Hypoxia determines survival outcomes of bacterial infection through HIF-1alpha dependent re-programming of leukocyte metabolism.

Hypoxia and bacterial infection frequently co-exist, in both acute and chronic clinical settings, and typically result in adverse clinical outcomes. To ameliorate this morbidity, we investigated the interaction between hypoxia and the host response. In the context of acute hypoxia, both S. aureus and S. pneumoniae infections rapidly induced progressive neutrophil mediated morbidity and mortality, with associated hypothermia and cardiovascular compromise. Preconditioning animals through longer exposures to hypoxia, prior to infection, prevented these pathophysiological responses and profoundly dampened the transcriptome of circulating leukocytes. Specifically, perturbation of HIF pathway and glycolysis genes by hypoxic preconditioning was associated with reduced leukocyte glucose utilisation, resulting in systemic rescue from a global negative energy state and myocardial protection. Thus we demonstrate that hypoxia preconditions the innate immune response and determines survival outcomes following bacterial infection through suppression of HIF-1α and neutrophil metabolism. The therapeutic implications of this work are that in the context of systemic or tissue hypoxia therapies that target the host response could improve infection associated morbidity and mortality.

Hypoxia causes IL-8 secretion, Charcot Leyden crystal formation, and suppression of corticosteroid-induced apoptosis in human eosinophils.

BACKGROUND: Inflamed environments are typically hypercellular, rich in pro-inflammatory cytokines, and profoundly hypoxic. While the effects of hypoxia on neutrophil longevity and function have been widely studied, little is known about the consequences of this stimulus on eosinophils. OBJECTIVE: We sought to investigate the effects of hypoxia on several key aspects of eosinophil biology, namely secretion, survival, and their sensitivity to glucocorticosteroids (GCS), agents that normally induce eosinophil apoptosis. METHODS: Eosinophils derived from patients with asthma/atopy or healthy controls were incubated under normoxia and hypoxia, with or without glucocorticoids. Activation was measured by flow cytometry, ELISA of cultured supernatants, and F-actin staining; apoptosis and efferocytosis by morphology and flow cytometry; and GCS efficacy by apoptosis assays and qPCR. RESULTS: Hypoxic incubation (3 kPa) caused (i) stabilization of HIF-2α and up-regulation of hypoxia-regulated genes including BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3) and GLUT1 (glucose transporter 1); (ii) secretion of pre-formed IL-8, and Charcot Leyden crystal (CLC) formation, which was most evident in eosinophils derived from atopic and asthmatic donors; (iii) enhanced F-actin formation; (iv) marked prolongation of eosinophil lifespan (via a NF-κB and Class I PI3-kinase-dependent mechanism); and (v) complete abrogation of the normal pro-apoptotic effect of dexamethasone and fluticasone furoate. This latter effect was evident despite preservation of GCS-mediated gene transactivation under hypoxia. CONCLUSION AND CLINICAL RELEVANCE: These data indicate that hypoxia promotes an eosinophil pro-inflammatory phenotype by enhancing eosinophil secretory function, delaying constitutive apoptosis, and importantly, antagonizing the normal pro-apoptotic effect of GCS. As eosinophils typically accumulate at sites that are relatively hypoxic, particularly during periods of inflammation, these findings may have important implications to understanding the behaviour of these cells in vivo.

Distinct responses to hypoxia in subpopulations of distal pulmonary artery cells contribute to pulmonary vascular remodeling in emphysema.

We have shown previously that hypoxia inhibits the growth of distal human pulmonary artery smooth muscle cells (PASMC) isolated under standard normoxic conditions (PASMC(norm)). By contrast, a subpopulation of PASMC, isolated through survival selection under hypoxia was found to proliferate in response to hypoxia (PASMC(hyp)). We sought to investigate the role of hypoxia-inducible factor (HIF) in these differential responses and to assess the relationship between HIF, proliferation, apoptosis, and pulmonary vascular remodeling in emphysema. PASMC were derived from lobar resections for lung cancer. Hypoxia induced apoptosis in PASMC(norm) (as assessed by TUNEL) and mRNA expression of Bax and Bcl-2, and induced proliferation in PASMC(hyp) (as assessed by (3)H-thymidine incorporation). Both observations were mimicked by dimethyloxallyl glycine, a prolyl-hydroxylase inhibitor used to stabilize HIF under normoxia. Pulmonary vascular remodeling was graded in lung samples taken from patients undergoing lung volume reduction surgery for severe heterogenous emphysema. Carbonic anhydrase IX expression in the medial compartment was used as a surrogate of medial hypoxia and HIF stabilization and increased with increasing vascular remodeling. In addition, a mixture of proliferation, assessed by proliferating-cell nuclear antigen, and apoptosis, assessed by active caspase 3 staining, were both higher in more severely remodeled vessels. Hypoxia drives apoptosis and proliferation via HIF in distinct subpopulations of distal PASMC. These differential responses may be important in the pulmonary vascular remodeling seen in emphysema and further support the key role of HIF in hypoxic pulmonary hypertension.

Effects of the cyclin-dependent kinase inhibitor R-roscovitine on eosinophil survival and clearance.

BACKGROUND: Eosinophils are pro-inflammatory cells implicated in the pathogenesis of asthma and atopy. Apoptosis has been proposed as a potential mechanism underlying the resolution of eosinophilic inflammation and studies have indicated the ability of interventions that induce human eosinophil apoptosis to promote the resolution of eosinophilic inflammation. Recently, the cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to enhance neutrophil apoptosis and promote the resolution of neutrophilic inflammation. OBJECTIVE: The purpose of this study was to examine the expression of CDKs in human blood eosinophils, the effects of R-roscovitine on eosinophil survival in vitro and whether R-roscovitine could influence eosinophilic lung inflammation in vivo. METHODS: Eosinophils were isolated from human peripheral blood and the effects of R-roscovitine on apoptosis, degranulation and phagocytic uptake examined in vitro. The effects of R-roscovitine on eosinophilic lung inflammation in vivo were also assessed using an ovalbumin mouse model. RESULTS: Our data demonstrate that human eosinophils express five known targets for R-roscovitine: CDK1, -2, -5, -7 and -9. R-roscovitine induced eosinophil apoptosis in a time- and concentration-dependent manner but also accelerated transition to secondary necrosis as assessed by microscopy, flow cytometry and caspase activation. In addition, we show that R-roscovitine can override the anti-apoptotic signals of GM-CSF and IL-5. We report that the pro-apoptotic effect of R-roscovitine is associated with suppression of Mcl-1L expression and that this compound enhanced phagocytic clearance of eosinophils by macrophages. Finally, we show that R-roscovitine induces apoptosis in murine peripheral blood and spleen-derived eosinophils; despite this, R-roscovitine did not modulate the tissue and lumen eosinophilia characteristic of the ovalbumin mouse model of airway eosinophilia. CONCLUSION AND CLINICAL RELEVANCE: These data demonstrate that R-roscovitine is capable of inducing rapid apoptosis and secondary necrosis in eosinophils but does not affect the onset or improve the resolution of eosinophilic airway inflammation in vivo.

Cystic fibrosis neutrophils have normal intrinsic reactive oxygen species generation.

Previous studies have identified abnormalities in the oxidative responses of the neutrophil in cystic fibrosis (CF), but it is unclear whether such changes relate to loss of membrane cystic fibrosis transmembrane conductance regulator (CFTR) or to the inflammatory environment present in this disease. The aim of the present study was to determine whether neutrophils from CF patients demonstrate an intrinsic abnormality of the respiratory burst. The respiratory burst activity of neutrophils isolated from stable DeltaF508 homozygote CF patients and matched healthy controls was quantified by both chemiluminscence and cytochrome C reduction. Expression of NADPH oxidase components and CFTR was determined by Western blotting and RT-PCR. The oxidative output from neutrophils from CF in response to receptor-linked and particulate stimuli did not differ from that of controls. Expression of NADPH oxidase components was identical in CF and non-CF neutrophils. While low levels of CFTR mRNA could be identified in the normal human neutrophil, we were unable to detect CFTR protein in human neutrophil lysates or immunoprecipitates. CFTR has no role in controlling neutrophil oxidative activity; previously reported differences in neutrophil function between CF and non-CF subjects most likely relate to the inflammatory milieu from which the cells were isolated.

Idiopathic pulmonary fibrosis in a Christmas Island nuclear test veteran.

We describe the case of a 71-year-old man with idiopathic pulmonary fibrosis (usual interstitial pneumonia (UIP) pattern) diagnosed on clinical, radiological and lung function criteria, in accordance with the American Thoracic Society/European Respiratory Society consensus criteria (2000), who had been in close proximity to three atmospheric nuclear bomb blasts during military service in 1957. He does not have clubbing and clinically and radiologically his lung disease is stable. He also has bladder carcinoma and carotid arteriosclerosis, both recognised consequences of radiation injury. This is the first reported case of UIP in a nuclear test veteran. Awareness of this potential association is important given the current attempts of the British Nuclear Test Veterans Association to gain compensation for claimed injuries.

Kinetics of pulmonary neutrophil recruitment and clearance in a natural and spontaneously resolving model of airway inflammation.

BACKGROUND: Neutrophil apoptosis and phagocytic clearance have been proposed as key determinants affecting the resolution of airway inflammation. Objective To determine the kinetics of neutrophil priming, recruitment, activation and subsequent clearance in a naturally occurring equine disease model of neutrophilic pulmonary inflammation. METHODS AND RESULTS: A 5 h mouldy hay/straw challenge in hypersensitive horses induced transient pulmonary dysfunction lasting 4 days. At 24 h circulating neutrophils were primed and displayed delayed rates of spontaneous apoptosis in vitro. Neutrophil numbers in the airspaces peaked at 5 h and then fell abruptly, returning to pre-challenge levels by 4 days. Airspace neutrophils demonstrated increased respiratory burst activity compared with circulating cells and equine neutrophil elastase 2A concentrations increased in parallel with neutrophil numbers indicating in vivo priming and degranulation. The number of apoptotic neutrophils and proportion of alveolar macrophages containing phagocytosed apoptotic neutrophils increased significantly at 24 h and 4 days post-challenge corresponding to the period of most rapid neutrophil clearance. CONCLUSION: This is the first demonstration of spontaneous neutrophil apoptosis and phagocytic removal in a natural disease model of airway inflammation and provides critical kinetic data to support the hypothesis that this clearance pathway plays a central role in the resolution of neutrophilic inflammation.

Efficacy and tolerability of anti-immunoglobulin E therapy with omalizumab in patients with poorly controlled (moderate-to-severe) allergic asthma.

BACKGROUND: Patients with poorly controlled asthma have greater morbidity and mortality. This study evaluated the efficacy and tolerability of omalizumab in patients with poorly controlled, moderate-to-severe allergic asthma. METHODS: This was a randomized, open-label, multicentre, parallel-group study. A total of 312 patients (12-73 years) receiving >/=400 microg/day (adolescent) or >/=800 microg/day (adult) inhaled beclomethasone dipropionate, or equivalent were included. Patients received best standard care (BSC) with or without omalizumab [at least 0.016 mg/kg/IgE (IU/ml) every 4 weeks] for 12 months. RESULTS: The annualized mean number of asthma deterioration-related incidents was reduced from 9.76 with BSC alone (n = 106) to 4.92 per patient-year with omalizumab (n = 206) (P < 0.001). Mean clinically significant asthma exacerbation rates were 2.86 and 1.12 per patient-year, respectively (P < 0.001). Omalizumab-treated patients (41.4%) required rescue medication <1 day/week compared with 20.7% for BSC alone (P < 0.001). Omalizumab improved absolute forced expiratory volume in 1 s (FEV(1)) compared with BSC alone (2.48 and 2.28 l, respectively; P < 0.05) and reduced symptom scores relative to BSC alone (decrease of 6.5 and 0.7 respectively; P < 0.001). Omalizumab was well-tolerated. CONCLUSIONS: Omalizumab administered as add-on therapy to BSC benefits patients with poorly controlled, moderate-to-severe allergic asthma.

Characterization of the survival effect of tumour necrosis factor-alpha in human neutrophils.

Granulocyte apoptosis has been proposed as a fundamental, injury-limiting granulocyte-clearance mechanism. As such, inhibition of this process may prevent the resolution of inflammation. Our previous studies have shown that TNFalpha (tumour necrosis factor-alpha) has a bi-modal influence on the rate of constitutive neutrophil apoptosis in vitro, causing early acceleration and late inhibition of this process. The pro-apoptotic effect is uniquely TNFR1 (TNF receptor 1) and TNFR2-dependent and the latter survival process is mediated via phosphoinositide 3-kinase and NF-kappaB (nuclear factor-kappaB) activation. In the present study, we show that, in contrast with GM-CSF (granulocyte/macrophage colony-stimulating factor), the delayed addition (i.e. at 6 h) of TNFalpha increases its survival effect despite substantial loss of neutrophil TNFR1 and TNFR2 at that time. This paradox was resolved using PBMC (peripheral blood mononuclear cell)-deplete and 5% PBMC-replete neutrophil cultures, where the enhanced survival effect observed after delayed TNFalpha addition was shown to be PBMC-dependent. TNFR2-blocking antibodies had no effect on the late survival effect of TNFalpha, implying a TNFR1-dependent process. Finally, I-kappaBalpha (inhibitory kappaB-alpha) and NF-kappaB time-course studies demonstrated that the survival effects of both GM-CSF and TNFalpha could be explained by maintenance of functional NF-kappaB.

What can we learn from highly purified neutrophils?

Neutrophil purification has traditionally been performed by centrifugation of leucocytes through density gradients. These reliable methods produce populations that are typically >95% pure neutrophils, and have allowed the widespread study of the function of these cells. Our recent work has suggested that residual monocytes may play a more important role than has been previously realized, and suggest that for some functional experiments, further purification of cells is required to understand fully the neutrophil phenotype.

Hypoxic regulation of neutrophil apoptosis role: of reactive oxygen intermediates in constitutive and tumor necrosis factor alpha-induced cell death.

Activation of the NADPH oxidase system to generate reactive oxygen species (ROS) plays a key role in bacterial killing by human neutrophils. However, the involvement of such radicals in spontaneous and TNFalpha-driven neutrophil apoptosis remains uncertain. While incubation of cells under anoxic conditions attenuated the pro-apoptotic effect of TNFalpha, full activation of the respiratory burst using PAF followed by fMLP, or the addition of physiologically relevant concentrations of H(2)O(2), had no effect on the rate of apoptosis. Furthermore, the phosphoinositide 3-kinase inhibitor, LY294002, which abolishes receptor-mediated activation of the NADPH oxidase, and five discrete anti-oxidants all failed to affect apoptotic thresholds. Thus ROS do not appear to modulate constitutive apoptosis in neutrophils or appear sufficient to mediate the pro-apoptotic effect of TNFalpha.