This program explores innate anti-microbial defense and inflammatory mechanisms involving the host's ability to deliberately produce reactive oxygen species (ROS). Neutrophils and other circulating phagocytes generate high levels of ROS in response to infectious or inflammatory stimuli in a process known as the respiratory burst. The response is attributed to the activity of NADPH oxidase, which produces superoxide, a precursor of ROS that are important microbicidal agents and mediators of inflammation. Patients with chronic granulomatous disease (CGD) suffer from NADPH oxidase deficiencies, resulting in enhanced susceptibility to microbial infections and aberrant inflammatory responses. This project explores the cellular mechanisms regulating the respiratory burst oxidase in phagocytes (phox or Nox2-based system) and is characterizing related oxidant-generating NADPH oxidases expressed in non-immune cells (Nox1, Nox3, Nox4, Nox5, Duox1, Duox2), now known as """"""""Nox family"""""""" NADPH oxidases. We are studying sources of ROS in several non-myeloid tissues, notably colon, kidney, liver thyroid and salivary glands, mucosal surfaces (lung and gastrointestinal tract), brain, and vascular tissues. Our recent evidence indicates several of these non-phagocytic Nox enzymes also serve in host defense and inflammatory processes, since they are expressed predominately on apical surfaces of epithelial cells and are induced or activated by pro-inflammatory cytokines or recognition of microbial factors. ROS produced by these enzymes also provide redox signals that can affect gene expression patterns during differentiation, cellular senescence, programmed cell death (apoptosis), oxygen sensing, or responses to infection, growth factors, cytokines, or hormones. ? ? In the last year, we have explored the functional importance of dual oxidases (Duox1 and Duox2) in human airway epithelial cells and have developed the means for their reconstitution in transfected cell models. Duox targeted to the apical surfaces of bronchial epithelial cells provides extracellular hydrogen peroxide needed to support the well-documented anti-microbial activities of lactoperoxidase. We examined airway epithelial cell Duox expression and activity in response to differentiation, pathogen exposure, and pro-inflammatory cytokines. We demonstrated Duox- and lactoperoxidase-dependent microbial killing of Pseudomonas aeruginosa, Burkholderia Cepacia, and Staphylococcus aureus, organisms that commonly infect airways of immunocompromised patients, including those with Cystic Fibrosis. We explored host-pathogen interactions involved in oxidative antimicrobial resistance as well as the adaptive microbial counter-defenses to these oxidants. We identified specific effects of the Pseudomonas aeruginosa virulence factor, pyocynin, showing that it can act as a cell permeable, redox-active inhibitor of Duox activity and expression, as it consumes intracellular NADPH and imposes oxidative stress on airway cells. The findings provide novel insight on the adaptive capabilities of this opportunistic pathogen and shed light on pathology of chronic infections encountered in Cystic Fibrosis. We confirmed that airway Duox isozymes are induced by interferon-gamma, IL-4, and IL-13, suggesting roles in airway viral and microbial infection and in inflammatory airway disease (i.e., asthma, chronic obstructive pulmonary disease). We are also exploring determinants enabling delivery of active Duox to the plasma membrane, where it supports extracellular antimicrobial peroxidases. Active recombinant forms of Duox co-expressed along with essential maturation factors have been produced in whole transfected cells. We identified multiple splice variants of the Duox1 maturation factor (DuoxA1) capable of targeting Duox1 to distinct subcellular sites. These recombinant systems will be used to screen for Duox and maturation factor genetic polymorphisms associated with altered oxidase targeting and function.? ? In efforts aimed at exploring functional roles of the renal oxidase (Nox4 or Renox), we are characterizing mouse strains in which the Nox4 gene is deleted. We are investigating the proposed role of Nox4 in renal oxygen sensing and erythropoiesis, since ROS are thought to provide feedback signals regulating renal erythropoietin synthesis. The renal oxidase is a constitutively active enzyme, consistent with its proposed role as an oxygen-sensing enzyme. Surprisingly, Nox4-deficient mice exhibit a normal phenotype in the unstressed state. Hematology as well as serum and urine chemistries (i.e., urine hydrogen peroxide levels) are normal in these animals. Related gene microarray studies are focused on identifying alterations in other oxidant generating or scavenging systems to explore mechanisms maintaining normal redox homeostasis in Nox4-deficient mice. Nox4 levels respond directly to Transforming Growth Factor-beta (TGF-beta) or hypoxia in renal cells and to hepatitis C virus (HCV) in hepatic cells. Furthermore, the Nox4 promoter, fused to Nox4 cDNA or to other reporters, responds to hypoxia, TGF-beta and HCV. Based on these findings, future work will examine the responses of Nox4-deficient mice to these factors to assess potential roles of Nox4 in fibrotic disease (cirrhosis) and redox homeostasis related to hypoxia (angiogenesis, anemia). In studies aimed at defining the sources of ROS responsive to angiotensin II-mediated cell stimulation, we used reconstituted Nox1 and Nox2 transfected cell models to show involvement of a cell signaling pathway comprising AT(1)R, Galpha(q/11), phospholipase C-beta, and protein kinase C. ? ? Finally, our long-standing interests in the phagocytic oxidase are focused on the activating roles of cytosolic regulators, p40phox and cytosolic phospholipase A2 (cPLA2). We have shown that cPLA2 is recruited to the membrane during cellular activation through direct interactions with the p47phox oxidase component, involving the cPLA2 C2 domain and the p47phox PX domain. Peptides designed from these interacting domains were shown to be effective inhibitors of the oxidase in intact neutrophils. We also explored the role of p40phox as a positive oxidase regulator in cells undergoing phagocytosis, as it functions as an adaptor promoting retention of other regulators (p47phox and p67phox) on phagosomes. We also showed that p40phox recruitment to phagosomal membranes involves arachidonate-dependent exposure of its membrane-binding PX domain. Finally, we also obtained evidence for direct interactions between SH3 domains of p47phox and the catalytic gp91phox (Nox2) component. These findings broaden our understanding of the mechanisms of phagocytic oxidase activation and may suggest novel therapeutic strategies for modifying oxidase activity.
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