Atopic dermatitis and skin disease
Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice

https://doi.org/10.1016/j.jaci.2012.01.068Get rights and content

Background

Loss-of-function mutations in filaggrin are major predisposing factors for atopic dermatitis. Although various reports suggest a critical role for filaggrin in stratum corneum (SC) barrier formation, the lack of filaggrin-null (Flg−/−) mice has hampered detailed in vivo analysis of filaggrin’s functions.

Objective

We sought to generate Flg−/− mice and to assess the effect of filaggrin loss on SC barrier function and percutaneous immune responses.

Methods

We generated Flg−/− mice using gene targeting and assessed the morphology, hydration, mechanical strength, and antigen permeability of their SC. Percutaneous immune responses were evaluated through irritant- and hapten-induced contact hypersensitivity studies and by measuring humoral responses to epicutaneous sensitization with protein antigen.

Results

Newborn Flg−/− mice exhibited dry scaly skin. Despite marked decreases in natural moisturizing factor levels, which are filaggrin degradation products, SC hydration and transepidermal water loss were normal. Microscopic analyses suggested premature shedding of SC layers, and indeed, increased desquamation under mechanical stress was demonstrated. Loss of keratin patterns, which are critical for corneocyte stabilization, is likely attributable to fragility in the Flg−/− SC. Antigens penetrated the Flg−/− SC more efficiently, leading to enhanced responses in hapten-induced contact hypersensitivity and higher serum levels of anti-ovalbumin IgG1 and IgE.

Conclusion

Complete filaggrin deficiency led to altered barrier integrity and enhanced sensitization, which are important factors in early-phase atopic dermatitis. Flg−/− mice should provide a valuable tool to further explore additional factors the dysfunction of which leads to uncontrolled inflammation in patients with atopic diseases.

Section snippets

Generation of Flg−/− mice

Flg, which is located on chromosome 3, consists of 3 exons and shares unique characteristics with its human counterpart. Exon 1 consists of 5′ untranslated region (UTR) sequences, exon 2 contains the translation start site, and the unusually large exon 3 encodes nearly identical filaggrin repeats (see Fig E1, A, in this article’s Online Repository at www.jacionline.org). Large open reading frames and repetitive sequences complicate the strategy by which the genes are targeted. Nevertheless, we

Generation of Flg−/− mice

Flg−/− mice were generated by means of homologous recombination (see the Methods section and Fig E1 in this article’s Online Repository). Mice were backcrossed to both C57BL/6 and BALB/c backgrounds to minimize phenotypic variation. Immunoblotting and immunohistochemical analyses revealed that Flg-targeted mice were completely deficient for both profilaggrin and filaggrin (Fig 1), which is in contrast to ft/ma mice, in which abundant amounts of truncated profilaggrin and low but detectable

Discussion

Filaggrin has been demonstrated to function in SC integrity as a structural protein that organizes keratin filaments.6 It is also a major source of the hygroscopic amino acids in SC referred to as NMFs.8 NMFs are believed to maintain SC hydration levels.8 Although the biology and functions of filaggrin have been modeled through in vitro experiments, its in vivo functions have remained elusive because of the absence of mice that completely and specifically lack filaggrin. This was due to

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    Supported by a Health Labour Sciences Research Grant for Research on Allergic Diseases and Immunology from the Ministry of Health, Labour and Welfare of Japan; Grants-in-Aid for Scientific Research; the “Promotion of Environmental Improvement for Independence of Young Researchers” program and a Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Research Grants for Life Sciences and Medicine from Keio University Medical Science Fund; and a Keio University Grant-in-Aid for Encouragement of Young Medical Scientists.

    Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

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