**4.2** *Candida albicans* **gut colonization**

It has been hypothesized that excessive colonization by *C. albicans* in the gastrointestinal tract may constitute an aggravating factor in AD, but this remains controversial (Faergemann et al., 2002; Lacour et al., 2002; Nikkels & Pierard, 2003). To date, laboratory and clinical investigations have demonstrated that IgE mediated food allergy plays a pathogenic role in a subset of AD patients (Eigenmann et al., 1998; Lever et al., 1998; van Reijsen et al., 1998). Some reports have shown increased gastrointestinal permeability in

Fungus as an Exacerbating Factor

**4.3 Skin barrier dysfunction** 

*C. albicans* gut colonization in immune diseases in humans.

of Atopic Dermatitis, and Control of Fungi for the Remission of the Disease 147

(2011) examined whether *C. albicans* gut colonization aggravates immune diseases in mice. Mice were inoculated intragastrically with *C. albicans* to establish chronic and latent *C. albicans* gut colonization. Allergic diarrhea was induced by repeated intragastric administration of ovalbumin in BALB/c mice. Contact hypersensitivity was evaluated by measuring ear swelling after topical application of 2, 4-dinitrofluorobenzene in NC/Nga mice, which are often used as a mouse model of AD (Jin et al., 2011; Orita et al., 2010). Arthritis was induced by intradermal injection of bovine type-II collagen emulsified with complete Freund's adjuvant in DBA/1J mice. *C. albicans* gut colonization increased the incidence of allergic diarrhea, which was accompanied by gut hyperpermeability, as well as increased infiltration of inflammatory cells in the colon. Contact hypersensitivity was also exacerbated by *C. albicans* gut colonization, as demonstrated by increased swelling, myeloperoxidase activity, and proinflammatory cytokines in ear auricles. Furthermore, *C. albicans* gut colonization promoted limb joint inflammation in collagen-induced arthritis in an animal model of rheumatoid arthritis (Setoguchi et al., 2010; Takagi et al., 2009). These findings suggest that *C. albicans* gut colonization in mice aggravates inflammation in allergic and autoimmune diseases, and evokes the necessity of investigating the pathogenic role of

Skin barrier dysfunction (Ogawa et al., 1993; Cork et al., 2006 & 2009; Elias et al., 2008; Palmer et al., 2006) has emerged as a critical driving force in the initiation and exacerbation of AD with a recent major breakthrough in the genetics of AD (O'Regan et al., 2009; Hudson et al., 2006; Brown SJ, McLean, 2009). For instance, as addressed by Ogawa et al. (1993), dryness of the skin is an important component of the atopic diathesis, thereby reflecting possible skin barrier dysfunction. When the two abnormalities, dry skin/barrier dysfunction and allergy/immunological dysfunction, are considered as the major underlying defects of AD, the wide range of clinical manifestations seen in AD can be more easily comprehended. A defect of the mucocutaneous barrier readily allows penetration of multiple antigens or haptens, which enhances allergic inflammation. On the other hand, an allergic inflammation derived from the immunological abnormalities damages barrier functions. This sequence cycle could answer the question as to why AD patients show IgE production against, and contact hypersensitivity to, various antigens or haptens. A set of protective/defensive functions generated in the epidermis is likely mediated by its unique differentiation end product, the stratum corneum (Elias 2005; Elias & Choi, 2005). Basically, a markedly increased transepidermal water loss and a markedly decreased water holding capacity of the stratum corneum were reported in AD patients (Watanabe et al., 1991). In addition, since the patients showed a higher transepidermal water loss following irritant exposure, the susceptibility to irritants in AD patients seemed to be closely related with a breakdown in the barrier function of the stratum corneum (Tupker et al., 1990). More recently, it has been proposed that AD is a multifactorial, heterogenous disease that arises as a result of the interaction between both environmental and genetic factors (Cork et al., 2009). Changes in at least three groups of genes encoding structural proteins, epidermal proteases, and protease inhibitors make AD patients prone to a defective epidermal barrier, resulting in increased risk of developing AD. Loss-offunction mutations found within the FLG gene, which encodes the structural protein, filaggrin, could be the most significant genetic factor toward AD. In addition, enhanced protease activity and decreased synthesis of the lipid lamellae lead to exacerbated

AD patients (Jackson et al., 1981; Majamaa et al., 1996; Pike et al., 1986). Hyperpermeability of the gastrointestinal mucosal barrier results in enhanced transport of intact and degraded antigens across the gastrointestinal mucosal barrier, which could induce food protein sensitization and food allergy in susceptible individuals (Farhadi et al., 2003) (Fig. 2). Yamaguchi et al. (2006) therefore hypothesized that gastrointestinal colonization by *C albicans* may be involved in aggravation of AD by affecting the mucosal barrier in a manner that results in increased permeation of food allergens and subsequent manifestation of a food allergy. Using mice, they examined whether gastrointestinal colonization by *C. albicans* contributes to the aggravation of AD. *Candida* colonization was establised by intragastric inoculation with *C. albicans*, and then mice were intragastrically administered ovalbumin every other day for nine weeks. As a result, ovalbumin specific IgG and IgE titres were higher in BALB/c mice with *Candida* colonization than in normal mice, suggesting that gastrointestinal permeation of ovalbumin was enhanced by colonization in the mice. Histological examination showed that colonization promoted infiltration and degranulation of mast cells.

Fig. 2. *Candida albicans* gut colonization as an aggravating factor in atopic dermatitis. Excessive colonization by *C. albicans* in the gastrointestinal tract induces hyperpermeability of the gastrointestinal mucosal barrier, resulting in enhanced transport of intact and degraded antigens across the gastrointestinal mucosal barrier. This induces food protein sensitization and food allergy in susceptible individuals.

*Candida* colonization did not enhance ovalbumin permeation in mast cell deficient W/Wv mice but did in congenic littermate control +/+ mice. Reconstitution of mast cells in W/Wv mice by transplantation of bone marrow-derived mast cells restored the ability to increase ovalbumin permeation in response to *Candida* colonization. These results suggest that gastrointestinal *Candida* colonization promotes sensitization against food antigens, at least partly due to mast cell-mediated hyperpermeability in the gastrointestinal mucosa of mice. To confirm that gut colonization of *C. albicans* aggravates atopic dermatitis, Sonoyama et al. (2011) examined whether *C. albicans* gut colonization aggravates immune diseases in mice. Mice were inoculated intragastrically with *C. albicans* to establish chronic and latent *C. albicans* gut colonization. Allergic diarrhea was induced by repeated intragastric administration of ovalbumin in BALB/c mice. Contact hypersensitivity was evaluated by measuring ear swelling after topical application of 2, 4-dinitrofluorobenzene in NC/Nga mice, which are often used as a mouse model of AD (Jin et al., 2011; Orita et al., 2010). Arthritis was induced by intradermal injection of bovine type-II collagen emulsified with complete Freund's adjuvant in DBA/1J mice. *C. albicans* gut colonization increased the incidence of allergic diarrhea, which was accompanied by gut hyperpermeability, as well as increased infiltration of inflammatory cells in the colon. Contact hypersensitivity was also exacerbated by *C. albicans* gut colonization, as demonstrated by increased swelling, myeloperoxidase activity, and proinflammatory cytokines in ear auricles. Furthermore, *C. albicans* gut colonization promoted limb joint inflammation in collagen-induced arthritis in an animal model of rheumatoid arthritis (Setoguchi et al., 2010; Takagi et al., 2009). These findings suggest that *C. albicans* gut colonization in mice aggravates inflammation in allergic and autoimmune diseases, and evokes the necessity of investigating the pathogenic role of *C. albicans* gut colonization in immune diseases in humans.

### **4.3 Skin barrier dysfunction**

146 Atopic Dermatitis – Disease Etiology and Clinical Management

AD patients (Jackson et al., 1981; Majamaa et al., 1996; Pike et al., 1986). Hyperpermeability of the gastrointestinal mucosal barrier results in enhanced transport of intact and degraded antigens across the gastrointestinal mucosal barrier, which could induce food protein sensitization and food allergy in susceptible individuals (Farhadi et al., 2003) (Fig. 2). Yamaguchi et al. (2006) therefore hypothesized that gastrointestinal colonization by *C albicans* may be involved in aggravation of AD by affecting the mucosal barrier in a manner that results in increased permeation of food allergens and subsequent manifestation of a food allergy. Using mice, they examined whether gastrointestinal colonization by *C. albicans* contributes to the aggravation of AD. *Candida* colonization was establised by intragastric inoculation with *C. albicans*, and then mice were intragastrically administered ovalbumin every other day for nine weeks. As a result, ovalbumin specific IgG and IgE titres were higher in BALB/c mice with *Candida* colonization than in normal mice, suggesting that gastrointestinal permeation of ovalbumin was enhanced by colonization in the mice. Histological examination showed that colonization promoted

Fig. 2. *Candida albicans* gut colonization as an aggravating factor in atopic dermatitis. Excessive colonization by *C. albicans* in the gastrointestinal tract induces hyperpermeability of the gastrointestinal mucosal barrier, resulting in enhanced transport of intact and degraded antigens across the gastrointestinal mucosal barrier. This induces food protein

*Candida* colonization did not enhance ovalbumin permeation in mast cell deficient W/Wv mice but did in congenic littermate control +/+ mice. Reconstitution of mast cells in W/Wv mice by transplantation of bone marrow-derived mast cells restored the ability to increase ovalbumin permeation in response to *Candida* colonization. These results suggest that gastrointestinal *Candida* colonization promotes sensitization against food antigens, at least partly due to mast cell-mediated hyperpermeability in the gastrointestinal mucosa of mice. To confirm that gut colonization of *C. albicans* aggravates atopic dermatitis, Sonoyama et al.

sensitization and food allergy in susceptible individuals.

infiltration and degranulation of mast cells.

Skin barrier dysfunction (Ogawa et al., 1993; Cork et al., 2006 & 2009; Elias et al., 2008; Palmer et al., 2006) has emerged as a critical driving force in the initiation and exacerbation of AD with a recent major breakthrough in the genetics of AD (O'Regan et al., 2009; Hudson et al., 2006; Brown SJ, McLean, 2009). For instance, as addressed by Ogawa et al. (1993), dryness of the skin is an important component of the atopic diathesis, thereby reflecting possible skin barrier dysfunction. When the two abnormalities, dry skin/barrier dysfunction and allergy/immunological dysfunction, are considered as the major underlying defects of AD, the wide range of clinical manifestations seen in AD can be more easily comprehended. A defect of the mucocutaneous barrier readily allows penetration of multiple antigens or haptens, which enhances allergic inflammation. On the other hand, an allergic inflammation derived from the immunological abnormalities damages barrier functions. This sequence cycle could answer the question as to why AD patients show IgE production against, and contact hypersensitivity to, various antigens or haptens. A set of protective/defensive functions generated in the epidermis is likely mediated by its unique differentiation end product, the stratum corneum (Elias 2005; Elias & Choi, 2005). Basically, a markedly increased transepidermal water loss and a markedly decreased water holding capacity of the stratum corneum were reported in AD patients (Watanabe et al., 1991). In addition, since the patients showed a higher transepidermal water loss following irritant exposure, the susceptibility to irritants in AD patients seemed to be closely related with a breakdown in the barrier function of the stratum corneum (Tupker et al., 1990). More recently, it has been proposed that AD is a multifactorial, heterogenous disease that arises as a result of the interaction between both environmental and genetic factors (Cork et al., 2009). Changes in at least three groups of genes encoding structural proteins, epidermal proteases, and protease inhibitors make AD patients prone to a defective epidermal barrier, resulting in increased risk of developing AD. Loss-offunction mutations found within the FLG gene, which encodes the structural protein, filaggrin, could be the most significant genetic factor toward AD. In addition, enhanced protease activity and decreased synthesis of the lipid lamellae lead to exacerbated

Fungus as an Exacerbating Factor

hyperpermeability in the gastrointestinal mucosa.

**5. Conclusion** 

**6. References** 

0007-4888

6749

of Atopic Dermatitis, and Control of Fungi for the Remission of the Disease 149

Well-known representative fungi that exacerbate AD are the resident fungi in the skin, *Malassezia* spp. such as *M. furfur*, *M. globosa* and *M. restricta*, and the resident fungus in the intestinal tract, *C. albicans*. The lipophilic fungus *M. furfur* indigenously inhabits the seborrheic region of the body such as the face, cervical part, and upper part of back. It was also reported that the fungus may be implicated in rosacea-like dermatitis and edematous erythema, which are chronic and intractable symptoms characteristic to the face in adulttype AD. Regarding the underlying mechanism by which clinical manifestation of AD is affected in the presence of *M. furfur*, the following points have been proposed: 1) antigenspecific inflammation caused via activation of antigen-specific T cells, and 2) dysfunction of skin barrier. A defect of skin barrier readily allows penetration of multiple antigens or haptens, which enhances allergic inflammation, and vice versa. That is, an allergic inflammation derived from the immunological abnormalities damages barrier functions. This sequence cycle could answer the question as to why AD patients show IgE production against, and contact hypersensitivity to, various antigens or haptens. Gut colonization of *C. albicans* is also regarded as the other fungal factor exacerbating AD by promoting sensitization against food antigens, at least partly due to mast cell-mediated

Abeck, D. & Mempel, M. (1998). *Staphylococcus aureus* colonization in atopic dermatitis

Aizawa, T., Kano, R., Nakamura, Y., Watanabe, S. & Hasegawa, A. (2001). The genetic

*Medical Mycology*. Vol.39, No.4, (August 2001), pp.329-334, ISSN 1369-3786 Arzumanyan, G., Magarshak, O. & Semenov, B. (2000). Yeast fungi in patients with

Bäck, O., Scheynius, A. & Johansson, G. (1995). Ketoconazole in atopic dermatitis:

Bayrou, O., Pecquet, C., Flahault, A., Artigou, C., Abuaf, N. & Leynadier, F. (2005). Head

*Dermatology* Vol.211, No.2, (August 2005), pp.107-113, ISSN 1018-8665 Boguniewicz, M., Schmid-Grendelmeier, P. & Leung, Y. Atopic dermatitis. *The Journal of* 

*Immunology*, Vol.144, No.1, (April 2006), pp.1-9, (ISSN 0009-9104)

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and its therapeutic implications. *British Journal of Dermatology*, Vol.139,

diversity of clinical isolates of Malassezia pachydermatis from dogs and cats.

allergic diseases: species variety and sensitivity to antifungal drugs. *Bulletin of Experimental Biology and Medicine*, Vol.129, No.6, (June 2000) pp.601–604, ISSN

therapeutic response is correlated with decrease in serum IgE. *Archives of dermatological research*, Vol.287, No.5, (May 1995) pp.448-451, ISSN 0340-3696 Baker, S. (2006). The role of microorganisms in atopic dermatitis. *Clinical and Experimental* 

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breakdown of the epidermal barrier. It can be summarized that these functions include the permeability barrier, which prevents transcutaneous evaporative water loss, and an antimicrobial barrier, which simultaneously encourages colonization by nonpathogenic ''normal'' flora (Elias, 2007). According to the report by Selander et al. (2009), approximately 50% of adult AD patients have allergen-specific IgE reactivity to the skin commensal yeast *Malassezia* spp. Due to the ruptured skin barrier in AD, it is likely that *Malassezia* come into contact with mast cells, which are known to be involved in AD. Since mast cells are located in the superficial dermis close to blood vessels, they are advantageously positioned to react with allergens diffusing through a ruptured epidermis. They are, therefore, recognized as key effector cells during IgE-associated Th2 type immune responses (Galli et al., 2005), and cross-linking of the high-affinity IgE receptor (FcεRI) leads to release of potent inflammatory mediators (Turner & Kinet, 1999) such as histamine, proteases, chemotactic factors, cytokines, and metabolites of arachidonic acid (Henz et al., 2001). Mast cells have a wide variety of cell surface receptors that can interact directly with pathogens, including Toll-like receptors (TLRs), which are involved in innate immune recognition of invading microorganisms (Qiao et al., 2006). Fungal products such as zymosan can activate mast cells through TLR2 (Marshall, 2004). 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 (Qiao et al., 2006) (Fig. 3). Although both a defective epidermal permeability (Sugarman et al., 2003; Seidenari & Giusti, 1995; Proksch et al., 2006; Chamlin et al., 2002; Eberlein-Konig et al., 2000) and a propensity to secondary infection (Boguniewicz et al., 2006; Baker, 2006) are well-recognized features of AD, these abnormalities have been widely assumed to reflect downstream consequences of a primary immunologic abnormality.

Fig. 3. Skin barrier dysfunction in combination with skin indigenous *Malassezia* as an exacerbating factor in atopic dermatitis (AD). Due to the ruptured skin barrier in AD, it is likely that *Malassezia* and/or its products come into contact with mast cells which have a wide variety of cell surface receptors that interact directly with pathogens, including Tolllike receptors (TLRs). A synergistic activation between TLR and IgE receptor (Fcε RI) can occur in mast cells, resulting in increased production of inflammatory cytokines.
