Combined application of dinitrofluorobenzene and ovalbumin induced AD-like dermatitis with an increase in helper T-cell cytokines and a prolonged Th2 response

Abstract

Background: The progression of acute-to-chronic atopic dermatitis is accompanied by multiple helper T-cell cytokine responses, but the mechanisms and relative importance of these changes remain unclear. There is no animal model for atopic dermatitis that recapitulates these cytokine responses.

Objective: We sought to build a novel mouse model for atopic dermatitis (AD) that recapitulates these helper T-cell responses and some dynamic changes in cytokine responses in the progression of AD.

Methods: Female BALB/c mice were subjected to the application of dinitrofluorobenzene (DNFB) and ovalbumin (OVA) to induce AD-like dermatitis. Skin lesions and serum were collected from mice in the acute and chronic phases to detect changes in cytokine responses and other features of AD.

Results: Combined application of DNFB and OVA successfully induced AD-like dermatitis and histological changes as well as epidermal barrier dysfunction. In the acute phase of AD-like dermatitis, Th2-associated cytokines were mainly increased in serum and skin lesions. In the chronic phase of AD-like dermatitis, Th2-associated cytokines were still highly expressed, while Th1- and Th17-associated cytokines were also gradually increased. Compared with the acute phase, the JAK-STAT signaling pathway was highly expressed in the chronic phase of AD-like dermatitis.

Conclusion: The combined application of DNFB and OVA could be used to build a new mouse model for atopic dermatitis. This mouse model recapitulates the helper T-cell responses and some dynamic changes in cytokine responses in the progression of acute-to-chronic in human AD. The JAK-STAT signaling pathway plays a pivotal role in the chronicity of AD.

Keywords: Atopic dermatitis; BALB/c mice; Dinitrofluorobenzene; Ovalbumin.

Fig. 1

Typical AD-like dermatitis and scratching behavior were induced by DNFB + OVA. A Morphological changes of skin lesion during four weeks. B Skin lesion scorings. The total scores of day 24 and day 28 were compared between D + O group and DNFB group. C Scratching behavior examination. D, E The serum total IgE and OVA-specific IgE in each group. F Serum IgE concentration of acute phase and chronic phase in the DNFB + OVA group. (Date shows mean ± SD, significance levels of data were denoted as *P < 0.05, **P < 0.01, and ***P < 0.001, ****P<0.0001)

Fig. 2

DNFB + OVA induced typical AD-like phenotypic and histologic changes in BALB/C mice. A Representative images of H&E and Toluidine blue stained sections from each group; scale bar = 100 μm. B, C Comparison of the epidermal and full thickness of skin in mice subjected to different treatments. D The average numbers of mast cells per field. Data are representative of five randomly selected fields from each section. E Comparison of histological changes between acute phase and chronic phase in the DNFB + OVA group. (Date shows mean ± SD, significance levels of data were denoted as *P < 0.05, **P < 0.01, and ***P < 0.001, ****P<0.0001)

Fig. 3

DNFB + OVA induced epidermal barrier dysfunction and higher FLG mRNA expression. A TEWL values in each group. B The comparison of the expression levels of FLG gene between each group. C Representative sections of anti-filaggrin immunohistochemistry staining in each group. Typical positively stained cells are marked with arrows. D (d1) The comparison of positively stained area between acute lesions and chronic lesions. (d2,d3) The comparison of FLG mRNA expression levels and TEWL values between acute lesions and chronic lesions. (Data shows mean ± SD. Scale bar = 50 μm. significance levels of data were denoted as *P < 0.05, **P < 0.01, and ***P < 0.001, ****P<0.0001)

Fig. 4

Combined application of DNFB + OVA aggravates the expression levels of Th2 and Th2 related cytokines both in serum and skin lesions. A–C The gene expression levels of TSLP, IL4, IL13, respectively. D, E Serum concentrations of IL4 and IL13 in each group. F Comparison of Th2 related cytokines between acute phase and chronic phase in the DNFB + OVA group. (Data shows mean ± SD. significance levels of data were denoted as *P < 0.05, **P < 0.01, and ***P < 0.001, ****P<0.0001)

Fig. 5

Upregulation of Th1 and Th17 related cytokines were observed in DNFB + OVA group. A, B The gene expression levels of Th1 related cytokines. C Serum concentration of IFN-γ. D, E Gene expression levels of IL17A and IGFL1 in each group. F Gene expression level of IL19 in each group. G Comparison of Th1/Th17 related cytokines expression between acute phase and chronic phase in the DNFB + OVA group. (Data shows mean ± SD. significance levels of data were denoted as *P < 0.05, **P < 0.01, and ***P < 0.001, ****P<0.0001)

Fig. 6

Comparisons of the JAK-STAT signaling pathway expression levels in mice subjected to different treatments. A–C The expression levels of JAK-STAT pathway in each group. D Comparison of the expression levels of JAK-STAT pathway between acute phase and chronic phase in the DNFB + OVA group. (Data shows mean ± SD. Significance levels of data were denoted as *P < 0.05, **P < 0.01, and ***P < 0.001, ****P<0.0001)

Fig. 7

Sensitization and induction protocol. Mice were randomly divided into four groups, listed as below. Control group (n = 4): mice were given solvent or saline, no hapten, allergen or drugs were given to them. DNFB only group (n = 4): DNFB(100ul each time) and saline were given to them,5 times in total for each single week. DNFB + OVA group (n = 8): DNFB and OVA were alternately given to the mice 5 times in total for each week, four mice of this group were euthanized at day14 (acute phase) by cervical dislocation, the other four mice were euthanized by the same way at day28 (chronic phase). OVA only group (n = 4): OVA and solvent were alternately given to the mice 5 times in total each week