**3. Immunological aspects of** *Malassezia* **colonizaiton**

#### **3.1** *Malassezia* **specific IgE antibody**

Specific IgE antibodies against skin *Malassezia* are present in the serum of AD patients whereas no anti-*Malassezia* specific IgE antibody is found in the serum of healthy individuals (Sugita *et al*. 2001). Many studies have reported on anti-*Malassezia* specific IgE antibodies in AD patients (Zargari *et al*. 2003; Kato *et al*. 2006). Using an enzyme-linked immunosorbent assay (ELISA), Kato *et al*. (2006) quantified specific IgE antibodies against soluble proteins of eight *Malassezia* species in mechanically disrupted extracts of serum samples from AD patients. The level of IgE specific for *M. restricta* was greater than that against other *Malassezia* species (*M. dermatis*, *M. furfur*, *M. globosa*, *M. obtusa*, *M. pachydermatis*, *M. slooffiae*, and *M. sympodialis*) (Fig. 7); however, a competitive inhibition ELISA revealed that *M. restricta* contained species-specific as well as shared antigens.

N=24

Fig. 7. The species-specific IgE values of eight *Malassezia* species in sera from patients with atopic dermatitis determined using an ELISA.

proportion of ceramide 1, which is a carrier of linoleate and responsible for the water-barrier function of the skin, is significantly lower in patients with AD (Yamamoto *et al*. 1991).

Specific IgE antibodies against skin *Malassezia* are present in the serum of AD patients whereas no anti-*Malassezia* specific IgE antibody is found in the serum of healthy individuals (Sugita *et al*. 2001). Many studies have reported on anti-*Malassezia* specific IgE antibodies in AD patients (Zargari *et al*. 2003; Kato *et al*. 2006). Using an enzyme-linked immunosorbent assay (ELISA), Kato *et al*. (2006) quantified specific IgE antibodies against soluble proteins of eight *Malassezia* species in mechanically disrupted extracts of serum samples from AD patients. The level of IgE specific for *M. restricta* was greater than that against other *Malassezia* species (*M. dermatis*, *M. furfur*, *M. globosa*, *M. obtusa*, *M. pachydermatis*, *M. slooffiae*, and *M. sympodialis*) (Fig. 7); however, a competitive inhibition

ELISA revealed that *M. restricta* contained species-specific as well as shared antigens.

Fig. 7. The species-specific IgE values of eight *Malassezia* species in sera from patients with

Therefore, the composition of sebum may also affect the fungal microbiota.

**3. Immunological aspects of** *Malassezia* **colonizaiton** 

**3.1** *Malassezia* **specific IgE antibody** 

N=24

atopic dermatitis determined using an ELISA.

The precise mechanisms by which *Malassezia* colonization induces IgE antibody production and the inflammatory cascades that lead to AD remain unclear. The presence of IgE antibodies has been implicated in the production of Th2-type cytokines such as interleukins (IL)-4, -5, -6, -10, and -13, the promotion of IgE antibody production, the differentiation of mast cells, and the growth, migration, and activation of eosinophils (Hamid *et al*. 1994; Leung *et al*. 2000; Chen *et al*. 2004). Keratinocytes, the major cell type in the epidermis, have roles in both skin structural and immunological defense (Esche *et al*. 2004; Albanesi *et al*. 2005). Keratinocytes produce a range of proinflammatory and immune cytokines in response to microorganisms and/or skin damage (Grone *et al*. 2002; Watanabe *et al*. 2001). A recent study has demonstrated that keratinocytes secrete several Th2-type cytokines that are critical in the pathogenesis of AD (Ishibashi *et al*. 2006). Cytokine secretion profiling by antibody array analysis has revealed that *M. globosa* and *M. restricta* induce the secretion of distinct Th2-type cytokines by human keratinocytes: *M. globosa* induces IL-5, IL-10, and IL-13 secretion, while *M. restricta* induces IL-4 secretion. These findings have been confirmed by cDNA microarray analysis showing that *M. globosa* and *M. restricta* upregulate the transcription of the *IL-5* and *IL-4* genes, respectively, in keratinocytes. These observations provide evidence of a possible relationship between *Malassezia* colonization and increased IgE production in AD. It is possible that *M. globosa* and *M. restricta* play a synergistic role in triggering a Th2-shifted humoral immune response in AD. Another important connection between *Malassezia* colonization and AD relates to the increased secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) and cutaneous T-cellattracting chemokine (CTACK) by keratinocytes (Ishibashi *et al*. 2006). *Malassezia globosa* is capable of stimulating keratinocytes to secrete GM-CSF, which primarily contributes to the maintenance of the chronic inflammatory process in AD by enhancing the antigenpresenting capacity of Langerhans cells and dendritic cells (Witmer-Pack *et al*. 1987). *Malassezia restricta* induces the secretion of CTACK by keratinocytes. CTACK selectively attracts cutaneous lymphocyte antigen-positive memory T cells to inflammatory sites (Morales *et al*. 1999) and is upregulated in AD patients (Kakinuma *et al*. 2003). The above findings suggest the following possible mechanism by which *Malassezia* species induce an IgE-immune response in patients with AD: a skin barrier dysfunction facilitates skin penetration by colonized *Malassezia*, allowing interactions between *Malassezia* and epidermal Langerhans cells, dendritic cells, and keratinocytes, which subsequently present *Malassezia* antigens, thereby inducing an immune response. This may be augmented by keratinocyte-derived GM-CSF. *Malassezia*-stimulated keratinocytes produce Th2 cytokines, including IL-4 and IL-13, which may in turn stimulate B cells to undergo IgE class switching and produce *Malassezia-*specific IgE. In addition, keratinocyte-derived IL-5 may attract and locally activate eosinophils in lesions of AD.

#### **3.2** *Malassezia* **allergens**

Many *Malassezia* allergens have been identified, including Mala f2-4, Mala s1, and Mala s5-13. Several researchers have attempted to produce recombinant *Malassezia* allergens (rMala s1 and rMala s5–11) for diagnostic purposes (Schmidt *et al*. 1997; Schmid-Grendelmeier *et al*. 2005, 2006; Limacher *et al*. 2007) (Table 2). Recently, proteomics analysis has been applied to identify major allergens of *M. globosa* (Ishibashi *et al*. 2009). The IgE-reactive component of *M. globosa*, with a molecular mass of 42 kDa and designated as MGp42, has been identified by twodimensional immunoblotting and partially sequenced by matrix-assisted laser desorption ionization time of flight mass spectrometry with post-source decay of the peptide digest. The

Atopic Dermatitis and Skin Fungal Microorganisms 133

There was also a significant correlation between the level of *Malassezia* specific IgE antibody and clinical severity criteria, as reflected by the SCORAD index (*p* < 0.0001, *r2* = 0.55), whereas total IgE showed only a slight correlation with severity criteria (*p* < 0.001, *r2*=0.29). No correlation was found between age or gender, and specific or total IgE. Based on pricktest results and specific IgE antibody levels, treatment of HANAD patients with antifungal

Compared with the plethora of antibacterial agents, only a small number of antifungal agents are available, which limits the treatment options for HANAD. Ketoconazole and itraconazole are highly effective *in vitro* (Sugita *et al*. 2005, Miranda *et al*. 2007, Sancak *et al*. 2005, Velegraki *et al*. 2004). In a large-scale study using 125 strains of 11 *Malassezia* species, all of the *Malassezia* species were highly susceptible to both itraconazole and ketoconazole, with minimum inhibitory concentrations (MICs) ranging from 0.016 to 0.25 mg/ml; approximately 80% of the strains had a MIC of ≤0.03 mg/ml (Sugita *et al*. (2005). This efficacy is not specific to these species, but applies to all members of the genus *Malassezia*. To our knowledge, no resistant strain has been detected. Ketoconazole and itraconazole are chemically classified as azole compounds, but other azole agents, fluconazole, voriconazole,

A calcineurin inhibitor, topical tacrolimus, is widely used to treat AD. This compound had antifungal activity against half of the known *Malassezia* strains, with MICs of 16–32 mg/mL (Sugita *et al*. 2005). The immunosuppressive drugs cyclosporine and tacrolimus target calcineurin and are also toxic to *Candida albicans* and *Cryptococcus neoformans* (Cruz *et al*. 2001). A combination of either ketoconazole or itraconazole and tacrolimus had a synergistic effect against *Malassezia* strains, based on a fractional inhibitory index of 0.245 to 0.378. These observations follow earlier reports on the effectiveness of a combination of tacrolimus and fluconazole against *C. albicans* and *C. neoformans* strains. The combination of topical tacrolimus and an azole agent can simultaneously treat AD and reduce the number of exacerbating *Malassezia* cells colonizing the skin surface. Although the synergistic mechanism of this combination is not known, Maesaki *et al*. (1998) demonstrated that tacrolimus increases the intracellular concentration of the azole agent in *C. albicans*. This observation may provide the basis for future clinical trials of these agents aimed at reducing the number of *Malassezia* cells colonizing the skin of AD patients (more details are provided

Devos and Valk, 2000 HANAD 100% (n=22) Non-HANAD 14% (n=22) Johansson et al. 2003 HANAD 55% (n=98) Non-HANAD 19% (n=33) Jensen-Jarolim et al. 1992 HANAD 68% (n=80)

Table 3. *Malassezia* IgE antibodies in sera of patient with atopic dermatitis

agents has been recommended for the previous two decades.

**4.2 Susceptibility of** *Malassezia* **to drug treatment** 

and terbinafine, cannot inhibit the growth of *Malassezia*.

in the following section).

**Authors Patients Production of anti-***Malassezia*

**specific IgE antibodies** 

full-length cDNA encoding MGp42 has been cloned and sequenced by the rapid amplification of cDNA ends method. MGp42 exhibits properties similar to those of heat shock protein (hsp) family members, and evidence indicates that MGp42 may be a cleavage product of intact HSP70. However, no IgE cross-reactivity has been observed between MGp42 and recombinant human HSP70, suggesting that the epitopes of MGp42 recognized by serum IgE of AD patients are masked by steric hindrance in the presence of intact HSP70 and become exposed as a result of conformational changes during HSP70 cleavage.


Table 2. *Malassezia* allergens
