Chronic Granulomatous Disease Neutrophils Research Articles

TNFα: TNFR1 signaling inhibits maturation and maintains the pro-inflammatory programming of monocyte-derived macrophages in murine chronic granulomatous disease.

Loss of NADPH oxidase activity results in proinflammatory macrophages that contribute to hyperinflammation in Chronic Granulomatous Disease (CGD). Previously, it was shown in a zymosan-induced peritonitis model that gp91phox-/- (CGD) monocyte-derived macrophages (MoMacs) fail to phenotypically mature into pro-resolving MoMacs characteristic of wild type (WT) but retain the ability to do so when placed in the WT milieu. Accordingly, it was hypothesized that soluble factor(s) in the CGD milieu thwart appropriate programming. We sought to identify key constituents using ex vivo culture of peritoneal inflammatory leukocytes and their conditioned media. MoMac phenotyping was performed via flow cytometry, measurement of efferocytic capacity and multiplex analysis of secreted cytokines. Addition of exogenous TNFα, TNFα neutralizing antibody and TNFR1-/- MoMacs were used to study the role of TNFα: TNFR1 signaling in MoMac maturation. More extensive phenotyping defined normal MoMac maturation and demonstrated failure of maturation of CGD MoMacs both ex vivo and in vivo. Protein components, and specifically TNFα, produced and released by CGD neutrophils and MoMacs into conditioned media was identified as critical to preventing maturation. Exogenous addition of TNFα inhibited WT MoMac maturation, and its neutralization allowed maturation of cultured CGD MoMacs. TNFα neutralization also reduced production of IL-1β, IL-6 and CXCL1 by CGD cells though these cytokines played no role in MoMac programming. MoMacs lacking TNFR1 matured more normally in the CGD milieu both ex vivo and following adoptive transfer in vivo. These data lend mechanistic insights into the utility of TNFα blockade in CGD and to other diseases where such therapy has been shown to be beneficial.

Modulation of Host Defense in Chronic Granulomatous Disease (CGD) Via Adoptive Transfer of Neutrophil Progenitor Cells

Ectopic expression of transcription factor HoxB8 in murine bone marrow stem cells programs cells to stably expand exponentially in culture. When HoxB8 expression is regulated through a conditional promoter, cells are capable of expansion through HoxB8 induction or differentiation into neutrophil-like cells through HoxB8 withdrawal. We have previously demonstrated that some derivations of HoxB8 progenitor cells are capable of engraftment in an unconditioned murine host leading to rapid formation of a transient population of donor neutrophils in the periphery. Donor derived neutrophils (ddN) function comparably to host wild-type (WT) neutrophils ex vivo. Based on these findings, we hypothesize that conditionally immortalized HoxB8 progenitor cells have the potential to serve as a novel neutrophil driven cellular therapy. To investigate the use of HoxB8 progenitor cells as a cellular therapy for disease, we utilized a well characterized murine model of the congenital disorder CGD. Individuals with CGD are particularly susceptible to infections with Staphylococcus aureus due to loss of reactive oxygen species (ROS) production via the NADPH oxidase. Impaired neutrophil function leads to increased inflammation and decreased microbial killing. We found that prophylactic treatment of CGD mice with an adoptive transfer (AT) of WT HoxB8 progenitor cells protected them from an otherwise lethal pulmonary infection (Figure 1). Mice which received an AT of progenitor cells derived from a CGD animal did not have improved outcomes. This suggests the effector functions of the WT ddN were responsible for the change in survival. To better understand the mechanism of protection derived from the AT, we developed a novel fluorescent killing assay. This assay demonstrated the positive influence of the WT ddN on the host CGD neutrophils intracellular killing capacity in vivo. In addition to prophylactic treatment, post infectious treatment with the progenitor cells improved survival to >50% (Figure 2). Interestingly, twelve hours post adoptive transfer, donor derived cells represent a small population of the BAL cellular environment (<1%). Evaluation of the BAL and serum cytokine environment at this time-point provide insight to the immuno-modulatory role of the donor derived cells. Results from this study demonstrate the promise of a neutrophil progenitor based cellular therapy. This approach is unique from those currently employed as progenitor cells engraft without conditioning, expand and differentiate in vivo unlike granulocyte transfusions, and may be genetically modified in culture to potentially address the challenges of microbial resistance genes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Improving the function of neutrophils from chronic granulomatous disease patients using mesenchymal stem cells’ exosomes

In chronic granulomatous disease (CGD) patients, reactive oxygen species (ROS) production by neutrophils is impaired. So, they are susceptible to infections. Studies showed that, mesenchymal stem cells (MSCs) have protective effects on the function of neutrophils and an approach that MSCs use to apply their effects, is secreting soluble factors and exosomes. So, we investigated the effects of MSC-exosomes and MSC-conditioned media (MSC-CM) on the function and apoptosis of neutrophils in CGD patients.In this study, neutrophils were isolated from healthy donors and CGD patients and then incubated with exosomes or CM that were prepared from MSCs. Then, neutrophil respiratory burst, apoptosis and phagocytosis capacity were evaluated by NBT assay, Annexin V-PI method and Giemsa staining.It was demonstrated that both MSC-exosomes and CM could improve the phagocytosis capacity and ROS production of neutrophils in CGD patients and healthy donors. In contrast to the healthy group, in CGD patients, exosomes significantly reduced the percentage of viable neutrophils.This report indicated that MSC exosomes and CM could increase the function of the neutrophils isolated from CGD patients. But decreasing the number of the living cells is one of the limitations of them. However, it is hoped that this intervention will be developed in future studies to minimize its limitations.

Downregulation of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) in a co-culture system with human stimulated X-linked CGD neutrophils.

Phagocytes in patients with chronic granulomatous disease (CGD) do not generate reactive oxidative species (ROS), whereas nitric oxide (NO) production is increased in response to the calcium ionophore A23187 in CGD phagocytes compared with healthy phagocytes. Recently, patients with X-linked CGD (X-CGD) have been reported to show higher flow-mediated dilation, suggesting that endothelial cell function is affected by NO production from phagocytes. We studied NOS3 and EDN1 mRNA and protein expression in human umbilical vein endothelial cells (HUVECs) in a co-culture system with neutrophils from X-CGD patients. HUVECs were co-cultured for 30 minutes with human neutrophils from X-CGD or healthy participants in response to A23187 without cell-to-cell contact. The expression of NOS3 and EDN1 mRNA in HUVECs was quantified by real-time polymerase chain reaction. Moreover, we demonstrated the protein expression of eNOS, ET-1, and NFκB p65, including phosphorylation at Ser1177 of eNOS and Ser536 of NFκB p65. Neutrophils from X-CGD patients showed significantly higher NO and lower H2O2 production in response to A23187 than healthy neutrophils in vitro. Compared with healthy neutrophils, X-CGD neutrophils under A23187 stimulation exhibited significantly increased NO and decreased H2O2, and promoted downregulated NOS3 and EDN1 expression in HUVECs. The total expression and phosphorylation at Ser1177 of eNOS and ET-1 expression were significantly decreased in HUVECs co-cultures with stimulated X-CGD neutrophils. Also, phosphorylation at Ser536 of NFκB p65 were significantly decreased. In conclusions, eNOS and ET-1 significantly down-regulated in co-culture with stimulated X-CGD neutrophils through their excessive NO and the lack of ROS production. These findings suggest that ROS generated from neutrophils may mediate arterial tone affecting eNOS and ET-1 expression via their NO and ROS production.

NADPH oxidase controls pulmonary neutrophil infiltration in the response to fungal cell walls by limiting LTB4

AbstractLeukocyte reduced NADP (NADPH) oxidase plays a key role in host defense and immune regulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by recurrent bacterial and fungal infections and aberrant inflammation. Key drivers of hyperinflammation induced by fungal cell walls in CGD are still incompletely defined. In this study, we found that CGD (CYBB−) neutrophils produced higher amounts of leukotriene B4 (LTB4) in vitro after activation with zymosan or immune complexes, compared with wild-type (WT) neutrophils. This finding correlated with increased calcium influx in CGD neutrophils, which was restrained in WT neutrophils by the electrogenic activity of NADPH oxidase. Increased LTB4 generation by CGD neutrophils was also augmented by paracrine cross talk with the LTB4 receptor BLT1. CGD neutrophils formed more numerous and larger clusters in the presence of zymosan in vitro compared with WT cells, and the effect was also LTB4- and BLT1-dependent. In zymosan-induced lung inflammation, focal neutrophil infiltrates were increased in CGD compared with WT mice and associated with higher LTB4 levels. Inhibiting LTB4 synthesis or antagonizing the BLT1 receptor after zymosan challenge reduced lung neutrophil recruitment in CGD to WT levels. Thus, LTB4 was the major driver of excessive neutrophilic lung inflammation in CGD mice in the early response to fungal cell walls, likely by a dysregulated feed-forward loop involving amplified neutrophil production of LTB4. This study identifies neutrophil LTB4 generation as a target of NADPH oxidase regulation, which could potentially be exploited therapeutically to reduce excessive inflammation in CGD.

Mitochondrial permeability transition pore is involved in oxidative burst and NETosis of human neutrophils

Neutrophils release neutrophil extracellular traps (NETs) in response to numerous pathogenic microbes as the last suicidal resource (NETosis) in the fight against infection. Apart from the host defense function, NETs play an essential role in the pathogenesis of various autoimmune and inflammatory diseases. Therefore, understanding the molecular mechanisms of NETosis is important for regulating aberrant NET release. The initiation of NETosis after the recognition of pathogens by specific receptors is mediated by an increase in intracellular Ca2+ concentration, therefore, the use of Ca2+ ionophore A23187 can be considered a semi-physiological model of NETosis. Induction of NETosis by various stimuli depends on reactive oxygen species (ROS) produced by NADPH oxidase, however, NETosis induced by Ca2+ ionophores was suggested to be mediated by ROS produced in mitochondria (mtROS).Using the mitochondria-targeted antioxidant SkQ1 and specific inhibitors of NADPH oxidase, we showed that both sources of ROS, mitochondria and NADPH oxidase, are involved in NETosis induced by A23187 in human neutrophils. In support of the critical role of mtROS, SkQ1-sensitive NETosis was demonstrated to be induced by A23187 in neutrophils from patients with chronic granulomatous disease (CGD). We assume that Ca2+-triggered mtROS production contributes to NETosis either directly (CGD neutrophils) or by stimulating NADPH oxidase. The opening of the mitochondrial permeability transition pore (mPTP) in neutrophils treated by A23187 was revealed using the electron transmission microscopy as a swelling of the mitochondrial matrix. Using specific inhibitors, we demonstrated that the mPTP is involved in mtROS production, NETosis, and the oxidative burst induced by A23187.

Next Generation Proteomics of Human Neutrophil Granulocytes in Monogenic Disease

Background and objectiveNeutrophil granulocytes (NG) are critical mediators of innate immunity and tissue regeneration. Few monogenic diseases affecting their differentiation and/or function have been described, yet a systems-biology perspective has not yet been opened. We set out to define the proteome and transcriptome of NG from healthy individuals and exome-sequenced patients aiming to develop a multi-dimensional perspective for diagnostic purposes.Patients and methodsWe analyzed highly purified peripheral blood neutrophil granulocytes from 68 healthy individuals, 6 children with severe congenital neutropenia (SCN) associated with ELANE mutations, 5 children with chronic granulomatous disease (CGD) with CYBA (2) or CYBB (3) mutations and 3 children with leukocyte adhesion deficiency (LAD, 2x ITGB2 mutation, 1x SLC35C1 mutation). In addition we analyzed proteomes of 2 children with genetically undetermined neutrophil defects. Cells were isolated from fresh venous blood using magnetic bead-based negative selection (purity >99%). Whole cell proteome analysis was done by data-independent acquisition using a Thermo Fisher QExactive HF mass spectrometer.ResultsWe quantified a total of ~4300 proteins in each sample. This is the first study to analyze highly purified neutrophil granulocytes from a large cohort of healthy individuals as well as from patients with rare diseases affecting differentiation and/or function of NG. Intraindividual reproducibility was shown for one donor sampled at 3 different time points with a correlation coefficient of ~0.89. Principal component analysis demonstrated separation of the proteome of healthy and diseased cells. Interestingly, only 6 proteins constituted for 50% of the neutrophils proteome with the antimicrobial proteins DEF3A and S100A8 being the most abundant.Stringent differential expression analysis (p<0.01) showed specific differences between the neutrophil proteome of healthy individuals and patients with monogenic diseases. In patients with LAD, only few proteins were found to be present in decreased (7 proteins) or increased (5 proteins) abundance. Of note, proteins with lowest expression levels in LAD1 (ITGB2 mutation) were the integrins ITGB2, ITGAM and ITGAX while in LAD2 (SLC35C1 mutation) integrin expression was not affected. In contrast we noticed lack of PRCP, HDHD2 and XRCC4 protein. In patients with CGD, CYBA and CYBB were decreased in expression, independent of the underlying genotype. In contrast, the other members of the NADPH oxidase complex (NCF1/2/4, Rac1/2) were not found to be differentially expressed. This suggests that both membrane-bound proteins CYBB and CYBA stabilize each other. In total, CGD neutrophils showed limited aberrations with 23 overexpressed and 11 underexpressed proteins. In striking contrast to LAD and CGD, SCN patients with ELANE mutations had a much higher number of proteins with increased (93 proteins) or decreased (47 proteins) expression levels. It is currently unknown whether these imbalances are due to the immaturity of peripheral blood neutrophils in SCN or rather secondary to unfolded protein response and cellular stress mechanisms.In ongoing studies, we analyze the transcriptome of healthy neutrophil granulocytes in correlation to the proteome. Preliminary data indicate that there is poor correlation (PCC 0.11) between protein and mRNA levels, indicating that granule proteins are synthesized during earlier steps of differentiation.In two patients where exome sequencing did not reveal any underlying mutation, proteome analysis showed decreased expression of NCF1 and RAB27A, respectively. In both patients we were able to use targeted sequencing of amplified DNA to show the causative mutations in NCF1 and RAB27A. Multi-omic analysis may therefore be superior to unidimensional genomic sequencing to establish a molecular diagnosis.ConclusionIn-depth mass spectrometry-based proteomics provides new insights into disease-specific alterations of neutrophil granulocytes and may help to refine molecular diagnostics. DisclosuresNo relevant conflicts of interest to declare.

Genetic Phagocyte NADPH Oxidase Deficiency Enhances Nonviable Candida albicans-Induced Inflammation in Mouse Lungs.

Patients with chronic granulomatous disease (CGD) have mutated phagocyte NADPH oxidase, resulting in reduced production of reactive oxygen species (ROS). While the mechanism underlying hyperinfection in CGD is well understood, the basis for inflammatory disorders that arise in the absence of evident infection has not been fully explained. This study aimed to evaluate the effect of phagocyte NADPH oxidase deficiency on lung inflammation induced by nonviable Candida albicans (nCA). Mice deficient in this enzyme (CGD mice) showed more severe neutrophilic pneumonia than nCA-treated wild-type mice, which exhibited significantly higher lung concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and keratinocyte-derived chemokine (KC). Neutralization of these proinflammatory mediators significantly reduced neutrophil infiltration. In vitro, production of IL-1β and TNF-α from neutrophils and that of KC from macrophages was enhanced in nCA-stimulated neutrophils from CGD mice. Expression of IL-1β mRNA was higher in the stimulated CGD neutrophils than in the stimulated wild-type cells, concomitant with upregulation of nuclear factor (NF)-κB and its upstream regulator extracellular-signal regulated kinase (ERK) 1/2. Pretreatment with an NADPH oxidase inhibitor significantly enhanced IL-1β production in the wild-type neutrophils stimulated with nCA. These results suggest that lack of ROS production because of NADPH oxidase deficiency results in the production of higher levels of proinflammatory mediators from neutrophils and macrophages, which may at least partly contribute to the exacerbation of nCA-induced lung inflammation in CGD mice.

298. Transfection of Neutrophils from X-Linked Chronic Granulomatous Disease Patients with gp91phox mRNA Restores Oxidase Activity with High Efficiency and Viability

Chronic granulomatous disease (CGD) is an inherited immune deficiency due to defective phagocyte NADPH oxidase required for production of antimicrobial oxidants. Fungal or bacterial infections in CGD may persist despite intensive and prolonged antibiotic therapy. In settings where conventional antibiotic therapy and/or surgical debridement do not control infection, use of allogeneic unmatched donor granulocyte transfusions has been therapeutic. However, CGD patients treated with granulocyte transfusions may develop high titers of anti-HLA antibodies, restricting use of allogeneic granulocyte transfusions and further complicating potentially curative bone marrow transplant. Using the CGD patient as an autologous donor for granulocyte transfusions would be a helpful solution.Here we demonstrate highly efficient functional correction of the functional defect in CGD by transfection of patient's own granulocytes with mRNA while retaining viability using a scalable, cGMP-compliant MaxCyte® GT™ electroporation system capable of handling clinically relevant numbers of cells (>1010cells).Freshly collected granulocytes from normal volunteers were electroporated using the MaxCyte electroporation system with eGFP-mRNA on the day of collection. Flow cytometry of demonstrated that >90% of neutrophils expressed eGFP with viability exceeding 90% at 24 hrs after electroporation. Phagocyte NADPH oxidase activity of the normal neutrophils was retained as well as bactericidal activity against Burkolderia cepacia, a CGD bacterial pathogen. Antibacterial activity of electroporated granulocytes was comparable to unmanipulated granulocytes confirming retention of cellular function following the electroporation. Finally, electroporated eGFP positive human neutrophils injected into CGD mice were shown to remain in circulation as well as migrate to the peritoneal cavity following thioglycollate IP injection and there were no adverse effects observed in the mice.Electroporation of gp91phox mRNA into human X-linked CGD (gp91phox-deficient) patient neutrophils resulted in expression of gp91phox protein in >70% of cells with >90% viability at 24 hours after treatment. Using the dihydrorhodamine assay of phagocyte NADPH oxidase activity, >70% of neutrophils remained functionally corrected 2 days after transfection. Other studies of cellular function in vitro and in vivo with electroporated corrected CGD neutrophils are in progress. In summary, we show the feasibility of restoring the function of CGD patient-autologous neutrophils via electroporation of mRNA encoding the defective gene product. This approach may have potential clinical utility in the management of severe chronic bacterial and fungal infections in CGD but also short-term correction of other hematopoietic gene diseases.

Excessive Nitric Oxide Production of CGD Neutrophils Induces the Down-Regulation of NOS3 and EDN1 Expression in Human Endothelial Cells

Introduction: Chronic granulomatous disease (CGD) is an inherited immunodeficiency due to a mutation in genes that encode the subunits of NADPH oxidase of phagocytes. Phagocytes of CGD can not generate the reactive oxidative species (ROS), whereas nitric oxide (NO) production of CGD phagocytes is increased in response to calcium ionophore, A23187, compared with that of phagocytes from healthy subjects. Recently, X-linked CGD (X-CGD) patients showed lower oxidative stress and higher flow-mediated dilation, suggesting that endothelial cell function is affected by NO production of phagocytes. We studied the effects of NO on the regulation of endothelial gene expression related to dilator and constrictive effect of blood vessels, such as NOS3 and EDN1 using neutrophils from X-CGD patients.Methods: Eighteen X-CGD patients and 18 age-matched healthy male subjects were enrolled in this study from 2009 to 2013. NO production of phagocytes was assessed by flow cytometry using DAF2/DA fluorescent probe. Human umbilical vein endothelial cells (HUVECs) were co-cultured with human neutrophils of X-CGD or healthy subjects in response to calcium ionophore,A23187 without cell to cell contact using the Transwell-permeable support systems. The expression of endothelial NOS3 and EDN1 mRNA gene in HUVECs were quantified by real-time PCR.Results: Neutrophils of X-CGD patients showed significantly higher production of NO in response to A23187 for 30 to 60 minutes than those of normal subjects detected by flow cytometric analyses. NO concentration artificially generated by NO donor was significantly decreased by ROS produced by xanthine and xanthine oxidase. Similarly, neutrophils from healthy subjects, but not from X-CGD stimulated with A23187 induced the decrease in the concentration of NO. These results strongly suggest that the lack of ROS generation in CGD neutrophils leads to the increase of NO production in response to A23187. HUVECs were incubated with NO generation system, ROS generation system alone or with both systems. The expression of NOS3 and EDN1 was significantly decreased depending on the concentration of extracellular NO. In contrast, both NOS3 and EDN1 expression was significantly up-regulated under the ROS generation system without NO. Next, HUVECs were incubated by X-CGD neutrophils or by control neutrophils with or without A23187 for 30 minutes using the wells divided by membrane (0.45μm) to prevent cell to cell contact. Both NOS3 and EDN1 expression of HUVECs incubated with X-CGD neutrophils was significantly down-regulated under A23187 stimulation compared with normal neutrophils (112±117 vs. 34.5±39.9, n=8, p<0.05, 0.94± 0.24 vs. 0.61±0.24, n=5, p<0.05, respectively).Conclusion: This study demonstrated that the stimulated X-CGD neutrophils induced the decreased endothelial NOS3 and EDN1 gene expression through the excessive generation of NO due to the lack of ROS production. These findings suggest that ROS generated by phagocytes may modulate arterial tone affecting the amount of NO, a potent vasodilator molecule produced by endothelial cells. DisclosuresNo relevant conflicts of interest to declare.

Assessment of NETosis in patients with primary immunodeficiencies: evidence for a ROS‐independent pathway (1046.6)

Neutrophil‐mediated host defenses against pathogens include the extrusion of a lattice of DNA and microbicidal enzymes known as Neutrophil Extracellular Traps (NETs). Since the host response of extravasated neutrophils to mycotic infections within tissues necessitates contact with extracellular matrices, this study also identified a novel and significant regulatory role for the ubiquitous matrix component fibronectin (Fn) in NET release. We report that recognition of the purified fungal‐associated molecular pattern, β‐glucan, by normal human neutrophils causes rapid (蠄 30 mins) homotypic aggregation and NET release by a mechanism that requires Fn. NET formation to Candida albicans hyphae depends on β‐glucan recognition by CR3 (CD11b/CD18), requires Fn and ERK but not production of ROS, and results in hyphal destruction. To better understand the genotypic and phenotypic correlations of NET disgorgement in the host defense we assayed responses to Fn + β‐glucan by neutrophils from various patient populations including: GATA2 deficiency, Chronic Granulomatous Disease (CGD), Leukocyte Adhesion Deficiency (LAD), STAT3 loss‐of‐function mutation, STAT1 gain‐of‐function mutation, IL‐12R β2 chain mutation, IFNγ receptor mutation, IRAK 4 deficiency, and neonatal PMNs. Surprisingly, and in contrast with previous reports, CGD and neonatal patient neutrophils formed aggregates and NETs even when treated with DPI to block residual ROS production. These data support our previous findings that the respiratory burst is dispensable for PMN aggregation and NET extrusion. Mechanisms involving STAT1, STAT3 and GATA2 may potentially mediate Fn + β‐glucan‐induced NETosis or aggregation, whereas IRAK4, IL‐12 and IFNγ do not appear to play a major role. Further studies will challenge patient cells with C. albicans to better understand mediators of phagocyte responses and help elucidate the mechanisms in rapid release of NETs to fungal pathogens.Grant Funding Source: Supported by NIH Grant GM066194; A.S. Byrd is supported by the UNCF/Merck Graduate Fellowship

Enhanced generation of reactive oxygen species by interferon-γ may have contributed to successful treatment of invasive pulmonary aspergillosis in a patient with chronic granulomatous disease

Invasive pulmonary aspergillosis (IPA) is a life-threatening complication of chronic granulomatous disease (CGD), a rare inherited disorder of phagocytes that is characterized by a defect in the production of reactive oxygen species (ROS) caused by mutations in NADPH oxidase 2. Here, we report a case of successful treatment of IPA complicated with CGD by the administration of interferon-γ (IFN-γ) in combination with voriconazole. The patient carried a splice site mutation in the CYBB gene, and the neutrophils could produce a certain amount of ROS. In this case, augmentation of ROS generation in the patient's neutrophils was observed after in vivo IFN-γ treatment, which may be attributable to the induction of a normal CYBB gene in the myeloid progenitor cells. This treatment, in combination with voriconazole, may have contributed to the reversal of IPA in this patient. These results suggest that the in vivo use of IFN-γ may augment ROS generation in CGD neutrophils, thus leading to the successful treatment of severe IPA.

Pyocyanin-Enhanced Neutrophil Extracellular Trap Formation Requires the NADPH Oxidase

Beyond intracellular killing, a novel neutrophil-based antimicrobial mechanism has been recently discovered: entrapment and killing by neutrophil extracellular traps (NETs). NETs consist of extruded nuclear DNA webs decorated with granule proteins. Although NET formation is an important innate immune mechanism, uncontrolled NET release damages host tissues and has been linked to several diseases including cystic fibrosis (CF). The major CF airway pathogen Pseudomonas aeruginosa establishes chronic infection. Pseudomonas imbedded within biofilms is protected against the immune system, but maintains chronic inflammation that worsens disease symptoms. Aberrant NET release from recruited neutrophils was found in CF, but the underlying mechanisms remain unclear. One of the most important Pseudomonas virulence factors is pyocyanin, a redox-active pigment that has been associated with diminished lung function in CF. Here we show that pyocyanin promotes NET formation in a time- and dose-dependent manner. Most CF Pseudomonas clinical isolates tested produce pyocyanin in vitro. Pyocyanin-derived reactive oxygen species are required for its NET release. Inhibitor experiments demonstrated involvement of Jun N-terminal Kinase (JNK) and phosphatidylinositol 3-Kinase (PI3K) in pyocyanin-induced NET formation. Pyocyanin-induced NETs also require the NADPH oxidase because NET release in chronic granulomatous disease neutrophils was greatly reduced. Comparison of neutrophils from gp91phox- and p47phox-deficient patients revealed that pyocyanin-triggered NET formation is proportional to their residual superoxide production. Our studies identify pyocyanin as the first secreted bacterial toxin that enhances NET formation. The involvement of NADPH oxidase in pyocyanin-induced NET formation represents a novel mechanism of pyocyanin toxicity.

Deletion of OLFM4 Rescues Defective Host Defense Against Staphylococcus Aureus, but Not Aspergillus Fumigatus in Murine X-Linked Chronic Granulomatous Disease

AbstractAbstract 3276 Introduction:Chronic granulomatous disease (CGD) patients have recurrent life-threating bacterial and fungal infections due to the mutation of one of four subunits of the respiratory burst oxidase (NADPH-oxidase). Currently, the overall fatality rate in CGD patients remains high, making it necessary to better understand the basic biological processes governing host defense against bacteria and fungi in CGD. Olfactomedin 4 (OLFM4) is a neutrophil granule protein, which has been recently identified as a negative regulator of host innate immunity against bacteria infection in mice through modulation of neutrophil protease activity. The goal of this study was to evaluate the impact of OLFM4 deletion on host innate immunity against Staphylococcus aureus and Aspergillus fumigatus, two major pathogens encountered in CGD patients, in a murine X-linked CGD model. Results:We created gp91phox-and OLFM4-double deficient mice and investigated the mice defense against S. aureus and A. fumigatus infection. We found that neutrophil intracellular killing and in vivo clearance of S. aureus have been significantly increased in gp91phox- and OLFM4-double deficiency mice compared with CGD mice. The mice survival to S. aureus sepsis in gp91phox- and OLFM4-double deficiency mice has also been significantly prolonged compared with CGD mice. Our study has shown that the CGD mice immune deficiency against S. aureus has been totally corrected by additional loss of OLFM4 gene. To explore the mechanism that OLFM4 deletion rescued the bactericidal activities of CGD neutrophils, we analyzed cathepsin C and its downstream protease (neutrophil elastase and cathepsin G) activities in the mice neutrophils. Cathepsin C activities in OLFM4 deficient as well as double deficient mice neutrophils were significantly higher than those in WT mouse neutrophils. Cathepsin C activities in the neutrophils of CGD were similar to those in WT mice. Accordingly, the elastase and cathepsin G activities in the neutrophils of OLFM4 deficient and double deficient mice were also substantially higher than those in WT mice as well as CGD mice. However, we have not observed enhanced innate immunity against A. fumigatus in OLFM4 deficiency mice compared with wild-type mice using a lung infection model. The lung histopathology showed similar inflammation and fungal burden in the OLFM4 deficiency mice compared with wild-type mice. Correspondingly, mice survival to severe A. fumigatus infection did not show significant difference in gp91phox- and OLFM4-double deficiency mice compared with CGD mice, suggesting that OLFM4 may not play a role in mice host defense against A. fumigatus. Conclusion:1. The damaged neutrophil bacterial killing and host innate immunity against S. aureus in CGD mice due to oxidative mechanism deficiency could be successfully rescued by deletion of OLFM4. 2. These results showed that the granule protease activities in CGD neutrophils could be substantially enhanced above the level in normal neutrophils by deletion of OLFM4, suggesting that the increased of serine proteinase activities due to OLFM4 deletion is NADPH-independent. 3. OLFM4 may not play a role in mice host defense against pulmonary A. fumigatus infection. 4. OLFM4 might prove to be an important target in CGD patients to augment host defense against bacterial infection. Disclosures:No relevant conflicts of interest to declare.

PEGylated d-amino acid oxidase restores bactericidal activity of neutrophils in chronic granulomatous disease via hypochlorite

Chronic granulomatous disease (CGD) causes impaired hydrogen peroxide (H(2)O(2)) generation. Consequently, neutrophils in patients with CGD fail to kill infecting pathogens. We expected that supplementation with H(2)O(2) would effectively restore the bactericidal function of neutrophils in CGD. Here, we used polyethylene glycol-conjugated D-amino acid oxidase (PEG-DAO) as an H(2)O(2) source. The enzyme DAO generates H(2)O(2) by using D-amino acid and oxygen as substrates. PEG-DAO plus D-amino acid indeed exerted bacteriostatic activity against Staphylococcus aureus via H(2)O(2) in vitro. Furthermore, use of PEG-DAO plus D-amino acids, which increased the amount of intracellular H(2)O(2), restored bactericidal activity of neutrophils treated with diphenylene iodonium, in which nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was defective. This restoration of bactericidal activity was mediated by myeloperoxidase, with concomitant production of H(2)O(2) by PEG-DAO plus D-Ala. We also confirmed that PEG-DAO treatment restored bactericidal activity of congenitally defective neutrophils from patients with CGD. These results indicate that PEG-DAO can supply additional H(2)O(2) for defective NADPH oxidase of neutrophils from patients with CGD, and thus neutrophils regain bactericidal activity.

Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils

Neutrophils enter sites of infection, where they can eliminate pathogenic bacteria in an oxidative manner. Despite their predominance in active tuberculosis lesions, the function of neutrophils in this important human infection is still highly controversial. We observed that virulent Mycobacterium tuberculosis survived inside human neutrophils despite prompt activation of these defence cells' microbicidal effectors. Survival of M. tuberculosis was accompanied by necrotic cell death of infected neutrophils. Necrotic cell death entirely depended on radical oxygen species production since chronic granulomatous disease neutrophils were protected from M. tuberculosis-triggered necrosis. More, importantly, the M. tuberculosis ΔRD1 mutant failed to induce neutrophil necrosis rendering this strain susceptible to radical oxygen species-mediated killing. We conclude that this virulence function is instrumental for M. tuberculosis to escape killing by neutrophils and contributes to pathogenesis in tuberculosis.

Transducible form of p47phox and p67phox compensate for defective NADPH oxidase activity in neutrophils of patients with chronic granulomatous disease

Protein delivery to primary cells by protein transduction domain (PTD) serves as a novel measure for manipulation of the cells for biological study and for the treatment of various human conditions. Although the method has been employed to modulate cellular function in vitro, only limited reports are available on its application in the replacement of deficient signaling molecules into primary cells. We examined the potential of recombinant proteins to compensate for defective cytosolic components of the NADPH oxidase complex in chronic granulomatous disease (CGD) neutrophils in both p47phox and p67phox deficiency. The p47phox or p67phox protein linked to Hph-1 PTD was effectively expressed in soluble form and transduced into human neutrophils efficiently without eliciting unwanted signal transduction or apoptosis. The delivered protein was stable for more than 24h, expressed in the cytoplasm, translocated to the membrane fraction upon activation, and, most importantly able to restored reactive oxygen species (ROS) production. Although research on human primary neutrophils using the protein delivery system is still limited, our data show that the protein transduction approach for neutrophils may be applicable to the control of local infections in CGD patients by direct delivery of the protein product.

Singlet Oxygen Is Essential for Neutrophil Extracellular Trap Formation,

Abstract 3209Neutrophil extracellular traps (NETs), which capture microbes in extracellular structures consisting of DNA fibers and antimicrobial granule proteins, are pivotal for innate host defense, but the mechanism of NET formation remains unclear. Previous observation in neutrophils of chronic granulomatous disease (CGD) patients, which defect NADPH oxidase (Nox) and fail to produce reactive oxygen species (ROS), demonstrated that ROS are important for NET formation. However, the active species were not identified. Neutrophils first generate superoxide by Nox activation, and this superoxide is converted to hydrogen peroxide (H2O2) by superoxide dismutase. Then, hypochlorous acid is produced from H2O2 by myeloperoxidase (MPO), and reacts with H2O2 to form singlet oxygen (1O2). In the current study, we revealed that 1O2, one of the ROS, is essential for NET formation. We first examined the effect of 1O2 scavengers, a-phenyl-N-tert-butyl nitrone (PBN), 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), or uric acid (UA), on Nox-dependent NET formation upon stimulation with phorbol myristate acetate (PMA). We previously showed that PBN and edaravone suppressed 1O2 release from activated neutrophils, but did not affect superoxide release (Sommani et al. J Pharmacol Sci 2007, Kawai et al. J Pharmacol Sci 2008). We also showed that PBN is a more specific 1O2 scavenger, since it neither affected MPO activity, nor reacted with hypochlorous acid (Kawai et al. J Pharmacol Sci 2008). UA is a well-known 1O2 scavenger. Human neutrophils were isolated from peripheral blood by sedimentation through two-step Percoll gradients. The CGD patient was a 24-year-old male with gp91-phox deficiency with a G-to-A point mutation at nucleotide 389 in exon 10. Healthy volunteers and the patient provided written informed consent for participation in an institutional review board-approved protocol at Kyoto University Hospital. NET formation by PMA was visualized by fluorescence microscopy with Sytox green, a cell-impermeable DNA dye, and scanning electron microscopy (SEM). NET formation was observed at 3 h after PMA stimulation (100 ng/ml) in healthy neutrophils. The treatment of edaravone (10 μM), PBN (4 mM), or UA (5 μg/ml) significantly suppressed NET formation, suggesting that 1O2 is involved in NET formation. In contrast, CGD neutrophils did not exhibit NET formation, which is consistent with previous reports (Fuchs et al. J Cell Biol 2007. Bianchi et al. Blood 2009). We next explored the direct effect 1O2 in NET formation. First, we detected 1O2 generation by the most specific method with irradiation of porfimer sodium (Photofrin) in a cell-free system, by direct analysis of near-infrared luminescence at 1270 nm using Raman spectroscopy. The treatment of edaravone (400 μM) almost completely suppressed the 1O2 spectrum, as well as azide (400 μM), a well-known 1O2 scavenger. Next, we observed the accumulation of Photofrin in neutrophils by fluorescence confocal microscopy after incubation with Photofrin (10 μg/ml) for 1 h. These findings indicated that 1O2 was generated in neutrophils by Photofrin with light irradiation. NET formation by Photofrin (10 μg/ml, for 1 h) with irradiation (LED lamp, λmax=660 nm, for 5 min) was analyzed by fluorescence microscopy and SEM. Interestingly, NET formation was observed in CGD neutrophils, as well as healthy neutrophils. It is noteworthy that CGD neutrophils did not exhibit Nox-dependent NET formation by PMA stimulation. This was suppressed by the treatment of 1O2 scavengers, edaravone (10 μM) or PBN (4 mM). These results suggested that 1O2 directly induced NET formation in both healthy and CGD neutrophils, independent of Nox activation. Taken together, our findings demonstrated that 1O2 is essential for NET formation, which supported a recent report that MPO is required for NET formation (Metzler et al. Blood, 2011), since 1O2 is generated downstream of MPO. The modulation of NET formation should be effective in the control of infectious or inflammatory disorders. From the current study, the suppression of 1O2 could be an optimal target for regulating NET formation, minimizing impairment of innate host defense. Disclosures:No relevant conflicts of interest to declare.

Singlet oxygen is essential for neutrophil extracellular trap formation

Neutrophil extracellular traps (NETs) that bind invading microbes are pivotal for innate host defense. There is a growing body of evidence for the significance of NETs in the pathogenesis of infectious and inflammatory diseases, but the mechanism of NET formation remains unclear. Previous observation in neutrophils of chronic granulomatous disease (CGD) patients, which defect NADPH oxidase (Nox) and fail to produce reactive oxygen species (ROS), revealed that ROS contributed to the formation of NETs. However, the active species were not identified. In this study, we discovered that singlet oxygen, one of the ROS, mediated Nox-dependent NET formation upon stimulation with phorbol myristate acetate. We also revealed that singlet oxygen itself could induce NET formation by a distinct system generating singlet oxygen with porfimer sodium (Photofrin) in CGD neutrophils, as well as healthy neutrophils. This was independent of Nox activation. These results show that singlet oxygen is essential for NET formation, and provide novel insights into the pathogenesis of infectious and inflammatory diseases.

Chronic Granulomatous Disease: Lessons from a Rare Disorder

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency with X-linked or autosomal recessive inheritance involving defects in genes encoding phox proteins, which are the subunits of the phagocyte NADPH oxidase. This results in failure to produce superoxide anion and downstream antimicrobial oxidant metabolites and to activate antimicrobial proteases. Affected patients are susceptible to severe, life-threatening bacterial and fungal infections and excessive inflammation characterized by granulomatous enteritis resembling Crohn's disease and genitourinary obstruction. Early diagnosis of CGD and rapid treatment of infections are critical. Prophylaxis with antibacterial and mold-active antifungal agents and the administration of interferon-γ has significantly improved the natural history of CGD. Currently, the only cure is allogeneic hematopoietic cell transplant (HCT), although there remains controversy as to which patients with CGD should get a transplant. Allele-based HLA typing of alternative donors, improved supportive care measures, and use of reduced toxicity conditioning have resulted in event-free survival (EFS) of at least 80% even with an unrelated donor and even better when the patient has no active infections/inflammation. Gene correction of CGD would eliminate the risks of graft-versus-host disease (GVHD) and the immunoablative chemotherapy required for allogeneic HCT. Based on gene therapy trials in patients with SCID-X1, ADA-SCID, and the early experience with CGD, it is clear that at least some degree of myeloablation will be necessary for CGD as there is no inherent selective growth advantage for gene-corrected cells. Current efforts for gene therapy focus on use of lentivector constructs, which are thought to be safer from the standpoint of insertional mutagenesis and more efficient in transducing hematopoietic stem cells (HSCs).