Phagocyte NADPH Oxidase Research Articles

Infected wound repair correlates with collagen I induction and NOX2 activation by cold atmospheric plasma

Cold atmospheric plasma (CAP) is a promising complement to tissue repair and regenerative medicine approaches. CAP has therapeutic potential in infected cutaneous wounds by mechanisms which remain enigmatic. Here, CAP is shown to activate phagocyte NADPH oxidase complex NOX2. CAP induced increased intracellular reactive oxygen species, alleviated by NOX2 inhibitors. Genetic and pharmacological inhibitions of NOX2 in macrophages and bioengineered skin infected with Staphylococcus aureus and treated with CAP reduced intracellular oxidants and increased bacterial survival. CAP triggered Rac activation and phosphorylation of p40phox and p47phox required for NOX2 assembly and activity. Furthermore, CAP induced collagen I expression by fibroblasts. Infection and healing kinetics showed that murine skin wounds infected with S. aureus and treated with CAP are characterized by decreased bacterial burden, increased length of neoepidermis and extracellular matrix formation. Collectively, our findings identify mechanisms triggered by CAP that subdue infection and result in enhanced repair following skin injury.

Structural profiles of the full phagocyte NADPH oxidase unveiled by combining computational biology and experimental knowledge.

The phagocyte NADPH oxidase (NOX2) is an enzyme, crucial for innate immune defense, producing reactive oxygen species necessary for pathogen destruction. Its activation requires the assembly of soluble proteins (p47phox, p40phox, p67phox, and Rac) with the membrane-bound flavocytochrome b558 (cytb558). We combined circular-dichroism analyses, with decades of experimental data, to filter structural models of the NADPH oxidase complex generated by the artificial intelligence program AlphaFold2 (AF2). The predicted patterns tend to closely resemble the active states of the proteins, as shown by the compact structure of the cytb558, whose dehydrogenase domain is stabilized closer to the membrane. The modeling of the interaction of p47phox with cytb558, which is the initial assembly and activation steps of the NADPH oxidase, enables us to describe how the C-terminus of p47phox interacts with the cytb558. Combining the AF2 cytb558 -p47phox model and its classical molecular dynamics simulations, we highlighted new hydrophobic lipid insertions of p47phox, particularly at residues Trp80-Phe81 of its PX domain. The AF2 models also revealed the implications of intrinsically disordered regions, such as the fragment between the PX domain and the SH3 regions of p47phox, in ensuring distant protein-protein and membrane-protein interactions. Finally, the AF2 prediction of the cytb558-Trimera model highlighted the importance of leaving Rac1 as a separate protein to reach an active state of the NADPH oxidase complex. Altogether, our step-by-step approach provides a structural model of the active complex showing how disordered regions and specific lipid and protein interactions can enable and stabilize the multi-subunit assembly.

Clinical presentation, diagnosis, and treatment of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is caused by an impaired respiratory burst reaction in phagocytes. CGD is an X-linked (XL) (caused by pathogenic variants in CYBB) or autosomal recessive inborn error of immunity (caused by pathogenic variants in CYBA, NCF1, NCF2, or CYBC1). Female carriers of XL-CGD and unfavorable lyonization may present with the partial or full picture of CGD. Patients with CGD are at increased risk for invasive bacterial and fungal infections of potentially any organ, but especially the lymph nodes, liver, and lungs. Pathogens most frequently isolated are S. aureus and Aspergillus spp. Autoinflammation is difficult to control with immunosuppression, and patients frequently remain dependent on steroids. To diagnose CGD, reactive oxygen intermediates (O2 - or H2O2) generated by the NADPH oxidase in peripheral blood phagocytes are measured upon in vitro activation with either phorbol-12-myristate-13-acetate (PMA) and/or TLR4 ligands (E. coli or LPS). Conservative treatment requires strict hygienic conduct and adherence to antibiotic prophylaxis against bacteria and fungi, comprising cotrimoxazole and triazoles. The prognosis of patients treated conservatively is impaired: for the majority of patients, recurrent and/or persistent infections, autoinflammation, and failure to thrive remain lifelong challenges. In contrast, cellular therapies (allogeneic stem cell transplantation or gene therapy) can cure CGD. Optimal outcomes in cellular therapies are observed in individuals without ongoing infections or inflammation. Yet cellular therapies are the only curative option for patients with persistent fungal infections or autoinflammation.

In vivo upstream factors of mouse hepatotoxic mechanism with sustained hepatic glutathione depletion: Acetaminophen metabolite-erythrocyte adducts and splenic macrophage-generated reactive oxygen species

Investigation of acetaminophen (APAP)-induced liver damage recently indicated the significance of phagocytic NADPH oxidase (NOX)-derived reactive oxygen species (ROS) and ferroptosis in the liver. Here, we focused on phagocytosis by iron-containing erythrocyte-devouring splenic macrophages and explored upstream factors of known APAP hepatotoxic mechanisms in vivo. Splenectomy did not alter hepatic cytochrome P450 (CYP) 2E1 activity or hepatic glutathione (GSH) content. APAP injection into splenectomized mice almost completely suppressed increases in plasma alanine aminotransferase levels and centrilobular hepatic necrosis showing the spleen to be a critical tissue in APAP-induced liver damage. Hepatic GSH was recovered to approximately 50 % content at 8 h. In non-splenectomized mice, liver damage was dramatically suppressed by a sensitive redox probe (DCFH-DA), macrophage-depleting clodronate (CL), and a NOX2 inhibitor. APAP treatment resulted in markedly stronger fluorescence intensity from DCFH-DA due to excessive ROS around splenic macrophages, which was lost upon co-treatment with a CYP inhibitor and CL. Deformed erythrocytes disappeared in mice co-treated with DCFH-DA, CL, the NOX2 inhibitor, and the CYP inhibitor. Simultaneously, these four compounds significantly improved APAP-depleted GSH levels. The CYP inhibitor also prevented the formation of APAP-cell adducts in the blood and spleen. In the spleen, CL co-treatment markedly reduced the number of adducts. Splenic ferrous iron levels were significantly elevated by APAP. Therefore, we demonstrated that splenic macrophages devoured APAP metabolite-erythrocyte adducts and subsequently splenic macrophage-related ROS caused sustained hepatic GSH depletion and excessive erythrocyte deformation around 7 h. Our data indicate in vivo upstream factors of known APAP hepatotoxic mechanisms.

Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91phox), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently identified cause for chronic granulomatous disease, a genetic disorder with recurrent bacterial and fungal infections. Here, we report a cryo-EM structure of the EROS-NOX2-p22phox heterotrimeric complex at an overall resolution of 3.56Å. EROS and p22phox are situated on the opposite sides of NOX2, and there is no direct contact between them. EROS associates with NOX2 through two antiparallel transmembrane (TM) α-helices and multiple β-strands that form hydrogen bonds with the cytoplasmic domain of NOX2. EROS binding induces a 79° upward bend of TM2 and a 48° backward rotation of the lower part of TM6 in NOX2, resulting in an increase in the distance between the two hemes and a shift of the binding site for flavin adenine dinucleotide (FAD). These conformational changes are expected to compromise superoxide production by NOX2, suggesting that the EROS-bound NOX2 is in a protected state against activation. Phorbol myristate acetate, an activator of NOX2 in vitro, is able to induce dissociation of NOX2 from EROS with concurrent increase in FAD binding and superoxide production in a transfected COS-7 model. In differentiated neutrophil-like HL-60, the majority of NOX2 on the cell surface is dissociated with EROS. Further studies are required to delineate how EROS dissociates from NOX2 during its transport to cell surface, which may be a potential mechanism for regulation of NOX2 activation.

Membrane potential dynamics of C5a-stimulated neutrophil granulocytes

Neutrophil granulocytes play a crucial role in host defense against invading pathogens and in inflammatory diseases. The aim of this study was to elucidate membrane potential dynamics during the initial phase of neutrophil activation and its relation to migration and production of reactive oxygen species (ROS). We performed ROS production measurements of neutrophils from healthy C57BL/6J mice after TNFα-priming and/or C5a stimulation. The actin cytoskeleton was visualized with fluorescence microscopy. Furthermore, we combined migration assays and measurements of membrane potential dynamics after stimulating unprimed and/or TNFα-primed neutrophils with C5a. We show that C5a has a concentration-dependent effect on ROS production and chemokinetic migration. Chemokinetic migration and chemotaxis are impaired at C5a concentrations that induce ROS production. The actin cytoskeleton of unstimulated and of ROS-producing neutrophils is not distributed in a polarized way. Inhibition of the phagocytic NADPH oxidase NOX2 with diphenyleneiodonium (DPI) leads to a polarized distribution of the actin cytoskeleton and rescues chemokinetic migration of primed and C5a-stimulated neutrophils. Moreover, C5a evokes a pronounced depolarization of the cell membrane potential by 86.6 ± 4.2 mV starting from a resting membrane potential of -74.3 ± 0.7 mV. The C5a-induced depolarization occurs almost instantaneously (within less than one minute) in contrast to the more gradually developing depolarization induced by PMA (lag time of 3—4 min). This initial depolarization is accompanied by a decrease of the migration velocity. Collectively, our results show that stimulation with C5a evokes parallel changes in membrane potential dynamics, neutrophil ROS production and motility. Notably, the amplitude of membrane potential dynamics is comparable to that of excitable cells.

NADPH oxidase exerts a B cell-intrinsic contribution to lupus risk by modulating endosomal TLR signals.

Genome-wide association studies in systemic lupus erythematosus (SLE) have linked loss-of-function mutations in phagocytic NADPH oxidase complex (NOX2) genes, including NCF1 and NCF2, to disease pathogenesis. The prevailing model holds that reduced NOX2 activity promotes SLE via defective efferocytosis, the immunologically silent clearance of apoptotic cells. Here, we describe a parallel B cell-intrinsic mechanism contributing to breaks in tolerance. In keeping with an important role for B cell Toll-like receptor (TLR) pathways in lupus pathogenesis, NOX2-deficient B cells exhibit enhanced signaling downstream of endosomal TLRs, increased humoral responses to nucleic acid-containing antigens, and the propensity toward humoral autoimmunity. Mechanistically, TLR-dependent NOX2 activation promotes LC3-mediated maturation of TLR-containing endosomes, resulting in signal termination. CRISPR-mediated disruption of NCF1 confirmed a direct role for NOX2 in regulating endosomal TLR signaling in primary human B cells. Together, these data highlight a new B cell-specific mechanism contributing to autoimmune risk in NCF1 and NCF2 variant carriers.

Linalool, a Fragrance Compound in Plants, Protects Dopaminergic Neurons and Improves Motor Function and Skeletal Muscle Strength in Experimental Models of Parkinson's Disease.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the gradual loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), resulting in reduced dopamine levels in the striatum and eventual onset of motor symptoms. Linalool (3,7-dimethyl-1,6-octadien-3-ol) is a monoterpene in aromatic plants exhibiting antioxidant, antidepressant, and anti-anxiety properties. The objective of this study is to evaluate the neuroprotective impacts of linalool on dopaminergic SH-SY5Y cells, primary mesencephalic and cortical neurons treated with 1-methyl-4-phenylpyridinium ion (MPP+), as well as in PD-like mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Cell viability, α-tubulin staining, western blotting, immunohistochemistry and behavioral experiments were performed. In MPP+-treated SH-SY5Y cells, linalool increased cell viability, reduced neurite retraction, enhanced antioxidant defense by downregulation of apoptosis signaling (B-cell lymphoma 2 (Bcl-2), cleaved caspase-3 and poly ADP-ribose polymerase (PARP)) and phagocyte NADPH oxidase (gp91phox), as well as upregulation of neurotrophic signaling (brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF)) and nuclear factor-erythroid 2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. In MPP+-treated primary mesencephalic neurons, linalool enhanced the expressions of tyrosine hydroxylase (TH), Sirtuin 1 (SirT1), and parkin. In MPP+-treated primary cortical neurons, linalool upregulated protein expression of SirT1, γ-Aminobutyric acid type A-α1 (GABAA-α1), and γ-Aminobutyric acid type B (GABAB). In PD-like mice, linalool attenuated the loss of dopamine neurons in SNpc. Linalool improved the motor and nonmotor behavioral deficits and muscle strength of PD-like mice. These findings suggest that linalool potentially protects dopaminergic neurons and improves the impairment symptoms of PD.

Structure of human phagocyte NADPH oxidase in the activated state.

Phagocyte NADPH oxidase, a proteincomplex 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.

Anaerobic respiration of host-derived methionine sulfoxide protects intracellular Salmonella from the phagocyte NADPH oxidase

Intracellular Salmonella experiencing oxidative stress downregulates aerobic respiration. To maintain cellular energetics during periods of oxidative stress, intracellular Salmonella must utilize terminal electron acceptors of lower energetic value than molecular oxygen. We show here that intracellular Salmonella undergoes anaerobic respiration during adaptation to the respiratory burst of the phagocyte NADPH oxidase in macrophages and in mice. Reactive oxygen species generated by phagocytes oxidize methionine, generating methionine sulfoxide. Anaerobic Salmonella uses the molybdenum cofactor-containing DmsABC enzymatic complex to reduce methionine sulfoxide. The enzymatic activity of the methionine sulfoxide reductase DmsABC helps Salmonella maintain an alkaline cytoplasm that supports the synthesis of the antioxidant hydrogen sulfide via cysteine desulfuration while providing a source of methionine and fostering redox balancing by associated dehydrogenases. Our investigations demonstrate that nontyphoidal Salmonella responding to oxidative stress exploits the anaerobic metabolism associated with dmsABC gene products, a pathway that has accrued inactivating mutations in human-adapted typhoidal serovars.

A novel mutation in EROS (CYBC1) causes chronic granulomatous disease

Chronic Granulomatous Disease (CGD) is an inborn error of immunity characterised by opportunistic infection and sterile granulomatous inflammation. CGD is caused by a failure of reactive oxygen species (ROS) production by the phagocyte NADPH oxidase. Mutations in the genes encoding phagocyte NADPH oxidase subunits cause CGD. We and others have described a novel form of CGD (CGD5) secondary to lack of EROS (CYBC1), a highly selective chaperone for gp91phox. EROS-deficient cells express minimal levels of gp91phox and its binding partner p22phox, but EROS also controls the expression of other proteins such as P2X7. The full nature of CGD5 is currently unknown. We describe a homozygous frameshift mutation in CYBC1 leading to CGD. Individuals who are heterozygous for this mutation are found in South Asian populations (allele frequency = 0.00006545), thus it is not a private mutation. Therefore, it is likely to be the underlying cause of other cases of CGD.

The methylglyoxal pathway is a sink for glutathione in Salmonella experiencing oxidative stress.

Salmonella suffer the cytotoxicity of reactive oxygen species generated by the phagocyte NADPH oxidase in the innate host response. Periplasmic superoxide dismutases, catalases and hydroperoxidases detoxify superoxide and hydrogen peroxide (H2O2) synthesized in the respiratory burst of phagocytic cells. Glutathione also helps Salmonella combat the phagocyte NADPH oxidase; however, the molecular mechanisms by which this low-molecular-weight thiol promotes resistance of Salmonella to oxidative stress are currently unknown. We report herein that Salmonella undergoing oxidative stress transcriptionally and functionally activate the methylglyoxal pathway that branches off from glycolysis. Activation of the methylglyoxal pathway consumes a substantial proportion of the glutathione reducing power in Salmonella following exposure to H2O2. The methylglyoxal pathway enables Salmonella to balance glucose utilization with aerobic respiratory outputs. Salmonella take advantage of the metabolic flexibility associated with the glutathione-consuming methylglyoxal pathway to resist reactive oxygen species generated by the enzymatic activity of the phagocyte NADPH oxidase in macrophages and mice. Taken together, glutathione fosters oxidative stress resistance in Salmonella against the antimicrobial actions of the phagocyte NADPH oxidase by promoting the methylglyoxal pathway, an offshoot metabolic adaptation of glycolysis.

Role of the phospholipid binding sites, PX of p47phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2

In phagocytes, superoxide anion (O2−), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b558 (cyt b558) and four cytosolic components: p40phox, p47phox, p67phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b558 to form the active enzyme.To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47phox. We also used a fused chimera consisting of p47phox(aa 1–286), p67phox(aa 1–212) and Rac1Q61L, as well as mutated versions in the p47phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b558. They also have an impact on O2.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.

Arginine Metabolism Powers Salmonella Resistance to Oxidative Stress.

Salmonella invades host cells and replicates inside acidified, remodeled vacuoles that are exposed to reactive oxygen species (ROS) generated by the innate immune response. Oxidative products of the phagocyte NADPH oxidase mediate antimicrobial activity, in part, by collapsing the ΔpH of intracellular Salmonella. Given the role of arginine in bacterial resistance to acidic pH, we screened a library of 54 single-gene mutants in Salmonella that are each involved in, but do not entirely block, arginine metabolism. We identified several mutants that affected Salmonella virulence in mice. The triple mutant ΔargCBH, which is deficient in arginine biosynthesis, was attenuated in immunocompetent mice, but recovered virulence in phagocyte NADPH oxidase deficient Cybb-/- mice. Furthermore, ΔargCBH Salmonella was profoundly susceptible to the bacteriostatic and bactericidal effects of hydrogen peroxide. Peroxide stress led to a larger collapse of the ΔpH in ΔargCBH mutants than occurred in wild-type Salmonella. The addition of exogenous arginine rescued ΔargCBH Salmonella from peroxide-induced ΔpH collapse and killing. Combined, these observations suggest that arginine metabolism is a hitherto unknown determinant of virulence that contributes to the antioxidant defenses of Salmonella by preserving pH homeostasis. In the absence of phagocyte NADPH oxidase-produced ROS, host cell-derived l-arginine appears to satisfy the needs of intracellular Salmonella. However, under oxidative stress, Salmonella must additionally rely on de novo biosynthesis to maintain full virulence.

The cardiovascular effects of alternative tobacco products and the role of phagocytic NADPH oxidase (NOX-2)

The cardiovascular effects of alternative tobacco products and the role of phagocytic NADPH oxidase (NOX-2)

Non-infectious skin manifestations in patients with Chronic Granulomatous Disease: A report of 4 cases

BackgroundChronic granulomatous disease (CGD) is a monogenic inborn error of immunity caused by defects in the phagocytic NADPH oxidase complex. These defects lead to an increased risk of severe infection, autoimmunity, widespread granuloma formation and dysregulated inflammation. In individuals with CGD, immune dysregulation and autoimmune manifestations may impact almost every organ system with atypical dermatologic findings frequently reported. Case PresentationsHere we describe four cases of inflammatory skin lesions in patients with genetically confirmed CGD. Patient A is a 4-year-old male who presented with diffuse alopecia totalis preceded by a widespread skin eruption composed of polycyclic, annular, erythematous plaques and prominent facial erythema and edema. Whole exome sequencing was obtained due to concern for ectodermal dysplasia, which identified a variant in CYBB (CYBB c.901G>T; pVal301Phe). Patient B is a 4-year-old male with X-CGD (CYBB c.1016 C > A, p.Pro339His) who developed annular to polycyclic indurated red plaques on his face and arms. Patient C is a 19-year-old female with autosomal recessive CGD (NCF1 c.75_76delGT) and persistent hidradenitis suppurativa-like lesions in the axilla, inguinal areas, and popliteal fossa. Patient D is a 12-year-old female with autosomal recessive CGD (p47phox deficiency) and psoriasiform dermatitis of the scalp.Patient A had persistence of lesions that required chronic systemic corticosteroids and methotrexate. He underwent hematopoietic stem cell transplantation using a matched sibling donor with improvement in his dermatitis. Skin lesions in patient B resolved with the use of topical corticosteroids. Patient C improved with a combination of isotretinoin therapy and both intralesional and systemic corticosteroid treatment. Patient D had improvement in her psoriasiform dermatitis with topical corticosteroid solution and salicylic acid shampoo. ConclusionsThis case series expands our understanding of cutaneous non-infectious eruptions that can occur in patients with both X-linked and autosomal recessive forms of CGD. Each of these cases illustrates a unique clinical history, histologic findings and treatment plan in patients who developed cutaneous lesions secondary to immune dysregulation in the context of CGD. This series provides additional insights that may assist providers in identifying and managing non-infectious dermatologic manifestations of CGD.

Lupus nephritis with clinical and laboratory features of humoral immune deficiency in a 29-year-old female with a novel NCF1 variant consistent with autosomal recessive chronic granulomatous disease

Chronic Granulomatous Disease (CGD) is a rare, inherited disorder of the innate immune system characterized by defects in the phagocyte NADPH oxidase complex. This results in poor phagocyte function causing frequent infections (bacterial and fungal) and chronic inflammation. Here we discuss a rare presentation of lupus nephritis and antibody deficiency in an adult female patient with a novel homozygous nonsense mutation in the NCF-1 gene causing autosomal recessive CGD.Our patient is a 29-year-old female with history of ITP, biopsy-confirmed lupus nephritis, systemic lupus erythematosus (SLE), idiopathic avascular necrosis of the hip, and previous diagnosis of common variable immune deficiency (CVID) treated with Immunoglobulin replacement therapy (IVIG). She presented to our clinic to establish care in the setting of persistent skin abscesses/cellulitis, poor wound healing, necrotic lymphadenitis, and recurrent urinary tract infections and pneumonia despite years of IVIG therapy. She had near-normal total IgG, absent IgA and IgE, and protective vaccine antibody titers (pneumococcus, diphtheria, and tetanus) prior to the start of IVIG. Immunophenotyping demonstrated quantitatively normal number of total B cells, absent CD27+ memory B cells, and normal T cell subsets. She had reassuring lymphocyte proliferation with mitogen stimulation and normal TLR function. Dihydrorhodamine (DHR) flow cytometric assay demonstrated abnormally low neutrophil oxidative burst activity. Gene sequencing revealed homozygous nonsense mutation in the NCF-1 gene (c.75-76del; pY26Hfs*26) and a pathogenic heterozygous variant in FANCM (c.3732-3735dup; p. Leu1246Asnfs*8). She was started on daily prophylactic trimethoprim-sulfamethoxazole and itraconazole. She has marked decrease in infections/hospitalizations and her lupus nephritis is well controlled with hydroxy-chloroquine. She is being referred to the NIH for HSCT evaluation. The majority of CGD cases in the United States are due to X-linked CGD (70%) and AR variants in NCF1 (20%). While rare, discoid lupus erythematosus and SLE have been documented with X-linked forms of CGD. Similarly, there are few case reports of antibody deficiency in patients with CGD. This is the first documented case of AR CGD with SLE/lupus nephritis and humoral immune defects. Additionally, we report a disease-causing, novel variant in the NCF1 gene. This case report adds to the existing literature on CGD and associated diseases.

Gre factors help Salmonella adapt to oxidative stress by improving transcription elongation and fidelity of metabolic genes.

Detoxification, scavenging, and repair systems embody the archetypical antioxidant defenses of prokaryotic and eukaryotic cells. Metabolic rewiring also aids with the adaptation of bacteria to oxidative stress. Evolutionarily diverse bacteria combat the toxicity of reactive oxygen species (ROS) by actively engaging the stringent response, a stress program that controls many metabolic pathways at the level of transcription initiation via guanosine tetraphosphate and the α-helical DksA protein. Studies herein with Salmonella demonstrate that the interactions of structurally related, but functionally unique, α-helical Gre factors with the secondary channel of RNA polymerase elicit the expression of metabolic signatures that are associated with resistance to oxidative killing. Gre proteins both improve transcriptional fidelity of metabolic genes and resolve pauses in ternary elongation complexes of Embden-Meyerhof-Parnas (EMP) glycolysis and aerobic respiration genes. The Gre-directed utilization of glucose in overflow and aerobic metabolism satisfies the energetic and redox demands of Salmonella, while preventing the occurrence of amino acid bradytrophies. The resolution of transcriptional pauses in EMP glycolysis and aerobic respiration genes by Gre factors safeguards Salmonella from the cytotoxicity of phagocyte NADPH oxidase in the innate host response. In particular, the activation of cytochrome bd protects Salmonella from phagocyte NADPH oxidase-dependent killing by promoting glucose utilization, redox balancing, and energy production. Control of transcription fidelity and elongation by Gre factors represent important points in the regulation of metabolic programs supporting bacterial pathogenesis.

New insights in the molecular regulation of the NADPH oxidase 2 activity: Negative modulation by Poldip2.

Poldip2 was shown to be involved in oxidative signaling to ensure certain biological functions. It was proposed that, in VSMC, by interaction with the Nox4-associated membrane protein p22phox, Poldip2 stimulates the level of reactive oxygen species (ROS) production. In vitro, with fractionated membranes from HEK393cells over-expressing Nox4, we confirmed the up-regulation of NADPH oxidase 4 activity by the recombinant and purified Poldip2. Besides Nox4, the Nox1, Nox2, or Nox3 isoforms are also established partners of the p22phox protein raising the question of their regulation by Poldip2 and of the effect in cells expressing simultaneously different Nox isoforms. In this study, we have addressed this issue by investigating the potential regulatory role of Poldip2 on NADPH oxidase 2, present in phagocyte cells. Unexpectedly, the effect of Poldip2 on phagocyte NADPH oxidase 2 was opposite to that observed on NADPH oxidase 4. Using membranes from circulating resting neutrophils, the ROS production rate of NADPH oxidase 2 was down-regulated by Poldip2 (2.5-fold). The down-regulation effect could not be correlated to the interaction of Poldip2 with p22phox but rather, to the interaction of Poldip2 with the p47phox protein, one of the regulatory proteins of the phagocyte NADPH oxidase. Our results show that the interaction of Poldip2 with p47phox constitutes a novel regulatory mechanism that can negatively modulate the activity of NADPH oxidase 2 by trapping the so-called "adaptor" subunit of the complex. Poldip2 could act as a tunable switch capable of specifically regulating the activities of NADPH oxidases. This selective regulatory role of Poldip2, positive for Nox4 or negative for Nox2 could orchestrate the level and the type of ROS generated by Nox enzymes in the cells.

Continuous Measurement of Reactive Oxygen Species Formation in Bacteria-infected Bone Marrow-derived Macrophages Using a Fluorescence Plate Reader.

Macrophages are at the center of innate immunity and are the main target cells of the intracellular pathogen Salmonella enterica serovar Typhi. The production of reactive oxygen and nitrogen species (ROS/RNS) is the host's early response to invading microbes, as oxidative stress is highly toxic for bacteria. Adequate ROS/RNS production in infected macrophages is critical for the clearance of intracellular pathogens; this is achieved by several enzymes, including inducible NADPH phagocyte oxidase (NOX) and nitric oxide synthase (iNOS), respectively. The pro-inflammatory cytokine interferon gamma (IFNγ), primarily produced by activated natural killer cells and T-helper cells type 1, is a potent inducer of iNOS. Therefore, it is crucial for infection control through oxidative microbicidal activity. To characterize the early oxidative stress response via ROS formation, which is critical for the reduction of Salmonella proliferation within macrophages, we established an in vitro model of murine macrophages infected with Salmonella enterica serovar Typhimurium (S.tm). This serovar induces a systemic infection in mice that is frequently used as a model for typhoid fever, which, in human subjects, is caused by Salmonella Typhi. We generated bone marrow-derived macrophages (BMDM) from C57BL/6N wildtype mice using macrophage colony-stimulating factor (M-CSF) stimulation for six days. Thereafter, we infected BMDM withS. tm for one hour. Shortly before infection, cells were stained with CellROXTM Deep Red reagent. In its reduced form, CellROXTM is non-fluorescent. As a result of oxidation by ROS, this reagent exhibits strong fluorescence and persists within the cells. Subsequently, changes as a result of the oxidative stress response can be measured with a TECAN Spark microplate reader over time. We designed this protocol to measure oxidative stress in macrophages through the course of an infection with an intracellular bacterium. The protocol has several advantages over established techniques. First, it allows to continuously monitor and quantify ROS production in living cells from the very start of the infection to the final clearance of the intracellular pathogen. Second, this protocol enables efficient ROS detection without stressing the cells by detaching or staining procedures. Graphical abstract.