Frauke Pedersen

Neutrophil extracellular trap formation and extracellular DNA in sputum of stable COPD patients

INTRODUCTION Chronic obstructive pulmonary disease (COPD) is characterized by neutrophilic airway inflammation. Neutrophil extracellular trap (NET) formation - a meshwork of neutrophil DNA components and neutrophil enzymes are involved in innate immunity and inflammation. Little is known about the presence of these structures in induced sputum from stable COPD patients. METHODS Induced sputum samples of 23 COPD patients and 10 healthy controls were collected. Sputum cells were harvested, cultivated and stained for NET components. Extracellular DNA was quantified using a NanoDrop 2000 spectrophotometer. RESULTS NET formation was markedly upregulated in COPD sputum compared with healthy controls, irrespective of sputum purulence or smoking status. NET formation was associated with significantly higher concentration of extracellular DNA in sputum supernatant (484 ng/μl in COPD versus 268 ng/μl in controls, p = 0.013). Log-transformed extracellular DNA correlated with log-transformed absolute neutrophil numbers in sputum (r = 0.60; p < 0.001) and airway obstruction (r = -0.43; p = 0.013). CONCLUSION NET formation associated with higher concentrations of extracellular DNA may be a pathobiological feature of COPD-derived sputum neutrophils.

The anti-IL-17A antibody secukinumab does not attenuate ozone-induced airway neutrophilia in healthy volunteers

To the Editor: Within the lung the T-helper cell interleukin (IL)-17 (also referred to as IL-17A) is recognised as one of the principal cytokines that links the activation of T-lymphocytes to the chronic accumulation of neutrophils. It has been proposed that IL-17A is a relevant pharmacological target in inflammatory lung diseases with sustained mobilisation of neutrophils [1]. Secukinumab (AIN457; Novartis Healthcare Pvt. Ltd, Basel, Switzerland) is a recombinant high-affinity fully human monoclonal IL-17A antibody of the immunoglobulin (Ig)G1κ isotype, which binds to human IL-17A and neutralises the bioactivity of this cytokine [2]. Ozone exposure in healthy volunteers induces an acute and reproducible neutrophilic airway inflammation, which can be utilised as a pharmacological model to test anti-inflammatory drugs in early development [3–5]. We performed a single centre, phase II, double-blind, placebo-controlled, parallel-group study with an open label reference arm, investigating the ability of secukinumab to attenuate airway neutrophilia in induced sputum 24 h and 48 h following ozone exposure in healthy volunteers. Subjects underwent a 3 h ozone challenge, and induced sputum samples were collected 24 h and 48 h after the challenge. Subjects with an inflammatory response to ozone (as reflected by an increase in the total number of sputum neutrophils by at least 50% compared with the screening visit) were then randomised to receive either secukinumab (10 mg·kg−1 bodyweight) or placebo, in a double blind fashion administered as an infusion over 120 mins or to an open-label single-dose oral corticosteroid treatment (OCS, 50 mg) in the ratio of 2:1:1 (secukinumab:placebo:OCS). The secukinumab/placebo treatment period consisted of a single dosing visit on day 8 followed by an ozone challenge 1 week later on day 15 and subsequent …

Multi-analyte profiling of inflammatory mediators in COPD sputum--the effects of processing.

Prior to using a new multi-analyte platform for the detection of markers in sputum it is advisable to assess whether sputum processing, especially mucus homogenization by dithiothreitol (DTT), affects the analysis. In this study we tested a novel Human Inflammation Multi Analyte Profiling® Kit (v1.0 Luminex platform; xMAP®). Induced sputum samples of 20 patients with stable COPD (mean FEV1, 59.2% pred.) were processed in parallel using standard processing (with DTT) and a more time consuming sputum dispersion method with phosphate buffered saline (PBS) only. A panel of 47 markers was analyzed in these sputum supernatants by the xMAP®. Twenty-five of 47 analytes have been detected in COPD sputum. Interestingly, 7 markers have been detected in sputum processed with DTT only, or significantly higher levels were observed following DTT treatment (VDBP, α-2-Macroglobulin, haptoglobin, α-1-antitrypsin, VCAM-1, and fibrinogen). However, standard DTT-processing resulted in lower detectable concentrations of ferritin, TIMP-1, MCP-1, MIP-1β, ICAM-1, and complement C3. The correlation between processing methods for the different markers indicates that DTT processing does not introduce a bias by affecting individual sputum samples differently. In conclusion, our data demonstrates that the Luminex-based xMAP® panel can be used for multi-analyte profiling of COPD sputum using the routinely applied method of sputum processing with DTT. However, researchers need to be aware that the absolute concentration of selected inflammatory markers can be affected by DTT.

HOPE-preservation of paraffin-embedded sputum samples–A new way of bioprofiling in COPD

Induced sputum is a non-invasive sampling technique for the analysis of airway inflammation in various lung diseases and comprises valuable potential for the identification of biomarkers and therapeutic targets by molecular methods. In the context of biobanking with preservation of induced sputum samples for subsequent analyses we applied the HEPES-glutamic acid buffer-mediated organic solvent protection effect (HOPE)-technique for preparation of induced sputum samples. Induced sputum samples of 20 patients with moderate to severe chronic obstructive pulmonary disease (COPD) and 12 healthy controls were collected. Cell pellets of induced sputum samples were preserved with HOPE and subsequently embedded in paraffin. Immunostaining of paraffin-block sections for interleukin-8, interleukin-17, myeloperoxidase, matrixmetalloproteinase-9, CD68, and CD8 revealed distinct signals without antigen retrieval. Moreover, RNA was extracted and successfully used for transcription microarray analysis. Sputum samples preserved by the HOPE-technique display a tool to address scientific approaches in pulmonary research, which can enable the identification of new biomarkers and therapeutic targets in respiratory diseases.

Effects of the CXCR2 antagonist AZD5069 on lung neutrophil recruitment in asthma

Increased airway neutrophils with defective apoptosis are prevalent in a subset of patients with severe asthma [1], and excessive neutrophil recruitment and activation via CXCR2 receptors may contribute to the disease pathophysiology [2]. CXCR2 antagonism has been shown to reduce sputum neutrophilia in various human airway challenge models [3,4] and in patients with bronchiectasis [5], COPD [6] and asthma [7]. However, it is currently unknown, whether the decrease of sputum neutrophils is accompanied by reduction of neutrophils in the lung tissue. AZD5069 is a selective small-molecule antagonist of the human CXCR2 chemokine receptors with >100-fold selectivity over CXCR1 receptors [8], that does not adversely affect neutrophilmediated host immunity [9,10]. Here, we studied the distribution of neutrophils in three distinct compartments, namely bronchial mucosal tissue, induced sputum and blood after CXCR2 blockade by AZD5069, in patients with moderate persistent neutrophilic asthma defined by a sputum neutrophil cut-off of >50%. We performed an exploratory, single centre, open-label, noncontrolled, pilot study with oral twice daily (BD) administration of 45 mg AZD5069 (90 mg/day) over 4 weeks. Co-primary endpoints were neutrophil scores/cell counts in bronchial mucosa, induced sputum, and blood (ClinicalTrials.gov Identifier: NCT01890148). Patients with persistent neutrophilic asthma (>50% neutrophils in sputum) on medium or high dose inhaled corticosteroids (fluticasone 250 to 1000 mg) plus long-acting b2-agonist were recruited. The study was approved by the Ethics Committee of the Medical Chamber of Schleswig-Holstein (119/13 II) and prior written informed consent was obtained. Endobronchial biopsies (3 from lobar carinae, 3 from segmental carinae) were taken preand post-dose (28 days). Biopsies were scored blind for the presence of immunostained neutrophils on a 5-graded scale (minimal to abundant), according to histological compartment (epithelial/ subepithelial/submucosal). Sputum was induced and processed at screening, day 1 (pre-treatment), 8, 22, and 29 for cell counts, biomarker measurements in supernatant, and transcriptome analysis (pre-treatment and day 22) in sputum cell pellets [11]. Blood neutrophil counts were assessed in routine laboratory tests. Data was summarized by descriptive statistics. 5 asthmatic patients (median age [interquartile range [IR]], 48 years [38e55 years]; 4 male patients; median FEV1 [IR], 76%

Longitudinal measurement of airway inflammation over one year in children and adults with intermittent asthma

BackgroundAsthma is an inflammatory disease of the airways, but in clinical practice inflammation is rarely monitored. The aim of this study was to assess the level of airway inflammation in steroid naïve adult and pediatric patients with intermittent asthma over one year.Methods54 children and 50 adults with intermittent asthma (GINA step 1) were included. On up to 6 visits lung function, airway hyperresponsiveness to methacholine (PC20FEV1), sputum eosinophils and exhaled nitric oxide (FeNO) were assessed.Results36 pediatric and 34 adult patients were able to produce at least three adequate sputum samples over the study period and were included into the analysis.In 8 children (22%) the percentage of sputum eosinophils was always below 2.5%. A higher level of eosinophils (>2.5%) was found on at least one visit in 16 (44%) and always >2.5% in 12 children (33%). In the adult group the respective numbers were 14 patients (41%) with always low (<2.5%), 17 (50%) with at least once over 2.5% and three patients (9%) were always above the threshold of 2.5% sputum eosinophils.ConclusionThese results demonstrate that a substantial number of children and adults with intermittent asthma under ß-agonist treatment only, have variable or persistently high levels of eosinophilic airway inflammation. Long-term studies are needed to observe the progression of asthma severity in such patient populations.

Neutrophil Extracellular Trap Formation is regulated by CXCR2 in COPD neutrophils

Chronic obstructive pulmonary disease (COPD) is characterised by persistent neutrophilic inflammation of the airways, which is associated with enhanced neutrophil extracellular trap (NET) formation of airway neutrophils [1–3]. NETs are web-like extracellular structures consisting of neutrophil DNA components and degradative enzymes (e.g. neutrophil elastase and myeloperoxidase) which are released from azurophilic granules by activated neutrophils [4]. Initially found to immunoregulate host defence responses during bacterial infections, emerging data indicate that exaggerated NET formation and/or delayed NET clearance may inflict tissue damage and organ dysfunction in a range of human inflammatory diseases independent of infections [5, 6]. Recently, we found NET formation of sputum neutrophils and extracellular DNA levels in sputum supernatant to be significantly enhanced in patients with stable COPD irrespective of the current smoking state or the purulence of the sputum samples [1]. Several studies, including our own, show that the amount of NET formation and/or level of extracellular DNA in patients with stable COPD correlate with the airflow limitation, suggesting that NET formation by airway neutrophils may contribute to the lung tissue damage observed in COPD [1–3]. However, the underlying mechanisms inducing NET formation in COPD neutrophils are largely unexplored. Furthermore, it is unknown whether neutrophils undergo NETosis following migration into the lung tissues or whether neutrophils are constitutively poised to undergo this response in circulation during COPD-related inflammation. It is notable that there is an increase in the release of cell-free DNA and myeloperoxidase that are active constituents of NETs in peripheral blood in COPD patients [7–9]. Here, we aimed to compare NET formation in autologous neutrophils derived from peripheral blood and sputum from COPD patients ex vivo. We also investigated whether the selective CXCR2 antagonist AZD5069, that has been shown to block trafficking of neutrophils from blood into airways in bronchiectasis patients [10] and more recently in a small cohort of neutrophilic asthma patients [11], could dampen NET formation in COPD-derived neutrophils ex vivo. CXCR2 might have a crucial role in neutrophil extracellular trap formation in COPD neutrophils http://ow.ly/LRst30iE1Bw