The Neutrophil Granule Protein Olfactomedin-4 Relates to Bronchiectasis Severity and Alters Mucociliary Clearance

RATIONALE: Bronchiectasis is a chronic respiratory disease characterised by airway infection, inflammation and reduced mucociliary clearance. A previous proteomic study in patients with bronchiectasis revealed olfactomedin-4 (OLFM-4) as a dominant protein defining a cluster of patients with severe disease and frequent exacerbations. OLFM-4 is a glycoprotein found in the specific granules of a subset of human neutrophils. The aim of this study was to validate these proteomic findings in a large European cohort and to identify potential mechanism for the association with disease severity through assessment of the impact of OLFM-4 on mucociliary clearance.METHODS: Sputum samples from 227 patients with bronchiectasis from the international BRIDGE cohort study were included. OLFM-4 concentration (ug/ml) measured by ELISA was compared to Bronchiectasis Severity Index (BSI), FEV1% predicted and frequency of exacerbation. Neutrophil elastase activity, neutrophil extracellular trap assay (H1-Elastase) and the chemokine IL-8 were also measured. Cilia Beat Frequency (CBF) and mucociliary clearance were analysed on primary human nasal epithelial cells from healthy donors, cultured at an air liquid interface (n=4). CBF was recorded using high speed video microscopy and mucociliary clearance was recorded by transport of fluorescent microbeads before, during and after OLFM-4 addition at physiological concentrations (5ug/ml). RESULTS: OLFM4 is present in bronchiectasis sputum and increases with bronchiectasis severity, MILD (n=57, median (IQR) OLFM-4 conc=0.68 (0.25-1.72)), MODERATE (n=90, 1.02 (0.28-2.06) and SEVERE (n=80, 1.82(0.81-2.69). Lung function measured by FEV1% showed significant correlation to OLFM-4 concentration (r=-0.20, p<0.0001) and increased OLFM4 in sputum was related to increased number of exacerbations.OLFM4 levels correlated with the neutrophilic cytokine IL-8 and neutrophil extracellular trap assay (H1-Elastase) (r=0.54, p<0.001), (r=0.55, p <0.001) respectively. OLFM-4 addition to epithelial cells resulted in no significant acute change in CBF. However, following removal of OLFM4 by washing with phosphate buffered saline the range of CBF values significantly increased to 273.4% above baseline. Assessment of mucociliary clearance by particle image velocimetry revealed that OLFM-4 increases the cilia driven flow average velocity compared to control, OLFM-4 (33.04 +/-30.57 um/s), PBS (2.61 2.81 m/s) (n=4). CONCLUSION: Patients with severe bronchiectasis, poor lung function and high exacerbation frequency have increased sputum OLFM-4 compared to those with milder disease. Surprisingly this neutrophil glycoprotein increases ciliary beat frequency and promotes mucociliary clearance. Future work will assess the impact of OLFM-4 on neutrophil and epithelial cell function to further understand its role in bronchiectasis.