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.

Cutaneous exposure to hypoxia does not affect skin perfusion in humans.

AIM: Experiments have indicated that skin perfusion in mice is sensitive to reductions in environmental O2 availability. Specifically, a reduction in skin-surface PO2 attenuates transcutaneous O2 diffusion, and hence epidermal O2 supply. In response, epidermal HIF-1α expression increases and facilitates initial cutaneous vasoconstriction and subsequent nitric oxide-dependent vasodilation. Here, we investigated whether the same mechanism exists in humans. METHODS: In a first experiment, eight males rested twice for 8 h in a hypobaric chamber. Once, barometric pressure was reduced by 50%, while systemic oxygenation was preserved by O2 -enriched (42%) breathing gas (HypoxiaSkin ), and once barometric pressure and inspired O2 fraction were normal (Control1 ). In a second experiment, nine males rested for 8 h with both forearms wrapped in plastic bags. O2 was expelled from one bag by nitrogen flushing (AnoxiaSkin ), whereas the other bag was flushed with air (Control2 ). In both experiments, skin blood flux was assessed by laser Doppler on the dorsal forearm, and HIF-1α expression was determined by immunohistochemical staining in forearm skin biopsies. RESULTS: Skin blood flux during HypoxiaSkin and AnoxiaSkin remained similar to the corresponding Control trial (P = 0.67 and P = 0.81). Immunohistochemically stained epidermal HIF-1α was detected on 8.2 ± 6.1 and 5.3 ± 5.7% of the analysed area during HypoxiaSkin and Control1 (P = 0.30) and on 2.3 ± 1.8 and 2.4 ± 1.8% during AnoxiaSkin and Control2 (P = 0.90) respectively. CONCLUSION: Reductions in skin-surface PO2 do not affect skin perfusion in humans. The unchanged epidermal HIF-1α expression suggests that epidermal O2 homoeostasis was not disturbed by HypoxiaSkin /AnoxiaSkin , potentially due to compensatory increases in arterial O2 extraction.

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.

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.

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.

Are cysteinyl leukotrienes involved in allergic responses in human skin?

BACKGROUND: Cysteinyl leukotrienes have been suggested to be involved in producing the symptoms of both the early and late phases of the allergic response in the lung and other tissues. OBJECTIVE: To use scanning laser Doppler imaging, microdialysis and immunocytochemistry to explore the mediator and cellular mechanisms of the dermal allergic response. METHODS: Thirteen atopic volunteers received intradermal injections into the forearm of grass pollen or D. pteronyssinus extract. Changes in dermal blood flow up to 8 h were monitored by scanning laser Doppler imaging. The release of histamine, PGD2 and LTC4/D4/E4 was assessed by dermal microdialysis. Skin biopsies were taken at 6 h to determine numbers of mast cells, eosinophils, basophils, Langerhans' cells, and monocytes/macrophages, and the expression of COX-1, COX-2, 5-LO and FLAP. RESULTS: Allergen provocation produced an immediate weal and flare response followed by an erythematous induration peaking at 6 h. During the first hour, c. 84 pmoles of histamine and c. 0.3 pmoles of PGD2 were recovered by microdialysis (both P < 0.001) but LTC4/D4/E4 was undetectable. No histamine, PGD2 or LTC4/D4/E4 was detectable at later times. Immunocytochemical examination of biopsies taken at 8 h showed increased numbers of eosinophils and basophils and in COX-2, 5-LO and FLAP, but not COX-1. Expression of 5-LO and FLAP was associated primarily with eosinophils. CONCLUSIONS: These findings suggest that inflammatory cells recruited to the site of allergen injection are not activated to release detectable amounts of cysteinyl leukotrienes. Hence, it is unlikely that the late-phase erythematous induration is mediated by this autocoid.

Phosphoinositide 3-kinase: a critical signalling event in pulmonary cells.

Phosphoinositide 3-kinases (PI-3Ks) are enzymes that generate lipid second messenger molecules, resulting in the activation of multiple intracellular signalling cascades. These events regulate a broad array of cellular responses including survival, activation, differentiation and proliferation and are now recognised to have a key role in a number of physiological and pathophysiological processes in the lung. PI-3Ks contribute to the pathogenesis of asthma by influencing the proliferation of airways smooth muscle and the recruitment of eosinophils, and affect the balance between the harmful and protective responses in pulmonary inflammation and infection by the modulation of granulocyte recruitment, activation and apoptosis. In addition they also seem to exert a critical influence on the malignant phenotype of small cell lung cancer. PI-3K isoforms and their downstream targets thus provide novel therapeutic targets for intervention in a broad spectrum of respiratory diseases.

IL-5 increases expression of 5-lipoxygenase-activating protein and translocates 5-lipoxygenase to the nucleus in human blood eosinophils.

Cysteinyl-leukotrienes are potent bronchoconstrictor mediators synthesized by the 5-lipoxygenase (5-LO) pathway. Eosinophilopoietic cytokines such as IL-5 enhance cysteinyl-leukotriene synthesis in eosinophils in vitro, mimicking changes in eosinophils from asthmatic patients, but the mechanism is unknown. We hypothesized that IL-5 induces the expression of 5-LO and/or its activating protein FLAP in eosinophils, and that this might be modulated by anti-inflammatory corticosteroids. Compared with control cultures, IL-5 increased the proportion of normal blood eosinophils immunostaining for FLAP (65 +/- 4 vs 34 +/- 4%; p < 0.0001), enhanced immunoblot levels of FLAP by 51 +/- 14% (p = 0.03), and quadrupled ionophore-stimulated leukotriene C4 synthesis from 5.7 to 20.8 ng/106 cells (p < 0.02). IL-5 effects persisted for 24 h and were abolished by cycloheximide and actinomycin D. The proportion of FLAP+ eosinophils was also increased by dexamethasone (p < 0.0001). Neither IL-5 nor dexamethasone altered 5-LO expression, but IL-5 significantly increased 5-LO immunofluorescence localizing to eosinophil nuclei. Compared with normal subjects, allergic asthmatic patients had a greater proportion of circulating FLAP+ eosinophils (46 +/- 6 vs 27 +/- 3%; p < 0.03) and a smaller IL-5-induced increase in FLAP immunoreactivity (p < 0.05). Thus, IL-5 increases FLAP expression and translocates 5-LO to the nucleus in normal blood eosinophils in vitro. This is associated with an enhanced capacity for cysteinyl-leukotriene synthesis and mimics in vivo increases in FLAP expression in eosinophils from allergic asthmatics.

Overexpression of leukotriene C4 synthase in bronchial biopsies from patients with aspirin-intolerant asthma.

Aspirin causes bronchoconstriction in aspirin-intolerant asthma (AIA) patients by triggering cysteinyl-leukotriene (cys-LT) production, probably by removing PGE2-dependent inhibition. To investigate why aspirin does not cause bronchoconstriction in all individuals, we immunostained enzymes of the leukotriene and prostanoid pathways in bronchial biopsies from AIA patients, aspirin-tolerant asthma (ATA) patients, and normal (N) subjects. Counts of cells expressing the terminal enzyme for cys-LT synthesis, LTC4 synthase, were fivefold higher in AIA biopsies (11.5+/-2.2 cells/mm2, n = 10) than in ATA biopsies (2.2+/-0.7, n = 10; P = 0. 0006) and 18-fold higher than in N biopsies (0.6+/-0.4, n = 9; P = 0. 0002). Immunostaining for 5-lipoxygenase, its activating protein (FLAP), LTA4 hydrolase, cyclooxygenase (COX)-1, and COX-2 did not differ. Enhanced baseline cys-LT levels in bronchoalveolar lavage (BAL) fluid of AIA patients correlated uniquely with bronchial counts of LTC4 synthase+ cells (rho = 0.83, P = 0.01). Lysine-aspirin challenge released additional cys-LTs into BAL fluid in AIA patients (200+/-120 pg/ml, n = 8) but not in ATA patients (0. 7+/-5.1, n = 5; P = 0.007). Bronchial responsiveness to lysine-aspirin correlated exclusively with LTC4 synthase+ cell counts (rho = -0.63, P = 0.049, n = 10). Aspirin may remove PGE2-dependent suppression in all subjects, but only in AIA patients does increased bronchial expression of LTC4 synthase allow marked overproduction of cys-LTs leading to bronchoconstriction.