CD47-SIRPα Controls ADCC Killing of Primary T Cells by PMN Through a Combination of Trogocytosis and NADPH Oxidase Activation.

Immunotherapies targeting the "don't eat me" myeloid checkpoint constituted by CD47 SIRPα interaction have promising clinical potential but are limited by toxicities associated with the destruction of non-tumor cells. These dose-limiting toxicities demonstrate the need to highlight the mechanisms of anti-CD47-SIRPα therapy effects on non-tumor CD47-bearing cells. Given the increased incidence of lymphopenia in patients receiving anti-CD47 antibodies and the strong ADCC (antibody-dependent cellular cytotoxicity) effector function of polymorphonuclear cells (PMNs), we investigated the behavior of primary PMNs cocultured with primary T cells in the presence of anti-CD47 mAbs. PMNs killed T cells in a CD47-mAb-dependent manner and at a remarkably potent PMN to T cell ratio of 1:1. The observed cytotoxicity was produced by a novel combination of both trogocytosis and a strong respiratory burst induced by classical ADCC and CD47-SIRPα checkpoint blockade. The complex effect of the CD47 blocking mAb could be recapitulated by combining its individual mechanistic elements: ADCC, SIRPα blockade, and ROS induction. Although previous studies had concluded that disruption of SIRPα signaling in PMNs was limited to trogocytosis-specific cytotoxicity, our results suggest that SIRPα also tightly controls activation of NADPH oxidase, a function demonstrated during differentiation of immature PMNs but not so far in mature PMNs. Together, our results highlight the need to integrate PMNs in the development of molecules targeting the CD47-SIRPα immune checkpoint and to design agents able to enhance myeloid cell function while limiting adverse effects on healthy cells able to participate in the anti-tumor immune response.

Neutrophil-Derived Tumor Necrosis Factor Drives Fungal Acute Lung Injury in Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) results from deficiency of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase and impaired reactive oxygen species (ROS) generation. This leads to impaired killing of Aspergillus and, independently, a pathologic hyperinflammatory response to the organism. We hypothesized that neutrophil-derived ROS inhibit the inflammatory response to Aspergillus and that acute lung injury in CGD is due to failure of this regulation. Mice with gp91phox deficiency, the most common CGD mutation, had more severe lung injury, increased neutrophilinfiltration, and increased lung tumor necrosis factor (TNF) after Aspergillus challenge compared with wild-types. Neutrophils were surprisingly the predominant source of TNF in gp91phox-deficient lungs. TNF neutralization inhibited neutrophil recruitment in gp91phox-deficient mice and protected from lung injury. We propose that, in normal hosts, Aspergillus stimulates TNF-dependent neutrophil recruitment to the lungs and neutrophil-derived ROS limit inflammation. In CGD, in contrast, recruited neutrophils are the dominant source of TNF, promoting further neutrophil recruitment in a pathologic positive-feedback cycle, resulting in progressive lung injury.

Spatial Properties of Reactive Oxygen Species Govern Pathogen-Specific Immune System Responses.

Significance: Reactive oxygen species (ROS) are often considered to be undesirable toxic molecules that are generated under conditions of cellular stress, which can cause damage to critical macromolecules such as DNA. However, ROS can also contribute to the pathogenesis of cancer and many other chronic inflammatory disease conditions, including atherosclerosis, metabolic disease, chronic obstructive pulmonary disease, neurodegenerative disease, and autoimmune disease. Recent Advances: The field of ROS biology is expanding, with an emerging paradigm that these reactive species are not generated haphazardly, but instead produced in localized regions or in specific subcellular compartments, and this has important consequences for immune system function. Currently, there is evidence for ROS generation in extracellular spaces, in endosomal compartments, and within mitochondria. Intriguingly, the specific location of ROS production appears to be influenced by the type of invading pathogen (i.e., bacteria, virus, or fungus), the size of the invading pathogen, as well as the expression/subcellular action of pattern recognition receptors and their downstream signaling networks, which sense the presence of these invading pathogens. Critical Issues: ROS are deliberately generated by the immune system, using specific NADPH oxidases that are critically important for pathogen clearance. Professional phagocytic cells can sense a foreign bacterium, initiate phagocytosis, and then within the confines of the phagosome, deliver bursts of ROS to these pathogens. The importance of confining ROS to this specific location is the impetus for this perspective. Future Directions: There are specific knowledge gaps on the fate of the ROS generated by NADPH oxidases/mitochondria, how these ROS are confined to specific locations, as well as the identity of ROS-sensitive targets and how they regulate cellular signaling.

Thioredoxin-Interacting Protein Promotes Phagosomal Acidification Upon Exposure to Escherichia coli Through Inflammasome-Mediated Caspase-1 Activation in Macrophages.

In host defense, it is crucial to maintain the acidity of the macrophage phagosome for effective bacterial clearance. However, the mechanisms governing phagosomal acidification upon exposure to gram-negative bacteria have not been fully elucidated. In this study, we demonstrate that in macrophages exposed to Escherichia coli, the thioredoxin-interacting protein (TXNIP)-associated inflammasome plays a role in pH modulation through the activated caspase-1-mediated inhibition of NADPH oxidase. While there was no difference in early-phase bacterial engulfment between Txnip knockout (KO) macrophages and wild-type (WT) macrophages, Txnip KO macrophages were less efficient at destroying intracellular bacteria in the late phase, and their phagosomes failed to undergo appropriate acidification. These phenomena were associated with reactive oxygen species production and were reversed by treatment with an NADPH oxidase inhibitor or a caspase inhibitor. In line with these results, Txnip KO mice were more susceptible to both intraperitoneally administered E. coli and sepsis induced by cecum ligation and puncture than WT mice. Taken together, this study suggests that the TXNIP-associated inflammasome-caspase-1 axis regulates NADPH oxidase to modulate the pH of the phagosome, controlling bacterial clearance by macrophages.

Does Pioglitazone Lead to Neutrophil Extracellular Traps Formation in Chronic Granulomatous Disease Patients?

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme complex responsible for reactive oxygen species (ROS) production, is defective in chronic granulomatous disease (CGD) patients. This enzyme helps in antimicrobial host defense by phagocytes. CGD patients are unable to form neutrophil extracellular traps (NETs), which are composed of granule-derived proteins from neutrophils decorated with decondensed chromatin. Mitochondria have gained attention, being a rich source of flavochrome enzymes due to the presence of several sites for superoxide production. Recently, PPARγ agonists, a mitochondrial ROS inducer, induce mitochondrial ROS formation post-treatment in murine NADPH oxidase knockout models. Mitochondrial ROS is also essential for NOX-independent NETosis. Our study for the first time detects induction of NETosis independent of NADPH oxidase post-treatment with agonists such as pioglitazone and rosiglitazone in CGD subjects. Neutrophils isolated from CGD subjects were treated with pioglitazone and rosiglitazone. After treatment, qualitative analysis of NET formation was done using confocal microscopy after staining with DAPI. Quantitative estimation of extracellular DNA was performed using Sytox green. Mitochondrial ROS production with PPARγ agonist-treated/untreated neutrophils was detected using MitoSOX red. Pioglitazone and rosiglitazone induce significant NET formation in CGD patients. Our data clearly signify the effect of PPARγ agonists in induction of NET formation in CGD cases. Apart from the proposed experimental studies regarding the detailed mechanism of action, controlled trials could provide valuable information regarding the clinical use of pioglitazone in CGD patients as curative HSCT remains challenging in developing countries.

Inflammatory consequences of inherited disorders affecting neutrophil function.

Primary immunodeficiencies affecting the function of neutrophils and other phagocytic leukocytes are notable for an increased susceptibility to bacterial and fungal infections as a result of impaired leukocyte recruitment, ingestion, and/or killing of microbes. The underlying molecular defects can also impact other innate immune responses to infectious and inflammatory stimuli, leading to inflammatory and autoimmune complications that are not always directly related to infection. This review will provide an update on congenital disorders affecting neutrophil function in which a combination of host defense and inflammatory complications are prominent, including nicotinamide dinucleotide phosphate oxidase defects in chronic granulomatous disease and ß2 integrin defects in leukocyte adhesion deficiency.

The roles of NADPH oxidase in modulating neutrophil effector responses.

Neutrophils are phagocytic innate immune cells essential for killing bacteria via activation of a wide variety of effector responses and generation of large amounts of reactive oxygen species (ROS). Majority of the ROS in neutrophils is generated by activation of the superoxide-generating enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Independent of their anti-microbial function, NADPH oxidase-derived ROS have emerged as key regulators of host immune responses and neutrophilic inflammation. Data from patients with inherited defects in the NADPH oxidase subunit alleles that ablate its enzyme function as well as mouse models demonstrate profound dysregulation of host inflammatory responses, neutrophil hyper-activation and tissue damage in response to microbial ligands or tissue trauma. A large body of literature now demonstrates how oxidants function as essential signaling molecules that are essential for the regulation of neutrophil responses during priming, degranulation, neutrophil extracellular trap formation, and apoptosis, independent of their role in microbial killing. In this review we summarize how NADPH oxidase-derived oxidants modulate neutrophil function in a cell intrinsic manner and regulate host inflammatory responses. In addition, we summarize studies that have elucidated possible roles of oxidants in neutrophilic responses within the oral mucosa and periodontal disease.

NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages.

LC3-associated phagocytosis (LAP) is an emerging non-canonical autophagy process that bridges signaling from pattern-recognition receptors (PRRs) to autophagic machinery. LAP formation results in incorporation of lipidated LC3 into phagosomal membrane (termed LAPosome). Increasing evidence reveals that LAP functions as an innate defense mechanism against fungal pathogens. However, the molecular mechanism involved and the consequence of LAP in regulating anti-fungal immune response remain largely unexplored. Here we show that Histoplasma capsulatum is taken into LAPosome upon phagocytosis by macrophages. Interaction of H. capsulatum with Dectin-1 activates Syk and triggers subsequent NADPH oxidase-mediated reactive oxygen species (ROS) response that is involved in LAP induction. Inhibiting LAP induction by silencing LC3α/ß or treatment with ROS inhibitor impairs the activation of MAPKs-AP-1 pathway, thereby reduces macrophage proinflammatory cytokine response to H. capsulatum. Additionally, we unravel the importance of NLRX1 in fungus-induced LAP. NLRX1 facilitates LAP by interacting with TUFM which associates with autophagic proteins ATG5-ATG12 for LAPosome formation. Macrophages from Nlrx1-/- mice or TUFM-silenced cells exhibit reduced LAP induction and LAP-mediated MAPKs-AP-1 activation for cytokine response to H. capsulatum. Furthermore, inhibiting ROS production in Nlrx1-/- macrophages almost completely abolishes H. capsulatum-induced LC3 conversion, indicating that both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. Our findings reveal new pathways underlying LAP induction by H. capsulatum for macrophage cytokine response.

Park 7: A Novel Therapeutic Target for Macrophages in Sepsis-Induced Immunosuppression.

Sepsis remains a serious and life-threatening condition with high morbidity and mortality due to uncontrolled inflammation together with immunosuppression with few therapeutic options. Macrophages are recognized to play essential roles throughout all phases of sepsis and affect both immune homeostasis and inflammatory processes, and macrophage dysfunction is considered to be one of the major causes for sepsis-induced immunosuppression. Currently, Parkinson disease protein 7 (Park 7) is known to play an important role in regulating the production of reactive oxygen species (ROS) through interaction with p47phox, a subunit of NADPH oxidase. ROS are key mediators in initiating toll-like receptor (TLR) signaling pathways to activate macrophages. Emerging evidence has strongly implicated Park 7 as an antagonist for sepsis-induced immunosuppression, which suggests that Park 7 may be a novel therapeutic target for reversing immunosuppression compromised by sepsis. Here, we review the main characteristics of sepsis-induced immunosuppression caused by macrophages and provide a detailed mechanism for how Park 7 antagonizes sepsis-induced immunosuppression initiated by the macrophage inflammatory response. Finally, we further discuss the most promising approach to develop innovative drugs that target Park 7 in patients whose initial presentation is at the late stage of sepsis.

Lysophosphatidylcholine induces cyclooxygenase-2-dependent IL-6 expression in human cardiac fibroblasts.

Lysophosphatidylcholine (LysoPC) has been shown to induce the expression of inflammatory proteins, including cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6), associated with cardiac fibrosis. Here, we demonstrated that LysoPC-induced COX-2 and IL-6 expression was inhibited by silencing NADPH oxidase 1, 2, 4, 5; p65; and FoxO1 in human cardiac fibroblasts (HCFs). LysoPC-induced IL-6 expression was attenuated by a COX-2 inhibitor. LysoPC-induced responses were mediated via the NADPH oxidase-derived reactive oxygen species-dependent JNK1/2 phosphorylation pathway, leading to NF-κB and FoxO1 activation. In addition, we demonstrated that both FoxO1 and p65 regulated COX-2 promoter activity stimulated by LysoPC. Overexpression of wild-type FoxO1 and S256D FoxO1 enhanced COX-2 promoter activity and protein expression in HCFs. These results were confirmed by ex vivo studies, where LysoPC-induced COX-2 and IL-6 expression was attenuated by the inhibitors of NADPH oxidase, NF-κB, and FoxO1. Our findings demonstrate that LysoPC-induced COX-2 expression is mediated via NADPH oxidase-derived reactive oxygen species generation linked to the JNK1/2-dependent pathway leading to FoxO1 and NF-κB activation in HCFs. LysoPC-induced COX-2-dependent IL-6 expression provided novel insights into the therapeutic targets of the cardiac fibrotic responses.

Resveratrol prevents nanoparticles-induced inflammation and oxidative stress via downregulation of PKC-α and NADPH oxidase in lung epithelial A549 cells.

BACKGROUND: Exposure to carbon black nanoparticles (CBNPs), a well-known industrial production, promotes pulmonary toxicity through inflammation and oxidative stress. Recent studies show that some polyphenols exert their antioxidant properties through regulation of protein kinase C-α (PKC-α) and NADPH oxidase (Nox) signaling. Resveratrol, a dietary polyphenol in fruits, possesses various health beneficial effects including anti-inflammatory and antioxidative properties. In this study, we aimed to elucidate the involvement of PKC-α and Nox in CBNPs-induced inflammation and oxidative stress, and to investigate the protective effects of resveratrol on CBNP-induced inflammation and oxidative stress in human lung epithelial A549 cells. METHODS: The production of reactive oxygen species (ROS) and the change of mitochondrial membrane potential (ΔΨm) were measured by flow cytometry. Nitric oxide (NO) was measured using the Griess reagent, and prostaglandin E2 (PGE2) production was detected by ELISA, while protein expressions were measured by Western blotting analysis. RESULTS: In lung epithelial A549 cells, CBNPs significantly enhanced oxidative stress by upregulation of Nox2 and membrane expression of p67phox accompanied with increase of ROS production. CBNPs also increased inflammatory factors, including iNOS, COX-2, NO and PGE2. However, resveratrol attenuated the above effects induced by CBNPs in A549 cells; additionally, CBNPs-induced activation of PKC-α was observed. We found that PKC-α inhibitor (Gö6976) could attenuate CBNPs-induced inflammation by down-regulation of ROS, NO and PGE2 production in A549 cells, suggesting PKC-α might be involved in CBNPs-induced oxidative stress and inflammation. Our results also found resveratrol was able to inhibit protein expression of PKC-α induced by CBNPs. Moreover, ROS scavenger (NAC) and Nox inhibitor (DPI) attenuated CBNPs-induced expressions of iNOS and COX-2. DPI could also attenuate CBNPs-induced ROS, NO and PGE2 production. CONCLUSIONS: Resveratrol attenuated CBNPs-induced oxidative and inflammatory factors in lung epithelial A549 cells, at least in part via inhibiting PKC-α- and Nox-related signaling.

Monoclonal antibody 7D5 recognizes the R147 epitope on the gp91phox , phagocyte flavocytochrome b558 large subunit.

Human phagocyte flavocytochrome b558 (Cyt b), the catalytic center of nicotinamide adenine dinucleotide phosphate oxidase, consists of a heavily glycosylated large subunit (gp91phox ; Nox2) and a small subunit (p22phox ). Cyt b is a membrane-spanning complex enzyme. Chronic granulomatous disease (CGD) is predominantly caused by a mutation in the CYBB gene encoding gp91phox on the X-chromosome. Because the phagocytes of patients with CGD are not able to generate the superoxide anion, these patients are susceptible to severe infections that can be fatal. It has been suggested that the extracellular region of gp91phox is necessary for and critical to forming the epitope of mAb 7D5 and that 7D5 provides a useful tool for rapid screening of X-linked CGD by FACS. To further elucidate the mAb 7D5 epitope on human gp91phox , chimeric DNA expressed human and mouse gp91phox recombinant protein were constructed. The fusion proteins were immunostained for mAb 7D5 and analyzed by FACS and western blot analysis. The 143 ELGDRQNES151 region was found to reside at the extracellular surface on human gp91phox and to be an important epitope for the interaction with mAb 7D5, as analyzed by FACS analysis. In particular, amino acid R147 is a unique epitope on the membrane-associated Cyt b for mAb 7D5. In conclusion, it is proposed that FACS analysis using mAb 7D5 is a valuable tool for early diagnosis of CGD.

The Macrophage Mannose Receptor Regulate Mannan-Induced Psoriasis, Psoriatic Arthritis, and Rheumatoid Arthritis-Like Disease Models.

The injection of mannan into mice can result in the development of psoriasis (Ps) and psoriatic arthritis (PsA), whereas co-injection with antibodies toward collagen type II leads to a chronic rheumatoid-like arthritis. The critical event in all these diseases is mannan-mediated activation of macrophages, causing more severe disease if the macrophages are deficient in neutrophil cytosolic factor 1 (Ncf1), i.e., lack the capacity to make a reactive oxygen species (ROS) burst. In this study, we investigated the role of one of the receptors binding mannan; the macrophage mannose receptor (MR, CD206). MR is a C-type lectin present on myeloid cells and lymphatics. We found that mice deficient in MR expression had more severe mannan-induced Ps, PsA as well as rheumatoid-like arthritis. Interestingly, the MR-mediated protection was partly lost in Ncf1 mutated mice and was associated with an type 2 macrophage expansion. In conclusion, these results show that MR protects against a pathogenic inflammatory macrophage response induced by mannan and is associated with induction of ROS.

ROS and glutathionylation balance cytoskeletal dynamics in neutrophil extracellular trap formation.

The antimicrobial defense activity of neutrophils partly depends on their ability to form neutrophil extracellular traps (NETs), but the underlying mechanism controlling NET formation remains unclear. We demonstrate that inhibiting cytoskeletal dynamics with pharmacological agents or by genetic manipulation prevents the degranulation of neutrophils and mitochondrial DNA release required for NET formation. Wiskott-Aldrich syndrome protein-deficient neutrophils are unable to polymerize actin and exhibit a block in both degranulation and DNA release. Similarly, neutrophils with a genetic defect in NADPH oxidase fail to induce either actin and tubulin polymerization or NET formation on activation. Moreover, neutrophils deficient in glutaredoxin 1 (Grx1), an enzyme required for deglutathionylation of actin and tubulin, are unable to polymerize either cytoskeletal network and fail to degranulate or release DNA. Collectively, cytoskeletal dynamics are achieved as a balance between reactive oxygen species-regulated effects on polymerization and glutathionylation on the one hand and the Grx1-mediated deglutathionylation that is required for NET formation on the other.

NOX2-dependent immunosuppression in chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a myeloproliferative and myelodysplastic neoplasm with few treatment options and dismal prognosis. The role of natural killer (NK) cells and other antileukemic lymphocytes in CMML is largely unknown. We aimed to provide insight into the mechanisms of immune evasion in CMML with a focus on immunosuppressive reactive oxygen species (ROS) formed by the myeloid cell NADPH oxidase-2 (NOX2). The dominant population of primary human CMML cells was found to express membrane-bound NOX2 and to release ROS, which, in turn, triggered extensive PARP-1-dependent cell death in cocultured NK cells, CD8+ T effector memory cells, and CD8+ T effector cells. Inhibitors of ROS formation and scavengers of extracellular ROS prevented CMML cell-induced lymphocyte death and facilitated NK cell degranulation toward Ab-coated, primary CMML cells. In patients with CMML, elevation of immature cell counts (CD34+) in blood was associated with reduced expression of several NK cell-activating receptors. We propose that CMML cells may use extracellular ROS as a targetable mechanism of immune escape.

Platelet CD36 promotes thrombosis by activating redox sensor ERK5 in hyperlipidemic conditions.

Atherothrombosis is a process mediated by dysregulated platelet activation that can cause life-threatening complications and is the leading cause of death by cardiovascular disease. Platelet reactivity in hyperlipidemic conditions is enhanced when platelet scavenger receptor CD36 recognizes oxidized lipids in oxidized low-density lipoprotein (oxLDL) particles, a process that induces an overt prothrombotic phenotype. The mechanisms by which CD36 promotes platelet activation and thrombosis remain incompletely defined. In this study, we identify a mechanism for CD36 to promote thrombosis by increasing activation of MAPK extracellular signal-regulated kinase 5 (ERK5), a protein kinase known to be exquisitely sensitive to redox stress, through a signaling pathway requiring Src kinases, NADPH oxidase, superoxide radical anion, and hydrogen peroxide. Pharmacologic inhibitors of ERK5 blunted platelet activation and aggregation in response to oxLDL and targeted genetic deletion of ERK5 in murine platelets prevented oxLDL-induced platelet deposition on immobilized collagen in response to arterial shear. Importantly, in vivo thrombosis experiments after bone marrow transplantation from platelet-specific ERK5 null mice into hyperlipidemic apolipoprotein E null mice showed decreased platelet accumulation and increased thrombosis times compared with mice transplanted with ERK5 expressing control bone marrows. These findings suggest that atherogenic conditions critically regulate platelet CD36 signaling by increasing superoxide radical anion and hydrogen peroxide through a mechanism that promotes activation of MAPK ERK5.

Interleukin-27 Enhances the Potential of Reactive Oxygen Species Generation from Monocyte-derived Macrophages and Dendritic cells by Induction of p47phox.

Interleukin (IL)-27, a member of the IL-12 cytokine family, plays an important and diverse role in the function of the immune system. We have previously demonstrated that IL-27 is an anti-viral cytokine which inhibits HIV-1, HIV-2, Influenza virus and herpes simplex virus infection, and enhances the potential of reactive oxygen species (ROS) generating activity during differentiation of monocytes to macrophages. In this study, we further investigated the mechanism of the enhanced potential for ROS generation by IL-27. Real time PCR, western blot and knock down assays demonstrate that IL-27 is able to enhance the potential of superoxide production not only during differentiation but also in terminally differentiated-macrophages and immature dendritic cells (iDC) in association with the induction of p47phox, a cytosolic component of the ROS producing enzyme, NADPH oxidase, and the increase in amounts of phosphorylated p47phox upon stimulation. We also demonstrate that IL-27 is able to induce extracellular superoxide dismutase during differentiation of monocytes but not in terminal differentiated macrophages. Since ROS plays an important role in a variety of inflammation, our data demonstrate that IL-27 is a potent regulator of ROS induction and may be a novel therapeutic target.

LPS-induced renal inflammation is prevented by (-)-epicatechin in rats.

This work investigated the capacity of (-)-epicatechin to prevent the renal damage induced by LPS administration in rats. Male Sprague Dawley rats were fed for 4 days a diet without or with supplementation with (-)-epicatechin (80mg/kg BW/d), and subsequently i.p. injected with lipopolysaccharide (LPS). Six hours after injection, LPS-treated rats exhibited increased plasma creatinine and urea levels as indicators of impaired renal function. The renal cortex of the LPS-treated rats showed: i) increased expression of inflammatory molecules (TNF-α, iNOS and IL-6); ii) activation of several steps of NF-κB pathway; iii) overexpression of TLR4, and iv) higher superoxide anion production and lipid peroxidation index in association with increased levels of gp91phox and p47phox (NOX2) and NOX4. Pretreatment with dietary (-)-epicatechin prevented the adverse effects of LPS challenge essentially by inhibiting TLR4 upregulation and NOX activation and the consequent downstream events, e.g. NF-kB activation.

Phosphoinositol 3-phosphate acts as a timer for reactive oxygen species production in the phagosome.

Production of reactive oxygen species (ROS) in the phagosome by the NADPH oxidase is critical for mammalian immune defense against microbial infections and phosphoinositides are important regulators in this process. Phosphoinositol 3-phosphate (PI(3)P) regulates ROS production at the phagosome via p40phox by an unknown mechanism. This study tested the hypothesis that PI(3)P controls ROS production by regulating the presence of p40phox and p67phox at the phagosomal membrane. Pharmacologic inhibition of PI(3)P synthesis at the phagosome decreased the ROS production both in differentiated PLB-985 cells and human neutrophils. It also releases p67phox, the key cytosolic subunit of the oxidase, and p40phox from the phagosome. The knockdown of the PI(3)P phosphatase MTM1 or Rubicon or both increases the level of PI(3)P at the phagosome. That increase enhances ROS production inside the phagosome and triggers an extended accumulation of p67phox at the phagosome. Furthermore, the overexpression of MTM1 at the phagosomal membrane induces the disappearance of PI(3)P from the phagosome and prevents sustained ROS production. In conclusion, PI(3)P, indeed, regulates ROS production by maintaining p40phox and p67phox at the phagosomal membrane.

STAT5A-mediated NOX5-L expression promotes the proliferation and metastasis of breast cancer cells.

NADPH oxidase (NOX) generates reactive oxygen species (ROS) and has been suggested to mediate cell proliferation in some cancers. Here, we show that an increase in the expression of NOX5 long form (NOX5-L) is critical for tumor progression in breast tumor tissues. Immunostaining of clinical samples indicated that NOX5 was overexpressed in 41.1% of breast ductal carcinoma samples. NOX5-L depletion consistently suppressed cell proliferation, invasion, and migration in vitro. Antibody-mediated neutralization of NOX5-L attenuated tumor progression in a mouse xenograft model. Promoter analysis revealed that NOX5-L expression is regulated by STAT5A in breast cancer cells. Based on our novel findings, we suggest that inhibition of NOX5-L may be a promising therapeutic strategy that exerts anti-cancer effects via the modulation of ROS-mediated cell signaling.