Phagocyte NADPH Oxidase Research Articles

Myeloid-Derived Suppressor Cells Modulate Immune Responses Independently of NADPH Oxidase in the Ovarian Tumor Microenvironment in Mice

The phagocyte NADPH oxidase generates superoxide anion and downstream reactive oxidant intermediates in response to infectious threat, and is a critical mediator of antimicrobial host defense and inflammatory responses. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are recruited by cancer cells, accumulate locally and systemically in advanced cancer, and can abrogate anti-tumor immunity. Prior studies have implicated the phagocyte NADPH oxidase as being an important component promoting MDSC accumulation and immunosuppression in cancer. We therefore used engineered NADPH oxidase-deficient (p47phox−/−) mice to delineate the role of this enzyme complex in MDSC accumulation and function in a syngeneic mouse model of epithelial ovarian cancer. We found that the presence of NADPH oxidase did not affect tumor progression. The accumulation of MDSCs locally and systemically was similar in tumor-bearing wild-type (WT) and p47phox−/− mice. Although MDSCs from tumor-bearing WT mice had functional NADPH oxidase, the suppressive effect of MDSCs on ex vivo stimulated T cell proliferation was NADPH oxidase-independent. In contrast to other tumor-bearing mouse models, our results show that MDSC accumulation and immunosuppression in syngeneic epithelial ovarian cancer is NADPH oxidase-independent. We speculate that factors inherent to the tumor, tumor microenvironment, or both determine the specific requirement for NADPH oxidase in MDSC accumulation and function.

The NADPH oxidase Nox2 regulates VEGFR1/CSF‐1R‐mediated microglial chemotaxis and promotes early postnatal infiltration of phagocytes in the subventricular zone of the mouse cerebral cortex

The phagocyte NADPH oxidase Nox2 generates superoxide ions implicated in the elimination of microorganisms and the redox control of inflammatory signaling. However, the role of Nox2 in phagocyte functions unrelated to immunity or pathologies is unknown. During development, oriented cell migrations insure the timely recruitment and function of phagocytes in developing tissues. Here, we have addressed the role of Nox2 in the directional migration of microglial cells during development. We show that microglial Nox2 regulates the chemotaxis of purified microglia mediated by the colony stimulating factor-1 receptor (CSF-1R) and the vascular endothelial growth factor receptor-1 (VEGFR1). Stimulation of these receptors triggers activation of Nox2 at the leading edge of polarized cells. In the early postnatal stages of mouse brain development, Nox2 is activated in macrophages / microglial cells in the lateral ventricle or the adjacent subventricular zone (SVZ). Fluorescent microglia injected into the lateral ventricle infiltrate the dorso-caudal SVZ through a mechanism that is blocked by pretreatment of the injected cells with an irreversible Nox inhibitor. Infiltration of endogenous microglia into the caudal SVZ of the cerebral cortex is prevented by (1) Nox2 gene deficiency, (2) treatment with a Nox2 inhibitor (apocynin), and (3) invalidation of the VEGFR1 kinase. We conclude that phagocytes move out of the lateral ventricle soon after birth and infiltrate the cortical SVZ through a mechanism requiring microglial Nox2 and VEGFR1 activation. Nox2 therefore modulates the migration of microglia and their development.

Evolutionary Dynamics of the Human NADPH Oxidase Genes CYBB, CYBA, NCF2, and NCF4: Functional Implications

The phagocyte NADPH oxidase catalyzes the reduction of O2 to reactive oxygen species with microbicidal activity. It is composed of two membrane-spanning subunits, gp91-phox and p22-phox (encoded by CYBB and CYBA, respectively), and three cytoplasmic subunits, p40-phox, p47-phox, and p67-phox (encoded by NCF4, NCF1, and NCF2, respectively). Mutations in any of these genes can result in chronic granulomatous disease, a primary immunodeficiency characterized by recurrent infections. Using evolutionary mapping, we determined that episodes of adaptive natural selection have shaped the extracellular portion of gp91-phox during the evolution of mammals, which suggests that this region may have a function in host-pathogen interactions. On the basis of a resequencing analysis of approximately 35 kb of CYBB, CYBA, NCF2, and NCF4 in 102 ethnically diverse individuals (24 of African ancestry, 31 of European ancestry, 24 of Asian/Oceanians, and 23 US Hispanics), we show that the pattern of CYBA diversity is compatible with balancing natural selection, perhaps mediated by catalase-positive pathogens. NCF2 in Asian populations shows a pattern of diversity characterized by a differentiated haplotype structure. Our study provides insight into the role of pathogen-driven natural selection in an innate immune pathway and sheds light on the role of CYBA in endothelial, nonphagocytic NADPH oxidases, which are relevant in the pathogenesis of cardiovascular and other complex diseases.

Vascular angiotensin II type 2 receptor attenuates atherosclerosis via a kinin/NO-dependent mechanism

The angiotensin II (Ang II) type 1 receptor exerts pro-atherogenic action by augmenting oxidative stress, whereas the Ang II type 2 receptor (AT2)-mediated effect on atherosclerosis remains controversial. AT2 transgenic (AT2-Tg) mice, which overexpress AT2 in their vascular smooth muscle cells, were crossed with apoE-deficient (apoE(-/-)) mice to generate AT2 transgenic apoE(-/-) mice (AT2-Tg/apoE(-/-)). A subpressor dose of Ang II infusion exaggerated atherosclerosis development in apoE(-/-) mice, which was markedly suppressed in AT2-Tg/apoE(-/-) mice. Inhibitors of nitric oxide (NO) synthase (L-NAME) or bradykinin type 2 receptor completely abolished AT2-mediated anti-atherogenic actions. The vascular cell adhesion molecule-1 expression levels and degree of monocyte/macrophage accumulation in the intima were also considerably reduced in AT2-Tg/apoE(-/-) mice; these phenomena were completely reversed by L-NAME treatment. Ang II infusion significantly enhanced the accumulation of dihydroethidium-positive mononuclear cells in the intima and mRNA expression levels of Nox2, a phagocytic cell-type NADPH oxidase subunit in apoE(-/-) mice, which was completely inhibited in AT2-Tg/apoE(-/-) mice. Vascular AT2 stimulation exerts anti-atherogenic actions in an endothelial kinin/NO-dependent manner, and its anti-oxidative effect is likely to be exerted by inhibiting the accumulation of superoxide-producing mononuclear leukocytes.

Apocynin regulates cytokine production of CD8+ T cells

Apocynin is known to suppress the production of reactive oxygen species (ROS) by inhibiting NADPH oxidases, specifically phagocytic NADPH oxidase (PHOX or NOX2). Given the pro-inflammatory effects of ROS, apocynin has been studied extensively for its use as a therapeutic agent in various disease models. While the effects of apocynin on neutrophils and monocytes have been investigated, it remains to be elucidated whether apocynin modulates the effector function of T cells. In the present study, we examined the effect of apocynin on CD8(+) T cells and further investigated its mechanism of action. We found that apocynin directly inhibited the production of pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL-2 in anti-CD3/anti-CD28-stimulated CD8(+) T cells. The action of apocynin was upstream of the protein kinase C and calcium signaling in the T cell receptor signaling pathway because apocynin did not inhibit cytokine production in phorbol 12-myristate 13-acetate/ionomycin-stimulated CD8(+) T cells. Electrophoretic mobility shift assays revealed that apocynin attenuated anti-CD3/anti-CD28-induced NF-κB activation in CD8(+) T cells. In the experiments with NOX2-deficient mice, we demonstrated that apocynin inhibited TNF-α production of CD8(+) T cells in a NOX2-independent manner. Taken together, we demonstrated that apocynin, a well-known NOX2 inhibitor, suppressed the cytokine production of CD8(+) T cells. We also showed the NOX2-independent action of apocynin in the inhibition of TNF-α production in CD8(+) T cells.

Inhibition of reactive oxygen species production attenuates highly pathogenic avian influenza H5N1-induced inflammation via up-regulation of suppressor of cytokine signalling 3 (P6188)

Abstract Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased ROS production and acute lung injury. Pro-inflammatory stimuli induce intracellular ROS by activating NADPH oxidase (NOX). Nox2 is the catalytic subunit of the phagocyte NOX. It has been shown that Nox2-containing NOX is the major source of ROS induced by influenza A virus infection in mice. Apocynin, a NOX inhibitor, reduced virus replication and accumulation of inflammatory cells in the lungs of X31- or PR8- infected mice. As hypercytokinemia is thought to contribute to severe disease pathogenesis of high pathogenicity avian influenza (HPAI) H5N1 infection, it is hypothesized that apocynin could reduce HPAI H5N1-induced cytokine production. A panel of airway epithelial and immune cells from mammals and chickens were infected with low pathogenicity avian influenza H5N3 (A/duck/Victoria/0305-2/2012), HPAI H5N1 (A/chicken/Vietnam/0008/2004), or PR8 H1N1 virus in the absence or presence of apocynin. Quantitative real-time PCR showed that apocynin inhibited influenza-induced cytokines (IL-6, IFN-β, CXCL10 and CCL5) and ROS production, although viral replication was not significantly altered. Interestingly, apocynin treatment significantly increased SOCS3 expression, enhancing negative regulation of cytokine signaling. Therefore, apocynin or its derivatives could be used as a therapeutic agent for reducing hypercytokinemia, resulting in amelioration of disease.

Induction of genes encoding NADPH oxidase components and activation of IFN regulatory factor-1 by prolactin in fish macrophages

The role played by prolactin (PRL) in fish immunity is scant. We report here that stimulation of the Atlantic salmon monocytic cell line SHK-1 with native salmon PRL resulted in activation of the respiratory burst and induction of the expression of the genes encoding the phagocyte NADPH oxidase components p47phox, p67phox and gp91phox, and the transcription factor IFN regulatory factor-1 (IRF-1). Interestingly, the pharmacologic inhibition of the Jak/Stat signaling pathway with AG490 blocked reactive oxygen species (ROS) production, and the induction of genes encoding the NADPH oxidase components and IRF-1 in PRL-activated SHK-1 cells. In addition, PRL promoted the phosphorylation of Stat and induced the DNA binding activity of IRF-1. These results, together with the presence of several consensus target motifs for Stat and IRF-1 in the promoter of the tilapia p47phox gene, suggest that PRL regulates p47phox gene expression in fish through the activation of these two key transcription factors. Taken together, our results demonstrate that PRL induces the expression of the genes encoding the major phagocyte NADPH oxidase components and ROS production in fish macrophages via the JAK2/Stat/IRF-1 signaling pathway.

Molecular mechanism of voltage sensing in voltage-gated proton channels

Voltage-gated proton (Hv) channels play an essential role in phagocytic cells by generating a hyperpolarizing proton current that electrically compensates for the depolarizing current generated by the NADPH oxidase during the respiratory burst, thereby ensuring a sustained production of reactive oxygen species by the NADPH oxidase in phagocytes to neutralize engulfed bacteria. Despite the importance of the voltage-dependent Hv current, it is at present unclear which residues in Hv channels are responsible for the voltage activation. Here we show that individual neutralizations of three charged residues in the fourth transmembrane domain, S4, all reduce the voltage dependence of activation. In addition, we show that the middle S4 charged residue moves from a position accessible from the cytosolic solution to a position accessible from the extracellular solution, suggesting that this residue moves across most of the membrane electric field during voltage activation of Hv channels. Our results show for the first time that the charge movement of these three S4 charges accounts for almost all of the measured gating charge in Hv channels.

Diabetes, Renal and Cardiovascular Disease in p47 phox−/− Chronic Granulomatous Disease

Chronic granulomatous disease is a rare immunodeficiency due to defects in the phagocyte NADPH oxidase. The X-linked form (gp91 (phox) deficiency) accounts for about 70 % of cases; autosomal recessive p47 (phox) deficiency accounts for about 25 % of cases. We identified a 10 % incidence of diabetes in p47 (phox) deficient CGD, but none in X-linked CGD. Renal and cardiovascular diseases were also higher in p47 (phox) deficiency. p47 (phox) deficient CGD has non-infectious morbidities distinct from those in X-linked CGD.

Abstract A11: Cancer cell-secreted CXCL1 chemokine acts on neutrophils to support metastasis

Abstract Innate immune cells such as macrophages, monocytes and neutrophils can be recruited to tumors by chemokines, which act on chemokines receptors on these cells. Macrophages and monocytes have well-established roles in promotion of metastasis. However, neutrophils have been reported to have both pro- and anti-metastatic effects. In this study, we used the metastatic 4T1 and non-metastatic 4T07 murine breast carcinoma cells lines (originating from the same tumor) to study effects of neutrophils in breast metastasis. Primary tumors from 4T1 cells, orthotopically injected into syngeneic mice, had more infiltrating neutrophils than did primary tumors from 4T07 cells. The increased numbers of neutrophils, which expressed the chemokine receptor CXCR2, was associated with significantly higher levels of cancer cell-secreted CXCL1, a chemokine ligand for CXCR2. To test whether cancer cell-secreted CXCL1 acted on neutrophils to regulate metastasis, we used shRNAs to reduce CXCL1 expression in the 4T1 cells. Consistent with the lack of CXCR2 expression by the cancer cells, this did not influence cancer cell proliferation or migration in vitro. However, in vivo, tumor infiltration of neutrophils and lung metastasis were both reduced. Moreover, when parental 4T1 cells were orthotopically injected into syngeneic Cxcr2-/-mice, neutrophil infiltration and lung metastasis were significantly decreased as compared to tumors in wild type littermate. These results suggest that paracrine CXCL1 by cancer cells facilitates neutrophil infiltration via CXCR2 and that neutrophils in turn promote lung metastasis. To examine the mechanisms by which interactions between cancer cells and neutrophils might promote lung metastasis, we performed co-culture experiments with cancer cells and neutrophils. We found that neutrophils directly stimulated 4T1 cell invasion through matrigel. Interestingly, cancer-released CXCL1 also promoted the formation of neutrophil extraceullar traps (NETs), which is an expulsion of DNA and proteases from the neutrophils. Both invasion and NET formation were blocked by inhibition of phagocyte NADPH oxidase. Moreover, when mice with 4T1 tumors were treated with a phagocyte NADPH oxidase inhibitor, tumor growth and lung metastasis was reduced. Finally, decreased levels of the phosphorylated form of p47phox, a cytosolic component of phagocyte NAPDH oxidase that is activated by phosphorylation, was decreased in the tumor lysates from 4T1 injected Cxcr2-/- mice as compared to Cxcr2+/- mice. In conclusion, our study revealed that cancer cell-secreted CXCL1 regulates neutrophil infiltration and extracellular trap formation, and that phagocyte NADPH oxidase activity in these neutrophils promoted cancer cell invasion and metastasis. Citation Format: Juwon Park, Jing Qiu, Elizabeth S. Nakasone, Mikala Egeblad. Cancer cell-secreted CXCL1 chemokine acts on neutrophils to support metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A11.

Myeloperoxidase: a front-line defender against phagocytosed microorganisms

Successful immune defense requires integration of multiple effector systems to match the diverse virulence properties that members of the microbial world might express as they initiate and promote infection. Human neutrophils--the first cellular responders to invading microbes--exert most of their antimicrobial activity in phagosomes, specialized membrane-bound intracellular compartments formed by ingestion of microorganisms. The toxins generated de novo by the phagocyte NADPH oxidase and delivered by fusion of neutrophil granules with nascent phagosomes create conditions that kill and degrade ingested microbes. Antimicrobial activity reflects multiple and complex synergies among the phagosomal contents, and optimal action relies on oxidants generated in the presence of MPO. The absence of life-threatening infectious complications in individuals with MPO deficiency is frequently offered as evidence that the MPO oxidant system is ancillary rather than essential for neutrophil-mediated antimicrobial activity. However, that argument fails to consider observations from humans and KO mice that demonstrate that microbial killing by MPO-deficient cells is less efficient than that of normal neutrophils. We present evidence in support of MPO as a major arm of oxidative killing by neutrophils and propose that the essential contribution of MPO to normal innate host defense is manifest only when exposure to pathogens overwhelms the capacity of other host defense mechanisms.

Pulmonary manifestations of chronic granulomatous disease

Chronic granulomatous disease (CGD) is an inherited disorder, characterized by defects in superoxide-generating NADPH oxidase of phagocytes. The genetic defects in CGD induce failure to activate the respiratory burst in the phagocytes, leading to severe recurrent infections and unexplained prolonged inflammatory reactions that may produce granulomatous lesions. A noble advance in curative therapy for CGD is hematopoietic stem cell transplantation. Since the most common site of involvement in CGD is the lung, the pulmonologists (pediatrics or adult) may be among the first to recognize the pattern of infection, inflammation and granuloma formation, leading to diagnosis of CGD. Pulmonologists need to be aware of different lung manifestations of CGD.

Nitroarachidonic acid prevents NADPH oxidase assembly and superoxide radical production in activated macrophages

Nitration of arachidonic acid (AA) to nitroarachidonic acid (AANO2) leads to anti-inflammatory intracellular activities during macrophage activation. However, less is known about the capacity of AANO2 to regulate the production of reactive oxygen species under proinflammatory conditions. One of the immediate responses upon macrophage activation involves the production of superoxide radical (O2•−) due to the NADPH-dependent univalent reduction of oxygen to O2•− by the phagocytic NADPH oxidase isoform (NOX2), the activity of NOX2 being the main source of O2•− in monocytes/macrophages. Because the NOX2 and AA pathways are connected, we propose that AANO2 can modulate macrophage activation by inhibiting O2•− formation by NOX2. When macrophages were activated in the presence of AANO2, a significant inhibition of NOX2 activity was observed as evaluated by cytochrome c reduction, luminol chemiluminescence, Amplex red fluorescence, and flow cytometry; this process also occurs under physiological mimic conditions within the phagosomes. AANO2 decreased O2•− production in a dose- (IC50=4.1±1.8μM AANO2) and time-dependent manner. The observed inhibition was not due to a decreased phosphorylation of the cytosolic subunits (e.g., p40phox and p47phox), as analyzed by immunoprecipitation and Western blot. However, a reduction in the migration to the membrane of p47phox was obtained, suggesting that the protective actions involve the prevention of the correct assembly of the active enzyme in the membrane. Finally, the observed in vitro effects were confirmed in an in vivo inflammatory model, in which subcutaneous injection of AANO2 was able to decrease NOX2 activity in macrophages from thioglycolate-treated mice.

CD11b/CD18 (Mac-1) Is a Novel Surface Receptor for Extracellular Double-Stranded RNA To Mediate Cellular Inflammatory Responses

During viral infection, extracellular dsRNA is a potent signaling molecule that activates many innate immune cells, including macrophages. TLR3 is a well-known receptor for extracellular dsRNA, and internalization of extracellular dsRNA is required for endosomal TLR3 activation. Preserved inflammatory responses of TLR3-deficient macrophages to extracellular dsRNA strongly support a TLR3-independent mechanism in dsRNA-mediated immune responses. The present study demonstrated that CD11b/CD18 (Mac-1 [macrophage-1 Ag]), a surface integrin receptor, recognized extracellular dsRNA and induced macrophage immune responses. CD11b deficiency reduced inflammatory cytokine induction elicited by polyinosinic:polycytidylic acid (poly I:C; a synthetic dsRNA) in mouse sera and livers, as well as in cultured peritoneal macrophages. dsRNA-binding assay and confocal immunofluorescence showed that Mac-1, especially the CD11b subunit, interacted and colocalized with poly I:C on the surface of macrophages. Further mechanistic studies revealed two distinct signaling events following dsRNA recognition by Mac-1. First, Mac-1 facilitated poly I:C internalization through the activation of PI3K signaling and enhanced TLR3-dependent activation of IRF3 in macrophages. Second, poly I:C induced activation of phagocyte NADPH oxidase in a TLR3-independent, but Mac-1-dependent, manner. Subsequently, phagocyte NADPH oxidase-derived intracellular reactive oxygen species activated MAPK and NF-κB pathways. Our results indicate that extracellular dsRNA activates Mac-1 to enhance TLR3-dependent signaling and to trigger TLR3-independent, but Mac-1-dependent, inflammatory oxidative signaling, identifying a novel mechanistic basis for macrophages to recognize extracellular dsRNA to regulate innate immune responses. This study identifies Mac-1 as a novel surface receptor for extracellular dsRNA and implicates it as a potential therapeutic target for virus-related inflammatory diseases.

Chronic Alcohol Ingestion Changes the Landscape of the Alveolar Epithelium

Similar to effects of alcohol on the heart, liver, and brain, the effects of ethanol (EtOH) on lung injury are preventable. Unlike other vital organ systems, however, the lethal effects of alcohol on the lung are underappreciated, perhaps because there are no signs of overt pulmonary disorder until a secondary insult, such as a bacterial infection or injury, occurs in the lung. This paper provides overview of the complex changes in the alveolar environment known to occur following both chronic and acute alcohol exposures. Contemporary animal and cell culture models for alcohol-induced lung dysfunction are discussed, with emphasis on the effect of alcohol on transepithelial transport processes, namely, epithelial sodium channel activity (ENaC). The cascading effect of tissue and phagocytic Nadph oxidase (Nox) may be triggered by ethanol exposure, and as such, alcohol ingestion and exposure lead to a prooxidative environment; thus impacting alveolar macrophage (AM) function and oxidative stress. A better understanding of how alcohol changes the landscape of the alveolar epithelium can lead to improvements in treating acute respiratory distress syndrome (ARDS) for which hospitalized alcoholics are at an increased risk.

Identification of a region in p47phox/NCF1 crucial for phagocytic NADPH oxidase (NOX2) activation

A point mutation in the mouse Ncf1(m1J) gene decreases production of ROS by the phagocytic NOX2 complex. Three mRNA splice variants are expressed, but only one is expressed as a protein, although at lower levels than the WT NCF1 (also known as p47phox). Our aim was to investigate whether the mutant p47phox, lacking 8 aa, is active, but as a result of its low expression, ROS production is decreased in Ncf1(m1J) mice, or whether the mutant p47phox completely lacks the capability to activate the NOX2 complex. The p47phox mutant (Δ228-235), which was equal to the protein in Ncf1(m1J) mice, failed to activate the NOX2 complex. When the deleted region was narrowed down to 2 aa, the p47phox protein remained inactive and failed to translocate to the membrane upon activation. Single amino acid substitutions revealed Thr233 to be vital for ROS production. Residues Tyr231 and Val232 also seemed to be important for p47phox function, as p47phox_Y231G and p47phox_V232G resulted in a >50% decrease in ROS production by the NOX2 complex. In addition, we identified the epitope of the D-10 anti-p47phox mAb. In conclusion, the p47phox protein variant expressed in Ncf1(m1J) mice is completely defective in activating the NOX2 complex to produce ROS, and the effect is dependent on SH3 region amino acids at positions 231-233, which are vital for the proper assembly of the NOX2 complex.

Peroxiredoxin 6 Enhances Phagocyte NADPH Oxidase (phox) Activity in Response to Agonists Acting Via Cell Surface Receptors and Intracellular Signaling Pathways.

Abstract 2139 IntroductionThe phagocyte NADPH oxidase (phox) is a multiprotein, transmembrane-enzyme-complex found in diverse professional phagocytes including neutrophils. Phox in its inactive state consists of the membrane-associated subunit cytochrome b558 (a dimer of gp91phox/Nox2 and p22phox) and cytoplasmic subunits which include p47phox, p67phox, p40phox and Rac1 or Rac2. Phosphorylation of phox proteins promotes tight interaction of the cytoplasmic phox components with cytochrome b558 and the resulting active complex catalyzes transmembrane electron transfer from cytoplasmic NADPH to molecular oxygen forming superoxide anion (O2−). O2− and other reactive oxygen species formed from it are microbicibal and crucial to pathogen killing by neutrophils.Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with a peroxiredoxin activity that reduces oxidized lipids and H2O2 and a PLA2 activity that cleaves the sn2 position of phospholipids forming the corresponding free fatty acid and lysophospholipid.Previous work in our laboratory has identified Prdx6 as a binding partner of p67phox and as an enhancer of O2− production by active phox; we have shown that this enhancement is due to products of the PLA2 activity of the peroxiredoxin but the precise mechanism by which these molecules act is unclear. Here we present evidence that Prdx6 causes maximal activation of phox when the complex is activated by physiological agonists (the formylated peptide fMLP and the particulate agonist serum opsonized zymosan (SOZ)) but not when it is activated by the non-physiological agonist phorbol 12-myristate 13-acetate (PMA). Because fMLF and SOZ interact with cell surface receptors but PMA enters the cell and activates protein kinase C (PKC) enzymes that directly phosphorylate phox proteins our data provides a clue as to the mode of action of Prdx6 in phox activation. MethodsPLB985 cells were used to model neutrophils. This myeloid cell line can be terminally differentiated to a neutrophil like state with phox activity by exposure to DMSO. Prdx6 was suppressed in these cells by stable expression of an shRNA resulting in an approximately 70% reduction of the protein. Following maturation of the cells they were stimulated with fMLP, SOZ or PMA and O2− production was measured by superoxide dismutase (SOD) inhibitable luminescence of Diogenes fluorophore. For fMLP, due to the transient nature of the O2- production, the entire amount of O2− production was measured. SOZ and PMA induced prolonged (>1hr) O2− production and O2− production over 1hr was thus measured. ResultsIn cells with Prdx6 suppressed, SOD inhibitable O2− production in response to fMLP was reduced by 53% ± 3% (SEM), n=34, which was significant (p<0.001). O2− produced in response to SOZ was reduced by 37% ± 9% (SEM), n = 4, which was also significant (p<0.05).In contrast, the response to PMA was not altered (p>0.5, n of at least 4) at a range of PMA concentrations from one causing slight activation of phox (10 ng/ml) to one causing maximal O2- production (1 μg/ml). ConclusionsfMLP binds to receptors on neutrophils and triggers intracellular signaling cascades that ultimately lead to phosphorylation and activation of phox. Similarly, opsonins of particulate stimuli bind cell surface complement receptors that trigger intracellular signaling cascades that activate phox. PMA on the other hand enters cells and activates PKC enzymes that directly phosphorylate phox proteins; it thus bypasses the physiological signaling pathways that mediate the fMLP and SOZ response.Since Prdx6 enhances the SOZ and fMLF mediated phox response but not the PMA mediated response we propose that in myeloid cells, products of the PLA2 activity of Prdx6 enhance the SOZ and PMA mediated signaling cascades. This may be via activation of signaling molecules in these pathways which would be consistent with the known ability of the fatty acid arachidonate to activate PKC enzymes, MAP kinases, PLA2 enzymes and a class Ia phosphatidylinositol 3-kinase and to mobilize Ca2+. Disclosures:No relevant conflicts of interest to declare.

Bcr-Abl Represses IRF8 Gene Transcription in a Stat5-Dependent Manner

Abstract 3497The interferon consensus sequence binding protein is a member of the interferon regulatory factor family of transcription factors (referred to as Icsbp or Irf8). The first described functions for Icsbp involved regulation of phagocyte and B-cell effector genes, including genes encoding components of the phagocyte NADPH-oxidase, Toll-like receptors and interleukin receptors. However, subsequent studies in murine models and human disease indicated that Icsbp also functions as a myeloid leukemia suppressor. For example, decreased Icsbp expression is found in chronic myeloid leukemia (CML) in association with uncontrolled disease, drug resistance and progression to blast crisis (BC). Decreased Icsbp expression is also found in the bone marrow of subjects with some subtypes of acute myeloid leukemia (AML). Consistent with this clinical correlative data, IRF8−/− mice exhibit a myeloproliferative neoplasm that is similar to CML and progresses to BC over time. However, the mechanism for decreased Icsbp expression in leukemia is not known, although preliminary studies indicate that DNA-methylation of the IRF8 locus is not altered. Therefore, in these studies, we investigate the effects of Bcr-abl on IRF8 transcription. This is clinically relevant, because previous studies in our laboratory identified a set of Icsbp-target-genes that contribute to the pathogenesis of CML. We find that Bcr-abl decreases expression of Icsbp mRNA and protein in a kinase dependent manner. Since it is unlikely that Bcr-abl directly binds to the promoter to regulate gene transcription, we hypothesized that Bcr-abl regulates IRF8 through an intermediary transcription factor. In this study, we determine that Stat5 negatively regulates IRF8 transcription through a proximal promoter cis-element. We also find that Stat5 repression activity is necessary for Bcr-abl dependent regulation of IRF8. Bcr-abl is known to phosphorylate and activate Stat5 in CML. In our studies, we find that Stat5 protein (but not mRNA) is also increased in Bcr-abl+ cells. Stat5 is a known substrate for calpain; a serine protease. We previously demonstrated that Icsbp regulates calpain protease activity through repression of the gene encoding Gas2; an endogenous calpain inhibitor. Consistent with this, our current studies demonstrate that Stat5 protein stability is increased in Bcr-abl+ cells in an Icsbp/Gas2/calpain-dependent manner. These results identify novel mechanisms by which Bcr-abl-kinase activity controls a positive feedback loop that leads to decreased Icsbp expression and stabilization of Stat5 protein. These studies suggest that targeting Gas2/calpain might be a novel therapeutic approach to CML. Disclosures:No relevant conflicts of interest to declare.

Yuwen02f1 suppresses LPS-induced endotoxemia and adjuvant-induced arthritis primarily through blockade of ROS formation, NFkB and MAPK activation

Phagocytes release inflammatory mediators to defense harmful stimuli upon bacterial invasion, however, excessive inflammatory reaction leads to tissue damage and manifestation of pathological states. Therefore, targeting on uncontrolled inflammation seems feasible to control numerous inflammation-associated diseases. Under the drug screening process of synthetic diphenylpyrazole derivatives, we discovered compound yuwen02f1 possesses anti-inflammatory effects in decreasing the release of pro-inflammatory cytokines including TNFα and IL-6, nitric oxide, reactive oxygen species (ROS) as well as inhibiting migration of LPS-stimulated phagocytes. In addition, we observed that the molecular mechanism of yuwen02f1-mediated anti-inflammation is associated with decreasing phosphorylation of MAPK molecules including ERK1/2, JNK and p38, and attenuating translocation of p47phox and p67phox to the cell membrane. Yuwen02f1 also reverses IκBα degradation and attenuates the expression of NFκB-related downstream inducible enzymes like iNOS and COX-2. Furthermore, we found that yuwen02f1 attenuates some pathological syndromes of LPS-induced sepsis and adjuvant-induced arthritis in mice, as evidenced by decreasing the cytokine production, reversing thrombocytopenic syndrome, protecting the mice from tissue injury in septic mice, and attenuating paw edema in arthritic mice as well. These results suggest that yuwen02f1 is a potential anti-inflammatory agent for alleviating syndromes of acute and chronic inflammatory diseases as evidenced by attenuating the generation of cytokines and down-regulating the expression of iNOS and COX-2 through the blockade of ROS generation and NADPH oxidase, NFκB and MAPK activation pathways in LPS-stimulated phagocytes.

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response (1). Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury. Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47(phox), p67(phox), and p40(phox)) and Rac to a membrane-bound flavocytochrome (composed of a gp91(phox) and p22(phox) heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91(phox) -deficient mice and p47(phox)-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense (2, 13). In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase. Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion (14). Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species (15, 16). Kielland et al. (16) showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47(phox)-deficient mice. We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47(phox-/-) mice (2) in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47(phox-/-) mice enables us to delineate the specific contribution of ROS generated by p47(phox)-containing NADPH oxidase to the bioluminescent signal in these models. Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47(phox-/-) mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.