**2.2 Filaggrin and altered skin barrier function**

AD is characterized by eczematous skin lesion, dry skin, pruritus, increased TEWL, and enhanced percutaneous penetration of both lipophilic and hydrophilic compounds (Jakasa, et al., 2011, Wollenberg &Bieber, 2000). The skin barrier defect is one of the primary events that initiate disease pathogenesis, allowing the entrance of numerous antigens into the epidermis in patients with AD (Onoue, et al., 2009, Osawa, et al., 2011). The *FLG* mutation carriers have demonstrated elevated TEWL (Jungersted, et al., 2010, Kezic, et al., 2008), basal erythema, skin hydration, increased skin pH (Jungersted, et al., 2010, Nemoto-Hasebe, et al., 2009), SC thickness (Nemoto-Hasebe, et al., 2009), impaired SC integrity upon repeated tape stripping (Angelova-Fischer, et al., 2011), and increased diffusivity of PEG 370 (Jakasa, et al., 2011) compared to healthy donors. Nevertheless, these alterations found in *FLG* mutation carriers are not consistently correlated with AD since AD patients without *FLG* mutation might also share some similar features. (Hubiche, et al., 2007, Jakasa, et al., 2011, Jungersted, et al., 2010, Kezic, et al., 2008). It is, therefore, suggested that other factors besides *FLG* lossof-function mutations modulate skin barrier integrity, especially in AD.

Since the skin barrier is related to intercellular lipid bilayers of the SC, it might be interesting to examine the composition and the organization of intercellular lipids of the SC in AD patients in relation to *FLG* genotype and disease severity (Jakasa, et al., 2011). Carriers of *FLG* mutations showed significantly reduced levels of NMF in the SC (Kezic, et al., 2008). Similar lipid composition of *FLG* mutation carriers and individuals with normal filaggrin was observed (Angelova-Fischer, et al., 2011, Jungersted, et al., 2010), but a lower cermide/cholesterol ratio was detected in the former group (Angelova-Fischer, et al., 2011). Filaggrins proteolytically degraded into a pool of free amino acids including histidine and glutamine which are further converted to, respectively, urocanic acid (UCA) and 2-

Flaky Tail Mouse as a Novel Animal Model of Atopic Dermatitis:

**3.1 Origin of flaky tail mice** 

Presland, et al., 2000).

copies of the filaggrin repeat (Fallon, et al., 2009).

two to four weeks (Jarret A, 1957, Searle A.G., 1957).

et al., 2010, Presland, et al., 2000) (Fig.1).

Possible Roles of Filaggrin in the Development of Atopic Dermatitis 7

same result could be seen among HLA-DRB1\*1501 (a HLA class II complex which is immunodominant in individuals with AD (Ardern-Jones, et al., 2007)) positive adult individuals with moderate-to-severe AD and *FLG* mutations (McPherson, et al., 2010).

The above findings indicate the involvement of filaggrin in the development of AD. Therefore, the impact of filaggrin deficiency on cutaneous biological functions *in vivo* should

Flaky tail mice (*Flgft*), first introduced in 1958, are spontaneously mutated mice with smaller ears, tail constrictions, and a flaking tail skin appearance (Lane, 1972). *Flgft* mice were outcrossed onto B6 mice at Jackson Laboratory (Bar Harbor, ME, USA) (Lane, 1972, Presland, et al., 2000) (Note: Although this strain was crossed with B6, it is not a B6 congenic strain but rather a hybrid stock that is probably semi-inbred). Homozygous *Flgft* mice have dry, flaky skin which expresses reduced amounts of profilaggrin mRNA and abnormal profilaggrin protein that is not processed to filaggrin monomers (Fallon, et al., 2009,

Recently, it has been revealed that the gene responsible for the characteristic phenotype of *Flgft* mice is a single nucleotide deletion at position 5303 in exon 3 (5303delA) of the profilaggrin gene, resulting in a frameshift mutation and premature truncation of the predicted protein product. The copy number of the filaggrin repeat contained within this gene varies depending on the background strain. This mutant occurs in an allele with 16

*Flgft* mouse carries double gene mutation, *Flg* and matted (*ma*) in which the locations of the mutated genes are within close linkage to one another (Lane, 1972). The *ma* gene characteristic reported by Searle & Spearman (1957) causes the body-hair of affected mice to be brittle and inflexible, which results in longitudinal splitting and breaking due to friction against the cage and other objects. This mutation is a fully penetrant recessive house-mouse mutant which belongs to the "naked" category (i.e., a house-mouse with baldness resulting from the breaking of hairs or from hereditary hairlessness). This mutation can be identified morphologically by (1) erection of hairs, (2) matting of hair in clumps, (3) a tendency towards baldness, (4) a change from black- to brown-colored melanin in old hairs. The age at which this mutant is first identified based on external appearance varies from between

Recognition of the features of this mouse is more evident between 5 and 14 days of age when constricted, flaking tail skin and thickened short pinna of the ears are observed. In addition, *Flgft* mice are often smaller than their normal siblings at this age. Routine histological sections stained with hematoxylin and eosin showed that the stratum granulosum in *Flgft* mice at 1, 2, 4, and 8 days of age does not contain as many granular layers as that of non-*Flgft* mice (Lane, 1972). Mice of the *Flgft* genotype express an abnormal profilaggrin polypeptide that does not form normal keratohyalin F-granules and is not proteolytically processed to filaggrin. Therefore, filaggrin is absent from the cornified layers in the epidermis of the *Flgft* mouse (Fallon, et al., 2009, Presland, et al., 2000, Scharschmidt, et al., 2009). Consistently, we and others have described that *Flgft* mice express a truncated and smaller profilaggrin protein that is not processed to filaggrin (Fallon, et al., 2009, Moniaga,

**3. Flaky tail mouse as a novel animal model of atopic dermatitis** 

be analyzed in detail. To address this issue, animal models are of great value.

pyrrolidone-5-carboxylic acid (PCA). The concentrations of UCA and PCA in SC in the carriers of *FLG* mutations were significantly lower than those in healthy donors (Kezic, et al., 2009). Therefore, filaggrin deficiency is sufficient to impair epidermal barrier formation.

An *in vitro* experiment using filaggrin knocked down human organotypic skin cultures showed enhanced penetration of hydrophilic dye Lucifer yellow, smaller lamellar bodies, and deficiency of their typical lamellae without altered lipid composition (Mildner, et al., 2010). In addition, UCA, one of the filaggrin-derived free amino acids and as an important UV absorbent within SC, was decreased following filaggrin knocked down, leading to increased sensitivity to UVB-induced keratinocyte (KC) damage (Mildner, et al., 2010).

#### **2.3 Filaggrin and altered immunobiology**

The SC serves as a biosensor of the external environment and a link between innate and adaptive immune systems (Vroling, et al., 2008). The critical association between the abnormal barrier in AD and Th2 polarization may in part be explained by the production of the cytokine, thymic stromal lymphopoietin (TSLP) (Ebner, et al., 2007). TSLP is expressed by epithelial cells, with the highest levels seen in lung-derived and skin-derived epithelial cells (Soumelis, et al., 2002, Ziegler, 2010), and is highly detected in the lesional skin of AD (Soumelis, et al., 2002) Inducible TSLP transgene specifically in the skin leads to the development of a spontaneous Th2-type skin inflammatory disease with the hallmark features of AD (Yoo, et al., 2005).

TSLP has been shown to activate dendritic cells to drive Th2 polarization, through upregulation of the co-stimulatory molecules CD40, CD80, and OX40L, triggering the differentiation of allergen-specific naïve CD4+ T cells to Th2 cells that produce IL-4, IL-5, and IL-13 (Ebner, et al., 2007, Soumelis, et al., 2002).

Patients with Netherton syndrome (NS), a severe ichthyosis in which affected individuals experience a significant predisposition for AD, have elevated levels of TSLP in their skin. Upregulated kallikrein (KLK) 5 in the skin of NS patients directly activates proteinaseactivated receptor 2 (PAR-2) and induces nuclear factor kappaB-mediated overexpression of TSLP, intercellular adhesion molecule 1, TNF-α, and IL-8. This phenomenon occurs independently of the environment, adaptive immune system and underlying epithelial barrier defect (Briot, et al., 2009, Briot, et al., 2010). *In vitro* study using human keratinocyte cell line HaCaT cells and reconstituted human epidermal layers transfected with filaggrin siRNA showed increased production of TSLP via toll-like receptor (TLR) 3 stimulation (Lee, et al., 2011). These findings suggest that reduced filaggrin levels may influence innate immune response via TLR stimuli and elevate TSLP, leading to AD-like skin lesions.

AD is one of the emerging diseases in which epidermal dysfunction increases allergen and microbial penetration in the skin, with the consequent development of adaptive Th2 immune responses (Kondo, et al., 1998) within regional lymphoid tissue. The resultant Th2 cells may then home back to the skin or lungs, where they recognize allergen in the skin (McPherson, et al., 2010), which leads to local Th2 inflammation, reduced antimicrobial peptide expression (Nomura, et al., 2003), and filaggrin downregulation (Howell, et al., 2007). Indeed, the induction of circulating allergen-specific CD4+ T cells may be an important prerequisite underlying the pathogenesis of the atopic march (O'Regan, et al., 2009). Among moderate-to-severe AD patients, the *FLG* mutation carriers showed a greater number of house dust mite Der p1-specific IL-4 producing CD4+ T cells, suggesting that filaggrin mutations predispose to the development of allergen-specific CD4+ Th2 cells. The same result could be seen among HLA-DRB1\*1501 (a HLA class II complex which is immunodominant in individuals with AD (Ardern-Jones, et al., 2007)) positive adult individuals with moderate-to-severe AD and *FLG* mutations (McPherson, et al., 2010).
