Experimental Chemotherapy in Paracoccidioidomycosis Using Ruthenium NO Donor

Mycopathologia (2011) 172:95–107 DOI 10.1007/s11046-011-9416-8

Experimental Chemotherapy in Paracoccidioidomycosis Using Ruthenium NO Donor Wander Roge´rio Pavanelli • Jean Jerley Nogueira da Silva • Carolina Panis • Thiago Mattar Cunha • Ivete Conchon Costa • Maria Claudia Noronha Dutra de Menezes Francisco Jose´ de Abreu Oliveira • Luiz Gonzaga de Franc¸a Lopes • Rubens Cecchini • Fernando de Queiroz Cunha • Maria Ange´lica Ehara Watanabe • Eiko Nakagawa Itano

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Received: 14 September 2010 / Accepted: 6 March 2011 / Published online: 25 March 2011 Ó Springer Science+Business Media B.V. 2011

Abstract Paracoccidioidomycosis (PCM) is a granulomatous disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb). To determine the influence of nitric oxide (NO) on this disease, we tested cis-[Ru(bpy)2(NO)SO3](PF6), ruthenium nitrosyl, which releases NO when activated by biological reducing agents, in BALB/c mice infected intravenously with Pb 18 isolate. In a previous study by our group, the fungicidal activity of ruthenium nitrosyl was evaluated in a mouse model of acute PCM, by measuring the immune cellular response (DTH), histopathological characteristics of the granulomatous

This paper is based on part of the post-doctoral work of WR Pavanelli.

Electronic supplementary material The online version of this article (doi:10.1007/s11046-011-9416-8) contains supplementary material, which is available to authorized users. W. R. Pavanelli  C. Panis  I. C. Costa  M. C. N. D. de Menezes  F. J. de Abreu Oliveira  R. Cecchini  M. A. E. Watanabe  E. N. Itano Department of Pathology Science, CCB, State University of Londrina-UEL, Londrina, PR, Brazil J. J. N. da Silva Institute of Physics of Sa˜o Carlos, University of Sa˜o Paulo-USP, Sa˜o Paulo, Brazil

lesions (and numbers), cytokines, and NO production. We found that cis-[Ru(bpy)2(NO)SO3](PF6)-treated mice were more resistant to infection, since they exhibited higher survival when compared with the control group. Furthermore, we observed a decreased influx of inflammatory cells in the lung and liver tissue of treated mice, possibly because of a minor reduction in fungal cell numbers. Moreover, an increased production of IL-10 and a decrease in TNF-a levels were detected in lung tissues of infected mice treated with cis-[Ru(bpy)2(NO)SO3](PF6). Immunohistochemistry showed that there was no difference in the number of VEGF- expressing cells. The animals treated with cis-[Ru(bpy)2(NO)SO3](PF6) showed high NO levels at 40 days after infection. These results show that NO is effectively involved in the mechanism that regulates the immune response in lung of Pb-infected mice. These data suggest that NO is a resistance factor during paracoccidioidomycosis by L. G. de Franc¸a Lopes Department of Organic and Inorganic Chemistry, Federal University of Ceara´, Fortaleza, Brazil W. R. Pavanelli (&) Universidade Estadual de Londrina-UEL-Rodovia Celso Garcia Cid, Campus Universita´rio, Cx. Postal 6001, Londrina, PR 86051-990, Brazil e-mail: [email protected]

T. M. Cunha  F. de Queiroz Cunha Department of Pharmacology, Faculty Medicine of Ribeira˜o Preto-USP, Ribeira˜o Preto, Brazil

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controlling fungal proliferation, influencing cytokine production, and consequently moderating the development of a strong inflammatory response. Keywords Ruthenium nitrosyl  NO donors  Nitric oxide  Paracoccidioidomycosis

Introduction Paracoccidioidomycosis (PCM), a deep mycosis endemic in Latin America, is caused by the thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) that develops as a yeast at body temperature and as a mycelium at room temperature. Pb causes natural infections by inhalation of conidia or mycelial elements [1]. Most exposed subjects develop an asymptomatic infection, although some individuals present with clinical manifestations that can vary from benign and localized to severe and disseminated forms [2]. Patients with benign PCM usually present with cellular immune response to the Th1 pattern with the production of IFN-c and TNF-a and basal levels of IL-4, IL-5, and IL-10, while those with the disseminated disease typically show immune response to the Th2 pattern with high production of IL-4, IL-5, and IL-10 and impaired secretion of IFN-c [2]. No consensus exists regarding the determinants of a Th1 or Th2 immune response in PCM [3]. However, several factors may contribute to this process, such as type of cytokine produced in the microenvironment of the infection and antigenpresenting cells, host genetic background, and dose of infectious microorganism [4, 5]. Beside these factors, previous studies have suggested that nitric oxide (NO), generated from the amino acid L-arginine by the inducible isoform of NO synthase (iNOS or NOS2), was involved in the immune response and consequent elimination of P. brasiliensis [6]. Killing of pathogens mediated by NO has been described in several infectious and parasitic diseases including those caused by Mycobacterium tuberculosis [7], Leishmania major [8], Schistosoma mansoni [9], and Histoplasma capsulatum [10]. Fungicidal and immunosuppressive properties of NO were described by studies with P. brasiliensis infection. In experimental models of PCM, some authors suggested a protective role of NO, whereas

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others showed that its persistent production is associated with susceptibility, depending on the level and persistence of NO production during infection [6, 11, 12]. A dual role of NO was shown in susceptible and resistant mice infected with P. brasiliensis, suggesting a protective effect in the early phase of infection, but a deleterious action associated with a more severe disease with time [12]. There is evidence that inflammatory cytokines and nitric oxide play an important role in the genesis and control of PCM. Therefore, a ruthenium NO donor, trans-[RuII(NO?)(NH3)4L]3?, was chosen as a good model for assessing fungicidal activity in vivo. Besides its low toxicity, it shows good water solubility and stability in aqueous media in the presence of oxygen, and the NO released by these types of compounds at the site of action can be controlled through the judicious selection of the trans ligand (L) [11]. Additionally, these compounds are activated to release NO by reducing agents present in a biological milieu [13]. Hence, the features shown by these types of compounds are quite promising for designing metallopharmaceuticals, especially to combat infectious diseases where the NO concentration has to be high enough to prevent the development of the microorganisms but not so high as to cause immunosuppression, inhibition of respiratory complexes and acotinase, DNA modifications, or apoptosis in the host cells [14]. In fact, studies have demonstrated that ruthenium complexes [Ru(Ctz)2(H2O)2](PF6)2 and [Ru(Ktz)2(H2O)Cl3] acting in synergism with anti-fungal drugs are more active than the corresponding free ligands [15, 16]. Moreover, previous studies [15, 16] have shown that ruthenium complexes are able to inhibit 70% of the proliferation of epimastigote forms of T. cruzi [17]. Following this approach, the [Ru(NH3)4L]n? moiety has been successfully tested as a NO carrier in vitro and in vivo [18, 19], because the trypanocidal effect of activated macrophages has been ascribed to NO production [18]. In addition, in a recent related work, trans-[Ru(NO)(NH3)4L]n?, where L = N-heterocyclic, SO32-, or P(OEt)3 exhibited not only low cytotoxicity but also anti-T. cruzi activity due to NO action [19]. However, in the present study, we proposed to determine the influence of a ruthenium donor of nitric oxide on the inflammatory response induced against P. brasiliensis.

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Materials and Methods Animals Female BALB/c mice, aged 6–8 weeks, were bred and maintained in microisolator cages in the animal housing facility of the Department of Pathology Science, CCB, State University of Londrina-UEL, in accordance with the local protocols of ethics in animal care. The procedures involving animals and their care were conducted in agreement with national and international policies. Fungus and Murine Infection Yeast cells of virulent strain P. brasiliensis 18 were cultured at 36°C in Fava-Netto’s medium for 7 days. The yeast cells were harvested and washed three times in phosphate-buffered saline (PBS), pH 7.2. The viability of yeast cells was determined as previously described [20]. The animals (n = 5) were infected intravenously (i.v.) with 1 9 106 viable yeast cells in 100 ll of PBS. Negative controls (n = 5) were inoculated i.v. with 100 ll of vehicle [21]. For survival determination, the animals (n = 5) were infected intravenously (i.v.) with 1 9 108 viable yeast cells in 100 ll of PBS and treated with 100 lM cis-[Ru(bpy)2(NO)SO3](PF6) or not (control group) and were followed for up to 60 days of infection. Chemicals, Drugs, and Reagents Ruthenium trichloride from Aldrich Chemical Company (ACC) was the starting material for the synthesis of the ruthenium complex described here. All solvents were purified following known procedures [22] and double-distilled water was used throughout. The synthesis and all manipulations were carried out under argon atmosphere [23].

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spectrophotometer, Hitachi (model Z-8100), with a Hitachi Hollow Cathode Lamp, 12 mA, and k = 349.9 nm. UV visible measurements were performed in a 1.0 cm quartz cell in a Hewlett-Packard diode array model 8452A spectrophotometer. IR spectra were recorded with a Bomem FTIR, model MB-102, spectrophotometer in the 400–4,000 cm-1 range, with the sample supported in potassium bromide pellets. A polarographic analyzer/stripping voltammeter model 264A from Princeton Applied Research attached to a microcomputer and employing Microquı´mica Eletrochemical software was used for the electrochemical measurements. The electrochemical cell used was a conventional three-electrode type with an aqueous saturated calomel electrode as a reference electrode and a glassy carbon and platinum wire as working and auxiliary electrodes, respectively. DTH Reaction The delayed-type hypersensitivity reaction (DTH) was evaluated by injecting 25 ll of exoantigen (EXO) derived from Pb18 (2 mg/ml) into the footpads of mice on days 19 and 39 after infection by P. brasiliensis. The size of the swelling was determined on days 20 and 40 after infection and with a caliper (Mitutoyo Corporation, Tokyo, Japan). Histological Analysis Groups of 5 mice were euthanized after 20 and 40 days of infection with P. brasiliensis. The lung and liver were fixed in 10% formaldehyde in PBS, embedded in paraffin, sectioned, stained with hematoxylin eosin, and examined by light microscopy. The number of granulomatous lesions in the lung and liver were quantified by histocytometry using an image analyzer (BioScan/OPTIMAS; Media Cybernetics, Silver Spring, MD). Values were expressed as the mean ± SEM of triplicate sections.

Synthesis and Instrumentation Organ Colony-Forming Units The ruthenium NO donor cis-[Ru(bpy)2(NO)SO3] (PF6) was synthesized and characterized following published procedures [24, 25]. Elemental analysis of hydrogen, carbon, and nitrogen was carried out using an EA 1110 CHNS-O CE instrument. Analysis of ruthenium was performed as described elsewhere [26], using a Polarized Zeeman atomic absorption

The lung and liver fractions were removed, weighed, homogenized, and washed three times in phosphate buffered, and the resuspended pellet was plated on brain–heart infusion (BHI) agar supplemented with 4% fetal serum and 5% spent culture medium from P. brasiliensis as a growth factor. Gentamicin and

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chloramphenicol were added at 60 and 100 lg/ml, respectively. The plates were incubated at 35°C and read after 8 days. The results were expressed as the number of P. brasiliensis CFUs per mg of tissue per mouse. Cytokine Assays Concentrations of cytokines in tissue homogenates from lung were measured by ELISA. IFN-c and IL-10 (OpTEIA, BD Bioscience, San Diego-CA, USA) and IL-4 (Duoset R&D Systems, Minneapolis-MN, USA) were assayed following the manufacturer’s instructions. The reaction was revealed with peroxidase-conjugated streptavidin (Vector Laboratories, Burlingame-CA, USA) followed by the substrate containing TMB (Promega, Madison-WI, USA) as a chromogen. Optical densities (O.D.) of samples were then read at 450 nm, and the concentrations of cytokines were determined by extrapolation from a standard curve of each recombinant cytokine. Immunohistochemical Staining of Vascular Endothelial Growth Factor

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Determination of Nitrite Levels Sample nitrite was determined according to Panis et al., 2010 [27] with some modifications. Briefly, serum aliquots were deproteinized by adding 50 lL of 75 mM ZnSO4 and 70 lL of NaOH, shaken, and centrifuged at 10,000 rpm for 5 min, 25°C. The clear supernatant was recovered and diluted in glycine buffer (45 g/L, pH 9.7). Cadmium granules were rinsed in sterile distilled water for 5 min and added to a 5 mM CuSO4 solution in glycine-NaOH buffer (15 g/l, pH 9.7), and the copper-coated cadmium granules were used within 10 min. The activated granules were added to glycine buffer-diluted supernatant and stirred for 10 min. Aliquots of 200 lL were recovered in appropriate tubes for nitrite determination and the same volume of Griess reagent was added. After an incubation of 10 min at room temperature, the tubes were centrifuged at 10,000 rpm, for 2 min at 25°C, and the resuspended pellet added to 96-well microplates in triplicate. A calibration curve was prepared by dilution of NaNO2, and the absorbance was determined at 505 nm in a microplate reader. Statistical analysis

Immunohistochemistry for VEGF was performed on 3 lm-thick paraffin-embedded sections from lung in both group by the labeled streptavidin–biotin method using an LSAB kit (DAKO Japan, Kyoto, Japan) with microwave antigen retrieval. The paraffin-embedded sections were heated for 30 min at 65°C, deparaffinized in xylene, and rehydrated through a graded ethanol series at room temperature. Incubations were performed in a humidified chamber. Sections were treated for 40 min at room temperature with 2% BSA and incubated overnight at 4°C with primary antibody (anti-VEGF rabbit polyclonal antibody diluted 1:50, Sigma). Horseradish peroxidase activity was visualized by treatment with H2O2 and 3,30 -diaminobenzidine (DAB) for 5 min. In the last step, the sections were weakly counterstained with Harry’s hematoxylin (Merck). For each case, negative controls were performed on serial sections. On the control sections, incubation with the primary antibodies was omitted. Intensity and localization of immunoreactivities against all primary antibodies used were examined on all sections using a photomicroscope (Leica DM 2500) and the score determined.

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Results are expressed as mean ± SEM of two independent experiments. Student’s t test was used to analyze the statistical significance of the observed differences. The Kaplan–Meier method was used to compare survival times of the groups studied. Values of P B 0.05 were considered significant.

Results Influence of Treatment with Ruthenium NO Donor on the Survival of P. brasiliensis-Infected mice To determine the effect of ruthenium NO donor on mortality during the acute phase of PCM, mice were treated for 20 days with 100 lM cis-[Ru(bpy)2(NO)SO3](PF6) or vehicle. We observed that 100% of infected mice treated with cis-[Ru(bpy)2(NO)SO3](PF6) survived up to 60 days p.i. The mortality of the infected mice treated with vehicle (control group) was 40% on day 19 p.i. and was as high as 80% on day 35 p.i. (Fig. 1).

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Days after infection Fig. 1 Survival curves of BALB/c mice treated with cis[Ru(bpy)2(NO)SO3](PF6) or control. BALB/c mice were infected i.v. with 1 9 108 viable yeast cells of P. brasiliensis and treated with cis-[Ru(bpy)2(NO)SO3](PF6) or vehicle for 20 days. The mortality of these mice was also evaluated. Data are representative of two independent experiments with 5 mice per group. The data shown represent the mean ± SEM of the results obtained. *P \ 0.05, cis-[Ru(bpy)2(NO)SO3](PF6)treated mice versus control mice

Treatment with NO Donor Improves Cellular Immune Response In attempt to determine whether the treatment with ruthenium NO donor improved cellular immune response in PCM, we performed the DTH test (delayed type hypersensitivity reaction). The treated animals showed a 2-fold increase in cellular immune response when compared to untreated infected animals (control mice), only on the 20th day after infection (Fig. 2). The Influence of cis-[Ru(bpy)2(NO)SO3](PF6) Administration on Inflammatory Response in Lung and Liver of P. brasiliensis-Infected Mice NO is implicated in the regulation of inflammation where it augments or decreases cell migration to inflamed tissue [28]. Therefore, we evaluated the inflammatory response in lung and liver of P. brasiliensis-infected mice. The animals treated with cis[Ru(bpy)2(NO)SO3](PF6) showed reduced and focal inflammation in lung and liver (Figs. 3a, 4a) when compared to control mice (vehicle). In fact, quantitative analysis clearly showed in both organs of treated animals a decreased number of granulomas (Figs. 3b, 4b). Accordingly, this difference was most evident at 40 days after infection. These results indicated that NO

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Days after infection Fig. 2 Evaluation of delayed type hypersensitivity reaction (DTH). BALB/c mice were infected i.v. with 1 9 106 viable yeast cells of P. brasiliensis and treated with cis-[Ru(bpy)2(NO)SO3](PF6) or vehicle for 20 days. DTH was evaluated by inoculating 25 ll of exoantigen (EXO) derived from Pb18 into the footpads of mice on days 19 and 39 after infection by P. brasiliensis. Data are representative of two independent experiments with 5 mice per group. The data shown represent the mean ± SEM of the results obtained. *P \ 0.05, cis[Ru(bpy)2(NO)SO3](PF6)-treated mice verus control mice

released in the lung and liver of P. brasiliensis-infected mice contributed to the abrogation of an inflammatory response, consequently reducing tissue injury. Fungicidal Activity of cis-[Ru(bpy)2(NO)SO3](PF6) in PCM We observed that at 40 days post-infection, mice treated with NO donor showed lesions with compact granulomas containing a small number of cells (CFUs) of P. brasiliensis in the lung and liver (Fig. 5a, b), without spreading to the spleen (data not shown). This result demonstrates that treatment with the ruthenium NO donor was able to eliminate the fungus at an early stage of infection. Detection of VEGF-Positive Cells in Lung of P. brasiliensis-Infected Mice Treated with cis-[Ru(bpy)2(NO)SO3](PF6) Vascular endothelial growth factor (VEGF) is widely regarded as a potent stimulator of angiogenesis, edema, inflammation, and vascular remodeling [29]. Immunohistochemistry was performed to detect VEGF cells, with positive immunostaining found present in lung sections from mice treated with cis[Ru(bpy)2(NO)SO3](PF6) or vehicle. The results

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Days after infection Fig. 3 NO release lessens histological alteration in the lung of mice infected with P. brasiliensis. BALB/c mice were infected i.v. with 1 9 106 viable yeast cells of P. brasiliensis and treated with cis-[Ru(bpy)2(NO)SO3](PF6) or vehicle, and the lung removed, fixed, and examined using photomicrography. a Representative photomicrography of lung pathology of BALB/c mice treated with cis-[Ru(bpy)2(NO)SO3](PF6) or

vehicle at days 20 and 40 p.i. b Quantification of inflammatory score. Data are representative of two independent experiments with 5 mice per group. The data shown represent the mean ± SEM of the results obtained. *P \ 0.05, cis[Ru(bpy)2(NO)SO3](PF6)-treated mice versus control mice. Scale bars = 50 lm

demonstrated that the treatment with NO donor did not change the distribution of VEGF-positive cells at the two times examined when compared with the control group (Fig. 6a). In fact, quantitative analysis revealed no differences between the treated and control mice (Fig. 6b).

Effect of Ruthenium NO Donor Administration on Cytokine Production in P. brasiliensis-Infected Mice

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Several works show the importance of a different profile of cytokines during PCM. Therefore, we

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Days after infection Fig. 4 NO release lessens histological alteration in the liver of mice infected with P. brasiliensis. BALB/c mice were infected i.v. with 1 9 106 viable yeast cells of P. brasiliensis and treated with cis-[Ru(bpy)2(NO)SO3](PF6) or vehicle, and the liver removed, fixed, and examined using photomicrography. a Representative photomicrography of liver pathology of BALB/c mice treated with cis-[Ru(bpy)2(NO)SO3](PF6) or

vehicle on days 20 and 40 p.i. b Quantification of inflammatory score. Data are representative of two independent experiments with 5 mice per group. The data shown represent the mean ± SEM of the results obtained. *P \ 0.05, cis[Ru(bpy)2(NO)SO3](PF6)-treated mice versus control mice. Scale bars = 50 lm

determined the kinetics of cytokine production (TNF-a, IL-4 and IL-10) in the lung of infected mice treated with ruthenium NO donor or vehicle (controls). The cis-[Ru(bpy)2(NO)SO3](PF6) treatment caused a significant decrease in TNF-a levels in the lung by 20 days p.i., compared to control mice (Fig. 7a). However, the production of IL-10 in treated animals was higher in both periods analyzed when compared with control mice (Fig. 7b). However, the production of IL-4 was significantly higher in the control group than in NO-treated mice (Fig. 7c). Therefore, these results demonstrate that NO delivery directly induced an anti-inflammatory condition, but not a Th2 profile.

Effect of Ruthenium NO Donor Administration on Nitric Oxide Production in Infected Mice Nitric oxide (NO) is one of the most important mediators involved in fungicidal mechanisms during P. brasiliensis infection [6, 11, 12]. Therefore, we measured NO levels in the serum of mice treated with cis-[Ru(bpy)2(NO)SO3](PF6) or vehicle. At 40 days after infection, the animals treated with NO donor showed higher serum levels of NO when compared to control mice (Fig. 8). This finding suggests that the elevated production of NO in mice treated with cis[Ru(bpy)2(NO)SO3](PF6) was responsible for the elimination of P. brasiliensis, principally in the liver.

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Days after infection Fig. 5 Fungicidal activity of cis-[Ru(bpy)2(NO)SO3](PF6) in BALB/c mice with PCM, treated with NO donor or not. BALB/c mice were infected i.v. with 1 9 106 viable yeast cells of P. brasiliensis and treated with cis-[Ru(bpy)2(NO)SO3](PF6) or vehicle for 20 days; the lung and liver were removed, fixed, stained with gray nitrate, and examined using photomicrography. Figure 5a and b showed the numbers of viable fungal cells in lung and liver, respectively. Data are representative of two independent experiments with 5 mice per group. The data shown represent the mean ± SEM of the results obtained. *P \ 0.05 and **P \ 0.01, cis-[Ru(bpy)2(NO)SO3](PF6)-treated mice versus control mice

Discussion In human and experimental PCM, several findings suggest that nitric oxide (NO) plays an essential role in host defense against P. brasiliensis. In fact, in evaluating the influence of treatment with cis-[Ru(bpy)2(NO)SO3](PF6) (NO donor) in experimental PCM, we found that NO release induced host resistance against P. brasiliensis infection, since 100% of treated mice survived for at least 60 days (Fig. 1). Animals that are resistant to these microorganisms release high concentrations of this mediator and

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become susceptible to infection when treated with NOS inhibitors, as observed also in animals that are genetically deprived of iNOS [7, 30, 31]. These results suggest that endogenously produced NO is involved in the control of fungal infection. Confirming this notion, Bocca et al. (1998) [11] observed that treating the infected animals daily with Nx-nitro-Larginine (nitro-Arg) blocked NO production and caused a significant increase in the lung fungal burden by day 60 of infection, and the time to death of the Nitro-Arg-treated animals was sorted compared with untreated animals. In our laboratory, using a model of T. cruzi infection, the treatment for infected animals with an NO donor, similar to that used in this study, markedly increased survival (to 60%) [19]. It is known that one of the most prominent functions of NO in the immune system is its participation in protective immunity, which may directly and indirectly modulate the inflammatory response. The immunosuppressor activity of NO has been reported in vitro and in several in vivo models of infection, including T. gondii [32], L. monocytogenes [33], and P. vinckei [34]. Teixeira et al. (1987) [35] previously showed that P. brasiliensis infection in susceptible mice leads to the suppression of humoral and cellular responses. Later, it was found that the low lymphoproliferative response of B6 mice infected with P. brasiliensis was prevented by treatment with Nx-nitro-L-arginine, an inhibitor of NOS1 and NOS2, during infection [11]. In our studies, we found no differences in cellmediated immune response between the groups analyzed (Fig. 2). This effect has been associated with the low concentration of the compound utilized in treatment. Knowing that the lung is one of the most compromised organs in PCM disease, with consequent deterioration of pulmonary function, we then evaluated the inflammatory response to this organ and observed that NO release induced a mild and focal inflammatory response (Fig. 3a) In the animals that were treated with NO donor, these lesions were present in smaller numbers and were well defined, compact, and circumscribed, mainly consisting of epithelioid cells surrounding most of the fungi present in the center of the granuloma. The number of granulomas was reduced in the lung and liver of the cis-[Ru(bpy)2(NO)SO3](PF6)-treated animals

Mycopathologia (2011) 172:95–107 Fig. 6 Vascular endothelial growth factor (VEGF) immunoreactivity in lung of BALB/c mice, treated with NO donor and control. BALB/c mice were infected i.v. with 1 9 106 viable yeast cells of P. brasiliensis and treated with cis [Ru(bpy)2(NO)SO3](PF6) or vehicle for 20 days, and the lung removed, fixed, and examined using photomicrography. a Representative photomicrography of VEGF presence in lung of BALB/c mice treated with NO donor or vehicle at days 20 and 40 p.i. b Percentage of VEGF per area in lung. Data are representative of two independent experiments with 5 mice per group. The data shown represent the mean ± SEM of the results obtained

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(Fig. 4a). In fact, the quantification (inflammatory index) of the inflammatory cells confirmed these data. Our results are in consonance with a previous report showing that NOS2-deficient mice were more susceptible to P. brasiliensis infection, as revealed by extensive granulomatous lesions in the lungs and parenchyma of the liver in these animals [11, 35]. It is possible that this attenuated inflammatory response found in the lung of animals treated with an NO donor is due in part to increased production of IL-10 (Fig. 6a), which has immunomodulatory

activity. A known function of IL-10 is its ability to modulate the production of several proinflammatory mediators, including cytokines and chemokines, thus reducing the severity of the inflammatory response to P. brasiliensis infection. In fact, the suppressor effects of IL-10 on protective immune responses against fungi have also been detected in several models of murine infections with C. albicans [36–38], Histoplasma capsulatum [39], Aspergillus fumigatus [37], and Coccidioides immitis [40].

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Days after infection Fig. 7 Kinetics of cytokine production during P. brasiliensis infection. BALB/c mice were infected i.v. with 1 9 106 viable yeast cells of P. brasiliensis and treated with cis-[Ru(bpy)2(NO)SO3](PF6) or vehicle for 20 days; the animals were euthanized at different time points after infection. The concentrations of TNF-a (7A), IL-10 (7B), and IL-4 (7C) were determined in tissue homogenate by ELISA. Data are representative of two independent experiments with 5 mice per group. The data shown represent the mean ± SEM of the results obtained. *P \ 0.05, cis-[Ru(bpy)2(NO)SO3](PF6)treated mice versus control mice. The dashed line represents values obtained on day zero for mice not infected and treated

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In addition, we examined the production of TNFa, an important cytokine responsible for the increased expression of vascular endothelial growth factor (VEGF) in various tissues [41–43], and the recruitment/activation of leukocytes during the immune response, contributing to the formation of a functional granuloma [44]. In our investigation, we found that cis-[Ru(bpy)2(NO)SO3](PF6)-treated mice showed reduced levels of TNF-a, compared to untreated mice (Fig. 6b), but no difference was found regarding VEFG staining in the lung (Fig. 7a, b). These results together showed that treatment with the NO donor reduced cell migration (inflammatory response), by increasing the production of the immunomodulatory cytokine IL-10 and decreasing the synthesis of proinflammatory TNF-a. Interestingly, this modulating effect of the inflammatory response had already been seen in a model of T. cruzi infection [45]. These findings were associated with reduced or absent damage (fibrosis) in lung and liver during experimental PCM (data not show). Several experimental infection models demonstrated that fibrosis induced by S. mansoni, M. tuberculosis [46], and M. avium [47] are regulated by differential ARG-1 and iNOS expression. Thus, an anti-fibrotic role was attributed to NO. Nitric oxide could decrease the accumulation of ECM deposits by modulating the expression and activity of zinc-dependent matrixdegrading enzymes, called MMPs. These proteases are produced as inactive forms, called zymogens or pro-MMPs and require a proteolytic cleavage to become active MMPs [48]. Since cis-[Ru(bpy)2(NO)SO3](PF6)-treated mice had high levels of circulating nitric oxide, as shown in this Fig. 8, we evaluated the microbicidal activity of this agent. We found that treatment with the NO donor reduced the fungal cell numbers in both organs examined (Fig. 5a, b) at 20 days p.i., showing that circulating NO is able to kill fungi and hence control parasite multiplication. In accordance with our data, published studies in the literature [7, 12] also report this phenomenon. Nascimento et al. (2002) [12] also found that in both mouse strains (BALB/c and B6) transient NOS2 inhibition exacerbated the infection, i.e., the infection spread extensively to the lungs and liver. These data suggest that regardless of the mouse strain, NO plays an essential role in the control of fungal dissemination.

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105

*

Control cis-[Ru(bpy)2(NO)SO3](PF6)

Nitrite Levels/serun

40 35 30 25 20 15 10 5 0

----------------------------------------------20.0

40.0

Days after infection Fig. 8 Kinetics of nitric oxide production in serum of BALB/c mice, treated with NO donor and control. BALB/c mice were infected i.v. with 1 9 106 viable yeast cells of P. brasiliensis and treated with cis-[Ru(bpy)2(NO)SO3](PF6) or vehicle for 20 days. Serum levels of nitrite/nitrate are shown for BALB/c mice treated with cis-[Ru(bpy)2(NO)SO3](PF6) or vehicle at days 20 and 40 p.i. Data are representative of two independent experiments with 5 mice per group. The data shown represent the mean ± SEM of the results obtained. *P \ 0.05, cis[Ru(bpy)2(NO)SO3](PF6)-treated mice versus control mice. The dashed line represents values obtained on day zero for mice not infected and treated

In general, when the fungus enters a host, it has to interact with various effector cells (macrophage, polymorphonuclear leukocytes, and monocytes), and for successful colonization, it needs to resist their microbiostatic and microbicidal mechanisms. Several groups have studied the interactions of murine and human effector cells, with both the infective conidia and the parasitic yeast forms of P. brasiliensis [49]. Gonzales et al. (2000) (6) revealed that in vitro the inhibitory and/or killing mechanism used by activated murine macrophage against this pathogen involves NO production, confirming the results obtained in vivo by Bocca et al. (1998) [11] and suggesting that NO is important in fungal killing. The cellular diffusion and half-life of the NO molecule are important factors in a better understanding of the fungicidal activity of NO. NO molecules released by our NO donors probably do not act only at the site of release but also at considerable distances. This NO property is an important factor to be considered as charged, inorganic, water-soluble NO scavengers should remain preferentially in the bloodstream instead of crossing lipophilic host cell membranes [50]. It is likely that the NO donor studied may

also face some resistance in crossing these membranes, but the extracellularly released NO could still diffuse into the cell [14]. This explains the results obtained from analyses of the lung and liver photomicrographs. These micrographs showed that the compound cis-[Ru(bpy)2(NO)SO3](PF6) was able to eliminate intracellular as well as extracellular yeast cells of P. brasiliensis, thus reducing the inflammatory infiltrates in the tissues. Thus, it is clear that NO plays a fundamental role in the effector mechanism underlying resistance and may act intra- and extracellularly through different metabolites. Our results clearly show that NO donor treatment for infected mice resulted in fungal killing (blocking parasite multiplication), amelioration of the inflammatory response in the lung and liver, protection of the mice against tissue damage, and significant increase in survival. In conclusion, this work showed that the compound modulates the immune response against the fungus by regulating the influx of inflammatory cells, consequently reducing fungal burden. Acknowledgments The authors would like to acknowledge the financial support from Fundac¸a˜o Arauca´ria/SETI-PR, Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Special Program for Research and Training in Tropical Diseases (TDR/WHO). Dr. A. Leyva provided English editing of the manuscript. Conflict of Interest interest.

The authors have no financial conflict of

References 1. Restrepo A, Tobo0 n AM. Paracoccidioides brasilensis. In: Mandell GL, Bennet JE, Dollin R, editors. Principles and practice of infectious diseases. Philadelphia: Elsevier; 2005. p. 3062–8. 2. Borges-Walmsley MI, Chen D, Shu X, Walmsley AR. The pathobiology of Paracoccidioides brasiliensis. Trends Microbiol. 2002;10:80–7. 3. Mamoni RL, Blotta MHSL. Kinetics of cytokines and chemokines gene expression distinguishes Paracoccidioides brasiliensis infection from disease. Cytokine. 2005;32:20–9. 4. Pulendran B. Modulating Th1/Th2 responses with microbes, dendritic cells, and pathogen recognition receptors. Immunol Res. 2004;29:187–96. 5. Corthay A. A three-cell model for activation of naı¨ve T helper cells. Scand J Immunol. 2006;64:93–6. 6. Gonzalez A, De Gregori W, Velez D, Restrepo A, Cano LE. Nitric oxide participation in the fungicidal mechanism

123

106

7.

8.

9.

10.

11.

12.

13. 14.

15.

16.

17.

18.

19.

Mycopathologia (2011) 172:95–107 of interferon-activated murine macrophages against Paracoccidioides brasiliensis conidia. Infect Immun. 2000;68: 2546–52. MacMicking JD, Nathan C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, Stevens K, Xie Q, Sokol K, Hutchinson N, Chen H, Mudgett JS. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell. 1995;81:641. Liew FY, Millot S, Parkinson C, Palmer RMJ, Moncada S. Macrophage killing of Leishmania parasitic in vivo is mediated by nitric oxide from L-arginine. J Immunol. 1990;144:4794–7. James SL, Glaven J. Macrophage citotoxicity against schistosomula of Schistosoma mansoni involved argininedependent production of reactive nitrogen intermediates. J Immunol. 1989;143:4208–12. Lane TE, Otero GC, Wa-Hsieh B, Howard D. Expression of inducible nitric oxide synthase by stimulated macrophages correlates with their antihistoplasma activity. Infect Immun. 1994;62:1940–5. Bocca AL, Hayashi EE, Pinheiro AG, Furlanetto AB, Campanelli AP, Cunha FQ, Figueiredo F. Treatment of Paracoccidioides brasiliensis-infected mice with a nitric oxide inhibitor prevents the failure of cell-mediated immune response. J Immunol. 1998;161:3056. Nascimento FRF, Calich VLG, Rodrı´guez D, Russo M. Dual role for nitric oxide in paracoccidioidomycosis: essential for resistance, but overproduction associated with susceptibility. J Immunol. 2002;168:4593–600. Bogdan C. Nitric oxide and the immune response. Nat Immun. 2001;10:907–16. Zanichelli PG, Sernaglia RL, Franco DW. Immobilization of the [RuII(edta)NO?] ´ıon on surface of functionalized sı´lica gel. Langmuir. 2006;22:203–8. Sa´nchez-Delgado RA, Navarro M, Lazardi K, Atencio R, Capparelli M, Vargas F, Urbina JA, Bouillez A, Noels AF, Mais D. Toward a novel metal-based chemotherapy against tropical diseases. Part 4. Synthesis and characterization of new metal–clotrimazole complexes and evaluation of their activity against trypanosoma cruzi. Inorg Chim Acta. 1998; 39:528–540. Navarro MT, Lahmann EJ, Cisneros-Fajardo A, Fuentes RA, Sa´nchez-Delgado P, Silva JA. Toward a novel metalbased chemotherapy against tropical diseases. Part 5. Synthesis and characterization of new Ru(II) and Ru(III) clotrimazole and ketoconazole complexes and evaluation of their activity against trypanosoma cruzi polyhedron. 2000;19:2319–2325. Sanchez-Delgado RA, Anzellotti A. Metal complexes as chemotherapeutic agents against tropical diseases: trypanosomiasis, malaria and leishmaniasis mini. Rev Med Chem. 2004;4:23–30. Tfouni E, Krieger M, McGarvey B, Franco DW. Structure, chemical and photochemical reactivity and biological activity of some ruthenium nitrosyl complexes. Coord Chem Rev. 2003;236:57–69. Silva JJN, Osakabe AL, Pavanelli WR, Silva JS, Franco DW. In vitro and in vivo antiproliferative and trypanocidal activities of ruthenium NO donors Br. J Pharmacol. 2007; 152:112–21.

123

20. Calich VLG, Purchio A, Paulo CR. A new fluorescent viability test for fungi cells. Mycopathologia. 1979;66:175–7. 21. Souto JT, Figueiredo F, Furlanetto A, Pfeffer K, Rossi MA, Silva JS. IFN-c and TNF-a determine resistance to Paracoccidioides brasiliensis infection in mice. Am J Pathol. 2000;156:811–1820. 22. Perrin DD, Armarego WLF, Perrin DR. Purification of laboratory chemicals. Elmsford, USA: Pergamon Press; 1980. 23. Shriver DF. The manipulation of air-sensitive compound. McGraw-Hill: New York; 1969. 24. Borges SSS, Davanzo CU, Castellano EE, Z-Zchpector J, Silva SC, Franco DW. Ruthenium nitrosyl complexes with N-heterocyclic ligands. Inorg Chem. 1998;37:2670–7. 25. Cerecetto H, Gonza´lez M. Chemotherapy of Chagas’ disease: status and new development. Curr Top Med Chem. 2002;2:1187–213. 26. Clarke MJ. Electrochemistry, synthesis, and spectra of pentaammineruthenium(III) complexes of cytidine, adenosine, and related ligands. J Am Chem Soc. 1978;100:5068–75. 27. Panis C, Mazzuco TL, Costa CZ, Victorino VJ, Tatakihara VL, Yamauchi LM, Yamada-Ogatta SF, Cecchini R, Rizzo LV, Pinge-Filho P. Trypanosoma cruzi: effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on levels of cytokines, nitric oxide and iNOS expression in cardiac tissue in the acute phase of infection in mice. Exp Parasitol. 2010. (In press). 28. Mariano FS, Gutierrez FR, Pavanelli WR, Milanezi CM, Cavassani KA, Moreira AP, Ferreira BR, Cunha FQ, Cardoso CR, Silva JS. The involvement of CD4?CD25? T cells in the acute phase of Trypanosoma cruzi infection. Microbes Infect. 2008;10:825–33. 29. Furuta T, Kimura M, Watanabe N. Elevated levels of vascular endothelial growth factor (VEGF) and soluble vascular endothelial growth factor receptor (VEGFR)-2 in human malaria. Am J Trop Med Hyg. 2010;82:136–9. 30. Vespa GNR, Cunha FQ, Silva JS. Nitric oxide is involved in control of Trypanosoma cruzi-induced parasitemia and directly kills the parasite in vitro. Infect Immun. 1994;62: 5177. 31. Wei X, Charles IG, Smith A, Ure J, Feng GJ, Huang FP, Xu D, Muller W, Moncada S, Liew FY. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature. 1995;375:408. 32. Candolfi E, Hunter CA, Remington JS. Mitogen- and antigen-specific proliferation of T-cells in murine toxoplasmosis is inhibited by reactive nitrogen intermediates. Infect Immun. 1994;62:1995. 33. Gregory SH, Wing EJ, Hoffman RA, Simmons RL. Reactive nitrogen intermediates suppress the primary immunologic response to Listeria. J Immunol. 1993;150:2901. 34. Rockett KA, Auburu MM, Rockett EJ, Coride WB, Clark I. Possible role of nitric oxide in malarial immunosuppression. Parasite Immunol. 1994;16:243. 35. Teixeira HC, Calich VLG, Singer-Vermes LM, D’Impe´rio Lima MR, Russo M. Experimental paracoccidioidomycosis: early immunosuppression occurs in susceptible mice after infection with pathogenic fungi. Braz J Med Biol Res. 1987;20:587. 36. Tonnetti L, Spaccapelo R, Cenci E, Mencacci A, Puccetti P, Coffman RL, Bistoni F, Romani L. Interleukin-4 and 10

Mycopathologia (2011) 172:95–107

37.

38.

39.

40.

41.

42.

43.

exacerbate candidiasis in mice. Eur J Immunol. 1995;25: 1559–65. Del Sero G, Mencacci A, Cenci E, d’Ostiani CF, Montagnoli C, Bacci A, Mosci P, Kopf M, Romani L. Antifungal type 1 response are upregulated in IL-10-deficient mice. Microbes Infect. 1999;1:1169–80. Vazquez-Torres J, Jones-Carson RD, Wagner T, Balish E. Early resistance of interleukin-10 knockout mice to acute systemic candidiasis. Infect Immun. 1999;67:670–4. Deepe GS, Gibbons RS. Protective and memory immunity to Histoplasma capsulatum in the absence of IL-10. J Immunol. 2003;171:5353–62. Fierer J, Walls L, Eckmann L, Yamamoto T, Kirkland TN. Importance of interleukin-10 in genetic susceptibility of mice to Coccidioides immitis. J Immunol. 1998;66:4397–402. Ohba T, Haro H, Ando T, Wako M, Suenaga F, Aso Y, Koyama K, Hamada Y, Nako A. TNF-alpha-induced NFkappaB signaling reverses age-related declines in VEGF induction and angiogenic activity in intervertebral disc tissues. J Orthop Res. 2009;27(2):229–35. Kim JE, Son JE, Jung SK, Kang NJ, Lee CY, Lee KW, Lee HJ. Cocoa polyphenols suppress TNF-alpha-induced vascular endothelial growth factor expression by inhibiting phosphoinositide 3-kinase (PI3 K) and mitogen-activated protein kinase-1 (MEK1) activities in mouse epidermal cells. Br J Nutr. 2010;16:1–8. Zhen G, Xue Z, Zhao J, Gu N, Tang Z, Xu Y, Zhang Z. Mesenchymal stem cell transplantation increases expression of vascular endothelial growth factor in papain-

107

44.

45.

46.

47.

48.

49.

50.

induced emphysematous lungs and inhibits apoptosis of lung cells. Cytotherapy. 2010;29. Abbas AK, Lichtman AH. Citocinas, Imunologia celular e molecular. 5th ed. Brazil: Elsevier; 2005. p. 260–2. pp. 270, 276–278. Guedes PM, Oliveira FS, Gutierrez FR, da Silva GK, Rodrigues GJ, Bendhack LM, Franco DW, Do Valle Matta MA, Zamboni DS, da Silva RS, Silva JS. Nitric oxide donor trans-[RuCl([15]aneN)NO] as a possible therapeutic approach for Chagas’ disease. Br J Pharmacol. 2010; 160(2):270–82. Hesse M, Modolell M, La Flamme AC, Schito M, Fuentes JM, Cheever AW, Pearce EJ, Wynn TA. Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by pattern of L-argininie metabolism. J Immunol. 2001;167:6533–44. Lousada S, Flo´rido M, Appelberg R. Regulation of granuloma fibrosis by nitric oxide during Mycobacterium avium experimental infection. Int J Exp Pathol. 2006;87:307–15. Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol. 2001;17: 463–516. Brummer E. Interaction of Paracoccidioides brasiliensis with host defense cells. In: Franco M, Silva Lacaz C, Restrepo Moreno A, del Negro, editors. Paracoccidioidomycosis. Boca Raton: CRC Press; 1994. p. 213–223. Fricker SP. Ruthenium, nitric oxide and disease. Platinum Metal Rev. 1995;59:150–9.

123