CTSS contributes to airway neutrophilic inflammation in mixed granulocytic asthma

Abstract

Background: Mixed granulocytic asthma (MGA) is usually associated with poor response to corticosteroid therapy and a high risk of severe asthma. Cathepsin S (CTSS) has been found to play an important role in various inflammatory diseases. This study was aimed to investigate the role of CTSS in MGA.

Methods: Induced sputum was obtained from healthy subjects and asthma patients. Two murine models of MGA were established using either TDI (toluene diisocyanate) alone or OVA emulsified in CFA. LY3000328, a specific antagonist of CTSS, was therapeutically given to BALB/c mice after airway challenge with TDI or OVA. The effects of recombinant CTSS was tested in vivo, and Akt inhibition was used to explore a possible mechanism for CTSS-induced airway inflammation.

Results: MGA patients have a significant higher sputum CTSS level than the health and subjects with other inflammatory phenotypes, which was positively correlated with sputum level of soluble E-cadherin (sE-cadherin), sputum neutrophils, FeNO, FEF25-75% and glucocorticoid dosage. Allergen exposure markedly increased CTSS level and pharmacological antagonism of CTSS with LY3000328 decreased airway hyperresponsiveness, airway neutrophil accumulation, as well as the release of IL-17 and sE-cadherin in murine models of MGA, yet had no effects on eosinophilic inflammation nor type 2 inflammatory cytokines (IL-4 and IL-5). In addition, intratracheal instillation of recombinant CTSS leads to neutrophil recruitment and overproduction of sE-cadherin in the lung tissues, which could be attenuated by inhibition of Akt signaling.

Conclusion: Our data suggested that CTSS contributes to airway neutrophilic inflammation in MGA through an Akt-dependent pathway.

Keywords: Akt; CTSS; Mixed granulocytic asthma.

Fig. 1

CTSS was increased in MGA patients. Sputum CTSS (A) and sE-cadherin (B) levels were detected in healthy control (HC), and patients with mixed granulocyte asthma (MGA), paucigranulocytic asthma (PGA), eosinophilic asthma (EA) and neutrophilic asthma (NA). The data were analyzed using Kruskal-Wallis test. No significant differences of CTSS and sE-cadherin were observed among the four phenotype groups

Fig. 2

CTSS was associated with neutrophilic airway inflammation and lung function in asthma. A ~ D, Significant positive correlations were detected between sputum CTSS and the dose of inhaled corticosteroids (ICS), sputum sE-cadherin, neutrophils (Neu), FeNO. E, CTSS level was negatively correlated with the lung function parametre FEF_{25 − 75}. The data were analyzed using nonparametric Spearman correlation

Fig. 3

CTSS was upregulated in experimental models of MGA. BALF CTSS levels was increased in both TDI-esposed (A) and OVA/CFA-exposed (D) mice. n = 6 ~ 10. Whole lung homogenates from TDI (B, C) and OVA/CFA (E, F) models were subjected to western blot for analysis of CTSS expression. Its relative expression was normalized to GAPDH. n = 6. The allergen-induced upregulation of CTSS could be inhibited by LY3000328 (LY), which is a sellective antagonist of CTSS

Fig. 4

Pharmacological inhibition of CTSS with LY3000328 (LY) alleviated neutrophilic inflammation in toluene diisocyanate (TDI) -induced asthma model. A, Representative H&E stained lung sections of mice in vehicle, TDI and TDI + LY groups, as well as semi-quantitative scoring of airway inflammation (B, data analyzed using Kruskal-Wallis test), n = 8 ~ 10. Scale bar = 200 μm. C, Airway hyperresponsiveness was measured by resistance. Results were shown as percentage of baseline value. n = 4 ~ 5. D ~ F, Total cell in BALF were counted, as well as the numbers of eosinophils and neutrophils. n = 8 ~ 10. G ~ J, Levels of IL-4, IL-5, IL-17 and sE-cadherin in BALF were tested. n = 8 ~ 10. *: p < 0.05; ***: p < 0.001; ns: none significance. Data were analysed using One way ANOVA unless otherwise indicated

Fig. 5

Pharmacological inhibition of CTSS with LY3000328 (LY) alleviated airway neutrophilic inflammation in OVA/CFA -induced asthma. A, Representative H&E stained lung sections of mice in control, OVA/CFA and OVA/CFA + LY groups, as well as semi-quantitative scoring of airway inflammation (B, data analyzed using Kruskal-Wallis test), n = 8 ~ 10. Scale bar = 200 μm. C, Airway hyperresponsiveness was measured by resistance. Results were shown as percentage of baseline value. n = 4 ~ 5. D ~ F, Total cell in BALF were counted, as well as the numbers of eosinophils and neutrophils. n = 8 ~ 10. G ~ J, Levels of IL-4, IL-5, IL-17 and sE-cadherin in BALF were tested. n = 8 ~ 10. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: none significance. Data were analysed using One way ANOVA unless otherwise indicated

Fig. 6

Exogenous CTSS induce pulmonary neutrophilia through activation of Akt signaling in mice. Recombinant mouse CTSS (5 µg/mouse) were given intratracheally to the mice, with or without the Akt antagonist MK-2206 2HCl. 24 h later, mice were sacrificed for analysis of airway inflammation. A, Representative HE-stained cytospin samples from BALF (left panel, Scale bar = 50 μm) and lung sections from different treatment groups (right panel, Scale bar = 200 μm). Each represented images from 4 ~ 6 fields of at least 8 mice. B, Semi-quantitative scoring of airway inflammation in H&E-stained lung sections. The data were analyzed using Kruskal-Wallis test. n = 8 ~ 10. C ~ D, The numbers of total inflammatory cells, and neutrophils in BALF were determined. n = 8 ~ 10. E, The level of sE-cadherin in BALF were determined. n = 8 ~ 10. F, Phosphor-Akt (Ser473) was detected via western blot analysis by using whole lung homogenates from mice treated with recombinant mouse CTSS. Its relative expression was normalized to total Akt (G). n = 6. The data were analyzed using one-way ANOVA unless otherwise indicated. **: p < 0.01; ***: p < 0.001