Transgenic expression of interleukin-13 in the skin induces a pruritic dermatitis and skin remodeling

Abstract

IL-13 has been implicated in the pathogenesis of allergic diseases, including atopic dermatitis (AD). However, a direct role of IL-13 in AD has not been established. We aimed to develop an inducible transgenic model in which IL-13 can be expressed in the skin and to define the resulting dermal phenotype and mechanisms involved. The keratin 5 promoter was used with a tetracycline-inducible system to target IL-13 to the skin. The clinical manifestations, dermal histology, cytokine gene regulation, and systemic immune responses in the transgenic mice were assessed. IL-13 was produced exclusively in the skin and caused a chronic inflammatory phenotype characterized by xerosis and pruritic eczematous lesions; dermal infiltration of CD4+ T cells, mast cells, eosinophils, macrophages, and Langerhans cells; upregulation of chemokine and cytokine genes, including thymic stromal lymphopoietin; and skin remodeling with fibrosis and increased vasculature. The dermal phenotype was accompanied by elevated serum total IgE and IgG1 and increased production of IL-4 and IL-13 by CD4+ cells from lymphoid tissues and peripheral blood mononuclear cells. IL-13 is a potent stimulator of dermal inflammation and remodeling and this transgenic model of AD is a good tool for investigating the underlying mechanisms in the pathogenesis of AD.

Conflict of interest statement

CONFLICT OF INTEREST

The authors state no conflict of interest.

Figures

Figure 1. Targeting IL-13 to the skin

(a) Schematic construct of TRE-Tight-IL-13. (b) Tissue specificity of IL-13 expression in Tg(+) mice as determined by RT–PCR. The experiments were started by withdrawing Dox and total RNA from different tissues was analyzed for IL-13 mRNA expression. (c) IL-13 mRNA expression in the skin of Tg(+) mice in comparison with Tg(−) mice 6 weeks off Dox. (d) IL-13 protein in the skin extract 8 weeks after Dox withdrawal (n = 36, each group).

Figure 2. IL-13-induced AD

(a) Gross comparison of a Tg(+) mouse and a Tg(−) mouse off Dox for 8 weeks. Tg(+) mice developed inflammatory skin lesions in the back and abdomen areas with hair loss, dry skin, excoriation, crusting, and bacterial pyoderma. Clinical scores of dermatitis in a representative group of Tg(+) mice (n = 7) after induction of IL-13 transgene for varying time. Tg(−) mice and Tg(+) mice without induction did not show any clinical signs of dermatitis. (b) Skin sections (H&E) showing thickening of epidermal and dermal layers, spongiosis, and infiltration of eosinophils (arrows) and mononuclear cells in Tg(+) mice. Scale bars: left and middle panels = 100 µm, right = 20 µm. IHC analyses of infiltrating cells (c) MBP + eosinophils (dark brown). Bar = 50 µm; (d) F4/80 + cells (brown). Bar = 50 µm; and (e) CD4 + cells (brown). Bar = 50 µm.

Figure 3. Mast cells and mast-cell mediators in the skin

(a) Toluidine blue staining of skin samples from Tg(−) and Tg(+) mice that were off Dox for 8 weeks. Scale bars: left and middle panels = 100 µm, right = 20 µm. (b) Total tissue histamine and (n = 7 per group). (c) β-hexosaminidase content in the skin extracts (n = 12 per group).

Figure 4. Chemokine mRNA expression in the skin

Total RNA purified from skin samples from K5-tTA-IL-13 Tg(+) mice and Tg(−) controls was analyzed using gene-specific primers. β-Actin gene was used as an internal control. At least two independent experiments were performed for each of the genes.

Figure 5. Upregulation of TSLP expression in the skin

(a) RT–PCR for TSLP mRNA in the skin. (b) TSLP protein in the skin extracts (n = 6 for each group). (c) IHC identification of cell types expressing TSLP. Skin sections from Tg(+) and Tg(−) mice were stained using anti-TSLP. Bar = 50 µm. Shown are representative slides of three pairs of stained samples.

Figure 6. IL-13-induced fibrosis and upregulation of TGF-β1

(a) Trichrome staining of skin sections from Tg(+) and Tg(−) mice after Dox off for 8 weeks. Bar = 100 µm. (b) Quantitative analysis (Sircol Assay) of collagen content in the skin (n = 6, each group). (c) TGF-β1 mRNA and (d) TGF-β1 protein (n = 6, each group).

Figure 7. IL-13-induced increased vasculature and upregulation of VEGF

(a) Increased numbers of blood vessels were present in the skin of Tg(+) mice as compared with that of Tg(−) mice (H&E). Bars: upper panels = 200 µm and lower panels = 50 µm. (b) ELISA analysis of VEGF protein in skin extracts (n = 6 each group).

Figure 8. Dermal expression of IL-13 resulted in altered serum immunoglobulins

Serum samples from Tg(+) and Tg(−) mice off Dox for 8 weeks were collected and analyzed by ELISA. Shown are serum total IgE, IgG1, and IgG2a (n = 25 each group).

Figure 9. Cytokine production by PBMC and lymphocytes

PBMC and lymphocytes from cutaneous draining lymph nodes were isolated from Tg(+) and Tg(−) mice 8 weeks after Dox withdrawal and stimulated with anti-CD3/CD28 for 3 days in culture. (a) IL-13 and IFN-γ production by peripheral LN CD4 + cells (n = 14 per group). (b) IL-13 and IFN-γ production by PBMC (n = 5 per group).