Circulating BMP9 Protects the Pulmonary Endothelium during Inflammation-induced Lung Injury in Mice.

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.

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.

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-ß 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.

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.

Acute Respiratory Distress Syndrome Neutrophils Have a Distinct Phenotype and Are Resistant to Phosphoinositide 3-Kinase Inhibition.

RATIONALE: Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease's complex pathophysiology, yet these cells have been little studied. OBJECTIVES: To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition. METHODS: Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays. MEASUREMENTS AND MAIN RESULTS: Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures. CONCLUSIONS: Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase-dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.

The neutrophil in chronic obstructive pulmonary disease.

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.