inhibit ROS production, macrophages were treated with nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin and dissolved in DMSO (0.5 M) before use. The inhibitor was further diluted in complete RPMI medium and added to cultures at different concentrations, 30 min before LTB4 (107 M) treatment and infection with promastigotes. Parasite burden was determined as mentioned above. 2.10. Statistical analysis Data are presented as median [minimum and maximum] and interquartile values. The ManneWhitney and Wilcoxon matched pair tests were used, and the level of significance was set at p < 0.05. The analyses were performed using GraphPad Prism 5.0 Software (San Diego, CA, USA).
Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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3. Results 3.1. Leukotriene B4 is generated by human macrophages infected with L. (V.) braziliensis potentiating macrophages leishmanicidal activity Initially, we investigated the infection pattern of the human isolate of L. (V.) braziliensis (IMG3) in human monocytederived macrophages. Our data showed that macrophages were susceptible to IMG3 L. (V.) braziliensis infection. Parasites were detected in parasitophorous vacuoles at all time points tested. Fig. 1A shows intracellular amastigotes in macrophages 48 h after infection. Even though we observed high individual variability, there was a significant increase in the parasite burden from 4 h to 48 h (Fig. 1B). Since LTB4 has been previously shown to increase leishmanicidal activity in murine macrophages through its high affinity receptor BLT1 [9], we first investigated whether human macrophages express
this receptor. We found that they did express BLT1 mRNA and protein (Fig. 1C and D, respectively). Fig. 1 also shows that Leishmania infection down regulates BLT1 expression (52% inhibition of protein expression; Fig 1D). Next, we measured LTB4 in the supernatant of the infected macrophages and found that Leishmania infection induces LTB4 production already after 30 min of infection, and it remains elevated at 4 h (Fig. 1E). To determine the role of endogenously produced leukotrienes on L. (V.) braziliensis infection macrophages were pretreated with the FLAP inhibitor MK0591 before infection. This compound was effective to inhibit leukotriene production at 106 M concentration (118.0 [9.8e220.1] pg/mL vs. 541.0 [360.0e653.0] pg/mL, 30 min, n ¼ 8; p < 0.05). Fig. 2A shows that 106 M MK0591 significantly increased the parasite burden at 48 h of infection. Moreover, treatment with the selective antagonist of LTB4 receptor BLT1, the compound CP-105,696, at 106 M concentration also increased the
Fig. 1. L. (V.) braziliensis induces release of LTB4 and modulates BLT1 expression in human macrophages. Monocyte-derived macrophages (2 105 MNC) were cultured for 4 h with promastigotes (2 106 parasites; 6th day of culture). After washing out the excess of parasites (4 h), cultures were incubated for 48 h (36 C). In A, photomicrography showing infected macrophages and amastigotes are indicated (arrow; bar represents 10 mm). B, The number of parasites per 100 macrophages; each line represents an individual assessed in experiments performed in duplicate (n ¼ 19 individuals in 5 independent experiments); horizontal broken lines are the medians, *p < 0.05. In C and D, Macrophages (1 107) were incubated in the absence (Control) or presence of parasites (1 108) for 6 h to evaluate mRNA BLT1 expression by qRT-PCR (C, expression of mRNA BLT1/GAPDH relative to uninfected macrophages) or 24 h to assess BLT1 expression by using Western blotting (D, arbitrary units of the ratio between the optical densities obtained for BLT1/b-actin relative to uninfected macrophages). *p < 0.05 (medians and interquartile from 9 independent experiments in duplicates). E, Macrophages (1 106) were incubated with parasites (1 107), and after 0.5 h or 4 h supernatants were assayed to LTB4 concentration by using EIA. Control: uninfected macrophages. *p < 0.05 (vs. control), #p < 0.05 (vs. 30 min). E, Data represent median, interquartile and the minimal and maximal values (n ¼ 6 individuals in 2 independent experiments). Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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parasite burden (Fig. 2B). The BLT1 antagonist increased both the percentage of infected macrophages (61.5% [51.0e75.3%] vs. 56.0% [25.5e76.0%], n ¼ 9; p < 0.05) and the number of parasites per cell (7.1% [3.6e11.2%] vs. 4.8% [1.5e5.9%], n ¼ 9; p < 0.05). The addition of LTB4 to macrophage cultures together parasites had similar effects compared with the endogenously produced LTB4. Fig. 2C shows the parasite load after 48 h of infection and LTB4 treatment during the first 4 h. LTB4 decreased dose-dependently the parasite burden in infected macrophages. Both the percentage of infected macrophages (48.0% [17.0e58.0%] vs. 72.4% [29.6e95.0%], n ¼ 18; p < 0.05) and number of parasites per cell (3.9% [1.7e8.7%] vs. 6.8% [3.1e16.7%], n ¼ 18; p < 0.05) were decreased by LTB4 treatment. CP-105,696 at 106 M was effective to block LTB4 effects (Supplementary Fig. 1). Next, we investigated whether LTB4 would also increase leishmanicidal activity when infection is already established. Macrophages were infected with promastigotes and simultaneously treated with 107 M of LTB4 (Fig. 3A), or treated 4 h (Fig. 3B) or 24 h (Fig. 3C) after parasite addition. The parasite load was determined after 48 h. While the addition of LTB4 at the same time of parasites or 4 h after significantly decreased the parasite load (Fig. 3A, B) LTB4 caused no significant change when it was added 24 h after parasites. These results allow the conclusion that human macrophages produce LTB4 upon infection with Leishmania, which by autocrine or paracrine activation of the BLT1 receptor potentiates the leishmanicidal activity of human macrophages. 3.2. ROS generation is involved in LTB4-enhanced leishmanicidal activity of human macrophages
Fig. 2. LTB4 decreases the infection of human macrophages with Leishmania (V.) braziliensis. A and B, Monocyte-derived macrophages were treated 30 min before infection with different concentrations of MK0591 (A) or CP-105,696 (B), infected for 4 h, washed, then re-incubated for 48 h; Control: vehicle treated macrophages (A, B, C). Data represent median, interquartile and the minimal and maximal values (n ¼ 8 (A), n ¼ 9 (B) individuals in 3 independent experiments), *p < 0.05. C, Macrophages were incubated with parasites for 4 h in the absence (Control) or presence of LTB4 in different concentrations. After washing, medium was replaced and cultures were incubated until 48 h. Data represent median, interquartile and the minimal and maximal values (n ¼ 18 individuals in 6 independent experiments), *p < 0.05 (vs. Control).
Since human macrophages infected with L. (V.) braziliensis produce superoxide [5] and LTB4 has been shown to increase ROS production [21,22], we measured ROS in human macrophages infected with L. (V.) braziliensis and the involvement of LTB4 in ROS production. We found that human macrophages produce ROS during infection with Leishmania (Fig. 4). The addition of LTB4 to the infected macrophages potentiated ROS production (Fig. 4). This was further confirmed when macrophages showing strong NBT reactions (dark blue precipitates) were counted among the NBT-positive cells. This semi-quantitative evaluation revealed a higher frequency of cells with strong ROS reactions in LTB4-treated macrophages than in untreated cells (Control vs. Leishmania vs. Leishmania plus LTB4: 0% vs. 1.45% [0%e7.9%] vs. 9.95% [1.6%e25.5%], p ¼ 0.06, n ¼ 6). Inhibition of LT synthesis by MK0591 or blocking of BLT1 by CP-105,696 both significantly reduced ROS production by infected macrophages (Fig. 4). The effect of exogenous LTB4 was blocked by CP-105,696 (Fig. 4). Leishmania-induced ROS production was inhibited by 104 M of the NADPH oxidase inhibitor, apocynin (Fig. 4) and in this situation, the macrophage infection markedly increased (Fig. 5A). That LTB4 utilizes ROS to control Leishmania infection was evidenced by the lack of a significant LTB4 effect in apocynin-treated cells (Fig. 5B). This
Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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Fig. 4. Production of ROS in human macrophages infected with Leishmania (V.) braziliensis is dependent on LTB4. Monocyte-derived macrophages were exposed to different treatments and parasites (, absence; þ, presence), and after 1 h of incubation, the NBT assay was performed to evaluate ROS production. Parasites were added to macrophages at the same time as 107 M LTB4; Apocynin (104 M), 106 M MK0591, 106 M CP-105,696 were added 30 min before parasite addition. Data represent median, interquartile and the minimal and maximal values. *p < 0.05 (vs. Medium, -); $p < 0.05 (vs. Leishmania); #p < 0.05 (vs. Leishmania þ LTB4); n ¼ 7 individuals in two independent experiments.
Fig. 3. Leishmania (Viannia) braziliensis promastigote infection decreases the response to LTB4. Monocyte-derived macrophages were infected with promastigotes for 4 h, excess of parasites was washed out, and cultures were incubated for 48 h. A, LTB4 (107 M, n ¼ 15) was added at the same time of parasites; B, LTB4 (107 M, n ¼ 9) was added 4 h after infection; C, LTB4 (107 M, n ¼ 9) was added 24 h after infection. Data represent individual values before (Control) and after treatment (107 M LTB4) from 6 (A) or 3 (B, C) independent experiments; horizontal broken lines are the medians, *p < 0.05.
further supports the data obtained with the LTs antagonist/ inhibitor indicating that LTs increase the leishmanicidal activity by potentiating ROS production. 4. Discussion In this study, we showed for the first time that LTB4 is produced by human monocyte-derived macrophages infected
with L. (V.) braziliensis and contributes to parasite control by increasing ROS production. In our studies, a clinical field isolate called MHOM/BR/2003/IMG (IMG3) was used. This strain was obtained from a patient with cutaneous leishmaniasis, extensively characterized as L. (V.) braziliensis [17]. We used a high IMG3 parasites:macrophage ratio to determine the parasite burden because in low ratios it was observed a huge individual variability among the percentage of infected cells associated with the low number of IMG3 parasites internalized by macrophages of healthy donors lead to inconsistent results (data not shown). When the number of parasites was increased, the results were more homogeneous and consistent. Besides, clumps of parasites and absence of synchronization of phagocytosis by using centrifugation of the cultures after addition of the parasites can also explain the variability observed with low MOI. That human macrophages are permissive to L. (V.) braziliensis has been previously demonstrated in both human monocytic lineages [17,23] and human monocyte-derived macrophages [5,24]. Our data showed that L. (V.) braziliensis IMG3 can be found in human macrophages up to 96 h after infection (data not shown). The mechanisms underlying human macrophage permissiveness represent an active research program, and further studies are under way. To evaluate the role of LTB4 during L. (V.) braziliensis infection in human macrophages, we first search for BLT1 expression and LTB4 production. Indeed, human macrophages express BLT1, which is down-modulated during infection, and release LTB4. The early production of LTB4 after stimulation
Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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Fig. 5. The inhibition of ROS production increases macrophage infection and reverses the effect of LTB4 on Leishmania (V.) braziliensis infection. A, Monocyte-derived macrophages were treated 30 min before infection with 104 M apocynin. Control: untreated macrophages. Parasites were added, and after 4 h cultures were washed, apocynin was added again, and cells were incubated until 48 h. Each line represents an individual assayed in tests in duplicate, and broken lines are medians (n ¼ 11 individuals in 3 independent experiments); *p < 0.05 (vs. control). B, In another series of experiments, macrophages were cultured in the absence () or presence (þ) of LTB4 and apocynin for 48 h. Data represent median, interquartile and the minimal and maximal values (n ¼ 9 individuals in 3 independent experiments), *p < 0.05 (vs. control); #p < 0.05 (vs. LTB4).
of human monocytes or monocyte-derived macrophages has been reported before [25,26], achieving similar concentrations as showed here. In a previous study, we also demonstrated the release of LTB4 by murine macrophages infected with L. (L.) amazonensis. We showed that the production of LTB4 was higher in macrophages from resistant mice than from susceptible animals, suggesting a role of LTB4 in resistance to infection [9]. To our knowledge, this is the first report showing BLT1 inhibition by L. (V.) braziliensis. Pharmacological inhibition of leukotrienes was achieved using MK0591 (FLAP inhibitor). LT inhibition increased intracellular parasite load in a drug concentration-dependent manner after 48 h of incubation. These results indicate that LTB4 was produced during infection and plays a role in controlling parasite growth. We had previously showed that endogenous leukotrienes are important to control murine L. (L.) amazonensis infection in vivo or in vitro [9]. Together, these results provide new evidence that leukotrienes can be involved in the control of New World Leishmania infections.
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However, these studies did not rule out which classes of leukotrienes are involved in the enhancement of in vivo and in vitro leishmanicidal activity. By employing a BLT1 antagonist, our data unveiled an essential role of LTB4 in controlling the infection. Along those lines, addition of LTB4 decreased L. (V.) braziliensis infection in human macrophages. In our previous studies, exogenous LTB4 added to murine or rat macrophages enhanced the clearance of different pathogens, including L. (L.) amazonensis [9,27,28]. It has been shown that LTB4 increases phagocytosis of the complement and IgG antibody-opsonized pathogens by murine and human neutrophils [6,7,12,28,29] and that BLT1 physically associates with Fcg receptor I to increase phagocytosis of IgG-opsonized Klebsiella pneumonia [30]. Here we did not address the question of LTB4 effect on phagocytosis of L. (V.) braziliensis. To this purpose different protocols and methodologies should be used. To investigate the mechanisms by which LTB4 enhanced leishmanicidal activity of human macrophages, we analyzed the involvement of ROS, since it is known that LTB4 induces ROS production [31] and that Leishmania is sensitive to ROS [24,32]. In fact, we showed that L. (V.) braziliensis induced ROS that was further enhanced by LTB4. Previously, it has been demonstrated that L. tropica and L. donovani induce superoxide anion production in murine macrophages [33]; it has also been shown that ROS is induced in human macrophages infected with L. (L.) amazonensis or L. infantum [16,24]. We showed here that L. (V.) braziliensis infection induces ROS dependent on endogenous LTB4. We have previously demonstrated that LTB4/BLT1 enhances different aspects of NADPH oxidase activation, including increases in p47phox expression and phosphorylation, and membrane translocation in a PKC-d-dependent manner, resulting in ROS production [27,34]. Furthermore, BLT1 signaling activates Rac kinase-ERK to induce ROS [35,36]. The relevance of ROS in L. (V.) braziliensis infection seems to be dependent on the host Rocha et al. [37] showed that NADPH oxidase is not relevant in murine infection control. On the other hand, it has been shown that L. (V.) braziliensis killing is mediated by ROS in human macrophages [24]. Therefore, more detailed studies concerning the role of ROS in L. (V.) braziliensis infection are needed. Next, pharmacological inhibition of NADPH oxidase, in the present study, increased the parasite load, indicating that ROS are indeed produced after human macrophage infection with L. (V.) braziliensis and that it is an important component involved in leishmanicidal activity. Yet, we showed that inhibition of ROS decreased LTB4-mediated macrophage leishmanicidal activity. These results are in agreement with previous studies showing the relevance of ROS to control Leishmania spp. infection in both murine and human macrophages [24,32,33,38]. Our data suggest that LTB4 acts during the initial contact of human macrophage with L. (V.) braziliensis promastigote to increase leishmanicidal activity and therefore prevent infection establishment. Our experiments showed that the timing of LTB4 stimulation can be critical to the outcome of human macrophage
Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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infection with L. (V.) braziliensis. Treatment with LTB4 concomitantly with the parasite or even after 4 h of infection leads to a significant decrease in parasite load. However, when LTB4 was added 24 h after parasite addition, this effect was more subtle, suggesting that L. (V.) braziliensis decreases the response of macrophages to LTB4. These observations lead us to speculate that Leishmania controls LTB4/BLT1 actions. Indeed, we observed that L. (V.) braziliensis promastigotes decreases BLT1 expression in macrophages, as a possible evasion mechanism to prevent ROS secretion and therefore establish the infection. More detailed studies are needed to further dissect the molecular programs involved in the inhibition of BLT1 expression in human macrophages and its consequences in the establishing of Leishmania infection. In addition, whether this expression profile also extends to other types of pathogens is of interest. Based on our results, we hypothesize that human macrophages infected with L. (V.) braziliensis fast produce LTB4, which in turn controls parasite loads during the first hours of infection in a ROS-dependent manner. In order to evade LTB4 effects, the promastigotes down-modulate BLT1 expression decreasing further macrophage response to LTB4, allowing parasite survival and the establishment of the infection. Whether during in vivo chronic infection amastigote forms also inhibit BLT1 expression must be investigated. The infection did not completely abolish BLT1 expression in vitro, so it is also important to determine whether BLT1 signaling pathway is compromised leading to a state of nonresponse to LTB4 treatment. To find new strategies to treat leishmaniasis, it is essential to understand the pathogenicity and control mechanisms of L. (V.) braziliensis infection. This is the most prevalent species of Leishmania in the New World, and it can cause severe disease involving tissue destruction of the oral, nasal and pharynx mucosa. In spite of the importance of L. (V.) braziliensis, the immunobiology of this parasite species has been poorly investigated, especially in human macrophage infection. The present study greatly contributes to understanding the relationship between human macrophages and L. (V.) braziliensis and the role of LTB4 in this interaction. Our results suggest that LTB4 can be used in immunotherapeutic strategies to activate human phagocytes to control L. (V.) braziliensis infection and therefore opens new perspectives for therapies of ATL. Acknowledgments The authors are grateful to Dr. Maria Imaculada MunizJunqueira (UnB, Brasília, DF, Brazil) for technical contributions with the NBT assay. Also, we thank the Blood Banks of Hospital das Clínicas/UFG and INGOH (Goi^ania, GO, Brazil). This study was funded by Fundaç~ao de Amparo a Pesquisa do Estado de Goias (FAPEG, Brazil; Grant 200810267000072 to Dr. Fatima Ribeiro-Dias) and NIH R00HL-103777-03, NIH R56AI065543 and the Showalter Foundation (USA; Grant to Dr. Carlos Henrique Serezani). Dr. Fatima Ribeiro-Dias and Sonia Jancar are research fellows of CNPq, Brazil; Camila I. Morato and Arissa F. Borges were fellows of CAPES, Brazil, and Ildefonso A. da Silva, Jr. was a fellow of CNPq, Brazil.
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[19] Serezani CH, Kane S, Collins L, Morato-Marques M, Osterholzer JJ, Peters-Golden M. Macrophage dectin-1 expression is controlled by leukotriene B4 via a GM-CSF/PU.1 axis. J Immunol 2012;189:906e15. [20] Muniz-Junqueira MI, de Paula-Coelho VN. Meglumine antimonate directly increases phagocytosis, superoxide anion and TNF-alpha production, but only via TNF-alpha it indirectly increases nitric oxide production by phagocytes of healthy individuals, in vitro. Int Immunopharmacol 2008;8:1633e8. [21] Serezani CHC, Aronoff DM, Jancar S, Mancuso P, Peters-Golden M. Leukotrienes enhance the bactericidal activity of alveolar macrophages against Klebsiella pneumoniae through the activation of NADPH oxidase. Blood 2005;106:1067e75. [22] Woo CH, Yoo MH, You HJ, Cho SH, Mun YC, Seong CM, et al. Transepithelial migration of neutrophils in response to leukotriene B4 is mediated by a reactive oxygen species-extracellular signal-regulated kinase-linked cascade. J Immunol 2003;170:6273e9. [23] Ogunkolade BW, Colomb-Valet I, Monjour L, Rhodes-Feuillette A, Abita JP, Frommel D. Interactions between the human monocytic leukaemia THP-1 cell line and old and new world species of Leishmania. Acta Trop 1990;47:171e6. [24] Khouri R, Bafica A, Silva MdaP, Noronha A, Kolb JP, Wietzerbin J, et al. IFN-beta impairs superoxide-dependent parasite killing in human macrophages: evidence for a deleterious role of SOD1 in cutaneous leishQ3 maniasis. J Immunol 2009;182:2525e31. [25] Coffey MJ, Phare SM, Cinti S, Peters-Golden M, Kazanjian PH. Granulocyte-macrophage colony-stimulating factor upregulates reduced 5lipoxygenase metabolism in peripheral blood monocytes and neutrophils in acquired immunodeficiency syndrome. Blood 1999;94:3897e905. [26] Finney-Hayward TK, Bahra P, Li S, Poll CT, Nicholson AG, Russell REK, et al. Leukotriene B4 release by human lung macrophages via receptor-not voltage-operated Caþ channels. Eur Respir J 2 2009;33:1105e12. [27] Serezani CHC, Aronoff DM, Jancar S, Peters-Golden M. Leukotriene B4 mediates p47phox phosphorylation and membrane translocation in polyunsaturated fatty acid-stimulated neutrophils. J Leukoc Biol 2005;78:976e84.
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[28] Morato-Marques M, Campos MR, Kane S, Rangel AP, Lewis C, Ballinger MN, et al. Leukotrienes target F-actin/cofilin-1 to enhance alveolar macrophage anti-fungal activity. J Biol Chem 2011;286:28902e13. [29] Secatto A, Rodrigues LC, Serezani CH, Ramos SG, Dias-Baruffi M, Faccioli LH, et al. 5-Lipoxygenase deficiency impairs innate and adaptive immune responses during fungal infection. PLoS One 2012;7:e31701. [30] Serezani CH, Aronoff DM, Sitrin RG, Peters-Golden M. FcgammaRI ligation leads to a complex with BLT1 in lipid rafts that enhances rat lung macrophage antimicrobial functions. Blood 2009;114:3316e24. [31] Cho KJ, Seo JM, Kim JH. Bioactive lipoxygenase metabolites stimulation of NADPH oxidases and reactive oxygen species. Mol Cells 2011;32:1e5. [32] Murray HW, Cartelli DM. Killing of intracellular Leishmania donovani by human mononuclear phagocytes. Evidence for oxygen-dependent and -independent leishmanicidal activity. J Clin Invest 1983;72:32e44. [33] Murray HW. Susceptibility of Leishmania to oxygen intermediates and killing by normal macrophages. J Exp Med 1981;153:1302e15. [34] Mancuso P, Lewis C, Serezani CH, Goel D, Peters-Golden M. Intrapulmonary administration of leukotriene B4 enhances pulmonary host defense against pneumococcal pneumonia. Infect Immun 2010;78:2264e71. [35] Woo CH, Eom YW, Yoo MH, You HJ, Han HJ, Song WK, et al. Tumor necrosis factor-alpha generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade. J Biol Chem 2000;275:32357e62. [36] Woo CH, You HJ, Cho SH, Eom YW, Chun JS, Yoo YJ, et al. Leukotriene B4 stimulates Rac-ERK cascade to generate reactive oxygen species that mediates chemotaxis. J Biol Chem 2002;277:8572e8. [37] Rocha FJS, Schleicher U, Mattner J, Alber G, Bogdan C. Cytokines, signaling pathways, and effector molecules required for the control of Leishmania (Viannia) braziliensis in mice. Infect Immun 2007;75:3823e32. [38] Murray HW, Nathan CF. Macrophage microbicidal mechanisms in vivo: reactive nitrogen versus oxygen intermediates in the killing of intracellular visceral Leishmania donovani. J Exp Med 1999;189:741e6.
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Microbes and Infection xx (2014) 1e9 www.elsevier.com/locate/micinf
Original article
Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages Camila I. Morato a, Ildefonso A. da Silva Jr. a, Arissa F. Borges a, Miriam L. Dorta a, Milton A.P. Oliveira a, Sonia Jancar b, Carlos H. Serezani c, Fatima Ribeiro-Dias a,*
Q4
a
Tropical Pathology and Public Health Institute, Federal University of Goias, Goi^ania, Goias, Brazil b Institute of Biomedical Sciences, University of S~ao Paulo, S~ao Paulo, Brazil c Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA Received 1 March 2014; accepted 27 August 2014
Abstract Although Leishmania (Viannia) braziliensis is the most prevalent species that cause American tegumentary leishmaniasis (ATL), the immune response against this parasite has been poorly investigated. Upon activation, macrophages produce a series of pro-inflammatory molecules, including the lipid mediator leukotriene B4 (LTB4). LTB4 has been shown to enhance several macrophage functions, but its role in human macrophages is less known. Here, we investigated the role of LTB4 on human monocyte-derived macrophages infected with human isolate of L. (V.) braziliensis (IMG3). It was found that human macrophages produce LTB4 upon infection with Leishmania, which by autocrine or paracrine activation of its high affinity receptor BLT1, potentiates macrophage leishmanicidal activity. This LTB4 effect is mediated by increased secretion of reactive oxygen species (ROS). Moreover, Leishmania infection decreased the expression of BLT1, leading to the speculation that this could represent a parasite escape mechanism to establish a chronic inflammatory infection. Therefore, our data suggest that LTB4 could be used in therapeutic strategies to control Leishmania infection. © 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur.
Keywords: Leishmania (V.) braziliensis; LTB4; Human; Macrophage; ROS
1. Introduction American tegumentary leishmaniasis (ATL) is a disease caused by a protozoa parasite of the genus Leishmania, characterized by a broad clinical spectrum ranging from localized or disseminated cutaneous lesions and mucosal lesions affecting oral, nasal and pharynx mucosa. Several species can cause ATL, but in South America, the most prevalent species is Leishmania (Viannia) braziliensis [1,2]. Although
* Corresponding author. Setor de Imunologia, Instituto de Patologia Tropical e Sau´de Pu´blica, Universidade Federal de Goias, Rua 235 S/N, Setor Universitario, Goi^ania 74605-050, GO, Brazil. Tel.: þ55 62 3209 6116; fax: þ55 62 3209 6363. E-mail addresses: [email protected], [email protected] (F. RibeiroDias).
the immune response during ATL has been extensively investigated [3,4], the mechanisms underlying the pathogenesis of the disease remain to be understood. It is well established that macrophages bear the parasites, but the biology of human macrophages in leishmaniasis is still incompletely understood. In particular, in the case of L. (V.) braziliensis, it was shown that human macrophages are able to control the infection via cytokine/chemokine production [5]. Lipid mediators such as leukotrienes (LTs), and LTB4 in particular, are essential in the control of infections by several pathogens [6e13]. Leukotrienes are eicosanoids derived from the 5-lipoxygenase metabolism of arachidonic acid (AA) to form LTB4 and the cysteinyl LTs, LTC4, LTD4 and LTE4. Both classes of LTs act on their respective G protein-coupled receptors, BLT1 or BLT2 and CysLT1 or CysLT2 [14]. The role of LTs in leishmaniasis has been investigated in in vivo and
http://dx.doi.org/10.1016/j.micinf.2014.08.015 1286-4579/© 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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in vitro murine models of infection caused by Leishmania donovani or Leishmania (L.) amazonensis [9,15]. We have demonstrated that LTB4 production is associated with patterns of resistance and susceptibility in a murine model of L. (L.) amazonensis infection, suggesting that LTB4 can be an important endogenous mediator involved in the control of ATL [9]. Recently, Lefevre et al. [16] demonstrated that LTB4 is produced by human monocyte-derived macrophages infected with Leishmania infantum, which causes visceral leishmaniasis. Studies concerning the production and actions of LTs either in patients with ATL or in human macrophages infected with L. (V.) braziliensis are lacking. Here, we sought to investigate the role of LTB4 in the control of L. (V.) braziliensis infection in human macrophages. To this purpose we first investigated the infection pattern of human monocytederived macrophages infected with a human isolate of L. (V.) braziliensis (IMG3). Then we found that LTB4 is produced by the infected macrophages, which also express the receptor BLT1. Studies using selective receptor antagonists and inhibitors of LT synthesis showed that LTB4 is a critical mediator involved in human leishmanicidal activity by enhancing the generation of ROS.
2.3. Human macrophage differentiation and culture All protocols were approved by the Ethics Committee of the Hospital das Clínicas/UFG (n. 190/2011). Venous blood samples (~10 ml) were obtained from healthy blood donors (n ¼ 77) and collected in vacuum tubes (Greiner bio-one, Vacuette) containing EDTA solution. Blood was centrifuged at 200 g for 5 min, and the plasma was discarded. The pellet was diluted (v/v) in phosphate-buffered saline (PBS) containing 0.01 mM EDTA, pH 7.3 and layered on Percoll gradient (GE Healthcare). Mononuclear cells (MNC) were suspended in RPMI 1640 medium supplemented with 15% FCS, 11 mM sodium bicarbonate, 2 mM L-glutamine, 100 U/ ml penicillin and 100 mg/ml streptomycin (complete RPMI medium). Cells (2 105) were distributed into 24-well plates (TPP, Techno Plastic Products) on 13-mm round glass coverslips (Deckglaser). Complete RPMI medium was replaced every 48 h for 5 days of culture (36 C, 5% CO2), allowing non-adherent cells to be removed and adherent monocytes to differentiate into macrophages. 2.4. Infection of monocyte-derived macrophages and quantification of parasite burden
2. Materials and methods 2.1. Reagents Cell culture reagents were from SigmaeAldrich (St. Louis, USA), unless otherwise stated. Fetal calf serum (FCS) was purchased from Cripion Biotechnology to be used with parasites, and from Gibco Life Technology for using with the cells. The FLAP inhibitor MK0591 was from Merck KgaA; the BLT1 antagonist, CP-105, 696 was from Pfizer. The LTB4 and LTB4 enzyme immunoassay (EIA) kits were from Cayman Chemical Co., and the antibodies against BLT1 were purchased from Enzo Life Science. The peroxidase-conjugated secondary antibody (anti-rabbit-HRP conjugate) and chemiluminescence system were from Santa Cruz Biotechnology, and the Trizol reagent was from Invitrogen. The Revert Aid TM First Strand cDNA Synthesis kit was from Thermo Scientific. 2.2. Parasite culture Throughout this study we employed the human clinical isolate MHOM/BR/2003/IMG L. (V.) braziliensis (IMG3) that was well characterized by us [17]. The isolate was obtained from a patient with localized cutaneous leishmaniasis and stored in the Leishbank (Central West's Bank of Leishmania sp., Federal University of Goias (UFG), Brazil). Parasites were cultured in Grace's Insect Medium supplemented with 20% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin, at 26 C. Passages of parasites were performed every three days, always starting the culture with 5 105 parasites/ml, up to a maximum of six times. To infect macrophages, promastigotes were used on the 6th day of growth (stationary phase).
Concentrations of 1 108 promastigotes/ml were suspended in complete RPMI medium and added to macrophages (2 106/well). Considering that monocytes represent ~5e20% of MNC, the MOI varied between ~50:1 to 200:1 (parasites: macrophage). After parasite addition plates were not centrifuged to synchronize the phagocytosis. Cultures were incubated for 4 h (36 C, 5% CO2), washed three times with warmed complete RPMI medium to remove extracellular parasites, and the coverslips were then removed to stain the cells and analyze the parasite burden at the first 4 h. In another wells, complete RPMI medium was added again, cultures were re-incubated and coverslips harvested at different time points (24 h, 48 h, 72 h). Cells were fixed with methanol, stained with the commercial Instant Prov kit (Newprov) and analyzed under light microscopy (1000; Nikon) to determine the parasite burden. The percentage of infected cells (300 cells counted in random fields) and the mean number of intracellular amastigotes per infected cell (at least 50 infected cells were counted) were obtained by blinded counts. The parasite burden was calculated as: Parasites/ 100 macrophages ¼ percentage of infected cells mean number of parasites per infected cell. All experiments were done in duplicate. 2.5. Pharmacological treatment of monocyte-derived macrophages Inhibition of leukotriene biosynthesis or actions was achieved by treating macrophages with the 5-lipoxygenase-activating protein (FLAP) inhibitor MK0591 or the leukotriene B4 receptor 1 antagonist (BLT1) CP-105, 696, respectively. The compounds (103 M) were dissolved in DMSO (vehicle) and stored at 80 C until use. For the experiments, compounds
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were diluted in complete RPMI medium and added to the macrophages in different concentrations, 30 min before infection. The vehicle control was added at the same final concentration used in the experimental compounds. The compounds were added back or not after 4 h of incubation (when cultures were washed to eliminate extracellular parasites), as indicated in the figure legends. After 4 h or 48 h of incubation, coverslips were removed, and the parasite burden was evaluated as described above. In another set of experiments, macrophages were treated or not with LTB4, dissolved in ethanol (3 104 M) and further diluted in PBS. We have done three different treatment protocols: 1) macrophages were stimulated with LTB4 plus parasites; 2) LTB4 was added 4 h after the infection; and 3) LTB4 was added 24 h after the infection. In all circumstances, coverslips were harvested 48 h after the initial 4 h of infection. Vehicle control (ethanol) was added in all time points tested. None of the compounds or vehicles (MK0591, CP-105,696, DMSO and ethanol) was toxic to cells or parasites (data not shown). A MTT assay was used to evaluate cytotoxicity [18]. To check the activity of MK0591 (106 M), macrophages were incubated in absence or presence of MK0591 for 30 min before parasite addition. After 30 min, LTB4 was measured as described below (at 2.6), showing that MK0591 was inhibiting LTB4 production. To check the ability of CP-105,696 to block BLT1 receptors, macrophages were incubated in the presence or absence of CP-105,696 (106 M) before parasite and LTB4 addition. Results showed that CP-105,696 blocked the macrophage leishmanicidal activity induced by LTB4 (Supplementary Fig. 1). 2.6. Measurement of LTB4 As the amount of adherent cells in each MNC preparation is variable and the number of macrophages is low, to measure LTB4 production after 4 days of culture (2 107 MNC/well, 6-well plates) adherent cells were scrapped from the plates, counted and then plated again (1 106/well). After 24 h, macrophages were incubated or not with MK059, followed by promastigote parasites (MOI: 10 parasites:1 macrophage) and after 0.5 h or 4 h, the supernatants were collected. LTB4 was determined by enzyme immunoassay (EIA) kits according to manufacturer's instructions. The results were expressed in pg/ mL, and the detection limit was 13 pg/mL. 2.7. Quantification of BLT1 expression Human macrophages were scrapped from the plates (2 107 MNC/well, 6-well plates), counted and then plated again (107 cells/well, 6-well plates). They were infected as described above (MOI: 10:1), and BLT1 expression was evaluated by real-time polymerase chain reaction (qPCR, 6 h) and Western blotting (24 h), as previously described [19]. After 6 h of macrophageeparasite interaction, macrophages were washed 3 with warmed PBS (37 C) and RNA was extracted using Trizol. cDNA synthesis was performed with the Revert Aid TM First Strand cDNA Synthesis kit, according
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to the manufacturer's instructions. Human BLT1 specific primers (sense primer 50 - TATGTCTGCGGAGTCAGCATGTACGC-30 and antisense 50 CCTGTAGCCGACG CCCTATGTCCG-30 ) and the housekeeping gene 50 -GAPDH (GAGTCAACGGAT TTGGTCGT-30 sense primer, antisense 50 -TTGATTTTG GAGGGATCTCG-30 ) were used. qPCR was performed using the Stratagene cycler Mx3005PTM QPCR Systems according to the manufacturer's instructions. Gene expression was analyzed by 2-DDCT method and expressed as the percentage of uninfected macrophages. For immunoblotting experiments, infected macrophages were washed 3 with warmed PBS and lysed, and protein content was measured using a BCA kit (Pierce). Proteins (20 mg) were separated in 10% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After blocking, membranes were incubated with anti-BLT1 followed by peroxidase-conjugated secondary antibody (anti-rabbit-HRP conjugate) for 2 h at room temperature. Visualization was performed with chemiluminescence system. Densitometric analysis was performed, and the intensity of the protein band was divided by that of the bactin. This ratio was expressed relative to that of the uninfected macrophages (100%). 2.8. Measurement of ROS To measure ROS production, we employed the NBT (nitro blue tetrazolium) assay, adapted from Muniz-Junqueira & Paula-Coelho [20]. Briefly, macrophages were placed in glass coverslips and infected with parasites. After 1 h, macrophages were incubated with Tris-Hanks containing 0.05% NBT for 20 min at 36 C, 5% CO2. Coverslips were washed, fixed with methanol, and stained with a solution of 1.4% safranin and 28.6% glycerol in distilled water. The percentage of ROSpositive macrophages was evaluated microscopically. In one protocol, the percentage of macrophages that were strongly positive (dark blue staining) among ROS-positive cells were counted. 2.9. Inhibition of ROS production To inhibit ROS production, macrophages were treated with nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin and dissolved in DMSO (0.5 M) before use. The inhibitor was further diluted in complete RPMI medium and added to cultures at different concentrations, 30 min before LTB4 (107 M) treatment and infection with promastigotes. Parasite burden was determined as mentioned above. 2.10. Statistical analysis Data are presented as median [minimum and maximum] and interquartile values. The ManneWhitney and Wilcoxon matched pair tests were used, and the level of significance was set at p < 0.05. The analyses were performed using GraphPad Prism 5.0 Software (San Diego, CA, USA).
Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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3. Results 3.1. Leukotriene B4 is generated by human macrophages infected with L. (V.) braziliensis potentiating macrophages leishmanicidal activity Initially, we investigated the infection pattern of the human isolate of L. (V.) braziliensis (IMG3) in human monocytederived macrophages. Our data showed that macrophages were susceptible to IMG3 L. (V.) braziliensis infection. Parasites were detected in parasitophorous vacuoles at all time points tested. Fig. 1A shows intracellular amastigotes in macrophages 48 h after infection. Even though we observed high individual variability, there was a significant increase in the parasite burden from 4 h to 48 h (Fig. 1B). Since LTB4 has been previously shown to increase leishmanicidal activity in murine macrophages through its high affinity receptor BLT1 [9], we first investigated whether human macrophages express
this receptor. We found that they did express BLT1 mRNA and protein (Fig. 1C and D, respectively). Fig. 1 also shows that Leishmania infection down regulates BLT1 expression (52% inhibition of protein expression; Fig 1D). Next, we measured LTB4 in the supernatant of the infected macrophages and found that Leishmania infection induces LTB4 production already after 30 min of infection, and it remains elevated at 4 h (Fig. 1E). To determine the role of endogenously produced leukotrienes on L. (V.) braziliensis infection macrophages were pretreated with the FLAP inhibitor MK0591 before infection. This compound was effective to inhibit leukotriene production at 106 M concentration (118.0 [9.8e220.1] pg/mL vs. 541.0 [360.0e653.0] pg/mL, 30 min, n ¼ 8; p < 0.05). Fig. 2A shows that 106 M MK0591 significantly increased the parasite burden at 48 h of infection. Moreover, treatment with the selective antagonist of LTB4 receptor BLT1, the compound CP-105,696, at 106 M concentration also increased the
Fig. 1. L. (V.) braziliensis induces release of LTB4 and modulates BLT1 expression in human macrophages. Monocyte-derived macrophages (2 105 MNC) were cultured for 4 h with promastigotes (2 106 parasites; 6th day of culture). After washing out the excess of parasites (4 h), cultures were incubated for 48 h (36 C). In A, photomicrography showing infected macrophages and amastigotes are indicated (arrow; bar represents 10 mm). B, The number of parasites per 100 macrophages; each line represents an individual assessed in experiments performed in duplicate (n ¼ 19 individuals in 5 independent experiments); horizontal broken lines are the medians, *p < 0.05. In C and D, Macrophages (1 107) were incubated in the absence (Control) or presence of parasites (1 108) for 6 h to evaluate mRNA BLT1 expression by qRT-PCR (C, expression of mRNA BLT1/GAPDH relative to uninfected macrophages) or 24 h to assess BLT1 expression by using Western blotting (D, arbitrary units of the ratio between the optical densities obtained for BLT1/b-actin relative to uninfected macrophages). *p < 0.05 (medians and interquartile from 9 independent experiments in duplicates). E, Macrophages (1 106) were incubated with parasites (1 107), and after 0.5 h or 4 h supernatants were assayed to LTB4 concentration by using EIA. Control: uninfected macrophages. *p < 0.05 (vs. control), #p < 0.05 (vs. 30 min). E, Data represent median, interquartile and the minimal and maximal values (n ¼ 6 individuals in 2 independent experiments). Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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parasite burden (Fig. 2B). The BLT1 antagonist increased both the percentage of infected macrophages (61.5% [51.0e75.3%] vs. 56.0% [25.5e76.0%], n ¼ 9; p < 0.05) and the number of parasites per cell (7.1% [3.6e11.2%] vs. 4.8% [1.5e5.9%], n ¼ 9; p < 0.05). The addition of LTB4 to macrophage cultures together parasites had similar effects compared with the endogenously produced LTB4. Fig. 2C shows the parasite load after 48 h of infection and LTB4 treatment during the first 4 h. LTB4 decreased dose-dependently the parasite burden in infected macrophages. Both the percentage of infected macrophages (48.0% [17.0e58.0%] vs. 72.4% [29.6e95.0%], n ¼ 18; p < 0.05) and number of parasites per cell (3.9% [1.7e8.7%] vs. 6.8% [3.1e16.7%], n ¼ 18; p < 0.05) were decreased by LTB4 treatment. CP-105,696 at 106 M was effective to block LTB4 effects (Supplementary Fig. 1). Next, we investigated whether LTB4 would also increase leishmanicidal activity when infection is already established. Macrophages were infected with promastigotes and simultaneously treated with 107 M of LTB4 (Fig. 3A), or treated 4 h (Fig. 3B) or 24 h (Fig. 3C) after parasite addition. The parasite load was determined after 48 h. While the addition of LTB4 at the same time of parasites or 4 h after significantly decreased the parasite load (Fig. 3A, B) LTB4 caused no significant change when it was added 24 h after parasites. These results allow the conclusion that human macrophages produce LTB4 upon infection with Leishmania, which by autocrine or paracrine activation of the BLT1 receptor potentiates the leishmanicidal activity of human macrophages. 3.2. ROS generation is involved in LTB4-enhanced leishmanicidal activity of human macrophages
Fig. 2. LTB4 decreases the infection of human macrophages with Leishmania (V.) braziliensis. A and B, Monocyte-derived macrophages were treated 30 min before infection with different concentrations of MK0591 (A) or CP-105,696 (B), infected for 4 h, washed, then re-incubated for 48 h; Control: vehicle treated macrophages (A, B, C). Data represent median, interquartile and the minimal and maximal values (n ¼ 8 (A), n ¼ 9 (B) individuals in 3 independent experiments), *p < 0.05. C, Macrophages were incubated with parasites for 4 h in the absence (Control) or presence of LTB4 in different concentrations. After washing, medium was replaced and cultures were incubated until 48 h. Data represent median, interquartile and the minimal and maximal values (n ¼ 18 individuals in 6 independent experiments), *p < 0.05 (vs. Control).
Since human macrophages infected with L. (V.) braziliensis produce superoxide [5] and LTB4 has been shown to increase ROS production [21,22], we measured ROS in human macrophages infected with L. (V.) braziliensis and the involvement of LTB4 in ROS production. We found that human macrophages produce ROS during infection with Leishmania (Fig. 4). The addition of LTB4 to the infected macrophages potentiated ROS production (Fig. 4). This was further confirmed when macrophages showing strong NBT reactions (dark blue precipitates) were counted among the NBT-positive cells. This semi-quantitative evaluation revealed a higher frequency of cells with strong ROS reactions in LTB4-treated macrophages than in untreated cells (Control vs. Leishmania vs. Leishmania plus LTB4: 0% vs. 1.45% [0%e7.9%] vs. 9.95% [1.6%e25.5%], p ¼ 0.06, n ¼ 6). Inhibition of LT synthesis by MK0591 or blocking of BLT1 by CP-105,696 both significantly reduced ROS production by infected macrophages (Fig. 4). The effect of exogenous LTB4 was blocked by CP-105,696 (Fig. 4). Leishmania-induced ROS production was inhibited by 104 M of the NADPH oxidase inhibitor, apocynin (Fig. 4) and in this situation, the macrophage infection markedly increased (Fig. 5A). That LTB4 utilizes ROS to control Leishmania infection was evidenced by the lack of a significant LTB4 effect in apocynin-treated cells (Fig. 5B). This
Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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Fig. 4. Production of ROS in human macrophages infected with Leishmania (V.) braziliensis is dependent on LTB4. Monocyte-derived macrophages were exposed to different treatments and parasites (, absence; þ, presence), and after 1 h of incubation, the NBT assay was performed to evaluate ROS production. Parasites were added to macrophages at the same time as 107 M LTB4; Apocynin (104 M), 106 M MK0591, 106 M CP-105,696 were added 30 min before parasite addition. Data represent median, interquartile and the minimal and maximal values. *p < 0.05 (vs. Medium, -); $p < 0.05 (vs. Leishmania); #p < 0.05 (vs. Leishmania þ LTB4); n ¼ 7 individuals in two independent experiments.
Fig. 3. Leishmania (Viannia) braziliensis promastigote infection decreases the response to LTB4. Monocyte-derived macrophages were infected with promastigotes for 4 h, excess of parasites was washed out, and cultures were incubated for 48 h. A, LTB4 (107 M, n ¼ 15) was added at the same time of parasites; B, LTB4 (107 M, n ¼ 9) was added 4 h after infection; C, LTB4 (107 M, n ¼ 9) was added 24 h after infection. Data represent individual values before (Control) and after treatment (107 M LTB4) from 6 (A) or 3 (B, C) independent experiments; horizontal broken lines are the medians, *p < 0.05.
further supports the data obtained with the LTs antagonist/ inhibitor indicating that LTs increase the leishmanicidal activity by potentiating ROS production. 4. Discussion In this study, we showed for the first time that LTB4 is produced by human monocyte-derived macrophages infected
with L. (V.) braziliensis and contributes to parasite control by increasing ROS production. In our studies, a clinical field isolate called MHOM/BR/2003/IMG (IMG3) was used. This strain was obtained from a patient with cutaneous leishmaniasis, extensively characterized as L. (V.) braziliensis [17]. We used a high IMG3 parasites:macrophage ratio to determine the parasite burden because in low ratios it was observed a huge individual variability among the percentage of infected cells associated with the low number of IMG3 parasites internalized by macrophages of healthy donors lead to inconsistent results (data not shown). When the number of parasites was increased, the results were more homogeneous and consistent. Besides, clumps of parasites and absence of synchronization of phagocytosis by using centrifugation of the cultures after addition of the parasites can also explain the variability observed with low MOI. That human macrophages are permissive to L. (V.) braziliensis has been previously demonstrated in both human monocytic lineages [17,23] and human monocyte-derived macrophages [5,24]. Our data showed that L. (V.) braziliensis IMG3 can be found in human macrophages up to 96 h after infection (data not shown). The mechanisms underlying human macrophage permissiveness represent an active research program, and further studies are under way. To evaluate the role of LTB4 during L. (V.) braziliensis infection in human macrophages, we first search for BLT1 expression and LTB4 production. Indeed, human macrophages express BLT1, which is down-modulated during infection, and release LTB4. The early production of LTB4 after stimulation
Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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Fig. 5. The inhibition of ROS production increases macrophage infection and reverses the effect of LTB4 on Leishmania (V.) braziliensis infection. A, Monocyte-derived macrophages were treated 30 min before infection with 104 M apocynin. Control: untreated macrophages. Parasites were added, and after 4 h cultures were washed, apocynin was added again, and cells were incubated until 48 h. Each line represents an individual assayed in tests in duplicate, and broken lines are medians (n ¼ 11 individuals in 3 independent experiments); *p < 0.05 (vs. control). B, In another series of experiments, macrophages were cultured in the absence () or presence (þ) of LTB4 and apocynin for 48 h. Data represent median, interquartile and the minimal and maximal values (n ¼ 9 individuals in 3 independent experiments), *p < 0.05 (vs. control); #p < 0.05 (vs. LTB4).
of human monocytes or monocyte-derived macrophages has been reported before [25,26], achieving similar concentrations as showed here. In a previous study, we also demonstrated the release of LTB4 by murine macrophages infected with L. (L.) amazonensis. We showed that the production of LTB4 was higher in macrophages from resistant mice than from susceptible animals, suggesting a role of LTB4 in resistance to infection [9]. To our knowledge, this is the first report showing BLT1 inhibition by L. (V.) braziliensis. Pharmacological inhibition of leukotrienes was achieved using MK0591 (FLAP inhibitor). LT inhibition increased intracellular parasite load in a drug concentration-dependent manner after 48 h of incubation. These results indicate that LTB4 was produced during infection and plays a role in controlling parasite growth. We had previously showed that endogenous leukotrienes are important to control murine L. (L.) amazonensis infection in vivo or in vitro [9]. Together, these results provide new evidence that leukotrienes can be involved in the control of New World Leishmania infections.
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However, these studies did not rule out which classes of leukotrienes are involved in the enhancement of in vivo and in vitro leishmanicidal activity. By employing a BLT1 antagonist, our data unveiled an essential role of LTB4 in controlling the infection. Along those lines, addition of LTB4 decreased L. (V.) braziliensis infection in human macrophages. In our previous studies, exogenous LTB4 added to murine or rat macrophages enhanced the clearance of different pathogens, including L. (L.) amazonensis [9,27,28]. It has been shown that LTB4 increases phagocytosis of the complement and IgG antibody-opsonized pathogens by murine and human neutrophils [6,7,12,28,29] and that BLT1 physically associates with Fcg receptor I to increase phagocytosis of IgG-opsonized Klebsiella pneumonia [30]. Here we did not address the question of LTB4 effect on phagocytosis of L. (V.) braziliensis. To this purpose different protocols and methodologies should be used. To investigate the mechanisms by which LTB4 enhanced leishmanicidal activity of human macrophages, we analyzed the involvement of ROS, since it is known that LTB4 induces ROS production [31] and that Leishmania is sensitive to ROS [24,32]. In fact, we showed that L. (V.) braziliensis induced ROS that was further enhanced by LTB4. Previously, it has been demonstrated that L. tropica and L. donovani induce superoxide anion production in murine macrophages [33]; it has also been shown that ROS is induced in human macrophages infected with L. (L.) amazonensis or L. infantum [16,24]. We showed here that L. (V.) braziliensis infection induces ROS dependent on endogenous LTB4. We have previously demonstrated that LTB4/BLT1 enhances different aspects of NADPH oxidase activation, including increases in p47phox expression and phosphorylation, and membrane translocation in a PKC-d-dependent manner, resulting in ROS production [27,34]. Furthermore, BLT1 signaling activates Rac kinase-ERK to induce ROS [35,36]. The relevance of ROS in L. (V.) braziliensis infection seems to be dependent on the host Rocha et al. [37] showed that NADPH oxidase is not relevant in murine infection control. On the other hand, it has been shown that L. (V.) braziliensis killing is mediated by ROS in human macrophages [24]. Therefore, more detailed studies concerning the role of ROS in L. (V.) braziliensis infection are needed. Next, pharmacological inhibition of NADPH oxidase, in the present study, increased the parasite load, indicating that ROS are indeed produced after human macrophage infection with L. (V.) braziliensis and that it is an important component involved in leishmanicidal activity. Yet, we showed that inhibition of ROS decreased LTB4-mediated macrophage leishmanicidal activity. These results are in agreement with previous studies showing the relevance of ROS to control Leishmania spp. infection in both murine and human macrophages [24,32,33,38]. Our data suggest that LTB4 acts during the initial contact of human macrophage with L. (V.) braziliensis promastigote to increase leishmanicidal activity and therefore prevent infection establishment. Our experiments showed that the timing of LTB4 stimulation can be critical to the outcome of human macrophage
Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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infection with L. (V.) braziliensis. Treatment with LTB4 concomitantly with the parasite or even after 4 h of infection leads to a significant decrease in parasite load. However, when LTB4 was added 24 h after parasite addition, this effect was more subtle, suggesting that L. (V.) braziliensis decreases the response of macrophages to LTB4. These observations lead us to speculate that Leishmania controls LTB4/BLT1 actions. Indeed, we observed that L. (V.) braziliensis promastigotes decreases BLT1 expression in macrophages, as a possible evasion mechanism to prevent ROS secretion and therefore establish the infection. More detailed studies are needed to further dissect the molecular programs involved in the inhibition of BLT1 expression in human macrophages and its consequences in the establishing of Leishmania infection. In addition, whether this expression profile also extends to other types of pathogens is of interest. Based on our results, we hypothesize that human macrophages infected with L. (V.) braziliensis fast produce LTB4, which in turn controls parasite loads during the first hours of infection in a ROS-dependent manner. In order to evade LTB4 effects, the promastigotes down-modulate BLT1 expression decreasing further macrophage response to LTB4, allowing parasite survival and the establishment of the infection. Whether during in vivo chronic infection amastigote forms also inhibit BLT1 expression must be investigated. The infection did not completely abolish BLT1 expression in vitro, so it is also important to determine whether BLT1 signaling pathway is compromised leading to a state of nonresponse to LTB4 treatment. To find new strategies to treat leishmaniasis, it is essential to understand the pathogenicity and control mechanisms of L. (V.) braziliensis infection. This is the most prevalent species of Leishmania in the New World, and it can cause severe disease involving tissue destruction of the oral, nasal and pharynx mucosa. In spite of the importance of L. (V.) braziliensis, the immunobiology of this parasite species has been poorly investigated, especially in human macrophage infection. The present study greatly contributes to understanding the relationship between human macrophages and L. (V.) braziliensis and the role of LTB4 in this interaction. Our results suggest that LTB4 can be used in immunotherapeutic strategies to activate human phagocytes to control L. (V.) braziliensis infection and therefore opens new perspectives for therapies of ATL. Acknowledgments The authors are grateful to Dr. Maria Imaculada MunizJunqueira (UnB, Brasília, DF, Brazil) for technical contributions with the NBT assay. Also, we thank the Blood Banks of Hospital das Clínicas/UFG and INGOH (Goi^ania, GO, Brazil). This study was funded by Fundaç~ao de Amparo a Pesquisa do Estado de Goias (FAPEG, Brazil; Grant 200810267000072 to Dr. Fatima Ribeiro-Dias) and NIH R00HL-103777-03, NIH R56AI065543 and the Showalter Foundation (USA; Grant to Dr. Carlos Henrique Serezani). Dr. Fatima Ribeiro-Dias and Sonia Jancar are research fellows of CNPq, Brazil; Camila I. Morato and Arissa F. Borges were fellows of CAPES, Brazil, and Ildefonso A. da Silva, Jr. was a fellow of CNPq, Brazil.
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Please cite this article in press as: Morato CI, et al., Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.015
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