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M. sympodialis can activate mast cells (Paper III)

4 Results and Discussion

4.3 M. sympodialis can activate mast cells (Paper III)

The number of MCs is increased in the lesional skin of AE patients (Damsgaard et al., 1997; Horsmanheimo et al., 1994) and can thus be suspected to contribute to the chronic inflammation. Due to the ruptured skin barrier in AE, it is likely that compounds from Malassezia can come into contact with MCs in the skin. We were therefore interested in exploring this proposed interaction and hypothesized that Malassezia can activate MCs.

In order to assess this question we decided to analyze the effect of M.

sympodialis extract on bone marrow-derived MCs (BMMCs) from C57BL/6 mice. We chose to work with MCs derived from mice, since we then could use gene deleted mice as a well-established model system to explore possible activation mechanisms. The yeast extract was chosen as a tool to investigate the possible interaction between Malassezia and MCs, since it contains a variety of allergens known to be recognized by serum from AE patients sensitized to M. sympodialis. Furthermore, we find it more likely that the MCs in the skin would come in contact with components from Malassezia diffusing through a ruptured epidermis rather than the intact yeast. The extract was first tested for its ability to activate non-sensitized Wt BMMCs and we analyzed signs of activation as increased degranulation, release of cysteinyl leukotrienes and production of the inflammatory cytokine IL-6 and the chemokine MCP-1. We could not observe any degranulation or release of IL-6 and MCP-1 from non-sensitized Wt BMMCs following addition of M. sympodialis extract. We found,

however, that the extract stimulated non-sensitized Wt BMMCs to release cysteinyl leukotrienes in a dose-dependent manner. Olynych et al. have previously shown that the fungal product zymosan can induce release of cysteinyl leukotrienes from MCs, by signaling through the fungal recognition receptor Dectin-1 (Olynych et al., 2006). In contrast, in our study the observed release of cysteinyl leukotrienes was independent of Dectin-1, since Dectin-1 knockout BMMCs released cysteinyl leukotrienes in a similar fashion to Wt BMMCs following culture with M. sympodialis extract. Since Dectin-1 recognizes charbohydrates, and the M. sympodialis extract used in this study mainly is composed of proteins and only low amounts of carbohydrates, this could provide a possible explanation for the independence of Dectin-1 in our study.

We next investigated the effect of M. sympodialis extract on IgE-sensitized MCs, since MCs in the skin of AE patients express IgE on their surface (Grabbe et al., 1996). BMMCs from Wt mice were sensitized with IgE-anti-trinitrophenyl (TNP) and thereafter treated with 0.01-100 µg/mL M. sympodialis extract. The highest concentration of M. sympodialis extract induced MC degranulation, release of MCP-1 but no IL-6 production. Chemokine production in MCs is mediated through MAPK signaling (Wong et al., 2006), and we therefore investigated if M. sympodialis could cause activation of MAPK in IgE-sensitized MCs.

We determined that IgE-sensitized Wt BMMCs, treated with the highest concentration of M. sympodialis extract, activated phosphorylation of the MAPK ERK 1/2, indicating that M. sympodialis can activate MAPK signaling in IgE-sensitized MCs. This enhanced MAPK activation may be connected to the MCP-1 release from IgE-sensitized MCs cultured with M. sympodialis extract. Additionally, M. sympodialis extract induced release of cysteinyl leukotrienes from IgE-sensitized MCs in a dose-dependent manner. Interestingly, the levels of cysteinyl leukotrienes released were increased in IgE-sensitized MCs compared to non-sensitized MCs. This indicates that the IgE-sensitization increases the MCs’ susceptibility to release cysteinyl leukotrienes in response to M. sympodialis activation. These findings concord with a study by Genovese et al., which demonstrates that the interaction between IgE on MCs and bacterial antigens results in an enhanced release of cysteinyl leukotrienes (Genovese et al., 2000). In agreement with our results from non-sensitized MCs, the release of cysteinyl leukotriens from IgE-sensitized MCs cultured with M. sympodialis extract, was not dependent on signaling through Dectin-1.

As BMMCs express several TLRs (Marshall, 2004) we explored if M.

sympodialis could interact with MCs through a TLR-dependent pathway that mediates degranulation and MCP-1 release. BMMCs from MyD88 knockout mice were generated, since MyD88 is a protein involved in the signaling pathway of most TLRs (Takeda et al., 2003). IgE-sensitized MyD88 knockout BMMCs were after culture with M. sympodialis extract activated in a similar fashion to Wt BMMCs, indicating a MyD88-independent activation of M. sympodialis-exposed IgE-sensitized MCs. This observed antigen-independent activation of MCs suggests that some components of the M. sympodialis extract acts on IgE-anti-TNP-sensitized MCs through what might be described as an ‘IgE-superantigen-like’ effect. Proteins from bacteria have previously been demonstrated to bind to FcİRI-bound IgE and thereby act as IgE superantigens (Genovese et al., 2003).

It has been reported that E. coli can interfere with MC responses and can negatively affect IgE-mediated activation (Kulka et al., 2006). We therefore studied the

effect of M. sympodialis extract on the FcİRI expression of Wt BMMCs. We noted similar FcİRI expression after 24 h of incubation with or without M. sympodialis extract. We further analyzed if M. sympodialis would affect MC activation induced by aggregation of IgE receptors. IgE-anti-TNP-sensitized Wt BMMCs were therefore activated by addition of TNP-BSA together with increasing amounts of M. sympodialis extract. The M. sympodialis extract significantly enhanced the IgE-mediated degranulation and modified the IL-6 release in a dose-dependent manner, however, no increase in release of cysteinyl leukotrienes or MCP-1 was detected in IgE-receptor cross-linked MCs upon addition of M. sympodialis extract.

To determine if activation through PRRs could be the cause of the increased degranulation, we assessed the activation of BMMCs from Dectin-1, TLR-2, TLR-4 and MyD88 knockout mice, respectively, following their co-activation with TNP-BSA and M. sympodialis extract. However, all the deficient BMMCs exhibited equivalent degranulation reactivity to M. sympodialis extract as Wt BMMCs. Thus, the cause of the observed increase in degranulation can not be explained by signaling through either Dectin-1, TLR-2, TLR-4 or MyD88.

As stated above M. sympodialis extract affected IgE-receptor cross-linked BMMCs to alter their IL-6 production in a dose-dependent manner. 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. A synergistic activation through FcİRI and either of TLR-2 or TLR-4 has been reported to aggravate IL-6 production in BMMCs (Qiao et al., 2006), and we thus proceeded to investigate how M. sympodialis extract influenced IgE-receptor cross-linked BMMCs from TLR-2, TLR-4 and MyD88 knockout mice, respectively. The IL-6 release from TLR-4 knockout BMMCs was influenced in a similar fashion as Wt BMMCs by addition of M. sympodialis extract. In contrast, M. sympodialis extract exerted no significant effect on IL-6 production in IgE-receptor cross-linked BMMCs derived from TLR-2 or MyD88 knockout mice, respectively. This indicates a dependency on signaling through the TLR-2/MyD88 pathway and a possible synergistic effect between TLR-2 and FcİRI. These findings corroborate with the work of Baroni et al., which showed that human keratinocytes increase their gene expression of TLR-2 and MyD88 following activation with M. furfur (Baroni et al., 2006).

Since cytokine production in MCs has been shown to require MAPK signaling (Qiao et al., 2006), we next studied if M. sympodialis extract influenced activation of MAPK in IgE-receptor cross-linked Wt BMMCs cultured with or without M. sympodialis extract. We found that higher concentrations of M. sympodialis extract inhibited phosphorylation of the MAPK ERK 1/2, indicating that the modulation of the IL-6 release might be mediated through the ERK 1/2 pathway.

Figure 9. A schematic model for activation of MCs by M. sympodialis extract. Non-sensitized MCs release cysteinyl leukotrienes after activation with M. sympodialis extract (A). IgE-sensitized MCs release cysteinyl leukotrienes, degranulate and produce MCP-1 but not IL-6 upon addition of M. sympodialis extract, as indicated in bold letters (B-C). M. sympodialis extract enhances the degranulation of IgE-receptor cross-linked MCs and by signaling through TLR-2 modifies their IL-6 production, as indicated in bold letters (D-F).

Taken together, our results demonstrate that M. sympodialis extract causes release of cysteinyl leukotrienes from non-sensitized MCs. In IgE-sensitized MCs, the extract induced release of cysteinyl leukotrienes, degranulation, MCP-1 production and activation of the MAPK ERK 1/2. Moreover, M. sympodialis extract enhanced IgE-dependent MC activation, inhibited activation of the MAPK ERK 1/2 and altered IL-6 production in a dose-dependent manner through the TLR-2/MyD88 pathway. Our findings (summarized in figure 9) imply that the effects of Malassezia on MCs might exacerbate the inflammation in AE.

4.4 MAST CELLS FROM PATIENTS WITH AE SHOW AN ENHANCED

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