Anti-inflammatory Deficiencies in Neutrophilic Asthma

Reduced Galectin-3 and IL-1RA/IL-1 Beta

Peng Gao; Peter G Gibson; Katherine J Baines; Ian A Yang; John W Upham; Paul N Reynolds; Sandra Hodge; Alan L James; Christine Jenkins; Matthew J Peters; Jie Zhang; Jodie L Simpson

Disclosures

Respiratory Research. 2015;16(5) 

In This Article

Methods

Participant Population

Eligible adults with asthma that was sub-optimally controlled were recruited from tertiary care centers around Australia. A group of 80 adults with asthma were assessed and sputum collected for the measurement of a panel of biomarkers of inflammatory phenotype including gal-3, gal-3BP, IL-1RA, IL-1β, IL-6 and IL-8. Serum was available in a sub-group of participants for the assessment of gal-3 and gal-3BP (n = 57).

The diagnosis of asthma was established using the American Thoracic Society guidelines based upon current episodic respiratory symptoms (past 12 months), clinical diagnosis and evidence of variable airflow obstruction.[22] Participants were all prescribed inhaled corticosteroids (ICS) or combination inhaled corticosteroid/long acting bronchodilator therapy (ICS/LABA)but remained not well controlled (asthma control questionnaire 6 (ACQ6) >0.7) despite receiving this therapy. All participants underwent a clinical assessment which included history of smoking, respiratory symptoms, skin prick allergy testing and sputum induction and gave written informed consent. Ethical approval was granted by Hunter New England Human Research Ethics Committee approval number 08/11/19/3.03.

Participants were excluded if they had a post-bronchodilator FEV1 < 40% predicted, were a current smoker or an ex-smoker who had ceased smoking within the last year. Those with significant smoking related airspace disease (ex-smokers with more than 10 pack year history and DLCO/VA <70% predicted OR a smoking history >10 pack years with an exhaled carbon monoxide >10 ppm) were also excluded. Participants were assessed during a stable phase of disease with no treatment with oral corticosteroids or antibiotics, no exacerbations and no change in asthma medications over the previous four weeks.

Sputum and Blood Collection

Sputum induction and processing were performed as previously described.[23] Briefly, a fixed sputum induction time of 15 minutes with hypertonic saline (4.5%) was used for all participants. For inflammatory cell count, sputum cells were dispersed using dithiothreitol (DTT) and cells resuspended in phosphate-buffered saline (PBS).[23] The suspension was filtered and a total cell count (TCC) of leucocytes and cell viability was performed. Cytospins were prepared, stained (May-Grunwald Giemsa) and a differential cell count obtained from 400 non-squamous cells. The quality of induced sputum samples was assessed and considered adequate for samples with fewer than 50% squamous epithelial cells and more than 40% cell viability.

Blood was collected in a 9 mL EDTA tube, mixed gently and then centrifuged at 700 g for 10 minutes at room temperature and serum was stored at -80°C.

Biomarkers of Neutrophilic Inflammation

The levels of sputum IL-1β, IL-1RA, IL-6, IL-8, gal-3 (R&D Systems; Minneapolis, MS, USA) and gal-3BP (eBioscience; San Diego, CA, USA) were measured by ELISA according to the manufacturer's instructions. We have established the validity of IL-RA, gal-3 and gal-3BP and IL-6 assays for the use in induced sputum and IL-8 and IL-1β validations have been reported elsewhere.[24,25] The addition of DTT to the commercial standard showed no effect on the ELISA and all mediators showed better than 80% recovery in spiking experiments. These data are available in Additional file 1 http://respiratory-research.com/content/16/1/5/additional.

Asthma Subtype Classification

The granulocyte cut-off values used were 3% for sputum eosinophils and 61% for sputum neutrophils.[26,27] Individual patients were classified as eosinophilic asthma (EA) with sputum eosinophils ≥3% of total cells, as neutrophilic asthma (NA) with neutrophils ≥61%, as paucigranulocytic asthma (PGA) with eosinophils ≤3% and neutrophils <61% and as mixed granulocytic asthma (MGA) with eosinophils ≥3% and neutrophils ≥61%.

Sputum Immunocytochemistry

Sputum immunocytochemistry was performed as previously described (25). Briefly, cytospins were fixed in PLP fixative, dried and coated in 15% sucrose and stored at -20°C. Thawed cytospins were washed, permeabilized and blocked. Primary antibodies anti-galectin-3 (EP2775Y) and anti-LGALS3BP (3G8) (Abcam, Cambridge, UK) were added followed by secondary donkey Alexa Fluor® antibodies matched for species (Life Technologies, Carlsbad, CA, USA). Cells were mounted using ProLong® Gold Antifade Mountant with DAPI (Life Technologies). Slides were observed on an Axio Imager A1 epifluorescence microscope (Carl Zeiss MicroImaging Inc, Thornwood, NY, USA) under fluorescent optics and pictures taken using an Olympus DP70 digital microscope camera (Olympus America, Centre Valley, PA, USA). Pictures were observed visually with the inflammatory mediators represented by the colours green (gal-3) and red (gal-3BP). Colocalization was assessed as both gal-3 and gal-3BP being present in the same location identified by the presence of a yellow colour.

Statistics

IBM SPSS Statistics 17.0 was used for statistical analysis. Normally distributed data were summarized as the mean and standard deviation (SD) and more than two groups compared using ANOVA with least significant difference (LSD) post hoc testing or Student's t-test for two groups. All levels of inflammatory mediators were log-transformed and normal distribution was checked thereafter and the analysis was conducted by using analysis of variance with LSD post hoc test and adjusted for age and body mass index (BMI) because there were significant correlations between age, BMI and some inflammation mediators, such as gal-3BP and IL-1β. Non-parametric data were reported as the median and interquartile range (IQR) and analyzed by Kruskal-Wallis test followed by Bonferroni correction or the Mann-Whitney U test. Spearman's rank correlation coefficient was used to test correlations and also adjusted for age and BMI. Categorical variables were analyzed by Chi-squared test. A p value of <0.05 was considered statistically significant.

Logistic regression was used to establish whether the ratio of gal-3 to gal-3BP was a predictor of asthmatic phenotypes. We examined seven variables on the basis of the strength of the univariate associations (p < 0.2): atopy, FEV1, FVC and ratio of gal-3 to gal-3BP. Age, sex and BMI were included and other variables were added stepwise in a multivariable logistic regression model.

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