Mycopathologia (2011) 171:161–169 DOI 10.1007/s11046-010-9359-5
Cyclosporin A Treatment and Decreased Fungal Load/Antigenemia in Experimental Murine Paracoccidioidomycosis T. Y. C. Massuda • L. A. Nagashima • P. C. Leonello • M. S. Kaminami • M. S. Mantovani • A. Sano • J. Uno • E. J. Venancio • Z. P. Camargo • E. N. Itano
Received: 1 June 2010 / Accepted: 25 August 2010 / Published online: 11 September 2010 Ó Springer Science+Business Media B.V. 2010
Abstract Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb). The cyclosporin A (CsA) is an immunosuppressant drug that inhibits calcineurin and has been described as a potential antifungal drug. The present study investigated the effect of CsA on the immune response, fungal load/antigenemia in experimental murine PCM. It was used four groups of BALB/c mice: (a) infected with 1 9 105 Pb18 yeast cells (Pb), (b) infected and treated with CsA every other day 10 mg/kg of CsA (s.c.) during 30 days (Pb/CsA), (c) treated with CsA (CsA) and (d) no infected/treated (PBS). The immune response was evaluated by lymphocyte proliferation, DTH assays to exoAgs, ELISA for IgG anti-gp43 (specific immune
T. Y. C. Massuda L. A. Nagashima P. C. Leonello M. S. Kaminami M. S. Mantovani E. J. Venancio E. N. Itano (&) Department of Pathology Sciences, State University of Londrina, CCB, Universidade Estadual de Londrina, Campus Universita´rio, Londrina, PR 86051-970, Brazil e-mail:
[email protected]
responses) and cytokine serum levels (IFN-c, TNF-a, IL-4 and IL-10). Fungal load was determined by lung colony-forming units (CFU) counts, lung and liver histopathology analysis and antigenemia determined by inhibition-ELISA. As expected, CsA was able to inhibit the specific cellular and humoral immune response (P \ 0.05), with decrease in serum IFN-c, TNF-a and IL-4 levels (P \ 0.05). Cyclosporin A treatment also resulted in significantly decreased lung Pb CFU (P \ 0.05) as well as a lower number of yeasts in the lung and liver (P \ 0.05) by histopathology. In concordance, the decreased antigenemia was observed in Pb/CsA group (P \ 0.05). In conclusion, even with immunosuppressive action, treatment with CsA results in decreased lung fungal load/ antigenemia in experimental PCM in BALB/c mice. Further study is required to determine whether this represents less severe disease or protection by CsA. Keywords Immunosuppression Paracoccidioides brasiliensis Antifungal drug Calcineurin Cytokines Lymphoproliferative response
A. Sano J. Uno Medical Mycology Research Center, Chiba University, Chiba, Japan
Introduction
Z. P. Camargo Department of Microbiology, Immunology and Parasitology, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil
Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is one of the most important systemic mycoses in Latin
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America [1, 2]. It is believed that airborne fungal propagules, consisting of conidia or hyphal fragments, begin the infection and undergo conversion to the yeast phase, the infective stage of P. brasiliensis, in the lungs [3]. According to McEwen et al. [4], about 10 million people are infected with the fungus, and 2% of them may develop the disease. There are two forms of the disease: the acute form (AF) and chronic form (CF). The CF is more frequent in men older than 40 years, with multiple forms, ranging from benign and localized (unifocal) to severe and disseminated (multifocal) disease [5]. Itraconazole and amphotericin B are common drugs applied for antifungal therapy, the former for mild and moderate forms and the latter for severe forms [6, 7]. Unfortunately, these antifungal agents frequently present toxicity at some level [8, 9]. Also, because the treatment is usually long, patients abandon treatment before being fully cured [7]. Thus, the search of alternative therapies and the development of more effective and more specific antifungal drugs or combination of drugs could shorten the treatment time. In this context, the calcineurin signaling pathway (CaN) inhibitors combined with current antifungal drugs have been shown promising for the treatment of various invasive fungal infections [10]. The immunosuppressant drug cyclosporine A (CsA) and tacrolimus (FK506), calcineurin pathway inhibitors, can in vitro change the development, survival and growth of several species of pathogenic fungi [11–13]. This antifungal effect is dependent on the binding of CsA and FK506 to their respective yeast immunophilins inactivating calcineurin [14, 15]; however, according to Campos et al. [16], interestingly, only CsA, not FK506, produces significant in vitro effects on P. brasiliensis’s growth and morphology [16]. The murine models for some fungal infections have shown that CsA is able to reduce fungal burden in organs tissue [10, 17, 18], and the synergistic effect of the treatment with conventional antifungal agents and calcineurin inhibitors has significantly improved the effectiveness of antifungal therapy [19–21]. Considering the in vitro effects of CsA on P. brasiliensis [16] and the lack of in vivo investigations, in the current study, we evaluated the effect of CsA on immune response, fungal load/antigenemia in experimental murine PCM.
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Materials and Methods Fungal Isolates Yeast cells of P. brasiliensis strain 18 (Pb 18) were firstly inoculated into Swiss mice to restore virulence, re-isolated and then used for antigen production and experimental infection. Pb18 was cultured in Sabouraud agar (Micromed, Rio de Janeiro, RJ, Brazil) and maintained by subculturing at 35°C at 5-day intervals. Exoantigen (ExoAg) and gp43 Purification A lyophilized exoAg was prepared from a yeast-phase culture of P. brasiliensis according to Camargo et al. [22] and gp43 purification as previously described [23]. Mouse Infection and Treatment BALB/c mice (6- to 8-week-old males) were fed ad libitum and kept in a 12-h light–dark cycle at 25°C. The animals were randomly divided into four groups with six animals each for experiment: (a) Pb (infected and treated with PBS), (b) Pb/CsA (infected and treated with 10 mg/kg of CsA), (c) CsA (noninfected and treated with CsA) and (d) PBS (inoculated only with phosphate-buffered saline). The experiment was performed twice (total n = 12). Infected groups were inoculated with 100 ll of P. brasiliensis virulent strain Pb18 (1 9 106 viable yeast cells/ml in PBS) by the intravenous route, and CsA groups received every other day 10 mg/kg of CsA (100 ll, s.c.) (SANDIMMUNÒ, 50 mg/ml, Novartis Farma– Produtos Farmaceˆuticos, SA). CsA dosage and route of administration used were in accordance with del Pozo et al. and Wassef et al. [24, 25], respectively. The treatment with CsA started 1 day before the infection. Mice were killed on day 30 after treatment, and their blood and organs were aseptically removed. All the procedures applied to animals in this study were approved by the Animal Care and Use Committee of the State University of Londrina (CEEA no. 67/08). Pb Colony-Forming Units The CsA antifungal effects were assessed by pulmonary fungal burden by colony-forming unit
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(CFU) counts. The lung of each animal was aseptically removed, rinsed and homogenized in sterile PBS, pH 7.4 (0.2 g of tissue/ml). The homogenates were cultured on BHI agar (Acumedia, Baltimore, MD) supplemented with 4% fetal bovine serum and 5% spent culture medium from P. brasiliensis as a growth factor. Gentamycin was added, and the plates were incubated at 35°C, and Pb CFUs were counted on day 15 post-plating, and the results were expressed as CFUs per mg of tissue, in logarithmic scale. Histopathology The organs were fixed in 10% paraformaldehyde solution and prepared by routine techniques for paraffin embedding. Histological 5-lm sections were stained with hematoxylin/eosin (HE) or silver methenamine (Grocott) to detect the mycotic structures using standard protocols, and the slides were evaluated by light microscopy. The organs’ injury was evaluated by the presence of both fungi cells and inflammatory cells within tissue granulomatous lesions. Inhibition-ELISA for Detection of P. brasiliensis Circulating Antigen in Serum Inhibition-ELISA (Inh-ELISA) was performed as previously described [23]. Briefly, inhibition standard curve was constructed by adding different concentrations of P. brasiliensis gp43 (from 1 ng to 30 lg/ml) in 100 ll of normal serum and then adding 100 ll of the standardized concentration of monoclonal antibody (MAb) anti-gp43 (10 lg/ml). Serum samples (100 ll) were added to 100 ll of MAb anti-gp43. Normal serum was used as a negative control. Polystyrene plates (Corning Costar Co., Corning, USA) were coated with 500 ng of gp43 in carbonate buffer (pH 9.6) per well (100 ll/well) overnight at 4°C. After, the plates were blocked by incubation with 200 ll of 1% bovine serum albumin in PBS per well for 1 h at 37°C and washed 3 times, and 100 ll from inhibition standard curve, samples and controls was added per well and allowed to stand for 2 h at 37°C. After washings (39), 100 ll of goat antimouse immunoglobulin G-peroxidase (Sigma) was added, and the plates were incubated for 1 h at 37°C. After further washings, the reaction was developed with a solution of o-phenylenediamine (Sigma) and
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H2O2. The reaction was stopped with 4 N H2SO4 after 10 min of incubation in the dark, and the absorbance on an EX microplate reader (Labsystems, Helsinki, Finland) was 492 nm. Concentrations of gp43 in the serum were calculated based on the inhibition standard curve. Proliferation Assay Spleen cells from all mice were removed aseptically, teased and the erythrocytes lysed with trisammonium chloride solution. In 96-well flat-bottom culture plates, 100 ll of the spleen cells (1 9 106 cells/ml) and 100 ll of the exoAg (50 lg/ml) were cultured in triplicate wells in RPMI 1640 (containing L-glutamine, 10% fetal calf serum). As a control, the exoAg was replaced by RPMI medium. The cells were cultured for 116 h at 37°C with 5% CO2, and then 50 ll of 3-(4,5-dimethylthiazol-2-ly)-2,5-diphenyltetrazolium bromide (5 mg/ml MTT in PBS, Sigma) was added in each well, and plates were further incubated at 37°C for 4 h. The plates were centrifuged, and the supernatant from each well was aspirated carefully, and formazan crystals were solubilized by adding 100 ll DMSO. The optical density was measured by a Multskan EX microplate reader (Labsystems, Helsinki, Finland) at 550 nm, and the proliferation index (P.I.) calculated as the triplicate of stimulated cells/nonstimulated cells. Delayed-Type Hypersensitivity Assay Delayed-type hypersensitivity (DTH) reactions were evaluated by employing a footpad test previously described [26]. Briefly, mice were inoculated with 25 ll of exoAg, and footpad thickness was measured with a caliper (Mitutoyo Corporation, Tokyo, Japan) immediately before and 18 h after antigen inoculation (10 lg/ml). The increase in footpad thickness was calculated and expressed in millimeters. Cytokine Assays IFN-c, TNF-a, IL-4 e IL-10 serum levels were determined by commercial ELISA kits from BioSource International, Inc (Camarillo, California, USA), according to the manufacturer’s instructions. The reaction was revealed with peroxidase-conjugated streptavidin (Sigma) followed by the substrate
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mixture containing hydrogen peroxide and TMB (Zymed, California, USA) as a chromogen. The absorbance was read into an ELISA microplate reader (ELX-800 Universal, Bio-tek instruments, INC) at 450 nm. The absolute cytokine level was calculated based on a standard curve provided by the manufacturer.
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Results Lung Pb Colony-Forming Units
Immunoplates were coated with gp43 (25 lg/ml), incubated with serum samples (1/10) at 37°C for 1 h and then with goat anti-mouse IgG labeled with peroxidase, and the absorbance was read at 492 nm, as previously described [27].
The lung fractions (0.2 g of tissue/ml) from all groups of mice were homogenized, plated on BHI agar culture medium and incubated at 35°C during 15 days. Colony-forming units (CFUs) were counted, and the results were expressed as CFUs per mg of tissue. The lung fungal burden by CFU resulted in a significant reduction in viable colonies of the Pb/CsA (51.0 ± 7.39) when compared with Pb group (232.0 ± 24.5), P \ 0.05. The CFU was negative in control groups (CsA and PBS). The results in logarithmic scale are shown in Fig. 1a.
Statistical Analysis
Histopathology Analysis
Data were expressed as means ± SEM. Two-way analysis of variance was applied to analyze the interaction between variables infection and treatment. The Tukey’s post hoc test was used to identify differences between the groups. For comparison between Pb and Pb/CsA groups, Student’s t-test was performed. In both cases, significance was assumed at P \ 0.05. All statistical analyzes were performed using GraphPad Prism v. 5.00 for Windows (GraphPad Software, San Diego California USA).
Histological lung and liver analysis (stained with HE) shows a large number of granulomas, with extensive areas of tissue destruction in infected animals (Pb) 30 days post-infection. However, no inflammatory response or granuloma was found in the infected and CsA-treated mice (Pb/CsA) (Fig. 2a, c). Grocott staining shows a large number of Pb yeast cells in the organs of the infected animals and a much lower number in CsA-treated group (Fig. 2b, d). Table 1 shows mean granulomas numbers and the presence of P. brasiliensis in the lung and liver.
Fig. 1 Colony-forming units (CFU) and gp43 seric levels (antigenemy). a The homogenized lung tissue samples from group of mice infected with Pb18; 1 9 105 yeast cells (Pb), infected and treated with 10 mg/kg of CsA (Pb/CsA) every other day and control groups were plated on BHI agar culture medium (5 ml) at 35°C, during 15 days (n = 12 each group). CFUs were counted, and the results expressed as CFUs per mg of tissue, in logarithmic scale. The lung fungal burden by CFU resulted in a significant reduction in viable colonies of the Pb/CsA when compared with Pb group. P \ 0.05 (b). The CFU
was negative in control groups (treated only with CsA and only with PBS). b Circulating gp43 levels were determined by inhibition-ELISA (n = 12 each group), and the results expressed as lg/ml. Lower seric gp43 levels were observed in group of animals infected and CsA treated (Pb/CsA) when compared to the infected group (Pb) (P [ 0.05). No difference was found between the not infected groups (CsA and PBS) whose data were used to calculate the cutoff value. The line represents the average of the controls without infection. * P \ 0.05
Anti-gp43 IgG Analysis by ELISA
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Fig. 2 Lung histopathology. Histological lung stained with HE in the group of infected (1 9 105 Pb18) (Pb) (a) and infected plus treated with 10 mg/kg of CsA (Pb/CsA) (c) every
other day. Histological lung stained with Grocott in group Pb (b) and in Pb/CsA group (d), after 30 days of infection
Table 1 Granuloma number and presence of the P. brasiliensis in the organsa Organs
Granuloma number
Presence of the P. brasiliensis
Lung
Liver
Lung
Liver
29.2 (± 9.5) 0.0
4.4 (± 2.3) 0.0
(12/12) (10/12)
(12/12) (8/12)
Groups Pb Pb/CsA a
The mean granuloma number with SD and number of mice with fungi in lung and liver in group of mice infected (Pb) and in infected plus treated with CsA (Pb/CsA). Pb = Infected with 1 9 105 Pb18 and Pb/CsA = infected plus treated with 10 mg/kg of CsA every other day, 30 days post-infection
Soluble gp43 in Serum (Antigenemia) Inhibition-ELISA (Inh-ELISA) was performed for antigenemia analysis. Soluble gp43, in lg gp43/ml, was significantly lower in the animal’s sera from the infected and CsA-treated group (Pb/CsA) (14.06 ± 1.65 lg gp43/ml) when compared to the infected group (Pb) (22.26 ± 4.15 lg gp43/ml) (P [ 0.05). No difference was found between the noninfected
groups (CsA and PBS) whose data were used to calculate the cutoff value (Fig. 1b). Proliferation Assay The spleen cells from mice at 30 days post-infection were stimulated in vitro with P. brasiliensis antigens (exoAg) for evaluation of cell immune response. A higher proliferative response was found in the
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Fig. 3 Lymphoproliferation. Proliferation index (PI) of lymphocytes from spleens of group of mice infected with 1 9 105 yeast cells of Pb18 (Pb), infected and treated with 10 mg/kg of CsA every other day (Pb/CsA) and controls only treated with CsA (CsA) and treated only with PBS (PBS), stimulated with Pb exoAg (n = 12 each group). A higher proliferative response was found in the infected group (Pb) in relation to the infected and CsA-treated (Pb/CsA) or control groups. Mean values ± SD of the proliferation index (OD of test well/OD of control well) of triplicate cultures. (a, P \ 0.05 compared to all other groups)
spleenocytes stimulated with exoAg in infected group (Pb) in relation to infected and CsA-treated (Pb/CsA) or control groups (CsA and PBS) (P \ 0.05), showing that treatment with CsA reduces the rate of lymphocytes proliferation to levels similar to controls (CsA and PBS). Means values ± SD of the proliferation index (OD of test well/OD of control well) of triplicate cultures were shown in Fig. 3. DTH Assay As shown in Fig. 4, infected and CsA-treated animals (Pb/CsA) developed low or absent cutaneous reactivity to Pb exoAg at 30 post-infection. In contrast, in the same period, only infected animals presented positive DTH responses (P \ 0.05). No significant differences were detected between control groups (Fig. 4). Cytokine Levels The IFN-c, TNF-a and IL-4 serum levels, expressed in pg/ml, were decreased in the infected and CsA-treated groups in relation to infected group: IFN-c = Pb: 9953 ± 661; Pb/CsA: 8083 ± 350; IL-4 = Pb: 761 ± 131; Pb/CsA: 235 ± 44; and TNF-a = Pb: 2059 ± 33; Pb/CsA: 1704 ± 25 (P \ 0.05). The
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Fig. 4 DTH reactions. DTH reactions were evaluated employing a footpad test by exoAg inoculation (25 ll, 10 lg/ml) and footpad thickness measured 18 h after. The footpad thickness was expressed in millimeters in the group of mice infected (Pb), infected and treated with CsA (Pb/CsA), after 30 days of infection, treated only with CsA (CsA) and treated only with PBS (PBS) (n = 12 each group). (a, P \ 0.05 compared to all other groups)
IL-10 level was similar in the infected and in the infected and treated with CsA group (Pb: 1309 ± 42; Pb/CsA: 1418 ± 30, P [ 0.05) (Fig. 5). Level of IgG Anti-gp43 Humoral immune response to gp43 was evaluated by ELISA. The mean ELISA results in OD at 492 nm showed statistically high levels of anti-gp43 IgG in Pb group (0.494 ± 0.107) in relation to Pb/CsA group (0.355 ± 0.077) and to control groups (CsA: 0.046 ± 0.006; PBS: 0.044 ± 0.004, P \ 0.05) (Fig. 6).
Discussion The cellular immune response is considered the main defense against Pb [5], and it could be expected that CsA treatment induces the greatest disease severity because it is a drug that primarily suppresses the cellular response [28]. Surprisingly, in this study, treatment with CsA was able to reduce Pb lung CFU, and also the histopathological analysis showed a significant decrease in the number of yeasts in the lung and liver in experimental murine PCM. In concordance, decreased circulating gp43, the main Pb antigen [5], was observed in CsA-treated animals. These parameters are used to assess the greater or lesser severity of PCM illness [29, 30].
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Fig. 5 Cytokines profile in the serum. The IFN-c (a), IL-4 (b), TNF-a (c) and IL-10 (d) levels analyzed by capture ELISA and expressed in pg/ml in the serum of group of mice infected (1 9 105 Pb18) and infected plus treated with 10 mg/kg of CsA (Pb/CsA) every other day, after 30 days of infection (n = 12 each group). * P \ 0.05
Fig. 6 Levels of IgG anti-gp43. Humoral immune response to gp43 evaluated by ELISA in the group of infected mice (1 9 105 Pb18) (Pb), infected plus treated with 10 mg/kg of CsA (Pb/CsA) every other day, after 30 days of infection, treated only with CsA (CsA) and treated only with PBS (PBS). The results are expressed as means and standard error deviation in optical density (OD) at 492 nm (n = 12 each group). P \ 0.05: Pb vs. Pb/CsA and controls (CsA or PBS) vs. Pb or Pb/CsA
Although other organ studies are needed, the lowest level of gp43 suggests a lower overall fungal load and therefore perhaps the disease is less severe in animals treated with CsA, at least in the period investigated. The CsA is an immunosuppressive drug with great effect over the T lymphocytes [28], and in accordance with literature, the present work also demonstrated the T-cell function suppression. The significant decrease in in vitro lymphoproliferation and in vivo DTH response to Pb exoAg was observed in CsA-treated group.
As expected, the cytokines IFN-c, TNF-a and IL-4 levels were also significantly decreased in serum of the PCM mouse treated with CsA. These reductions are due to CsA mechanism of action by blocking the activity of calcineurin, then inhibiting the transcription of IL-2, IL-4, IFN-c and TNF-a genes and therefore decreasing the secretion of these cytokines [28, 31]. The levels of IL-10 remained unchanged, and this result can be explained by the no effect of the CsA on the regulatory lymphocytes, which are described as a major source of this cytokine [32]. The IFN-c has protective effect in experimental PCM [33], and its absence is associated with host susceptibility [34]. In accordance with cytokine levels data, histopathological analysis showed an intense granulomatous response, with the formation of granulomas containing numerous giant cells in the infected animals. However, there was no evidence of inflammatory or granulomatous response in the infected and CsA-treated animals. The formation of granuloma is considered a protective response in PCM [35], but even in the absence of granuloma and low IFN-c levels, only few yeast Pb cells were found in CsA treated mice. These results suggest that the CsA antifungal action overrides the immunosuppressive effect on murine PCM. The humoral immune response is not considered protective in PCM, and high levels of antibodies are observed in severe cases of PCM [29, 36]. In this study, decreased IgG anti-gp43 serum levels were observed in animals treated with CsA. This result could also be due to CsA action in cytokines
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regulation and indirect action on B cells as demonstrated in mice immune response to sheep red blood cells [37], and it could also be due to the neutralization by increased circulating gp43. Although the results show lower antigenemia and fungal burden, therefore possibly even lower disease severity, the presence of the some remaining viable Pb, requires additional studies, extending the treatment time and also for a longer period after cessation of treatment. Considering the nephrotoxicity of the antifungal agents in current use for PCM treatment [8, 9], the other antifungal investigations become interesting. As already considered by Steinbach et al. [10], as calcineurin has a role in the development, survival and growth of the fungi [12, 13], calcineurin pathway inhibitors have a potential as a therapeutic target for the treatment of several mycoses [10, 18–21], and our in vivo results are in agreement with the in vitro effect of the CsA on Pb observed by Campos et al. [16]. In conclusion, even with immunosuppressive action, treatment with CsA results in decreased lung fungal load/antigenemia in experimental PCM in BALB/c mice. Further study is required to determine whether this represents less severe disease or protection by CsA. Acknowledgments This work was supported by Secretaria de Estado da Ciencia, Tecnologia e Ensino Superior (SETI/PR), Fundac¸a˜o Arauca´ria/PR, Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES). We thank Dr. Everson A. Nunes for his careful correction of the English language.
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