**2.2** *Malassezia* **colonization and severity of AD**

The *Malassezia* microbiota of the skin is also associated with the severity of AD. Fifty-six adult neck and head AD patients (21 mild, 18 moderate, and 17 severe cases) and 32 healthy individuals were examined for skin *Malassezia* microbiota, using a real-time PCR assay (Kaga *et al*. 2011). The level of colonization by *Malassezia* was almost identical among the mild and moderate AD patients and the healthy individuals, while *Malassezia* colonization in the severe AD cases was approximately 2- to 5-fold that in the mild and moderate AD patients and healthy individuals (Fig. 5A). Two major species, *M*. *globosa* and *M*. *restricta*, accounted for more than 80% of all *Malassezia* colonization in AD patients of all severities, but their proportions differed with severity. In the mild and moderate cases, *M. restricta* predominated over *M. globosa* (*p* < 0.05), whereas the proportions of *M. globosa* and *M. restricta* were almost identical (*p* > 0.05) in the severe patients (Fig. 5B).

Atopic Dermatitis and Skin Fungal Microorganisms 129

that both *M. globosa* and *M. restricta* were the predominant species regardless of disease severity, with a detection rate of 57.5–70.4% in all clones analyzed. However, the ratio of *M. globosa* to *M. restricta* in the mild and moderate cases (*M. restricta/M. globosa*: 3.1–3.4 in mild and 2.1–4.1 in moderate cases) differed from that in the severe cases (1.1–1.4). Figure 6 shows the phylogenetic distribution between AD patients and healthy individuals, based on principal coordinates analysis. Patients with mild or moderate symptoms of AD constituted a single cluster, and patients with severe disease formed a separate cluster. Similarly, the

Closed triangle, patients with mild symptoms; closed square, patient with moderate symptoms; closed

Differences in microbiota are thought to be attributable to differences in the physiological condition of the skin between patients with AD and healthy subjects. For example, skin pH may change skin microbiota (Seidenari and Giusti, 1995). *Staphylococcus epidermidis* is present in the skin microbiota of healthy individuals, whereas *S. aureus* is not. The level of colonization by *S. aureus* increases according to the severity of AD. In contrast, the level of colonization by *S. epidermidis* decreases gradually with increasing AD severity. Healthy skin is weakly acidic, whereas the skin pH in patients with AD is near neutral, which facilitates invasion by exogenous microorganisms, including *S. aureus* (Higaki *et al*. 1999; Hoeger *et al*. 1992). The expression levels of antimicrobial peptides may also affect the fungal microbiota (Howell 2007). The antimicrobial peptides known as defensins and cathelicidins are deficient in the skin of AD patients, and thus the fungal microbiota should be different between AD patients and healthy individuals. Sebum is a growth medium for skin microorganisms and consists of squalene, cholesterol esters, wax esters, triglycerides, free fatty acids, cholesterol, ceramides, cholesterol sulfate, and phospholipids. Of these, the

Fig. 6. Principal coordinates analysis (PCA) score plot of the sequence profiles for the

circle, patients with severe symptoms; open circle, healthy individuals

predominant skin fungi

healthy individuals clustered independently.

AD, patients with atopic dermatitis; HS, healthy subjects.

Fig. 4. Phylogenetic tree of *M. globosa* colonizing the skin surface of AD patients and healthy subjects based on DNA sequences of the IGS 1 region

Fig. 5. Level of *Malassezia* colonization in patients with atopic dermatitis and in healthy individuals (A). Ratio of the two major *Malassezia* species, *M. globosa* and *M. restricta*, in patients with atopic dermatitis and in healthy individuals (B)

In a comprehensive analysis using an rRNA gene clone library method, Zhang *et al*. (2011) found that not only *Malassezia* but also the overall fungal microbiota differed according to AD severity. Their analysis of 3,647 clones of the fungal rRNA gene in scale samples from nine AD patients (3 mild, 3 moderate, and 3 severe cases) and 10 healthy individuals revealed 58 fungi and seven unknown phylotypes. *Malassezia* predominated, representing 63–86% of the clones identified from each subject. The number of clones had no noticeable relationship to disease severity, with the mild, moderate, and severe cases accounting for 67.8 ± 2.2, 70.7 ± 2.8, and 64.9 ± 1.8% of the clones, respectively. The study also confirmed

Fig. 4. Phylogenetic tree of *M. globosa* colonizing the skin surface of AD patients and healthy

Fig. 5. Level of *Malassezia* colonization in patients with atopic dermatitis and in healthy individuals (A). Ratio of the two major *Malassezia* species, *M. globosa* and *M. restricta*, in

In a comprehensive analysis using an rRNA gene clone library method, Zhang *et al*. (2011) found that not only *Malassezia* but also the overall fungal microbiota differed according to AD severity. Their analysis of 3,647 clones of the fungal rRNA gene in scale samples from nine AD patients (3 mild, 3 moderate, and 3 severe cases) and 10 healthy individuals revealed 58 fungi and seven unknown phylotypes. *Malassezia* predominated, representing 63–86% of the clones identified from each subject. The number of clones had no noticeable relationship to disease severity, with the mild, moderate, and severe cases accounting for 67.8 ± 2.2, 70.7 ± 2.8, and 64.9 ± 1.8% of the clones, respectively. The study also confirmed

patients with atopic dermatitis and in healthy individuals (B)

AD, patients with atopic dermatitis; HS, healthy subjects.

subjects based on DNA sequences of the IGS 1 region

that both *M. globosa* and *M. restricta* were the predominant species regardless of disease severity, with a detection rate of 57.5–70.4% in all clones analyzed. However, the ratio of *M. globosa* to *M. restricta* in the mild and moderate cases (*M. restricta/M. globosa*: 3.1–3.4 in mild and 2.1–4.1 in moderate cases) differed from that in the severe cases (1.1–1.4). Figure 6 shows the phylogenetic distribution between AD patients and healthy individuals, based on principal coordinates analysis. Patients with mild or moderate symptoms of AD constituted a single cluster, and patients with severe disease formed a separate cluster. Similarly, the healthy individuals clustered independently.

Closed triangle, patients with mild symptoms; closed square, patient with moderate symptoms; closed circle, patients with severe symptoms; open circle, healthy individuals

Fig. 6. Principal coordinates analysis (PCA) score plot of the sequence profiles for the predominant skin fungi

Differences in microbiota are thought to be attributable to differences in the physiological condition of the skin between patients with AD and healthy subjects. For example, skin pH may change skin microbiota (Seidenari and Giusti, 1995). *Staphylococcus epidermidis* is present in the skin microbiota of healthy individuals, whereas *S. aureus* is not. The level of colonization by *S. aureus* increases according to the severity of AD. In contrast, the level of colonization by *S. epidermidis* decreases gradually with increasing AD severity. Healthy skin is weakly acidic, whereas the skin pH in patients with AD is near neutral, which facilitates invasion by exogenous microorganisms, including *S. aureus* (Higaki *et al*. 1999; Hoeger *et al*. 1992). The expression levels of antimicrobial peptides may also affect the fungal microbiota (Howell 2007). The antimicrobial peptides known as defensins and cathelicidins are deficient in the skin of AD patients, and thus the fungal microbiota should be different between AD patients and healthy individuals. Sebum is a growth medium for skin microorganisms and consists of squalene, cholesterol esters, wax esters, triglycerides, free fatty acids, cholesterol, ceramides, cholesterol sulfate, and phospholipids. Of these, the

Atopic Dermatitis and Skin Fungal Microorganisms 131

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.

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

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

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