**3.6 Flaky tail mouse denotes human AD**

12 Atopic Dermatitis – Disease Etiology and Clinical Management

Fig. 6. Amount of FITC in the skin of B6 and *Flgft* mice (left panel) and fluorescence

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

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

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

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

intensities of FITC of the skin (right panel) after topical application.

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

nonatopic individuals is still controversial (Mailhol, et al., 2009).

the ear of *Flgft* mice was higher than that of B6 mice.

at a possible mechanism involved in human AD.

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

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.

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

clarify these relationships.

**5. References** 

needs to be clarified in future studies.

25), 1225-1232

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*Med,* 206, 5, (May 11), 1135-1147

Possible Roles of Filaggrin in the Development of Atopic Dermatitis 15

Since *Flgft* mice are not a homogenous C57BL/6 background, two papers with spontaneous eczematous skin lesion on *Flgft* mice compared their outcomes with other mouse strains, such as C57BL6 and BALB/c mice as controls (Oyoshi, et al., 2009); these two strains lie on opposite ends of the spectrum of T helper responses. Nevertheless, the skin inflammation and susceptibility to EC sensitization of non-tape stripped skin observed in *Flgft* mice were not observed in other strains. In the second paper, they observed immune responses in mice of other genotypes, such as BALB/c and C3H, as controls, but both of these lines exhibited much less severe CHS responses compared to *Flgft* mice (Moniaga, et al., 2010). These data suggested that the enhanced responses seen in *Flgft* mice were not solely due to their genetic background. In addition, other studies used the *Flgft* mice which were backcrossed four generations to a B6 strain (a background coding sequence showed 99.3% identity between B6 and *Flgft)*, and similar enhanced

Furthermore, unlike human AD patients, most of whom are heterozygous for the *FLG* mutation, the heterozygous mice intercrossed with *Flgft* mice and B6 mice did not develop spontaneous dermatitis (Moniaga, et al., 2010). Similar results were obtained with the OVA-induced AD model, where homozygous, but not heterozygous (crossed with B6 mice) *Flgft* mice, showed enhanced susceptibility to cutaneous exposure to OVA (Fallon, et al., 2009). Not only human studies but also additional mouse studies will be required to

Since *Flgft* mice express a hair phenotype (matted), one cannot eliminate the possibility that some of the observations could have been influenced by the concurrent *ma* mutation (Scharschmidt, et al., 2009). Nevertheless, one study indeed removed the matted hair allele (*ma*) early in the course of backcrossing with B6 mice, and showed enhanced antigen (OVA) ingress in mice with the same *Flg* mutation, but no *ma* mutation in their background (Fallon, et al., 2009). The effect of the *ma* mutation in relation to the *Flg*  mutation in commercially available *Flgft* mice in the development of AD-like skin lesions

(1998). Worldwide variation in prevalence of symptoms of asthma, allergic

Aioi, A., et al. (2001). Impairment of skin barrier function in NC/Nga Tnd mice as a possible

Angelova-Fischer, I., et al. (2011). Distinct barrier integrity phenotypes in filaggrin-related

Ardern-Jones, M. R., et al. (2007). Bacterial superantigen facilitates epithelial presentation of allergen to T helper 2 cells. *Proc Natl Acad Sci U S A,* 104, 13, (Mar 27), 5557-5562

Briot, A., et al. (2009). Kallikrein 5 induces atopic dermatitis-like lesions through PAR2-

model for atopic dermatitis. *Br J Dermatol,* 144, 1, (Jan), 12-18

Bieber, T. (2008). Atopic dermatitis. *N Engl J Med,* 358, 14, (Apr 3), 1483-1494

rhinoconjunctivitis, and atopic eczema: ISAAC. The International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee. *Lancet,* 351, 9111, (Apr

atopic eczema following sequential tape stripping and lipid profiling. *Exp Dermatol,* 

mediated thymic stromal lymphopoietin expression in Netherton syndrome. *J Exp* 

responses to OVA-induced AD models were observed (Fallon, et al., 2009).

Histological examination of H&E-stained sections of involved *Flgft* skin after 16 applications showed acanthosis, elongation of rete ridges, and dense lymphocyte and neutrophil infiltration in the dermis, accompanied by an increased number of mast cells in the dermis. Consistently, scratching behavior, TEWL, and Dp-specific IgE levels were significantly higher in *Flgft* mice than in B6 mice (Fig.8) (Moniaga, et al., 2010). Thus the treatment of *Flgft* mice with Dp ointment, even without prior barrier disruption, remarkably enhanced both the clinical manifestations and the laboratory findings that correspond to indicators of human AD.

Fig. 8. TEWL and mite-specific serum IgE levels of *Flgft* mice and control mice after the last application.
