Pathogenic Mechanism of Der p 38 as a Novel Allergen Homologous to RipA and RipB Proteins in Atopic Dermatitis

Abstract

Atopic dermatitis (AD) is a chronic relapsing pruritic disease encompassing skin inflammation and barrier dysfunction. House dust mites are key allergens that augment the development of atopic dermatitis. We aimed to investigate the pathogenic mechanism of AD due to Der p 38, recently identified by us. The frequency of IgE reactivity to Der p 38 in AD subjects was 52.6% (10/19) in the skin prick test and 57.9% (11/19) in the dot blot assay. In human keratinocyte HaCaT cells, Der p 38 triggered the impairment of filaggrin expression and induced pro-inflammatory cytokines such as IL-6, IL-8 and MCP-1 through TLR4, PI3K, AKT, c-Jun N-terminal kinase (JNK) and NF-κB pathway. Supernatants from Der p 38-treated cells blocked filaggrin expression and neutrophil apoptosis. The anti-apoptotic effect of the Der p 38-released molecules on neutrophils was accomplished by inhibition of the caspase 9/3 pathway, and by increased MCL-1 expression and BCL-2/BAX expression ratio. In C57BL/6 wild type (WT) mice, Der p 38 induced a dose-dependent increase of AD-like skin lesions, with enhanced expressions of total and Der p 38-specific IgE. Der p 38 also diminished the expressions of skin barrier proteins and induced JNK activation. However, the AD-like features following cutaneous Der p 38 exposure were observed to be reduced in the TLR4 knockout (KO) group, as compared to the WT group. Skin infiltration of neutrophils, eosinophils and mast cells was increased in the WT mice, but was not portrayed in the TLR4 KO mice. These findings indicate that Der p 38 is a novel mite allergen that triggers AD by lowering skin barrier proteins and increasing inflammatory cells. Results of this study have thereby paved the way to unveil the pathogenic mechanisms of AD.

Keywords: Der p 38; TLR4; atopic dermatitis; filaggrin; skin barrier.

Figure 1

Der p 38 shows IgE reactivity in AD subjects. (A) Der p 38 amino acid sequence is aligned with the RipA and RipB proteins. Red box (strict identity). Blue frame (similarity across groups). Red character (similarity in a group). (B) Der p 38 cDNA were amplified by PCR using gene specific primer. The cDNA was cloned to the vector and transformed into E coli. Recombinant Der p 38 protein was produced, separated by SDS-PAGE, and stained with Coomassie brilliant blue. (C) Natural Der p 38 protein in DP extract was detected by Western blotting using antibodies against Der p 38. (D) Protease activity of Der p 38 was measured by a protease activity kit. (E) Dot blot assay was performed with sera of healthy and AD subjects.

Figure 2

Der p 38 diminishes filaggrin expression via TLR4, PI3K, AKT, JNK and NF-κB in human keratinocyte HaCaT cells. (A, C, F) HaCaT cells were treated with 10 μg/mL Der p 38 for the indicated duration. The harvested cells were lysed, and filaggrin, loricrin, involucrin (A), phospho-JNK (C), and phospho-AKT (F) were analyzed by Western blotting. (B, D) The cells were pretreated with 2 μM TLR4i, 10 μM LY294002 (LY), 10 μM AKTi, 10 μM SP600125 (SP) or 10 μM BAY-11-7085 (BAY) for 1 h, following Der p 38 treatment for 48 h (B) or 30 min (D). Filaggrin and phospho-JNK were analyzed by Western blotting. (E) The cells were pretreated with 10 μM LY294002, 10 μM AKTi or 10 μM SP600125, after which they were treated with Der p 38 for 48 h. Filaggrin was analyzed by Western blotting. ERK2 was used as an internal control. (G, H) The cells were treated with 10 μg/mL Der p 38 in a time dependent manner (G), or 10 μg/mL Der p 38 after pretreatment with 2 μM TLR4i, 10 μM LY294002, 10 μM AKTi, or 10 μM SP600125 (H). After lysis of the harvested cells, NF-κB activity in the lysates was evaluated by the luciferase assay. The data are presented relative to the control, which was set at 1 as the mean ± S.D (n=3). **p < 0.01 indicates a significant difference between untreated and Der p 38-treated groups, and ^# p < 0.05 and ^## p < 0.01 indicate significant difference between the Der p 38-treated group and the inhibitor-treated groups.

Figure 3

Der p 38 increases cytokine secretion in human keratinocyte HaCaT cells, thereby preventing filaggrin expression and neutrophil apoptosis. (A) HaCaT cells were treated with 10 μg/mL Der p 38 for 48 h. Supernatant (Sup) was collected, and the other cells were treated with the supernatant for 48 h. The harvested cells were lysed in lysis buffer and the homogenate was centrifugated, and the supernatant was collected as total lysate. Following separation of the protein samples by 10% SDS-PAGE, the transferred nitrocellulose membrane was sequentially incubated with anti-filaggrin (1:1,000) and secondary antibody (1:3,000) for 1 h at room temperature, and developed using the Enhanced Chemiluminescence Western blotting Detection System. ERK2 expression is used as an internal control. (B, C) Cells were treated with 10 μg/mL Der p 38 for the indicated time (B), or for 48 h after pretreatment with 2 μM TLR4i, 10 μM LY294002 (LY), 10 μM AKTi, 10 μM SP600125, and 10 μM BAY-11-7085 (BAY) (C). The supernatant was collected and analyzed by ELISA. (D) Neutrophils and eosinophils were treated with the supernatant after treatment with 10 μg/mL Der p 38. Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. The data are presented relative to the control, which was set at 100% as the mean ± S.D (n=3). *p < 0.05 and **p < 0.01 indicate a significant difference between the untreated and Der p 38-treated groups, and ^# p < 0.05 and ^## p < 0.01 indicate significant difference between the Der p 38-treated group and the inhibitor-treated groups. (E, F) Activation and expression of the indicated signal proteins were evaluated by Western blotting. Lower panel (F) is the ratio of BCL-2/BAX using densitometry.

Figure 4

Der p 38 induces AD-like phenotypes via TLR4 in the mice. C57BL/10ScNJ wild type (WT) and C57BL/10ScNJ TLR4 knockout (KO) mice were used in this experiment. The mice were dorsally administered Der p 38 (20, 50, or 100 μg/mice) or DP (100 μg/mice) using a 2 x 2 cm patch of sterile gauze. The mice were wrapped with a band aid. After 6 days, the patch was removed, and 24 h later a new patch was applied, for a total of five patches over a 5 weeks period. Clinical skin severity score (A), histological features using hematoxylin and eosin stain (B), layers of epidermis (C) and dermis (D), total IgE (E), and Der p 38-specific IgE (F) were evaluated as described in the Materials and Methods section. The data are presented as a mean ± SD (n=3). *p < 0.05 and **p < 0.01 indicate a significant difference between the control group and stimulator-treated groups, and ^# p < 0.05 and ^## p < 0.01 indicate significant difference between the WT and the TLR4 KO groups.

Figure 5

Der p 38 decreases the expression of filaggrin and increases neutrophils, eosinophils and mast cells in the skin of mice. (A, B, E, F) For skin barrier proteins and inflammatory cell analysis, skin sections of wild type (WT) (A, E) and TLR4 KO mice (B, F) were fixed, embedded in paraffin, and incubated with primary antibodies against filaggrin, loricrin, involucrin, Ly6G, eosinophil peroxidase (EPX) and tryptase. The samples were examined by light microscopy (magnification, ×200). Filaggrin, loricrin, involucrin (C), and phospho-JNK (D) in tissue lysates were analyzed by Western blotting.

Figure 6

Proposed mechanism of filaggrin downregulation due to Der p 38 in AD. Der p 38 suppresses filaggrin expression and induces cytokine release through TLR4, PI3K, AKT, JNK, and NF-κB, which blocks neutrophil apoptosis.