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BackgroundPutative genetic risk loci for atherosclerotic vascular disease includeSMARCA4, a chromatin remodeling gene important for gene activation. Its causal role in atherosclerosis has been uncertain. Intraplaque hemorrhage (IPH) is a late event in atherosclerosis that is linked to plaque destabilisation and increased inflammation. IPH is countered by Mhem macrophages, which are directed by hemin-mediated induction of Heme Oxygenase 1 (HMOX1) via Activating Transcription Factor 1 (ATF1).Atf1deficiencyin vivoimpairs hematoma clearance, promoting inflammation and oxidative stress. Like its homologue cyclic-adenosine monophosphate response element binding protein 1 (CREB1), ATF1 is normally cyclic-AMP activated.HypothesisHemin-directed chromatin remodelling by SMARCA4 regulates specificity of ATF1 gene-binding, thereby switching between leukocyte disposal and erythrocyte disposal, contributing to its role in atherosclerosis.ResultsWe here show thatSMARCA4is genetically independent of the adjacentLDLRlocus (pHMOX1enhancer. si-RNA-mediatedSMARCA4-knockdown suppressed p-ATF1 binding toHMOX1but increased its binding to cyclic-AMP responsive genesFOSandNR4A2, with corresponding changes in mRNA levels. This functionally correlated withSMARCA4-knockdown switching hemin to mimic prostacyclin (PGI2), for induced genes and phagocytic disposal of leukocytes rather than erythrocytes.ConclusionsThese data establishSMARCA4as an independent atherosclerosis risk gene and reveal a novel mechanism in which it switches between disposal of leukocytes or erythrocytes, with important clinical implications for atherosclerotic inflammation and intraplaque hemorrhage including treatment by histone deacetylase inhibitors.

Background/Aims Real Time-Deformability Cytometry (RT-DC) is a novel technique able to identify morpho-rheological characteristics of individual cells such as size, deformability, and elasticity using only 50µl of whole blood. Cells in suspension flow through a microfluidic channel while hydrodynamic force is applied, leading to reversible deformation of individual cells from shear stress and pressure gradients. Cell brightness and area can be used to identify individual cell types. The morpho-rheological characteristics of immune cells in ANCA associated vasculitis (AAV) are unknown. Methods Whole blood from healthy controls (HC), patients with active AAV, or patients with AAV in remission was analysed using RT-DC. The diagnosis of AAV was based upon positive ANCA testing, with either (i) pauci-immune glomerulonephritis, or (ii) typical clinical features of extra-renal AAV. Patients with active disease may have received steroids prior to sampling but those who received other immunosuppression or cytotoxics were excluded. Remission was defined as BVAS of 0 with prednisolone dose ≤7.5mg/day. Blood was collected into K2 EDTA and diluted 1:20 in 1xPBS/0.6% methylcellulose. Cell suspensions were flowed across a Flic20 polydimethylsiloxane microfluidic chip, containing reservoirs connected by a measurement channel with a 20x20 μm2 cross-section. RT-DC measurements were collected at a flow rate of 0.12μL/s using a high-speed CMOS camera to capture images at a rate of 2000 frames/second. ShapeOut software was used to calculate cell size, deformation, and elasticity. Results There was no difference in neutrophil size between patients with active AAV (n = 15), patients with AAV in remission (n = 31), and HC (n = 15). Neutrophils from patients with active AAV were significantly stiffer than patients in remission or HC, displaying decreased deformation (median 0.084, 0.085, and 0.078 au respectively, p = 0.0068) and increased elasticity (median 0.973, 0.968 and 1.006 au respectively, p = 0.0196). In patients with active AAV, there was strong inverse correlation between neutrophil deformation and disease activity measured by BVAS score (r=-0.573, p = 0.032). Neutrophils isolated from healthy donors exhibited a trend towards decreased deformability when primed with TNF and stimulated with MPO-ANCA IgG compared to unstimulated or cells treated with healthy donor IgG (median 0.086, 0.085, and 0.083 for unstimulated, control IgG, and MPO-ANCA IgG respectively, ns). Conclusion Neutrophils from patients display distinct physical properties in active AAV which correlate with disease activity. Similar results are seen when neutrophils are stimulated in vitro with MPO-ANCA IgG. This phenotype of increased cell stiffness may lead to increased neutrophil retention in pulmonary and renal microvasculature, thus increasing the potential for neutrophil-endothelial cell interactions and microvascular damage. Morphorheological parameters can be rapidly measured using a small volume of whole blood; thus, RT-DC may be a useful technique to aid identification of disease activity in AAV and to guide treatment. Disclosure N. Pisacano: None. S.P. McAdoo: None. J. Guck: None. C.D. Pusey: None. E.R. Chilvers: None. A.C. Cowburn: None. K.M. Lodge: None. M.F. Prendecki: None.

OBJECTIVE: Syk is a cytoplasmic protein tyrosine kinase that plays a role in signaling via B cell and Fc receptors (FcR). FcR engagement and signaling via Syk is thought to be important in antineutrophil cytoplasm antibody (ANCA) IgG-mediated neutrophil activation. This study was undertaken to investigate the role of Syk in ANCA-induced myeloid cell activation and vasculitis pathogenesis. METHODS: Phosphorylation of Syk in myeloid cells from healthy controls and ANCA-associated vasculitis (AAV) patients was analyzed using flow cytometry. The effect of Syk inhibition on myeloperoxidase (MPO)-ANCA IgG activation of cells was investigated using functional assays (interleukin-8 and reactive oxygen species production) and targeted gene analysis with NanoString. Total and phosphorylated Syk at sites of tissue inflammation in patients with AAV was assessed using immunohistochemistry and RNAscope in situ hybridization. RESULTS: We identified increased phosphorylated Syk at critical activatory tyrosine residues in blood neutrophils and monocytes from patients with active AAV compared to patients with disease in remission or healthy controls. Syk was phosphorylated in vitro following MPO-ANCA IgG stimulation, and Syk inhibition was able to prevent ANCA-mediated cellular responses. Using targeted gene expression analysis, we identified up-regulation of FcR- and Syk-dependent signaling pathways following MPO-ANCA IgG stimulation. Finally, we showed that Syk is expressed and phosphorylated in tissue leukocytes at sites of organ inflammation in AAV. CONCLUSION: These findings indicate that Syk plays a critical role in MPO-ANCA IgG-induced myeloid cell responses and that Syk is activated in circulating immune cells and tissue immune cells in AAV; therefore, Syk inhibition may be a potential therapeutic option.

Rationale: Patients with chronic obstructive pulmonary disease (COPD) experience excess cardiovascular morbidity and mortality, and exacerbations further increase the risk of such events. COPD is associated with persistent blood and airway neutrophilia and systemic and tissue hypoxia. Hypoxia augments neutrophil elastase release, enhancing capacity for tissue injury. Objective: To determine whether hypoxia-driven neutrophil protein secretion contributes to endothelial damage in COPD. Methods: The healthy human neutrophil secretome generated under normoxic or hypoxic conditions was characterized by quantitative mass spectrometry, and the capacity for neutrophil-mediated endothelial damage was assessed. Histotoxic protein concentrations were measured in normoxic versus hypoxic neutrophil supernatants and plasma from patients experiencing COPD exacerbation and healthy control subjects. Measurements and Main Results: Hypoxia promoted PI3Kγ-dependent neutrophil elastase secretion, with greater release seen in neutrophils from patients with COPD. Supernatants from neutrophils incubated under hypoxia caused pulmonary endothelial cell damage, and identical supernatants from COPD neutrophils increased neutrophil adherence to endothelial cells. Proteomics revealed differential neutrophil protein secretion under hypoxia and normoxia, and hypoxia augmented secretion of a subset of histotoxic granule and cytosolic proteins, with significantly greater release seen in COPD neutrophils. The plasma of patients with COPD had higher content of hypoxia-upregulated neutrophil-derived proteins and protease activity, and vascular injury markers. Conclusions: Hypoxia drives a destructive "hypersecretory" neutrophil phenotype conferring enhanced capacity for endothelial injury, with a corresponding signature of neutrophil degranulation and vascular injury identified in plasma of patients with COPD. Thus, hypoxic enhancement of neutrophil degranulation may contribute to increased cardiovascular risk in COPD. These insights may identify new therapeutic opportunities for endothelial damage in COPD.

Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome. Circulating BMP9 (bone morphogenetic protein 9) is emerging as an important regulator of pulmonary vascular homeostasis. Objectives:To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity and whether loss of endogenous BMP9 occurs during LPS challenge. Methods: A BMP9-neutralizing antibody was administrated to healthy adult mice, and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human lung endothelial cells. The impact of BMP9 administration was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and from endotoxemic mice. Measurements and Main Results: Subacute neutralization of endogenous BMP9 in mice (N = 12) resulted in increased lung vascular permeability (P = 0.022), interstitial edema (P = 0.0047), and neutrophil extravasation (P = 0.029) compared with IgG control treatment (N = 6). In pulmonary endothelial cells, BMP9 regulated transcriptome pathways implicated in vascular permeability and cell-membrane integrity. Augmentation of BMP9 signaling in mice (N = 8) prevented inhaled LPS-induced lung injury (P = 0.0027) and edema (P < 0.0001). In endotoxemic mice (N = 12), endogenous circulating BMP9 concentrations were markedly reduced, the causes of which include a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human patients with sepsis (N = 10), circulating concentratons of BMP9 were also markedly reduced (P < 0.0001). Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial-protective factor, downregulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury.

RationalePulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome (ARDS), which carries a high mortality. Circulating bone morphogeneic protein 9 (BMP9) is emerging as an important regulator of pulmonary vascular homeostasis.ObjectiveTo determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity, and whether loss of endogenous BMP9 occurs during lipopolysacharride (LPS)-induced lung inflammation and permeability.MethodsA BMP9-neutralizing antibody was administrated to healthy adult mice and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human primary lung endothelial cells. Impact of BMP9 was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and endotoxemic mice.Main ResultsSubacute neutralization of endogenous BMP9 in mice resulted in increased lung vascular permeability, interstitial edema and neutrophil extravasation. In lung endothelial cells, BMP9 regulated a programme of gene expression and pathways controlling vascular permeability and cell membrane integrity. Augmentation of BMP9 signalling in mice with exogenous BMP9 prevented inhaled LPS-caused lung injury and edema. In endotoxemic mice, endogenous BMP9 levels were markedly reduced, due to a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human sepsis patients, circulating levels of BMP9 were also markedly reduced.ConclusionsEndogenous circulating BMP9 is a pulmonary endothelial protective factor, down-regulated during inflammation. Supplementation with exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in the setting of lung injury.Short summaryScientific Knowledge on the SubjectIncreased pulmonary endothelial permeability is a major factor in the development of acute respiratory distress syndrome (ARDS). Evidence is emerging that circulating BMP9, secreted from the liver, might protect the pulmonary endothelium from injury. For example, loss of BMP9 levels or signalling receptor contributes to the development of pulmonary arterial hypertension. The role of endogenous BMP9 in endothelial permeability remains unclear.What This Study Adds to the FieldHere we show that subacute neutralization of endogenous BMP9 leads to lung vascular injury, including enhanced permeability and neutrophil extravasation. BMP9 levels were markedly reduced in the setting of inflammation in mice and humans. Conversely, exogenous supplementation of BMP9 protected the lung from LPS-induced injury. This study suggests that exogenous BMP9 could offer a novel approach to prevent increased pulmonary endothelial permeability in the setting of lung injury and ARDS.

Neutrophils are key effector cells of innate immunity, rapidly recruited to defend the host against invading pathogens. Neutrophils may kill pathogens intracellularly, following phagocytosis, or extracellularly, by degranulation and the release of neutrophil extracellular traps; all of these microbicidal strategies require the deployment of cytotoxic proteins and proteases, packaged during neutrophil development within cytoplasmic granules. Neutrophils operate in infected and inflamed tissues, which can be profoundly hypoxic. Neutrophilic infiltration of hypoxic tissues characterises a myriad of acute and chronic infectious and inflammatory diseases, and as well as potentially protecting the host from pathogens, neutrophil granule products have been implicated in causing collateral tissue damage in these scenarios. This review discusses the evidence for the enhanced secretion of destructive neutrophil granule contents observed in hypoxic environments and the potential mechanisms for this heightened granule exocytosis, highlighting implications for the host. Understanding the dichotomy of the beneficial and detrimental consequences of neutrophil degranulation in hypoxic environments is crucial to inform potential neutrophil-directed therapeutics in order to limit persistent, excessive, or inappropriate inflammation.

Background: Neutrophilic airway infiltration is central to chronic obstructive pulmonary disease (COPD) pathogenesis. COPD patient sputum contains high levels of neutrophil-derived microvesicles (NMVs). NMVs can be internalised by and activate target cells, however, little is known about their interaction with the lung epithelium. Methodology: NMVs were isolated from human neutrophils treated with PBS, 10µM fMLP, or 50% cigarette smoke extract (CSE) for 1h. Matrix metalloproteinase-9 (MMP-9) levels were determined by western blotting (n=4). Internalisation of fluorescently-labelled NMVs by BEAS-2B bronchial epithelial cells after 2h was quantified using confocal microscopy and flow cytometry (n=5), and IL-8 and MCP-1 secretion after 24h was quantified by ELISA (n=3). Plasma MVs from exacerbating COPD patients (n=6) and age-matched controls (n=5) were analysed by multi-colour flow cytometry to determine cellular origin and surface MMP-9 expression. Results: CSE generated more NMVs than fMLP. NMVs from stimulated cells had high levels of active MMP-9, were internalised by BEAS-2B cells (34.1±15.0%) and significantly increased epithelial IL-8 (>12-fold; p6-fold; p In patient plasma, NMVs represented 22.5% of the total MVs, and surface expression of MMP-9 was detected (117.4±75.8 MMP-9+NMVs/µl), however, levels did not differ between patients and controls. Conclusion: NMVs contain proteases associated with COPD progression and induce pro-inflammatory activation of lung epithelial cells, indicating a potential pathogenic role in the disease. Systemic NMV levels may not correlate with levels in the lungs and local production of NMVs may be key.

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.

The inflamed bronchial mucosal surface is a profoundly hypoxic environment. Neutrophilic airway inflammation and neutrophil-derived proteases have been linked to disease progression in conditions such as COPD and cystic fibrosis, but the effects of hypoxia on potentially harmful neutrophil functional responses such as degranulation are unknown.Following exposure to hypoxia (0.8% oxygen, 3 kPa for 4 h), neutrophils stimulated with inflammatory agonists (granulocyte-macrophage colony stimulating factor or platelet-activating factor and formylated peptide) displayed a markedly augmented (twofold to sixfold) release of azurophilic (neutrophil elastase, myeloperoxidase), specific (lactoferrin) and gelatinase (matrix metalloproteinase-9) granule contents. Neutrophil supernatants derived under hypoxic but not normoxic conditions induced extensive airway epithelial cell detachment and death, which was prevented by coincubation with the antiprotease α-1 antitrypsin; both normoxic and hypoxic supernatants impaired ciliary function. Surprisingly, the hypoxic upregulation of neutrophil degranulation was not dependent on hypoxia-inducible factor (HIF), nor was it fully reversed by inhibition of phospholipase C signalling. Hypoxia augmented the resting and cytokine-stimulated phosphorylation of AKT, and inhibition of phosphoinositide 3-kinase (PI3K)γ (but not other PI3K isoforms) prevented the hypoxic upregulation of neutrophil elastase release.Hypoxia augments neutrophil degranulation and confers enhanced potential for damage to respiratory airway epithelial cells in a HIF-independent but PI3Kγ-dependent fashion.

A 34-year-old man presented with a 12-month history of progressive tiredness and weight loss. Further questioning elicited symptoms of polyuria, polydipsia, reduced libido and erectile dysfunction. He had a 20-pack-a-year smoking history. Physical examination was notable for sparse androgenic hair, loss of muscle bulk and 15 ml testes bilaterally. Biochemical profiling excluded diabetes mellitus, but confirmed partial anterior and posterior hypopituitarism [LH 0.2 U/l (RR 1.5–9.3), FSH 0.9 U/l (RR 1.4–8.1), testosterone 1.1 nmol/l (RR 7.4–25.7), FT4 5.9 pmol/l (RR 5.0–19.6), TSH 2.25 mU/l (RR 0.49–3.95), post-Synacthen® cortisol 463 nmol/l (RR > 550), water deprivation test urine osmolalities: baseline 132 mOsm/kg; 8 hr 147 mOsm/kg; post-desmopressin (2 mcg) 616 mOsm/kg]. Magnetic resonance imaging (MRI) of the brain showed infundibular thickening with contrast enhancement (Figure 1A) and absence of the posterior pituitary bright spot, but no other lesions. A screen for inflammatory and infiltrative disorders [ANCA, ANA, immunoglobulins (including IgG4), serum ACE, ESR and CRP] proved unremarkable. Cerebrospinal …

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).