Sodium nitroprusside has leishmanicidal activity independent of iNOS

Introduction: Leishmaniasis is a zoonotic disease caused by protozoa of the genus Leishmania. Cutaneous leishmaniasis is the most common form, with millions of new cases worldwide each year. Treatments are ineffective due to the toxicity of existing drugs and the resistance acquired by certain strains of the parasite. Methods: We evaluated the activity of sodium nitroprusside in macrophages infected with Leishmania (Leishmania) amazonensis. Phagocytic and microbicidal activity were evaluated by phagocytosis assay and promastigote recovery, respectively, while cytokine production and nitrite levels were determined by ELISA and by the Griess method. Levels of iNOS and 3-nitrotyrosine were measured by immunocytochemistry. Results: Sodium nitroprusside exhibited in vitro antileishmanial activity at both concentrations tested, reducing the number of amastigotes and recovered promastigotes in macrophages infected with L. amazonensis. At 1.5μg/mL, sodium nitroprusside stimulated levels of TNF-α and nitric oxide, but not IFN-γ. The compound also increased levels of 3-nitrotyrosine, but not expression of iNOS, suggesting that the drug acts as an exogenous source of nitric oxide. Conclusions: Sodium nitroprusside enhances microbicidal activity in Leishmania-infected macrophages by boosting nitric oxide and 3-nitrotyrosine.

Leishmaniasis, a group of infectious diseases found worldwide, is caused by protozoa of the genus Leishmania.Approximately 20 to 40 thousand deaths each year are attributed to this disease, which can manifest in various forms with different symptoms depending on the infecting species and the host immune response.The main forms of the disease are cutaneous, mucocutaneous, and visceral, of which the cutaneous form is the most common, with 0.7 to 1.2 million new cases each year in 98 countries (1) .Pentavalent antimonials are the standard treatments, of which meglumine antimoniate and sodium stibogluconate are the most frequently used (2) .However, these drugs require long treatment regimens and parenteral or intralesional administration (3) , and cause numerous side effects, including pancreatitis, hepatitis, and cardiotoxicity (3) (4) .
In addition, some strains of the parasite have acquired resistance to these drugs (5) .
Macrophages mount various mechanisms to combat parasites, including oxidative burst, acidifi cation of vesicles, and expression of inducible nitric oxide synthase (iNOS) (6) .iNOS synthesizes nitric oxide, a highly reactive, membranediffusible molecule used to control various pathogens (7) .Nitric oxide reacts with reactive oxygen species to generate reactive nitrogen species such as peroxynitrite (8) , which damage DNA, inhibit enzymes, and peroxidize lipids (9) .
Leishmania spp.has evolved several mechanisms to evade macrophage activity.For instance, parasites suppress nitric oxide production by taking up L-arginine, a required substrate for iNOS, as well as by inhibiting the release of IFN-γ and TNF-α, proinfl ammatory cytokines that stimulate expression of this enzyme (10) .These observations suggest that exogenous sources of nitric oxide may potentially be used to control leishmaniasis.

METHODS
an inorganic compound (16) active against promastigotes and axenic amastigotes of Leishmania (Leishmania) amazonensis (17) .In this report, we demonstrate that sodium nitroprusside enhances the microbicidal activity of Leishmania-infected macrophages by enhancing production of nitric oxide and 3-nitrotyrosine.

Culture of peritoneal macrophages and phagocytosis assay
Macrophages were obtained from the peritoneal cavity of BALB/c mice, re-suspended in RPMI 1640 medium pH 7.2 (Gibco BRL), and incubated for 2h at 37ºC and 5% CO 2 in 24-well plates (5 × 10 5 cells per well) with 13mm glass coverslips with 200µL RPMI 1640 medium.Adherent cells were infected for 2h with L. amazonensis promastigotes at a ratio of 1:5, washed with phosphate-buffered saline to remove non-phagocytized parasites, and treated for 24h at 37ºC and 5% CO 2 with RPMI 1640 (control) or with 0.5 and 1.5µg/mL sodium nitroprusside.Subsequently, cells were stained with Giemsa, and 200 cells per sample were imaged at 1,000× under a CX31RBSFA light microscope (Olympus Optical Co. Ltd., Tokyo, Japan) to quantify the number of infected macrophages and the average number of amastigotes per macrophage.

Promastigote recovery
Promastigote recovery was performed as previously described (18) .Briefl y, peritoneal macrophages (5 × 10 5 cells) were infected with L. amazonensis, treated for 24h at 37ºC and 5 % CO 2 with 0.5 and 1.5µg/mL sodium nitroprusside, washed with phosphate-buffered saline, and incubated at 24°C with 199 medium to induce differentiation of intracellular amastigotes into promastigotes.Promastigotes recovered were counted daily in a Neubauer chamber for three days after infection.

Cytokine levels
Supernatants were collected from cultures of infected macrophages 24h after treatment with sodium nitroprusside, centrifuged at 460 ×g for 7 min at 4°C, and stored at -80°C until analysis.TNF-α, IFN-γ, and IL-12-p70 were determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (eBiosciences®, USA).Plates were read at 450nm using a plate reader (Thermo-TP-Reader).

Nitrite levels
Nitrite was also determined in the supernatant of cultures of infected macrophages treated with sodium nitroprusside.Nitric oxide was measured with the Griess reagent according to published methods (19) .Briefl y, aliquots were diluted in 45g/L glycine pH 9.7, and treated for 10 min with cadmium granules previously activated with 5mM CuSO 4 .Subsequently, 200µL of this mixture reacted for 10 min at room temperature with an equal volume of Griess reagent.Tubes were then centrifuged at 10,845 ×g for 2 min at 25°C, and transferred to 96-well microplates in triplicate.Absorbance at 550nm was determined in a microplate reader.A calibration curve was constructed using dilutions of NaNO 2 .

Immunocytochemistry for iNOS and 3-nitrotyrosine
Slides with adherent macrophages were prepared in triplicate as described for the phagocytic assay, and labeled by the streptavidin-biotin method (Universal Dako LSAB ® + System-HRP Kit, DAKO Japan, Kyoto, Japan) without microwave pretreatment.Slides were then treated with 10% Triton-X for 15 min, washed with phosphate-buffered saline, and incubated in 1% fetal bovine serum for 30 min.Subsequently, slides were probed overnight at 4 ºC with 1:300 dilutions of rabbit polyclonal primary antibodies against iNOS and 3-nitrotyrosine (Santa Cruz Biotechnology), and then with biotinylated anti-rabbit, anti-mouse, and anti-goat IgG (LSAB+ System-HRP, DAKO, Japan, Kyoto, Japan) for 2h at room temperature.Negative controls were performed omitting the primary antibodies.Horseradish peroxidase activity was visualized with H 2 O 2 and 3,3'-diaminobenzidine for 5 min, and cells were counterstained with Harris hematoxylin (Merck).Finally, slides were digitally imaged in color at 400× using a BX41 photomicroscope (Olympus Optical Co. Ltd., Tokyo, Japan).Representative fi elds of view from 10 images of each replicate were scored semi-quantitatively using color deconvolution in ImageJ (NIH, USA).Pixels with intensity 0-255 were categorized as strongly positive (3+, intensity 0-60), positive (2+, intensity 61-120), weakly positive (1+, intensity 121-170), and negative (0, intensity 171-230), as previously described (20) .Slides that were not probed with primary antibody were used as negative control.

Statistical analysis
Data are reported as mean ± standard error of the mean.Duplicate datasets from three independent experiments with three animals per experiment were analyzed in Prism GraphPad 5.00 (GraphPad Software, Inc., USA).Data were found to be normally distributed by Kolmogorov-Smirnov test, and variances were found to be homogeneous by F test.Treatments were compared by Student's t-test or analysis of variance followed by Tukey's test for multiple comparisons.Differences were considered statistically signifi cant when p < 0.05.

Ethics statement
Female BALB/c mice weighing approximately 25-30g and aged 6-8 weeks were housed in pathogen-free conditions according to protocols approved by the Institutional Animal Care and Use Committee at Londrina State University.This study was approved by the Londrina State University Ethics Committee for Animal Experimentation (33064.2012.42).

Sodium nitroprusside alters phagocytic capacity and increases microbicidal activity
To characterize the impact of sodium nitroprusside on phagocytic and microbicidal activity, macrophages were treated with different concentrations of the compound for 24h after infection.We found that exposure to sodium nitroprusside for 24h did not signifi cantly affect the number of infected macrophages.However, the number of amastigotes per macrophage was signifi cantly reduced in cells treated with 0.5µg/mL (p = 0.0188) and 1.5µg/mL (p = 0.0409) sodium nitroprusside (Figure 1A and 1B).Accordingly, sodium nitroprusside also reduced the number of promastigotes recovered, with p < 0.0001 for both concentrations 72h after exposure (Figure 1C).The data indicate that treatment with sodium nitroprusside for 24h enhanced leishmanicidal activity in macrophages.

Sodium nitroprusside increases nitric oxide and favors formation of 3-nitrotyrosine
Exposure to 1.5µg/mL sodium nitroprusside for 24h signifi cantly increased nitric oxide (p = 0.0304, Figure 2A) FIGURE 1 -Peritoneal macrophages from BALB/c mice were infected in vitro with Leishmania amazonensis and treated for 24h with 0.5 and

A B C
in macrophages infected with L. amazonensis.Accordingly, there was increased immunostaining for 3-nitrotyrosine (p = 0.0467, Figure 2B), implying that sodium nitroprusside acts as an exogenous source of nitric oxide.Notably, 3-nitrotyrosine colocalized with the parasite in some cells.

Sodium nitroprusside does not induce iNOS expression
Semi-quantitative immunocytochemistry demonstrated that sodium nitroprusside exposure was not associated with elevated expression of iNOS (Figure 2B), indicating that the increase in nitric oxide was exogenous.

DISCUSSION
Nitric oxide is well known to be a key effector in clearing Leishmania (21) , although the parasite is capable of suppressing nitric oxide production via several mechanisms (22) (23) .Thus, drugs that release nitric oxide, including sodium nitroprusside (24) , may enhance leishmanicidal activity in macrophages.The pharmacological characteristics of these drugs were established in 1955 (25) , and clinical application has since widened (26) (27) .Indeed, sodium nitroprusside remains in use due to its effectiveness and rapid action (26) (28) , despite reports of cyanide toxicity.
A previous study in vitro demonstrated that sodium nitroprusside decreased the number of L. amazonensis promastigotes and axenic amastigotes in a dose-dependent manner (17) .In accordance with this result, we observed that the drug reduced the number of intracellular amastigotes, and, consequently, the number of promastigotes recovered (Figure 1B and 1C).Thus, we investigated the mechanism by which sodium nitroprusside enhanced leishmanicidal activity.We found that nitric oxide levels increased in the supernatant of cultured macrophages exposed to 1.5µg/mL sodium nitroprusside (Figure 2A).Consequently, 3-nitrotyrosine was generated, indicating enhanced formation of reactive nitrogen radicals (29) .Notably, 3-nitrotyrosine and nitrated proteins accumulated in phagosomes and in intracellular parasites (Figure 2B), reinforcing the idea that parasite clearance depends on reactive nitrogen species.Indeed, 3-nitrotyrosine peaks early in infection in leishmaniasis-resistant C57BL6 mice, presaging a subsequent decline in parasitosis.In contrast, 3-nitrotyrosine peaks late in leishmaniasis-susceptible BALB/c mice (30) , implying that 3-nitrotyrosine formation is a relevant indicator of antiparasitic activity.
In contrast, sodium nitroprusside stimulated levels of TNF-α (Figure 3A), a key cytokine involved in macrophage expression of iNOS (37) .However, the increase in TNF-α did not stimulate iNOS expression (Figure 2B), in line with published data demonstrating that exogenous sources of nitric oxide suppress expression this enzyme (38) (39) .Thus, we that the antileishmanial activity of sodium nitroprusside depends on its properties as a source of nitric oxide (40) .
In summary, we have demonstrated in vitro that sodium nitroprusside enhances leishmanicidal activity in macrophages infected with L. amazonensis via release of nitric oxide.Even though the drug has some toxicity and is challenging to administer, the results provide a rationale for further studies in vivo, in light of the serious limitations of current therapies, which have limited effi cacy and signifi cant toxicity, and require long treatment regimens.

Rev
FIGURE 1 -Peritoneal macrophages from BALB/c mice were infected in vitro with Leishmania amazonensis and treated for 24h with 0.5 and 1.5μg/mL sodium nitroprusside (SNP).A) Number of infected macrophages.B) Number of amastigotes per macrophage.Dashed line indicates the number of (A) infected macrophages and (B) the amount of internalized parasites after infection period (2 h).C) Leishmania amazonensis promastigotes recovered over three days after infection.*p < 0.05; **p < 0.01; ***p < 0.001 vs. control, by one-way ANOVA followed by Tukey test.