Absence of functional TLR4 impairs response of macrophages after Candida albicans infection

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1 Medical Mycology December 2010, 48, Original Articles Absence of functional TLR4 impairs response of macrophages after Candida albicans infection THA Í S HELENA GASPAROTO *,1, VANESSA TESSAROLLI *,1, THIAGO POMPERMAIER GARLET, S É RGIO APARECIDO TORRES *, GUSTAVO POMPERMAIER GARLET *, JO Ã O SANTANA DA SILVA & ANA PAULA CAMPANELLI * * Department of Biological Sciences, Bauru Dental School University of São Paulo, Bauru, SP, Brazil, Department of Biochemistry and Immunology, and Department of Pharmacology, School of Medicine of Ribeir ã o Preto University of S ã o Paulo, Ribeir ã o Preto, SP, Brazil Candida albicans is recognized by phagocytic cells through a set of recognition receptors patterns. Recently, we showed the importance of TLR2 in the regulation of neutrophil survival after C. albicans infection. In the present work, we analyzed the involvement of TLR4 in the recognition of C. albicans by neutrophils and macrophages. Our results show that the absence of functional TLR4 resulted in lower chemotaxis of neutrophils to the site of infection, lower levels of TNF- α, CXCL1 and nitric oxide, and dissemination and persistence of the pathogen in lymph nodes and spleen. In vitro, the phagocytic activity, nitric oxide production and myeloperoxidase activity, CXCL1, IL-1 β production by neutrophils from TLR4-defective mice were not changed. In contrast, macrophages from TLR4-defective mice demonstrated lower phagocytosis and lower levels of CXCL1, IL-1 β and TNF-α. Together, these data demonstrate that TLR4 signals are important for the recognition of C. albicans by macrophages and their absence allows persistence of the infection. Keywords TLR4, innate response, Candida albicans Introduction Candida is one of the most frequently recovered human fungal pathogens. Candida albicans is the most common fungal pathogen found as part of the normal microbiota of the mucosal surfaces in the oral, gastrointestinal and genitourinary tracts of most healthy individuals [1], and as a result, is the most important factor contributing to fungal infections, particularly in immunocompromised patients [2]. The resistance to candidiasis depends on coordinated actions of the innate and acquired immunity mechanisms, where phagocytes are responsible for the initial recognition and response against yeast. Lymphocytes act subsequently 1 These researchers collaborated equally in this study. Received 7 November 2009 ; Received in final revised form 7 February 2010; Accepted 25 March 2010 Correspondence: Ana Paula Campanelli, Bauru Dental School, Department of Biological Sciences University of S ã o Paulo, Al. Oct á vio Pinheiro Brisolla, 9 75, CEP Bauru, S ã o Paulo, Brazil. Tel: ; fax: ; apcampan@usp.br to provide the signals to amplify and sustain a satisfactory defense [3,4]. The influx of polymorphonuclear neutrophils to the site of infection is critical to the control of fungal infection, and is thought to be the major factor that limits C. albicans dissemination. Neutrophils are essential for the initiation and execution of the acute inflammatory response, and indeed, the absence or defective functioning of neutrophils is the primary cause of disseminated candidiasis [5,6]. The family of Toll-like receptors (TLR) enables the host to recognize a diverse array of conserved pathogenassociated microbial products of a large number of pathogen-associated molecular patterns, such as bacterial lipopolysaccharides, viral RNA, CPG-containing DNA, among others. Surface components of the wall in yeasts, such as mannan and phospholipomannan, also interact with host cells by means of the TLRs [7]. Isolated components of C. albicans have been shown to activate TLR2 and TLR4 on neutrophils and macrophages [8 10]. TLR signaling of phagocytes results in their activation, leading to increased phagocytosis and cytokine production [11]. Both 2010 ISHAM DOI: /

2 1010 Gasparoto et al. TLR2 and TLR4 have been implicated in the host defense against C. albicans [7]. TLR4 was initially identified as the molecule responsible for recognizing lipopolysaccharide (LPS). Later the absence of TLR4-mediated signals was described to cause an increased susceptibility to disseminated candidiasis [12]. This effect was shown to be associated with a decreased release of the chemokines keratinocyte-derived chemokine (KC, CXCL1) and macrophage inflammatory protein (MIP)-2 (CXCL2), in addition to impairing neutrophils recruitment to the site of infection. Furthermore, polymorphisms of TLR4 are involved with susceptibility to severe candidiasis in humans [13]. However, the literature concerning recognition of C. albicans by TLRs lacks information about cells lacking the TLR4 determinate susceptibility to infection [10]. Therefore, the present study was undertaken to characterize the functional activity of TLR4 in macrophages and neutrophils in response to C. albicans infection. Material and methods Animals Experimental groups (8 mice per group, four groups) comprised of eight-week-old male C3H/HePas mice (TLR4lps- n -expressing a functional TLR4; WT mice), and C3H/HeJ mice with targeted disruption of TLR4 (TLR- 4lps- d -expressing a nonfunctional TLR4; TLR4-defective mice) were bred (breeding pairs obtained from Jackson Laboratory, Bar Harbor, Maine, USA) and maintained in the animal facilities of the Department of Biological Science-FOB/USP. The experimental protocols were approved by the Institutional Committee for Animal Care and Use. Candida albicans C. albicans (ATCC 10231) was used in all experiments. Yeast cells were kept on Sabouraud dextrose agar (SDA; Difco, Becton Dickinson, Le Point de Claix, France), at 28 C, and were grown in Sabouraud dextrose broth (SDB; Difco) for 24 h at room temperature before inoculation into the experimental animals. Yeast cells were then harvested by centrifugation (700 g ), and suspended at 10 8 cells/ml in RPMI medium (GIBCO Life Technologies, Grand Island, NY, USA) containing 5% of heat inactivated fetal calf serum FCS (GIBCO) or saline, depending on the assay. The viability of yeast cells was determined as previously described [14]. For the in vitro assays, yeast cells were incubated for 5 h with Fluorotag FITC (Sigma Aldrich, St Louis, USA) followed by two saline washings (350 g for 10 min at 4 C). C. albicans infection model WT and TLR4-defective mice were injected intraperitoneally (i.p.) with 10 7 C. albicans cells suspended in 100 μl of saline, and the animals were euthanized 24 h after inoculation. Negative controls consisted of mice which received saline without yeast, and non-infected animals. Isolation of leukocytes from peritoneal exudates Isolation of neutrophils and macrophages was performed as described previously [6]. Briefly, peritoneal exudate cells were harvested by washing the peritoneal cavity with sterile RPMI medium containing 5% FCS (GIBCO), 24 h after the mice were injected with C. albicans, RPMI medium or thioglycolate (3%). The peritoneal fluid was then collected through an incision in the abdominal wall and subjected to centrifugation at 350 g for 10 min at 4 C. The supernatant was removed and frozen at 20 C and the pellet cell was suspended in RPMI medium, and counted in a haemcytometer. The cell viability was 95% as determined by Trypan blue exclusion. The percentage of neutrophils and macrophages was determined by Turk staining of the cytocentrifuged preparations. Analysis of phagocytosis by neutrophils and macrophages WT and TLR4-defective mice were injected i.p. with 1 ml of thioglycolate (3% w/v) and the peritoneal cells were harvested after 6 h ( 85 % of neutrophils) or 72 h ( 90% of macrophages) by washing the cavities with RPMI 1640 (10 ml), and the cell viability was 98% (Trypan blue exclusion). Cells obtained after 6 h were cultivated for 1 h at 37 C in antibiotic-free RPMI 1640, and the nonadherent cells were used as neutrophils ( 94%). After the purification of each cell group, morphology of the cells was assessed by Turk staining. For the phagocytosis assay, neutrophils or macrophages were incubated with FITC stained C. albicans (at 1:10 ratio of phagocyte: yeast) at 37 C with gentle shaking. After 1 h, the cells were washed with PBS at 4 C and centrifuged at 350 g, fixed with 1% formaldehyde PBS and the percentage of neutrophils and macrophages phagocytosing C. albicans were analyzed by flow cytometry (FACScalibur) using CellQuest software (BD Biosciences). Extra cellular yeast cells were quenched by Trypan blue before flow cytometer analysis. The phagocytosis assays were visualized and photographed by confocal laser scanning microscopy (TCS model, SPE, Leica, Mannheim, Germany). For ex vivo phagocytosis investigation, assays were stained by the Giemsa method [14]. Nitric oxide production For the determination of in vivo NO production by inflammatory cells, the production of nitrite (NO 2 ) was measured in freshly isolated peritoneal fluid. For the determination of in vitro NO production, the production

3 Infl uence of TLR4 in the recognition of Candida albicans 1011 of NO 2 was measured in the supernatants of cultivated neutrophils and macrophages as described below. Neutrophils or macrophages ( cells) were incubated with medium or C. albicans for 24 h at 37 C and 5% CO 2. The supernatant was collected and the total amount of nitrite was determined by the Griess method [15]. Briefly, 50 μl of supernatant samples were incubated with an equal volume of the Griess reagent at room temperature. The absorbance was measured on a plate scanner (Spectra Max 250; Molecular Devices) at 540 nm. The NO 2 concentration was determined using a NaNO 2 standard curve at a concentration range from μm. In vivo and in vitro cytokine and chemokine production The in vivo levels of chemokines and cytokines were analyzed in the peritoneal exudate obtained by washing their peritoneal cavity of WT or TLR4-defective infected mice with 5% FCS RPMI For the in vitro analyses of chemokines and cytokines, neutrophils or macrophages ( cells) from WT or TLR4-defective mice were isolated and incubated with thioglycolate, medium, LPS from Escherichia coli (0.1 μg/ml; Sigma, St Louis, MO) or C. albicans for 24 h at 37 C under 5% CO 2. In vivo or in vitro production of TNF- α, CXCL1, CCL3, CXCL2, CCL5 and IL-1 β were determined by ELISA using commercially available kits (R&D Systems), as follows: TNFα (sensitivity 3.4 pg/ml), CXCL1 ( 2 pg/ml), CCL3 ( 1.5 pg/ml), CCL5 ( 2 pg/ml), CXCL2 ( 7.8 pg/ml) and IL-1 β ( 4.2 pg/ml). All assays were carried out according to the manufacturer s instructions. The results were expressed as picograms of cytokine per ml ( SD) and the experiments were performed in triplicate. Colony-forming units assay To determine the growth and dissemination of C. albicans, the amount of colony-forming units (CFUs) recovered from the spleen, inguinal lymph nodes and peritoneal exudate were analyzed at 24 hours and 30 days after infection. The organs were aseptically removed, weighed, homogenized in sterile phosphate buffer saline (PBS) using tissue grinders, and 100 μl of the samples diluted in 900 μl of PBS. Aliquots of 100 μl of each organ and the exudate were dispensed onto Petri dishes containing SDA. The plates were incubated at 37 C, the colonies counted 2 to 7 days later, and the number of CFUs/g of tissue (organs) and per ml (peritoneal exudate) were calculated. Statistical analysis The results are expressed as mean standard error (SD). Statistical analysis was performed using analysis of variance (ANOVA) followed by the parametric Tukey- Kramer test (INSTAT software: GraphPad, San Diego, CA, USA). Values of P 0.05 were considered statistically significant. Results Absence of functional TLR4 results in impaired neutrophils recruitment, nitric oxide production and phagocytosis after Candida albicans infection We first evaluated the role of TLR4 in the recruitment of macrophages and neutrophils after C. albicans infection. The induction of inflammatory cell migration in TLR4- defective mice ( ) was significantly ( P 0.05) lower than that observed in WT mice ( ) (Fig. 1A). The total number of infiltrating neutrophils was reduced in the peritoneal cavity of TLR4-defective mice (Fig. 1B). In spite of the significantly lower number of neutrophils in TLR4-defective mice, the number of macrophages was similar in both groups of mice. These results indicate a possible involvement of TLR4 signals in neutrophil chemotaxis after C. albicans infection. To evaluate the impact of TLR4-mediated Candida phagocytosis, we isolated inflammatory cells from C. albicans- infected mice 6 h after inoculation. Our results show that a significantly lower percentage of cells were phagocytosing yeasts in TLR4-defective infected mice ( %) than in WT mice ( %) (Fig. 1C). In addition, the peritoneal fluid of infected and non-infected animals was recovered to determine NO production. NO production was significantly lower ( P 0.001) in the peritoneal fluid of TLR4-defective mice ( μm) when compared with WT mice ( μm) (Fig. 1D). Role of TLR4 in Candida albicans -induced TNF- α and CXCL1 production In view of the decreased inflammatory cell migration to the peritoneal cavity of TLR4-defective mice after C. albicans infection, we investigated the levels of chemokines and cytokines in the peritoneal cavity of TLR4-defective and WT-infected mice (Fig. 2A F). We found a significantly lower production of TNF-α in the peritoneal cavity of TLR4-defective mice ( pg/ml) as compared with WT mice ( pg/ml) (Fig. 2A). In addition, CXCL1 production was reduced in TLR4-defective mice ( pg/ml) (Fig. 2B, C). However, the levels of CXCL2, CCL3, CCL5 and IL1- β did not differ between the two groups of infected mice (Fig. 2C F). Cytokines and chemokines production were significantly lower in the peritoneal fluid from TLR4-defective mice after LPS stimulation (Fig. 2).

4 1012 Gasparoto et al. Fig. 1 Role of TLR4 in inflammatory cells recruitment, phagocytosis and nitric oxide production in response to Candida albicans. TLR4-defective ( ) and WT ( ) mice were injected i.p. with 10 7 viable C. albicans yeasts, and the neutrophils or macrophages from the peritoneal exudate were harvested 24 h later. (A) Total number of inflammatory peritoneal cells, (B) Total number of neutrophils and macrophages from TLR4-defective or WT mice, (C) Percentage of phagocytosis in TLR4-defective and WT mice, (D) NO levels (measured as the nitrite production by the Griess reagent method) detected in the peritoneal fluid from TLR4-defective and WT mice. The data are represented as mean SD for three experiments with 10 mice/group. *, * * and * * * indicate significant differences ( P 0.05, 0.01 and 0.001, respectively) between WT and TLR4- defective mice. TLR4-defective macrophages display a lower production of pro-infl ammatory cytokines and chemokines In order to determine the role of TLR4 signals in cytokines and chemokines production after C. albicans stimulation, we isolated neutrophils and macrophages and stimulated these cells in vitro with C. albicans. Although there was a reduction in TNF-α, CXCL1 and IL-1 β production by TLR4-defective neutrophils, only TNF-α was significantly lower when compared to WT mice (Fig. 3A C). Lower levels of TNF-α, IL1- β and CXCL1 were detected in macrophage cultures from TLR4-defective mice (Fig. 3D F). Absence of functional TLR4 infl uences macrophages activation after C. albicans infection Neutrophils and macrophages were stimulated in vitro with C. albicans and the phagocytic activity and NO production were determined. Our results showed that neutrophils from WT mice presented similar phagocytic activity compared with cells from TLR4-defective mice (Fig. 4A). However, macrophages from TLR4-defective mice presented significantly lower C. albicans phagocytosis (23.9 3%) as compared to WT mice ( %) (Fig. 4A). These data indicate the role of TLR4 in the recognition and phagocytosis of C. albicans by macrophages. As indicated in Fig. 4B, no differences in nitric oxide production by neutrophils were observed. Our results showed that macrophages from TLR4-defective mice produced lower levels of NO ( μm) than cells isolated from WT mice (Fig. 4C). Enhanced dissemination of Candida albicans in TLR4-defective mice The smaller number of leucocytes in the peritoneal cavity of TLR4-defective-infected mice suggested the possible facilitation of fungal dissemination. In order to confirm this hypothesis, we determined the fungal load 24 h and 30 days after C. albicans infection. The results showed a significantly higher concentration of yeasts in the lymph nodes and spleen of TLR4-defective mice than in WT mice (Fig. 5B, C). Although CFU counts were reduced 30 days after infection, a significantly higher number of yeasts was detected in draining lymph node, spleen and peritoneal exudates of TLR4-defective mice (Fig. 5D F). These results demonstrate that absence of TLR4 activation predisposes to the longer persistence of C. albicans infection.

5 Infl uence of TLR4 in the recognition of Candida albicans 1013 Fig. 2 In vivo down-regulation of cytokines and chemokines production in response to Candida albicans in the absence of functional TLR4. The figure shows the levels of cytokines and chemokines measured at the peritoneal exudates of TLR4-defective ( ) and WT ( ) mice stimulated with C. albicans yeasts or LPS from Escherichia coli (0.1μ g/ml). The results are expressed as mean SD for three experiments. Significant differences between strains are indicated by * ( P 0.05) and * * (P 0.01). Discussion The involvement of TLR in the protective immune response against C. albicans has been extensively studied [16]. Mannosyl residues from cell walls of C. albicans are recognized by TLR4 [10]. Although many aspects of the role of TLR4 in the innate defense mechanisms after C. albicans infections have been established, there are still many contradictions [10,17,18]. We first evaluated the role of TLR4 in the recruitment of neutrophils, and our findings demonstrate that in the absence of TLR4 the influx of neutrophils to the peritoneal cavity was significantly reduced. These results are in accord with a previous study that showed impaired macrophage chemokines production and subsequent low neutrophils recruitment 4 h after infection [12]. The decreased inflammatory reaction in the peritoneal cavity of TLR4- defective mice seems to be due to insufficient activation and signalization of resident macrophages. In the absence of this receptor, macrophages showed lower C. albicans phagocytosis (in vivo and in vitro). In fact, it has been shown that TLR4 activation triggers intracellular cascades that culminate in the augmentation of phagocytic receptors expressed by these cells [19]. TLR recognition of pathogenic microbes orchestrates the innate immune responses through the induction of chemokines and inflammatory cytokines that coordinate the recruitment of neutrophils in response to the activation of macrophages. This, in turn, leads to direct killing of the invading pathogens, primarily by phagocytosis [20 22]. Therefore, cytokines and chemokines levels were analyzed from inflammatory exudates and in supernatants from cultured neutrophils or macrophages stimulated by C. albicans. We observed a marked reduction of TNF- α and CXCL1 levels in the peritoneal exudates from

6 1014 Gasparoto et al. Fig. 3 In vitro down-regulation of Candida-induced cytokines and chemokines production by neutrophils and macrophages in the absence of functional TLR4. In this experiment, neutrophils (A C) and macrophages (D F) were isolated from the peritoneal cavity of TLR4-defective ( ) and WT ( ) mice and incubated with viable C. albicans. The figure depicts the levels of TNF-α, CXCL1 and IL-1β produced by inflammatory cells after 24 h incubation with the yeast. The results are expressed as means SD for three experiments. Significant differences between strains are indicated by *(P 0.05) and **(P 0.01). TLR4-defective infected mice as compared to WT mice (Fig. 2A, B). This is in agreement with the lower influx of neutrophils to the infection site in these mice. Both TNF- α and CXCL1 are known as potent neutrophils chemoattractants [23]. Although TLR4-defective neutrophils infected with C. albicans were able to produce, in vitro, CXCL1 and IL-1 β at similar rate of the WT neutrophils, the secretion of TNF- α (Fig. 3A C) was impaired. There is data in the literature supporting a direct role for TLR4 in the protection against epithelial cell injury by C. albicans as a result of blocking TLR4 and TNF- α that could override the protective influence of neutrophil and restore C. albicans -induced cell damage [24]. Accordingly, our results support the concept that the absence of TLR4- mediated signaling leads to inappropriate macrophage activation and the consequent lower production of TNF- α and CXCL1. These events might be critical to neutrophil migration to the infection focus. In addition to decreased leukocyte migration to the peritoneal cavity, TLR4 deficiency affected the function and activation of macrophages. In fact, our results showed that TLR4-defective macrophages had, beyond lower phagocytic activity of yeasts, reduced production of TNF- α, CXCL1 and IL-1 β, as compared with WT mice (Fig. 3D F and 4A). IL-1 β is the major cytokine secreted by triggering the inflammatory cascade and defects in its production by macrophages are related to increased susceptibility of Candida infection [25,26]. However, neutrophil microbicidal mechanisms in response to C. albicans infection remained intact, as MPO activity, candidacidal activity (data not shown) and NO secretion were not found in the present study. Equally, apoptosis rate of neutrophils from TLR4-defective and

7 Infl uence of TLR4 in the recognition of Candida albicans 1015 Fig. 4 Phagocytosis and NO production by Candida albicans stimulated-macrophages are less efficient in the absence of functional TLR4. (A) Neutrophils and macrophages were isolated from TLR4-defective ( ) and WT ( ) mice, incubated with viable C. albicans, and phagocytosis was assessed by flow cytometry. The results are expressed as means SD for three experiments. Significant differences are indicated by **(P 0.001). (B) Neutrophils and (C) macrophages from TLR4-defective or WT mice were incubated for 24 h with viable C. albicans and nitric oxide levels were estimated in culture supernatants through the Griess method. The results are expressed as means SD for three experiments. Significant differences between strains are indicated by *(P 0.05) and **(P 0.01). WT was similar (data not shown). Such results differ from those observed in neutrophils from TLR2 / and WT mice [6]. Perhaps, signaling triggers by other receptors on neutrophils (e.g., TLR2, Dectin-1 or FcγR) could be activating neutrophils to perform their role against C. albicans [10,27]. In fact, our data showed that IL-1 β and CXCL2 were at the same level in both infected mice groups, and both these factors were produced in the presence or absence of the functional TLR4 receptor. IL-1 β and CXCL2 have been largely related to activation and long time survival of neutrophils [28,29]. Considering that neutrophils from TLR4-defective mice showed unaltered function, and the infection was not controlled (higher CFUs were detected in lymph nodes, spleen and exudates) it is possible that, in spite of lower neutrophil infiltration, insufficient activation of TLR4- defective macrophages allowed faster and more persistent C. albicans dissemination. Neither a higher rate of apoptotic phagocytes nor a different number of macrophages recruited to the peritoneal cavity was found in the absence of TLR4. However, low levels of NO, a potent reagent principally produced by macrophages in response to pathogens [30], and deficient candidacidal activity (data not shown) were detected. This result reinforces the hypothesis of insufficient macrophage activation in the absence of TLR4 [20]. On the other hand, NO production by neutrophils was similar in TLR4-defective and WT mice in response to C. albicans, indicating that TLR4 signaling does not seem to be fundamental in the neutrophils response against C. albicans. Previous studies have shown that stimulation of TLRs may trigger the expression of a number of phagocytic receptors in macrophages, which are mediated through cytokines that act in an autocrine fashion in these cells [31,32]. In addition, MyD88 / mice demonstrated impaired phagocytosis and intracellular killing of C. albicans by macrophages indicating that MyD88 signaling is involved in Candida phagocytosis. MyD88 serves as an adapter protein for TLRs signal to NF-κB translocation and, ultimately, affects the production of proinflammatory cytokines that effectively participate in phagocytosis by macrophages [32,33]. Thus, absence of TLR4 could be impairing the activation mechanism and producing an inefficient yeast ingestion/elimination. The increased fungal load in the organs of the TLR4- defective C3H/HeJ mice also could be associated with a blunted production of CXC chemokines CXCL1, which resulted in a reduced recruitment of neutrophils to the infection site and led to overwhelming multiplication of the yeasts. This result is similar to that in earlier studies that showed that, translocation of NF-kB to the nucleus, via MyD88 signaling, is required for the transcription of CXCL1 (KC) gene under the control of TLR4 [12]. As CFUs remain significantly higher in TLR4-defective mice 30 days after C. albicans infection our result reinforces the hypothesis that the impaired macrophage activation is deleterious in controlling the C. albicans infection. Our results demonstrate that TLR4 is important for macrophage activation after C. albicans infection. The

8 1016 Gasparoto et al. Fig. 5 Enhanced dissemination of Candida albicans in TLR4-defective mice. TLR4-defective ( ) and WT ( ) mice were injected i.p. with viable C. albicans yeast cells, and were euthanized 24 h (A, B and C) and 30 days (D, E and F) post-infection to assess the infection of the organs. The peritoneal exudates, lymph nodes and spleen were collected and distributed on Sabouraud dextrose Agar. Each column represents the number (means SD) of CFU/ml of exudates or gram of tissue. **P 0.01 and ***P 0.001, when compared with the control group. absence of TLR4 was shown to be detrimental to early events of inflammatory responses against C. albicans, as the absence of TLR4 signals impairs the control and restriction of C. albicans infection by macrophages. In addition, the production of TNF- α and CXCL1 involved with macrophage activation and neutrophil recruitment were diminished with the lack of functional TLR4. Although neutrophil antimicrobial mechanisms have not been shown to be altered in the present study, our results showed that TLR4- defective mice were more susceptible to C. albicans infection which persisted for longer than 30 days after inoculation. In conclusion, we have demonstrated that TLR4 is important for the initiation of the inflammatory response against C. albicans. The lack of TLR4 signaling impairs phagocytosis, survival, release of cytokines, chemokines and antimicrobial mediators by macrophages, whereas it does not modify the function of neutrophils after C. albicans stimulation. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References 1 Biswas S, Van Dijck P, Datta A. Environmental sensing and signal transduction pathways regulating morphopathogenic determinants of Candida albicans. Microbiol Mol Biol Rev 2007; 71 : Castón-Osorio JJ, Rivero A, Torre-Cisneros J. Epidemiology of invasive fungal infection. Int J Antimicrob Agents 2008; 2 : S Shoham S, Levitz SM. The immune response to fungal infections. Br J Haematol 2005; 129 :

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