A 24 kDa excretory-secretory protein of Anisakis simplex larvae could elicit allergic airway inflammation in mice

Abstract

We have reported that a 24 kDa protein (22U homologous; As22U) of Anisakis simplex larvae could elicit several Th2-related chemokine gene expressions in the intestinal epithelial cell line which means that As22U may play a role as an allergen. In order to determine the contribution of As22U to allergic reactions, we treated mice with 6 times intra-nasal application of recombinant As22U (rAs22U). In the group challenged with rAs22U and ovalbumin (OVA), the number of eosinophils in the bronchial alveolar lavage fluid (BALF) was significantly increased, as compared to the group receiving only OVA. In addition, mice treated with rAs22U and OVA showed significantly increased airway hyperresponsiveness. Thus, severe inflammation around the airway and immune cell recruitment was observed in mice treated with rAs22U plus OVA. The levels of IL-4, IL-5, and IL-13 cytokines in the BALF increased significantly after treatment with rAs22U and OVA. Similarly, the levels of anti-OVA specific IgE and IgG1 increased in mice treated with rAs22U and OVA, compared to those treated only with OVA. The Gro-α (CXCL1) gene expression in mouse lung epithelial cells increased instantly after treatment with rAs22U, and allergy-specific chemokines eotaxin (CCL11) and thymus-and-activation-regulated-chemokine (CCL17) gene expressions significantly increased at 6 hr after treatment. In conclusion, rAs22U may induce airway allergic inflammation, as the result of enhanced Th2 and Th17 responses.

Keywords: Anisakis simplex; As22U; allergic airway inflammation; excretory secretory protein.

Fig. 1

Induction of eosinophilic airway inflammation after rAs22U treatment. (A) The differential immune cells were counted in BALF. The number of immune cells, especially eosinophils and lymphocytes were increased in BALF of mice treated with rAs22U. (B) A thin section of the lung stained with hematoxylin-eosin (Bar=50 µm, ×100). In the lungs of mice treated with rAs22U, massive peribronchial infiltration with immune cells and hyperplasis of bronchial epithelial cells were observed. Also, thickened bronchial epithelial cells were observed in rAs22U-treated mice. (C) Enhanced pause (PenH) was increased at baseline and after treatment with increasing doses of aerosolized methacholine (0 to 50 mg/ml). The PenH value of rAs22U-treated mice was significantly higher than that of the only OVA-treated mice (^*, P<0.05; ^**, P<0.01).

Fig. 2

Cytokine levels in BALF. The level of IL-4, IL-5, IL-13, and IL-17A in BALF were determined using sandwich ELISA. Levels of all cytokines were significantly increased after rAs22U treatment. The data for cytokine levels in the BALF are expressed as means±SD of values from individual mice (^**, P<0.01; ^***, P<0.001).

Fig. 3

The level of OVA-specific immunoglobulin in serum. OVA-specific IgE, IgG1, and IgG2a levels in serum were measured. The 96-well plates were incubated with OVA (final concentration 10 µg/ml) for 16 hr at 4℃. After blocking with 1% BSA, sera ere diluted with PBS to 1:10⁴-10⁶ for IgG1 and IgG2a measurement, and the levels of immunoglobulins were measured. The rAs22U treated mice had significantly increased serum levels of OVA-specific IgE and IgG1 (^**, P<0.01; ^***, P<0.001).

Fig. 4

Chemokine gene expression in mouse lung epithelial cells after rAs22U treatment. After treatment with 10 µg/ml (final concentration) rAs22U to MLE12 cells, the expression of several chemokine genes was evaluated every 2 hr until 10 hr after treatment (^*, P<0.05; ^**, P<0.01, compare to the level of gene expression at 0 hr).