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Systemic Upregulation of Neutrophil A-Defensins and Serine Proteases In Asthma Systemic upregulation of neutrophil a-defensins and Thorax: first published as 10.1136/thx.2010.157719 on 23 July 2011. Downloaded from serine proteases in neutrophilic asthma Katherine J Baines,1,2 Jodie L Simpson,1,2 Lisa G Wood,1,2 Rodney J Scott,3 Peter G Gibson1,2 1Priority Research Centre for ABSTRACT Asthma and Respiratory Background The well-characterised airway inflammatory Key messages Diseases, The University of phenotypes of asthma include eosinophilic, neutrophilic, Newcastle, Callaghan, Australia 2Department of Respiratory and mixed eosinophilic/neutrophilic and paucigranulocytic What is the key question? Sleep Medicine, Hunter Medical asthma, identified based on the proportion of sputum < Are systemic gene expression profiles different Research Institute, John Hunter granulocytes. Systemic inflammation is now recognised between inflammatory phenotypes of asthma? Hospital, New Lambton, as an important part of some airway diseases, but the Australia 3 involvement of systemic inflammation in the What is the bottom line? Priority Research Centre of < Information Based Medicine, pathogenesis of airway inflammatory phenotypes of The neutrophilic phenotype of asthma is Hunter Medical Research asthma remains unknown. associated with increased systemic gene Institute, The University of Methods Induced sputum samples and peripheral blood expression of neutrophil a-defensins and prote- Newcastle, Callaghan, Australia were collected from participants with asthma (n¼36). ases elastase and cathepsin G. Airway inflammatory cell counts were performed from Correspondence to Why read on? Dr Katherine J Baines, Level 3, induced sputum and inflammatory phenotype assigned < HMRI, John Hunter Hospital, based on the airway eosinophil and neutrophil cut-offs of This study explores the relationship between Locked Bag 1, Hunter Region 3% and 61%, respectively. RNA was extracted from systemic gene expression, airway inflammation Mail Centre, Newcastle, NSW whole blood and gene expression profiles were and asthma phenotypes, and discovers novel 2310, Australia; katherine. systemic markers of neutrophilic airway inflam- [email protected] generated (Illumina Humanref-8 V3) and analysed using GeneSpring GX11. mation in asthma. Received 5 January 2011 Results There were six genes classified as differentially Accepted 16 June 2011 expressed between the four asthma phenotypes, Published Online First a et al4 23 July 2011 including the -defensins (DEFA) 1, 1B, 3 and 4 and have recently reported the presence of similar neutrophil proteases cathepsin G (CTSG) and elastase inflammatory phenotypes of asthma based on the fl (ELA2). Systemic expression of DEFA1,1B,3,4,CTSG and in ammatory cell numbers present in peripheral http://thorax.bmj.com/ ELA2 was significantly higher in the neutrophilic asthma blood, suggesting there may also be systemic (NA) phenotype. Microarray results of the a-defensins manifestations of the airway inflammatory and neutrophil proteases were successfully validated response in asthma. using real-time PCR. Plasma elastase was significantly The underlying biology of asthma inflammatory increased in people with neutrophilic airway phenotypes has not been fully characterised. A Th2- inflammation. mediated defect forms part of EA, and it is thought Conclusion There is systemic upregulation of that dysregulation of the innate immune response a-defensin and neutrophil protease expression in NA, involving neutrophils is important in the patho- on September 23, 2021 by guest. Protected copyright. which may represent proinflammatory effects on the genesis of NA.5 6 We have previously shown that bone marrow and the release of immature neutrophils NA and EA can be identified by distinct gene into the circulation. This demonstrates a systemic expression profiles in peripheral blood neutrophils, inflammatory component in NA that further differentiates and that enhanced systemic neutrophil migration this from other asthma phenotypes and indicates and survival may be involved in the pathogenesis of different mechanisms in NA. NA.5 In addition to identifying a neutrophil defect in asthma, these studies also suggested that there may be systemic inflammation in asthma. In other airway diseases such as chronic obstructive INTRODUCTION pulmonary disease, low-grade systemic inflamma- Asthma is now well recognised to be a heteroge- tion is recognised as a key pathogenic feature of the neous disease, whether assessed using clinical disease.7 factors, causative triggers, response to treatments or However, it is unclear if there is a relationship e types of inflammation present.1 3 The measure- between airway and systemic inflammation in ment of cellular patterns of inflammation present in asthma, and whether systemic inflammation is induced sputum samples has proved to be useful in related to certain inflammatory phenotypes of identifying inflammatory phenotypes of asthma. asthma. It is thought that airway inflammation We have previously described four distinct cellular can ‘spillover’ into the circulation and this can subtypes of asthma based on the presence or produce low-grade systemic inflammation.8 absence of sputum granulocytesdnamely, eosino- However, this concept is challenged by several philic asthma (EA), neutrophilic asthma (NA), studies in chronic obstructive pulmonary disease mixed eosinophilic and neutrophilic asthma (ME/ that found no correlation between mediator levels NA) and paucigranulocytic asthma (PGA).3 Nadif in the sputum and plasma,9 suggesting that 942 Thorax 2011;66:942e947. doi:10.1136/thx.2010.157719 Asthma DEFA1 DEFA1B DEFA3 pulmonary and systemic responses may be modulated sepa- , and owing to the high homology Thorax: first published as 10.1136/thx.2010.157719 on 23 July 2011. Downloaded from rately. Furthermore, the range of markers used to characterise between the genes. PCR primers and probes were combined systemic inflammation has been limited predominantly to C with Taqman gene expression master mix according to the reactive protein and fibrinogen. Better characterisation of manufacturer’s instructions in Singleplex real-time PCRs (7500 systemic pathways involved in asthma may identify potentially Real-Time PCR System, Applied Biosystems). The results were ÀDD important mechanisms. This project investigated gene expres- calculated using 2 Ct relative to both the housekeeping gene sion profiles of the inflammatory cells of the blood and related (b-actin) and the mean DCt of all samples. these to inflammatory phenotypes of asthma defined by airway inflammation. Quantification of protein levels of plasma neutrophil elastase Levels of active neutrophil elastase (NE) were measured in METHODS corresponding available plasma samples (n¼26) using the Inno- Participants zyme Human Neutrophil Elastase Immunocapture Activity Assay Adults with stable asthma were recruited from the John Hunter Kit (Calbiochem-EMD4biosciences, San Diego, California, USA) Hospital Asthma Clinic, NSW, Australia. Asthma was diagnosed according to the manufacturer’s instructions. The standard curve according to American Thoracic Society guidelines based upon used to detect plasma NE activity ranged from 31.25 pg/ml to current (past 12 months) episodic respiratory symptoms, 2000 pg/ml; samples were run in duplicate and the fluorescence doctor’s diagnosis (ever) and demonstrated evidence of airway was measured at 360 nm (excitation) and 440 nm (emission). hyper-responsiveness to hypertonic saline. Exclusion criteria included recent (past month) respiratory tract infection, recent Asthma subtype classification asthma exacerbation, recent unstable asthma or change in The granulocyte cut-off values used were 3% for sputum maintenance therapy and current smoking (or past smoking eosinophils and 61% for sputum neutrophils.3 10 Participants within 6 months of cessation). All participants gave written with a sputum eosinophil count of >3% alone were classified as informed consent prior to their inclusion in the study and the having EA, those with sputum eosinophils >3% and sputum Hunter New England Area Health Service and The University of neutrophils >61% were classified as having ME/NA, participants Newcastle Research ethics committees approved the study. with sputum neutrophils >61% and sputum eosinophils <3% were classified as having NA and those with sputum eosinophils Sputum induction and analysis <3% and sputum neutrophils <61% were classified as having Spirometry (KoKo PD Instrumentation, Louisville, Colorado, PGA. USA) and sputum induction with hypertonic saline (4.5%) were performed. A fixed sputum induction time of 15 min was used Statistical analysis for all participants. For inflammatory cell counts, selected Clinical and cell count data were analysed using Stata 9 (Stata sputum (sputum portions separated from saliva) was dispersed Corporation, College Station, Texas, USA) and reported as mean using dithiothreitol. The suspension was filtered and a total (SD) for normally distributed data and median (Q1, Q3) for non- http://thorax.bmj.com/ cell count of leucocytes and cell viability was performed. parametric data. Statistical comparisons were performed using Cytospins were prepared, stained (MayeGrunwald Giemsa) and the multiple comparison ANOVA test for normally distributed a differential cell count obtained from 400 non-squamous cells. parametric data (p<0.05 considered significant) with post-hoc tests between the four groups adjusting for multiple compari- Whole genome gene expression microarrays sons using the Bonferroni method (p<0.013). The Krus- Four millilitres of blood
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