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.

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.

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.

Thomas et al. show that a novel protein, Eros, controls the abundance of components of the phagocyte NADPH oxidase, making it essential for the phagocyte respiratory burst and defense against common infections.

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.

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.

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.

Introduction CT pulmonary angiography (CTPA) is the recommended imaging modality for suspected pulmonary embolism (PE). Current NICE guidelines recommend using clinical prediction scoring systems to estimate the probability of PE and guide further investigation [i]. A low or intermediate probability score, coupled with a negative D-dimer, reliably excludes PE, thereby avoiding the need for CTPA. Objectives We undertook a retrospective audit to examine adherence to NICE guidelines for diagnosis of suspected PE in patients admitted to a district general hospital, and identify patients who may have undergone unnecessary CTPA. Methods We obtained a list of all CTPAs undertaken in our hospital between December 2012 and February 2013. D-dimer tests are poorly specific within hospitalised patients; therefore, we excluded post-surgical and obstetric patients, and pre-existing inpatients where primary admission was not for suspected PE. We also excluded outpatient CTPAs. We searched the records for contemporaneous PE probability scores and D-dimer results. For patients without a probability score result, we reviewed the clinical notes and calculated a probability score retrospectively using a local scoring system adapted from BTS guidelines. Results There were 115 CTPAs during the study period -36 were excluded and 4 patients' case notes were unavailable. 75 patients fulfilled the inclusion criteria (mean age 68.2 years), and PE was confirmed in 20%. 50 patients (66.7%) had a contemporaneous documented clinical probability score. There were 5 patients (6.7%) with a low/intermediate probability score and negative D-dimer, who underwent unnecessary CTPA (PE excluded in each case). There were 9 patients (12%) with retrospectively calculated low/intermediate clinical probability scores and no D-dimer result, who may have avoided CTPA had Ddimer been undertaken (CTPA excluded PE in each case). Conclusions In our district general hospital, the underuse of clinical probability scoring and D-dimer testing in patients with suspected PE is contributing to unnecessary CTPAs. Introducing mandatory documentation of PE clinical probability score on CTPA request forms may reduce the number of unnecessary CTPAs.