This program explores innate anti-microbial defense and inflammatory mechanisms involving deliberate reactive oxygen species (ROS) production by the host. Circulating phagocytes generate high levels of ROS that serve as important microbicidal agents in response to infectious or inflammatory stimuli, which is attributed to NADPH oxidase activation. Patients with chronic granulomatous disease (CGD) suffer from NADPH oxidase (Nox2- or phox-based) deficiencies, resulting in enhanced susceptibility to microbial infections and aberrant inflammatory responses. Our current focus investigates cellular mechanisms regulating related Nox family NADPH oxidases expressed in non-phagocytic cells (Nox1, Nox4, Duox1, Duox2), notably on mucosal surfaces (lung and gastrointestinal tract), the liver, kidney, thyroid and salivary glands, brain, and vascular tissues. ROS produced by these oxidases provide redox signals that affect gene expression patterns during responses to infection, oxygen sensing, growth factors, hormones, cytokines, cell differentiation, cellular senescence, programmed cell death (apoptosis). Several non-phagocytic Nox enzymes also serve in host defense and inflammatory processes, as they are expressed predominately on apical surfaces of epithelial cells and are induced or activated by cytokines or by recognition of pathogen-associated molecular patterns. Recently, we found that mature ciliated airway epithelial cells produce sufficient Duox-derived hydrogen peroxide to support lactoperoxidase-mediated killing of several airway pathogens, and that freshly grown Pseudomonas aeruginosa elicits airway epithelial Duox activation in response to multiple microbial factors (lipopolysaccharide, flagellin and the type III secretion system). In contrast, overgrown Pseudomonas secretes a microbial toxin (pyocyanin) that competitively inhibits Duox activity as it produces intracellular superoxide and imposes oxidative stress on host cells. The latter process in this 'redox warfare'between host and microbe represents a counter-offensive adaptation of Pseudomonas during the establishment of biofilms. In 2011, we have studied in detail the importance of the redox-active Pseudomonas aeruginosa virulence factor, pyocyanin, in the pathogenesis of chronic Pseudomonas airway infections. This factor is produced in response to quorum signals when Pseudomonas is overgrown in biofilms of chronically infected lungs of immunocompromised individuals (i.e., cystic fibrosis patients). We showed the effects of (purified) pyocyanin-mediated oxidative stress imposed on isolated airway epithelial cells recapitulate many of the phenotypic features of advanced cystic fibrosis disease, including mucin hypersecretion (mucin5a and mucin2) and release of pro-inflammatory cytokines and inflammatory cell-stimulating and chemotactic agents. These responses were detected initially by microarray-based gene expression profiling, which identified some 286 genes upregulated by pyocyanin. The importance of many of the induced genes was confirmed by ELISA assays of secreted cytokines and putative EGF receptor ligands. Biochemical, pharmacological, and antibody-neutralizing approaches were then used to establish a mechanistic model for pyocyanin-induced mucin hypersecretion in which several secreted epidermal growth factor receptor (EGFR) ligands (IL-beta, IL-6, TGF-alpha, TNF-alpha, HB-EGF) were shown to act through autocrine or paracrine signaling pathways to promote mucin secretion. These findings suggest potential utility of therapies aimed at the effects of this toxin for treating advance cystic fibrosis disease, and they provide insights into recent findings showing that chronic pyocyanin administration into mouse airways produces a cystic fibrosis-like phenotype. In other collaborative studies, we examined innate immune responses of airway epithelial cells to polyinosinic-polycytidylic acid (poly I:C), as a mimic of viral double-stranded RNA signaling through TLR3 pathways. Poly I:C was shown to induce shedding of a soluble TNF receptor ectodomain (sTNFR1), which could down-regulating cellular responses to TNF-alpha. We demonstrated that receptor shedding requires two pathways: one involving Duox2-mediated hydrogen peroxide release and the other involving caspase-mediated apoptosis. Poly I:C triggered hydrogen peroxide production and sTNFR1 shedding from airway cells, which was suppressed by siRNA-mediated Duox2 knockdown, oxidase inhibitors, or antioxidants. These findings reveal novel mechanisms by which innate immune and inflammatory responses of airway epithelium to viral infection can be modulated. Our advances in Duox reconstitution technology have identified several Duox single nucleotide polymorphisms (SNPs) or mutations that alter oxidase function or cellular targeting, which may relate to congenital hypothyroidism or altered susceptibilities to airway infectious or inflammatory disease (cystic fibrosis, asthma, bacterial or viral infection). Other Duox polymorphisms identified between mouse strains that exhibit altered susceptibilities to inflammatory bowel disease are being investigated for alterations in oxidase function.
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