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Aaron S et al. Re-evaluation of diagnosis in adults with physician-diagnosed asthma. JAMA (1) Reviewed by Akhilesh Jha Asthma diagnosis is based on classical symptoms together with variable airflow limitation (2). Accuracy is essential to ensure appropriate long-term medication; misdiagnosis can lead to unnecessary drug-related adverse effects and medical expenditure. Lefaudeux D et al. U-BIOPRED clinical adult asthma clusters linked to a subset of sputum omics. J Allergy Clin Immunol (6) Reviewed by Martin Knolle Recent advances in asthma phenotyping (7-9) have enabled more effective and targeted asthma treatments. However, a mechanistic understanding of these inflammatory endotypes remains limited. To this end, the ‘Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes’ (U-BIOPRED) consortium has applied ‘multi-omics’ approaches to well-characterised asthma patient cohorts (10). Nair P et al. Oral glucocorticoid-sparing effect of benralizumab in severe asthma. N Engl J Med (15) Reviewed by Katharine M Lodge Patients with treatment-refractory asthma account for a large proportion of asthma healthcare costs and suffer substantial glucocorticoid-induced co-morbidities (16, 17). Type 2 immune response-driven eosinophilia is associated with severe and uncontrolled asthma (18). Interleukin 5 (IL-5), a pro-inflammatory cytokine produced by Th2 cells, promotes eosinophil recruitment and survival, and represents an important therapeutic target (19). Monoclonal antibodies against IL-5 (mepolizumab and reslizumab) or the IL-5 receptor (benralizumab) reduce exacerbation frequency in severe eosinophilic asthma, with potential for lung function and quality of life improvement (20-22).

Introduction COPD is a progressive neutrophilic lung disease associated with increased risk of cardiovascular complications. Neutrophil elastase (NE) is implicated in COPD pathogenesis but the precise mechanisms of neutrophil-mediated tissue damage are unknown, particularly with respect to systemic manifestations. Inflamed COPD airways are profoundly hypoxic. We therefore hypothesised that hypoxia synergises with inflammatory cytokines to promote a destructive neutrophil phenotype with enhanced capacity for tissue damage, both locally and systemically. Methods Neutrophils isolated from exacerbating COPD patients and age/sex-matched healthy volunteers were incubated under normoxia (21% O2) or hypoxia (0.8% O2) for 4 hours, before treatment with priming (PAF) and stimulating (fMLP) agents, with/without PI3Kinase inhibitors. NE activity was measured by Enzchek assay. Neutrophil supernatants were incubated with primary human pulmonary artery endothelial cells (HPAEC); cell damage was assessed by confocal microscopy. Normoxic/hypoxic neutrophil supernatants underwent tandem mass tag-labelled mass spectrometry (TMT-MS), and identified protein abundance was quantified. Neutrophil-derived microparticles (NDMPs) were isolated by ultra-centrifugation and quantified by NanoSight nanoparticle tracking technology. Results Hypoxia increased NE release in a PI3K-dependent manner, with significantly more NE secreted by hypoxic neutrophils from exacerbating COPD patients versus healthy controls (p Conclusions Hypoxia augments NE release in a PI3K-dependent manner, further increased during COPD exacerbations, and hypoxic neutrophil supernatants injure endothelial cells in vitro. Unbiased characterisation of hypoxic neutrophil secretomes identified several upregulated proteins which may contribute to cellular/tissue damage. In addition to degranulation, NDMP release may underpin differential protein secretion under hypoxia. Hypoxia engenders a neutrophil phenotype with potential to cause local and distant tissue damage in COPD; novel targets in the hypoxic neutrophil secretome may identify new therapeutic opportunities.

Introduction Evidence has implicated neutrophil elastase (NE), a proteolytic enzyme, as a key driver of the pulmonary vascular remodelling that underlies pulmonary arterial hypertension (PAH). Moreover, studies using animal models and explanted human lung tissue have demonstrated that inhibition of NE attenuates pulmonary hypertension. However, there has been little investigation into neutrophil function in PAH patients even though their azurophilic granules are the main physiological reservoir of NE. We investigated neutrophil phenotypes in patients with PAH versus healthy controls, with a focus on neutrophil degranulation. Methods Neutrophils were isolated from venous blood of PAH patients and healthy controls (HC) and treated with LPS; viability was assessed at 20 hours by morphology. Cell surface receptor expression was determined by flow cytometry. To evaluate degranulation, neutrophils were treated with priming agents, platelet activating factor (PAF, 1 µM) or tumour necrosis factor-α (TNF, 20 ng/ml), and subsequently stimulated with N-formylmethionyl-leucyl-phenylalanine (fMLP; 100 nM). NE release was measured by ELISA and released NE activity and myeloperoxidase (MPO) activity were determined by fluorogenic (Enzchek) and colorimetric (o-dianisidine oxidation) assays respectively. Results Neutrophil apoptosis 20 hours following stimulation with LPS was significantly lower in PAH (25.4%+/-2.2, n=12) versus HC samples (44.9%+/-4.7, n=9), p Conclusions Our Results indicate that neutrophil phenotype is altered in PAH, with a prolonged lifespan in response to a pro-inflammatory stimulus and increased release of NE. However, we did not detect a corresponding increase in NE activity, suggesting a concomitant increase in NE inhibitor release from PAH neutrophils. The potential role of this altered neutrophil phenotype in vascular remodelling requires further investigation.

Staphylococcal infection and neutrophilic inflammation can act in concert to establish a profoundly hypoxic environment. In this review we summarise how neutrophils and Staphylococcus aureus are adapted to function under hypoxic conditions, with a particular focus on the impaired ability of hypoxic neutrophils to effect Staphylococcus aureus killing.

Introduction Damage to host tissue from persistent neutrophilic inflammation is implicated in the pathogenesis of many diseases, including chronic obstructive pulmonary disease (COPD). Infected/inflamed tissues can be profoundly hypoxic; this state may synergise with inflammatory cytokines to promote a destructive neutrophil phenotype with enhanced potential for tissue damage. Methods Neutrophils isolated from COPD patients or healthy volunteers were incubated under normoxia (21% O2) or hypoxia (0.8% O2) before treatment with priming (GM-CSF/PAF/TNF-α) and stimulating (fMLP) agents, with/without PI3Kinase inhibitors (pan/γ/δ). Neutrophil elastase (NE) activity was measured by Enzchek® assay. Western blotting for total and phosphorylated Akt was performed using cell lysates. Neutrophil extracellular trap (NET) production was assessed by fluorescence absorbance. Neutrophil supernatants were incubated with primary human pulmonary artery endothelial cells (HPAEC); death and detachment were measured by MTT assay and confocal microscopy. Precipitated neutrophil supernatants were separated by SDS polyacrylamide gel electrophoresis (PAGE) and silver stained. S100A8/A9 homo- and heterodimer content of neutrophil supernatants was assessed by ELISA. Results Hypoxia increased NE release in an agonist- and PI3K-γ-dependent manner, with more pronounced hypoxic degranulation responses seen in exacerbating COPD patients. Hypoxia augmented resting and cytokine-stimulated Akt phosphorylation; PI3K-γ inhibition abrogated Akt phosphorylation and prevented the hypoxic uplift of NE release. Hypoxia did not increase NET production in resting or GM-CSF/fMLP treated cells. Hypoxic neutrophil supernatants induced extensive HPAEC detachment and death, which was prevented by co-incubation with alpha-1 antitrypsin. Silver stained protein bands from precipitated neutrophil supernatants separated by SDS-PAGE were identified by mass spectrometry, suggesting a hypoxic increase in damage associated molecular pattern (DAMP) proteins S100A8 and S100A9. When interrogated by ELISA, there was no difference between the amount of S100A8/A9 hetero- or homodimers in normoxic versus hypoxic supernatants. Conclusion Hypoxia augments neutrophil degranulation in an agonist- and PI3K-γ-dependent manner, which may be further increased during COPD exacerbations. Hypoxic neutrophil supernatants have enhanced capacity to damage endothelial cells in vitro, likely due to increased release of NE. The contribution of S100A8/A9 proteins to this damage is currently unclear. Hence, hypoxia promotes a destructive histotoxic neutrophil phenotype with potential relevance to diseases such as COPD.

Sites of infection and inflammation are profoundly hypoxic, requiring neutrophils to function under low oxygen tensions. Although neutrophils are well adapted and can rely on glycolytic metabolism, hypoxia still impairs the neutrophil oxidative burst, reduces bacterial killing and delays apoptosis. 1 As neutrophil proteases have been implicated in lung diseases such as COPD, we hypothesised that hypoxia might also promote neutrophil degranulation, with an enhanced potential for neutrophil-mediated tissue injury. Neutrophils isolated from healthy volunteers were subjected to normoxia (20 kPa) or hypoxia (3 kPa) and subsequently activated with GM-CSF (10 ng/ml) and the bacterial tri-peptide fMLP (100 nM). A549 cells and ciliated human primary bronchial epithelial (NHBE) cells were exposed to neutrophil supernatants, the extent of cellular damage was determined by MTT assay (A549 cells), EM ultrastructure and cleaved caspase 3 staining. Ciliary function was also investigated using video microscopy in the ciliated NHBE cells. Hypoxic incubation for 4 hours resulted in a 3–5 fold increase in neutrophil degranulation; this was evident for active elastase, MPO, MMP-9 and lactoferrin and hence occurred from all granule sub-types. Supernatants from hypoxic neutrophils induced 50% more cell death in A549 cells compared to supernatants from normoxic neutrophils. NHBE cells exposed to supernatants from hypoxic versus normoxic neutrophils suffered more cellular damage (EM; images were scored for cytoplasmic blebbing, vacuole formation and dead cells), an increase in LDH activity (from 35.7 ± 6 to 50.2 ± 0.7 nmol/min/ml, was indicative of cell death), increased cleaved caspase 3 staining was shown to be an indicator of apoptosis and there was a substantial increase in the proportion of dyskinetic and immotile cilia. In conclusion; hypoxia induced a destructive neutrophil phenotype with delayed apoptosis, impaired bacterial killing and increased release of histo-cytotoxic proteases. This phenotype may be important for understanding the mechanisms of chronic inflammatory diseases like COPD. Funded by the British Lung Foundation and NIHR Cambridge BRC. References McGovern, N. N. et al . Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in human neutrophils. J. Immunol. Baltim. Md 1950 186, 453–463 (2011).