NFκB signaling in <i>a/a ma ft/ma ft/J</i> mice.

<p><b>A.</b> P5 WT and <i>a/a ma ft/ma ft/J</i> mouse skin cryosections were immunostained with an anti-p50 antibody. Fluorescence was visualized with a 20x lens. Scale bar = 50 µm. (<b>B</b>). Epidermal protein extracts from P5 WT and <i>a/a ma ft/ma ft/J (ft/ft)</i>mice were submitted to WB, performed with anti-p50, anti-p-p65 and anti-β-actin antibodies, and detected by chemiluminescence. Quantification of p50 and p-p65 expression normalized to β-actin is represented in histograms. (<b>C</b>). Total RNAs were extracted from WT and <i>a/a ma ft/ma ft/J (ft/ft)</i> mice dorsal skin, and real-time PCRs were performed using primers specific for <i>Vcam</i>, <i>Icam</i>, and <i>Il6</i>. Ct values were normalized to <i>hprt</i>. The experiment was realized in groups of 10 mice and statistically significant differences were calculated with the student T-test (**p<0.1 and ***p<0.001).</p

Acanthosis and inflammation in 5-days-old <i>a/a ma ft/ma ft/J</i> skin.

<p><b>A.</b> Dorsal skin of P5 C57blk6/J wild type (WT) and flaky tail (<i>a/a ma ft/ma ft/J</i>) mice was collected, fixed in PFA 4% and embedded in paraffin. H&E staining was performed and sections were visualized with a 20x lens. Thickening of the epidermis, hypogranulosis (white arrowheads), lymphocytic exocytosis (arrows) and inflammatory infiltrates (black arrowheads) are observed. Magnification of two representative fields is presented in order to distinguish inflammatory infiltrates in the <i>a/a ma ft/ma ft/J</i> mouse skin. (<b>B</b> and <b>C</b>)<b>.</b> Dorsal skin biopsies were embedded in OCT and cryosections were prepared. Immunofluorescence was performed using antibodies against keratin 5 (KRT5) <b>(B)</b> and keratin 6 (KRT6) <b>(C)</b>. Fluorescence was visualized with a 20x lens (LSM 700 Zeiss confocal microscope (B) or Zeiss Axiovision microscope (C)). Scale bars = 50 µm.</p

Real time PCR analysis of gene expression in K14-TSLP tg mouse skin.

<p>cDNA was prepared from P5 WT and <i>K14-TSLP tg</i> mice, and real-time PCRs were performed using primers specific for (A) <i>Il13</i>, (B) <i>Il6</i>, (C) <i>Sprr2a</i> and (D) <i>Sprr2d</i>. The experiment was realized in groups of 5 WT and 5 <i>K14-TSLP</i> P5 mice and statistically significant differences were calculated with the student T-test (*p<0.05 and **p<0.01).</p

Increased Sprr2 expression in <i>a/a ma ft/ma ft/J</i> mouse epidermis.

<p>A. cDNA was prepared from P5 WT and <i>a/a ma ft/ma ft/J (ft/ft)</i> mice, real-time PCRs were performed using primers specific for <i>Sprr2a</i> and <i>Sprr2d</i>. Ct values were normalized with the <i>hprt</i> house-keeping gene. The experiment was realized in groups of 10 mice and statistically significant differences were calculated with the student T-test (***p<0.001). B. P5 WT and <i>a/a ma ft/ma ft/J</i> mouse skin cryosections were immunostained with an anti-SPRR2 antibody. Fluorescence was visualized with a 20x lens. Scale bar = 50 µm.</p

Spontaneous Atopic Dermatitis-Like Symptoms in a/a ma ft/ma ft/J Flaky Tail Mice Appear Early after Birth.

Loss-of-function mutations in human profilaggrin gene have been identified as the cause of ichthyosis vulgaris (IV), and as a major predisposition factor for atopic dermatitis (AD). Similarly, flaky tail (a/a ma ft/ma ft/J) mice were described as a model for IV, and shown to be predisposed to eczema. The aim of this study was to correlate the flaky tail mouse phenotype with human IV and AD, in order to dissect early molecular events leading to atopic dermatitis in mice and men, suffering from filaggrin deficiency. Thus, 5-days old flaky tail pups were analyzed histologically, expression of cytokines was measured in skin and signaling pathways were investigated by protein analysis. Human biopsies of IV and AD patients were analyzed histologically and by real time PCR assays. Our data show acanthosis and hyperproliferation in flaky tail epidermis, associated with increased IL1β and thymic stromal lymphopoietin (TSLP) expression, and Th2-polarization. Consequently, NFκB and Stat pathways were activated, and IL6 mRNA levels were increased. Further, quantitative analysis of late epidermal differentiation markers revealed increased Small proline-rich protein 2A (Sprr2a) synthesis. Th2-polarization and Sprr2a increase may result from high TSLP expression, as shown after analysis of 5-days old K14-TSLP tg mouse skin biopsies. Our findings in the flaky tail mouse correlate with data obtained from patient biopsies of AD, but not IV. We propose that proinflammatory cytokines are responsible for acanthosis in flaky tail epidermis, and together with the Th2-derived cytokines lead to morphological changes. Accordingly, the a/a ma ft/ma ft/J mouse model can be used as an appropriate model to study early AD onset associated with profilaggrin deficiency