**3.4 Flaky tail mouse showed enhanced percutaneous allergen priming**

Since the barrier dysfunction is a key element in the establishment of AD, it is necessary to evaluate outside-to-inside barrier function from the perspective of invasion of external stimuli. Scharschmidt et al. reported increased bidirectional paracellular permeability of water-soluble xenobiotes by ultrastructural visualization in *Flgft* mice suggesting a defect in the outside-to-inside barrier. The ultrastructural visualization of tracer perfusion was analyzed by water-soluble, low molecular weight, electron-dense tracer lanthanum nitrate or fluorophore calcium green with enhanced penetration in *Flgft* mice. The data demonstrated that filaggrin deficiency leads to alterations in basal barrier function through a defect in the SC extracellular matrix and greater permeability through the same paracellular pathway that is used by water itself when exiting the skin (Scharschmidt, et al., 2009).

A new method for evaluating outside-to-inside barrier function quantitatively by measuring the penetrance of fluorescein isothiocyanate isomer 1 (FITC) through the skin has been developed (Moniaga, et al., 2010). The epidermis of *Flgft* mice contained a higher amount of FITC than that of B6 mice did (Fig.6 left panel). Consistently, fluorescence intensities observation in the epidermis of both mice showed stronger fluorescence in *Flgft* mice (Fig.6 right panel). In addition, the *Flgft* embryo was entirely dye permeable to toluidine blue solution compared to its control littermate.

Another AD-like dermatitis model to test allergen priming of the skin in these mice was performed by application of ovalbumin (OVA) (Oyoshi, et al., 2009). Non tape-stripped skin of *Flgft* mice exposed to OVA exhibited significantly increased epidermal thickening, hyperkeratosis, spongiosis, acanthosis, and cellular infiltrates, as well as TEWL compared to control mice. mRNA levels for IL-17, IL-6, IL-23, IL-4, IFN-γ and CXCL2 but not IL-5 and IL-13 in the skin of *Flgft* mice after OVA exposure were significantly higher than those of control mice. The systemic immune response following cutaneous exposure revealed increased specific IgG and IgE to OVA, and splenocytes proliferated and produced OVAspecific Th1, Th2, Th17 and regulatory T cell cytokines (Fallon, et al., 2009, Oyoshi, et al., 2009). These findings demonstrate that *Flgft* mice tend to generate allergen-specific IgE and cytokine following cutaneous allergen challenge to the skin even without additional barrier disruption.

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

ointment as a control, induced no skin manifestation (Fig. 7).

histological changes (Scharschmidt, et al., 2009).

**3.6 Flaky tail mouse denotes human AD** 

Possible Roles of Filaggrin in the Development of Atopic Dermatitis 13

which provokes only marginal inflammation in wild-type mice, and displayed a reduced threshold for the development of hapten-induced acute allergic contact dermatitis by oxazolone (Ox). Repeated Ox challenges with lower doses of Ox revealed AD-like dermatitis in *Flgft* mice as shown by severe barrier abnormality (enhanced TEWL) and AD-like

Clinical studies have provided evidence that a house dust mite allergen plays a causative or exacerbating role in human AD (Kimura, et al., 1998), and that a strong correlation exists between *FLG* mutation patients and house dust mite-specific IgE (Henderson, et al., 2008). *Dermatophagoides pteronyssinus* (Dp) is a common mite aeroallergen, which is frequently involved in inducing human AD. Dp exhibits protease activities, and Der p1, Der p3, and Der p9, derived from Dp, are especially capable of activating the PAR-2 in human KC (Jeong, et al., 2008, Vasilopoulos, et al., 2007). A recent report has shown that activation of PAR-2 through Dp application significantly delays barrier recovery rate in barrier functionperturbed skin or otherwise compromised skin (Jeong, et al., 2008). Therefore, Dp may play a dual role in the onset of AD, both as an allergen and proteolytic signal and as a perturbation factor of the barrier function, leading to the persistence of eczematous skin lesions in AD (Jeong, et al., 2008, Roelandt, et al., 2008). It has also been reported that BALB/c and NC/Nga mice develop an allergic cutaneous immune response to mite antigens when they are applied to the skin after vigorous barrier disruption by means of tape-stripping or sodium dodecyl sulfate treatment (Kang, et al., 2006, Yamamoto, et al., 2007). Intriguingly, the application of Dp ointment to the skin without additional barrier disrupt induced dermatitis in *Flgft* mice, while this treatment did not induce any skin inflammation in control C57BL/6 mice (Fig.7). Petrolatum alone, used instead of Dp

Fig. 7. The mite-induced dermatitis model showed severe eczematous skin lesion after being

topically treated with Dp ointment in *Flgft* mice, as well as ear thickness change.

Fig. 6. Amount of FITC in the skin of B6 and *Flgft* mice (left panel) and fluorescence intensities of FITC of the skin (right panel) after topical application.

#### **3.5 Altered immunobiology response in flaky tail mouse**

The skin abnormality associated with AD is well known to be a predisposing factor to sensitive skin (Farage, et al., 2006, Willis, et al., 2001) and allergic contact dermatitis (Clayton, et al., 2006, Mailhol, et al., 2009). However, children with atopic dermatitis had lower PPD induration size compared to healthy donors, but this was not statistically significant (Gruber, et al., 2001, Yilmaz, et al., 2000). In humans, sensitive skin is defined as reduced tolerance to cutaneous stimulation, with symptoms ranging from visible signs of irritation to subjective neurosensory discomfort (Farage, et al., 2006, Willis, et al., 2001). The question of whether human AD patients are more prone to allergic contact dermatitis than nonatopic individuals is still controversial (Mailhol, et al., 2009).

Using phorbol myristate acetate (PMA) as an irritant, *Flgft* mice exhibited an enhanced ear swelling response compared to age-matched B6 mice throughout the experimental period (1 hr to 140 hrs). In addition, *Flgft* mice showed an increased skin-sensitized contact hypersensitivity (CHS) reaction to hapten, a form of classic Th1- and Tc1-mediated delayedtype hypersensitivity to haptens, emphasized by increased IFN-γ production, and terminated by regulatory T cells (Honda, et al., 2010, Mori, et al., 2008, Wang, et al., 2001). CHS is induced by epicutaneous sensitization and challenge. The ear thickness change was more prominent in *Flgft* mice than in B6 mice. In addition, the relative amount of IFN-γ in the ear of *Flgft* mice was higher than that of B6 mice.

To further assess the immune responses of *Flgft* mice, we elicited a delayed-type hypersensitivity (DTH) response through non-epicutaneous sensitization and challenge. Mice were immunized intraperitoneally with OVA, and challenged with a subcutaneous injection of OVA into the footpad. In contrast to the CHS response induced epicutaneously, the resulting footpad swelling in *Flgft* mice tended to be lower than that in wild-type mice. This finding is consistent with the observation on tuberculin tests in human. The levels of IFN-γ in the spleen were comparable between *Flgft* mice and wild-type mice. Thus, Th1/Tc1 immune responses were enhanced in *Flgft* mice only when the stimuli operated via the skin, suggesting that the enhanced immune responses seen in *Flgft* mice depend on skin barrier dysfunction and skin barrier function regulates cutaneous immune conditions, which hints at a possible mechanism involved in human AD.

A reduced threshold in *Flgft* mice for contact dermatitis was also reported. These mice showed enhanced propensity to irritant contact dermatitis via low-dose phorbol ester TPA which provokes only marginal inflammation in wild-type mice, and displayed a reduced threshold for the development of hapten-induced acute allergic contact dermatitis by oxazolone (Ox). Repeated Ox challenges with lower doses of Ox revealed AD-like dermatitis in *Flgft* mice as shown by severe barrier abnormality (enhanced TEWL) and AD-like histological changes (Scharschmidt, et al., 2009).
