Atopic eczema (AE) is a chronic inflammatory skin disease. Approximately 50% of adult AE patients have allergen-specific IgE reactivity to the skin commensal yeast Malassezia spp. Due to the ruptured skin barrier in AE, it is likely that Malassezia can come into contact with mast cells, which are known to be involved in AE. We therefore hypothesized that Malassezia spp. can activate mast cells. Bone marrow-derived mast cells (BMMCs) were generated from wild type, TLR2, TLR4, and MyD88 gene-deleted mice and cocultured with Malassezia sympodialis extract. We recorded that M. sympodialis induced release of cysteinyl leukotrienes in a dose-dependent manner in nonsensitized and IgE-anti-trinitrophenyl-sensitized BMMCs, respectively, with three times higher levels in the latter type of cells. IgE-sensitized BMMCs also responded by degranulation as assessed by release of β-hexosaminidase, increased MCP-1 production through a MyD88-independent pathway, and activated phosphorylation of the MAPK ERK1/2. Furthermore, M. sympodialis enhanced the degranulation of IgE receptor cross-linked wild-type BMMCs and altered the IL-6 release dose-dependently. This degranulation was independent of TLR2, TLR4, and MyD88, whereas the IL-6 production was dependent on the TLR2/MyD88 pathway and MAPK signaling. In conclusion, M. sympodialis extract can activate nonsensitized and IgE-sensitized mast cells to release inflammatory mediators, to enhance the IgE-mediated degranulation of mast cells, and to modulate MAPK activation and by signaling through the TLR2/MyD88 pathway to modify the IL-6 production of IgE receptor cross-linked mast cells. Collectively, these findings indicate that M. sympodialis can activate mast cells and might thus exacerbate the inflammatory response in AE.
Atopic eczema (AE)3 is a chronic inflammatory pruritic skin disorder for which the pathogenesis is not fully understood (1). The estimated prevalence of AE is 15–20% in children (2) and 1–3% in adults (3). Factors contributing to the clinical symptoms are genetic predisposition, environmental factors (such as microorganisms), and a ruptured skin barrier (1). Itching is a prominent symptom of AE pathology and often provokes scratching, increasing the local inflammation and resulting in development of secondary skin lesions (4). A defective permeability barrier facilitates penetration of environmental allergens into the skin (5). An elevated level of total serum IgE is frequently noted in AE patients, suggesting that allergens play a role in the underlying pathogenic mechanisms (1).
The lipophilic yeast Malassezia is part of the normal cutaneous flora but can elicit specific IgE and T cell reactivities in AE patients (6, 7). The genus presently comprises 13 species (8, 9) and Malassezia sympodialis is among the species most frequently isolated from both AE patients and healthy individuals (10). Several IgE-binding components in the 10- to 100-kDa molecular mass range have been identified in M. sympodialis extract (7). To date, 10 allergens from M. sympodialis, designated Mala s 1 and Mala s 5–13, have been cloned and sequenced (7). Interestingly, it has been demonstrated that M. sympodialis release more allergens when cultured at the increased pH of AE skin compared with culture at the pH of healthy skin (11).
The number of mast cells in AE patients is increased in lichenified skin and subacute lesions (12). Since mast cells are located in the superficial dermis close to blood vessels, they are uniquely positioned to react to allergens diffusing through a ruptured epidermis. They are recognized as key effector cells during IgE-associated Th2-type immune responses (13), and cross-linking of the high-affinity IgE receptor (FcεRI) leads to aggregation and release of potent inflammatory mediators (14) such as histamine, proteases, chemotactic factors, cytokines, and metabolites of arachidonic acid (15). Mast cells have a wide variety of cell surface receptors that can interact directly with pathogens, including TLRs, which are involved in innate immune recognition of invading microorganisms (16). Fungal products such as zymosan can activate mast cells through TLR2 (16). It has recently been reported that a synergistic activation between TLR2 and FcεRI can occur in mast cells, resulting in increased production of inflammatory cytokines (17).
Due to the ruptured skin barrier in AE, it is likely that Malassezia spp. can come into contact with mast cells in the skin, resulting in mast cell activation. Our aims were therefore to investigate: 1) whether Malassezia can activate mast cells and 2) if so, to define which receptor(s) and signaling pathway(s) through which the activation occurs. We determined that M. sympodialis can indeed activate mast cells, enhance the mast cell IgE response, and modulate MAPK activation and by signaling through the TLR2/MyD88 pathway alter IL-6 production in a dose-dependent manner.
Materials and Methods
Mast cell cultures
Bone marrow-derived mast cells (BMMCs) were obtained by culturing mouse bone marrow cells from wild-type (Wt), TLR2−/−, TLR4−/−, and MyD88−/− mice with the C57BL/6 genetic background (from the Karolinska Institutet mouse breeding facility, Department of Microbiology, Tumor and Cell Biology, Stockholm, Sweden), originally provided by Prof. Shizuo Akira (Osaka University, Osaka, Japan). The cells were cultured in Falcon culture flasks (BD Biosciences) at a concentration of 0.5 × 106 cells/ml in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated FCS (Invitrogen), 10% IL-3-conditioned medium (produced by X63/0 myeloma cells transfected with a mouse IL-3 expression construct) (18), 100 μg/ml penicillin/streptomycin (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 25 mM HEPES (Sigma-Aldrich), 4 mM l-glutamine (Sigma-Aldrich), 0.1 mM nonessential amino acids (Sigma-Aldrich), and 50 μM 2-ME (Sigma-Aldrich) for 6–12 wk at 37°C in a humidified incubator with 6% CO2 (in air) with the culture medium being changed weekly. Mast cell development was evaluated by toluidine blue staining (19) and the purity of mast cells used in the experiments exceeded 98%. Cell surface FcεRI expression was confirmed by flow cytometry analysis (FACSCalibur flow cytometer; BD Biosciences) using a hamster anti-mouse FcεRI-FITC Ab (BD Biosciences).
M. sympodialis extract
M. sympodialis extract was prepared from strain 42132 (American Type Culture Collection) according to a previously described protocol (20), with the modification of culture plates and incubation temperature. In brief, M. sympodialis yeast cells were harvested after 4 days of culture at 32°C on Dixon agar plates (21), freeze-dried, taken up in PBS, and sonicated. After overnight incubation at 4°C, centrifugation, and sterile filtration, the protein concentration was measured using the BCA Protein Assay Reagent (Pierce) according to the manufacturer’s instructions. The endotoxin content was <0.15 ng/mg protein as analyzed using the Limulus amebocyte lysate endochrome assay (Charles River Endosafe). The extract was stored at −20°C until use.
Mast cell activation
BMMCs were sensitized in a humidified incubator overnight at 37°C using a monoclonal mouse anti-trinitrophenyl (TNP) IgE Ab (IgEl-b4; American Type Culture Collection) in the form of a 15% hybridoma supernatant. IgE-sensitized or nonsensitized BMMCs were washed twice in PBS and seeded into 48-well plates (BD Biosciences) at a final concentration of 1 × 106 cells/ml in RPMI 1640 medium supplemented as described above. The nonsensitized cells were cultured in medium alone or subjected to treatment with different stimuli: 0.5 μM of the calcium ionophore calcimycin A23187 (Sigma-Aldrich), 1 μg/ml LPS from Escherichia coli O55:B5 (Sigma-Aldrich), 1 μg/ml zymosan from Saccharomyces cerevisiae (Sigma-Aldrich), or 0.01–100 μg/ml M. sympodialis extract. The IgE-sensitized cells were cultured with 0.01–100 μg/ml M. sympodialis extract either alone or in combination, for cross-linking, with 100 ng/ml TNP-BSA (coupling ratio 9; Biosearch Technologies). All cells were incubated at 37°C in a humidified incubator. Culture supernatants were harvested after 0.5 and 24 h after stimulation, respectively, and stored at −20°C until analyzed. The expression of FcεRI after 24 h of incubation with or without 0.01–100 μg/ml M. sympodialis extract was determined by flow cytometry analysis as described above.
N-acetyl-β-d-hexosaminidase release assay
To detect the granular enzyme β-hexosaminidase release into the supernatant, cell activation was performed for 0.5 h in RPMI 1640 medium (Sigma-Aldrich) supplemented with 100 μg/ml penicillin/streptomycin (Sigma-Aldrich), 4 mM l-glutamine (Sigma-Aldrich), 50 μM 2-ME, and 0.5% BSA (Sigma-Aldrich) at 37°C in a humidified incubator. An enzymatic colorimetric assay was used to analyze the amount of β-hexosaminidase as previously described (22). Briefly, 60 μl of supernatant was added in duplicates into a 96-well plate (BD Biosciences) and mixed with an equal volume of substrate solution (7.5 mM p-nitrophenyl-N-acetyl-β-d-glucosaminide (Sigma-Aldrich) dissolved in 80 mM citric acid, pH 4.5) and incubated with gentle agitation (200 rpm) for 2 h at 37°C. As a standard, the corresponding amount of lysed mast cells was used and medium alone served as a negative control. The incubation was stopped by the addition of 120 μl of glycine (0.2 M, pH 10.7) to each well, and the absorbances were measured at 405 nm and 490 nm using a Multiskan RC reader (Labsystems). Results are expressed as the percentage of total N-acetyl-β-d-hexosaminidase content mean + SEM.
Cysteinyl leukotriene enzyme immunoassay
An enzyme immunoassay (Amersham Biosciences) with a sensitivity of 10 pg/ml was used to determine the release of cysteinyl leukotrienes into the supernatants after 0.5 h of culture. All assays were set up in duplicates and the results are presented as mean + SEM.
Cytokine and chemokine ELISAs
To analyze MAPK activation by M. sympodialis, IgE-sensitized Wt BMMCs were cultured with medium alone or 0.01–100 μg/ml M. sympodialis
The Wilcoxon-matched pairs test was performed using the Statistica 7.1 software package (StatSoft Scandinavia). Values of p < 0.05 were considered to be statistically significant.
M. sympodialis induces release of cysteinyl leukotrienes, but not degranulation, IL-6, or MCP-1 release from nonsensitized mast cells
M. sympodialis extract contains a variety of proteins, a number of which have been identified as IgE-binding allergens (7, 20, 23). The extract was first tested for its ability to induce degranulation of Wt BMMCs as assessed by release of β-hexosaminidase. Wt BMMCs were treated with increasing concentrations of M. sympodialis extract (0.01–100 μg/ml) for 0.5 h. As depicted in Fig. 1⇓A, we could not observe any degranulation of Wt BMMCs following addition of either M. sympodialis extract, LPS, or zymosan, respectively. The cells responded as expected to the positive control A23187 (Fig. 1⇓A).
The M. sympodialis extract was next tested for its ability to induce release of cysteinyl leukotrienes in Wt BMMCs. In contrast to the lack of degranulation induction, the extract stimulated the release of cysteinyl leukotrienes in a dose-dependent manner (Fig. 1⇑B). Since mast cells have the capacity to secrete a variety of inflammatory cytokines and chemokines, we also measured the effect of M. sympodialis on the secretion of IL-6 and MCP-1. Both IL-6 and MCP-1 have potential roles in AE, IL-6 as a proinflammatory cytokine with Th2-promoting effects (24) and MCP-1 as a potent monocyte attractor (25). We did not observe any increase of IL-6 or MCP-1 secretion 24 h after addition of the M. sympodialis extract, in contrast to the expected LPS-induced IL-6 production and the positive response to A23187 (Fig. 1⇑, C and D).
Mast cells sensitized with IgE are activated by M. sympodialis extract through a MyD88-independent pathway
We next investigated the effect of M. sympodialis on IgE-sensitized mast cells, since mast cells in the skin of AE patients express IgE on their surface (26). BMMCs from Wt mice were sensitized with IgE-anti-TNP and thereafter treated with 0.01–100 μg/ml M. sympodialis extract. The highest concentration of M. sympodialis extract induced a significant mast cell degranulation (Fig. 2⇓A). Moreover, M. sympodialis extract induced within 0.5 h release of cysteinyl leukotrienes from IgE-sensitized Wt BMMCs in a dose-dependent manner (Fig. 2⇓B). Notably, the amount of cysteinyl leukotrienes released from IgE-sensitized mast cells stimulated with M. sympodialis extract was approximately three times higher than the levels released from nonsensitized mast cells following stimulation with M. sympodialis extract (Figs. 1⇑B and 2⇓B). We also measured the effect of M. sympodialis on the secretion of IL-6 and MCP-1. No significant increase of IL-6 secretion was detected following addition of the M. sympodialis extract (Fig. 2⇓C). In contrast, similar to the degranulation response, we could measure a significant release of MCP-1 upon addition of the highest concentration of M. sympodialis extract (100 μg/ml; Fig. 2⇓D). Chemokine production in mast cells is mediated through MAPK signaling (27). We therefore investigated whether M. sympodialis could cause activation of MAPK in IgE-sensitized mast cells. We determined that IgE-sensitized Wt BMMCs treated for 10 min with the highest concentration of M. sympodialis extract activated phosphorylation of the MAPK ERK1/2 (Fig. 2⇓E), indicating that M. sympodialis can activate MAPK signaling in IgE-sensitized mast cells.
Because BMMCs express several TLRs (16), we explored whether M. sympodialis could interact through a TLR-dependent pathway that mediates degranulation and MCP-1 release. BMMCs from MyD88−/− mice were generated, since MyD88 is a protein involved in the signaling pathway of most TLRs (28). We recorded that IgE-sensitized MyD88−/− mast cells degranulated, released MCP-1, and did not produce IL-6 (Fig. 3⇓, A–C) in a similar fashion to Wt BMMCs (Fig. 2⇑, A, C, and D), indicating a MyD88-independent activation in M. sympodialis-exposed, IgE-sensitized mast cells.
M. sympodialis enhances the IgE receptor cross-linked degranulation of mast cells independently of TLR2, TLR4, and MyD88
A recent study reported that E. coli can interfere with mast cell responses and can negatively affect IgE-mediated activation (29). We therefore studied the effect of M. sympodialis extract on the FcεRI expression of Wt BMMCs. We noted similar FcεRI expression after 24 h of incubation with or without M. sympodialis extract (data not included). We further analyzed whether M. sympodialis would affect mast cell degranulation induced by aggregation of IgE receptors. IgE-anti-TNP-sensitized Wt BMMCs were therefore activated by addition of TNP-BSA along with increasing amounts of M. sympodialis extract. The M. sympodialis extract significantly enhanced the IgE-mediated release of β-hexosaminidase in a dose-dependent manner (Fig. 4⇓A). As expected IgE receptor cross-linking resulted in release of cysteinyl leukotrienes and MCP-1 from Wt BMMCs, but in contrast to degranulation addition of M. sympodialis extract did not enhance this release (data not included).
To determine whether activation through TLRs could be the cause of the observed increase in degranulation, we assessed the activation of BMMCs from TLR2−/−, TLR4−/−, and MyD88−/− mice, respectively, following their coactivation with TNP-BSA and M. sympodialis extract. All of the deficient BMMCs exhibited equivalent degranulation reactivity to M. sympodialis extract as did Wt BMMCs (Fig. 4⇑, B–D). Since IgE-sensitized TLR4−/− BMMCs displayed the same degranulation pattern after coactivation with TNP-BSA and M. sympodialis extract as did Wt BMMCs, we can exclude that the increase in degranulation was due to LPS contamination. Thus, the cause of the observed increase in degranulation cannot be explained by signaling through TLR2, TLR4, or MyD88.
M. sympodialis enhancement of IL-6 secretion in IgE receptor cross-linked mast cells is dependent on the TLR2/MyD88 pathway
To address the question whether M. sympodialis could also influence the cytokine release of IgE receptor cross-linked mast cells, we investigated the effect of M. sympodialis extract on the secretion of the proinflammatory cytokine IL-6. Wt BMMCs presensitized with IgE-anti-TNP were coactivated with TNP-BSA and increasing amounts of M. sympodialis extract for 24 h, and the release of IL-6 was assessed by ELISA. M. sympodialis extract modified the production of IL-6 by IgE- and Ag-activated mast cells in a dose-dependent manner, whereby addition of low concentrations of M. sympodialis extract led to a significant increase in the IL-6 production and high concentrations led to a significant decrease (Fig. 5⇓A).
Since cytokine production in mast cells has been shown to require MAPK signaling (17), we next studied whether M. sympodialis extract influenced activation of MAPK in IgE receptor cross-linked Wt BMMCs cultured with or without M. sympodialis extract. We determined that higher concentrations of extract inhibited phosphorylation of the MAPK ERK1/2 10 min after stimulation (Fig. 5⇑B), which could reflect the observed inhibitory effect of high doses of M. sympodialis extract on IL-6 release from IgE receptor cross-linked mast cells (Fig. 5⇑A).
A synergistic activation through FcεRI and either of TLR2 or TLR4 has been reported to facilitate IL-6 production in BMMCs (17), and we thus proceeded to investigate how M. sympodialis extract influenced IgE receptor cross-linked BMMCs from TLR2−/−, TLR4−/−, and MyD88−/− mice, respectively. Similarly to Wt BMMCs (Fig. 5⇑A), the IL-6 release from TLR4−/− BMMCs was influenced by the addition of M. sympodialis extract, excluding LPS contamination of the extract (Fig. 5⇑C). In contrast, M. sympodialis extract exerted no significant effect on IL-6 production in IgE receptor cross-linked BMMCs derived from TLR2−/− or MyD88−/− mice, respectively (Fig. 5⇑, D and E), indicating a dependence on signaling through the TLR2/MyD88 pathway and a possible synergistic effect between TLR2 and FcεRI coactivation.
We herein demonstrate that extract from the skin commensal yeast M. sympodialis activates nonsensitized, IgE-sensitized, and IgE receptor cross-linked mast cells. When culturing nonsensitized Wt BMMCs with M. sympodialis extract alone, the cells released cysteinyl leukotrienes, but we could not observe any activation in terms of degranulation, chemokine release, or cytokine release (Fig. 6⇓A). We also determined that IgE-sensitized mast cells degranulate, release cysteinyl leukotrienes, and produce the chemokine MCP-1 upon addition of M. sympodialis extract (Fig. 6⇓, B and C). Furthermore, our results indicate that M. sympodialis is able to activate IgE-sensitized mast cells through the MAPK pathway and by signaling through the TLR2/MyD88 pathway can modify the IL-6 production of IgE receptor cross-linked mast cells.
The ability of M. sympodialis to induce an increase in β-hexosaminidase release could play a role in AE, since it has been reported that β-hexosaminidase is released by mast cells in combination with the serine protease tryptase (30). Tryptase can induce itching through the PAR-2 receptor, which has been shown to be involved during AE itch (31). An increase in MCP-1 release from IgE-sensitized mast cells could exacerbate inflammation in AE by recruiting monocytes (25) to the site of M. sympodialis invasion. The levels of cysteinyl leukotrienes released after coculture with M. sympodialis extract were increased in IgE-sensitized mast cells compared with in nonsensitized mast cells, indicating that the IgE sensitization increases the mast cells’ susceptibility to release cysteinyl leukotrienes in response to M. sympodialis activation. These findings concord with a study by Genovese et al. (32) which demonstrates that the interaction between IgE on mast cells and bacterial Ags results in an enhanced release of cysteinyl leukotrienes. Interestingly, enhanced releasability of cysteinyl leukotrienes upon activation has previously been reported in leukocytes from patients with AE compared with healthy controls (33). The observed Ag-independent activation of the mast cells indicates that some component of the M. sympodialis extract acts on IgE-anti-TNP-sensitized mast cells through what might be described as an “IgE-superantigen-like” effect. This is supported by the fact that mast cells can be activated independently of pathogen-specific Abs through the action of pathogen-derived Ig-binding proteins (16, 32). Several bacterial proteins have also been demonstrated to bind to different domains of FcεRI-bound IgE and thereby to act as IgE superantigens (34), two examples being Staphylococcus aureus protein A and Peptostreptococcus magnus protein L (34).
When the M. sympodialis extract was added to IgE receptor cross-linked mast cells, we observed that it increased the degranulation of Wt, TLR2−/−, TLR4−/−, and MyD88−/− mast cells in a dose-dependent manner. M. sympodialis thus acted as an amplifier of the IgE-activated mast cells by increasing the degranulation response through a TLR2-, TLR4-, and MyD88-independent mechanism. To further elucidate the mechanism involved in the amplified degranulation of IgE receptor cross-linked BMMCs by M. sympodialis, we investigated the involvement of the fungal recognition receptor dectin-1. Olynych et al. (35) have previously demonstrated that the fungal product zymosan can induce dectin-1-dependent release of cysteinyl leukotrienes from mast cells. When IgE receptor cross-linked dectin-1−/− BMMCs (provided by Prof. Gordon D. Brown, University of Cape Town, Cape Town, South Africa) were cultured with increasing amounts of M. sympodialis extract, the enhanced degranulation was independent of dectin-1, as was the enhanced cysteinyl leukotriene production caused by M. sympodialis in nonsensitized and IgE-sensitized mast cells (data not included). Another explanation for the amplified degranulation of IgE receptor cross-linked BMMCs by the extract could be up-regulation of FcεRI expression since an earlier study by Kulka et al. (29) reported that E. coli can regulate FcεRI expression in mast cells. However, we observed no change in FcεRI expression following stimulation with M. sympodialis for 24 h, and therefore the increased degranulation observed in our study could not be due to enhanced FcεRI expression.
We also demonstrated that M. sympodialis extract affects IgE receptor cross-linked BMMCs to alter their IL-6 production in a dose-dependent manner by signaling through the TLR2/MyD88 pathway (Fig. 6⇑, D–F), implying that cytokine release could be affected by a synergistic coactivation of the high-affinity IgE receptor and TLR2. A synergistic coactivation of TLR2 and FcεRI was previously reported by Qiao et al. (17) with TLR2 ligands substantially enhancing Ag-induced production of cytokines from mast cells. Furthermore, the higher concentrations of M. sympodialis extract caused inhibition of phosphorylated-ERK1/2 in IgE receptor cross-linked mast cells, indicating that the modulation of the IL-6 release might be mediated through the ERK1/2 pathway. Our findings that M. sympodialis can activate mast cells via the TLR2/MyD88 pathway corroborate with the work of Baroni et al. (36) who showed that human keratinocytes increase their gene expression of TLR2 and MyD88 following activation with Malassezia furfur. In another study, suppressed degranulation was determined upon combined stimulation of BMMCs with increasing concentrations of the TLR2 ligand Pam3CSK4 and IgE receptor cross-linking (37). The mechanism underlying the suppression in that study was the electrostatic binding of DNP by Pam3CSK4, thereby reducing the amount of available Ag for IgE receptor cross-linking. Our findings demonstrate an inhibitory effect of M. sympodialis on IL-6 release from IgE receptor cross-linked BMMCs and this effect was fully abolished in TLR2- and MyD88-deficient BMMCs, thereby excluding a suppressive effect of the extract on TNP-BSA as Fehrenbach et al. (37) previously observed with Pam3CSK4 and DNP.
The observed difference in influence of M. sympodialis on mast cell IL-6 release and degranulation might be explained by considering what distinguishes the degranulation pathway from the pathway resulting in cytokine production in mast cells. An increase in cytosolic calcium levels is an essential signal for degranulation (17), whereas the generation of cytokines follows the activation of the MAPK pathway in murine mast cells (38). TLR ligands do not seem to influence the calcium levels or to cause degranulation (17), but they have been shown to stimulate the phosphorylation of MAPK (17). Our findings concur with this, since 1–100 μg/ml M. sympodialis extract could inhibit the phosphorylation of ERK1/2 in IgE receptor cross-linked BMMCs (Fig. 5⇑B), which follows the observed TLR2/MyD88-dependent inhibition of IL-6 caused by M. sympodialis extract in IgE receptor cross-linked BMMCs.
Mast cells migrate to the upper dermis in AE (39) and in the lesional skin of AE patients have even been found in the epidermis (40), thus possibly facilitating their encounter with Malassezia spp. The concentrations encountered by mast cells in vivo can only be estimated. However, the extract concentrations used in this study concord with amounts used for atopy patch tests (41) and for in vitro activation of human T cells (42, 43). Even if the concentration of M. sympodialis that reaches the IgE receptor cross-linked mast cells in the skin along with other allergens is not sufficient to increase mast cell degranulation, it might still result in IL-6 release, thus enabling an increased Th2 differentiation and thereby promoting allergic inflammation.
We have demonstrated that M. sympodialis extract causes release of cysteinyl leukotrienes from nonsensitized mast cells. In IgE-sensitized mast cells, the extract induced release of cysteinyl leukotrienes, degranulation, MCP-1 production, and activation of the MAPK ERK1/2. Furthermore, M. sympodialis enhances mast cell IgE-dependent activation, inhibits activation of the MAPK ERK1/2, and alters IL-6 production in a dose-dependent manner through the TLR2/MyD88 pathway. Our findings suggest that these effects of M. sympodialis on mast cells could contribute to AE by perpetuating the inflammation.
We thank Prof. Shizuo Akira (Osaka University) for providing us with the TLR2−/−, TLR4−/−, and MyD88−/− mice in this study, Prof. Gordon D. Brown (University of Cape Town) for providing us with bone marrow from dectin-1−/− mice, and Dr. Robert A. Harris (Karolinska Institutet) for linguistic revision.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 This work was supported by grants from the Swedish Research Council; by the Center for Allergy Research, Karolinska Institutet; through the regional agreement on medical training and clinical research (ALF) between the Stockholm County Council and the Karolinska Institutet; by the Consul Th C Bergh Foundation; by the Ellen, Walter and Lennart Hesselman Foundation; by the Åke Wiberg Foundation; and by the Magnus Bergvall Foundation.
↵2 Address correspondence and reprint requests to Christine Selander, Clinical Allergy Research Unit L2:04, Karolinska Institutet and University Hospital Solna, S-171 76 Stockholm, Sweden. E-mail address:
↵3 Abbreviations used in this paper: AE, atopic eczema; BMMC, bone marrow-derived mast cell; Wt, wild type; TNP, trinitrophenyl.
- Received March 18, 2008.
- Accepted January 22, 2009.
- Copyright © 2009 by The American Association of Immunologists, Inc.