Fungus as an Exacerbating Factor of Atopic Dermatitis, and Control of Fungi for the Remission of the Disease

Atopic dermatitis (AD) is a common, chronic fluctuating skin disease with prevalence in children (Williams, 2000; Williams & Wuthrich, 2000). The disease is an inflammatory skin disorder characterized by itching, and chronically relapsing course. Moreover, it also produces vulnerablity to surface infections caused by pathogenic bacteria, fungi and viruses. The most common skin infections in AD patients are caused by Staphylococcus aureus and herpes simplex virus (Ong & Leung, 2010). S. aureus is frequently detected in AD patients (Abeck & Mempel, 1998; Katsarou & Armenaka, 2011) and becomes an aggravating factor. In addition, toxins, such as staphylococcal enterotoxins and toxic shock syndrome toxin-1 (McFadden et al., 1993; Bunikowski et al., 1999), generated from S. aureus may act as superantigens (Herz et al., 1999; Niebuhr et al., 2011; Yeung et al., 2011). In AD patients, viral infection is most often caused by herpes simplex virus (HSV) (Wollenberg et al., 2003). Eczema herpeticum is a potentially life-threatening disseminated HSV type 1 or type 2 infection that occurs in 10% to 20% of AD patients (Peng et al., 2007). However, not only bacteria and viruses but also fungi, such as Malassezia species and Candida species, may play an important role as aggravation factors in AD patients. It has been reported that antifungal therapy is beneficial in the treatment of some AD patients (Back et al. 1995; Svejgaard et al. 2004; Broberg et al. 1995; Mayser et al., 2006). In addition, several candidate Malassezia antigens have been implicated in the pathogenesis of AD. In this chapter, the involvement of fungi in the pathogenesis of AD is discussed.


Introduction
Atopic dermatitis (AD) is a common, chronic fluctuating skin disease with prevalence in children (Williams, 2000;Williams & Wüthrich, 2000).The disease is an inflammatory skin disorder characterized by itching, and chronically relapsing course.Moreover, it also produces vulnerablity to surface infections caused by pathogenic bacteria, fungi and viruses.The most common skin infections in AD patients are caused by Staphylococcus aureus and herpes simplex virus (Ong & Leung, 2010).S. aureus is frequently detected in AD patients (Abeck & Mempel, 1998;Katsarou & Armenaka, 2011) and becomes an aggravating factor.In addition, toxins, such as staphylococcal enterotoxins and toxic shock syndrome toxin-1 (McFadden et al., 1993;Bunikowski et al., 1999), generated from S. aureus may act as superantigens (Herz et al., 1999;Niebuhr et al., 2011;Yeung et al., 2011).In AD patients, viral infection is most often caused by herpes simplex virus (HSV) (Wollenberg et al., 2003).Eczema herpeticum is a potentially life-threatening disseminated HSV type 1 or type 2 infection that occurs in 10% to 20% of AD patients (Peng et al., 2007).However, not only bacteria and viruses but also fungi, such as Malassezia species and Candida species, may play an important role as aggravation factors in AD patients.It has been reported that antifungal therapy is beneficial in the treatment of some AD patients (Bäck et al. 1995;Svejgaard et al. 2004;Broberg et al. 1995;Mayser et al., 2006).In addition, several candidate Malassezia antigens have been implicated in the pathogenesis of AD.In this chapter, the involvement of fungi in the pathogenesis of AD is discussed.

Fungi isolated from AD patients and treatment
The genus Malassezia has recently been shown to consist of fifteen species based on the database of National Center for Biotechnology Information (2011), one lipid-independent species, M. pachydermatis and fourteen lipid-dependent species, M. sympodialis, M. furfur, M. globosa, M. obtusa, M. restricta, M. slooffiae, M. caprae, M. equine, M. dermatis, M. equi, M. japonica, M. nana, M. yamatoensis and M. cuniculi.Malassezia species have been recognized as members of the microbiological flora of human and animal skin.M. globosa and M. restricta are frequently isolated from the skin scales of human AD (Sugita et al., 2001;Tajima et al., 2008;Kaga et al., 2009) and M. pachydermatis and M. nana are isolated from some animals (Aizawa et al., 2001;Hirai et al., 2004).Antifungal drugs, e.g.ketoconazole and itraconazole, are used in AD patients with signs of a fungal infection (Sugita et al., 2005;Bäck et al., 1995).Antifungal therapy may remit the severity of AD by controlling these Malassezia yeasts.

Related pathogenic fungi
The yeasts of the genus Malassezia are members of the normal cutaneous flora.However, Malassezia colonization on the skin of AD patients shows a different pattern from that on healthy skin (Faergemann, 2002;Gupta et al., 2001;Nakabayashi et al., 2000;Sandström et al., 2005;Sugita et al., 2004Sugita et al., , 2006) ) and may aggravate AD due to an allergic reaction, especially on the head and neck area in adults (Brehler & Luger, 2001;Broberg et al., 1992;Faergemann, 1999;Huang et al., 1995;Jensen-Jarolim et al., 1992;Lintu et al., 1997;Rokugo et al., 1990;Schmidt et al., 1997;Nakabayashi et al., 2000;Savolainen et al., 2001;Scalabrin et al., 1999).Scalabrin et al. (1999) measured total IgE and specific IgE to Malassezia furfur in 73 AD patients.In the AD patients, specific IgE to M. furfur was observed more frequently in adults than children.The reaction of specific IgE to M. furfur was 132 times higher than that in healthy subjects.This result suggests that Malassezia yeast is associated with IgE-mediated skin inflammation in AD.Culture-dependent methods have been used for the detection of Malassezia species from AD patients (Nakabayashi et al., 2000;Sandström et al., 2005).However, in recent years, many researchers have attempted the detection of Malassezia species from AD patients by means of a molecular-based culture-independent method that is not affected by the isolation medium, sampling method, or incubation period.Table 1 summarizes the three major studies applying molecular based PCR assay to detect Malassezia species from AD patients and healthy subjects, indicating that the number of detected Malassetia species was similar to AD patients and healthy subjects (Sugita et al., 2001;Tajima et al., 2008;Kaga et al., 2009).

Species
Sugita et al. control dogs (without Malassezia dermatitis or otitis) did not differ significantly from those in atopic dogs with Malassezia dermatitis.No significant correlation was found between the lymphocyte blastogenic response and the type-1 hypersensitivity response to M. pachydermatis within any of the groups, suggesting that modification of the dysregulated immune response toward M. pachydermatis may assist in the reduction of pathologic changes associated with an AD phenotype in dogs.In another study, Chen et al. (2002) compared IgE responses to separated proteins of M. pachydermatis in atopic dogs with Malassezia dermatitis and clinically normal dogs.The results of their study showed that the majority of atopic dogs with Malassezia dermatitis have a greater IgE response than normal dogs, suggesting an IgE-mediated immune response may be clinically important in the pathogenesis of the disease.In felines, Malassezia spp.have been more frequently isolated from healthy ear canals and skin in feline leukaemia (FeLV)-or feline immunodeficiency virus (FIV)-infected cats than in those noninfected (Sierra et al., 2000).In addition, Malassezia spp.overgrowth has been described in feline localized benign exfoliative skin diseases, such as chin acne and the idiopathic facial dermatitis of Persian cats (Jazic et al., 2006;Bond et al., 2000).Based on these findings, Ordeix et al. (2007) conducted a multicentre, retrospective and descriptive study to document Malassezia spp.overgrowth in allergic cats.Their results suggested that Malassezia spp.overgrowth may represent a secondary cutaneous problem in allergic cats particularly in those with greasy adherent brownish scales on their skin.The favorable response to treatment with antifungal agent alone suggests that, as in dogs, Malassezia spp.may be partly responsible for both pruritus and cutaneous lesions in allergic cats.

Mechanisms by which fungi act as an exacerbating factor for atopic dermatitis 4.1 Antigen-specific inflammation caused via activation of antigen-specific T cells
Allergy to fungi such as Candida spp.and Malassezia spp.has been implicated as an exacerbating or intractable factor in the symptoms of AD (Savolainen et al., 1993;Tanaka et al., 1994;Kitamura et al., 1997;Morita et al., 1999;Linder et al., 2000;Faergemann 2002;Kanda et al., 2002;Svejgaard et al., 2004).Candida spp.are indigenous fungi inhabiting the oral cavity, digestive tract and vagina.Healthy people are thought to acquire the Th1 type immunity against Candida spp.(Tanaka et al., 1994;Romani et al., 1995).For instance, the activation of Th1-type CD4+ cell induces phagocyte-dependent immunity, which apparently represents an important mechanism of anti-Candida resistance, and it was demonstrated that healthy subjects with a normal immune response show high peripheral blood lymphocyte proliferative responses as well as positive scarification patch tests to C. albicans antigen, suggesting the dominant presence of Thl type T cells specific to C. albicans antigen.It is well known that Thl clones secrete IL-2 and IFN-γ and preferentially induce delayed type hypersensitivity (Stout & Bottomly, 1989), while Th2 clones produce IL-4, IL-5 (Mosmann et al., 1986) and IL-10 (Fiorentino et al., 1989) and help to promote IgE production (Boom et al., 1988;Killar et al., 1987).In AD patients, Th1-type immunity has been shown to shift to Th2type (Fig. 1) since the patients immediately react to skin testing using Candida-antigen (Tanaka et al., 1994;Kitamura et al., 1997), and Candida-specific IgE increases with the severity of the symptoms of AD (Tanaka et al., 1994).Specifically, AD patients displayed a significantly lower incidence of positive patch test reactions to C. albicans allergen than the healthy control subjects, and the patients with negative C. albicans patch tests tended to have higher levels of total serum IgE including anti-C.albicans IgE antibody.In other words, the delayed-type hypersensitivity to C. albicans antigen, which is highly prevalent in atopics without dermatitis as well as non-atopics, was reduced in most of the AD patients.The lipophilic fungus M. furfur indigenously inhabits the seborrheic region of the body, such as head, neck and upper part of the back.It was also reported that the fungus may be implicated in rosacea-like dermatitis and edematous erythema, which are chromic and intractable symptoms characteristic to the face with adult-type AD (Mukai et al., 1997), and that Malassezia-specific IgE level is high in the head and neck of AD patients (Bayrou et al., 2005;Darabi et al., 2009).Regarding the 11 currently recognized Malassezia species as an exacerbating factor in AD, M. globosa and M. restricta are found to frequently colonize the skin of AD patients.For instance, specific IgE antibodies against eight Malassezia species (M.dermatitis, M. furfur, M. globosa, M. obtusa, M. pachydermatis, M. slooffiae, M. sympodialis, and M. restricta) i n s e r a f r o m A D p a t i e n t s w e r e e x a m i n e d u s i n g a n e n z y m e -l i n k e d immunosorbent assay, and it was found that the specific IgE value against M. restricta was greater than those against the other Malassezia species (Kato et al., 2006).

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 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.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.

Skin barrier dysfunction
Skin barrier dysfunction (Ogawa et al., 1993;Cork et al., 2006Cork et al., & 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 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 Th2type 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.

Conclusion
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 i s a l s o r e g a r d e d a s t h e o t h e r f u n g a l f a c t o r e x a c e r b a t i n g A D b y p r o m o t i n g sensitization against food antigens, at least partly due to mast cell-mediated hyperpermeability in the gastrointestinal mucosa.

Fig. 1 .
Fig. 1.Shift of Th1-type immunity to Th2-type immunity in allergic diseases including atopic dermatitis (AD).In healthy individuals, dendritic cells present fungal antigen to naive T cells which in turn differentiate to Th1 type cells, resulting in the cellular immune response.In AD patients, Th1-type immunity shifts to Th2-type immunity in which Th2 clones produce IL-4, IL-5 and IL-10 and induce IgE production.

Fig. 2 .
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.

Fig. 3 .
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.