For a long time, superoxide generation by an NADPH oxidase was considered as an oddity only found in professional phagocytes. Over the last years, six homologs of the cytochrome subunit of the phag...

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The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology

Recently, substantial advances in the understanding of the molecular pathogenesis of inflammatory bowel disease (IBD) have been made owing to three related lines of investigation. First, IBD has been found to be the most tractable of complex disorders for discovering susceptibility genes, and these have shown the importance of epithelial barrier function, and innate and adaptive immunity in disease pathogenesis. Second, efforts directed towards the identification of environmental factors implicate commensal bacteria (or their products), rather than conventional pathogens, as drivers of dysregulated immunity and IBD. Third, murine models, which exhibit many of the features of ulcerative colitis and seem to be bacteria-driven, have helped unravel the pathogenesis/mucosal immunopathology of IBD.

Unravelling the pathogenesis of inflammatory bowel disease

Genetic, biochemical, and clinical features of chronic granulomatous disease

Flavocytochrome b558 is the catalytic core of the respiratory-burst oxidase, an enzyme complex that catalyzes the NADPH-dependent reduction of O2 into the superoxide anion O2 in phagocytic cells. Flavocytochrome b558 is anchored in the plasma membrane. It is a heterodimer that consists of a large glycoprotein gp91phox (phox forphagocyte oxidase) (beta subunit) and a small protein p22phox (alpha subunit). The other components of the respiratory-burst oxidase are water-soluble proteins of cytosolic origin, namely p67phox, p47phox, p40phox and Rac. Upon cell stimulation, they assemble with the membrane-bound flavocytochrome b558 which becomes activated and generates O2-. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections, illustrating the role of O2- and the derived metabolites H2O2 and HOCl in host defense against invading microorganisms. The electron carriers, FAD and hemes b, and the binding site for NADPH are confined to the gp91phox subunit of flavocytochrome b558. The p22phox subunit serves as a docking site for the cytoso lic phox proteins. This review provides an overview of current knowledge on the structural organization of the O2(-)-generating flavocytochrome b558, its kinetics, its mechanism of activation and the regulation of its biosynthesis. Homologues of gp91phox, called Nox and Duox, are present in a large variety of non-phagocytic cells. They exhibit modest O2(-)-generating oxidase activity, and some act as proton channels. Their role in various aspects of signal transduction is currently under investigation and is briefly discussed.

The superoxide-generating NADPH oxidase: structural aspects and activation mechanism.

Neutrophils constitute the dominant cell in the circulation that mediates the earliest innate immune human responses to infection. The morbidity and mortality from infection rise dramatically in patients with quantitative or qualitative neutrophil defects, providing clinical confirmation of the important role of normal neutrophils for human health. Neutrophil-dependent anti-microbial activity against ingested microbes represents the collaboration of multiple agents, including those prefabricated during granulocyte development in the bone marrow and those generated de novo following neutrophil activation. Furthermore, neutrophils cooperate with extracellular agents as well as other immune cells to optimally kill and degrade invading microbes. This brief review focuses attention on two examples of the integrated nature of neutrophil-mediated anti-microbial action within the phagosome. The importance and complexity of myeloperoxidase-mediated events illustrate a collaboration of anti-microbial responses that are endogenous to the neutrophil, whereas the synergy between the phagocyte NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and plasma-derived group IIA phospholipase A(2) exemplifies the collective effects of the neutrophil with an exogenous factor to achieve degradation of ingested staphylococci.

How human neutrophils kill and degrade microbes: an integrated view.

Recombination events between the p47-phox gene and its highly homologous pseudogenes are the main cause of autosomal recessive chronic granulomatous disease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide as a severe pandemic. Although its seroprevalence is highly variable among territories, it has been reported at around 10%, but higher in health workers. Evidence regarding cross-neutralizing response between SARS-CoV and SARS-CoV-2 is still controversial. However, other previous coronaviruses may interfere with SARS-CoV-2 infection, since they are phylogenetically related and share the same target receptor. Further, the seroconversion of IgM and IgG occurs at around 12 days post onset of symptoms and most patients have neutralizing titers on days 14-20, with great titer variability. Neutralizing antibodies correlate positively with age, male sex, and severity of the disease. Moreover, the use of convalescent plasma has shown controversial results in terms of safety and efficacy, and due to the variable immune response among individuals, measuring antibody titers before transfusion is mostly required. Similarly, cellular immunity seems to be crucial in the resolution of the infection, as SARS-CoV-2-specific CD4+ and CD8+ T cells circulate to some extent in recovered patients. Of note, the duration of the antibody response has not been well established yet.

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Antibody Responses in COVID-19: A Review.

The phagocytic NADPH-oxidase is a multiprotein system activated during the inflammatory response to produce superoxide anion (O2−), which is the substrate for formation of additional reactive oxygen species (ROS). The importance of this system for innate immunity is established by chronic granulomatous disease (CGD), a primary immunodeficiency caused by defects in the NADPH oxidase. In this review, we present and discuss recent knowledge about p40phox, the last NADPH oxidase component to be identified. Furthermore, its interaction with cellular pathways outside of the NADPH oxidase is discussed. Described in this review is evidence that p40phox participates in NADPH oxidase dynamics within cells, what is known about its role in the oxidase, the possibility that p40phox participates in non-NADPH oxidase processes in phagocytic and non-phagocytic cells and whether p40phox could mediate a similar function in other NADPH oxidases. An improved understanding of p40phox should provide new insights about NADPH oxidase, the physiology of phagocytic cells and the innate immune system.

p40phox: the last NADPH oxidase subunit.

Neutrophils are completely differentiated cells that die in tissues a few days after they migrate from the vascular compartment as a consequence of a rigouous apoptotic program. Many of the mediators produced during an inflammatory response delay neutrophil apoptosis allowing a more efficient removal of microorganisms but also favoring the tissue damage by reactive oxygen species (ROS) and lysosomal proteins released by neutrophils. Glucocorticoids delay the apoptosis of neutrophils but the mechanisms are not completely understood. To investigate the inhibition of glucocorticoids on neutrophil apoptosis we have used the glucose/glucose oxidase (G/GO) system as a constant source of hydrogen peroxide. When neutrophils are incubated in the presence of the G/GO system, a significant acceleration of their apoptotic response is observed. Preincubation with 10−6 M, 10−7 M, 10−8 M or 10−9 M of dexamethasone, negatively modulated the spontaneous and G/GO induced apoptosis of neutrophils. Then the G/GO system is a useful model to simulate the oxidative stress of neutrophils, and that the effect of DXM on neutrophil apoptosis depends, at least in part, on blocking the proapoptotic effect of ROS.

Dexamethasone inhibits apoptosis of human neutrophils induced by reactive oxygen species.

Chronic granulomatous disease (CGD) is an uncommon inherited disorder of phagocytic cells in which a defective respiratory burst leads to severe recurrent bacterial and fungal infections. The disease is a consequence of mutations in one of the four molecules that constitute the NADPH oxidase system of electron transport, whose most critical component is an unusual flavocytochrome b localized in the plasma and specific granule membranes. Mutations in the CYBB gene (localized in the short arm of the X chromosome) encoding the beta-subunit of this flavocytochrome (gp91-phox), which is are responsible for 60-65% of all cases of CGD. In this paper, we report the molecular characterization of seven unrelated kindreds native from Colombia and Brazil with CGD caused by gp91-phox deficiency. The exons with the possible mutation were identified by single-strand conformational polymorphism (SSCP) of genomic DNA and then confirmed by DNA sequencing. In one patient we found a substitution of A to G in the penultimate nucleotide of intron 12 (IVS12-2A-->G). In four other cases, four different nonsense mutations were detected: R91X, W106X, R157X, and R290X and the other two patients showed missense substitutions: E225V and C244Y. In six of these kindreds, all mothers were carriers but one that did not present any change in the gp91-phox gene, which indicates a de novo mutation in this kindred. Then, these family-specific mutations in gp91-phox produce different structural defects that alter the expression or function of an essential component of phagocyte oxidase.

Pablo Javier Patiño

Papers

Recombination events between the p47-phox gene and its highly homologous pseudogenes are the main cause of autosomal recessive chronic granulomatous disease.

Antibody Responses in COVID-19: A Review.

p40phox: the last NADPH oxidase subunit.

Dexamethasone inhibits apoptosis of human neutrophils induced by reactive oxygen species.

Molecular analysis of chronic granulomatous disease caused by defects in gp91‐phox