Editor: Alex van Belkum
Chloroquine is therapeutic in murine experimental model of paracoccidioidomycosis
Article first published online: 23 APR 2007
FEMS Immunology & Medical Microbiology
Volume 50, Issue 1, pages 133–143, June 2007
How to Cite
Dias-Melicio, L. A., Calvi, S. A., Bordon, A. P., Golim, M. A., Peraçoli, M. T. S. and Soares, A. M. V. C. (2007), Chloroquine is therapeutic in murine experimental model of paracoccidioidomycosis. FEMS Immunology & Medical Microbiology, 50: 133–143. doi: 10.1111/j.1574-695X.2007.00243.x
- Issue published online: 23 APR 2007
- Article first published online: 23 APR 2007
- Received 5 December 2006; revised 5 February 2007; accepted 2 March 2007.First published online April 2007.
- Paracoccidioides brasiliensis;
- hydrogen peroxide;
- nitric oxide;
Chloroquine, due to its basic properties, has been shown to prevent the release of iron from holotransferrin, thereby interfering with normal iron metabolism in a variety of cell types. We have studied the effects of chloroquine on the evolution of experimental paracoccidioidomycosis by evaluating the viable fungal recovery from lung, liver and spleen from infected mice and H2O2, NO production, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-10 levels and transferrin receptor (TfR) expression from uninfected and infected peritoneal macrophages. Chloroquine caused a significant decrease in the viable fungal recovery from all organs tested, during all periods of evaluation. Peritoneal macrophages from chloroquine-treated infected mice showed higher H2O2 production and TfR expression, and decreased levels of NO, endogenous and stimulated-TNF-α, IL-6 and IL-10 during the three evaluated periods. However, despite its suppressor effects on the macrophage function, the chloroquine therapeutic effect upon murine paracoccidioidomycosis was probably due to its effect on iron metabolism, blocking iron uptake by cells, and consequently restricting iron to fungus growth and survival.
Paracoccidioidomycosis is a human systemic mycosis that is endemic in Latin America, and constitutes one of the most prevalent deep mycoses in endemic regions. This disease is caused by Paracoccidioides brasiliensis, a thermally dimorphic fungus that develops as yeast at body temperature and as a mycelium at room temperature (Restrepo, 1985; Franco et al., 1989). Ingested conidia or yeast-form of P. brasiliensis readily multiply inside murine alveolar or peritoneal macrophages; however, if they were activated by cytokines, such as interferon (IFN)-γ, the multiplication was limited and conidia or yeast cells were killed (Brummer et al., 1989; Cano et al., 1992). Similarly, the interaction of human phagocytic cells and yeast-form of P. brasiliensis was studied in vitro and yeast cells were readily ingested by monocytes or monocyte-derived macrophages. However, these cells allowed P. brasiliensis to multiply, unless they were activated with IFN-γ or culture supernatants from concanavalin-A-stimulated mononuclear cells, suggesting that this activation could be responsible for iron restriction and consequent decrease in the survival of P. brasiliensis within cells (Moscardi-Bacchi et al., 1994).
The mechanisms used by P. brasiliensis yeasts to survive and to multiply within monocytes or macrophages are poorly understood. Iron has been considered an essential element for microbial growth and has also been associated with virulence by promoting microorganism intracellular growth (Lewin, 1984). Chloroquine, a clinical drug, is best known as an antimalarial drug (Wellems, 1992; Slater, 1993), but it also affects several functions of macrophages (Ziegler & Unanue, 1982; Antoni et al., 1986; Lang & Kaye, 1991; Zhu et al., 1993). More recently, chloroquine has been used in the treatment of diseases associated with increased secretion of pro-inflammatory cytokines, such as rheumatoid arthritis and systemic lupus erythematosus (Fox, 1993; Wallace, 1994). It acts as a lysosomotropic agent by increasing lysosomal pH (Ziegler & Unanue, 1982) and, due to its basic properties, has been shown to prevent release of iron from holotransferrin (HTF) and ferritin, thereby interfering with normal iron metabolism, which is dependent on an acidic environment (Forsbeck & Nilsson, 1983; Krogstad & Schlesinger, 1987; Sibille et al., 1989; Byrd & Horwitz, 1991).
Moreover, studies showed that both mycelial and yeast forms of P. brasiliensis have a metabolic requirement for iron (Arango & Restrepo, 1988) and the intracellular conidium-to-yeast transformation of P. brasiliensis ingested by murine macrophage was inhibited when cells were treated with the iron chelator deferoxamine or activated with spleen-cell-culture supernatants from immunized mice. The HTF reversed this inhibitory effect of both deferoxamine and activated macrophages on P. brasiliensis intracellular transformation, indicating that iron restriction is one of the mechanisms by which activated macrophages control the intracellular transformation of ingested P. brasiliensis (Cano et al., 1994). Likewise, studies in our laboratory have also demonstrated that deferoxamine or chloroquine inhibits the intracellular survival of P. brasiliensis yeasts within human monocytes by interfering with iron acquisition by the fungus (Dias-Melicio et al., 2005, 2006). In addition, chloroquine treatment of Pb 18-infected mice significantly reduced the viable fungal recovery from lungs (Dias-Melicio et al., 2006).
Based on these data, we undertook the current study to investigate chloroquine effects on the evolution of experimental paracoccidioidomycosis, as this drug prevents iron uptake by the cells and consequently makes iron unavailable to the intracellular fungus. For this, the viable fungal recovery from lung, liver and spleen from infected and chloroquine-treated infected mice was evaluated. Moreover, activity of peritoneal macrophages was evaluated by production of H2O2 and NO, levels of TNF-α, IL-6 and IL-10, and transferrin receptor (TfR–CD71) expression.
Materials and methods
Groups of six to eight BALB/c male mice (7–8 weeks old) were used for each period of infection. All the animals were purchased from the University of São Paulo (USP) and provided with water and sterilized food ad libitum throughout the experiment. All the procedures involving animals and their care were conducted in conformity with National and International Policies, and the Animals Ethics Committee of the Botucatu Medical School–UNESP approved the study.
The highly virulent P. brasiliensis strain 18 (Pb 18) was used throughout this study. To ensure virulence, the isolate was used after three serial animal passages. Pb 18 yeast cells were then maintained by weekly subcultivation in the yeast-form cells at 35°C on 2% glucose, 1% peptone, 0.5% yeast extract and 2% agar medium (GPY medium) and used on the sixth day of culture. Yeast cells were washed and suspended in 0.15 M phosphate-buffered saline (PBS), pH 7.2. To obtain individual cells, the fungal suspension was homogenized with glass beads in a Vortex homogenizer (three cycles of 10 s) (Peraçoli et al., 1999). Yeast viability was determined by phase contrast microscopy; bright yeast cells were counted as viable and dark cells as not viable (Soares et al., 2001). Fungal suspensions containing more than 95% viable cells were used in the experiments.
Paracoccidioides brasiliensis infection and chloroquine treatment
Mice were infected with 1 × 106 Pb 18 yeast cells in 100 μL of sterile PBS by the caudal intravenous route. Groups of uninfected and infected mice were treated daily with chloroquine (Sigma-Aldrich Inc., St Louis, MO), at two different doses (40 or 80 mg kg−1, prepared in sterile PBS and filtered) by intraperitoneal injection for 8 weeks. Uninfected and infected control groups received an intraperitoneal injection of sterile PBS. All the mice groups were evaluated at the 2nd, 4th and 8th weeks after infection. In these periods, the viable fungal recovery from lung, liver and spleen of infected and chloroquine-treated infected mice was evaluated. Moreover, H2O2 and NO production, TNF-α, IL-6 and IL-10 levels and TfR expression from peritoneal macrophages of all groups were assayed.
Viable fungal recovery
Groups of infected and treated infected mice were sacrificed chronologically in the 2nd, 4th and 8th weeks after infection, and the organs removed. Briefly, lungs, liver and spleen were put in sterile tubes, weighed separately, macerated and homogenized in 2.0 mL of sterile PBS. Aliquots of 100 μL of each homogenate were plated in triplicate on brain–heart infusion (BHI) agar medium (Difco Laboratories) containing 4% normal horse serum and 5%P. brasiliensis strain 192 culture filtrate (v/v), the latter being the source of growth-promoting factor (Singer-Vermes et al., 1992). Inoculated plates were incubated at 35°C in sealed plastic bags to prevent drying. After 10 days the number of CFU in each plate was counted, and the results were expressed as CFU per gram of tissue.
Peritoneal resident cells were obtained from uninfected, chloroquine-treated uninfected, infected and chloroquine-treated infected mice by washing their peritoneal cavities with 10 mL ice-cold RPMI 1640 (Sigma-Aldrich). The cells from each group of mice were centrifuged (180 g, 10 min, 4°C), and washed twice with RPMI-1640 tissue culture medium. After, the cells were prepared in complete tissue culture medium (CTCM) which consisted of RPMI 1640 tissue culture medium, supplemented with 2 mM l-glutamine (Sigma-Aldrich), 10% heat-inactivated foetal calf serum, 20 mM HEPES (Sigma-Aldrich) and 40 μg mL−1 gentamicin (Gibco Laboratories, Grand Island, NY). The macrophages and total cells were counted with neutral red (0.02%) and trypan blue staining respectively, and the concentrations were adjusted according to the different assays. Suspensions of 100 μL per well, containing 2 × 105 macrophages each, were dispensed into 96-well flat-bottomed plates (Nunc, Life Tech Inc., MD) for H2O2 and NO assays. Suspensions of 1000 μL well−1, containing 1 × 106 macrophages mL−1, were dispensed into in 24-well flat-bottomed plates (Nunc) for culture supernatant cytokine measurement assays. After incubation for 2 h at 37°C in 5% CO2, nonadhering cells from both plates were removed by aspiration and each well was rinsed twice with CTCM. After adherence, the macrophages were cultured in CTCM at 37°C in 5% CO2 for 18 h, alone or stimulated with lipopolysaccharide (20 μg mL−1). In all experiments the cell suspensions consisted of at least 98% macrophages.
Macrophage hydrogen peroxide (H2O2) release
The H2O2 production was measured by the modified HRP-dependent phenol red oxidation microassay according to Pick & Keisari (1980) and Pick & Mizel(1981). Briefly, after cells were cultured as described earlier, the supernatants were collected for the NO assay. To the macrophage monolayers were added a phenol red buffer with 100 μL of phenol red solution containing 5.5 mM dextrose, 0.56 mM phenol red (Sigma-Aldrich), and 0.01 mg mL−1 horseradish type II (HRP type II–Sigma-Aldrich). In cocultures, 10 μL phorbol myristate acetate (PMA) (1 μg mL−1; Sigma-Aldrich) was added. The plates were incubated in a humidified chamber for 1 h in 5% CO2 at 37°C. The reaction was stopped by addition of 10 μL of 1 N NaOH and the absorbance at 620 nm was determined by a micro-ELISA reader (MD 5000, Dynatech Laboratories Inc., Chantilly, VA). The conversion of absorbance to nanomolar concentrations of H2O2 was obtained from a standard curve. All determinations were performed in triplicate and expressed as nanomolar concentrations of H2O2.
Macrophage NO production
NO production was quantified by the accumulation of nitrite in the supernatants by the standard Griess reaction (Green, 1981). Briefly, 100 μL of the supernatants collected from 96-well macrophage cultures were added with an equal volume of Griess reagent containing 1% sulfanilamide (Sigma-Aldrich) diluted in 5% of H3PO4, and 0.1%N-(1-naphthyl)ethylenediamine (NEED–Sigma-Aldrich). The absorbance at 540 nm was determined by a micro-ELISA reader (MD 5000, Dynatech Laboratories). Conversion of absorbance to micromolar concentrations of NO was obtained from a standard curve using a known concentration of NaNO2 diluted in distilled H2O. All determinations were performed in triplicate and expressed as micromolar concentrations of NO.
Measurement of cytokines
Peritoneal macrophage culture supernatants were separated from cell debris by centrifugation at 1000 g for 15 min, and stored at −70°C. Next, the levels of TNF-α, IL-6 and IL-10 were measured by capture ELISA using Kit DuoSet (R&D Systems, Minneapolis, MN). The ELISA procedure was performed according to the manufacturer's protocol. The concentrations of cytokines were determined with reference to a standard curve for serial twofold dilutions of murine recombinant cytokines. The absorbance values were measured at 492 nm using a micro-ELISA reader (MD 5000, Dynatech Laboratories). The lower limit of each recombinant standard curve detection was 15.62, 7.8 and 15.62 pg mL−1 for TNF-α, IL-6 and IL-10, respectively.
The peritoneal total cell suspensions containing 1 × 106 total cells mL−1 were labeled with rat antimouse CD71-FITC (Caltag Laboratories, Burlingame, CA) and incubated for 1 h on ice in the dark. Nonspecific signals were calculated and attenuated by incubation with the rat IgG2a isotype control-FITC (Caltag Laboratories) as a secondary antibody in the absence of the primary antibody. After, the cells were washed in PBS, 100 μL of fixative solution consisting of 5% formaldehyde in buffer (Becton Dickinson, San Jose, CA) was added, and the solution analyzed by flow cytometry on a FACSCalibur Cytometer (Becton Dickinson) using win midi and cell quest software.
Results were statistically analyzed by anova using graphpad software (GraphPad Instat 3.05, San Diego, CA) and compared using the Tukey–Kramer test (Zar, 1984; Godfrey, 1985) with the level of significance set at P<0.05.
Chloroquine strongly decreases the number of viable fungi from lung, liver and spleen of Pb 18-infected mice
The treatment of mice with chloroquine reduced the numbers of P. brasiliensis in the lungs, liver and spleen during all evaluated periods (Fig. 1). At the first period of evaluation (2nd week), the infected group showed a high recovery of CFU from lung and the chloroquine-treated groups showed a great reduction in the viable fungal recovery at two doses used (40 and 80 mg kg−1), both statistically different from infected group (P<0.01) (Fig. 1a). Although infected mice showed a slight decrease in viable fungal recovery from lungs during the 4th and 8th weeks postinfection, the chloroquine effect on treated groups with two doses was more pronounced during these two periods (4th and 8th weeks) (P<0.01).
The levels of fungal recovery from liver were similar to those obtained from lungs at the 2nd week in infected groups (Fig. 1b). In this period, the chloroquine-treated infected mice showed a significant reduction in the CFU recovery, with doses of 80 mg kg−1 (P<0.01) and 40 mg kg−1 (P<0.05). At the 4th week, there was still a significant decrease in the number of viable fungal recovery from liver in the chloroquine-treated mice (P<0.05), although the infected group did show some reduction in fungal load. The results from about the 8th week showed a reduction of CFU in all groups, including the infected ones that did not receive chloroquine treatment. In this period (8th week), there was no difference between groups, indicating a tendency to resolution of infection in this organ.
In the spleen at the 2nd week, the levels of CFU detected in the infected group were two times higher than in the liver and lung (Fig. 1c). However, the chloroquine treatment at two doses significantly diminished this recovery (P<0.01). By the 4th week, chloroquine-treated groups still showed a significant reduction in CFU recovery (P<0.05), although the infected group had presented reduction in fungal load, too. The results around the 8th week showed a reduction of CFU in all groups, including the infected ones that did not receive chloroquine treatment, indicating a tendency to resolution of infection in this organ, too, although there was still a significant difference between infected and chloroquine-treated (40 and 80 mg kg−1) infected mice.
In relation to viable fungal recovery from infected treated mice, there were no differences in the groups treated with 40–80 mg kg−1 of chloroquine at any point of treatment, in any organ evaluated.
Chloroquine increases H2O2 production by peritoneal macrophages
At the 2nd week, macrophages from infected mice showed higher H2O2 levels compared with the uninfected mice (P<0.01) (Fig. 2). Macrophages from chloroquine-treated infected mice (40 and 80 mg kg−1) increased H2O2 production compared with the infected mice (P<0.01). Activation by lipopolysaccharide (20 μg mL−1; data not shown) and PMA (1 μg mL−1) increased the H2O2 production from uninfected and infected macrophages (P<0.01).
At the 4th week (Fig. 2), macrophages from infected mice maintained high H2O2 levels compared with the first period, and macrophages from chloroquine-treated infected mice increased the H2O2 production in relation to the infected ones and in relation to the first period (chloroquine 80 mg kg−1).
During the 8th week, the results were similar to those shown at the 4th week, with chloroquine-treated infected groups producing higher H2O2 levels than infected ones, and a slight decrease in all groups (data not shown).
The uninfected mice treated with two doses of chloroquine, during the three periods of evaluation, showed increases in H2O2 levels (P<0.01). Uninfected mice and chloroquine-treated uninfected mice (40 and 80 mg kg−1) showed a mean±SEM production of H2O2 of 0.45±0.03, 1.35±0.04*, and 3.69±0.4* nmol at the 2nd week, respectively; 0.66±0.08, 2.97±0.04* and 3.21±0.2* nmol at the 4th week, respectively; and 0.53±0.09, 3.9±0.2* and 4.2±0.7* nmol at the 8th week, respectively (asterisks indicate a significant difference). Activation by lipopolysaccharide (20 μg mL−1) and PMA (1 μg mL−1) increased the H2O2 production from all these groups.
Chloroquine diminishes NO production by peritoneal macrophages
Macrophages from infected mice, at the 2nd week, increased NO levels compared with the uninfected mice, as shown in Fig. 3 (P<0.01). However, macrophages from chloroquine-treated infected mice (40 and 80 mg kg−1) decreased the NO levels compared with the infected ones (P<0.01). Activation by lipopolysaccharide (20 μg mL−1) increased NO production in all groups (P<0.01).
At the 4th week (Fig. 3), macrophages from infected mice and from chloroquine-treated infected mice had decreased NO levels compared with the first period (P<0.05), and the difference between infected mice and from chloroquine-treated infected mice continued to be detected (P<0.01). The cells from all groups had decreased NO production when activated by lipopolysaccharide (20 μg mL−1) in relation to the first period (P<0.05).
During the 8th week, the results were similar to those in the 4th week, with infected and chloroquine-treated infected groups producing lower NO levels, and with a decrease in NO production when activated with lipopolysaccharide (20 μg mL−1) (data not shown).
The uninfected mice treated with two doses of chloroquine showed a slight increase in NO levels compared with uninfected mice in the 2nd and 4th weeks, and similar levels in the 8th week. Uninfected mice and chloroquine-treated uninfected mice (40 and 80 mg kg−1) showed a mean±SEM increase in NO of 2.7±0.02, 3.4±0.2 and 4.7±0.03* μmol at the 4th week, respectively; and 2.4±0.09, 2.6±0.2 and 2.7±0.02 μmol at the 8th week, respectively (asterisks indicate a significant difference). The chloroquine-treated uninfected mice (40 and 80 mg kg−1) produced lower NO levels than uninfected mice when activated with lipopolysaccharide (20 μg mL−1).
Figure 4 shows the results of macrophage TfR expression. Uninfected mice macrophages presented a higher CD71 expression percentage than did the infected ones (P<0.001) during the three different periods. The chloroquine-treated infected mice showed an increase in macrophage TfR expression at the 2nd and 4th weeks compared with infected ones (P<0.05). Data from the 8th week were similar to those presented in the 4th week (data not shown). However, the macrophages from chloroquine-treated uninfected mice presented a decreased TfR expression compared with uninfected mice, indicating that this modulation of CD71 expression is not directly due to the chloroquine effect. This modulation could be due to NO production that was negatively correlated with TfR expression (Fig. 5).
Chloroquine diminishes TNF-α and IL-6 levels
Figure 6 shows the effect of chloroquine treatment on endogenous and lipopolysaccharide-stimulated TNF-α (Fig. 6a), IL-6 (Fig. 6b) and IL-10 (Fig. 6c) levels, respectively, obtained from macrophage cultures. At the 2nd week, the infected mice macrophages did not produce endogenous TNF-α and these cells did not respond to lipopolysaccharide activation (Fig. 6a) (P<0.001). At the 4th and 8th weeks, infected mice macrophages presented and increase in TNF-α and responded to lipopolysaccharide (P<0.001). Chloroquine treatment significantly decreased the endogenous and lipopolysaccharide-stimulated TNF-α levels of infected mice macrophages, mainly at the 4th week (P<0.001) (Fig. 6a) and similar results were observed in the 8th week of evaluation (data not shown).
The uninfected mice treated with two doses of chloroquine showed decrease in TNF-α levels compared with uninfected mice during the three evaluation periods (P<0.05). Uninfected mice and chloroquine-treated uninfected mice (40 and 80 mg kg−1) showed a decrease in TNF-α (mean±SEM) of 42±1.7, 39±2 and 29.7±0.9* pg mL−1 at the 2nd week, respectively; 43.4±1.9, 32.5±0.5* and 25.3±0.9* pg mL−1 at the 4th week, respectively; and 42.7±2, 23.8±2.1* and 20.2±1.7* pg mL−1 at the 8th week, respectively. The chloroquine-treated uninfected mice (40 and 80 mg kg−1) produced lower TNF-α than uninfected mice when activated with lipopolysaccharide (20 μg mL−1).
In relation to IL-6, chloroquine significantly diminished the endogenous levels of this cytokine from infected mouse macrophages during the three periods and lipopolysaccharide-stimulated IL-6 levels in the second and third periods (Fig. 6b). The uninfected mice treated with two doses of chloroquine showed decrease in IL-6 levels compared with uninfected mice during the three evaluation periods.
The endogenous IL-10 levels were similarly low in uninfected and infected groups, and chloroquine treatment decreased infected macrophage IL-10 levels compared with only infected groups during the three periods (Fig. 6c). The uninfected mice treated with two doses of chloroquine showed decrease in IL-10 levels compared with uninfected mice only during the last evaluation period.
This study demonstrated for the first time that chloroquine was effective in decreasing the fungal load of lung, liver and spleen from infected P. brasiliensis mice. We believe that this effect was due to chloroquine action upon iron metabolism, as studies have shown that chloroquine diminishes the iron content in macrophages, liver and spleen of normal mice and iron-treated mice (Legssyer et al., 1999, 2003). We have previously demonstrated the role of iron in P. brasiliensis growth, showing that when monocytes were pretreated with deferoxamine, an iron chelator drug, P. brasiliensis survival was inhibited within cells and HTF was capable of reversing deferoxamine inhibitory effect by supplying iron to intracellular P. brasiliensis (Dias-Melicio et al., 2005). We also showed that chloroquine inhibits the intracellular survival of P. brasiliensis in human monocytes by interfering with iron acquisition by the fungus, reinforcing the hypothesis that availability of intracellular iron is clearly required for the survival of this pathogen. This inhibitory chloroquine effect on P. brasiliensis survival was reversed by FeNTA, an iron compound that is soluble in the neutral to alkaline pH range, but not by HTF, which requires an acidic pH to release ferric ions (Dias-Melicio et al., 2006).
By increasing lysosomal and endocytic vesicle pH, chloroquine interferes with intracellular iron metabolism in at least two major ways. First, this drug may inhibit the pH-dependent release of iron from HTF endocytized via transferrin receptors (TfR). Normally, TfR binds HTF on the cell surface, forming a complex that it is endocytized, and the endocytic vesicle is acidified. When the pH decreases to 5, all ferric ions dissociate from HTF and are transported across the vesicle membrane into the cytoplasm (Princiotto & Zapolski, 1975; Lestas, 1976; Byrd & Horwitz, 1989, 1991; Nunez et al., 1990; Lane et al., 1991; Newman et al., 1994). Thus, HTF becomes ATF, an unsaturated transferrin, that remains bound to the TfR and is recycled to the cell surface and dissociates from the TfR (Dautry-Varsat et al., 1983; Brummer et al., 1989; Byrd & Horwitz, 1989, 1991; Alford et al., 1991; Lane et al., 1991; Newman et al., 1994; Conrad et al., 1999). Upon translocation across the endocytic vesicle membrane, iron enters the cytosolic labile iron pool and reaches equilibrium with iron storage (Jacobs, 1977; Roberts & Bomford, 1988; Lane et al., 1991). Thus, chloroquine has been found to reduce markedly the release of iron from endocytized HTF in a variety of cell types, including mononuclear phagocytes and macrophages, without affecting receptor recycling (Octave et al., 1979; Forsbeck & Nilsson, 1983; Swaiman & Machen, 1986; Baynes et al., 1987).
The second way that chloroquine may interfere with intracellular iron metabolism is by blocking recycling of iron from ferritin. Studies have suggested that iron bound to ferritin is released to the intracellular intermediate labile iron pool after ferritin is degraded in lysosomes. By raising intralysosomal pH, chloroquine may block degradation of ferritin by acid proteases, by virtue of its ability to raise endocytic and lysosomal pH (Sibille et al., 1989), blocking iron release from ferritin.
Thus, by interfering with the release of iron from these two major sources to the intermediate labile iron pool of phagocytes, chloroquine may decrease the availability of iron to intracellular P. brasiliensis and inhibit survival and multiplication of this intracellular pathogen; subsequently it is killed, decreasing the fungal load.
Several studies have shown the modulation of intracellular iron pool by chloroquine and its inhibitory action on in vitro intracellular growth of Mycobacterium avium, Cryptococcus neoformans, Penicillium marneffei, Leishmania ssp., Histoplasma capsulatum, Mycobacterium tuberculosis and P. brasiliensis within murine and human phagocytes (Crowle & May, 1990; Newman et al., 1994; Levitz et al., 1997; Boelaert et al., 2001; Dias-Melicio et al., 2006). In vivo, chloroquine was tested in histoplasmosis and cryptococcosis murine models (Newman et al., 1994; Levitz et al., 1997), and the results were similar to those found in the present study, with a decrease in tissue fungal load also being obtained.
In our model, an important chloroquine effect detected was the increased H2O2 production by chloroquine-treated macrophages. These results could be explained by the effect of chloroquine on the inhibition of catalase activity (Legssyer et al., 1999). This process could be involved in the decrease of fungal load, as some studies have suggested that this metabolite is important for the killing of P. brasiliensis (Cano et al., 1995; Moreira et al., 2001; Calvi et al., 2003; Carmo et al., 2006).
Another important chloroquine effect was upon inhibition of NO production. These results are in agreement with studies that demonstrated the inhibitory effect of chloroquine on inducible nitric oxide synthase (iNOS), which is responsible for NO production (Hrabák et al., 1998; Park et al., 1999; Musial & Eissa, 2001; Utaisincharoen et al., 2002). An experimental paracoccidioidomycosis study has shown that this metabolite has a dual role in the experimental model; and it is essential for resistance, but overproduction is associated with susceptibility (Nascimento et al., 2002). Bocca et al.(1998) also showed that the activation of NO production in P. brasiliensis infection contributes to the occurrence of the immunosuppression observed during the course of the infection, although some studies demonstrated that NO is important for the killing of the fungi (Gonzalez et al., 2000). Thus, this inhibitory chloroquine effect on NO production, observed in our model, could have eliminated one of the major suppressor factors that influence the mice response, promoting the better response to infection.
An important role of NO in iron metabolism was described through its modulatory effect on the iron-regulatory proteins (IRP-1 and IRP-2). These are cytoplasmic proteins responsible for controlling cellular iron storage and uptake by interaction with specific nucleotide sequences, called iron-responsive elements (IREs), which are located in the 3′-untranslated region (UTR) of TfR mRNA as well as the 5′-UTRs of mRNA for ferritin. When iron in the intracellular transit pool is scarce, IRPs bind to IREs in the 5′-UTRs of the ferritin mRNA and 3′-UTR of the TfR mRNA. Such binding inhibits translation of ferritin mRNA and stabilizes the mRNA for TfR, whereas the opposite scenario develops when iron in the transit pool is plentiful (Hentze & Kuhn, 1996; Richardson & Ponka, 1997). Studies showed that NO causes a significant decrease in IRP-2 binding to the IREs followed by IRP-2 degradation that resulted in a dramatic decrease in TfR mRNA levels (Kim & Ponka, 1999, 2002). Thus, this NO modulatory action could explain our results, in which TfR expression was negatively correlated with NO levels. Studies have shown that IFN-γ reduces the TfR expression on macrophages, decreasing cellular iron uptake, and consequently the iron availability (Byrd & Horwitz, 1989, 1993; Lane et al., 1991). This process is mediated by an increase in NO levels from IFN-activated cells, and the NO effect on IRPs promotes the decrease in TfR expression (Kim & Ponka, 2000). However, we demonstrated that chloroquine-treated infected mice macrophages showed a decrease in NO levels that promoted an increase in TfR expression, which could induce an increase in intracellular iron. Even so, this process might be inhibited by the effect of chloroquine on the pH-dependent release of iron from HTF endocytized via transferrin receptors (TfR), with consequent decrease in iron intracellular content.
This study also investigated whether the chloroquine treatment was associated with changes in the levels of TNF-α, IL-6 and IL-10 produced by macrophages. Some studies have shown the inhibitory effect of chloroquine on cytokine production (Zhu et al., 1993; Hong et al., 2004; Rayne et al., 2004). Our data demonstrated that chloroquine treatment significantly decreased IL-6 production. This process could be important given that monocytes from paracoccidioidomycosis patients produce high IL-6 levels during the active disease, and this cytokine is capable of deactivating macrophages with consequent reduction in microbicidal activity against microorganisms (Blanchard et al., 1991; Bermudez et al., 1992). Furthermore, it has been shown that IL-6 increases Pb 18 growth in human monocytes compared with untreated monocytes (Siqueira et al., 2007), suggesting that this cytokine may contribute to the pathogenesis of infection with P. brasiliensis by promoting fungal growth during interaction with phagocytes.
Another cytokine that has a central role in paracoccidioidomycosis infection is TNF-α. Our results showed an eminent inhibitory chloroquine effect on TNF-α production, confirming previous studies showing that chloroquine is capable of diminishing macrophage TNF-α production (Hong et al., 2004; Rayne et al., 2004) by inhibiting TNF-αmRNA transcription (Zhu et al., 1993). Although this cytokine is associated with granulomatous reaction formation (Soler & Bernaudin, 1993), in paracoccidioidomycosis several authors report high levels of TNF-α in serum (Silva et al., 1995; Silva & Figueiredo, 1995; Fornari et al., 2001) and in peripheral blood mononuclear cell (PBMC) culture supernatants during active disease (Karhawi et al., 2000). Thus, the capacity to produce high TNF-α levels might be involved in the pathogenesis of the disease rather than in the resistance, even though several works have shown that TNF-α is very important for human monocyte activation and for effective killing of P. brasiliensis (Calvi et al., 2003). In experimental paracoccidioidomycosis models, persistent TNF-α production has been associated with resistance to P. brasiliensis infection, which is also essential for the control of fungal dissemination (Cano et al., 1995; Bocca et al., 1998; Souto et al., 2000; Nascimento et al., 2002). Considering these processes, in the present paper, the inhibitory chloroquine effect on TNF-α production could be one of the mechanisms by which chloroquine controls the infection promoting better control of the deleterious TNF-α effects on tissue, and the TNF-α participation in the resistance could be supplied by the effect of chloroquine on the iron metabolism interfering with iron acquisition by the fungus.
Our findings support the idea that the effect of chloroquine on murine experimental paracoccidioidomycosis is due to its capacity to interfere with intracellular iron availability that disabled its uptake by the fungus with consequent fungal death, as well as by its suppressor effects on the immune response. Up to now, no data have been available on the possible antifungicidal effect of chloroquine in the clinical situation. Thus, chloroquine could be utilized as a potential drug that could be administered in association with conventional paracoccidioidomycosis treatment.
This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), process no. 02/03680-8, Brazil.
We thank Professor Cesar Augusto Taconeli (Department of Biostatistics/IB–UNESP) for the correlation graphs, Luiz Henrique Alquati for helpful technical assistance, and Valéria Alves da Silva for helpful assistance in flow cytometry assays.
- 1991) Role of transferrin, transferrin receptors, and iron in macrophage listericidal activity. J Exp Med 174: 459–466. , & (
- 1986) Effect of emetine and chloroquine on phagocytic processes of rat macrophages. Biochem Pharmacol 35: 2869–2874. , & (
- 1988) Growth and production of iron chelants by Paracoccidioides brasiliensis mycelial and yeast forms. J Med Vet Mycol 26: 113–118. & (
- 1987) Transferrin receptors and transferrin iron uptake by cultured human blood monocytes. Eur J Cell Biol 43: 372–376. , , , & (
- 1992) Interleukin-6 antagonizes tumor necrosis factor-mediated mycobacteriostatic and mycobactericidal activities in macrophages. Infect Immun 60: 4245–4252. , , & (
- 1991) Mycobacterium avium-intracellulare induces interleukin-6 from human monocytes and large granular lymphocytes. Blood 77: 2218–2224. , , , & (
- 1998) Treatment of Paracoccidioides brasiliensis-infected mice with a nitric oxide inhibitor prevents the failure of cell-mediated immune response. J Immunol 161: 3056–3063. , , , , , & (
- 2001) Experimental results on chloroquine and AIDS-related opportunistic infections. J Acquir Immune Defic Syndr 26: 300–301. , , et al. (
- 1989) Intracellular multiplication of Paracoccidioides brasiliensis in macrophages: killing and restriction of multiplication by activated macrophages. Infect Immun 57: 2289–2294. , , & (
- 1989) Interferon-gamma activated human monocytes downregulate transferrin receptors and inhibit the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. J Clin Invest 83: 1457–1465. & (
- 1991) Chloroquine inhibits the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. A potential new mechanism for the therapeutic effect of chloroquine against intracellular pathogens. J Clin Invest 88: 351–357. & (
- 1993) Regulation of transferrin receptor expression and ferritin content in human mononuclear phagocytes. Coordinate upregulation by iron transferrin and downregulation by interferon gamma. J Clin Invest 91: 969–976. & (
- 2003) Effect of cytokines on the in vitro fungicidal activity of monocytes from paracoccidioidomycosis patients. Microbes Infect 5: 107–113. , , , , , & (
- 1992) Killing of P. brasiliensis conidia by pulmonary macrophages and the effect of cytokines. J Med Vet Mycol 30: 161–168. , , , , & (
- 1994) Inhibitory effect of deferoxamine or macrophage activation on transformation of Paracoccidioides brasiliensis conidia ingested by macrophages: reversal by holotransferrin. Infect Immun 62: 1494–1496. , , & (
- 1995) Pulmonary paracoccidioidomycosis in resistant and susceptible mice: relationship among progression of infection, bronchoalveolar cell activation, cellular immune response, and specific isotype patterns. Infect Immun 63: 1777–1783. , , , & (
- 2006) TNF-α activates human monocytes for Paracoccidioides brasiliensis killing by an H2O2-dependent mechanism. Med Mycol 44: 363–368. , , , & (
- 1999) Iron absorption and transport. Am J Med Sci 318: 213–229. , & (
- 1990) Inhibition of tubercle bacilli in cultured human macrophages by chloroquine used alone and in combination with streptomycin, isoniazid, pyrazinamide, and two metabolites of vitamin D3. Antimicrob Agents Chemother 34: 2217–2222. & (
- 1983) pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci USA 80: 2258–2262. , & (
- 2005) Inhibitory effect of deferoxamine on human monocytes Paracoccidioides brasiliensis survival: reversal by holotransferrin not by apotransferrin. Rev Inst Med Trop S Paulo 47: 263–266. , , & (
- 2006) Chloroquine inhibits Paracoccidioides brasiliensis survival within human monocytes by limiting the availability of intracellular iron. Microbiol Immunol 50: 307–314. , , & (
- 2001) High serum interleukin-10 and tumor necrosis factor alpha levels in chronic paracoccidioidomycosis. Clin Diagn Lab Immunol 8: 1036–1038. , , , , , & (
- 1983) Iron metabolism of established human hematopoietic cell lines in vitro. Exp Cell Res 14: 323–332. & (
- 1993) Mechanism of action of hydroxychloroquine as an antirheumatic drug. Semin Arthritis Rheum 23: 82–91. (
- 1989) Paracoccidioidomycosis. Bailliere's Clin Trop Med Commun Dis 4: 185–220. , , , & (
- 1985) Statistics in practice. Comparing the means of several groups. N Engl J Med 313: 1450–1456. (
- 2000) Nitric oxide participation in the fungicidal mechanism of gamma-interferon-activated murine macrophages against Paracoccidioides brasiliensis conidia. Infect Immun 68: 2546–2552. , , , & (
- 1981) Nitrite biosynthesis in man. Proc Natl Acad Sci 15: 106–122. (
- 1996) Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Natl Acad Sci USA 93: 8175–8182. & (
- 2004) Chloroquine protects mice from challenge with CpG ODN and lipopolysaccharide by decreasing proinflammatory cytokine release. Intern Immunopharm 4: 223–234. , , , , , , & (
- 1998) Action of chloroquine on nitric oxide production and parasite killing by macrophages. Eur J Pharm 354: 83–90. , , , , & (
- 1977) Low molecular weight intracellular iron transport compounds. Blood 50: 433–439. (
- 2000) Production of IFN-gamma is impaired in patients with paracoccidioidomycosis during active disease and is restored after clinical remission. Med Mycol 38: 225–229. , & (
- 1999) Control of transferrin receptor expression via nitric oxide-mediated modulation of iron-regulatory protein 2. J Biol Chem 274: 33035–33042. & (
- 2000) Effects of interferon-γ and lipopolysaccharide on macrophage iron metabolism are mediated by nitric oxide-induced degradation of iron-regulatory protein 2. J Biol Chem 275: 6220–6226. & (
- 2002) Nitric oxide-mediated modulation of iron regulatory proteins: implication for cellular iron homeostasis. Blood Cells Mol Dis 29: 400–410. & (
- 1987) Acid-vesicle function, intracellular pathogens, and the action of choroquine against Plasmodium falciparum. N Engl J Med 317: 542–549. & (
- 1991) Iron limitation and the gamma interferon-mediated antihistoplasma state of murine macrophages. Infect Immun 59: 2274–2278. , & (
- 1991) Presentation of Leishmania donovani promastigotes occurs via a brefeldin A-sensitive pathway. Eur J Immunol 21: 2407–2413. & (
- 1999) Effect of chronic chloroquine administration on iron loading in the liver and reticuloendothelial system and on oxidative responses by the alveolar macrophages. Biochem Pharmacol 57: 907–911. , , & (
- 2003) Changes in function of iron-loaded alveolar macrophages after in vivo administration of desferrioxamine and/or chloroquine. J Inorg Biochem 94: 36–42. , , , & (
- 1976) The effect of pH upon human transferrin: selective labeling of the two iron-binding sites. Br J Haematol 32: 341–350. (
- 1997) Chloroquine induces human mononuclear phagocytes to inhibit and kill Cryptococcus neoformans by a mechanism independent of iron deprivation. J Clin Invest 100: 1640–1646. , , & (
- 1984) How microorganisms transport iron. Science 225: 401–402. (
- 2001) Killing of Paracoccidioides brasiliensis by peritoneal macrophages activated by IFN-gamma or TNF-alpha is mediated by H2O2 and NO. Rev Soc Bras Med Trop 34: 145. , , , , & (
- 1994) Support of Paracoccidioides brasiliensis multiplication by human monocytes or macrophages: inhibition by activated phagocytes. J Med Microbiol 40: 159–164. , & (
- 2001) Inducible nitric-oxide synthase is regulated by the proteasome degradation pathway. J Biol Chem 276: 24268–24273. & (
- 2002) Dual role for nitric oxide in paracoccidioidomycosis: essential for resistance, but overproduction associated with susceptibility. J Immunol 168: 4593–4600. , , & (
- 1994) Chloroquine induces human macrophage killing of Histoplasma capsulatum by limiting the availability of intracellular iron and is therapeutic in a murine model of histoplasmosis. J Clin Invest 93: 1422–1429. , , & (
- 1990) Mobilization of iron from endocytic vesicles – the effects of acidification and reduction. J Biol Chem 265: 6688–6692. , , & (
- 1979) Transferrin protein and iron uptake by culture rat fibroblasts. FEBS Lett 108: 127–130. , , , & (
- 1999) Chloroquine inhibits inducible nitric oxide synthase expression in murine peritoneal macrophages. Pharmacol Toxicol 85: 188–191. , , , , & (
- 1999) Paracoccidioides brasiliensis isolated from armadillos is virulent to syrian hamsters. Mycopathol 148: 123–130. , , , & (
- 1980) A simple colorimetric method for measurement of hydrogen peroxidase produced by cells in culture. J Immunol Meth 38: 161–172. & (
- 1981) Rapid microassay for the measurement of superoxide and hydrogen peroxide production by macrophage in culture using an automatic enzyme immunoassay reader. J Immunol Meth 46: 211–226. & (
- 1975) Difference between the two iron-bindings sites of transferrin. Nature 255: 87–88. & (
- 2004) The ability of chloroquine to prevent Tat-induced cytokine secretion by monocytes is implicated in its in vivo anti-human immunodeficiency virus type 1 activity. J Virol 78: 12054–12057. , , & (
- 1985) The ecology of Paracoccidioides brasiliensis: a puzzle still unsolved. J Med Vet Mycol 23: 323–334. (
- 1997) The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochim Biophys Acta 1331: 1–40. & (
- 1988) Chelation of transferrin iron by desferioxamine in K562 cells – the partition of iron between ferrioxamine and ferritin. Biochem J 254: 869–875. & (
- 1989) Uptake of ferritin and iron bound to ferritin by rat hepatocytes: modulation by apotransferrin, iron chelators and chloroquine. Biochem Biophys Acta 1010: 204–208. , & (
- 1995) Tumor necrosis factor in paracoccidioidomycosis patients. J Infect Dis 164: 1033–1034. & (
- 1995) Differential correlation between interleukin patterns in disseminated and chronic human paracoccidioidomycosis. Clin Exp Immunol 101: 314–320. , , , , & (
- 1992) The source of growth-promoting factor(s) affects the plating efficiency of Paracoccidioides brasiliensis. J Med Vet Mycol 30: 261–264. , , , & (
- 2007) Interleukin-6 modulates cytokine production and increases growth of a virulent strain of Paracoccidioides brasiliensis in human monocytes. Cytokine, in press. , , & (
- 1993) Chloroquine: mechanism of drug action and resistance in Plasmodium falciparum. Pharmacol Ther 57: 203–235. (
- 2001) Modulatory effect of prostaglandins on human monocyte activation for killing of high- and low-virulence strains of Paracoccidioides brasiliensis. Immunol 102: 480–485. , , , , & (
- 1993) Physiology of granulomas. Rev Pneumol Clin 49: 257–261. & (
- 2000) Interferon-γ and tumor necrosis factor-α determine resistance to Paracoccidioides brasiliensis infection in mice. Am J Pathol 156: 1811–1820. , , , , & (
- 1986) Chloroquine reduces neuronal and glial iron uptake. J Neurochem 46: 652–654. & (
- 2002) CpG ODN activates NO and iNOS production in mouse macrophage cell line (RAW 264-7). Clin Exp Immunol 128: 467–473. , , , & (
- 1994) Antimalarial agents and lupus. Rheum Dis Clin North Am 20: 243–263. (
- 1992) Malaria. How chloroquine works. Nature 335: 108–109. (
- 1984) Biostatistical Analysis, 2nd edn. Englewood Cliffs, Prentice-Hall. (
- 1993) Chloroquine inhibits macrophage tumor necrosis factor-alpha mRNA transcription. Immunol 80: 122–126. , , , , & (
- 1982) Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of arginine presentation to T cells. Proc Natl Acad Sci USA 79: 175–178. & (