A homozygous frameshift mutation in the mouse Flg gene facilitates enhanced percutaneous allergen priming

Abstract

Loss-of-function mutations in the FLG (filaggrin) gene cause the semidominant keratinizing disorder ichthyosis vulgaris and convey major genetic risk for atopic dermatitis (eczema), eczema-associated asthma and other allergic phenotypes. Several low-frequency FLG null alleles occur in Europeans and Asians, with a cumulative frequency of approximately 9% in Europe. Here we report a 1-bp deletion mutation, 5303delA, analogous to common human FLG mutations, within the murine Flg gene in the spontaneous mouse mutant flaky tail (ft). We demonstrate that topical application of allergen to mice homozygous for this mutation results in cutaneous inflammatory infiltrates and enhanced cutaneous allergen priming with development of allergen-specific antibody responses. These data validate flaky tail as a useful model of filaggrin deficiency and provide experimental evidence for the hypothesis that antigen transfer through a defective epidermal barrier is a key mechanism underlying elevated IgE sensitization and initiation of cutaneous inflammation in humans with filaggrin-related atopic disease.

Figure 1. Murine filaggrin gene structure and mutation identification

(a) The mouse filaggrin gene (flg) resides within a cluster of 7 genes encoding closely related fused-S100 proteins, spanning ~350 kb on murine chromosomal band 3QF21. Like the human ortholog, flg has a 3 exon gene structure. Exon 1 consists of 5’UTR sequences only; exon 2 contains the ATG and encodes part of the N-terminal S100 calcium binding domain of profilaggrin; and exon 3 encodes the remainder of the S100 domain, the B-domain and the filaggrin polyprotein repeat domain. In the July 2007 mouse genome sequence, annotated here (Flg^C57), the latter consists of 16 near-perfect repeats encoding a 250 amino acid filaggrin protein (1–16) flanked by two imperfect repeats (0 and 17). Repeat 17 is followed by a short tail domain. The 485 bp 3’UTR contains a consensus polyadenylation signal. In ft mice, the exon 3 sequence determined here (Flg^ft) lacks one repeat due to a 750-bp in-frame deletion, as well as the frameshift mutation c.5303delA. In addition, there are several additional SNPs and small in-frame insertions/deletions in flg that differ between the two strains, so that their DNA sequences overall are 99.3% identical, excluding the repeat copy number variation. (b) Wild-type flg sequence, corresponding to codons 1765–1771, with amino acid translation. The A dinucleotide involved in the mutation is boxed (compare panel c). (c) Analogous flg sequence as shown in (b), derived from a homozygous ft/ft mouse, showing homozygous frameshift mutation designated c.5303delA, predicting truncation mutation p.Asn1768ThrfsX154. The single A nucleotide remaining at the site of the mutation is boxed (compare panel b). (d) Allele-specific genotyping of the parents and F1 offspring of a cross between an ft/ft homozygote and a wild-type mouse. M, molecular weight markers, upper band = 118 bp; lower band = 72 bp. The allele specific band is only amplified from mice carrying the ft mutation. (e) Genotyping for an Acc I restriction site polymorphism in filaggrin repeat 1, approximately 4 kb upstream of the 5303delA mutation. This assay is more convenient for scoring of heterozygotes. The 678 bp C57BL/6 allele does not digest; the ft allele cuts to yield fragments of 559 bp and 134 bp. M, molecular weight markers.

Figure 2. A truncated profilaggrin is expressed in flaky tail mouse skin, which lacks the C-terminus

Urea/Tris protein extracts from control wild type and ft/ft mice were fractionated by SDS/PAGE and immunoblotted with the peptide antibody developed to the C-terminus of mouse profilaggrin. (a) Coomassie brilliant blue-stained protein gel to control for loading, and (b), corresponding immunoblot probed with the C-terminal antibody. The antibody detects both full-length profilaggrin (P) and a putative C-terminal processing product (C) in wild type mouse extracts, however, these immunoreactive products are not detected in ft/ft mice (b). Note the lack of filaggrin (F) in ft/ft mice (a), observed previously. The mutant profilaggrin protein in ft/ft mice is faintly visible in (a) at an apparent molecular weight of ~215 kDa (*). This is readily detectable with a mouse filaggrin antibody as reported previously. Molecular weight marker sizes are shown at left.

Figure 3. Cutaneous inflammation in untreated ft/ft mice but not wt/wt or wt/ft animals

(a) Representative photomicrographs of skin sections from age- and sex-matched wt/wt, wt/ft and ft/ft mice. Ft/ft mice had orthokeratotic hyperkeratosis (arrow) with occasional foci of acanthosis (bracket) compared to wt/wt and ft/wt mice. (Original magnification x40.) (b). Numbers of skin infiltrating cells, lymphocytes, eosinophils and mononuclear cells were detected per high-power fields (HPF). Cells were counted on 15–20 HPF (x1,000) on hematoxylin and eosin stained sections of 5–6 wt/wt, wt/ft and ft/ft mice. Data represent the mean; error bars represent standard error of the mean. Student’s t-test, or corrected Welch corrected t-test, was used to determine statistical differences between groups. NS = Non-significant. (c) TEWL analysis of untreated wt/wt, wt/ft and ft/ft mice. Values are individual mice and Mean bars are shown.

Figure 4. Allergen exposure exacerbates skin inflammation ft/ft mice but not wt/wt or wt/ft animals

(a) Representative photomicrographs of skin sections from age- and sex-matched wt/wt, wt/ft and ft/ft mice exposed to OVA or PBS as a vehicle control. Ft/ft mice had diffuse mild acanthosis and marked increases in dermal cell infiltration when cutaneously challenged with OVA, compared to wt/wt and wt/ft mice. (Original magnification x40.) (b). Quantification of numbers of skin infiltrating cells, lymphocytes, eosinophils and mononuclear cells detected per high-power fields (HPF). Cells were counted at on 15–20 HPF (x1,000) on hematoxylin and eosin stained sections of 11–14 wt/wt, wt/ft and ft/ft mice. Data represent the mean; error bars represent standard error of the mean. Student’s t-test, or corrected Welch corrected t-test, was used to determine statistical differences between groups. NS = Non-significant. (c) TEWL analysis of skin of OVA exposed wt/wt, wt/ft and ft/ft mice. Values relate to individual mice and mean bars are shown.

Figure 5. Elevated OVA-specific immune response in allergen exposed ft/ft mice but not wt/wt or wt/ft animals

(a) ELISA detection of levels of total IgE, OVA-specific IgE and IgG in serum from wt/wt, wt/ft and ft/ft mice treated with PBS or OVA on skin. Data are Mean plus Standard Error of the Mean (SEM) from 7–12 mice. (b) Cell proliferation to OVA of spleen cells from wt/wt, wt/ft and ft/ft mice treated with PBS or OVA on skin. Data represent the mean of spleens from 3–4 individual mice per group; error bars represent standard error of the mean. (c) Cytokine production by spleen cells from OVA exposed wt/wt, wt/ft and ft/ft mice treated with PBS or OVA. Cells were cultured in media or OVA (500 µg/ml) and cytokines detected by ELISA. Data represent the mean of spleens from 3–4 individual mice per group; error bars represent standard error of the mean. Student’s t-test, or corrected Welch corrected t-test, was used to determine statistical differences between groups. NS = Non-significant.