Oxidative Burst Response Research Articles

INDUCTION OF ANTIOXIDANT ENZYMES AND ISOZYMES PROFILE OF CHICKPEA GENOTYPES UNDER COMPATIBLE AND INCOMPATIBLE INTERACTIONS WITH FUSARIUM WILT

Chickpea genotypes (ten desi and eight kabuli), differing in their reaction to Fusarium wilt, were grown in Fusarium infested soil and screened for wilt resistance and susceptibility. The root and leaf tissues of highly resistant (desi WR 315 and kabul KAK 2) and highly susceptible (desi JG 62 and kabul L 550) chickpea genotypes were collected during disease development stages under pathogen infestation and non-infestation conditions. They were analyzed for antioxidant enzyme activities and isozymes profiling. The specific activities of ROS scavenging enzymes superoxide dismutase (SOD), glutathione peroxidase (GPX), ascorbate peroxidase (APX), polyphenol oxidase (PPO) and catalase (CAT) were found higher in the root and leaf tissues under compatible interactions (infection) compared with incompatible (non-infested or non-infected?) during disease development stages. The susceptible compatible interactions of desi and kabuli chickpea genotypes induced systemic antioxidant bursts (SOD, GPX, APX, PPO, CAT) efficiently after Fusarium infection. The isozymes profiling of SOD, APX, PPO, CAT agreed with the enzyme activities and also showed differences in GPX zymogram pattern distinctive in the root of kabuli resistant KAK 2 during infection stage (S2). However, nativePAGE electrophoregram revealed distinctive bands for infected root tissues of desi resistant WR 315 (novel) and kabuli resistant KAK 2 (correlated with GPX isoforms) at S2 stage. The SOD, GPX, and PPO isoforms are more diverse and associated with Fusarium interactions during disease development stages. The results explained production of an oxidative burst and related responses during Fusarium wilt infection of desi and kabuli chickpea genotypes.

Elucidation of the Role of Lectin-Like oxLDL Receptor-1 in the Metabolic Responses of Macrophages to Human oxLDL.

Atherogenesis is the narrowing of arteries due to plaque build-up that results in cardiovascular disease that can lead to death. The macrophage lectin-like oxidized LDL receptor-1 (LOX-1), also called the oxidized low-density lipoprotein receptor 1 (OLR1), is currently thought to aid in atherosclerotic disease progression; therefore metabolic studies have potential to both provide mechanistic validation for the role of LOX-1 in disease progression and provide valuable information regarding biomarker strategies and clinical imaging. One such mechanistic study is the upregulation of LOX-1 by methylated bacterial DNA and deoxy-cytidylate-phosphate-deoxy-guanylate-DNA (CpG)-DNA exposure. CpG-DNA is known to promote oxidative burst responses in macrophages, due to its direct binding to toll-like receptor 9 (TLR9) leading to the initiation of an NF-κB mediated immune response. In addition to the upregulation of macrophage LOX-1 expression, these studies have also examined the macrophage metabolic response to murine LOX-1/OLR1 antibody exposure. Our data suggests the antibody exposure effectively blocks LOX-1 dependent oxLDL metabolic activation of the macrophage, which was quantified using the multianalyte microphysiometer (MAMP). Using the MAMP to examine metabolic fluctuations during various types of oxLDL exposure, LOX-1 upregulation and inhibition provide valuable information regarding the role of LOX-1 in macrophage activation of oxidative burst.

Early Changes in Monocyte Adhesion Molecule Expression and Tumor Necrosis Factor-α Levels in Chronic Kidney Disease - A 5-Year Prospective Study

Background: Despite the absence of clinical symptoms, patients with chronic kidney disease (CKD) exhibit elevated levels of pro-inflammatory markers. To investigate whether it is possible to detect inflammatory activity and altered monocyte function at an early stage of renal disease, we studied patients with CKD stages 2-3 over 5 years. Methods: The expression of adhesion molecules on monocytes at resting state and after stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP), as well as oxidative metabolism capacity was measured with flow cytometry in 108 CKD patients and healthy controls. Soluble markers of inflammation, such as cytokines, were analyzed using the Milliplex technique. Results: Patients showed significantly lower CD11b expression after stimulation during the 3rd (p = 0.002) and the 5th year (p < 0.001), together with a lower oxidative burst in response to fMLP over time (p = 0.02). The expression of CD62L on resting monocytes was lower during the 3rd (p = 0.001) and the 5th (p = 0.001) year in patients. Levels of tumor necrosis factor-α and RANTES were significantly increased (p = 0.001, p = 0.006) and interleukin-12 levels were also higher in CKD patients during the 5th year (p = 0.007). Conclusion: Monocytes in CKD stages 2-3 show emerging functional abrasions, with altered adhesion molecule expression and impaired fMLP response. These findings suggest that a transformation of monocyte function occurs at an early phase of renal impairment and may together with increased plasma levels of pro-inflammatory cytokines contribute to the higher vulnerability of CKD patients to comorbidities, such as infections and cardiovascular disease.

1,25-Dihydroxyvitamin D Modulates Antibacterial and Inflammatory Response in Human Cigarette Smoke-Exposed Macrophages.

Cigarette smoking is associated with increased inflammation and defective antibacterial responses in the airways. Interestingly, vitamin D has been shown to suppress inflammation and to improve antibacterial defense. However, it is currently unknown whether vitamin D may modulate inflammation and antibacterial defects in human cigarette smoke (CS)-exposed airways. To explore these unresolved issues, alveolar macrophages obtained from non-smoking and smoking subjects as well as human cigarette smoke extract (CSE)-treated THP-1 macrophages were stimulated with 1,25-dihydroxyvitamin D (1,25(OH)2D) to address inflammatory and antibacterial responses. Although basal levels of inflammatory cytokines and chemokines did not differ between non-smoking and smoking subjects, 1,25(OH)2D did reduce levels of IL-6, TNF-α and MCP-1 in alveolar macrophages in response to LPS/IFN-γ, although not statistically significant for TNF-α and IL-6 in smokers. CSE did not significantly alter vitamin D metabolism (expression levels of CYP24A1 or CYP27B1) in THP-1 macrophages. Furthermore, stimulation with 1,25(OH)2D reduced mRNA expression levels and/or protein levels of IL-8, TNF-α and MCP-1 in CSE-treated THP-1 macrophages. 1,25(OH)2D did not improve defects in phagocytosis of E. coli bacteria or the oxidative burst response in CSE-treated THP-1 macrophages or alveolar macrophages from smokers. However, 1,25(OH)2D significantly enhanced mRNA expression and/or protein levels of the antimicrobial peptide cathelicidin in alveolar macrophages and THP-1 macrophages, independently of CS exposure. In conclusion, our results provide the first evidence that vitamin D could be a new strategy for attenuating airway inflammation and improving antibacterial defense in CS-exposed airways.

Diacylglycerol kinases activate tobacco NADPH oxidase‐dependent oxidative burst in response to cryptogein

Cryptogein is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial-associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [33 P]-orthophosphate labelling of tobacco Bright Yellow-2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide-dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD-mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH-oxidase activity. Amongst cluster III DGKs, the expression of DGK5-like was up-regulated in response to cryptogein. Besides DGK5-like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid-mediated events in plant immunity.

Rapid Hydrogen Peroxide Release during Coral-Bacteria Interactions

Ocean warming has exacerbated the severity of coral diseases, many of which are mediated by pathogenic bacteria. In response to the presence of pathogens various organisms activate an oxidative burst response, involving strong and rapid generation of reactive oxygen species (ROS) that serve both as bactericides and as signals for other defense systems. While many components of the coral immune systems are being unveiled, an oxidative burst response, triggered by bacteria proximity, has not yet been reported. Hydrogen Peroxide (H2O2), one of the ROS produced during the oxidative burst, is often monitored as an indicator of this pathway. Here we report a yet undescribed release of H2O2 from the coral Stylophora pistillata upon contact with bacteria. In a series of short term experiments we monitored H2O2 concentrations in the vicinity of S. pistillata fragments prior to and following local administration of five bacteria isolates belonging to the genus Vibrio (obtained from healthy S. pistillata). In most experiments, rapid H2O2 release was recorded at the site of interaction within one minute from Vibrio addition and persisted for the remaining five minutes of the experiment. In some experiments, discrete or continuous drop of H2O2 to below background levels were seen following Vibrio addition. This H2O2 loss was quantitatively accounted for by the bacterial antioxidants and imply that Vibrio may at times offset the coral released H2O2 The total H2O2 released by the coral from the small interaction site (~10 mm2) was 200-600 pmol, which may build up to a concentration of ~20 µM in the coral diffusive boundary layer. H2O2 concentrations above 10 µM resulted in significant mortality of the coral pathogen Vibrio coralliilyticus, suggesting that the released H2O2 may act as bactericide. It is thus far unclear if this H2O2 release is part of an oxidative burst response, yet it strongly indicates that corals sense and chemically react towards bacteria.

Pleiotropic effects of 4-hydroxynonenal on oxidative burst and phagocytosis in neutrophils

Metabolic control of cellular function is significant in the context of inflammation-induced metabolic dysregulation in immune cells. Generation of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide are one of the critical events that modulate the immune response in neutrophils. When activated, neutrophil NADPH oxidases consume large quantities of oxygen to rapidly generate ROS, a process that is referred to as the oxidative burst. These ROS are required for the efficient removal of phagocytized cellular debris and pathogens. In chronic inflammatory diseases, neutrophils are exposed to increased levels of oxidants and pro-inflammatory cytokines that can further prime oxidative burst responses and generate lipid oxidation products such as 4-hydroxynonenal (4-HNE). In this study we hypothesized that since 4-HNE can target glycolysis then this could modify the oxidative burst. To address this the oxidative burst was determined in freshly isolated healthy subject neutrophils using 13-phorbol myristate acetate (PMA) and the extracellular flux analyzer. Neutrophils pretreated with 4-HNE exhibited a significant decrease in the oxidative burst response and phagocytosis. Mass spectrometric analysis of alkyne-HNE treated neutrophils followed by click chemistry detected modification of a number of cytoskeletal, metabolic, redox and signaling proteins that are critical for the NADPH oxidase mediated oxidative burst. These modifications were confirmed using a candidate immunoblot approach for critical proteins of the active NADPH oxidase enzyme complex (Nox2 gp91phox subunit and Rac1 of the NADPH oxidase) and glyceraldehyde phosphate dehydrogenase, a critical enzyme in the metabolic regulation of oxidative burst. Taken together, these data suggest that 4-HNE-induces a pleiotropic mechanism to inhibit neutrophil function. These mechanisms may contribute to the immune dysregulation associated with chronic pathological conditions where 4-HNE is generated.

Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae

Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4+/1013). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4+/1013 mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4+/1013 mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis.

Reduced Oxidative Burst by Primed Neutrophils in the Elderly Individuals Is Associated With Increased Levels of the CD16bright/CD62Ldim Immunosuppressive Subset.

The aim of our study was to analyze polymorphonuclear neutrophil (PMN) functions in elderly individuals compared with those in healthy young participants, directly in whole blood to avoid issues with data interpretation related to cell isolation procedures. Despite the presence of increased circulating levels of proinflammatory cytokines, resting PMNs from the elderly individuals were not activated as shown by normal CD62L and CD11b expression at the PMN surface and normal constitutive reactive oxygen species (ROS) production. However, suboptimal stimulation induced modulations of CD62L and CD11b expression, which positively correlated with the interleukin-6 circulating level, suggesting a possible in vivo preactivation of old PMNs by this cytokine. In addition, PMN phagocytosis of opsonized Escherichia Coli was decreased in elderly individuals. Furthermore, upon preincubation of elderly whole-blood samples with tumor necrosis factor-α or Toll Receptor agonists, we observed a reduced PMN oxidative burst in response to formyl peptides. Elderly participants also exhibited an increased percentage of the immunosuppressive CD16bright/CD62Ldim PMN subpopulation, which was characterized by a lower phagocytic index and a reduced ROS production compared with the CD16bright/CD62Lbright subset. Thus, the reduced phagocytosis and ROS production associated with an expansion of immunosuppressive CD16bright/CD62Ldim PMN subpopulation might be involved in the increased susceptibility to bacterial and fungal infections with old age.

Group A Streptococcus Prevents Initiation of the Oxidative Burst Response in Immune Cells

Group A Streptococcus (GAS) is a gram‐positive bacterium that produces infections ranging from mild self‐limiting infections to more severe infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. In response to pathogens such as GAS, the host innate immune system produces antimicrobial factors such as reactive oxygen species that target microbes directly via oxidative stress, or indirectly via the initiation of other innate immune responses. Some bacteria, such as Lactococcus lactis and Staphylococcus aureus, contain pigment or catalase to detoxify these oxygen radicals. While GAS lacks these enzymes, it is still able to survive in innate immune cells, indicating that GAS may inhibit the oxidative burst response altogether. To examine this possibility, we infected macrophages with M1 and M49 serotype GAS, Staphylococcus aureus, Lactococcus lactis, and Group B Streptococcus and measured hydrogen peroxide production. Cells infected with either GAS serotypes produced significantly less peroxide compared with cells infected with other bacterial species, indicating that GAS suppresses the oxidative burst response. The GAS pore‐forming toxin streptolysin O (SLO) has similarity to listerolysin O (LLO), which prevents the assembly of the multi‐subunit NADPH oxidase on phagosome membranes. NADPH oxidase assembly is necessary to initiate the oxidative burst response. Peroxide generation was restored when cells were infected with GAS mutant bacteria of both M1 and M49 serotypes lacking SLO. We are currently examining the mechanism by which GAS uses SLO to prevents the oxidative burst response. Activation of the NADPH oxidase p47 subunit was verified Western blot for phosphorylated p47. Assembly of the NADPH oxidase is currently being assessed by cell fractionation of phagosomes and mass spectrometry. Preliminary data indicate that several NADPH oxidase subunits are missing from the phagosome. Thus, similar to LLO, SLO may inhibit the proper assembly of the NADPH oxidase, and prevent the generation of harmful reactive oxygen species.

Real-Time Monitoring of Superoxide Anion Radical and Hydrogen Peroxide Generation in Higher Plants in Response to Wounding: In-Vivo Electrochemical Study

The growth and development in plants are deleteriously affected by various biotic and abiotic stress factors. Wounding in plants occurs with exposure to environmental mechanical stresses or via herbivory. The response of plants towards wounding have been categorized based on the response time; the fast response includes oxidative burst accompanied by generation of reactive oxygen species (ROS) or delayed response which includes transcriptional activation of wound-induced genes, deposition of callose, accumulation of proteinase inhibitors and hydrolytic enzymes etc. The oxidative burst in response to wounding is known to be associated with the formation of various ROS such superoxide anion radical (O2 •−), singlet oxygen and hydrogen peroxide (H2O2). In the current study, we provide experimental evidences and real time monitoring of O2 •− and H2O2 generation following wounding in leaves of spinach (Spinacia oleracea). Real-time monitoring of ROS generation was achieved using amperometric method utilizing different electrodes. Non-specific real-time measurements of ROS were done by monitoring the changes in reduction current using carbon electrode while qualitative and quantitative measurement of O2 •− and H2O2 generationwere done using newly developed iron-porphyrin based modified carbon electrodes and osmium-horseradish peroxidase (Os-HRP) based modified carbon electrodes, respectively. The results obtained showed continuous generation of O2 •− and H2O2 for minutes after injury followed by a decline. The activation of lipoxygenase after wounding; its involvements in the production of H2O2 and the contribution of H2O2 in generation of local response in injured plant tissue besides the initiation of systemic response caused by hormones such as jasmonic acid, ethylene, salicylic acid, and abscisic acid will be disscussed.

Habitual physical activity is associated with the maintenance of neutrophil migratory dynamics in healthy older adults

BackgroundDysfunctional neutrophils with advanced age are a hallmark of immunosenescence. Reduced migration and bactericidal activity increase the risk of infection. It remains unclear why neutrophil dysfunction occurs with age. Physical activity and structured exercise have been suggested to improve immune function in the elderly. The aim of this study was to assess a comprehensive range of neutrophil functions and determine their association with habitual physical activity. MethodPhysical activity levels were determined in 211 elderly (67±5years) individuals by 7-days of accelerometry wear. Twenty of the most physically active men and women were matched for age and gender to twenty of the least physically active individuals. Groups were compared for neutrophil migration, phagocytosis, oxidative burst, cell surface receptor expression, metabolic health parameters and systemic inflammation. Groups were also compared against ten young participants (23±4years). ResultsThe most active group completed over twice as many steps/day as the least active group (p<0.001), had lower BMI’s (p=0.007) and body fat percentages (p=0.029). Neutrophils migrated towards IL-8 better in the most active group compared to the least active (p<0.05) and was comparable to that of the young (p>0.05). These differences remained after adjusting for BMI, body fat and plasma metabolic markers which were different between groups. Correlations revealed that steps/day, higher adiponectin and lower insulin were positively associated with migratory ability (p<0.05). There was no difference in expression of the chemokine receptors CXCR1 or CXCR2 (p>0.05 for both). CD11b was higher in the most active group compared to the least active (p=0.048). No differences between activity groups or young controls were observed for neutrophil phagocytosis or oxidative burst in response to Escherichia coli (p>0.05). The young group had lower concentrations of IL-6, IL-8, MCP-1, CRP, IL-10 and IL-13 (p<0.05 for all) with no differences between the two older groups. ConclusionThese data suggest that impaired neutrophil migration, but not bactericidal function, in older adults may be, in part, the result of reduced physical activity. A 2-fold difference in physical activity is associated with better preserved neutrophil migratory dynamics in healthy older people. As a consequence increasing habitual physical activity may be beneficial for neutrophil mediated immunity.

Streptolysin O Rapidly Impairs Neutrophil Oxidative Burst and Antibacterial Responses to Group A Streptococcus.

Group A Streptococcus (GAS) causes a wide range of human infections, ranging from simple pharyngitis to life-threatening necrotizing fasciitis and toxic shock syndrome. A globally disseminated clone of M1T1 GAS has been associated with an increase in severe, invasive GAS infections in recent decades. The secreted GAS pore-forming toxin streptolysin O (SLO), which induces eukaryotic cell lysis in a cholesterol-dependent manner, is highly upregulated in the GAS M1T1 clone during bloodstream dissemination. SLO is known to promote GAS resistance to phagocytic clearance by neutrophils, a critical first element of host defense against invasive bacterial infection. Here, we examine the role of SLO in modulating specific neutrophil functions during their early interaction with GAS. We find that SLO at subcytotoxic concentrations and early time points is necessary and sufficient to suppress neutrophil oxidative burst, in a manner reversed by free cholesterol and anti-SLO blocking antibodies. In addition, SLO at subcytotoxic concentrations blocked neutrophil degranulation, interleukin-8 secretion and responsiveness, and elaboration of DNA-based neutrophil extracellular traps, cumulatively supporting a key role for SLO in GAS resistance to immediate neutrophil killing. A non-toxic SLO derivate elicits protective immunity against lethal GAS challenge in a murine infection model. We conclude that SLO exerts a novel cytotoxic-independent function at early stages of invasive infections (<30 min), contributing to GAS escape from neutrophil clearance.

Identification of Scedosporium boydii catalase A1 gene, a reactive oxygen species detoxification factor highly expressed in response to oxidative stress and phagocytic cells

Scedosporium boydii is an opportunistic filamentous fungus which may be responsible for a large variety of infections in both immunocompetent and immunocompromised individuals. This fungus belongs to the Scedosporium apiospermum species complex which usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF). Species of the S. apiospermum complex are able to chronically colonize the CF airways suggesting pathogenic mechanisms allowing persistence and growth of these fungi in the respiratory tract. Few putative virulence factors have been purified and characterized so far in the S. apiospermum complex including a cytosolic Cu,Zn-superoxide dismutase (SOD) and a monofunctional catalase (catalase A1). Upon microbial infection, host phagocytes release reactive oxygen species (ROS), such as hydrogen peroxide, as part of the antimicrobial response. Catalases are known to protect pathogens against ROS by degradation of the hydrogen peroxide. Here, we identified the S. boydii catalase A1 gene (CATA1) and investigated its expression in response to the environmental conditions encountered in the CF airways and to the oxidative stress. Results showed that S. boydii CATA1 gene expression is not affected by hypoxia, hypercapnia or pH changes. In contrast, CATA1 gene was overexpressed in response to a chemically induced oxidative stress with a relative gene expression 37-fold higher in the presence of 250 μM H2O2, 20-fold higher with 250 μM menadione and 5-fold higher with 2 mM paraquat. Moreover, S. boydii CATA1 gene expression progressively increased upon exposure to activated THP-1-derived macrophages, reaching a maximum after 12 h (26 fold). Activated HL60-derived neutrophils and activated human peripheral blood neutrophils more rapidly induced S. boydii CATA1 gene overexpression, a maximum gene expression level being reached at 75 min (17 fold) and 60 min (15 fold), respectively. In contrast expression of the gene encoding the Cu,Zn-SOD (SODC gene) was not affected by H2O2, menadione, paraquat or in co-culture with phagocytic cells. These results suggest that S. boydii CATA1 gene is highly stimulated by the oxidative burst response whereas SODC gene is constitutively expressed.

The effect of a selective CXCR2 antagonist (AZD5069) on human blood neutrophil count and innate immune functions.

The aim of the present study was to investigate whether selective antagonism of the cysteine-X-cysteine chemokine receptor-2 (CXCR2) receptor has any adverse effects on the key innate effector functions of human neutrophils for defence against microbial pathogens. In a double-blind, crossover study, 30 healthy volunteers were randomized to treatment with the CXCR2 antagonist AZD5069 (100 mg) or placebo, twice daily orally for 6 days. The peripheral blood neutrophil count was assessed at baseline, daily during treatment and in response to exercise challenge and subcutaneous injection of granulocyte-colony stimulating factor (G-CSF). Neutrophil function was evaluated by phagocytosis of Escherichia coli and by the oxidative burst response to E. coli. AZD5069 treatment reversibly reduced circulating neutrophil count from baseline by a mean [standard deviation (SD)] of -1.67 (0.67) ×10(9) l(-1) vs. 0.19 (0.78) ×10(9) l(-1) for placebo on day 2, returning to baseline by day 7 after the last dose. Despite low counts on day 4, a 10-min exercise challenge increased absolute blood neutrophil count, but the effect with AZD5069 was smaller and not sustained, compared with placebo treatment. Subcutaneous G-CSF on day 5 caused a substantial increase in blood neutrophil count in both placebo- and AZD5069-treated subjects. Superoxide anion production in E. coli-stimulated neutrophils and phagocytosis of E. coli were unaffected by AZD5069 (P = 0.375, P = 0.721, respectively vs. baseline, Day 4). AZD5069 was well tolerated. CXCR2 antagonism did not appear adversely to affect the mobilization of neutrophils from bone marrow into the peripheral circulation, phagocytosis or the oxidative burst response to bacterial pathogens. This supports the potential of CXCR2 antagonists as a treatment option for diseases in which neutrophils play a pathological role.

PTH-096 Monocyte oxidative burst defect in alcoholic hepatitis is resistant to interferon gamma

Introduction An important cause of mortality in patients with severe alcoholic hepatitis (SAH) is infection. We and others have found that the oxidative burst response of phagocytic cells to E. coli is impaired in SAH which may contribute to susceptibility to infection. This is analogous to Chronic Granulomatous Disease, an inherited condition caused by a mutation in the enzyme NADPH oxidase which leads to predisposition to recurrent life-threatening infections. Treatment with interferon gamma (IFN-γ) reduces the frequency of severe infections in these patients. We sought to investigate the molecular mechanism underlying the monocyte oxidative burst (mOB) defect in SAH and determine whether IFN-γ can restore this defect in vitro . Method Peripheral blood mononuclear cells (PBMCs) were isolated from 6 patients with SAH and 4 healthy controls (HCs). Monocytes were isolated from PBMCs using magnetic MACS beads and negative selection. Monocytes were incubated with 50 ng/ml IFN-γ supplemented by 10% healthy serum for 24 h and the mOB to E. coli was quantified by oxidation of fluorogenic rhodamine and flow cytometry as previously described. Western blotting was used to quantify protein using monoclonal antibodies to components of the IFN-γ signalling pathway (pSTAT-1 and negative regulator SOCS-1) and glucose-6-phosphate dehydrogenase (G6PD, which generates NADPH substrate required for NAPDH oxidase function), in the monocytes of HCs and AH patients. G6PD enzyme activity was measured using a colorimetric kit. Quantitative PCR was used to determine expression of STAT-1 in monocytes stimulated for 24 h in 50 ng/ml IFN-γ. Results Surprisingly, SAH mOB defect was resistant to 24 h’ stimulation by 50 ng/mlIFN-γ. The level of unstimulated phosphorylated STAT-1 protein, a transcription factor activated by IFN-γ signalling was significantly reduced in monocytes of SAH patients compared to healthy controls (pSTAT-1: housekeeping gene 0.90 HC vs 0.14 SAH, p .05). q-PCR confirmed STAT-1 expression following stimulation with IFN-γ was significantly reduced in SAH compared to HCs (ΔCT 0.97 HC vs SAH 0.54, p .05). The level of SOCS-1 protein was significantly increased in monocytes of SAH patients compared to HCs (SOCS-1: housekeeping gene 0.98 HC vs 2.24 AH, p .05). There was no difference in G6PD enzyme activity or protein expression between groups. Conclusion Our study suggests that elevated levels of the inhibitory factor SOCS-1 may reduce phosphorylation and activation of STAT-1. Reduced pSTAT-1 may explain the resistance to IFN-γ observed and hence the burst defect in SAH. This work provides a novel insight into the impaired mOB in SAH and furthers our knowledge of the predisposition to infection observed in this patient group. Disclosure of interest None Declared.

Effect of hen age and maternal vitamin D source on performance, hatchability, bone mineral density, and progeny in vitro early innate immune function

The metabolite 25-hydroxy vitamin D3 (25-OHD) can complement or replace vitamin D3 in poultry rations, and may influence broiler production and immune function traits. The effect of broiler breeder dietary 25-OHD on egg production, hatchability, and chick early innate immune function was studied. We hypothesized that maternal dietary 25-OHD would support normal broiler breeder production and a more mature innate immune system of young chicks. Twenty-three-week-old Ross 308 hens (n = 98) were placed in 4 floor pens and fed either 2,760 IU vitamin D3 (D) or 69 μg 25-OHD/kg feed. Hen weights were managed according to the primary breeder management guide. At 29 to 31 wk (Early), 46 to 48 wk (Mid), and 61 to 63 wk (Late), hens were artificially inseminated and fertile eggs incubated and hatched. Chicks were placed in cages based on maternal treatment and grown to 7 d age. Innate immune function and plasma 25-OHD were assessed at 1 and 4 d post-hatch on 15 chicks/treatment. Egg production, hen BW, and chick hatch weight were not affected by diet (P > 0.05). Total in vitro Escherichia coli (E. coli) killing by 25-OHD chicks was greater than the D chicks at 4 d for the Early and Mid hatches, and 1 and 4 d for the Late hatch. This can be partly explained by the 25-OHD chicks from the Late hatch also having a greater E. coli phagocytic capability. No consistent pattern of oxidative burst response was observed. Chicks from the Mid hatch had greater percent phagocytosis, phagocytic capability, and E. coli killing than chicks from Early and Late hatches. Overall, maternal 25-OHD increased hatchability and in vitro chick innate immunity towards E. coli. Regardless of treatment, chicks from Late and Early hens had weaker early innate immune responses than chicks from Mid hens. The hen age effect tended to be the greatest factor influencing early chick innate immunity, but maternal 25-OHD also increased several measures relative to D.

Oxidant trade-offs in immunity: an experimental test in a lizard.

Immune system functioning and maintenance entails costs which may limit investment into other processes such as reproduction. Yet, the proximate mechanisms and ‘currencies’ mediating the costs of immune responses remain elusive. In vertebrates, up-regulation of the innate immune system is associated with rapid phagocytic production of pro-oxidant molecules (so-called ‘oxidative burst’ responses). Oxidative burst responses are intended to eliminate pathogens but may also constitute an immunopathological risk as they may induce oxidative damage to self cells. To minimize the risk of infection and, at the same time, damage to self, oxidative burst activity must be carefully balanced. The current levels of pro- and antioxidants (i.e. the individual oxidative state) is likely to be a critical factor affecting this balance, but this has not yet been evaluated. Here, we perform an experiment on wild-caught painted dragon lizards (Ctenophorus pictus) to examine how the strength of immune-stimulated oxidative burst responses of phagocytes in whole blood relates to individual oxidative status under control conditions and during an in vivo immune challenge with Escherichia coli lipopolysaccharide (LPS). Under control conditions, oxidative burst responses were not predicted by the oxidative status of the lizards. LPS-injected individuals showed a strong increase in pro-oxidant levels and a strong decrease in antioxidant levels compared to control individuals demonstrating a shift in the pro-/antioxidant balance. Oxidative burst responses in LPS-injected lizards were positively related to post-challenge extracellular pro-oxidants (reflecting the level of cell activation) and negatively related to pre-challenge levels of mitochondrial superoxide (suggesting an immunoregulatory effect of this pro-oxidant). LPS-challenged males had higher oxidative burst responses than females, and in females oxidative burst responses seemed to depend more strongly on antioxidant status than in males. Our results confirm the idea that oxidative state may constrain the activity of the innate immune system. These constraints may have important consequences for the way selection acts on pro-oxidant generating processes.

Phosphoproteomics-based peptide ligand-receptor kinase pairing. Commentary on: "A peptide hormone and its receptor protein kinase regulate plant cell expansion".

Phosphoproteomics-based peptide ligand-receptor kinase pairing. Commentary on: "A peptide hormone and its receptor protein kinase regulate plant cell expansion".

Neisseria gonorrhoeae elicits extracellular traps in primary neutrophil culture while suppressing the oxidative burst.

ABSTRACT Neisseria gonorrhoeae (the gonococcus) causes gonorrhea and is uniquely adapted to survive within the human reproductive tract. Gonococci evade host immune surveillance in part by varying their pili and opacity-associated proteins. These variable surface antigens influence interactions with host epithelial and immune cells. A potent polymorphonuclear leukocyte (PMN) response is a hallmark of symptomatic gonococcal infection, with vast numbers of PMNs recruited to the site of infection. A large body of literature describes gonococcus-PMN interactions, but the factors driving the outcome of infection are not fully understood. Gonococci have been described to both induce and suppress the PMN oxidative burst, but we determined that gonococci differentially affect induction of the PMN oxidative burst depending on the multiplicity of infection (MOI). Infecting PMN at an MOI of <20 gonococci elicits an oxidative burst, while an MOI of >20 suppresses the burst. Oxidative burst in response to gonococci is enhanced by, but does not require, expression of pili or opacity proteins. Neutrophil extracellular traps (NETs) were observed in gonococcus-infected PMNs, a process which requires an oxidative burst, yet gonococci induced NETs under suppressing conditions. The NETs were unable to kill gonococci despite killing the common vaginal bacterium Lactobacillus crispatus. Thus, gonococci influence PMN biology to promote their own survival by suppressing the oxidative burst of PMNs and stimulating the formation of NETs, which do not effectively kill gonococci, illustrating how N. gonorrhoeae has evolved to modulate PMN responses to promote infection.