Interleukin-31 expression and relation to disease severity in human asthma

Abstract

Interleukin 31 (IL-31) is a novel T helper type 2 effector cytokine that plays an important role in the pathogenesis of allergic diseases. However, its role in human asthma remains unclear. The aim of this study was to measure IL-31 levels in the serum, bronchoalveolar lavage fluid (BALF) and bronchial tissue of asthmatics and healthy subjects, and identify its possible correlation to disease severity. We quantified IL-31 levels in the serum of patients with asthma (n = 44), as well as in controls (n = 22). Of these subjects, 9 asthmatics and five controls underwent bronchoscopy with endobronchial biopsy and BALF collection. Our data showed that serum and BALF IL-31 levels were significantly elevated in patients with asthma compared with controls. Expressions of IL-31 and IL-31 receptor (IL-31RA and OSMR) were more prominent in the bronchial tissue in severe compared to mild asthma and controls. Serum IL-31 levels correlated positively with Th2 related cytokines (IL-5, IL-13, and TSLP), asthma severity or total serum immunoglobulin E (IgE), and inversely with asthma control and the forced expiratory volume in 1 second (FEV1). The current data may provide insight into the underlying pathogenesis of asthma, in which IL-31 has an important pathogenic role.

Figure 1. Serum IL-31 levels in patients with asthma and controls.

The levels of serum IL-31 were assessed in patients with asthma (n = 44) and controls (n = 22) as measured by ELISA. (A) Serum IL-31 was increased in patients with asthma compared with the controls; (B) When patients were stratified according to the status of atopy, the serum IL-31 levels in patients with allergic asthma (n = 23) were higher than those in patients with nonallergic asthma (n = 21) and controls; (C) Serum IL-31 was increased in severe asthma patients (n = 22) compared with mild asthma patients (n = 22) and controls; (D) Receiver operating characteristic (ROC) curve for distinguishing patients with asthma from the controls. The area under the ROC curve was 0.791 (95% CI: 0.66 to 0.86). Data are presented as median with interquartile range. *p < 0.05, ***p < 0.001.

Figure 2. IL-31 levels in the bronchoalveolar lavage fluid (BALF).

(A) BALF samples were measured by ELISA and the BALF IL-31 levels in patients with asthma (n = 9) were significantly increased compared with the controls (n = 5); (B) Spearman’s rank correlation analysis showed the serum IL-31 levels correlated positively with BALF IL-31 levels (r = 0.63; p = 0.016). Data are presented as median with interquartile range. **p < 0.01.

Figure 3. Serum IL-31 levels in asthmatic patients correlate with the Th2 cytokines.

The classical Th2 cytokines such as IL-5, IL-13 and thymic stromal lymphopoietin (TSLP) were further measured by ELISA. The levels of serum IL-5, IL-13 and TSLP were significantly higher in patients with asthma (n = 44) compared with control subjects (n = 22) (A–C). Spearman’s rank correlation analysis showed a significant correlation between the serum IL-31 levels and the serum IL-5 levels (r = 0.34, p = 0.024), the serum IL-13 levels (r = 0.43, p = 0.003), the serum TSLP levels (r = 0.42, p = 0.004) (D–F). Data are presented as median with interquartile range. **p < 0.01, ***p < 0.001.

Figure 4. Serum IL-31 levels in relation to asthma control.

Patients were classified into three groups based on ACT scores. Patients with uncontrolled asthma (n = 15) had statistically higher serum IL-31 levels than patients with partly controlled (n = 18) and completely controlled asthma (n = 11) (A). The correlation between serum IL-31 levels and ACT score was assessed using Spearman’s rank correlation analysis (B). Data are presented as median with interquartile range. **p < 0.01, ***p < 0.001.

Figure 5. Serum IL-31 levels in relation to other clinical parameters.

Spearman’s rank correlation analysis showed a significant correlation between the serum IL-31 levels. (A) The total serum immunoglobulin (Ig) E levels (r = 0.437, p < 0.01); (B) forced expiratory volume in 1 s (FEV₁)/predicted value (r = −0.431, p < 0.01); (C) There was no significantly relationship between serum levels of IL-31 and percentage (%) of peripheral blood eosinophils (r = 0.254, p = 0.096).

Figure 6. Immunohistochemical analysis of IL-31.

Bronchial tissue obtained from healthy subjects (n = 5) and asthmatic patients (n = 9) who underwent fibreoptic bronchoscopy was processed immunohistochemically to show tissue IL-31 expression. Specific staining for IL-31 is brown (red arrows), whereas nuclei are stained blue (Magnification × 400). Representative photomicrographs are presented from (A) a healthy individual and (B) a patient with mild asthma and (C) patient with severe asthma and (D) the corresponding isotypic control. The expression of IL-31 in patients with severe asthma were higher than those in controls and paitents with mild asthma (E) Moreover, the percentage (%) of cells expressing IL-31 correlated positively with serum IL-31 levels (r = 0.60; p = 0.022, (F)) and BALF IL-31 levels (r = 0.61; p = 0.019, (G)). Data are presented as median with interquartile range. *p < 0.05, **p < 0.01.

Figure 7. Immunohistochemical analysis of IL-31 receptor (IL-31RA and OSMR).

Immunohistochemical staining was performed to assess the expression of IL-31RA (A–D) and OSMR (E–H). (Magnification × 400, red arrows). Representative photomicrographs are presented from a healthy individual (A,E), patient with mild asthma (B,F), patient with severe asthma (C,G) and the corresponding isotypic control (D,H). The expression of IL-31RA and OSMR in patients with severe asthma were higher than those in controls and patients with mild asthma (I,K). Moreover, the relationships between the serum IL-31 levels and percentage (%) of cells expressing of IL-31RA and OSMR were assessed by Spearman’s rank correlation analysis (J,L). Data are presented as median with interquartile range. *p < 0.05, **p < 0.01, ***p < 0.001.