K Hoenderdos

The Neutrophil in Chronic Obstructive Pulmonary Disease. Too Little, Too Late or Too Much, Too Soon?

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide and has few effective therapies. It is characterized by anomalous and persistent inflammation, both local and systemic. Neutrophilic inflammation predominates in the COPD airway wall and lumen, but, despite the presence of abundant innate immune cells, the progressive clinical course of the disease is punctuated by recurrent infection-driven exacerbations. An extensive body of evidence (from cell culture to murine models and finally to the susceptibility of human patients with α1-antitrypsin deficiency to develop COPD) implicates neutrophil elastase and other neutrophil-derived proteases as key mediators of the tissue damage and relentless decline in lung function that occurs in this condition. In addition to the well recognized role of cytokines in modulating neutrophil function and survival, it has recently become apparent that hypoxia can influence neutrophil function, with impaired killing of pathogenic bacteria, enhanced release of proteases, and delayed apoptosis. This destructive neutrophil phenotype is predicted to be highly detrimental in the setting of the COPD microenvironment.

Hypoxia upregulates neutrophil degranulation and potential for tissue injury

Background 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. Methods and results 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. Conclusion Hypoxia augments neutrophil degranulation and confers enhanced potential for damage to respiratory airway epithelial cells in a HIF-independent but PI3Kγ-dependent fashion.

Eros is a novel transmembrane protein that controls the phagocyte respiratory burst and is essential for innate immunity

The phagocyte respiratory burst is crucial for innate immunity. The transfer of electrons to oxygen is mediated by a membrane-bound heterodimer, comprising gp91phox and p22phox subunits. Deficiency of either subunit leads to severe immunodeficiency. We describe Eros (essential for reactive oxygen species), a protein encoded by the previously undefined mouse gene bc017643, and show that it is essential for host defense via the phagocyte NAPDH oxidase. Eros is required for expression of the NADPH oxidase components, gp91phox and p22phox. Consequently, Eros-deficient mice quickly succumb to infection. Eros also contributes to the formation of neutrophil extracellular traps (NETS) and impacts on the immune response to melanoma metastases. Eros is an ortholog of the plant protein Ycf4, which is necessary for expression of proteins of the photosynthetic photosystem 1 complex, itself also an NADPH oxio-reductase. We thus describe the key role of the previously uncharacterized protein Eros in host defense.

Bone Morphogenetic Protein 9 Enhances Lipopolysaccharide-Induced Leukocyte Recruitment to the Vascular Endothelium.

Bone morphogenetic protein (BMP)9 is a circulating growth factor that is part of the TGF-β superfamily and is an essential regulator of vascular endothelial homeostasis. Previous studies have suggested a role for BMP9 signaling in leukocyte recruitment to the endothelium, but the directionality of this effect and underlying mechanisms have not been elucidated. In this study, we report that BMP9 upregulates TLR4 expression in human endothelial cells and that BMP9 pretreatment 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. Small interfering RNA knockdown of activin receptor–like kinase 1 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 although 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.

P156 Neutrophil and redox dependent proteolysis of bone morphogenetic protein 9: potential role in the pathogenesis of pulmonary arterial hypertension

Introduction A critical reduction of bone morphogenetic protein type II receptor (BMPRII) in the pulmonary circulation, either due to the genetic loss-of-function mutations, heightened inflammation or prolonged hypoxia, is one of the major causes behind pulmonary arterial hypertension (PAH), a fatal disease with poor prognosis. BMPRII is highly expressed in the vascular endothelium and undergoes rapid turnover. Bone morphogenetic protein 9 (BMP9), the only active circulating BMP, signals via endothelial BMPRII, inducing BMPRII expression and maintaining endothelial homeostasis. Although BMPRII function has been studied extensively, factors that regulate BMP9 stability and activity remain unclear. Objective To investigate how BMP9 activity and stability are regulated and whether this regulation plays a role in pulmonary arterial hypertension. Results Two forms of BMP9 dimer could be co-purified, with (D-form) or without (M-form) intermolecular disulphide bond. M- and D-forms BMP9 are interchangeable with redox potential, but have different stability. While the M-form is more susceptible to redox-dependent cleavage and proteases present in serum, the D-form is a preferred substrate for neutrophil elastase. Freshly isolated human peripheral blood neutrophils, when activated by hypoxia or inflammatory stimuli, released elastase that cleaved BMP9 effectively. Conclusions and Discussions This study demonstrates a novel proteolytic regulation of BMP9 under physiological and pathological conditions, suggesting neutrophil elastase could be a potential link between inflammation/hypoxia and BMPRII signalling, and the recognised benefits of elastase inhibition in rodent models of PAH may be due in part to reduced degradation of BMP9 and preservation of endothelial BMPR-II signalling.

S114 Hypoxia drives neutrophil-mediated endothelial damage in copd

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<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. 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.

NBEAL2 is required for neutrophil and NK cell function and pathogen defense

Mutations in the human NBEAL2 gene cause gray platelet syndrome (GPS), a bleeding diathesis characterized by a lack of α granules in platelets. The functions of the NBEAL2 protein have not been explored outside platelet biology, but there are reports of increased frequency of infection and abnormal neutrophil morphology in patients with GPS. We therefore investigated the role of NBEAL2 in immunity by analyzing the phenotype of Nbeal2-deficient mice. We found profound abnormalities in the Nbeal2-deficient immune system, particularly in the function of neutrophils and NK cells. Phenotyping of Nbeal2-deficient neutrophils showed a severe reduction in granule contents across all granule subsets. Despite this, Nbeal2-deficient neutrophils had an enhanced phagocyte respiratory burst relative to Nbeal2-expressing neutrophils. This respiratory burst was associated with increased expression of cytosolic components of the NADPH oxidase complex. Nbeal2-deficient NK cells were also dysfunctional and showed reduced degranulation. These abnormalities were associated with increased susceptibility to both bacterial (Staphylococcus aureus) and viral (murine CMV) infection in vivo. These results define an essential role for NBEAL2 in mammalian immunity.

S43 Hypoxia upregulates PI3KINASE-dependent neutrophil degranulation and neutrophil-mediated tissue injury

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.

Role of Eros, a novel transmembrane protein, in regulation of host defence

Abstract Background Reactive oxygen species (ROS), generated via the phagocyte NADPH oxidase cytochrome b558, are essential for effective immune responses to common and serious pathogens. The phagocyte NADPH oxidase is a multisubunit protein complex and deficiency of either the membrane bound or cytoplasmic components leads to chronic granulomatous disease, a serious and often fatal illness characterised by recurrent infections and autoimmunity. Moreover, abnormal generation of ROS has been implicated in the pathogenesis of multigenic autoimmune diseases such as systemic lupus erythematosus. Eros (essential for reactive oxygen species), encoded by bc017643 , is a novel transmembrane protein that is highly expressed in the immune system and highly conserved in evolution but has no previously identified function. Eros is an orthologue of the plant protein Ycf4, necessary for expression of proteins of the photosynthetic photosystem 1 complex, an NADPH oxio-reductase complex. We elucidated its role in infection in mice. Methods ROS are essential for host defence against the serious bacterial pathogen Salmonella enterica serovar Typhimurium. We screened individual knockout mice (Wellcome Trust Knockout mouse project) for susceptibility to salmonella infection. Having identified mice deficient in Eros as being highly susceptible to salmonella, we used ex-vivo approaches including reactive oxygen burst assays and western blot, to characterise their defect further. Findings We found that Eros was essential for host defence to infection. Eros was crucial for generating reactive oxygen species through regulation of the essential NADPH oxidase components, gp91 and p22. Eros-deficient mice expressed almost no gp91 and p22 in neutrophils and macrophages secondary to accelerated degradation in the absence of Eros. As a result Eros-deficient mice died rapidly after infection with salmonella or listeria. Eros also regulated the ROS-dependent formation of neutrophil extracellular traps and melanoma metastases. Interpretation We have found a a key role for Eros in regulating host defence. The finding that Eros-deficient mice lack gp91 and p22 at the protein, though not mRNA, level shows how these key components of the reatcive oxygen burst are protected from degradation and furthers our understanding of reactive oxygen burst biology. Eros is highly conserved between mouse and man so it is likely that it also has a crucial role in human immunity. Funding Wellcome Trust, Academy of Medical Sciences starter grant.

S2 Vascular Quiescence Factor BMP9 is Regulated by Inflammation and Neutrophil Activation

Introduction Endothelial bone morphogenetic protein type II receptor (BMPR-II)-mediated signalling is essential for protecting vascular endothelium. Loss of BMPR-II predisposes human pulmonary artery endothelial cell (hPAEC) monolayers to apoptosis and increased permeability. In vivo, reduced BMPR-II function promotes endothelial permeability and the development of pulmonary arterial hypertension (PAH). Importantly, BMP9, the only confirmed active circulating BMP, signals preferentially via BMPR-II and induces BMPR-II expression to maintain endothelial integrity and homeostasis. It was recently shown that administration of recombinant BMP9 prevented LPS-induced lung vascular leakage in vivo and reversed established PAH in three rodent models.1 However, it is not known how circulating BMP9 is regulated during LPS-induced inflammation and in PAH. Objective To investigate whether BMP9 is regulated by inflammatory stimuli in vivo and in vitro. Results Intraperitoneal LPS challenge in mice led to a significant increase in circulating neutrophil elastase levels with a reciprocal reduction in BMP9 levels (both measured by ELISA) within 24 h. Since this reduction in BMP9 might be due to reduced BMP9 synthesis in the liver or cleavage of BMP9 by neutrophil-derived proteases, we quantified BMP9 synthesis in the liver after LPS challenge, as well as changes in alpha-1 antitrypsin, the major elastase inhibitor in man. Synthesis of BMP9 fell sharply 3 h after LPS-challenge but recovered completely by 18 h. No increase in the synthesis or levels of circulating active alpha-1 antitrypsin was observed. Supernatants from purified human peripheral blood neutrophils activated in vitro degraded recombinant BMP9. Inhibition studies confirmed that the BMP9-cleavage activity released by activated neutrophils was largely attributable to neutrophil elastase. Conclusions and discussions Synthesis of the endothelial protective factor BMP9 is actively regulated by inflammation, and BMP9 is subject to neutrophil elastase-mediated cleavage. Since inflammation has been shown to be a second hit in the pathogenesis of PAH, this study could provide a potential link between inflammation and reduced endothelial BMPR-II signalling. Reference 1 Long L, Ormiston ML, Yang X, et al. Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension. Nat. Med. 2015;21: 777–785