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.Supported by the British Lung Foundation, Papworth Hospital NHS Foundation Trust, BBSRC and the Cambridge NIHR-Biomedical Research Centre. CS was funded by Wellcome Trust Early Postdoctoral Research Fellowship for Clinician Scientists (WT101692MA).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by BMJ Publishing Group

Phosphoinositide-3 kinase inhibition modulates responses to rhinovirus by mechanisms that are predominantly independent of autophagy

Human rhinoviruses (HRV) are a major cause of exacerbations of airways disease. Aspects of cell signalling responses to HRV infection remain unclear, particularly with regard to signalling via PI3K, and the PI3K-dependent pathway, autophagy. We investigated the roles of PI3K and autophagy in the responses of epithelial cells to major and minor group HRV infection. The PI3K inhibitor 3-MA, commonly used to inhibit autophagy, markedly reduced HRV-induced cytokine induction. Further investigation of potential targets of 3-MA and comparison of results using this inhibitor to a panel of general and class I-selective PI3K inhibitors showed that several PI3Ks cooperatively regulate responses to HRV. Targeting by siRNA of the autophagy proteins Beclin-1, Atg7, LC3, alone or in combination, or targeting of the autophagy-specific class III PI3K had at most only modest effects on HRV-induced cell signalling as judged by induction of proinflammatory cytokine production. Our data indicate that PI3K and mTOR are involved in induction of proinflammatory cytokines after HRV infection, and that autophagy has little role in the cytokine response to HRV or control of HRV replication

The Aminopeptidase CD13 Induces Homotypic Aggregation in Neutrophils and Impairs Collagen Invasion.

Aminopeptidase N (CD13) is a widely expressed cell surface metallopeptidase involved in the migration of cancer and endothelial cells. Apart from our demonstration that CD13 modulates the efficacy of tumor necrosis factor-α-induced apoptosis in neutrophils, no other function for CD13 has been ascribed in this cell. We hypothesized that CD13 may be involved in neutrophil migration and/or homotypic aggregation. Using purified human blood neutrophils we confirmed the expression of CD13 on neutrophils and its up-regulation by pro-inflammatory agonists. However, using the anti-CD13 monoclonal antibody WM-15 and the aminopeptidase enzymatic inhibitor bestatin we were unable to demonstrate any direct involvement of CD13 in neutrophil polarisation or chemotaxis. In contrast, IL-8-mediated neutrophil migration in type I collagen gels was significantly impaired by the anti-CD13 monoclonal antibodies WM-15 and MY7. Notably, these antibodies also induced significant homotypic aggregation of neutrophils, which was dependent on CD13 cross-linking and was attenuated by phosphoinositide 3-kinase and extracellular signal-related kinase 1/2 inhibition. Live imaging demonstrated that in WM-15-treated neutrophils, where homotypic aggregation was evident, the number of cells entering IL-8 impregnated collagen I gels was significantly reduced. These data reveal a novel role for CD13 in inducing homotypic aggregation in neutrophils, which results in a transmigration deficiency; this mechanism may be relevant to neutrophil micro-aggregation in vivo.This work was funded by a Medical Research Council Research Training Fellowship to CAF (G0900329), Addenbrooke’s Charitable Trust (ACT), CUHNHSFT, Papworth Hospital NHS Foundation Trust and the NIHR Cambridge Biomedical Research Centre. CAF received a Raymond and Beverly Sackler Studentship.This is the final version of the article. It first appeared from the Public Library of Science via http://dx.doi.org/10.1371/journal.pone.016010

Alveolar macrophages isolated directly from human cytomegalovirus (HCMV)-seropositive individuals are sites of HCMV reactivation in vivo

Human cytomegalovirus (HCMV) causes significant morbidity in the immunocompromised host. Following primary infection, the virus establishes latent infection in progenitor cells of the myeloid lineage. These cells exhibit limited viral gene transcription and no evidence of de novo virion production. It is well recognized that differentiation of latently infected myeloid progenitor cells to dendritic or macrophage-like cells permits viral reactivation in vitro. This has been used to support the concept that viral reactivation in HCMV carriers routinely occurs from such terminally differentiated myeloid cells in vivo. However, to date this has not been shown for in vivo–differentiated macrophages. This study is the first to demonstrate that alveolar macrophages from HCMV carriers express immediate early lytic genes and produce infectious virus. This supports the view, until now based on in vitro data, that terminally differentiated myeloid cells in vivo are sites of HCMV reactivation and potential centers of viral dissemination in latently infected individuals with no evidence of virus disease or dissemination

Evasion of neutrophil extracellular traps by respiratory pathogens

The release of neutrophil extracellular traps (NETs) is a major immune mechanism intended to capture pathogens. These histone- and protease-coated DNA structures are released by neutrophils in response to a variety of stimuli, including respiratory pathogens, and have been identified in the airways of patients with respiratory infection, cystic fibrosis, acute lung injury, primary graft dysfunction, and chronic obstructive pulmonary disease. NET production has been demonstrated in the lungs of mice infected with Staphylococcus aureus, Klebsiella pneumoniae, and Aspergillus fumigatus. Since the discovery of NETs over a decade ago, evidence that “NET evasion” might act as an immune protection strategy among respiratory pathogens, including group A Streptococcus, Bordetella pertussis, and Haemophilus influenzae, has been growing, with the majority of these studies being published in the past 2 years. Evasion strategies fall into three main categories: inhibition of NET release by down-regulating host inflammatory responses; degradation of NETs using pathogen-derived DNases; and resistance to the microbicidal components of NETs, which involves a variety of mechanisms, including encapsulation. Hence, the evasion of NETs appears to be a widespread strategy to allow pathogen proliferation and dissemination, and is currently a topic of intense research interest. This article outlines the evidence supporting the three main strategies of NET evasion—inhibition, degradation, and resistance—with particular reference to common respiratory pathogens

Randomised controlled trial of GM-CSF in critically ill patients with impaired neutrophil phagocytosis

Background Critically ill patients with impaired neutrophil phagocytosis have significantly increased risk of nosocomial infection. Granulocyte-macrophage colony-stimulating factor (GM-CSF) improves phagocytosis by neutrophils ex vivo. This study tested the hypothesis that GM-CSF improves neutrophil phagocytosis in critically ill patients in whom phagocytosis is known to be impaired. Methods This was a multicentre, phase IIa randomised, placebo-controlled clinical trial. Using a personalised medicine approach, only critically ill patients with impaired neutrophil phagocytosis were included. Patients were randomised 1:1 to subcutaneous GM-CSF (3 μg/kg/day) or placebo, once daily for 4 days. The primary outcome measure was neutrophil phagocytosis 2 days after initiation of GM-CSF. Secondary outcomes included neutrophil phagocytosis over time, neutrophil functions other than phagocytosis, monocyte HLA-DR expression and safety. Results Thirty-eight patients were recruited from five intensive care units (17 randomised to GM-CSF). Mean neutrophil phagocytosis at day 2 was 57.2% (SD 13.2%) in the GM-CSF group and 49.8% (13.4%) in the placebo group, p=0.73. The proportion of patients with neutrophil phagocytosis≥50% at day 2, and monocyte HLA-DR, appeared significantly higher in the GM-CSF group. Neutrophil functions other than phagocytosis did not appear significantly different between the groups. The most common adverse event associated with GM-CSF was fever. Conclusions GM-CSF did not improve mean neutrophil phagocytosis at day 2, but was safe and appeared to increase the proportion of patients with adequate phagocytosis. The study suggests proof of principle for a pharmacological effect on neutrophil function in a subset of critically ill patients