Response of macrophage Toll-like receptor 4 to a Sporothrix schenckii lipid extract during experimental sporotrichosis

Abstract

Toll-like receptors have been implicated in the recognition of various pathogens, including bacteria, viruses, protozoa and fungi. However, no information is available about Toll-like receptor 4 (TLR4) participation in Sporothrix schenckii recognition and the consequent triggering of the immune response to this fungal pathogen. Following activation of TLRs by ligands of microbial origin, several responses are provoked, including reactions in immune cells that may lead them to produce signalling factors that trigger inflammation. The present study was designed to elucidate the role of TLR4 during the host response to S. schenckii. TLR4-deficient (C3H/HeJ) and control mice (C3H/HePas) were infected with S. schenckii yeast cells and immune response was assessed over 10 weeks by assaying production of pro-inflammatory mediator (nitric oxide and tumour necrosis factor-α) and anti-inflammatory cytokine (interleukin-10) by peritoneal macrophages and their correlation with apoptosis in peritoneal exudate cells. We found that both pro-inflammatory and anti-inflammatory mediators are reduced in TLR4-deficient mice, suggesting the involvement of this receptor in the recognition of this infectious agent. Translocation into the nucleus of nuclear transcription factor, nuclear factor-κB, was also evaluated and showed higher levels in TLR-4 normal mice, consistent with the results found for cytokine production. We are showing here, for the first time, the involvement of TLR4 in S. schenckii recognition. Taken together, our results demonstrate that the activation of peritoneal macrophages in response to S. schenckii lipid extracts has different responses in these two mouse strains which differ in TLR4 expression, suggesting an important role for TLR4 in governing the functions of macrophages in this fungal infection.

Introduction

Infection by the dimorphic fungus Sporothrix schenckii results in sporotrichosis in humans and animals, the most common subcutaneous fungal infection in South America.¹ Sporotrichosis has diverse clinical manifestations, of which the most frequent is the lymphocutaneous form. The disseminated cutaneous forms have mainly been observed among immunosuppressed patients, especially human immunodeficiency virus-positive individuals.^2,3 Both the pathogenic factors of the individual S. schenckii strains and the immunological status of the host determine the clinical manifestations of sporotrichosis.⁴

Recognition of invading fungi by the innate immune system is the first step in activating a rapid immunological response and ensuring survival after infection. The innate immune system allows rapid recognition of a wide spectrum of pathogens by using a limited repertoire of pattern recognition receptors (PRRs).⁵ An effective immune response depends on macrophages recognizing pathogen-associated molecular patterns (PAMPs) that distinguish the infectious agents from ‘self’ and also discriminate among pathogens.⁶

Immune responsiveness to many microbial pathogens depends on a family of PRR known as Toll-like receptors (TLRs), which are the major innate recognition system for microbial invaders in vertebrates.⁷ The known TLR family now consists of more than 13 members, each detecting distinct PAMPs that characterize various microbial pathogens, such as viruses, bacteria, protozoa and fungi.⁸ The TLRs are expressed on various immune cells, such as macrophages, dendritic cells (DCs), B cells and neutrophils, as well as on non-immune cells, such as fibroblasts, epithelial cells and keratinocytes. After recognition of PAMPs, TLRs activate signalling components that trigger the appropriate immune responses required for host defence.⁹

Macrophages initiate the innate immune response by recognizing pathogens, phagocytosing them and secreting inflammatory mediators. Ligand–TLR interactions trigger several adaptor molecules, such as MyD88. This is the first step of a signalling cascade that leads to the activation of various nuclear factors and, in particular, of nuclear factor-κB (NF-κB), which regulates the production of a large array of inflammatory molecules such as tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and IL-12,¹⁰ several cytokines, adhesion molecules, anti-apoptotic factors, chemokines, growth factors, and inducible enzymes such as cyclo-oxygenase 2 and inducible nitric oxide synthase,^11–13 which coordinate the recruitment of polymorphonuclear cells and activation of more macrophages that, in turn, kill the invading pathogen directly, primarily by phagocytosis.⁷

Recent studies have suggested that stimulation of TLRs can result in apoptosis by triggering pro-apoptotic signalling.^14–16 This TLR4-mediated signalling plays a dominant role in bacteria-stimulated apoptosis in macrophages.¹⁷ Pathogenic micro-organisms have evolved varied mechanisms to survive in the host environment and modulation of host-cell apoptosis is one of these. Escherichia coli¹⁸ and Candida albicans¹⁹ have been found to induce apoptosis in neutrophils, while Cryptococcus neoformans has been shown to induce apoptosis in inflammatory cells in granulomas of rats with cryptococcal meningitis.^20,21 For S. schenckii infection, there has been no information about apoptosis induction until this study.

Toll-like receptors have been implicated in host defence against various fungi.^22,23 The present study was undertaken to characterize, for the first time, the activity of TLRs in the macrophage response to S. schenckii infection. To this end, the involvement of TLR4 activation was assessed in murine macrophages from infected and healthy mice exposed to S. schenckii antigen. The ability of TLR4 to activate specialized antifungal effector functions in macrophages in response to S. schenckii infection suggests a possible involvement of TLR4 in governing the function of macrophages in sporotrichosis.

Materials and methods

Animals

Male, 6- to 10-week-old TLR4-deficient mice, strain C3H/HeJ, were obtained from the Animal House at the School of Pharmaceutical Sciences, UNESP (Araraquara, SP, Brazil), and the control mice, C3H/HePas, were purchased from the University of São Paulo (São Paulo, SP, Brazil) and maintained in the above-mentioned Animal House.

Procedures involving animals and their care were conducted in conformity with rules laid down by the Research Ethics Committee (# 43/2005), of the School of Pharmaceutical Sciences (UNESP, Araraquara, SP, Brazil).

Microorganisms and culture conditions

Sporothrix schenckii, strain 1099–18, was kindly provided by Dr Celuta Sales Alviano, Institute of Microbiology, Federal University of Rio de Janeiro, RJ, Brazil. This strain was isolated from a human case of sporotrichosis at the Mycology Section of the Department of Dermatology, Columbia University, New York, NY. The fungus was cultured at 37° for 8 days in brain–heart infusion broth (DIFCO Laboratories, Detroit, MI) with constant rotary shaking at 150 cycles/min, resulting in a suspension of yeast cells.

Lipid extraction

Lipid extract was prepared in the Laboratory of Fungal Surface Structures, Mycology Section, Department of Microbiology and Parasitology/Institute of Biomedical Science/Federal Fluminense University (UFF, RJ, Brasil), by Dr Diana Bridon da Graça Sgarbi. Lipids were extracted from S. schenckii cultures grown at 37° (yeast cells). The samples were subjected to lipid extraction with organic solvents and the solutions were fractionated by chromatographic techniques (column chromatography and thin-layer chromatography on silica gel and paper chromatography).

Infection method

For S. schenckii infection, a yeast suspension in phosphate-buffered saline (PBS, pH 7·4), containing 10⁷ cells/ml was prepared. Each animal was inoculated intraperitoneally with 0·10 ml of this suspension in the experimental group, while animals in the control group were injected with PBS alone. Mice were killed at different times after infection and peritoneal cells of infected animals and control group were collected and restimulated in vitro with the lipid extract of the S. schenckii yeast form.

Peritoneal macrophages

Thioglycollate-elicited peritoneal exudate cells (PECs) were harvested from C3H/HeJ and C3H/HePas mice in 5·0 ml of sterile PBS (pH 7·4). The cells were washed twice by centrifugation at 200 g for 5 min and resuspended in appropriate medium for each test.

Nitric oxide measurement

The PECs were resupended in RPMI-1640 medium (Sigma, St. Louis, MO) containing 100 U/ml penicillin, 100 μg/ml streptomycin, 5 × 10⁻² m mercaptoethanol and 5% inactivated fetal calf serum (Sigma) (RPMI-1640-C) at a concentration of 5 × 10⁶ cells/ml, and 100 μl of this suspension was added to each well of a 96-well flat bottom tissue culture plate along with 100 μl of lipid extract from S. schenckii yeast form (LEY), lipopolysaccharide (LPS) from E. coli O111:B in RPMI-1640-C for the positive control wells or RPMI-1640-C alone. The cells were incubated for 24 hr at 37° in a mixture of 95% air and 5% CO₂, and 50-μl aliquots of culture supernatant were mixed with 50 μl Griess reagent (1% weight/volume sulphanylamide, 0·1% weight/volume naphthylethylenediamine and 3% H₃PO₄), and incubated at room temperature for 10 min in the dark. The colour reaction was determined at 540 nm with a Multiskan Ascent Enzyme-Linked Immunosorbent Assay (ELISA) reader (Labsystems, Helsinki, Finland).²⁴

Cytokine assays

The PECs were resuspended in RPMI-1640-C at a concentration of 5 × 10⁶ cells/ml, and adherent cells were obtained by incubation for 1 hr at 37° in an atmosphere of air/CO₂ (95 : 5, volume/volume) (Forma Scientific, Marietta, OH). Non-adherent cells were removed by washing and adherent cells were incubated with LPS, LEY or RPMI-1640-C medium. The levels of the cytokines TNF-α and IL-10 in culture supernatants were determined by ELISA (Opt EIA; BD Biosciences, San Diego, CA), performed in 96-well microplates, according to the manufacturer’s instructions. Briefly, 96-well plates were coated overnight with 100 μl/well of a purified rat anti-mouse cytokine capture antibody diluted in sodium bicarbonate buffer (pH 9·5), or sodium phosphate buffer (pH 6·5), and incubated overnight at 4°. The plates were washed three times with 0·01 m PBS (pH 7·0) containing 0·05% Tween-20 (PBS-T). The plates were blocked with 300 μl/well of 10% fetal bovine serum (FBS) in PBS at room temperature for 1 hr. Plates were washed three times with PBS-T. Aliquots of 100 μl of samples (supernatant of macrophage cell culture) or standard murine cytokines were added to the appropriate wells. The plates were incubated at room temperature for 2 hr, washed five times with PBS-T, and 100 μl biotinylated goat anti-mouse cytokine detection monoclonal antibody plus streptavidin–horseradish peroxidase reagent, diluted in assay diluent (PBS + 10% FBS), was added to each well. The plates were incubated at room temperature for 1 hr, washed seven times with PBS-T and 100 μl of substrate solution (BD Pharmingen™, San Jose, CA, TMB Substrate Reagent Set – Cat. No. 555214) was added to each well, the plates being incubated at room temperature for 30 min in the dark. The reaction was stopped by adding 50 μl Stop Solution (1 m H₂SO₄) to each well. Absorbance was read at 450 nm, within 30 min of stopping the reaction, on a microplate reader (Multiskan Ascent, Labsystems) and cytokine concentrations were calculated from a curve of known concentrations of each cytokine standard. The results were expressed in pg/ml.

Measurement of nuclear translocation of NF-κB p65

Peritoneal exudate cells (2 × 10⁶) were obtained from S. schenckii-infected and healthy mice (C3H/HePas and C3H/HeJ). The relative increase of NF-κB p65 translocation into the nucleus was measured by an ELISA, following the manufacturer’s protocol (IMGENEX Corporation, San Diego, CA). In brief, the cells were centrifuged at 720 g for 5 min and washed with cold PBS. The cells were lysed in 1000 μl hypotonic buffer to which 50 μl detergent solution had been added. The mixture was centrifuged at 25 630 g for 30 seconds. The supernatant was discarded and 100 μl nuclear extraction buffer was added to the pellet and incubated at 4° for 30 min on a rocking platform. The suspension was centrifuged at 25 630 g for 1 min. The supernatant was used as nuclear extract. The anti-p65 antibody-coated plate captured nuclear p65 in samples (0·5–1 mg/ml of protein) and the amount of bound p65 was detected by adding the detecting antibody followed by alkaline phosphatase-conjugated secondary antibody. The absorbance value for each well was determined at 405 nm using a microplate reader (Multiskan Ascent, Labsystems).

Flow cytometry analysis of apoptosis/necrosis

The percentage of cells that underwent necrosis or apoptosis was determined by simultaneous labelling with an Annexin V–fluorescein isothiocyanate (FITC; BD Biosciences) and staining with propidium iodide (PI; BD Biosciences), according to the manufacturer’s instructions. The procedure consists of binding Annexin V–FITC to phosphatidylserine, which translocates from the inner to the outer surface of the cell membranes at the start of the apoptotic process, and then the binding of PI to DNA in cells where the membrane has been totally compromised.²⁵ The PECs (2 × 10⁶ cells/ml) were obtained as described above and washed twice with cold PBS. The cells were then resuspended in Annexin-V-binding buffer (BD Biosciences) and 1 ml of the cell suspension was transferred to 5-ml polystyrene tubes (BD Biosciences). Five microlitres of Annexin V–FITC conjugate and 10 μl PI solution were added to the to cell suspension and incubated in the dark at room temperature for 15 min. The relative level of apoptotic cells was detected by flow cytometry within 1 hr, using a FacsCanto flow cytometer (BD Biosciences, San Jose, CA) and FacsDiva software.

Statistical analysis

The Tukey test was used to determine the statistical significance of differences between experimental groups. Significance was declared at P < 0·05. The data reported are representative of three independent experiments and are presented as the mean ± SD of quadruplicate or triplicate observations. In vivo groups consisted of four to six animals.

Results

Reactive nitrogen intermediates induced by S. schenckii lipid extract

Nitric oxide (NO), together with oxygen radicals, contributes to the cytotoxic activity of phagocytic leucocytes (including macrophages) toward certain bacteria, protozoan parasites, fungi and viruses. Figure 1 shows NO production by PECs from S. schenckii-infected C3H/HeJ (Fig. 1d) and C3H/HePas (Fig. 1b) mice, over a period of 10 weeks in comparison to uninfected C3H/HeJ (Fig. 1c) and C3H/HePas (Fig. 1a) mice. The NO production was observed at high levels mainly in infected and healthy C3H/HePas mice (Fig. 1a,b, respectively) when challenged with LPS, which is known to be a potent macrophage activator. When the fungal antigen LEY was used in macrophage culture NO production was again high in infected C3H/HePas animals (Fig. 1b), showing higher NO concentrations between the 4th and 8th weeks of infection in C3H/HePas mice that could be related to NO suppressor activity, leading to a worsening of the disease.

Figure 1.

Sporothrix schenckiilipid extract induce higher levels of reactive nitrogen intermediates in Toll-like receptor 4 (TLR4) competent mice. Peritoneal exudate cells (5 × 10⁶ cells/ml) were cultured with lipopolysaccharide (LPS), lipid extract from yeast (LEY) or culture media. Supernatant was withdrawn after 24 hr stimulation with antigens from S. schenckii, LPS or culture media, and mixed with 50 μl Griess reagent. The colour reaction was determined at 540 nm with an automated microplate reader. Supernatants from triplicate cultures were assayed in four experiments and reported as the mean ± SD. The level of significance of the difference between control and experimental groups was set at P < 0·05.

Peritoneal cells from infected or healthy C3H/HeJ (Fig. 1c,d, respectively) mice produced much lower levels of NO than C3H/HePas mice, even when LPS was used as stimulus, as expected because C3H/HeJ mice have a defective response to this endotoxin, showing that TLR4 has an important role in S. schenckii LEY recognition and activation of this pathway of immune response.

Release of TNF-α

In the present study, TNF-α was produced in significantly greater amounts by cells from infected C3H/HePas mice (Fig. 2b) during the initial weeks of infection (2nd and 4th); it is an important cytokine to induce and regulate the immune response during this infection. During the final stages of infection (8th and 10th week), the low TNF-α concentration may be a result of the turning off of the inflammatory response and remission of the infection. This TNF-α production is clearly a result of the recognition of S. schenckii by TLR4, because the adherent peritoneal macrophages from C3H/HeJ produced only low concentrations of TNF-α (Fig. 2d) when compared with production by C3H/HePas, although with a similar profile.

Figure 2.

Tumour necrosis factor-α (TNF-α) production is also a result of Sporothrix schenckii lipid extract (LEY) interaction with Toll-like receptor 4 (TLR4). Peritoneal macrophage culture supernatants were drawn off after 24 hr stimulation with lipopolysaccharide (LPS), LEY or culture media, and used in the TNF-α assay by enzyme-linked immunosorbent assay (ELISA). The colour reaction was determined at 450 nm with an ELISA automated microplate reader. Results are reported as the mean ± SD. Differences between experimental and control groups were declared significant at P < 0·05.

Production of TNF-α by uninfected mice from both strains (C3H/HePas and C3H/HeJ) occurred in lower concentrations than in infected mice, as shown in Fig. 2(a,c), indicating TNF-α induction occurred during the immune response to this infectious agent.

Release of IL-10

In this study we observed higher production of IL-10 by cells from infected C3H/HePas mice (Fig. 3b) than by cells from infected C3H/HeJ mice (data not shown). Higher levels of IL-10 production were observed in the final stages of the study, showing its role as an anti-inflammatory mediator that may be responsible for inhibition of TNF-α production in the final weeks of infection observed in infected C3H/HePas mice. Peritoneal macrophages from infected and healthy C3H/HeJ mice do not show any detectable concentration of IL-10 (data not shown), reflecting the importance of TLR4 in S. schenckii recognition and in induction of the immune response.

Figure 3.

Interleukin-10 (IL-10) is produced only by C3H/HePas mice cells. Peritoneal macrophages culture supernatants were drawn off after 24 hr stimulation with lipopolysaccharide (LPS), lipid extract from yeast (LEY) or culture media, and used in the IL-10 assay by enzyme-linked immunosorbent assay (ELISA). The colour reaction was determined at 450 nm with an ELISA automated microplate reader. Results are reported as the mean ± SD. Differences between experimental and control groups were declared significant at P < 0·05.

Nuclear translocation of NF-κB p65

We measured the nuclear translocation of NF-κB p65 by ELISA in PECs from S. schenckii-infected and healthy C3H/HePas and C3H/HeJ mice (Fig. 4). As expected we observed higher NF-κB translocation to the nucleus in C3H/HePas mice than in C3H/HeJ mice because the cytokine production was also higher in TLR4 normal mice and NF-κB is an important factor for cytokine production. Results obtained for NF-κB followed a similar profile in C3H/HePas and C3H/HeJ mice, except that in C3H/HeJ mice there was a lower concentration of this factor in the nuclei of infected mouse cells, consistent with the lower NO and cytokine levels observed in this experimental group.

Figure 4.

Nuclear translocation of nuclear factor-κB (NF-κB) to the nucleus was also greater in Toll-like receptor 4 (TLR4) competent mice. Peritoneal exudate cells were obtained from Sporothrix schenckii infected or healthy C3H/HePas and C3H/HeJ mice and used for NF-κB extraction. The nuclear translocation of NF-κB was determined by enzyme-linked immunosorbent assay (ELISA). The colour reaction was determined at 405 nm with an ELISA automated microplate reader. Results are reported as the mean ± SD. The level of significance was set at P < 0·05, to compare the control and experimental groups.

Apoptosis/necrosis

Flow cytometry of Annexin V–FITC-labelled cells stained with PI allowed analysis of the early stages of apoptosis of peritoneal cells from S. schenckii-infected C3H/HePas and C3H/HeJ mice (Fig. 5). Results show that apoptosis occurred mainly during the 4th to 6th weeks of infection in C3H/HePas mice. In infected C3H/HeJ mice we observed a low percentage of apoptosis over the whole period studied. The highest percentage of apoptotic cells in infected C3H/HePas mice was observed during the initial stage of infection, the same period in which we observed high levels of NO and TNF-α, known to be inducers of apoptosis. Concomitantly with the high concentration of IL-10 in the final weeks of the experiment, we observed low levels of NO and TNF-α and a lower percentage of apoptotic cells in S. schenckii-infected C3H/HePas mice in those weeks. Consistent with the low levels of NO and TNF-α production by peritoneal cells from infected C3H/HeJ mice, we also observed a low percentage of apoptotic cells in this experimental group.

Figure 5.

Apoptosis may be involved in the immunosuppressive state observed between the 4th and 6th weeks in C3H/HePas mice. Apoptosis and necrosis were quantified in peritoneal exudate cells from mice infected with Sporothrix schenckii over a period of 10 weeks by Annexin V–fluorescein isothiocyanate labelling and propidium iodide staining and analysed by flow cytometry. Results are reported as the mean ± SD. Significant differences between experimental and control groups were declared as P < 0·05.

Discussion

Despite the major clinical consequences of fungal infections, current understanding of how immune cells recognize and respond to fungal pathogens is very limited. Although PRRs that recognize S. schenckii have not been identified, TLR2 and TLR4 are plausible candidates because many fungi such as C. albicans, Aspergillus niger, Aspergillus fumigatus and Saccharomyces cerevisiae are recognized through TLR2 and TLR4.^26,27

To elucidate the role of TLR4 in the host response to S. schenckii infection, we initially measured the concentrations of macrophage-derived mediators such as NO, TNF-α and IL-10 in the supernatants from cultures of peritoneal macrophages from normal mice and from mice that are genetically deficient in these PAMP receptors, over a period of 10 weeks of infection with this fungal pathogen.

Previous work performed in our laboratory²⁸ has demonstrated that S. schenckii crude lipid extract inhibits the macrophage phagocytosis process, an interesting result as it represents the first report of a compound from the fungus S. schenckii exhibiting this activity, influencing the immune response. In the present study we used lipid extract obtained from yeast cells of S. schenckii to assess immune stimulation during experimental infection. The immune response observed in S. schenckii-infected C3H/HePas (TLR4 normal) mice was stronger than that observed in C3H/HeJ (TLR4-defective) mice. We observed that LEY antigen induced high levels of NO production between the 4th and 8th weeks of infection, similar to previous results found in our laboratory demonstrating NO production stimulated by S. schenckii exoantigen during experimental sporotrichosis in Swiss mice.²⁹ The higher NO concentrations found in this period of infection in C3H/HePas mice in response to the lipid extract as stimulus could be related to NO suppressor activity, leading to a worsening of the disease, as has already been shown for S. schenckii infection in Swiss mice.²⁹ Although NO has been extensively recognized as a microbicidal molecule, its production can also be detrimental, even lethal, to the host.³⁰

Together with NO suppressor activity, the present results show a fall of TNF-α production after the 4th week which may be contributing to the immunosuppressed condition of the host, as already shown in another murine model tested in our laboratory.³¹ In this previous work,³¹ IL-1 and TNF-α production by adherent peritoneal cells from S. schenckii-infected Swiss mice was severely reduced from weeks 4 to 6 of infection and greater than normal from weeks 8 to 10; together with a depression of the delayed-type hypersensitivity response to a specific whole soluble antigen and an increase in fungal multiplication in the livers and spleens of infected mice between weeks 4 and 6 of infection. However, no increase in TNF-α production was observed in the final weeks of the present study. This result may be explained by the high IL-10 production in the final stage of infection on C3H/HePas mice. Recognition of fungi via TLR2 and TLR4 induces not only the release of pro-inflammatory cytokines such as IL-6 and TNF-α but can also mediate anti-inflammatory effects through the release of IL-10.^15,32–34

Fernandes et al.³⁵ have shown that early high levels of TNF-α detected in S. schenckii-yeast cell-infected wild-type mice can induce huge and spontaneous IL-10 production by the peritoneal macrophages, and while the in vitro inhibition of NO production by yeast cell-infected macrophages could be a result of IL-10 production.³⁵ In this report we observed that C3H/HePas mice showed higher levels of IL-10 production in the final stages of the study (Fig. 3), because the IL-10 is an anti-inflammatory cytokine, it is involved in the repression of TNF-α and IL-1 release.³⁶ In fact, Tripp et al.³⁷ observed that the production of interferon-γ and TNF-α was augmented when IL-10 was neutralized with monoclonal antibodies, demonstrating IL-10 to be a very potent inhibitor of macrophage cytokine.^37–39

Activation of TLR4 triggers an inflammatory cascade in macrophages, involving activation of mitogen-activated protein kinases and translocation of NF-κB,⁴⁰ a primary transcription factor controlling the expression of a series of cytokines with pro-inflammatory, pro-angiogenic and immunoregulatory activity, including TNF-α, IL-1α, IL-6 and IL-8.^41,42 In this way, the results found here of NF-κB tranlocation into the nucleus during S. schenckii infection showed higher concentrations in C3H/HePas mice than in C3H/HeJ mice (Fig. 4), which is similar to the results found for NO and cytokine production.

The mechanism of pathogen-induced cell death often involves modulation of the apoptotic response. Certain pathogens induce the death of immune cells, as a means of subverting normal host defence mechanisms, and of epithelial cells to enable invasion to deeper layers of an organ and the bloodstream. Killing of phagocytes impairs pathogen clearance and is detrimental to the host.⁴³ Results presented here showed an increase in PEC apoptosis level between the 4th and 6th weeks of infection of C3H/HePas mice (Fig. 5b), which may contribute to the suppressive state caused by disturbances in NO and TNF-α production.

Several researchers obtained similar results while studying other fungal pathogens. Zhao and Wu⁴⁴ investigated the underlying mechanisms of TLR2 and TLR4 regulating cytokine expression and their role in the innate defence of immortalized human corneal epithelial (THCE) cells, and found that Aspergillus fumigatus conidia induced the expression of TNF-α and IL-8 in a time-dependent manner, and pre-incubation of THCE cells with TLR4 neutralizing antibody significantly blocked A. fumigatus conidia-induced TNF-α and IL-8 secretion, demonstrating a TLR4-mediated mechanism of activating THCE cells to secrete pro-inflammatory cytokines. In another study, Gao et al. reported that A. fumigatus supernatant and hyphae stimulate the secretion of IL-8 and TNF-α from THCE cells in a TLR/NF-κB signalling pathway-dependent manner.^45,46 In a similar way, studies by Netea et al⁴⁷ showed that macrophage cells from TLR2-knockout mice or TLR4-deficient mice produced less TNF and IL-1β than did macrophages from control mice, when stimulated with A. fumigatus conidia.

Studies about TLR involvement in Cryptococcus neoformans recognition are controversial. Shoham et al.⁴⁸ demonstrated that glucuronoxylomannan, the major capsule polysaccharide of C. neoformans, interacts with CD14 and TLR4 to stimulate NF-κB nuclear translocation without mitogen-activated protein kinase activation, which ultimately results in the failure of TNF-α production by macrophages.⁴⁹ On the other hand, Nakamura et al.,⁵⁰ in their investigations, found no significant difference in the synthesis of IL-1β, IL-6, IL-12p40 and TNF-α in the sera or in the lungs of TLR2 and TLR4 knockout mice and control wild-type mice in host response against C. neoformans.

No information was available about PRR recognition during S. schenckii infection. We demonstrate, for the first time, a role for TLR4 during sporotrichosis. Our results show that the activation of peritoneal macrophages in response to S. schenckii was markedly different in two inbred mouse strains that differ in their TLR4 expression activating the antifungal state of peritoneal macrophages, playing an important role in governing the functions of macrophages in fungal infection and the associated inflammatory pathology. Moreover, other receptors like TLR2 and dectin-1 may also contribute to the immune response during S. schenckii infection, and could be the subject of future researches.

Disclosures

The authors have no financial conflict of interest.