Marie-Hélène Paclet

Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox

Neutrophils generate microbicidal oxidants through activation of a multicomponent enzyme called NADPH oxidase. During activation, the cytosolic NADPH oxidase components (p47phox, p67phox, p40phox, and Rac2) translocate to the membranes, where they associate with flavocytochrome b558, which is composed of gp91phox/NOX2 and p22phox, to form the active system. During neutrophil stimulation, p47phox, p67phox, p40phox, and p22phox are phosphorylated;however, the phosphorylation of gp91phox/NOX2 and its potential role have not been defined. In this study, we show that gp91phox is phosphorylated in stimulated neutrophils. The gp91phox phosphoprotein is absent in neutrophils from chronic granulomatous disease patients deficient in gp91phox, which confirms that this phosphoprotein is gp91phox. The protein kinase C inhibitor GF109203X inhibited phorbol 12‐myristate 13‐acetate‐induced phosphorylation of gp91phox, and protein kinase C (PKC) phosphorylated the recombinant gp91phox‐cytosolic carboxy‐terminal flavoprotein domain. Two‐dimensional tryptic peptide mapping analysis showed that PKC phosphorylated the gp91phox‐cytosolic tail on the same peptides that were phosphorylated on gp91phox in intact cells. In addition, PKC phosphorylation increased diaphorase activity of the gp91phox flavoprotein cytosolic domain and its binding to Rac2, p67phox, and p47phox. These results demonstrate that gp91phox is phosphorylated in human neutrophils by PKC to enhance its catalytic activity and assembly of the complex. Phosphorylation of gp91phox/NOX2 is a novel mechanism of NADPH oxidase regulation.—Raad, H., Paclet, M.‐H., Boussetta, T., Kroviarski, Y., Morel, F., Quinn, M. T., Gougerot‐Pocidalo, M.‐A., Dang, P. M.‐C., El‐Benna, J. Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox. FASEB J. 23, 1011–1022 (2009)

Biochemical and immunochemical properties of B lymphocyte cytochrome b558

Like neutrophils, Epstein-Barr virus (EBV)-immortalized B lymphocytes express all constituents of the NADPH oxidase complex necessary to generate superoxide anion O2-. The NADPH oxidase activity in EBV-B lymphocytes is only 5% of that measured in neutrophils upon PMA stimulation. Cytochrome b558 is the sole redox membrane component of NADPH oxidase; it is the protein core around which cytosolic factors assemble in order to mediate oxidase activity. In the present study, we have compared the structural and functional properties of cytochrome b558 from EBV-B lymphocytes and neutrophils. Cytochrome b558 from EBV-B lymphocyte plasma membrane, like that from neutrophils, is characterized by a heterodimeric structure with a highly glycosylated beta subunit, known as gp91-phox. While the amount of cytochrome b558 recovered after purification from EBV-B lymphocytes (approximately 0.24 nmol from 1010 cells) was low compared to that recovered from neutrophils (approximately 10 nmol), the biochemical properties of purified cytochrome b558 from both EBV-B lymphocytes and neutrophils were quite similar with respect to their differential spectra, redox potential, and FAD binding site. Once cytochrome b558 was extracted from the EBV-B lymphocyte membrane, it was able to mediate, in a reconstituted system of O2- production the same oxidase turnover as that found for cytochrome b558 extracted from neutrophils. A comparison between membrane bound and soluble cytochrome b558 suggested that the weak oxidase activity measured in intact EBV-B cells might be the result not only of the small amount of expressed cytochrome b558, but also of a defect of the activation process in lymphocyte membrane.

Localization of Nox2 N-terminus using polyclonal antipeptide antibodies

Nox2/gp91(phox) (where phox is phagocyte oxidase) is the catalytic membrane subunit of the granulocyte NADPH oxidase complex involved in host defence. The current model of membrane topology of Nox2 is based upon the identification of glycosylation sites, of regions that interact with the regulatory cytosolic factors and of the epitopes recognized by antibodies. So far, the localization of the N-terminus of Nox2 was only speculative. In order to clarify this localization, we raised a polyclonal antiserum against the N-terminal sequence M(1)GNWVAVNEGL(11). Purified antibodies recognize the mature protein as a broad band at 91 kDa (glycosylated form) or a band at 55 kDa after deglycosylation. Immunocytochemistry and flow-cytometry analysis show a strong binding of the anti-N-terminal antibodies to differentiated HL60 cells and neutrophils respectively, after permeabilization only. The N-terminus of Nox2 is therefore present in the mature protein and is located to the cytoplasmic side of the plasma membrane.

Regulation of phagocyte NADPH oxidase activity: identification of two cytochrome b558 activation states.

Activation of the phagocyte NADPH oxidase (phox) requires the association of cytosolic proteins (p67‐phox, p47‐phox, p40‐phox, and Rac1/2) with the membrane cytochrome b558, leading to a hemopro‐tein conformation change. To clarify this mechanism, the phagocyte NADPH oxidase complex was isolated through cytochrome b558 purification after three chromatographic steps. The purified neutrophil complex was constitutively active in the absence of an amphi‐phile agent with a maximum turnover (125 mol O2−•s−1τnol heme b−1), indicating that cytochrome b558 has been activated by cytosolic proteins and is in an “open conformation,” able to transfer a maximum rate of electrons. In contrast, the phox complex prepared with B lymphocyte cytosol shows a lower constitutive turnover (~50 mol O2− •s−1 Triol heme b−1). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67‐phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully “open” cyto‐chrome b558 conformation. Moreover, their effect was independent of p67‐phox. Data point out two potential cytochrome b558 activation states.—Paclet, M‐H., Berthier, S., Kuhn, L., Garin, J., Morel, F. Regulation of phagocyte NADPH oxidase activity: identification of two cytochrome b558 activation states. FASEB J. 21, 1244–1255 (2007)

NADPH Oxidase Is Internalized by Clathrin-coated Pits and Localizes to a Rab27A/B GTPase-regulated Secretory Compartment in Activated Macrophages

Background: Subcellular distribution of NADPH oxidase in macrophages is unclear. Results: In mature (activated) macrophages, NADPH oxidase is contained in a storage compartment and is internalized by clathrin-coated pits. Conclusion: NADPH oxidase trafficking is regulated by external factors. Significance: Subcellular localization of NADPH oxidase relates to pathogen eradication, nature, and severity of oxidative tissue stress as well as redox signaling. Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b558) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91phox and CeCl3 cytochemistry showed the presence of gp91phox and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b558 is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b558-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5′-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b558 under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNFα, and CD40L as physiological inducers of cyt b558 exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b558, which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b558 did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b558 depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell.

How Important are S100A8/S100A9 Calcium Binding Proteins for the Activation of Phagocyte NADPH Oxidase, Nox2

S100A8 and S100A9 are two soluble calcium-binding proteins highly expressed in myeloid cells, mainly neutrophils (45% of cytosolic proteins) or monocytes (1-5%) and also early differentiated macrophages. In neutrophils, they are believed to be expressed as a 1/1 non covalent heterodimer; the process of dimer and mainly tetramer formation is calcium dependent. The S100A8/S100A9 calcium loaded complex binds arachidonic acid and shuttles between cytosol and plasma membrane upon neutrophil stimulation. Neutrophils display, upon stimulation, a respiratory burst in which the cells catalyze NADPH oxidase activity through a redox membrane hemoprotein, cytochrome b558, which is constituted of 2 subunits: gp91-phox, the redox core and p22- phox the stabilizing partner. In neutrophils, this activity is transitory: to be active, regulatory cytosolic factors, p67-phox, p47-phox, p40-phox and Rac1/2 assemble with membrane cytochrome b558. Both S100A8 and S100A9 were recently introduced as partners for NADPH oxidase activation and associate with the cytosolic activating factors especially p67-phox and Rac1/2. Moreover, S100A8/S100A9 potentiates NADPH oxidase activity. This was observed ex vivo after co-transfection of genes encoding both S100A8 and S100A9 in B lymphocytes that express all the components of the phagocyte oxidase, but display a very low NADPH oxidase activity (in these cells, S100A8 and S100A9 are not present endogenously). In the biological function of S100A8/S100A9, S100A8 is a strategic protein that needs to be active in vivo as in vitro, its specific partner S100A9. New data introduce S100A8 and S100A9 as positive effectors in allosteric regulation of phagocyte NADPH oxidase activity.

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 workwas to study the relationship between PFK‐2 and NADPHoxidase 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 energeticmetabolismincrease thatwas induced by inflammatory conditions. PFK‐2 seems to be a strategic element that links NADPH oxidase activation and glycolysismodulation, and, as such, is proposedas 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 phagocyteNADPHoxidase in supporting hyperglycolysis in stimulated neutrophils:key role of 6‐phosphofructo‐2‐ kinase. FASEB J. 31, 663–673 (2017). www.fasebj.org

Calprotectin is not independent from baseline erosion in predicting radiological progression in early rheumatoid arthritis. Comment on ‘Calprotectin as a marker of inflammation in patients with early rheumatoid arthritis’ by Jonsson et al

We have read with great interest the article by Jonsson et al that was recently published online in ARD ,1 which suggested that calprotectin, also known as S100A8/S100A9 heterodimer, was associated with radiographic progression in early rheumatoid arthritis (RA). Calprotectin correlates significantly with inflammatory markers and disease activity score.2 Besides correlations between baseline calprotectin levels, Clinical Disease Activity Index and ultrasonography power Doppler, the authors showed that baseline calprotectin levels correlated with van der Heijde modified Sharp score (SHS) progression (defined as an increase ≥1 unit/year from 0 to 24 months), independently of age, gender, Clinical Disease Activity Index, erythrocyte sedimentation rate (ESR), C reactive protein (CRP) levels and rheumatoid factor positivity.1 We analysed the initial serum calprotectin among patients with early RA fulfilling American College of Rheumatology/European League Against Rheumatism 2010 of the French observational cohort Etude et Suivi des POlyarthrites Indifferenciees Recentes (ESPOIR). Calprotectin serum concentrations were assessed according to manufacturer method (Hycult, Frontstraat, Netherlands; …