BackgroundThe 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.Methods and resultsFollowing 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.ConclusionHypoxia augments neutrophil degranulation and confers enhanced potential for damage to respiratory airway epithelial cells in a HIF-independent but PI3Kγ-dependent fashion.

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.

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.

Bone morphogenetic protein 9 (BMP9) is a circulating growth factor that is part of the TGFβ superfamily, and an essential regulator of vascular endothelial homeostasis. Previous studies have suggested a role for BMP9 signalling in leukocyte recruitment to the endothelium, but the directionality of this effect and underlying mechanisms have not been elucidated. Here we report that BMP9 upregulates toll-like receptor 4 (TLR4) expression in human endothelial cells and that BMP9 pre-treatment synergistically increases human neutrophil recruitment to LPS-stimulated human endothelial monolayers in an in vitro flow adhesion assay. BMP9 alone did not induce neutrophil recruitment to the endothelium. We also show that E-selectin and VCAM-1, but not ICAM-1 are upregulated in response to BMP9 in LPS-stimulated human endothelial cells. siRNA knockdown of ALK1 inhibited the BMP9-induced expression of TLR4 and VCAM-1 and inhibited BMP9-induced human neutrophil recruitment to LPS-stimulated human endothelial cells. BMP9 treatment also increased leukocyte recruitment within the pulmonary circulation in a mouse acute endotoxemia model. These results demonstrate that whilst BMP9 alone does not influence leukocyte recruitment, it primes the vascular endothelium to mount a more intense response when challenged with LPS, through an increase in TLR4, E-selectin and VCAM-1 and ultimately through enhanced leukocyte recruitment.

Scientific knowledge on the subject: COPD is characterised by persistent neutrophilia in the setting of local and systemic hypoxia, and is associated with excess cardiovascular disease, even allowing for known risk factors. Neutrophils accumulating in areas of inflammation and microcirculatory impairment experience profound hypoxia, which prolongs their survival and increases their secretory responses. Thus, hypoxic neutrophils have increased potential to cause endothelial injury but their role in mediating the increased cardiovascular risk in COPD is poorly understood.What this study adds to the field: Herein we show that hypoxia augments the ability of neutrophils to selectively secrete a subset of histotoxic proteins capable of AJRCCM Articles in Press.

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.

Neutrophils are among the first immune cells to respond following infection or injury; this makes the possession of proficient pathogenkilling mechanisms essential. Phagocytosis, the process of detecting and engulfing particles into an organelle called the phagosome, is key to the ability of neutrophils to kill pathogens. In neutrophils, phagocytosis is a highly specialized and efficient event. Thus, neutrophils are the archetypal "professional" phagocyte, although monocytes, macrophages, eosinophils, and dendritic cells also display phagocytic ability to a somewhat lesser extent. 1 As well as killing ingested pathogens, innate immune phagocytes may present antigens to adaptive immune cells, highlighting the importance of phagocytosis for both arms of the immune system. 2 An intriguing neutrophil-dendritic cell hybrid phenotype has been identified in mice, exhibiting retained phagocytic and microbial-killing capacity as well as typical dendritic cell properties, such as antigen presentation. 3 The neutrophil phagosome is a distinctive organelle, formed from an invagination of the plasma membrane to completely enclose an engulfed particle. A host of complementary processes

IntroductionCOPD 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.MethodsNeutrophils 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.ResultsHypoxia increased NE release in a PI3K-dependent manner, with significantly more NE secreted by hypoxic neutrophils from exacerbating COPD patients versus healthy controls (p<0.0001). Supernatants generated from hypoxic, but not normoxic, stimulated neutrophils induced extensive HPAEC damage. Comparing the secretomes of supernatants derived from normoxic/hypoxic stimulated neutrophils, TMT-MS identified several additional proteins with potential to cause tissue damage as upregulated in hypoxia, including resistin and NGAL (neutrophil gelatinase-associated lipocalin). Notably, several of these proteins were not granule-associated, and some granule proteins were downregulated in hypoxia, indicating additional/alternative release mechanisms. Preliminary data show an increase in NDMP release under hypoxia, potentially contributing to the observed differential protein release.ConclusionsHypoxia 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.