Influence of TLR-2 in the immune response in the infection induced by fungus Sporothrix schenckii.

Immunological Investigations, 2014; 43(4): 370–390 ! Informa Healthcare USA, Inc. ISSN: 0882-0139 print / 1532-4311 online DOI: 10.3109/08820139.2013.879174

Influence of TLR-2 in the immune response in the infection induced by fungus Sporothrix schenckii

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Department of Clinical Analysis, Araraquara School of Pharmaceutical Sciences, Saõ Paulo State University, SP, Brazil, 2 Department of Preventive and Community Dentistry, School of Dentistry, Federal University of Rio Grande do Sul, RS, Brazil, 3 Department of Physiology and Pathology, Araraquara School of Dentistry, Saõ Paulo State University, SP, Brazil Toll-like receptors (TLRs) play an important role in immunity, since they bind to pathogen surface antigens and initiate the immune response. However, little is known about the role of TLR-2 in the recognition of S. schenckii and in the subsequent immune response. Therefore, the aim of this study was to evaluate the involvement of TLR-2 in the immune response induced by S. schenckii. C57BL/6 mice (WT) and C57BL/6 TLR-2 knockout (TLR-2 / ) were used to evaluate, over a period of 10 weeks of sporotrichotic infection, the influence of TLR-2 over macrophages production of IL-1b, IL-12 and TNF-a, their stimulation level by NO release and the production of IFN -g, IL-6, IL-17 and TGF-b by spleen cells. The results showed that the production of pro-inflammatory mediators and NO, TLR-2 interference is striking, since its absence completely inhibited it. IL-17 production was independent of TLR-2. The absence of Th1 response in TLR2 / animals was concomitant with IL-17 production. Therefore, it can be suggested that TLR-2 absence interferes with the course of the infection induced by the fungus S. schenckii. Keywords Cytokines, mice, Sporothrix schenckii, sporotrichosis, TLR-2

INTRODUCTION

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Thais de C. Negrini,1 Lucas S. Ferreira,1 Rodrigo A. Arthur,2 Pa ˆ mela Alegranci,1 Marisa C. P. Placeres,1 Luis C. Spolidorio,3 and Iracilda Z. Carlos1

Sporotrichosis is an infectious disease caused by the dimorphic fungus Sporothrix schenckii, which affects humans and other mammals (Kauffman, 1999). The disease has a worldwide distribution with focal areas in tropical and subtropical climates (Bustamante & Campos, 2001). The forms of this pathogen are commonly found in the soil where the fungus grows in association with plant debris, mold and wood (da Rosa et al., 2005). The infection generally results from inoculation of the fungus by small traumas during leisure and occupational activities such as floriculture, horticulture,

Correspondence: Iracilda Z. Carlos, Department of Clinical Analysis, Araraquara School of Pharmaceutical Sciences, Sao Paulo State University, Rua Expediciona´rios do Brasil, no 1621, Araraquara, SP, CEP 14.801-902. Brazil. E-mail: [email protected]


TLR-2 and immune response against S. schenckii

gardening and wood exploration (Lopes-Bezerra et al., 2006). Also, the role of felines in the transmission of this mycosis to humans has gained importance since the 1998 epidemic in Rio de Janeiro, Brazil, where several veterinarians, veterinary technicians, animal handlers and owners of cats were diagnosed with sporotrichosis (Barros et al., 2008; Nakamura et al., 1996; Schubach et al., 2008). This disease, in general, causes subcutaneous nodular and/or ulcerative lesions that spread through lymphatic vessels (Ramos-e-Silva et al., 2007). These lesions are usually restricted to the skin, with disseminated infection being unusual, although it has been associated with immunocompromised patients, such as HIV-infected patients, suggesting that S. schenckii is an emerging opportunistic pathogen in immunodeficient individuals (Hardman et al., 2005). The immunological mechanisms involved in prevention and control of sporotrichosis suggest that Th1 cell-mediated immunity plays an important role in protecting the host against S. schenckii (Alegranci et al., 2013; Carlos et al., 1992; Carlos et al., 1994; Maia et al., 2006). The primary defense mechanism against the fungus is the elimination of the microorganisms by macrophages via phagocytosis and production of toxic agents such as nitric oxide (NO), a potent mediator of inflammatory responses (Carlos et al. 2003). Moreover, IFN-g has also been described as participating in this process of protection, promotion an increase of neutrophils’ and macrophages’ activation (Romani, 2004). In 2003, Moseley et al. (2003) described a new phenotype of T helper lymphocytes known as Th17. The persistence and differentiation of Th17 cells have been associated with the pathology observed in some autoimmune diseases and chronic infections (Robinson et al., 2013; Weaver et al., 2006), besides being associated with the development of inflammatory responses and possible protection of the host against intracellular bacteria and fungi (McGeachy & McSorley, 2012; Weaver et al., 2007). These cells produce mainly IL-17 which, on macrophages, induces the expression of TNF-a and IL-6. Furthermore, it is suggested that IFN-g regulate negatively the differentiation of Th17 cells, while TGF-b stimulates the expression thereof (Korn et al. 2007; Lee et al., 2006). TGF-b represents a subpopulation of T lymphocytes important for maintaining the balance of immune responses. These cells suppress the production of proinflammatory cytokines preventing damage that could be caused by an exacerbated inflammatory response (Liu et al., 2010; Torre et al. 2002; Wing & Sakaguchi, 2012). Recognition of microorganisms by the immune system is the first and most important step for triggering of both innate and adaptive immune responses (Underhill & Ozinsky, 2002). Conserved throughout evolution, mammalian Toll-like receptors (TLRs) are thought to be part of a complex of innate recognition mechanisms against microbial pathogens and comprise a large family of at least 12 members (Kawai & Akira, 2010; Kondo et al., 2012). TLRs recognize a wide variety of pathogen associated molecular patterns (PAMPs) from bacteria, viruses and fungi as well as some host molecules (Akira et al., 2006; Kawai & Akira, 2007; Romani, 2004).According to their location, either on the cell membrane surface or intracellular, TLRs can be divided into two large groups. The components of the first group, TLRs-1, -2, -4, -5 and -6 are

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present on the cell surface and are mainly responsible for recognizing lipid structures (McGettrick & O’Neill, 2010). TLR-2, for example, recognize a variety of microbial components including lipoproteins/lipopeptides from various pathogens, lipoteichoic acid from Gram-positive bacteria and zymozan, a polysaccharide present in the cell wall of fungi (Takeda et al., 2003; Takeda & Akira, 2005). As for the components of the second group, TLRs-3, -7, -8 and -9 are present in the intracellular compartment and are mainly responsible for recognizing nucleic acids (McGettrick & O’Neill, 2010). In this context, it has been demonstrated that the recognition of several fungi is TLR-mediated. For instance, TLR-2 and TLR-4 are involved in the recognition of PAMPs from Aspergillus fumigatus and Candida albicans (Braedel et al., 2004; Chai et al., 2011; Meier et al., 2003; Underhill et al., 1999). Netea et al. (2002, 2004) showed that TLR-4-deficient mice had an increased susceptibility to C. albicans infection, whereas the absence of TLR-2 on phagocytes increased the resistance to candidiasis. Regarding S. schenckii, however, previous results from our group showed reduced synthesis of inflammatory mediators by S. schenckii-infected TLR-4-deficient mice when compared to wild-type mice (Sassa´ et al., 2009). Also, Negrini et al. (2013) evaluated the role of TLR-2 during the process of phagocytosis of S. schenckii, having verified that the absence of this receptor resulted in a lower rate of phagocytosis of this pathogen along with reduced production of TNF-a, IL-1b, IL-12 and IL-10 compared TLR-2-expressing controls. These results suggest a new concept of how the immune system by TLR-2, recognizes and induces the production of mediators in response to the fungus S. schenckii. Therefore, to better understand the role of TLR-2 in the host immune response against the fungus S. schenckii, TLR-2 knockout and control mice (C57BL/6) were infected with S. schenckii yeast cells and immune response was assessed over 10 weeks by assaying production of inflammatory mediators.

MATERIALS AND METHODS Microorganism and culture conditions Sporothrix schenckii, strain 1099-18, isolated from a human case of sporotrichosis at the Mycology Section of the Department of Dermatology, Columbia University, New York, NY, USA, was used on all experiments. This strain was provided by Dr. Celuta Sales Alviano, from the Institute of Microbiology, Federal University of Rio de Janeiro, RJ, Brazil. The fungus was grown at 37 C for 8 days in Brain–Heart Infusion broth (BHI; Difco Laboratories, Detroit, MI) under constant agitation of 150 cycles/min, in order to yield a high percentage of yeast conversion. Lipid antigen extraction Lipid antigen (LipAg) was extracted from yeast cells resuspended in 10 mL of sterile phosphate buffered saline (PBS, pH 7.4). This suspension was sonicated at 50 W (MSE Ultrasonic Disintegrator; Danbury, CT, USA) during 10 cycles of 20 minutes each with intervals of 2 minutes between each cycle and then centrifuged for 1 hour at 700 x g and 5 C (Hettich Zentrifugen, D-78532, Germany). LipAg was obtained from the resulting pellet by extraction with chloroform: methanol (2:1 v/v) for 2 h under constant stirring at room



temperature. The insoluble residue was separated by centrifugation at 5000 g for 1 h and then re-extracted 3 more times as before. The remaining residue was called LipAg (Carlos et al., 2003). For use on cell cultures, LipAg was dissolved in RPMI-1640C medium (containing 2b-mercaptoethanol 0.02 mM, penicillin 100 U/mL, streptomycin 100 U/ml, L-glutamine 2 mM and 5% fetal bovine serum). The antigen concentration used on cultured macrophages was assessed for its cytotoxicity potential by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, as described by Mosmann (1983), and resulted in a rate of cell viability above 70%. Animals Female C57BL/6 wild-type (WT) and C57BL/6 TLR-2 knockout (TLR-2 / ) mice (6 to 8 weeks-old, weighting from 20 to 25 grams) were obtained from the Ribeiraõ Preto School of Medicine, University of Saõ Paulo-USP, Brazil. The animals were kept in plastic cages with individual air filters in a room with controlled temperature (21 ± 1 C), humidity (65–75%) and light/dark cycles (12 h light – 12 h dark). The experimental protocol was approved by the Research Ethics Committee of the Araraquara Academic Center (# 940/2009) and was conducted in conformity with the standards of the Brazilian College of Animal Experimentation (COBEA). Infection method For S. schenckii infection, a yeast suspension in phosphate-buffered saline (pH 7.4) containing 109 cells/mL was prepared. Each animal in the experimental WT or TLR-2 / group was inoculated intraperitoneally with 0.10 mL of this suspension, while animals in the control groups of each lineage were injected with 0.10 mL of PBS alone. Mice were euthanized at different periods after inoculation (2, 4, 6, 8 and 10 weeks) according to the proposed experiments. Macrophages and culture conditions Macrophages were obtained from peritoneal exudates of WT and TLR-2 / animals. Briefly, a 3% aqueous solution of sodium thioglycollate (Difco) was inoculated into the peritoneal cavity of the animals. Three days later, the peritoneal exudates were collected, washed with 5 mL of sterile PBS (pH 7.4) and resuspended in RPMI-1640C medium. The macrophages were then counted in a Neubauer chamber (Boeco, Germany) and adjusted to a concentration of 5 106 macrophages/ml. Non-adherent cells (mostly neutrophils) were removed by incubating the suspension in cell culture plates for 1 h at 37 C in an atmosphere containing 5% CO2 (Forma Scientific, Marietta, OH) and then discarding the supernatant. The remaining adherent cells were incubated at 37 C, 5% CO2 for 24 h in the presence of bacterial lipopolysaccharide (Escherichia coli 0111 B) (LPS) (10 mg/mL), LipAg (57.6 mg/mL) or RPMI-1640C medium. After this period, supernatants were collected for measurement of nitric oxide (NO) and cytokines (IL-1b, IL-12 and TNF-a), by Griess assay and ELISA (enzyme-linked immunosorbent assay), respectively, as described elsewhere.

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Nitric oxide measurement First 50 mL aliquots of macrophage culture supernatants from all tested conditions were transferred to a flat bottom 96-well plate (Corning, Inc.) and added with the same volume of Griess reagent (Green et al., 1982). After 10 min of incubation in the dark at room temperature, the absorbance was read at 540 nm (Microplate Reader, Multiskan Ascent, Labsystems). NO concentration was calculated based on a standard curve of known sodium nitrite concentrations and results were expressed as mmols nitrite/105 cells. Extraction and weighting of the spleen Mice from both groups (infected and control) and lineages (WT and TLR-2 / ) were euthanized at different times during the course of the study and had the spleen aseptically removed. After extraction, the organ was weighed on an analytical balance (BOECO, Germany). Spleen cells and culture conditions Spleens were aseptically collected and placed in a Petri dish containing 3.0 mL of RPMI-1640C medium. A single-cell suspension was obtained by mincing each spleen. The obtained cell suspension was washed 3 times with RPMI-1640, counted on a Neubauer chamber and then resuspended to a concentration of 5 106 cells/mL in RPMI-1640C medium. After that, cells were cultivated in 24-well tissue culture plates at 5 106 cells per well in the presence of Concanavalin-A (Con-A) (0,5 mg/mL), LipAg (57.6 mg/mL) or RPMI-1640C medium at 37 C with 5% CO2 for 24 h. After this period, supernatants were collected for measurement of the cytokines IFN-g, IL-6, IL-17 and TGF-b by enzyme-linked immunosorbent assay (ELISA), as described below. Measurement of cytokines The levels of the cytokines IL-1b, IL-12, TNF-a, IFN-g, IL-6, IL-17 e TGF-b in culture supernatants were determined by ELISA (OptEIA; BD Biosciences, San Diego, CA) according to the manufacturer’s instructions on 96-well microplates. Cytokine concentrations were calculated from a standard curve of known cytokine concentrations and expressed in pg/mL. Statistical analysis Tukey’s test (GraphPad Prism 5 for Windows, San Diego, USA) was used to determine the statistical significance of differences between experimental groups. Significance was declared at p50.05. Results are representative of three independent experiments and are presented as the mean ± SD of duplicate observations. In vivo groups consisted of five animals.

RESULTS Production of IL-1b, IL-12 and TNF-a by peritoneal macrophages Ex vivo release of IL-1b (Figure 1), IL-12 (Figure 2) and TNF-a (Figure 3) was assayed in the supernatant from peritoneal macrophages obtained from noninfected or S. schenckii-infected WT and TLR-2 / mice and later exposed to LipAg, LPS (positive control) or RPMI-1640C alone (negative control).

Figure 1. IL-1b quantification in supernatants from peritoneal macrophages obtained from non-infected WT (A) or S. schenckii-infected WT mice (B) and from non-infected TLR-2 / (C) or S. schenckii-infected TLR-2 / mice (D). Supernatants were collected after 24-h stimulation of cultures with LPS (positive control), Lipid antigen (LipAg) or RPMI-1640C (negative control), and used to quantify IL-1b by a commercially available ELISA. The colour reaction was determined at 450 nm with an automated microplate reader. The results are expressed as pg/ml and are representative of three independent experiments. Each column represents the mean ± SD of 5 animals per experimental period (in weeks). Differences were declared significant at p50.05. * Means statistically significant difference compared with the respective week in non-infected mice. £Means statistically significant difference compared with week 4.



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Figure 2. IL-12 quantification in supernatants from peritoneal macrophages obtained non-infected WT (A) or S. schenckii-infected WT mice (B) and from noninfected TLR-2 / (C) or S. schenckii-infected TLR-2 / mice (D). Supernatants were collected after 24-h stimulation of cultures with LPS (positive control), Lipid antigen (LipAg) or RPMI-1640C (negative control), and used to quantify IL-12 by a commercially available ELISA. The colour reaction was determined at 450 nm with an automated microplate reader. The results are expressed as pg/ml and are representative of three independent experiments. Each column represents the mean ± SD of 5 animals per experimental period (in weeks). Differences were declared significant at p50.05. *Means statistically significant difference compared with the respective week in non-infected mice. £Means statistically significant difference compared with week 4.


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Figure 3. TNF-a quantification in supernatants from peritoneal macrophages obtained from non-infected WT (A) or S. schenckii-infected WT mice (B) and from non-infected TLR-2 / (C) or S. schenckii-infected TLR-2 / mice (D). Supernatants were collected after 24-h stimulation of cultures with LPS (positive control), Lipid antigen (LipAg) or RPMI-1640C (negative control), and used to quantify TNF-a by a commercially available ELISA. The colour reaction was determined at 450 nm with an automated microplate reader. The results are expressed as pg/mL and are representative of three independent experiments. Each column represents the mean ± SD of 5 animals per experimental period (in weeks). Differences were declared significant at p50.05. *Means statistically significant difference compared with the respective week in non-infected mice. £Means statistically significant difference compared with week 4.



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In general, there was an increased release of all three cytokines by cells from infected WT mice compared to non-infected mice, especially at the 2nd week post-infection. Interestingly, it was observed, at week 4, a decrease in the production of this mediator (p50.001 between 2nd and 4th weeks) for infected mice (Figures 1, 2 and 3). During the course of infection, the highest levels of IL-1b (Figure 1) and IL-12 (Figure 2) could be observed at the 2nd and 6th weeks post-infection, followed, at the 8th and 10th weeks, by subsequently declining levels. On the other hand, infected or non-infected TLR-2 / mice cells could not release detectable levels of any of the aforementioned cytokines in any culture condition (Figures 1, 2 and 3). Production of nitric oxide (NO) by peritoneal macrophages NO quantification was performed in supernatants from cultures of peritoneal macrophages from non-infected or S. schenckii-infected WT and TLR-2 / mice, cultured as previously described. In infected WT mice, NO production was ascending from the 2nd to the 6th week, reaching a sharp fall at the later phases of the disease (8th and 10th weeks) (Figure 4B) (p50.001 between 2nd and 10th weeks). In contrast, TLR-2 / mice were not able to produce detectable levels of this mediator in any of the culture conditions (Figures 4C and D). Weighting of the spleen Infected mice from both strains had increased spleen weight compared to their respective non-infected control (Figure 5). Such increase was significant at the 4th, 6th and 8th weeks post-infection within each strain (p50.001 in all cases), before returning to non-infected control-like values at the 10th week. When comparing infected WT and TLR2 / mice, weight differences were significant, in a period-to-period comparison, from the 2nd to the 8th week post-infection (p50.001 in all cases), but not at the 10th. Production of IFN-c, IL-6, IL-17 and TGF-b by spleen cells IFN-g, IL-6, IL-17 and TGF-b were assayed in the culture supernatant from spleen cells obtained from non-infected or S. schenckii-infected WT and TLR2 / mice and later exposed to LipAg, Concanavalin A (Con A) (positive control) or RPMI-1640C alone (negative control). Regarding IFN-g, peak release occurred at the 6th week post-infection in S. schenckii-infected WT mice, resembling the pattern found for IL-12 and NO. No significant difference was found between ConA- and LipAg-stimulated release of IFN-g (Figure 6B). As observed for macrophages’ cytokines (IL-1b, IL-12 and TNF-a), cultures from both non-infected and infected TLR-2 / mice showed no detectable release of IFN-g (Figures 6C and D). Unlike other cytokines showed above, release of IL-6 was observed in the culture supernatant of splenocytes from both WT and TLR-2 / mice (Figure 7). Moreover, analysis of cultures from infected WT mice revealed a constantly increasing release of IL-6 from the 2nd to the 10th week postinfection. At the latter, no significant difference was found between ConAand LipAg-stimulated release of IL-6 (Figure 7B). In cultures from infected TLR-2 / mice, peak release occurred at the 4th and 6th week post-infection (Figure 7D), coinciding with the peak release for IL-17.

Figure 4. NO quantification in supernatants from peritoneal macrophages obtained from non-infected WT (A) or S. schenckii-infected WT mice (B) and from non-infected TLR-2 / (C) or S. schenckii-infected TLR-2 / mice (D). Then, 50 mL aliquots of each supernatant were collected after 24-h stimulation of cultures with LPS (positive control), Lipid antigen (LipAg) or RPMI-1640C (negative control) and mixed with equal volume of Griess reagent. The colour reaction was determined at 540 nm with an automated microplate reader. The results are expressed as mmol of nitrite/5 106 cells and are representative of three independent experiments. Each column represents the mean ± SD of 5 animals per experimental period (in weeks). Differences were declared significant at p50.05. £means statistically significant difference compared with week 10.



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Figure 5. Spleen weight of control (C) or S. schenckii-infected WT and TLR-2 / mice. The results are expressed as mg and are representative of three independent experiments. Each column represents the mean ± SD of 5 animals per experimental period. Differences were declared significant at p50.05. *Means statistically significant difference compared with infected mice in weeks 4, 6 and 8. £Means statistically significant difference in the comparison between WT and TLR-2 / mice at the same week of infection.

Figure 8 shows the release of IL-17 in supernatants from spleen cells of noninfected or S. schenckii-infected WT and TLR-2 / mice. As was the case with IL-6, IL-17 release was detectable in the culture supernatant of splenocytes from both WT and TLR-2 / mice but, interestingly, it showed markedly higher levels in supernatants from TLR-2 / than WT mice (in a period-to-period comparison, p50.001 on all comparisons). Peak release for both mice strains occurred at the 6th week post-infection, higher in ConA- than in LipAgstimulated cultures (p50.001 and p50.01 for WT and TLR-2 / cultures, respectively. Figure 9 shows the release of TGF-b in the culture supernatants of spleen cells from WT and TLR-2 / mice. Surprisingly, TGF-b was detected only in cultures from infected TLR-2 / mice (Figure 9D). It is also important to note the inverse correlation between TGF-b’s lowest and IL-17’s highest release at the 6th week post-infection.

DISCUSSION Toll-like receptors (TLRs) are pattern-recognition receptors that recognize specific conserved components of pathogens and are responsible for enabling the innate immune system to induce an efficient adaptive response against the pathogen (Kondo et al., 2012). Several studies have demonstrated the importance of TLRs in fungal infections, pointing TLR-2, TLR-4 and TLR-9 as the main ones responsible for the recognition of these pathogens (Romani, 2011). Specifically for S. schenckii, previous studies from our group showed that TLR-4 participates in its recognition and immune response induction (Sassa´ et al., 2009; Sassa´ et al., 2012). In this work, we decided to investigate the role of TLR-2 using C57BL/6 TLR-2 knockout mice.

Figure 6. IFN-g quantification in supernatants from spleen cells obtained from non-infected WT (A) or S. schenckii-infected WT mice (B) and from non-infected TLR-2 / (C) or S. schenckii-infected TLR-2 / mice (D). Supernatants were collected after 24-h stimulation of cultures with ConA (positive control), Lipid antigen (LipAg) or RPMI-1640C (negative control), and used to quantify IFN-g by a commercially available ELISA. The colour reaction was determined at 450 nm with an automated microplate reader. The results are expressed as pg/ml and are representative of three independent experiments. Each column represents the mean ± SD of 5 animals per experimental period (in weeks). Differences were declared significant at p50.05.



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Figure 7. IL-6 quantification in supernatants from spleen cells obtained from non-infected WT (A) or S. schenckii-infected WT mice (B) and from non-infected TLR-2 / (C) or S. schenckii-infected TLR-2 / mice (D). Supernatants were collected after 24-h stimulation of cultures with ConA (positive control), Lipid antigen (LipAg) or RPMI-1640C (negative control), and used to quantify IL-6 by a commercially available ELISA. The colour reaction was determined at 450 nm with an automated microplate reader. The results are expressed as pg/ml and are representative of three independent experiments. Each column represents the mean ± SD of 5 animals per experimental period (in weeks). Differences were declared significant at p50.05. *Means statistically significant difference compared with LipAg in week 10.


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Figure 8. IL-17 quantification in supernatants from spleen cells obtained from non-infected WT (A) or S. schenckii-infected WT mice (B) and from non-infected TLR-2 / (C) or S. schenckii-infected TLR-2 / mice (D). Supernatants were collected after 24-h stimulation of cultures with ConA (positive control), Lipid antigen (LipAg) or RPMI-1640C (negative control), and used to quantify IL-17 by a commercially available ELISA. The colour reaction was determined at 450 nm with an automated microplate reader. The results are expressed as pg/mL and are representative of three independent experiments. Each column represents the mean ± SD of 5 animals per experimental period (in weeks). Differences were declared significant at p50.05. *Means statistically significant difference compared with WT infected mice. £Means statistically significant difference compared with LipAg in week 6.



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Figure 9. TGF-b quantification in supernatants from spleen cells obtained from non-infected WT (A) or S. schenckii-infected WT mice (B) and from noninfected TLR-2 / (C) or S. schenckii-infected TLR-2 / mice (D). Supernatants were collected after 24-h stimulation of cultures with ConA (positive control), Lipid antigen (LipAg) or RPMI-1640C (negative control), and used to quantify TGF-b by a commercially available ELISA. The colour reaction was determined at 450 nm with an automated microplate reader. The results are expressed as pg/ml and are representative of three independent experiments. Each column represents the mean ± SD of 5 animals per experimental period (in weeks). Differences were declared significant at p50.05.


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Li et al. (2012), attempting to determine if TLR-2, TLR-4 and NF-kB were involved in production of IL-6 and IL-8 by human keratinocytes challenged with S. schenckii conidia and yeast, pretreated the cells with anti-TLR-2, antiTLR-4 or a NF-kB inhibitor prior to challenge with the fungus. With that, the authors observed that production of the mediators was blocked using antiTLR-2 and anti-TLR-4 and neutralized with the use of the NF-kB inhibitor, suggesting that IL-6 and IL-8 are triggered by the activation of TLR-2 and TLR-4. To verify the role of TLR-2 on the immune response induced by S. schenckii, we challenged cells from infected and non-infected animals with the fungus’ lipid antigen and, as in previous works (Carlos et al., 2003; Sassa´ et al., 2009; Sassa´ et al., 2012), this antigen was able to effectively restimulate cells from infected animals ex vivo. This response was probably obtained by the ability of TLR-2 recognizes mainly lipid components present in the microorganisms (McGettrick & O’Neill, 2010; Takeda et al., 2003; Takeda & Akira, 2005). Our results showed that only WT animals’ cells were capable of releasing detectable levels of IL-1b, IL-12, TNF-a and IFN-g (Figures 1, 2, 3 and 6, respectively). A similar result was verified by Netea et al. (2002) who found that the production of TNF-a and IL-1b by mononuclear cells, obtained from C3H/HeJ mice deficient for TLR-4 and challenged with Candida albicans, was inhibited only when TLR-2 was blocked, suggesting a strong dependency on TLR-2. Villamon et al. (2004a) analyzed that the in vitro production of TNF-a by macrophages obtained from TLR2 / mice in response to C. albicans was significantly lower compared to control animals. In another study, these authors used a murine model of systemic infection by C. albicans in which resistance to reinfection was induced by prior exposure of TLR-2 / mice to a low virulence C. albicans strain. The results indicate that, despite presenting a very reduced production of Th1 cytokines (IFN-g, IL-12 and TNF-a) compared to WT animals, KO animals were still able to orchestrate a specific humoral response (Villamon et al., 2004b), suggesting that the absence of this receptor induces the immune system of these animals to use other means in an attempt to combat pathogens. In addition to not being able to release pro-inflammatory cytokines, TLR-2 / animals’ cells could not produce detectable levels of NO, as opposed to WT animals in which we observed a marked production throughout the infection period, peaking at the 6th and then declining over the following weeks (Figure 4). The large production of this mediator early on the initial weeks post-infection demonstrates a potent inflammatory response installed, since NO is a major cytotoxic mediator of immune effector cells capable of destroying microorganisms and even tumor cells (Dusse et al., 2003). Netea et al. (2004) demonstrated an increased IFN-g production by spleen cells in response to infection by C. albicans in mice deficient for TLR-2. In contrast, our results showed increased production of IFN-g only by spleen cells obtained from WT mice (Figure 6), especially in the 4th and 6th weeks of the infectious process. Another interesting fact to note is that in the same period of infection, there was increased production of IL-12 (Figure 2), which is the main inducer of IFN-g by T-cells (Szabo et al., 2003). Previous studies

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conducted in our laboratory with TLR-4-deficient C3H/HeJ mice showed an increased IL-12 ex vivo production by cells from S. schenckii-infected mice (Sassa´ et al., 2009). In fungal infections, despite numerous attempts to elucidate the role of TLRs in the production of pro-inflammatory cytokines in vitro, little is known about the actual role of these receptors in the contribution of the inflammatory pathology associated with infection in vivo. Although inflammation is a key component of the protective response against the fungal pathogens, a dysregulation in this mechanism can exacerbate the infectious process (Bozza et al., 2008). T helper 17 cells represent a new subtype of T effector cells that can act in an exacerbated inflammatory response, previously attributed to an uncontrolled Th1 response. Th17 lymphocytes produce, among other cytokines, IL-21, IL-22, IL-26 and cytokines of the IL-17 family which are potent inducers of inflammation, leading to cell infiltration and production of other inflammatory cytokines (Chen et al., 2007; Mesquita Jr. et al., 2009). Uncontrolled production of IL-17 is associated with various autoimmune conditions such as multiple sclerosis, inflammatory bowel disease, psoriasis and lupus erythematosus. On the other hand, Th17 lymphocytes have also been associated with protection in various infections (Chen et al. 2007; Mesquita Jr. et al., 2009), with IL-17 production being associated with the host’s defense mechanisms against several pathogens (Ouyang et al. 2008), including Gramnegative bacteria like Klebsiella pneumonia (Ye at al., 2001) and Citrobacter rodentium (Mangan et al., 2006) and Gram-positive bacteria such as Staphylococccus aureus (Joshi et al., 2012). In infections caused by fungi of the genus Coccidioides, it was found that production of IL-17 in the initial stage of infection promoted neutrophil recruitment into the lungs and tissue repair (Lin et al., 2010). Recent reports suggest that production of Th17 cytokines have a role in controlling Candida and Aspergillus infections, especially in conditions where Th1 response is impaired (Romani, 2011). Here we demonstrate that, in addition to not being able to release proinflammatory cytokines and NO, cells from TLR-2 / mice released higher levels of IL-17 when compared to that of WT mice. Thus our results demonstrated that the absence of Th1 cytokines in animals TLR2 / coincided with the production of IL-17 (Figure 8). Loures et al. (2009) in order to analyze the role of TLR-2 in an experimental model of chronic pulmonary infection induced by Paracoccidioides brasiliensis also observed that mouse macrophages TLR-2 / showed Th17 polarized response. TGF-b has been associated with the ability to inhibit the differentiation of Th1 and Th2 cells. Volpe et al. (2008) found that production of TGF-b, IL-23, IL-1b and IL-6 were essential for the differentiation of Th17 in humans and the absence of TGF-b induced transformation profile Th1 to Th17. A recent study of Ghoreschi et al. (2010). demonstrates that murine and human Th17 cells have the same development requirements. Furthermore, these authors demonstrated that Th17 differentiation can occur in the absence of TGF-b signaling. Therefore, the factors that drive the differentiation of Th17 pathway are still controversial. In the present study, only animals TLR-2 / produced TGF-b. This production was high at the beginning (week 2) and in the late stages of



infection (8th and 10th weeks) (Figure 9). TGF-b was inversely proportional to the production of IL-17 in these animals (Figure 8). Our results suggest that a high production of TGF-b inhibited the differentiation of Th17 cells, that can be deducted by the levels of IL-17 found during the experimental period. In relation to the WT mice, there was production of IL-17 independently of TGF-b, suggesting that the production of this cytokine was derived from other stimulus, probably by IL-6 (Figure 7) with IL-1b (Figure 1). Ghoreschi et al. (2010) demonstrated that IL-6 in combination with IL-1b effectively induced IL-17 production, independently of TGF-b. By weighting the spleen of the animals during the course of the infection, we have an indication of how infection is progressing. The increase in spleen weight until the 8th week post-infection suggests spleen cell proliferation as a result of the ongoing immune response. At the 10th week post-infection, spleens from infected animals of both strains had returned to non-infectedlevel weight, whereas most cytokines, although in decreasing paths, were still significantly increased. The present study suggests that, in sporotrichosis, TLR-2 absence prevents the development of a Th1 response, leading to a cytokine profile that favors Th17 cell development. Together with our previous reports regarding the influence of TLR-4 in immune response against sporotrichosis (Sassa´ et al. 2009; Sassa´ et al. 2012), it is clear that TLRs play an important role in determining how the immune system responds against S. schenckii, as it does to other fungi. The use of other signaling pathways in the absence of certain receptors underlines the immune system’s inherent plasticity to combat pathogens, but does not exclude the occurrence of such ‘‘secondary’’ paths in normal microenvironment conditions.

ACKNOWLEDGEMENTS The authors are grateful to Marisa Campos Polesi Placeres for technical support. This work was supported by grants from the The State of Sao Paulo Research Foundation (FAPESP) (Grants no. 2009/07529-1 and 2009/11999-3).

DECLARATION OF INTEREST The authors declare that there is no conflict of interest. The authors alone are responsible for the content and writing of the paper.

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The immune response against Candida spp. and Sporothrix schenckii.

Genome Sequence of the Pathogenic Fungus Sporothrix schenckii (ATCC 58251).

Chromosomal polymorphism in the Sporothrix schenckii complex.

Sporothrix schenckii scleritis.

Immune Response Induced by an Immunodominant 60 kDa Glycoprotein of the Cell Wall of Sporothrix schenckii in Two Mice Strains with Experimental Sporotrichosis.

Bone involvement by Sporothrix schenckii in an immunocompetent child.

Melanins Protect Sporothrix brasiliensis and Sporothrix schenckii from the Antifungal Effects of Terbinafine.

Sporothrix schenckii sensu stricto and Sporothrix brasiliensis Are Differentially Recognized by Human Peripheral Blood Mononuclear Cells.

In vitro photodynamic inactivation of Sporothrix schenckii complex species.

Sporothrix schenckii fungemia without disseminated sporotrichosis.

Molecular typing of Sporothrix schenckii isolates from cats in Malaysia.

Mouse bone marrow-derived dendritic cells can phagocytize the Sporothrix schenckii, and mature and activate the immune response by secreting interleukin-12 and presenting antigens to T lymphocytes.

Respiratory Failure due to Possible Donor-Derived Sporothrix schenckii Infection in a Lung Transplant Recipient.

Genotyping species of the Sporothrix schenckii complex by PCR-RFLP of calmodulin.

Dectin-1 expression by macrophages and related antifungal mechanisms in a murine model of Sporothrix schenckii sensu stricto systemic infection.

Δ(24)-Sterol Methyltransferase Plays an Important Role in the Growth and Development of Sporothrix schenckii and Sporothrix brasiliensis.

Cell wall proteins of Sporothrix schenckii as immunoprotective agents.

Long-chain fatty acids of Sporothrix (Sporotrichum) schenckii.

Antifungal activity of Pterocaulon species (Asteraceae) against Sporothrix schenckii.

O-glycosidically linked oligosaccharides from peptidorhamnomannans of Sporothrix schenckii.

Ex vivo determination of potentially virulent Sporothrix schenckii,.

Sporothrix schenckii complex biology: environment and fungal pathogenicity.

Comparative genomics of the major fungal agents of human and animal Sporotrichosis: Sporothrix schenckii and Sporothrix brasiliensis.

Potential Role of Carvedilol in the Cardiac Immune Response Induced by Experimental Infection with Trypanosoma cruzi.