Serum Zonulin Is a Biomarker for Severe Asthma

Abstract

Zonulin is a regulator of epithelial and endothelial barrier function. It regulates intestinal permeability through disrupting tight junctions. Defective epithelial barrier function is a hallmark of airway inflammation in asthma. This study aimed to investigate the role of zonulin in the pathogenesis of severe asthma. We enrolled 56 adult patients with asthma (29 severe asthma and 27 mild-to-moderate asthma) and 33 normal controls. The clinical data, sera, and lung tissues of the patients were provided by the Cohort for Reality and Evolution of adult Asthma in Korea (COREA) and the Biobank of Soonchunhyang University Bucheon Hospital, South Korea. Serum zonulin levels were estimated using an enzyme-linked immunosorbent assay, and zonulin expression in the bronchial tissue was evaluated by immunohistochemical staining. The serum zonulin levels were significantly higher in patients with severe asthma (51.98 ± 19.66 ng/mL) than in those with mild-to-moderate asthma and normal controls (26.35 ± 13.70 vs. 17.26 ± 10.29 ng/mL, P < 0.001). They significantly correlated with percent predicted forced expiratory volume in one second (%FEV1) (r = −0.35, P = 0.009). The zonulin expression in the bronchial epithelium was greater in patients with severe asthma. A serum zonulin cutoff value to distinguish between severe and mild-to-moderate asthmatics was 38.83 ng/mL. Zonulin may play an important role in the pathogenesis of severe asthma, and serum zonulin could be a potential biomarker for severe asthma.

INTRODUCTION

Asthma is a chronic airway inflammation characterized by reversible airflow obstruction, affecting over 300 million people worldwide.¹ Although it is well established that type 2 cell immunity plays an important role in asthma, defects in the epithelial barrier can also be key components involved in the pathogenesis of asthma. Functional damage to the airway epithelium increases the airway mucosal permeability, subsequently causing allergens to easily enter the barrier.²,³

Zonulin, a prehaptoglobulin protein, is a modulator of intercellular tight junctions and was first reported as a key regulator of intestinal permeability through disrupting tight junctions. Zonulin has been proposed to play a role in the pathogenesis of several chronic inflammatory diseases such as allergic, autoimmune, and metabolic diseases.⁴,⁵ Previous studies have demonstrated that an increase in serum zonulin level is associated with obesity, hyperlipidemia, and atopic dermatitis.⁶,⁷ A recent study has revealed high levels of serum zonulin in house dust mite-sensitized allergic asthmatic patients as well as a significant positive correlation between zonulin levels and asthma severity.⁸

In this study, we investigated the role of zonulin as a potential biomarker for severe asthma by measuring the serum zonulin concentration and expression in the bronchial epithelium of patients with severe and mild-to-moderate asthma and by comparing them with those of normal controls.

MATERIALS AND METHODS

Study design and patient selection

We conducted a retrospective analysis of prospectively collected data. This study enrolled 56 patients with asthma (29 with severe asthma and 27 with mild-to-moderate asthma) and 33 normal healthy controls (21.2% males; mean age ± standard deviation [SD], 36.88 ± 16.59 years). All asthmatic patients were clinically stable and without exacerbations for at least 1 month prior to enrollment. All normal controls were nonatopic without any allergic diseases. Clinical data, sera, and bronchial tissues of the patients were provided by the Cohort for Reality and Evolution of adult Asthma in Korea (COREA) and the Biobank of Soonchunhyang University Bucheon Hospital, South Korea, a member of the Korea Biobank Network (KBN). The COREA was established in 2005 and included 21 university hospitals in Korea. All patients were ethnic Koreans aged ≥18 years, and the COREA is a prospective and observational multicenter cohort that reflects the real-world situation without artificial interventions during the enrollment and follow-up periods. KBN is a national biobank network comprising the Korea Centers for Disease Control and Prevention, the National Biobank of Korea, and human bioresource regional banks located at 17 university hospitals. This study was approved by the Institutional Review Board of Hallym University Dongtan Sacred Heart Hospital, South Korea (HDT 2018-07-001).

Data collection and definitions

The diagnosis of asthma was based on a patient having a positive bronchodilator test of either at least 200 mL and 12% in forced expiratory volume in one second (FEV1) or at least a moderate bronchial hyperreactivity (provocative concentration resulting in 20% decrease in FEV1 [PC20] ≤ 16 mg/mL methacholine). We classified the severity of asthma into 2 groups (severe and mild-to-moderate), and compared them with normal controls. Patients with mild-to-moderate asthma were defined as those who were well-controlled with low-to-medium doses of inhaled corticosteroids (ICS) with or without long-acting inhaled beta₂-agonists (LABA) and/or leukotriene receptor antagonists (LTRA). Patients with severe asthma were defined as (1) those who required treatment with high-dose ICS and additional daily treatment with controller medications, such as LABA and/or LTRA, to maintain a well-controlled state of asthma during the previous year and (2) those whose asthma remained in a ‘uncontrolled’ state despite treatments. The cumulative dose of systemic corticosteroids was defined as the total prednisolone-equivalent dose taken by the patient for 1 year after the enrollment. Atopy was defined as ≥ 1 positive reactions to 12 common inhalant allergens by allergy skin prick tests or simultaneous multiple-allergen tests: Dermatophagoides pteronyssinus, Dermatopagoides farinae, birch, oak, grass mix, ragweed, mugwort, Japanese hop, Alternaria, Aspergillus, and dog/cat epithelium.

Measurement of serum zonulin levels and zonulin expression in the bronchial tissue

Serum zonulin levels were estimated using an enzyme-linked immunosorbent assay (ELISA) kit (Immundiagnostik AG, Bensheim, Germany) according to the manufacturer’s instructions.

Immunohistochemical (IHC) staining was performed for haptoglobin using the BenchMark XT automated immunostaining system (Ventana Medical System, Tucson, AZ, USA). Bronchial mucosal tissues from patients with severe asthma (n = 2), mild-to-moderate asthma (n = 2), and normal controls (n = 3) were obtained from the biobank (clinical data are summarized in Supplementary Table S1). The tissues collected were preserved in a defreezer at −70°C. Four-micrometer-thick sections from each formalin-fixed paraffin-embedded block were mounted on positively charged slides and dried for 1 hour at 60°C. After deparaffinization, pretreatment for antigen retrieval was performed for 60 minutes in Cell Conditioning 1 (Ventana #950-124, pH 8.0; Ventana Medical System) within an autostainer. The samples were incubated with anti-human haptoglobin primary antibody (rabbit polyclonal antibody, 1:300, #PA5-79391; Invitrogen, Carlsbad, CA, USA) for 16 minutes at 37°C and then treated with the OptiView DAB kit for 8 minutes at 37°C. The slides were counterstained with hematoxylin (Ventana #760-2021; Ventana Medical System) for 8 minutes at 37°C.

Statistical analysis

Data are expressed as mean ± SD or median and interquartile range after normality tests using the Shapiro–Wilk normality test. Analysis was performed using Student’s t-test or the Mann–Whitney U test for continuous variables and the Pearson χ² test or Fisher’s exact test for categorical variables for comparison between the mild-to-moderate and severe asthma groups. One-way analysis of variance test, followed by Games-Howell post hoc analysis, was used to compare the difference among the 3 groups. Spearman’s rank test was used to assess the correlations between variables. Logistic regression test was used for the control of age and sex as covariates. Receiver operating characteristic (ROC) curves were analyzed to establish an optimal cutoff value to distinguish between severe and mild-to-moderate asthmatics. All statistical analyses were performed with R version 4.0.2. Statistical significance was set at P < 0.05.

RESULTS

Characteristics of the study participants

The demographic and clinical characteristics of the study participants are shown in Table. The severe asthma group had significantly more male patients than the mild-to-moderate asthma group (51.7% vs. 25.9%; P = 0.048). The proportions of atopy were 44.8% and 57.7%, respectively, in the severe and mild-to-moderate asthma groups (P > 0.05). No significant differences in age, smoking history, prevalence of rhinitis and atopy, serum total immunoglobulin E (IgE), blood eosinophil counts, or body mass index were observed between the patient groups. In addition, there was no history of inflammatory bowel diseases in either patient group that could affect serum zonulin levels (data not shown).

Table. Clinical characteristics of the study population.

Variables		Severe asthma (n = 29)	Mild-to-moderate asthma (n = 27)	P value
Male		15 (51.7)	7 (25.9)	0.048
Age (yr)		50.03 ± 13.97	46.33 ± 14.85	0.341
BMI (kg/m2)		25.0 [22.5–28.4]	23.6 [21.8–25.9]	0.251
Smoking				0.063
	Current	2 (6.9)	6 (22.2)
	Former	8 (27.6)	2 (7.4)
	Never	19 (65.5)	19 (70.4)
Smoking intensity (pack-year)		12.20 ± 7.64	12.0 ± 9.29	0.961
Asthma duration (yr)		2.0 [0.5–10.0]	1.0 [0.1–3.0]	0.194
Number of asthma exacerbations (per year)		1.0 [1.0–3.0]	1.0 [0.0–1.0]	0.099
Rhinitis (%)		16 (55.2)	19 (70.4)	0.369
Atopy (%)		13/29 (44.8)	15/26 (57.7)	0.495
Serum total IgE (IU/mL)		498.9 [126.8–1116.0]	195.5 [99.6–322.4]	0.186
Blood eosinophil count (/mm3)		543.5 [209.0–760.0]	273.5 [111.0–470.0]	0.061
Pre-BD FVC (% pred)		80.0 [70.0–85.0]	91.0 [85.0–98.0]	< 0.001
Pre-BD FEV1 (% pred)		63.0 [46.0–74.0]	90.0 [76.5–98.0]	< 0.001
Pre-BD FEV1/FVC (%)		61.24 ± 13.73	75.85 ± 13.67	< 0.001
Delta FEV1 (%)		8.7 ± 15.8 (21)	4.9 ± 8.8 (21)	0.338
Amount of systemic corticosteroids (prednisolone equivalent, mg/year)		188.0 [90.0–321.0]	130.0 [0.0–210.0]	0.190
ICS dose				< 0.001
	High	29 (100.0)	0
	Medium	0	15 (55.6)
	Low	0	12 (44.4)
Asthma medication in addition to ICS
	LABA	27 (93.1)	25 (92.6)	1
	LTRA	25/28 (89.3)	21/24 (87.5)	1
	LAMA	13 (44.8)	3 (11.1)	0.013

Values are shown as numbers (%), means ± standard deviation, and median [interquartile range]. In cases of missing data, the number of patients for available was indicated in parentheses next to the value.

BMI, body mass index; BD, bronchodilator; Delta FEV1, change in FEV1 after bronchodilator; FEV1, forced expiratory volume in one second; FVC, forced vital capacity; ICS, inhaled corticosteroids; IgE, immunoglobulin E; LABA, long-acting bronchodilator; LTRA, leukotriene receptor antagonist; LAMA, long-acting muscarinic antagonist.

Patients with severe asthma had lower lung function parameters, such as forced vital capacity (FVC), %FEV1 (63.0% vs. 90.0%, P < 0.001), and FEV1/FVC. Although patients with severe asthma were treated with higher cumulative doses of systemic corticosteroids than those with mild-to-moderate asthma, this difference was not statistically significant. However, they were treated with more long-acting muscarinic antagonists in addition to inhaled corticosteroids (P = 0.013).

Serum zonulin levels in asthmatic patients

The serum zonulin levels were significantly higher in patients with severe asthma than in those with mild-to-moderate asthma (51.98 ± 19.66 vs. 26.35 ± 13.70 ng/mL, P < 0.001) and normal controls (17.26 ± 10.29 ng/mL, P < 0.001; Fig. 1). The serum zonulin levels were also significantly higher in mild-to-moderate asthmatics than in normal controls (P = 0.017, Fig. 1). The statistical significance remained after controlling for age, sex, and atopic status using logistic regression analysis (P < 0.001 for severe asthmatics vs. mild-to-moderate asthmatics; P < 0.001 for severe asthmatics vs. normal controls; P = 0.022 for mild-to-moderate asthmatics vs. normal controls).

Fig. 1. Serum zonulin levels in the study participants. Serum zonulin levels were significantly higher in patients with severe asthma than in those with mild-to-moderate asthma (P < 0.001) and in NC (P < 0.001).

NC, normal controls.

The serum zonulin levels negatively correlated with %FEV1 (r = −0.35, P = 0.009; Fig. 2). The statistical significance remained after controlling for age and sex using logistic regression analysis (P = 0.011). However, the serum IgE levels or blood eosinophil counts did not significantly correlate with the serum zonulin levels (P > 0.05).

Fig. 2. Correlation between FEV1 and serum zonulin levels in the study participants (n = 56; Spearman’s r = −0.35; P = 0.009).

FEV1, forced expiratory volume in one second; BD, bronchodilator.

Fig. 3 shows the ROC curve used to determine a zonulin cutoff level for severe asthma and the area under the curve 0.883 for zonulin. A serum zonulin level to distinguish between severe and mild-to-moderate asthmatics was 38.83 ng/mL, which had a sensitivity of 88.9% and a specificity of 72.4%.

Fig. 3. Receiver operating characteristic curve analysis to determine a serum zonulin cutoff value to distinguish between severe and mild-to-moderate asthmatics. The receiver operating characteristic curve analysis shows that a serum zonulin cutoff value to distinguish between severe and mild-to-moderate asthmatics was 38.83 ng/mL, with a sensitivity of 0.889 and a specificity of 0.724 (AUC, 0.883; 95% CI, 0.795–0.970; P = 0.008).

AUC, area under the curve; CI, confidence interval.

Zonulin expression in the bronchial epithelium

Zonulin expression was identified by IHC staining in the bronchial epithelium of patients with asthma (Fig. 4). Higher zonulin expression was found in patients with severe asthma than that in those with mild-to-moderate asthma, but the zonulin expression was undetectable in normal controls.

Fig. 4. Representative immunohistochemical staining micrographs of zonulin expression in the bronchial epithelium. The staining reveals no definite zonulin expression in the bronchial epithelium of normal control (A), weak expression for mild-to-moderate asthma (B), and strong expression (C) for severe asthma (×400).

DISCUSSION

Abnormality in the epithelium is a topic of interest in the pathogenesis of allergic diseases. Impaired barrier function caused by defects in epithelial tight junctions allows environmental triggers, such as allergens, microbes, and irritants, to pass more easily into the airways and then interact with the immune and inflammatory cells.⁹ Damaged epithelium leads to the release of growth factors that promote airway remodeling, leading to persistent chronic airway inflammation.²,³ Epithelial barrier dysfunction is a hallmark of airway inflammation in asthma, especially in severe asthma.

Zonulin is one of the few physiological mediators of paracellular intestinal permeability and has been linked with the development of several chronic inflammatory disorders (CIDs).¹⁰ The inappropriate upregulation of zonulin causes functional loss of the epithelial barrier, which leads to a break in tolerance with subsequent development of a CID,⁵ including inflammatory bowel disease, Crohn’s disease,¹¹ irritable bowel syndrome,¹² and type 1 diabetes.¹³,¹⁴ It has also been reported that patients with asthma have increased serum zonulin levels and intestinal permeability,¹⁵,¹⁶ which might activate the immune system leading to lung inflammation.¹⁷

Zonulin mediates disengagement of the protein zonula occludens 1 (ZO-1) from the tight junction protein complex.¹⁸ Based on bronchial biopsies, Boer et al.¹⁹ have previously reported lower expression of ZO-1 in patients with asthma than in normal controls. Upregulated zonulin reduces the expression of ZO-1 and induces tight junction disruption, thereby increasing the intracellular permeability. Baioumy et al.⁸ have demonstrated a significant positive correlation between serum zonulin levels and asthma severity in patients with house dust mite-sensitized asthma. The authors suggested that zonulin reflects an increase in intestinal permeability and acts as a biomarker of asthma severity. Consistent with previous research, we found that patients with asthma had a significantly higher level of serum zonulin than in normal controls. We also found a significant association between the serum zonulin level and asthma severity. Our results revealed a serum zonulin cutoff value of 38.83 ng/mL to distinguish between severe and mild-to-moderate asthmatics. Baioumy et al. ⁸ have recently reported the cutoff level of serum zonulin in patients with house dust mite-sensitized severe asthma as 198 ng/mL. The difference in sensitivity between the 2 studies could have been attributed to the ELISA kits from different manufacturers (with varying sensitivity thresholds) being used in the studies. Further studies are needed to validate and standardize the serum zonulin cutoff value as a biomarker of asthma severity.

Although a previous study reported a significant increase in serum zonulin levels in patients with asthma,⁸ few previous studies have shown zonulin expression in the bronchial epithelium. Sheen et al.⁷ have reported increased serum zonulin levels in patients with atopic dermatitis and zonulin expression in the epidermis of their skin lesions, which was related to the severity of atopic dermatitis. To our knowledge, this is the first report that zonulin expression was significantly increased in the bronchial epithelium of patients with asthma, which was especially stronger in those with severe asthma than in those with mild-to-moderate asthma. Although we did not investigate the intestinal permeability in this study, it is conceivable that the primary general mucosal defects presenting simultaneously in several organs are expressed clinically in a single organ after antigenic or environmental stimulation.¹⁵ Further studies are needed to elucidate these mechanisms.

The pathogenesis of asthma associated with barrier defects reveals novel approaches to treatments focused on improving the resistance of the airways to environmental allergens rather than simply controlling inflammation.²,²⁰ AT1001, a peptide inhibitor of zonulin, has been studied for the management of celiac disease and found to ameliorate intestinal barrier function, gastrointestinal symptoms, and production of proinflammatory cytokines, such as interferon-gamma.²¹,²² The levels of serum zonulin reflect not only intestinal secretion, but also secretion from several extra-intestinal tissues, including the lungs.²³ Therefore, a deeper understanding of the zonulin pathway may serve to identify novel therapeutic targets for restoration of airway epithelial integrity in asthmatics.

This study, however, has a few limitations. First, the number of study participants was relatively small. Secondly, the present study did not adjust for the confounding factors, including other allergic or autoimmune diseases. Diseases such as inflammatory bowel disease or Crohn’s disease, which are known to elevate zonulin expression, were not studied. Thirdly, although the expression of zonulin in the bronchial epithelium was analyzed using IHC staining, the intensity of zonulin expression was not quantified in this study. Finally, unlike in the previous studies, the intestinal barrier function was not evaluated.¹⁵

In this study, we demonstrated that serum zonulin levels were significantly increased in asthma and expressed in the bronchial epithelium of patients with asthma, and that the serum zonulin level and bronchial epithelial zonulin expression positively correlated with asthma severity. These results suggest that zonulin may play a substantial role in the pathogenesis of severe asthma, which can be a potential biomarker for severe asthma.

SUPPLEMENTARY MATERIAL

Supplementary Table S1

Clinical characteristics of the study participants