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. 2019 Feb 15;199(4):496-507.
doi: 10.1164/rccm.201802-0373OC.

Ezrin, a Membrane Cytoskeleton Cross-Linker Protein, as a Marker of Epithelial Damage in Asthma

Man Jia  1 , Xiaoyi Yan  1   2 , Xinyu Jiang  1 , Yunhui Wu  1 , Jiayan Xu  1 , Yaqi Meng  1 , Yi Yang  1 , Xia Shan  2 , Xiuwedi Zhang  2 , Shan Mao  3 , Wei Gu  3 , Stelios Pavlidis  4 , Peter J Barnes  5 , Ian M Adcock  5 , Mao Huang  1 , Xin Yao  1
Affiliations

Ezrin, a Membrane Cytoskeleton Cross-Linker Protein, as a Marker of Epithelial Damage in Asthma

Man Jia et al. Am J Respir Crit Care Med. .

Abstract

Rationale: Bronchial epithelial cell damage occurs in patients with bronchial asthma. Ezrin, a membrane-cytoskeleton protein, maintains cellular morphology and intercellular adhesion and protects the barrier function of epithelial cells.

Objectives: To study the role of ezrin in bronchial epithelial cells injury and correlate its expression with asthma severity.

Methods: Levels of ezrin were measured in exhaled breath condensate (EBC) and serum in patients with asthma and BAL fluid (BALF) from a mouse model of asthma by ELISA. The regulation of IL-13 on ezrin protein levels was studied in primary bronchial epithelial cells. Ezrin knockdown using shRNA was studied in human bronchial epithelial 16HBE cells.

Measurements and main results: Ezrin levels were decreased in asthmatic EBC (92.7 ± 34.99 vs. 150.5 ± 10.22 pg/ml, P < 0.0001) and serum (700.7 ± 55.59 vs. 279.2 ± 25.83 pg/ml, P < 0.0001) compared with normal subjects. Levels were much lower in uncontrolled (P < 0.001) and partly controlled patients (P < 0.01) compared with well-controlled subjects. EBC and serum ezrin levels correlated with lung function in patients with asthma and serum ezrin levels were negatively correlated with serum IL-13 and periostin. IL-13-induced downregulation of ezrin expression in primary bronchial epithelial cells was significantly attenuated by the Janus tyrosine kinase 2 inhibitor, TG101348. Ezrin knockdown changed 16HBE cell morphology, enlarged intercellular spaces, and increased their permeability. Ezrin expression was decreased in the lung tissue and BALF of "asthmatic" mice and negatively correlated with BALF IL-13 level.

Conclusions: Ezrin downregulation is associated with IL-13-induced epithelial damage and might be a potential biomarker of asthma control.

Keywords: IL-13; biomarker; bronchial asthma; bronchial epithelial cells.

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Figures

Figure 1.
Figure 1.
Ezrin expression was decreased in exhaled breath condensate (EBC) of patients with asthma and positively correlated with lung function. (A) Ezrin expression in EBC samples from patients with asthma (n = 56) and healthy control volunteers (n = 19) was detected by ELISA. Decreased levels of ezrin were associated with the degree of asthma symptom control according to the asthma control test scores. (B) The relationship between ezrin and FEV1, FEV1% predicted, and FEV1/FVC, n = 40–47. (C) Correlation between ezrin EBC levels and FEV1 and FEV1/FVC of patients (n = 6) before and after asthma symptom control using analysis of covariance (ANCOVA). The data were analyzed using Wilcoxon rank-sum test and Kruskal-Wallis test in A, Pearson’s correlation test in B. FEV1%pred = FEV1% predicted; ns = not significant. **P < 0.01 and ***P < 0.001 compared with respective control groups.
Figure 2.
Figure 2.
Reduced serum ezrin levels of patients with asthma correlate positively with lung function and negatively with serum IL-13 and periostin levels. (A) The expression of ezrin in serum was reduced in patients with asthma (n = 59) compared with healthy control subjects (n = 18). Serum levels of ezrin were decreased according to the degree of asthma control: well-controlled group (n = 19); partly controlled group (n = 13); and uncontrolled group (n = 23). (B) The correlation between ezrin levels in exhaled breath condensate (EBC) and in serum (n = 34). (C) Ezrin mRNA expression in blood cells in severe asthma compared with healthy control subjects, by cohort (adjusted P = 0.0022). Graphs are expressed as log2 intensity robust multiarray average signals. The differences between healthy control subjects and patients with severe asthma were analyzed using the Benjamini-Hochberg method for adjusted P value/false discovery rate. (D) The correlation between EZR expression and T2 signature gene expression in IL-13–stimulated epithelial cells from patients with asthma and healthy subjects (n = 147). Ezrin gene expression is presented as log2 intensity robust multiarray average signals and the expression of the IL-13 signature genes as an enrichment score. (E) The relationship between serum ezrin and FEV1, FEV1% predicted, and FEV1/FVC (n = 44–47). (F) The concentrations of serum IgE (asthma group, n = 18; control group, n = 14), IL-13 (asthma group, n = 29; control group, n = 13), and periostin (asthma group, n = 56; control group, n = 23) were measured using ELISA (upper panels), and their relationships with ezrin were also analyzed (lower panels). Data were quantified and expressed as mean ± SD. **P < 0.01 and ***P < 0.001 compared with respective control subjects. The data were analyzed using Wilcoxon rank-sum test (A), IgE and periostin (F), Student’s t test in IL-13 (F), and Pearson’s correlation test (B and D–F). EZR = ezrin; FEV1%pred = FEV1% predicted; HC = healthy nonsmoking control subjects; MMA = mild/moderate nonsmoking asthma; ns = not significant; SAn = severe nonsmoking asthma; SAs = smokers with severe asthma.
Figure 3.
Figure 3.
Ezrin is expressed on exosomes secreted by bronchial epithelial cells. (A) Electron microscopic observation of whole-mounted exosomes purified from 16HBE cells. White arrows indicate exosomes. Scale bars = 1 μm (left panel) and 200 nm (right panel). (B) Intensity and size distribution of exosomes derived from 16HBE cells were measured by nanoparticle tracking analysis. Graph showing the average percentage of particles within 50- to 350-nm size in exosome. (C) Western blot analysis showed the presence of ezrin as well as exosome markers, CD9 and CD63, in exosomes isolated from 16HBE cells (Ctrl) and the IL-13 (30 ng/ml)–treated group (IL-13).
Figure 4.
Figure 4.
IL-13 downregulates ezrin expression in bronchial epithelial cells via the JAK2 (Janus tyrosine kinase 2)/STAT6 (signal transducer and activator of transcription) pathway. (A) 16HBE cells were treated with IL-4 (20 ng/ml), IL-13 (30 ng/ml), and TNF-α (10 ng/ml) for 2, 6, and 12 hours, and ezrin mRNA expression was measured by quantitative RT-PCR. (B) 16HBE cells were exposed to IL-4 (20 ng/ml), IL-13 (30 ng/ml), and TNF-α (10 ng/ml) for 24, 48, and 72 hours, and ezrin protein production was determined by Western blotting. (C) Primary bronchial epithelial cells (PBECs) were stimulated with IL-13 (30 ng/ml) for 24, 48, and 72 hours. Ezrin protein was evaluated by Western blotting. PBECs were pretreated with the JAK2 inhibitor TG101348 (30 nM) for 1 hour before IL-13 (30 ng/ml) stimulation (1 h). The total phospho (p)-JAK2 protein level and p-STAT6 protein level in the nucleus were measured by Western blotting. The effect of TG101348 pretreatment on IL-13–regulated ezrin expression was evaluated by Western blotting. Data are presented as mean ± SEM of three independent experiments using one-way ANOVA followed by Student-Newman-Keuls post hoc analysis. ns = not significant. *P < 0.05, **P < 0.01 and ***P < 0.001, compared with control (Ctrl). TNF-α = tumor necrosis factor-α.
Figure 5.
Figure 5.
Ezrin depletion alters cell morphology and increases cellular permeability. (A) 16HBE cells were transfected with lentiviruses encoding for a control shRNA (Ctrl) or human ezrin-shRNA (EZR-shR1–3) tagged with GFP for 24 hours and then washed, and the cells were examined after a further 48 hours. GFP was detected by immunofluorescence (scale bar = 100 μm), quantitative RT-PCR, and Western blotting. ***P < 0.001 compared with the control group. (B) Phase-contrast images (original magnification, ×100 and ×400; scale bars = 50 μm) of 16HBE cells treated with control media (Ctrl), transfection control (Ctrl-shR), IL-13 (30 ng/ml), ezrin-shRNA (EZR-shR1–3), and IL-13 (30 ng/ml) + TG101348 (30 nM). White double-headed arrows show intercellular space enlargement; white single arrows show cellular protrusions. (C) Assessment of the permeability of the bronchial epithelium based on transepithelial electrical resistance (TER) (left panel) and fluorescein isothiocyanate (FITC)–dextran (right panel) in control media (Ctrl), transfection control (Ctrl-shR), IL-13 (30 ng/ml), ezrin-shRNA (EZR-shR2), and IL-13(30 ng/ml) + TG101348 (30 nM) groups. The data were analyzed using one-way ANOVA followed by Student-Newman-Keuls post hoc analysis. (C) Left panel: *P < 0.05 (IL-13 vs. control), #P < 0.05 (EZR-shR2 vs. Ctrl-shR); right panel: *P < 0.05 and **P < 0.01 compared with respective control subjects. Phase-contrast images are representative of those seen in three independent experiments.
Figure 6.
Figure 6.
Ezrin expression and epithelial cell–cell adhesion were decreased in an ovalbumin (OVA)-treated allergic mouse model of asthma and restored by anti–IL-13 treatment. (A) Hematoxylin and eosin (H&E) staining of lung tissue in “asthma mice” (black arrows indicate bronchial epithelial cells). Representative image of E-cadherin and ZO-1 immunostaining (black arrows in the middle and bottom panels indicate their expression on the bronchial epithelial cells) was examined in saline-exposed control mice (control), OVA-treated mice (OVA), OVA + anti-IgG antibody–treated mice (anti-IgG), and OVA + anti–IL-13 antibody–treated mice (anti–IL-13), and was analyzed by Image-Pro Plus 6.0. Scale bars, 50 μm. (B) Epithelial cell–cell adherence was determined by electron microscopy (scale bars, 1 μm; white arrow). (C) Immunohistochemical analysis of ezrin expression in saline-exposed control mice (control), OVA-treated mice (OVA), OVA + anti-IgG antibody–treated mice (anti-IgG), and OVA + anti–IL-13 antibody–treated mice (anti–IL-13) (original magnification, ×400; scale bar = 100 μm; black arrow) and scored (right graph). (D) The concentrations of ezrin in BAL fluid (BALF) of OVA-treated mice (OVA), OVA + anti-IgG antibody–treated mice (anti-IgG), and OVA + anti–IL-13 antibody–treated mice (anti–IL-13), and IL-13 of asthma mice and controls were measured using an ELISA. The data are presented as mean ± SEM and were analyzed by Student’s t test (control group, n = 8–15; asthma group; n = 8–17). The correlation between ezrin and IL-13 in BALF of mice was analyzed by Pearson’s correlation test. ns = not significant. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with respective controls.
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