Structure of human phagocyte NADPH oxidase in the activated state.

Phagocyte NADPH oxidase, a protein complex with a core made up of NOX2 and p22 subunits, is responsible for transferring electrons from intracellular NADPH to extracellular oxygen1. This process generates superoxide anions that are vital for killing pathogens1. The activation of phagocyte NADPH oxidase requires membrane translocation and the binding of several cytosolic factors2. However, the exact mechanism by which cytosolic factors bind to and activate NOX2 is not well understood. Here we present the structure of the human NOX2-p22 complex activated by fragments of three cytosolic factors: p47, p67 and Rac1. The structure reveals that the p67-Rac1 complex clamps onto the dehydrogenase domain of NOX2 and induces its contraction, which stabilizes the binding of NADPH and results in a reduction of the distance between the NADPH-binding domain and the flavin adenine dinucleotide (FAD)-binding domain. Furthermore, the dehydrogenase domain docks onto the bottom of the transmembrane domain of NOX2, which reduces the distance between FAD and the inner haem. These structural rearrangements might facilitate the efficient transfer of electrons between the redox centres in NOX2 and lead to the activation of phagocyte NADPH oxidase.

Quantitative live-cell imaging and 3D modeling reveal critical functional features in the cytosolic complex of phagocyte NADPH oxidase.

Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. However, uncontrolled ROS production contributes to inflammation, making NADPH oxidase a major drug target. It consists of two membranous (Nox2 and p22phox) and three cytosolic subunits (p40phox, p47phox, and p67phox) that undergo structural changes during enzyme activation. Unraveling the interactions between these subunits and the resulting conformation of the complex could shed light on NADPH oxidase regulation and help identify inhibition sites. However, the structures and the interactions of flexible proteins comprising several well-structured domains connected by intrinsically disordered protein segments are difficult to investigate by conventional techniques such as X-ray crystallography, NMR, or cryo-EM. Here, we developed an analytical strategy based on FRET-fluorescence lifetime imaging (FLIM) and fluorescence cross-correlation spectroscopy (FCCS) to structurally and quantitatively characterize NADPH oxidase in live cells. We characterized the inter- and intramolecular interactions of its cytosolic subunits by elucidating their conformation, stoichiometry, interacting fraction, and affinities in live cells. Our results revealed that the three subunits have a 1:1:1 stoichiometry and that nearly 100% of them are present in complexes in living cells. Furthermore, combining FRET data with small-angle X-ray scattering (SAXS) models and published crystal structures of isolated domains and subunits, we built a 3D model of the entire cytosolic complex. The model disclosed an elongated complex containing a flexible hinge separating two domains ideally positioned at one end of the complex and critical for oxidase activation and interactions with membrane components.

The NADPH Oxidase and the Phagosome.

The key purpose of phagocytosis is the destruction of pathogenic microorganisms. The phagocytes exert a wide array of killing mechanisms that allow mastering the vast majority of pathogens. One of these mechanisms consists in the production of reactive oxygen species inside the phagosome by a specific enzyme, the phagocyte NADPH oxidase. This enzyme is composed of 6 proteins that need to assemble to form a complex on the phagosomal membrane. Multiple signaling pathways tightly regulate the assembly. We briefly summarize key features of the enzyme and its regulation. We then focus on several related topics that address the activity of the NADPH oxidase during phagocytosis. Novel fluorescence microscopy techniques combined with fluorescent protein labeling of NADPH oxidase subunits opened the view on the structure and dynamics of these proteins in living cells. This combination revealed details of the role of anionic phospholipids in the control of phagosomal ROS production. It also added critical information to propose a 3D model of the complex between the cytosolic subunits prior to activation, in complement to other structural data on the oxidase.

Tissue Tropism in Streptococcal Infection: Wild-Type M1T1 Group A Streptococcus Is Efficiently Cleared by Neutrophils Using an NADPH Oxidase-Dependent Mechanism in the Lung but Not in the Skin.

Group A Streptococcus (GAS) commonly causes pharyngitis and skin infections. Little is known why streptococcal pharyngitis usually does not lead to pneumonia and why the skin is a favorite niche for GAS. To partially address these questions, the effectiveness of neutrophils in clearing wild-type (wt) M1T1 GAS strain MGAS2221 from the lung and from the skin was examined in murine models of intratracheal pneumonia and subcutaneous infection. Ninety-nine point seven percent of the MGAS2221 inoculum was cleared from the lungs of C57BL/6J mice at 24 h after inoculation, while there was no MGAS2221 clearance from skin infection sites. The bronchial termini had robust neutrophil infiltration, and depletion of neutrophils abolished MGAS2221 clearance from the lung. Phagocyte NADPH oxidase but not myeloperoxidase was required for MGAS2221 clearance. Thus, wt M1T1 GAS can be cleared by neutrophils using an NADPH oxidase-dependent mechanism in the lung. MGAS2221 induced robust neutrophil infiltration at the edge of skin infection sites and throughout infection sites at 24 h and 48 h after inoculation, respectively. Neutrophils within MGAS2221 infection sites had no nuclear staining. Skin infection sites of streptolysin S-deficient MGAS2221 ΔsagA were full of neutrophils with nuclear staining, whereas MGAS2221 ΔsagA infection was not cleared. Gp91phox knockout (KO) and control mice had similar GAS numbers at skin infection sites and similar abilities to select SpeB activity-negative (SpeBA-) variants. These results indicate that phagocyte NADPH oxidase-mediated GAS killing is compromised in the skin. Our findings support a model for GAS skin tropism in which GAS generates an anoxic niche to evade phagocyte NADPH oxidase-mediated clearance.

Unexpected function of the phagocyte NADPH oxidase in supporting hyperglycolysis in stimulated neutrophils: key role of 6-phosphofructo-2-kinase.

The phagocyte NADPH oxidase 2 (Nox2) is an enzymatic complex that is involved in innate immunity, notably via its capacity to produce toxic reactive oxygen species. Recently, a proteomic analysis of the constitutively active Nox2 complex, isolated from neutrophil fractions, highlighted the presence of 6-phosphofructo-2-kinase (PFK-2). The purpose of this work was to study the relationship between PFK-2 and NADPH oxidase in neutrophils. Data have underlined a specific association of the active phosphorylated form of PFK-2 with Nox2 complex in stimulated neutrophils. In its active form, PFK-2 catalyzes the production of fructose-2,6-bisphosphate, which is the main allosteric activator of phosphofructo-1-kinase, the limiting enzyme in glycolysis. Pharmacologic inhibition of PFK-2 phosphorylation and cell depletion in PFK-2 by a small interfering RNA strategy led to a decrease in the glycolysis rate and a reduction in NADPH oxidase activity in stimulated cells. Surprisingly, alteration of Nox2 activity impacted the glycolysis rate, which indicated that Nox2 in neutrophils was not only required for reactive oxygen species production but was also involved in supporting the energetic metabolism increase that was induced by inflammatory conditions. PFK-2 seems to be a strategic element that links NADPH oxidase activation and glycolysis modulation, and, as such, is proposed as a potential therapeutic target in inflammatory diseases.-Baillet, A., Hograindleur, M.-A., El Benna, J., Grichine, A., Berthier, S., Morel, F., Paclet, M.-H. Unexpected function of the phagocyte NADPH oxidase in supporting hyperglycolysis in stimulated neutrophils: key role of 6-phosphofructo-2-kinase.

NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes.

The balance between various cellular subsets of the innate and adaptive immune system and microbiota in the gastrointestinal tract is carefully regulated to maintain tolerance to the normal flora and dietary antigens, while protecting against pathogens. The intestinal epithelial cells and the network of dendritic cells and macrophages in the lamina propria are crucial lines of defense that regulate this balance. The complex relationship between the myeloid compartment (dendritic cells and macrophages) and lymphocyte compartment (T cells and innate lymphoid cells), as well as the impact of the epithelial cell layer have been studied in depth in recent years, revealing that the regulatory and effector functions of both innate and adaptive immune compartments exhibit more plasticity than had been previously appreciated. However, little is known about the metabolic activity of these cellular compartments, which is the basic function underlying all other additional tasks the cells perform. Here we perform intravital NAD(P)H fluorescence lifetime imaging in the small intestine of fluorescent reporter mice to monitor the NAD(P)H-dependent metabolism of epithelial and myeloid cells. The majority of myeloid cells which comprise the surveilling network in the lamina propria have a low metabolic activity and remain resting even upon stimulation. Only a few myeloid cells, typically localized at the tip of the villi, are metabolically active and are able to activate NADPH oxidases upon stimulation, leading to an oxidative burst. In contrast, the epithelial cells are metabolically highly active and, although not considered professional phagocytes, are also able to activate NADPH oxidases, leading to massive production of reactive oxygen species. Whereas the oxidative burst in myeloid cells is mainly catalyzed by the NOX2 isotype, in epithelial cells other isotypes of the NADPH oxidases family are involved, especially NOX4. They are constitutively expressed by the epithelial cells, but activated only on demand to ensure rapid defense against pathogens. This minimizes the potential for inadvertent damage from resting NOX activation, while maintaining the capacity to respond quickly if needed.

STIM1 for stimulation of phagocyte NADPH oxidase.

In this issue of Blood, Zhang et al show that mice lacking the stromal-interacting molecule 1 (STIM1) gene in bone marrow cells are more susceptible to bacterial infection but are resistant to ischemia/reperfusion injury because of defective activation of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.

STIM1 calcium sensor is required for activation of the phagocyte oxidase during inflammation and host defense.

The stromal-interacting molecule 1 (STIM1) is a potent sensor of intracellular calcium, which in turn regulates entry of external calcium through plasma membrane channels to affect immune cell activation. Although the contribution of STIM1 to calcium signaling in lymphocytes has been well studied, the role of this protein in neutrophil-mediated inflammation and host defense is unknown. We report that STIM1-deficient murine neutrophils show loss of store-operated calcium entry (SOCE) in response to both soluble ligands that activate G-proteins as well as Fcγ-receptor or integrin ligation that activates tyrosine kinase signaling. This results in modest defects in phagocytosis and degranulation responses but a profound block in superoxide production by the phagocyte oxidase. We trace the primary intracellular target of calcium to be protein kinase C isoforms α and ß (PKCα and PKCß), which in turn phosphorylate subunits of the oxidase leading to superoxide production. In vivo the loss of SOCE in stim1(-/-) chimeric mice results in marked susceptibility to bacterial infections but also protection from tissue injury in hepatic ischemia/reperfusion injury. These results demonstrate the critical role of STIM1-mediated SOCE and define major protein targets of calcium signaling in neutrophil activation during inflammatory disease.

Phagocyte NADPH oxidase restrains the inflammasome in ANCA-induced GN.

ANCA-activated phagocytes cause vasculitis and necrotizing crescentic GN (NCGN). ANCA-induced phagocyte NADPH oxidase (Phox) may contribute by generating tissue-damaging reactive oxygen species. We tested an alternative hypothesis, in which Phox restrains inflammation by downregulating caspase-1, thereby reducing IL-1ß generation and limiting NCGN. In an antimyeloperoxidase (anti-MPO) antibody-mediated disease model, mice transplanted with either gp91(phox)-deficient or p47(phox)-deficient bone marrow showed accelerated disease with increased crescents, necrosis, glomerular monocytes, and renal IL-1ß levels compared with mice transplanted with wild-type bone marrow. IL-1ß receptor blockade abrogated aggravated NCGN in gp91(phox)-deficient mice. In vitro, challenge with anti-MPO antibody strongly enhanced caspase-1 activity and IL-1ß generation in gp91(phox)-deficient and p47(phox)-deficient monocytes compared with wild-type monocytes. This enhanced IL-1ß generation was abrogated when caspase-1 was blocked. ANCA-induced superoxide and IL-1ß generation were inversely related in human monocytes. Furthermore, transplantation of gp91(phox)/caspase-1 double-deficient bone marrow rescued the accelerated NCGN phenotype in gp91(phox) bone marrow-deficient mice. These results suggest that Phox-generated reactive oxygen species downregulate caspase-1, thereby keeping the inflammasome in check and limiting ANCA-induced inflammation. IL-1 receptor blockade may provide a promising strategy in NCGN, whereas our data question the benefit of antioxidants.

Arachidonic acid induces direct interaction of the p67(phox)-Rac complex with the phagocyte oxidase Nox2, leading to superoxide production.

The phagocyte NADPH oxidase Nox2, heterodimerized with p22(phox) in the membrane, is dormant in resting cells but becomes activated upon cell stimulation to produce superoxide, a precursor of microbicidal oxidants. Nox2 activation requires two switches to be turned on simultaneously: a conformational change of the cytosolic protein p47(phox) and GDP/GTP exchange on the small GTPase Rac. These proteins, in an active form, bind to their respective targets, p22(phox) and p67(phox), leading to productive oxidase assembly at the membrane. Although arachidonic acid (AA) efficiently activates Nox2 both in vivo and in vitro, the mechanism has not been fully understood, except that AA induces p47(phox) conformational change. Here we show that AA elicits GDP-to-GTP exchange on Rac at the cellular level, consistent with its role as a potent Nox2 activator. However, even when constitutively active forms of p47(phox) and Rac1 are both expressed in HeLa cells, superoxide production by Nox2 is scarcely induced in the absence of AA. These active proteins also fail to effectively activate Nox2 in a cell-free reconstituted system without AA. Without affecting Rac-GTP binding to p67(phox), AA induces the direct interaction of Rac-GTP-bound p67(phox) with the C-terminal cytosolic region of Nox2. p67(phox)-Rac-Nox2 assembly and superoxide production are both abrogated by alanine substitution for Tyr-198, Leu-199, and Val-204 in the p67(phox) activation domain that localizes the C-terminal to the Rac-binding domain. Thus the "third" switch (AA-inducible interaction of p67(phox)·Rac-GTP with Nox2) is required to be turned on at the same time for Nox2 activation.

NADPH oxidase-driven phagocyte recruitment controls Candida albicans filamentous growth and prevents mortality.

Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis.

Phagocyte NADPH oxidase and specific immunity.

The phagocyte NADPH oxidase NOX2 produces reactive oxygen species (ROS) and is a well-known player in host defence. However, there is also increasing evidence for a regulatory role of NOX2 in adaptive immunity. Deficiency in phagocyte NADPH oxidase causes chronic granulomatous disease (CGD) in humans, a condition that can also be studied in CGD mice. Clinical observations in CGD patients suggest a higher susceptibility to autoimmune diseases, in particular lupus, idiopathic thrombocytopenic purpura and rheumatoid arthritis. In mice, a strong correlation exists between a polymorphism in a NOX2 subunit and the development of autoimmune arthritis. NOX2 deficiency in mice also favours lupus development. Both CGD patients and CGD mice exhibit increased levels of immunoglobulins, including autoantibodies. Despite these phenotypes suggesting a role for NOX2 in specific immunity, mechanistic explanations for the typical increase of CGD in autoimmune disease and antibody levels are still preliminary. NOX2-dependent ROS generation is well documented for dendritic cells and B-lymphocytes. It is unclear whether T-lymphocytes produce ROS themselves or whether they are exposed to ROS derived from dendritic cells during the process of antigen presentation. ROS are signalling molecules in virtually any cell type, including T- and B-lymphocytes. However, knowledge about the impact of ROS-dependent signalling on T- and B-lymphocyte phenotype and response is still limited. ROS might contribute to Th1/Th2/Th17 cell fate decisions during T-lymphocyte activation and might enhance immunoglobulin production by B-lymphocytes. In dendritic cells, NOX2-derived ROS might be important for antigen processing and cell activation.

IL-21 enhances phagocytosis in mononuclear phagocyte cells: identification of spleen tyrosine kinase as a novel molecular target of IL-21.

The biological significance of the IL-21/IL-21R system in human monocytes/macrophages is not well documented, and the expression of IL-21R is unclear and has been disputed. In this study, we showed for the first time, to our knowledge, that human monocyte-like THP-1 cells expressed the two IL-21R components, CD132 (γc) and IL-21Rα, on their cell surface, as assessed by flow cytometry. Moreover, IL-21 was found to enhance FcR-mediated phagocytosis, but not endocytosis. The ability of IL-21 to enhance phagocytosis was not associated with an increased expression of both IL-21R components at the cell surface, and IL-21 did not act in synergy with IL-15. IL-21 activated spleen tyrosine kinase (Syk), as evidenced by its ability to increase Syk phosphorylation. Using a pharmacological approach to inhibit Syk activity, and an antisense technique to downregulate Syk protein expression, we demonstrated the importance of Syk in IL-21-induced phagocytosis. In addition, both CD132 and IL-21Rα were expressed on the cell surface of naive monocytes, as well as in GM-CSF-monocyte-derived macrophages. Moreover, IL-21 also induced phagocytosis in these cells. We conclude that IL-21 possesses important biological effects in mononuclear phagocyte cells and that Syk is a novel molecular target of IL-21 that was previously unknown. Therefore, future development of therapeutic strategies targeting the IL-21/IL-21R system should consider that monocyte and macrophage cell physiology may be affected by this system.

Molecular diagnosis of chronic granulomatous disease.

Patients with chronic granulomatous disease (CGD) suffer from recurrent, life-threatening bacterial and fungal infections of the skin, the airways, the lymph nodes, liver, brain and bones. Frequently found pathogens are Staphylococcus aureus, Aspergillus species, Klebsiella species, Burkholderia cepacia and Salmonella species. CGD is a rare (∼1:250 000 births) disease caused by mutations in any one of the five components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. This enzyme generates superoxide and is essential for intracellular killing of pathogens by phagocytes. Molecular diagnosis of CGD involves measuring NADPH oxidase activity in phagocytes, measuring protein expression of NADPH oxidase components and mutation analysis of genes encoding these components. Residual oxidase activity is important to know for estimation of the clinical course and the chance of survival of the patient. Mutation analysis is mandatory for genetic counselling and prenatal diagnosis. This review summarizes the different assays available for the diagnosis of CGD, the precautions to be taken for correct measurements, the flow diagram to be followed, the assays for confirmation of the diagnosis and the determinations for carrier detection and prenatal diagnosis.

p38 MAPK inhibition suppresses the TLR-hypersensitive phenotype in FANCC- and FANCA-deficient mononuclear phagocytes.

Fanconi anemia, complementation group C (FANCC)-deficient hematopoietic stem and progenitor cells are hypersensitive to a variety of inhibitory cytokines, one of which, TNFα, can induce BM failure and clonal evolution in Fancc-deficient mice. FANCC-deficient macrophages are also hypersensitive to TLR activation and produce TNFα in an unrestrained fashion. Reasoning that suppression of inhibitory cytokine production might enhance hematopoiesis, we screened small molecules using TLR agonist-stimulated FANCC- and Fanconi anemia, complementation group A (FANCA)-deficient macrophages containing an NF-κB/AP-1-responsive reporter gene (SEAP). Of the 75 small molecules screened, the p38 MAPK inhibitor BIRB 796 and dasatinib potently suppressed TLR8-dependent expression of the reporter gene. Fanconi anemia (FA) macrophages were hypersensitive to the TLR7/8 activator R848, overproducing SEAP and TNFα in response to all doses of the agonist. Low doses (50nM) of both agents inhibited p38 MAPK-dependent activation of MAPKAPK2 (MK2) and suppressed MK2-dependent TNFα production without substantially influencing TNFα gene transcription. Overproduction of TNFα by primary FA cells was likewise suppressed by these agents and involved inhibition of MK2 activation. Because MK2 is also known to influence production and/or sensitivity to 2 other suppressive factors (MIP-1α and IFNγ) to which FA hematopoietic progenitor cells are uniquely vulnerable, targeting of p38 MAPK in FA hematopoietic cells is a rational objective for preclinical evaluation.

trans Arachidonic acid isomers inhibit NADPH-oxidase activity by direct interaction with enzyme components.

NADPH-oxidase is an enzyme that represents, when activated, the major source of non-mitochondrial reactive oxygen species. In phagocytes, this production is an indispensable event for the destruction of engulfed pathogens. The functional NADPH-oxidase complex consists of a catalytic membrane flavocytochrome b (Cytb(558)) and four cytosolic proteins p47(phox), p67(phox), Rac and p40(phox). The NADPH-oxidase activity is finely regulated spatially and temporally by cellular signaling events that trigger the translocation of the cytosolic subunits to its membrane partner involving post-translational modifications and activation by second messengers such as arachidonic acid (AA). Arachidonic acid in its natural cis-poly unsaturated form (C20:4) has been described to be an efficient activator of the enzyme in vivo and in vitro. In this work, we examined in a cell-free system whether a change of the natural cis geometry to the trans configuration, which could occur either by diet or be produced by the action of free radicals, may have consequences on the functioning of NADPH-oxidase. We showed the inability of mono-trans AA isomers to activate the NADPH-oxidase complex and demonstrated the inhibitory effect on the cis-AA-induced NADPH oxidase activation. The inhibition is mediated by a direct effect of the mono-trans AA which targets both the membrane fraction containing the cytb(558) and the cytosolic p67(phox). Our results suggest that the loss of the natural geometric feature (cis-AA) induces substantial structural modifications of p67(phox) that prevent its translocation to the complex.

HIV-1 reduces Abeta-degrading enzymatic activities in primary human mononuclear phagocytes.

The advent and wide introduction of antiretroviral therapy has greatly improved the survival and longevity of HIV-infected patients. Unfortunately, despite antiretroviral therapy treatment, these patients are still afflicted with many complications including cognitive dysfunction. There is a growing body of reports indicating accelerated deposition of amyloid plaques, which are composed of amyloid-ß peptide (Aß), in HIV-infected brains, though how HIV viral infection precipitates Aß accumulation is poorly understood. It is suggested that viral infection leads to increased production and impaired degradation of Aß. Mononuclear phagocytes (macrophages and microglia) that are productively infected by HIV in brains play a pivotal role in Aß degradation through the expression and execution of two endopeptidases, neprilysin (NEP) and insulin-degrading enzyme. In this study, we report that NEP has the dominant endopeptidase activity toward Aß in macrophages. Further, we demonstrate that monomeric Aß degradation by primary cultured macrophages and microglia was significantly impaired by HIV infection. This was accompanied with great reduction of NEP endopeptidase activity, which might be due to the diminished transport of NEP to the cell surface and intracellular accumulation at the endoplasmic reticulum and lysosomes. Therefore, these data suggest that malfunction of NEP in infected macrophages may contribute to acceleration of ß amyloidosis in HIV-inflicted brains, and modulation of macrophages may be a potential preventative target of Aß-related cognitive disorders in HIV-affected patients.

Phagocyte-derived catecholamines enhance acute inflammatory injury.

It is becoming increasingly clear that the autonomic nervous system and the immune system demonstrate cross-talk during inflammation by means of sympathetic and parasympathetic pathways. We investigated whether phagocytes are capable of de novo production of catecholamines, suggesting an autocrine/paracrine self-regulatory mechanism by catecholamines during inflammation, as has been described for lymphocytes. Here we show that exposure of phagocytes to lipopolysaccharide led to a release of catecholamines and an induction of catecholamine-generating and degrading enzymes, indicating the presence of the complete intracellular machinery for the generation, release and inactivation of catecholamines. To assess the importance of these findings in vivo, we chose two models of acute lung injury. Blockade of alpha2-adrenoreceptors or catecholamine-generating enzymes greatly suppressed lung inflammation, whereas the opposite was the case either for an alpha2-adrenoreceptor agonist or for inhibition of catecholamine-degrading enzymes. We were able to exclude T cells or sympathetic nerve endings as sources of the injury-modulating catecholamines. Our studies identify phagocytes as a new source of catecholamines, which enhance the inflammatory response.

Strategies for identifying synthetic peptides to act as inhibitors of NADPH oxidases, or "all that you did and did not want to know about Nox inhibitory peptides".

Phagocytes utilize reactive oxygen species (ROS) to kill pathogenic microorganisms. The source of ROS is an enzymatic complex (the NADPH oxidase), comprising a membrane-associated heterodimer (flavocytochrome b (558)), consisting of subunits Nox2 and p22(phox), and four cytosolic components (p47(phox), p67(phox), p40(phox), and Rac). The primordial ROS (superoxide) is generated by the reduction of molecular oxygen by NADPH via redox centers located on Nox2. This process is activated by the translocation of the cytosolic components to the membrane and their assembly with Nox2. Membrane translocation is preceded by interactions among cytosolic components. A number of proteins structurally and functionally related to Nox2 have been discovered in many cells (the Nox family) and these have pleiotropic functions related to the production of ROS. An intense search is underway to design therapeutic means to modulate Nox-dependent overproduction of ROS, associated with diseases. Among drug candidates, a central position is held by synthetic peptides reflecting domains in oxidase components involved in NADPH oxidase assembly. Peptides, corresponding to domains in Nox2, p22(phox), p47(phox), and Rac, found to be oxidase activation inhibitory in vitro, are reviewed. Usually, peptides are inhibitory only when added preceding assembly of the complex. Although competition with intact components seems most likely, less obvious mechanisms are, sometimes, at work. The use of peptides as inhibitory drugs in vivo requires the development of methods to assure cell penetration, resistance to degradation, and avoidance of toxicity, and modest successes have been achieved. The greatest challenge remains the discovery of peptide inhibitors acting specifically on individual Nox isoforms.

Towards specific NADPH oxidase inhibition by small synthetic peptides.

Reactive oxygen species (ROS) production by the phagocyte NADPH oxidase is essential for host defenses against pathogens. ROS are very reactive with biological molecules such as lipids, proteins and DNA, potentially resulting in cell dysfunction and tissue insult. Excessive NADPH oxidase activation and ROS overproduction are believed to participate in disorders such as joint, lung, vascular and intestinal inflammation. NADPH oxidase is a complex enzyme composed of six proteins: gp91phox (renamed NOX2), p22phox, p47phox, p67phox, p40phox and Rac1/2. Inhibitors of this enzyme could be beneficial, by limiting ROS production and inappropriate inflammation. A few small non-peptide inhibitors of NADPH oxidase are currently used to inhibit ROS production, but they lack specificity as they inhibit NADPH oxidase homologues or other unrelated enzymes. Peptide inhibitors that target a specific sequence of NADPH oxidase components could be more specific than small molecules. Here we review peptide-based inhibitors, with particular focus on a molecule derived from gp91phox/NOX2 and p47phox, and discuss their possible use as specific phagocyte NADPH oxidase inhibitors.