Nectin4 is a potential therapeutic target for asthma

Abstract

Background: Nectins comprise a family of cellular adhesion molecules involved in Ca²⁺-independent cellular adhesion. Neither the biological significance nor clinical potential of Nectin4 for asthma has been investigated.

Objectives: The aims of this study were to elucidate the role of Nectin4 in airway inflammation and to determine the relationship between Nectin4 and clinical variables in patients with asthma.

Methods: The relationship between Nectin4 levels in the blood of asthmatic patients and clinical variables was examined. Dermatophagoides pteronyssinus 1 (Der p1)-exposed normal human bronchial epithelial (NHBE) cells, and Nectin4-deficient (Nectin4^-/-) and wild-type (WT) mice sensitized/challenged with ovalbumin (OVA), were used to investigate the involvement of Nectin4 in the pathogenesis of bronchial asthma via the Src/Rac1 pathway.

Results: Plasma Nectin4 levels were significantly higher in asthmatic patients than controls and correlated with specific IgE D1, D2, lung function. The ROC curves for Nectin4 levels differed between asthma patients and controls. Nectin4/Afadin and Src/Rac1 levels were significantly increased in NHBE cells exposed to Der p1, but decreased in NHBE cells treated with Nectin4 siRNA. Airway obstruction and inflammation, as well as the levels of Th2 cytokines, Nectin4, and Src/Rac1, were increased in WT OVA/OVA mice compared with WT sham mice. Nectin4 knockdown resulted in lower levels of Afadin and Src/Rac1 in Nectin4^-/-OVA/OVA than WT OVA/OVA mice.

Conclusion: These results suggest that Nectin4 is involved in airway inflammation and may be a therapeutic target in patients with asthma.

Keywords: Nectin4; adherens junction protein; asthma; epithelial barrier; therapeutic target.

Figure 1

Specimens were obtained from control subjects (n = 60), subjects with non-steroid-treated asthma (n = 51), and subjects with steroid-treated asthma (n = 11) (A) Plasma Nectin4 levels in control subjects and asthma patients. *p<0.05, control vs. asthma patients. (B) Receiver operating characteristic (ROC) curves of the plasma Nectin4 levels. Nectin4 protein concentrations in asthma patients and controls (Area under the curve (AUC) =0.760, 95% CI 0.673-0.847, p<0.001). The diagonal line represents a hypothetical curve corresponding to a test affording no discriminatory power.

Figure 2

Relationship between plasma Nectin4 and clinical variables in asthma patients. (A) Specific IgE D1 or specific IgE D2 level for control subjects, steroid not treated group, and steroid treatment group for exacerbation. *p<0.05, control vs. asthma patients. (B) Correlation of plasma Nectin4 and clinical variables including FVC% predicted, FEV1% predicted, FEV1/FVC%, and PC20. * p<0.05, Spearman’s rank test.

Figure 3

Time courses of Dermatophagoides pteronyssinus 1 (Der p1) allergen-induced Nectin4/Afadin and Src/Rac1 pathway expression. (A) Nectin4/Afadin and Src/Rac1 pathway expression on western blots. (B) Densitometry of the bands obtained on three western blots. The values were normalized to those of β-actin and are expressed as the mean ± SEM. The results are representative of at least 3 independent experiments. *p<0.05 compared to the control.

Figure 4

The knockdown of Nectin4 alters the expression of Afadin and the Src/Rac1 pathway. (A) Nectin4 knockdown alters the expression of Afadin and the Src/Rac1 pathway in NHBE cells treated with Der p1. (B) The values were normalized to those of β-actin and are expressed as the means ± SDs. (C) Time-dependent increases in TEER in NHBE cells treated with Der p1 and decreases in NHBE cells treated with Der p1 and Nectin4 siRNA. The results are representative of at least 3 independent experiments. *p<0.05 compared to the control.

Figure 5

Absence of Nectin4 prevents development of OVA-induced asthma. (A) Scheme representing the acute ovalbumin-dependent asthma model of WT and Nectin4^-/- mice. (B) Airway hyperresponsiveness in response to methacholine (Mch) (n = 6-10 mice per group). (C) Inflammatory cells in BAL fluid. (D, E) Levels of IL-4, TNF-a, and IFN-g in BAL fluids and Lung proteins (n = 6-10 mice per group). (F) Representative images of H&E, PAS, Masson’s trichrome-stained, paraffin-embedded lung sections of asthmatic mice (scale bar = 50 μm). (G-I). Bar graphs summarize means ± SEMs of histological scoring of inflammation grading based on H&E staining, the number of goblet cells based on PAS staining and of fibrosis based on trichrome staining. * p<0.05 vs. WT sham. #p<0.05 vs. WT OVA/OVA.

Figure 6

Absence of Nectin4 controls the expression of Afadin and the Src/Rac1 pathway. (A) Expression of the Nectin4/Afadin and the Src/Rac1 pathway in ovalbumin-induced asthma model of WT and Nectin4^-/- mice. (B) Densitometry of the bands obtained on three different individuals western blots. The values were normalized to those of β-actin and are expressed as the mean ± SEMs (n = 6-10 mice/group). (C) The lung tissues of OVA-sensitized/challenged mice were analyzed for Nectin4/Afadin and Src/Rac1 pathway expression by immunohistochemical staining (scale bar = 50 μm). (D) Quantification of percentage of Nectin4/Afadin and Src/Rac1 pathway protein-positive areas. All quantitative analyses were performed using ImageJ software. The results are representative of at least 3 independent experiments. * p<0.05 vs. WT sham. #p<0.05 vs. WT OVA/OVA.

Figure 7

Double-staining immunofluorescence of Nectin4 (Green) and Rac1-GTP (Red) in ovalbumin-induced asthma model of WT and Nectin4^-/- mice lung. (A) Representative immunofluorescence images of Nectin4 and Rac1-GTP in lung of the mice (scale bar = 100 μm). (B) Quantitation of the fluorescence intensity of Nectin4 and Rac1-GTP. * p<0.05 vs. WT sham. #p<0.05 vs. WT OVA/OVA.