The novel house dust mite allergen Der p 39 exacerbates atopic dermatitis-like inflammation in mice by inducing skin barrier dysfunction

Abstract

Background: House dust mite (HDM) allergens can induce or exacerbate allergic inflammation, including atopic dermatitis (AD). Substances that damage the epithelial barrier can trigger or worsen AD. The mechanism by which the novel HDM allergen Der p 39 induces allergic inflammation remains unclear. Our aim was to investigate the effects of Der p 39 on AD-like inflammation and associated mechanisms.

Methods: Dinitrochlorobenzene (DNCB) and Der p 39 were utilized to establish AD model mice. Inflammation severity was evaluated with physiological and morphological assays. The effects of Der p 39 on inflammatory cytokine release and skin barrier protein expression were examined in HaCaT cells (human epidermal keratinocytes). Mitogen-activated protein kinase (MAPK) activation was examined by western blots. MAPK inhibitors were employed to assess MAPK involvement in filaggrin expression.

Results: Der p 39 worsened allergic inflammation (tissue thickness) in murine ears pretreated with 1% DNCB. Compared to controls, Der p 39-sensitized tissues showed epidermal and dermal thickening with elevated numbers of mast cells and eosinophils in inflammatory lesions. Der p 39 increased transcription and production of pro-inflammatory interleukins (ILs), down-regulated expression of the skin barrier proteins filaggrin and loricrin, and upregulated phosphorylation of ERK, JNK and p38 in HaCaT cells. Inhibition of MAPK signaling rescued filaggrin expression in Der p 39-treated cells.

Conclusions: The HDM allergen Der p 39 enhances allergic inflammation and promotes MAPK pathway-mediated epidermal barrier dysfunction, suggesting that Der p 39 may possess pathogenic and clinically relevant immunomodulatory potential.

Keywords: Atopic dermatitis; Der p 39; House dust mite; Skin barrier; Skin inflammation.

Fig. 1

Experimental design schematic and Der p 39 effects on AD-like inflammation. (A) Diagram of the study protocol (The figure was created by Figdraw). Mice were divided into 6 groups (n = 6 per group). AD-like skin inflammation lesions were induced with DNCB and/or Der p 39 as described in the Materials and Methods. (B) Representative images of tissues from each group. (C) Ears were weighed after euthanasia. (D) Changes in ear thickness were measured on the last day of the experiment. (E) DNCB/Der p 39 induced DS in mice. Data are expressed as means ± SD (n = 6). One-way ANOVA, Tukey's post-hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 vs. respective control

Fig. 2

Der p 39 induces histological changes and enhances leukocyte invasion in AD-like lesions. (A) Ear tissues were stained with H&E and TB. (B) Assessment of epidermal/dermal thicknesses in H&E stained sections. (C) Mast cell quantities were determined in TB-stained sections. (D) Eosinophil quantities were estimated in H&E stained sections. Data are expressed as means ± SEM (n = 6); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Data are expressed as means ± SD (n = 6). One-way ANOVA, Tukey's post-hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 vs. respective control

Fig. 3

Der p 39 modulates pro-inflammatory cytokine expression in HaCaT cells. CCK-8 assay analysis of HaCaT cell viability following Der p 39 treatment. (B) Following stimulation of HaCaT cells with Der p 39 for 0.5h and 1 h, expression levels of IL-6 and IL-8 transcripts were determined by RT-qPCR. Expression levels were normalized to GAPDH, and fold-change values are presented. (C) Secretion levels of IL-6 and IL-8 were determined by subjecting supernatants of HaCaT cells that had been stimulated with Der p 39 for 6 h, 12 h, or 24 h to ELISAs. Data are presented as means ± SD (n = 3). One-way ANOVA, Tukey's post-hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. untreated control

Fig. 4

Der p 39 diminishes filaggrin expression in HaCaT cells. (A) HaCaT cells were treated with 100 μg/mL Der p 39 for the indicated durations. (B, C) Quantified values of filaggrin and loricrine. (D) HaCaT cells were treated with a series of Der p 39 concentrations for 48 h. (E, F) Quantified values of filaggrin and loricrine. For both experiments, HaCaT cells were treated with TNF-α/IFN-γ (each 10 ng/mL) for 24 h as a positive control. Whole-cell lysates were used for Western blot analysis of filaggrin and loricrin levels. The results are expressed as means ± SD (n = 3). Unpaired Student’ s t-test, ##p < 0.01, ###p < 0.001 vs. respective control. One-way ANOVA, Tukey's post-hoc test. ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. respective control

Fig. 5

Der p 39 reduced filaggrin expression in HaCaT cells by activating MAPK signaling. (A) Effects of Der p 39 on MAPK activation in HaCaT cells. HaCaT cells were stimulated with Der p 39 (50, 100, and 200 μg/mL) for 15 min, and levels of p38, ERK, and JNK proteins were analyzed in western blots. (B–E) Effects of MAPK pathway inhibition on Der p 39-suppressed filaggrin expression. After being treated with Der p 39 for 48 h, the cells were exposed to an ERK inhibitor (C), JNK inhibitor (D), or p38 inhibitor (E) for 1 h. Filaggrin levels were analyzed in western blots. The results are expressed as means ± SD (n = 3). One-way ANOVA, Tukey's post-hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. respective control