Amidine Derivatives and Leishmania amazonensis: an Evaluation of the Effect of Nitric Oxide (NO) Production on the Parasite-macrophage Interaction

Previous work has demonstrated that N-N′-diphenyl-R-benzamidine was highly effective against Leishmania amazonensis promastigotes/axenic amastigotes and Trypanosoma evansi trypomastigotes and the compound with a methoxy substituent, was the most effective derivative in the parasite-macrophage interaction. Comparative analysis of the nitric oxide (NO) released from the culture infection's supernatant showed the amidine to be less effective than pentamidine Isethionate as a reference drug. Additionally, in order to verify if the methoxylated derivative interferes with NO production by L. amazonensis, the effect of the amidine on the constitutive nitric oxide synthase (cNOS) purified from parasites, was examined, but demonstrated less activity in comparison with the reference drug. This data contributes to studies concerning the metabolic targets present in Leishmania parasites for leishmanicidal drugs.     

Effect of amidine derivatives on nitric oxide production by Leishmania amazonensis promastigotes and axenic amastigotes.

The effects of pentamidine isethionate (reference drug) and N,N'-diphenyl-4-methoxy-benzamidine (test compound) on NO. production by Leishmania amazonensis promastigotes and axenic amastigotes were investigated by measuring nitrite, a by-product of nitric oxide released into culture supernatants. The NO. production by infective promastigotes was inhibited by OCH(3)-amidine in about 23.53% and by pentamidine in only 3.78%. In axenic amastigotes, the inhibition of NO. production by OCH(3)-amidine was significantly higher (52.94%; p=0.01) than that by pentamidine, which inhibited this radical production nonsignificantly (25.29%; p=0.1). The mechanism of amidine derivatives, as an antimicrobial agent, is unknown. However, other amidines, such as a diamidine (pentamidine), contain chemical structures shared by the guanidino group of the nitric oxide synthase substrate L-arginine, suggesting the possibility of an interaction with this enzyme or electronic factors (substituent constant) that alter physical and chemical properties significant for biological activity.     

Effect of L-arginine analogs and a calcium chelator on nitric oxide (NO) production by Leishmania sp

Leishmania amazonensis, L. braziliensis and L. chagasi promastigotes were grown in the presence of L-arginine analogs such as Nomega-nitro-L-arginine methyl ester (L-NAME), NG-nitro-L-arginine (L-NNA) and D-arginine (an inactive L-arginine isomer), besides an intracellular calcium chelator [ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid; EGTA] to verify the importance of L-arginine metabolism and the cofactors for these parasites. The parasite's growth curve was followed up and the culture supernatants were used to assay nitric oxide (NO) production by the Griess reaction. The results showed a significant effect of L-arginine analogs on NO production by all Leishmania species studied, especially L-NAME, an irreversible inhibitor of the constitutive nitric oxide synthase (cNOS). When L. amazonensis promastigotes were pre-incubated with L-NAME, the infection range of the murine macrophages was lowered to 61% in 24 h and 19% after 48 h. These data demonstrated that the parasite NO pathway is important to the establishment of the infection.     

Leishmania strains causing self-healing cutaneous leishmaniasis have greater susceptibility towards oxidative stress

The survival of Leishmania parasites within macrophages is influenced by generation of free radicals. To establish whether generation of free radicals influenced chemotherapeutic response, promastigotes from isolates causing self-healing or delayed/non-self-healing cutaneous leishmaniasis (CL) or visceral leishmaniasis (VL) were evaluated for their susceptibility to nitric oxide (NO), antimony and miltefosine. In a self-healing CL strain of Leishmania major (5ASKH), susceptibility to NO and antimony was higher than other species. Likewise, a Leishmania amazonensis strain, M2269, showed greater susceptibility to NO and antimony than other species but no such correlation was observed with miltefosine. Additionally, 5ASKH and M2269 showed poorer free radical scavenging capacity as also their thiol levels were lower than species causing VL. Collectively, our study suggests that self-healing isolates tend to be more susceptible to oxidative stress.     

Leishmanicidal activity of the Agaricus blazei Murill in different Leishmania species

Leishmaniasis is a major public health problem, and the alarming spread of parasite resistance underlines the importance of discovering new therapeutic products. The present study aims to investigate the in vitro leishmanicidal activity of an Agaricus blazei Murill mushroom extract as compared to different Leishmania species and stages. The water extract proved to be effective against promastigote and amastigote-like stages of Leishmania amazonensis, L. chagasi, and L. major, with IC(50) (50% inhibitory concentration) values of 67.5, 65.8, and 56.8 μg/mL for promastigotes, and 115.4, 112.3, and 108.4 μg/mL for amastigotes-like respectively. The infectivity of the three Leishmania species before and after treatment with the water extract was analyzed, and it could be observed that 82%, 57%, and 73% of the macrophages were infected with L. amazonensis, L. major, and L. chagasi, respectively. However, when parasites were pre-incubated with the water extract, and later used to infect macrophages, they were able to infect only 12.7%, 24.5%, and 19.7% of the phagocytic cells for L. amazonensis, L. chagasi, and L. major, respectively. In other experiments, macrophages were infected with L. amazonensis, L. chagasi, or L. major, and later treated with the aforementioned extract, presented reductions of 84.4%, 79.6%, and 85.3% in the parasite burden after treatment. A confocal microscopy revealed the loss of the viability of the parasites within the infected macrophages after treatment with the water extract. The applied extract presented a low cytotoxicity in murine macrophages and a null hemolytic activity in type O(+) human red blood cells. No nitric oxide (NO) production, nor inducible nitric oxide syntase expression, could be observed in macrophages after stimulation with the water extract, suggesting that biological activity may be due to direct mechanisms other than macrophage activation by means of NO production. In conclusion, the results demonstrate that the A. blazei Murill water extract can potentially be used as a therapeutic alternative on its own, or in association with other drugs, to treat Visceral and Cutaneous Leishmaniasis.     

Toxoplasma gondii exposes phosphatidylserine inducing a TGF-beta1 autocrine effect orchestrating macrophage evasion

Toxoplasmosis is a worldwide disease caused by Toxoplasma gondii. Activated macrophages control T. gondii growth by nitric oxide (NO) production. However, T. gondii active invasion inhibits NO production, allowing parasite persistence. Here we show that the mechanism used by T. gondii to inhibit NO production persisting in activated macrophages depends on phosphatidylserine (PS) exposure. Masking PS with annexin-V on parasites or activated macrophages abolished NO production inhibition and parasite persistence. NO production inhibition depended on a transforming growth factor-beta1 (TGF-beta1) autocrine effect confirmed by the expression of Smad 2 and 3 in infected macrophages. TGF-beta1 led to inducible nitric oxide synthase (iNOS) degradation, actin filament (F-actin) depolymerization, and lack of nuclear factor-kappaB (NF-kappaB) in the nucleus. All these features were reverted by TGF-beta1 neutralizing antibody treatment. Thus, T. gondii mimics the evasion mechanism used by Leishmania amazonensis and also the anti-inflammatory response evoked by apoptotic cells.     

Low and high-dose intradermal infection with Leishmania major and Leishmania amazonensis in C57BL/6 mice

A model of skin infection with Leishmania amazonensis with low doses of parasites is compared to infection with high doses of L. amazonensis and low and high doses of Leishmania major. C57BL/6 mice were infected with 103 or 10(6) parasites in the ear and the outcome of infection was assessed. The appearance of lesions in mice infected with 103 parasites was delayed compared to mice infected with 10(6) Leishmania and parasites were detectable at the infection site before lesions became apparent. Mice infected with L. amazonensis displayed persistent lesions, whereas infection with L. major spontaneously healed in all groups, although lymphocytes persisted at the site of infection after healing. Macrophages persisted only in L. amazonensis-infected mice. High-dose L. amazonensis-infected mice produced lower levels of IFN-γ and TNF than mice infected with L. major. No correlation between the persistence of parasites and IL-10 levels and the production of nitric oxide or urea by macrophages was found. We conclude that infection with low doses of L. amazonensis in the dermis changes the course of infection by delaying the appearance of lesions. However, low-dose infection does not change the outcomes of susceptibility and cytokine production described for subcutaneous infection with high numbers of parasites.     

Leishmanicidal activity of primary S-nitrosothiols against Leishmania major and Leishmania amazonensis: implications for the treatment of cutaneous leishmaniasis.

Nitric oxide (NO) is considered a key molecule in the defense against intracellular pathogens, particularly Leishmania. The expression of inducible nitric oxide synthase and consequent production of NO by infected macrophages has been shown to correlate with leishmaniasis resistance in the murine model as well as in human patients. Nitric oxide donors have been used successfully in the treatment of cutaneous leishmaniasis in humans, although their mechanisms of action are not fully understood. In the present work, the dose-dependent cytotoxic effects of the NO-donors S-nitroso-N-acetyl-l-cysteine (SNAC) and S-nitrosoglutathione (GSNO) against Leishmania were evaluated. GSNO inhibited the growth of Leishmania major and Leishmania amazonensis with in vitro 50% inhibitory concentrations (IC(50)) of 68.8+/-22.86 and 68.9+/-7.9 micromol L(-1), respectively. The IC(50) for SNAC against L. major and L. amazonensis were, respectively, 54.6+/-8.3 and 181.6+/-12.5 micromol L(-1). The leishmanicidal activity of GSNO, but not of SNAC, was reversed by ascorbic acid (AA) and dithiothreitol (DTT), suggesting that the mechanism of action of GSNO is related to the transnitrosation of parasite proteins. These results demonstrate that SNAC and GSNO have leishmanicidal activity, and are thus potential therapeutic agents against cutaneous leishmaniasis.     

Constitutive nitric oxide synthase-like enzyme in two species involved in cutaneous and mucocutaneous leishmaniasis

Leishmania is an obligate intracellular parasite that primarily inhabits macrophages. The destruction of the parasite in the host cell is a fundamental mechanism for infection control. In addition, inhibition of the leishmanicidal activity of macrophages seems to be related to the ability of some species to inhibit the production of nitric oxide (NO) by depleting arginine. Some species of Leishmania have the ability to produce NO from a constitutive nitric oxide synthase-like enzyme (cNOS-like). However, the localization of cNOS-like in Leishmania has not been described before. As such, this study was designed to locate cNOS-like enzyme and NO production in promastigotes of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. NO production was initially quantified by flow cytometry, which indicated a significant difference in NO production between L. (L.) amazonensis (GMFC = 92.17 +/− 4.6) and L. (V.) braziliensis (GMFC = 18.89 +/− 2.29) (P < 0.05). Analysis of cNOS expression by immunoblotting showed more expression in L. (L.) amazonensis versus L. (V.) braziliensis. Subsequently, cNOS-like immunolabeling was observed in promastigotes in regions near vesicles, the flagellar pocket and mitochondria, and small clusters of particles appeared to be fusing with vesicles suggestive of glycosomes, peroxisome-like-organelles that compartmentalize the glycolytic pathway in trypanosomatid parasites. In addition, confocal microscopy analysis demonstrated colocalization of cNOS-like and GAPDH, a specific marker for glycosomes. Thus, L. (L.) amazonensis produces greater amounts of NO than L. (V.) braziliensis, and both species present the cNOS-like enzyme inside glycosomes.     

Role of peroxynitrite in macrophage microbicidal mechanisms in vivo revealed by protein nitration and hydroxylation

The cytotoxins produced by phagocytic cells lacking peroxidases such as macrophages remain elusive. To elucidate macrophage microbicidal mechanisms in vivo, we compared the lesion tissue responses of resistant (C57Bl/6) and susceptible (BALB/c) mice to Leishmania amazonensis infection. This comparison demonstrated that parasite control relied on lesion macrophage activation with inducible nitric oxide synthase expression (iNOS), nitric oxide synthesis, and extensive nitration of parasites inside macrophage phagolysosomes at an early infection stage. Nitration and iNOS expression were monitored by confocal microscopy; nitric oxide synthesis was monitored by EPR. The main macrophage nitrating agent was shown to be peroxynitrite derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells (monitored as peroxidase activity) and was accompanied by protein hydroxylation (monitored as 3-hydroxytyrosine levels). In vitro studies confirmed that peroxynitrite is cytotoxic to parasites whereas nitric oxide is cytostatic. The results indicate that peroxynitrite is likely to be produced close to the parasites and most of it reacts with carbon dioxide to produce carbonate radical anion and nitrogen dioxide whose concerted action leads to parasite nitration. In parallel, some peroxynitrite decomposition to the hydroxyl radical should occur due to the detection of hydroxylated proteins in the healing tissues. Consequently, peroxynitrite and derived radicals are likely to be important macrophage-derived cytotoxins.     

Differential properties of CBA/J mononuclear phagocytes recovered from an inflammatory site and probed with two different species of Leishmania

While CBA/J mice fail to be permissive to Leishmania amazonensis-driven pathogenic processes, they heal easily following Leishmania major infection. The early-phase events are crucial to the outcome of Leishmania infection and it is known that macrophages (Mphi) are important in infection control. In the present study we investigated the role of Mphi in driving CBA/J susceptibility to L. amazonensis. We performed kinetic studies and compared the capacity of L. amazonensis and L. major to infect Mphi. There was no difference in percentages of infection or parasite burden for 6 h between the two groups. In contrast, after 12 h we observed that infection was about twice as high in L. amazonensis- than in L. major-infected Mphi. In addition, rIFN-gamma added to the cultures induced nitric oxide (NO) production, and did not modify L. amazonensis infection, although the percentage of L. major infection was significantly reduced. This reduction in L. major infection is a TNF-alpha dependent mechanism as L. major-infected Mphi expressed twice as much TNF-alpha mRNA as L. amazonensis-infected cells, and anti-TNF-alpha reversed the IFN-gamma effect. Moreover, rTNF-alpha plus IFN-gamma were able to significantly reduce the percentage of L. amazonensis-infected cells but not to the same extent as in L. major infection. Despite having higher NO production than IFN-gamma-treated cells, AMG addition to IFN-gamma-plus TNF-alpha-treated cells only partially reversed the inhibition in L. major, but not in L. amazonensis infection. Thus, in this study, we demonstrated that L. amazonensis both inactivated and resisted innate and IFN-gamma-induced Mphi killing mechanisms, indicating that the nature of the parasite and its interaction with Mphi could determine immune response polarization.     

Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from L-arginine

Peritoneal macrophages from CBA mice incubated with rIFN-gamma are effective in killing the protozoal parasite Leishmania major in vitro. This leishmanicidal activity can be completely inhibited by L-NG-monomethyl arginine (L-NMMA), a specific inhibitor of the L-arginine:nitric oxide (NO) pathway. The culture supernatants of macrophage activated by IFN-gamma contain increased levels of NO2-, the production of which is inhibited by L-NMMA, but not by its D-enantiomer. L. major promastigotes are killed when incubated at room temperature in PBS containing NO. These data demonstrate that NO is an effector mechanism in macrophage killing of intracellular protozoa. The importance of NO in vivo is demonstrated by the finding that CBA mice infected with L. major developed exacerbated disease when L-NMMA was injected into the lesions, resulting in 10(4)-fold increases in the number of parasites extractable from the lesions.     

Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during meta-cyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.     

Applications of recombinant Leishmania amazonensis expressing egfp or the beta-galactosidase gene for drug screening and histopathological analysis.

Leishmania amazonensis recombinants expressing the enhanced green fluorescent protein (egfp) gene or beta-galactosidase gene (lacZ) were constructed for drug screening and histopathological analysis. The egfp or lacZ in a leishmanial transfection vector, p6.5, was introduced into L. amazonensis promastigotes, and egfp or lacZ-carrying recombinant L. amazonensis, La/egfp and La/lacZ, respectively, were obtained. Expression of egfp or lacZ in both promastigotes and amastigotes could be clearly visualized by fluorescence microscopy or by light microscopy with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), respectively. Fluorescence signal and beta-galactosidase activity measured by a colorimetric reaction with chlorophenol red beta-D-galactopyranoside (CPRG) were well correlated to the numbers of these parasites. The inhibitory concentration (IC50) of a leishmanicidal drug, amphotericin B, in L. amazonensis promastigotes measured using La/egfp and La/lacZ was similar to that measured by conventional methods such as cell counting, thymidine incorporation and colorimetric assay. Furthermore, the fluorescence signal and absorbance of CPRG correlated well with the numbers of La/egfp and La/lacZ amastigotes in macrophages, respectively, suggesting La/egfp and La/lacZ can be a convenient and useful tool for drug screening not only in promastigotes, but also in amastigotes of L. amazonensis. La/lacZ collected from mouse tissues four weeks after the parasite infection were stained well with X-Gal. La/lacZ allowed parasite detection at high sensitivity in the tissues of infected mice and will be useful for following infections in macrophages in vivo. Thus, the marker-transfected Leishmania parasites constructed in this study will be useful for analyses of Leishmania parasites, especially at the intracellular stage.     

In vitro sodium nitroprusside-mediated toxicity towards Leishmania amazonensis promastigotes and axenic amastigotes

Leishmania parasites survive despite exposure to the toxic nitrosative oxidants during phagocytosis by the host cell. In this work, the authors investigated comparatively the resistance of Leishmania amazonensis promastigotes and axenic amastigotes to a relatively strong nitrosating agent that acts as a nitric oxide (NO) donor, sodium nitroprusside (SNP). Results demonstrate that SNP is able to decrease, in vitro, the number of L. amazonensis promastigotes and axenic amastigotes in a dose-dependent maner. Promastigotes, cultured in the presence of 0.25, 0.5, and 1 mmol L(-1) SNP for 24 h showed about 75% growth inhibition, and 97-100% when the cultures were treated with >2 mmol L(-1) SNP. In contrast, when axenic amastigotes were growing in the presence of 0.25-8 mM SNP added to the culture medium, 50% was the maximum of growth inhibition observed. Treated promastigotes presented reduced motility and became round in shape further confirming the leishmanicidal activity of SNP. On the other hand, axenic amastigotes, besides being much more resistant to SNP-mediated cytotoxicity, did not show marked morphological alteration when incubated for 24 h, until 8 mM concentrations of this nitrosating agent were used. The cytotoxicity toward L. amazonensis was attenuated by reduced glutathione (GSH), supporting the view that SNP-mediated toxicity triggered multiple oxidative mechanisms, including oxidation of thiols groups and metal-independent oxidation of biomolecules to free radical intermediates.     

Effect of hypoxia on macrophage infection by Leishmania amazonensis

In the present study, we compared the effect of 5% oxygen tension (hypoxia) with a normal tension of 21% oxygen (normoxia) on macrophage infection by the protozoan parasite Leishmania amazonensis. Macrophages from different sources (human cell line U937, murine cell line J774, and murine peritoneal macrophages) exposed to hypoxia showed a reduction of the percentage of infected cells and the number of intracellular parasites per cell. Observations on the kinetics of infection indicated that hypoxia did not depress L. amazonensis phagocytosis but induced macrophages to reduce intracellular parasitism. Furthermore, hypoxia did not act synergistically with gamma-interferon and bacterial lipopolysaccharides in macrophages to induce killing of parasites. Experiments also indicated no correlation between nitric oxide production and control of infection in macrophages under hypoxic condition. Thus, we have provided the first evidence that hypoxia, which occurs in various pathological conditions, can alter macrophage susceptibility to a parasitic infection.     

The role of interleukin-10 in susceptibility of BALB/c mice to infection with Leishmania mexicana and Leishmania amazonensis

Recent studies have demonstrated the critical role of IL-10 in susceptibility to cutaneous and visceral leishmaniasis caused by Leishmania major and Leishmania donovani, respectively. To determine whether IL-10 also plays a similar role in the susceptibility and pathogenesis of cutaneous leishmaniasis caused by the New World species, L. mexicana and L. amazonensis, we analyzed their course of infection in IL-10-deficient BALB/c mice and their wild-type counterparts. Although IL-10-deficient mice infected with either L. mexicana or L. amazonensis failed to control the lesion progression, we did observe consistently lower levels of infection in IL-10(-/-) mice compared with wild-type BALB/c mice. We also observed increased IFN-gamma and NO production and higher levels for IL-12p40 and IL-12Rbeta(2) mRNA in cells from IL-10(-/-) mice compared with cells from BALB/c mice. The mRNA levels for IL-4, which increased significantly in both IL-10(-/-) and BALB/c mice, were comparable. When treated with anti-IL-4 mAb, IL-10(-/-) mice resolved the infection more effectively and had significantly fewer parasites in their lesions compared with similarly treated BALB/c mice. These findings suggest that IL-10, although not the dominant mediator of susceptibility of BALB/c mice to infection with L. mexicana and L. amazonensis, does play a significant role in regulating the development of a protective Th1-type response. However, effective resolution of infection with these New World parasites requires neutralization of both IL-4 and IL-10.