Sporothrix schenckii yeasts induce ERK pathway activation and secretion of IL-6 and TNF-α in rat mast cells, but no degranulation

Abstract

Sporothrix schenckii is a dimorphic fungus that causes sporotrichosis, a subcutaneous mycosis found throughout the world in humans and other mammals. After contact with conidia, transition to the yeast stage is required for establishment of infection. Mast cells are one of the first components of the immune system to make contact with invading pathogens. They release potent mediators that are decisive in initiating and directing the course of immune and inflammatory responses in the host. It remains unknown whether or not yeast cells of S. schenckii activate mast cells. Our aim in this study was to evaluate the in vitro response of mast cells to S. schenckii yeasts cells. Mast cells became activated after interaction with the yeasts, although exocytosis of preformed mediators was not stimulated. Sporothrix schenckii yeasts induced the release of early response cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 and activation of the extracellular signal-regulated kinase (ERK) signaling pathway in mast cells. As TNF-α and IL-6 are considered crucial mediators in the defense of the host against fungal disease, the release of both mediators from mast cells may contribute to the overall response of the host immune system during S. schenckii infection.

Sporothrix schenckii, yeast, mast cell, cytokines, MAPK

Introduction

Sporothrix schenckii is considered to be a complex of closely related fungi composed of S. brasiliensis, S. globosa, S. schenckii sensu stricto, and S. luriei [1]. The traumatic inoculation of fungal particles through the skin as a result of contact with soil, plants, or organic matter contaminated with this fungus is the most common portal of entry for establishment of sporotrichosis, a chronic subcutaneous mycosis that affects both humans and animals worldwide. Fixed and lymphocutaneous forms are the most common types of skin infections. In sporotrichosis, S. schenckii exhibits dimorphism, which involves the transformation of mycelium to yeasts or vice versa [2]. The immune response developed by the host determines the degree of invasion by the fungus [3]. T cell– and macrophage-mediated immunity is important in limiting the extension of infection [4], and other components such as neutrophils [5], dendritic cells [6], and antibodies [7] also participate in the response to the fungus.

Mast cells are important effector cells in protective immune responses against pathogens [8]. These tissue-resident cells are commonly exposed to the external environment, including the skin, where they can initiate early response to environmental threats. Mast cells are a potential source of preformed products such as histamine and proteases. They also de novo synthesize mediators including metabolites of arachidonic acid, chemokines, growth factors, and early-response cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 [9]. The latter require the activation of different cell signaling pathways, as nuclear factor-κB and mitogen-activated protein kinase (MAPK), which modulate gene transcription [10]. Cytokine secretion is decisive in initiating and directing the course of immune and inflammatory responses in the host [9].

Recently, mast cells were found to be involved in the immune response to Paracoccidioides brasiliensis [11] and Malassezia sympodialis [12]. In vitro studies have demonstrated mast cell activation in response to Candida albicans yeasts [13], Aspergillus fumigatus hyphae [14], and M. sympodialis yeasts [15]. Recently, we found that mast cells become activated after interaction with conidia of S. schenckii and that they were involved in progression of sporotrichosis in mice [16]. Considering that the yeast stage dominates in sporotrichosis [17], we initiated this study in order to determine whether yeasts of S. schenckii activate mast cells and induce degranulation and/or cytokine secretion. We also analyzed MAPK pathway involvement in cellular activation.

Materials and methods

Fungal strain and growth conditions

The S. schenckii sensu stricto wild-type strain UAA-307 was obtained from a human lymphocutaneous sporotrichosis case in Huauchinango, Puebla, Mexico. The isolate's identity was confirmed by biochemical and morphological features [18] and polymerase chain reaction sequencing of the calmodulin gene. The sequence was submitted to GenBank with the accession number KJ921740. The isolate was subcultured from conidia until complete differentiation into the yeast form, followed by optical microscopy. Yeast cells were grown in brain–heart infusion agar for 6 days at 37°C, then washed three times with saline solution by centrifugation at 750 g and counted in a Neubauer chamber before all experiments.

Peritoneal mast cell isolation

Peritoneal mast cells were obtained from peritoneal exudate cells of adult male Wistar rats using a 30%–80% discontinuous Percoll gradient as described previously [16]. Preparations, with mast cell purity from 95% to 100% and viability >98%, were resuspended in 1 ml of supplemented Roswell Park Memorial Institute (RPMI)-1640 medium without phenol red (Sigma-Aldrich, St. Louis, MO, USA). All animals were used in accordance with the Institutional Normative Welfare standards and sacrificed by ether anesthesia.

Scanning electron microscopic study

Poly D-lysine–coated 24-well tissue culture plates were seeded with 8 × 10⁶S. schenckii yeasts in RPMI medium or 2.5 × 10⁴ mast cells in supplemented RPMI medium and incubated for 24 h at 4°C or 37°C. Then, 8 × 10⁵ mast cells were added in RPMI medium to wells containing yeast cells; the plate was incubated for 30 min at 37°C to allow interaction. RPMI medium with or without 5 μg/ml compound 48/80 (C48/80, Sigma-Aldrich) was added to wells with mast cells, and the plate was incubated for 30 min at 37°C to use as positive and negative stimulation controls. Samples were fixed overnight at 4°C with 2.5% glutaraldehyde in sterile phosphate-buffered saline pH 7.4. Samples were then washed, dehydrated, and followed by critical point drying with liquid carbon dioxide (Samadri-PVT-3D, TOUSIMIS Research Corporation, Rockville, MD, USA). Samples were coated with gold using a DESK II chamber and photographed with a JSM-5900LV scanning electron microscope (JEOL, Musashino, Tokyo, Japan).

Histamine release assay

Mast cells (1 × 10⁵) were stimulated with or without yeasts of S. schenckii (1 × 10⁶ or 5 × 10⁶) or with 5 μg/ml of C48/80 in supplemented RPMI medium for 30 min at 37°C. Supernatant and cell pellets were processed as previously described [16], and the histamine content was determined using the ophthaldialdehyde reaction with an LS 50B luminescence spectrometer (Perkin-Elmer, Beaconsfield, Buckinghamshire, UK). Histamine release was expressed as the percentage of the total cellular histamine content and calculated as follows: histamine in supernatant/(histamine in supernatant + histamine in pellet) × 100.

β-hexosaminidase release assay

Mast cells (5 × 10⁵) were stimulated with or without yeasts (5 × 10⁶) of S. schenckii or with 5 μg/ml of C48/80 in supplemented RPMI medium for 30 min at 37°C. After centrifugation, supernatants were collected and the remaining cells were lysed with cold RPMI medium plus 0.1% Triton X-100. Then, 50 μl of each sample was mixed with 100 μl of 2 mM p-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside in citrate buffer (62.5 mM citric acid monohydrate-100 mM anhydrous dibasic sodium phosphate [Sigma-Aldrich], pH 4.5) in 96-well plates, mixed gently, and then incubated at 37°C for 30 min. The reaction was stopped with 100 μl of 0.2 M glycine-NaOH (pH 10.7). The release of o-nitrophenol was measured at 405 nm in a microplate reader (iMark; Bio-Rad, Hercules, CA, USA). The percentage of β-hexosaminidase released was calculated as indicated for histamine.

Cytokine release assay

Mast cells (1 × 10⁶) were incubated with or without yeasts (1 × 10⁷) of S. schenckii at 37°C in supplemented RPMI medium with soybean trypsin inhibitor (0.1 mg/ml; Sigma-Aldrich) under an atmosphere of 5% CO₂ for 6 h and 24 h. Cell-free supernatants were collected and stored at −80ºC until assayed for TNF-α and IL-6. The concentrations were determined using rat TNF-α and IL-6 enzyme-linked immunosorbent assay kits (Invitrogen, Camarillo, CA, USA) according to the manufacturer's instructions. The results were expressed in pg/ml.

Western blot analysis

Peritoneal mast cells (2 × 10⁶) were stimulated with yeasts of S. schenckii (2 × 10⁷) in supplemented RPMI medium for 30 min at 37°C. Mast cells stimulated with or without 500 nM ionophore of calcium plus 2 μM phorbol 12-myristate 13-acetate were used as positive and negative controls. Cells were lysed with lysis buffer (150 mM NaCl, 50 mM HCl-Tris, 1 mM ethylene glycol tetraacetic acid, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.6 μM Aprotinin, and 2 μm Leupeptin; pH 8), incubated at 4°C with orbital agitation, and centrifuged at 13c200 g. Proteins from supernatants were quantified, and equal amounts were separated in 10% SDS-polyacrylamide gels. Membranes were blocked and incubated at 4°C overnight with rabbit polyclonal antibodies against phospho-extracellular signal-regulated kinase (pERK) 1/2 (1:2000, Santa Cruz Biotechnology, Dallas, Texas, USA) or mouse monoclonal antibodies against β-actin (1:1000, Sigma-Aldrich). After incubation with peroxidase-conjugated secondary antibody, proteins were detected using the chemiluminescence Western blot detection kit (Luminata Forte Western HRP substrate, Millipore). Quantification of protein bands was carried out by densitometry using Quantity One 1-D Software 4.6.3 (Bio-Rad, Hercules, CA, USA). Values were expressed as relative media of intensity of each band compared with that of the corresponding β-actin.

Statistical analysis

The data are presented as mean values ± standard error of the mean. Statistical analysis was performed using the Student t test. Differences were considered statistically significant when P < 0.05.

Results

Mast cells in contact with S. schenckii yeasts (Fig. 1A) showed a morphology that was different from unstimulated ones (Fig. 1B). Neither microvilli nor microfolds that cover the surface of unstimulated cells were apparent, and granules were protruding at the cell surface. However, granule matrices were not observed extracellulary, which was evident in mast cells degranulated by C48/80 (Fig. 1C). To confirm that yeasts of S. schenckii do not induce degranulation, the release of preformed mediators was analyzed. As shown in Figure 1D, histamine secreted by mast cells in response to different concentrations of yeasts was similar to that from spontaneous secretion. The increase in time of incubation did not modify the results. However, stimulation of mast cells by C48/80 resulted in a 3-fold increase in histamine release. To determine whether yeast cells of S. schenckii produce any substance that suppresses degranulation or degrades released histamine, we incubated cells simultaneously with the fungus and C48/80. Mast cells released equal percentages of histamine (48%) in response to C48/80 alone and in the presence of yeasts (Fig. 1E). Subsequently, we also examined the release of β-hexosaminidase (Fig. 1F), and no difference was shown when cells were stimulated with the fungus in relation to spontaneous release.

Figure 1.

Analysis of mast cell degranulation following exposure to yeasts from Sporothrix schenckii by scanning electron microscopy and quantification of released preformed mediators. Scanning electron micrographs of (A) peritoneal mast cells incubated with yeasts of S. schenckii (B) unstimulated peritoneal mast cells, and (C) peritoneal mast cells stimulated by C48/80 (5 μg/ml). All micrographs correspond to incubations for 30 min at 37°C. (D) Histamine released by mast cells after incubation with supplemented Roswell Park Memorial Institute (RPMI)-1640 medium (control), C48/80 (5 μg/ml), or yeasts of S. schenckii for 30, 60, or 90 min. (E) Histamine released by mast cells in response to C48/80 (5 μg/ml) following incubation with and without yeasts of S. schenckii for 30 min. (F) β-hexosaminidase released by mast cells following incubation with supplemented RPMI medium (control), C48/80 (5 μg/ml), or yeasts of S. schenckii (5 × 10⁶) for 30 min. Values of mediators released from cells incubated with RPMI medium or with C48/80 were indicative of spontaneous or degranulation-induced secretion. Results are expressed as mean ± standard error of the mean of three independent experiments performed in triplicate. *P < 0.001 C48/80 vs. control and yeasts of S. schenckii. This Figure is reproduced in color in the online version of Medical Mycology.

To assess de novo synthesis of early-response cytokines by mast cells in response to the fungus, we determined the secretion of TNF-α and IL-6. Significant levels of both cytokines were obtained at 24 h of stimulation with yeast cells (Fig. 2A, B), increasing the levels of TNF-α and IL-6 2.4-fold and 1.7-fold, respectively, in the medium in relation to the control. At 6 h, the values were similar to those of spontaneous secretion. Then, we examined the phosphorylation of ERK in mast cells in the presence and absence of S. schenckii yeasts. pERK1/2 was significantly induced (P < 0.05) in response to S. schenckii stimulation (Fig. 2C). The fungus increased ERK1 and ERK2 phosphorylation 1.7-fold and 1.5-fold, respectively, in relation to mast cells incubated without yeasts.

Figure 2.

De novo synthesis of mediators in mast cells following exposure to yeasts of Sporothrix schenckii. (A) Tumor necrosis factor alpha and (B) interleukin-6 released by mast cells incubated in supplemented Roswell Park Memorial Institute (RPMI)-1640 medium with or without 1 × 10⁷ yeasts of S. schenckii for 6 h and 24 h. Values of cytokines released from cells incubated with RPMI medium alone (control) was indicative of spontaneous secretion. (C) Western blot and densitometric analysis of the expression of p-extracellular signal-regulated kinase 1/2 in mast cells incubated with supplemented RPMI medium (control), yeasts of S. schenckii (2 ×10⁷), or ionophore of calcium–phorbol 12-myristate 13-acetate (500 mM–2 μM) for 30 min. In densitometric analysis, values are expressed as relative media of intensity (M.I.) and were calculated as the ratio of M.I of each protein to that of β-actin and normalized to that of control assay. Results are expressed as mean ± standard error of the mean of two (A and B) or three (C) independent experiments performed in duplicate. *P < 0.05 and +P < 0.01 S. schenckii vs. respective control. Abbreviations: CaI–PMA, ionophore of calcium–phorbol 12-myristate 13-acetate; IL, interleukin; pERK, phospho-extracellular signal-regulated kinase; TNF-α, tumor necrosis factor alpha. This Figure is reproduced in color in the online version of Medical Mycology.

Discussion

Mast cells, strategically located in skin and other portals of infection, are one of the first inflammatory cells to come in contact with pathogens [9]. In sporotrichosis, initial infection results from the transformation of S. schenckii conidia to the yeast form, likely triggered by exposure to host conditions [17]. We recently reported mast cell involvement in S. schenckii infection in mice and mast cell activation after interaction with conidia [16]. In the present study, we examined mast cells response to S. schenckii yeasts. Our results indicate that mast cells develop morphological changes related to activation through contact with the fungus yeast cells and the induction of TNF-α and IL-6 release and activate MAPK signaling events. No degranulation is induced.

Some recent works suggest that mast cells respond to yeasts or their components. Zymosan of yeast cell walls induces leukotriene production [19] and reactive oxygen species generation [20]. Yeast extract from M. sympodialis induces the release of cysteinyl leukotriene by mast cells [15] and increases the release of IL-6 from mast cells in atopic eczema patients [12]. However, no activation in terms of degranulation is obtained. Mast cells also have been involved in the immune response against skin lesions of P. brasiliensis apparently as a source of IL-10 [11]. We report no significant histamine or β-hexosaminidase release from peritoneal mast cells after stimulation by S. schenckii; however, cells are activated and secretion of cytokines is induced. Taken together, these findings suggest that yeast from pathogenic fungus can directly activate mast cells, primarily without inducing degranulation but by inducing de novo synthesis of inflammatory mediators.

It has been demonstrated that macrophages incubated with a lipid extract obtained from S. schenckii yeasts can produce TNF-α and IL-6 [21,22]. Dendritic cells derived from human monocytes also release TNF-α in response to yeast from S. schenckii [6], and marrow-derived dendritic cells produce TNF-α and IL-6 after exposure to live yeasts or high dosages of exoantigen [23]. Recently, human keratinocytes were reported to produce IL-6 when challenged with yeast cells [24]. Here, we identify mast cells as an additional source of both cytokines in response to S. schenckii yeasts. The time at which the secretion of both cytokines is detected and the absence of degranulation in response to yeast indicate that they are de novo synthesized. As TNF-α and IL-6 are considered crucial mediators in fungal defense [25], the release of both mediators from mast cells may contribute to the overall response to the host immune system during S. schenckii infection.

Fungi recognition in myeloid cells has been associated with an effector response involving secretion of cytokines [26], a process dependent on different signaling pathways including the MAPK pathway [27]. Infection of oral epithelial cells with C. albicans leads to activation of the three MAPK subfamilies, p38 MAPK, ERK, and c-Jun NH₂-terminal kinase (JNK), which orchestrates the production of proinflammatory mediators [28]. Malassezia sympodialis extract enhances IL-6 production by allergen-activated mast cells through ERK1/2 activation [15]. IL-6 and IL-8 secretion by human lung epithelial cells in response to P. brasiliensis is dependent on the activation of p38 MAPK and ERK1/2 [29]. In relation to S. schenckii, researchers have shown that yeasts induce a strong activation of JNK, ERK1/2, and p38 MAPK in dendritic cells in association with IL-6 and TNF-α secretion [6]. Thus, it is plausible that the ERK1/2 activation that we demonstrated in mast cells in response to yeasts of S. schenckii was related to the release of de novo synthesized IL-6 and TNF-α.

Previously, we showed that conidia from S. schenckii also induce de novo synthesis of cytokines [16]. However, the magnitude of the response was higher than what we reported for yeasts. Our results are consistent with data obtained relating to dendritic cells in which S. schenckii conidia are more potent than yeast in inducing TNF-α secretion [6]. Furthermore, macrophages induce a classic type I interferon transcriptional signature in response to infection with Histoplasma capsulatum conidia but not in response to infection with isogenic yeast cells [30]. Taken together, this data provide evidence that conidia are immunologically more active than yeasts. One explanation for the particular response induced by each morphotype is the differential expression of surface molecules in conidia and yeast that could be recognized by specific pattern-recognition receptors [31].

In summary, we provide the first description of the interaction of mast cells with S. schenckii yeasts. We show IL-6 and TNF-α secretion by mast cells in response to contact with S. schenckii yeasts, possibly by activating MAPK pathways without degranulation. Further studies are needed to evaluate the implication of this interaction during sporotrichosis.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.

This work was supported by grants PIBB 09–1 and 89642 from the Autonomous University of Aguascalientes and Consejo Nacional de Ciencia y Tecnología Y. Romo-Lozano had a doctoral fellowship from Consejo Nacional de Ciencia y Tecnología. The authors thank Biologist Karla Macías, Chemist Renata Roldán, Veterinary José Luis Ponce, Chemist María Luisa Rodríguez, and Chemist Érika Córdova-Martínez for their technical support; Dra. Claudia González-Espinosa for providing the p-ERK antibody; and Dr. Istvan Berczi for reviewing the paper.