Inducible nitric oxide synthase (iNOS) is a cytoplasmic protein responsible for the generation of nitric oxide (NO ⅐ ) in macrophages. In this work, we hypothesized that the intracellular localization of iNOS is significant for effective delivery of NO ⅐ to phagosomes containing ingested microorganisms. Using immunofluorescence microscopy and Western blot analysis, iNOS was shown to localize in the vicinity of phagosomes containing latex beads in stimulated macrophages. iNOS also localized to phagosomes containing Escherichia coli. The colocalization of iNOS with ingested latex beads was an actin-dependent process, since treatment with the actin microfilament disrupter cytochalasin D prevented iNOS recruitment to latex bead phagosomes. In contrast to E. coli and inert particle phagosomes, mycobacterial phagosomes did not colocalize with iNOS. This study demonstrates that (i) iNOS can be recruited to phagosomes; (ii) this recruitment is dependent on a functional actin cytoskeleton; (iii) certain microorganisms have the ability to prevent or reduce colocalization with iNOS; and (iv) spatial exclusion of iNOS may play a role in Mycobacterium tuberculosis pathogenesis.

The CFTR gene encodes a transmembrane conductance regulator, which is dysfunctional in patients with cystic fibrosis (CF). The mechanism by which defective CFTR (CF transmembrane conductance regulator) leads to undersialylation of plasma membrane glycoconjugates, which in turn promote lung pathology and colonization with Pseudomonas aeruginosa causing lethal bacterial infections in CF, is not known. Here we show by ratiometric imaging with lumenally exposed pH-sensitive green fluorescent protein that dysfunctional CFTR leads to hyperacidification of the trans-Golgi network (TGN) in CF lung epithelial cells. The hyperacidification of TGN, glycosylation defect of plasma membrane glycoconjugates, and increased P. aeruginosa adherence were corrected by incubating CF respiratory epithelial cells with weak bases. Studies with pharmacological agents indicated a role for sodium conductance, modulated by CFTR regulatory function, in determining the pH of TGN. These studies demonstrate the molecular basis for defective glycosylation of lung epithelial cells and bacterial pathogenesis in CF, and suggest a cure by normalizing the pH of intracellular compartments.

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The cystic fibrosis transmembrane conductance regulator (CFTR), which is aberrant in patients with cystic fibrosis, normally functions both as a chloride channel and as a pleiotropic regulator of other ion transporters. Here we show, by ratiometric imaging with luminally exposed pH-sensitive green fluorescent protein, that CFTR affects the pH of cellubrevin-labeled endosomal organelles resulting in hyperacidification of these compartments in cystic fibrosis lung epithelial cells. The excessive acidification of intracellular organelles was corrected with low concentrations of weak base. Studies with proton ATPase and sodium channel inhibitors showed that the increased acidification was dependent on proton pump activity and sodium transport. These observations implicate sodium efflux in the pH homeostasis of a subset of endocytic organelles and indicate that a dysfunctional CFTR in cystic fibrosis leads to organellar hyperacidification in lung epithelial cells because of a loss of CFTR inhibitory effects on sodium transport. Furthermore, recycling of transferrin receptor was altered in CFTR mutant cells, suggesting a previously unrecognized cellular defect in cystic fibrosis, which may have functional consequences for the receptors on the plasma membrane or within endosomal compartments.

The CFTR gene encodes a chloride channel with pleiotropic effects on cell physiology and metabolism. Here, we show that increasing cGMP levels to inhibit epithelial Na(+) channel in cystic fibrosis (CF) respiratory epithelial cells corrects several aspects of the downstream pathology in CF. Cell culture models, using a range of CF cell lines and primary cells, showed that complementary pharmacological approaches to increasing intracellular cGMP, by elevating guanyl cyclase activity though reduced nitric oxide, addition of cell-permeable cGMP analogs, or inhibition of phosphodiesterase 5 corrected multiple aspects of the CF pathological cascade. These included correction of defective protein glycosylation, bacterial adherence, and proinflammatory responses. Furthermore, pharmacological inhibition of phosphodiesterase 5 in tissues ex vivo or in animal models improved transepithelial currents across nasal mucosae from transgenic F508del Cftr(tm1Eur) mice and reduced neutrophil infiltration on bacterial aerosol challenge in Pseudomonas aeruginosa-susceptible DBA/2 mice. Our findings define phosphodiesterase 5 as a specific target for correcting a number of previously disconnected defects in the CF respiratory tract, now linked through this study. Our study suggests that phosphodiesterase 5 inhibition provides an opportunity for simultaneous and concerted correction of seemingly disparate complications in CF.

Progressive pulmonary disease and infections with Pseudomonas aeruginosa remain an intractable problem in cystic fibrosis (CF). At the cellular level, CF is characterized by organellar hyperacidification, which results in altered protein and lipid glycosylation. Altered pH of the trans-Golgi network (TGN) may further disrupt the protein processing and packaging that occurs in this organelle. Here we measured activity of the major TGN endoprotease furin and demonstrated a marked upregulation in human CF cells. Increased furin activity was linked to elevated production in CF of the immunosuppressive and tissue remodeling cytokine TGF-β and its downstream effects, including macrophage deactivation and augmented collagen secretion by epithelial cells. As furin is responsible for the proteolytic processing of a range of endogenous and exogenous substrates including growth factors and bacterial toxins, we determined that elevated furin-dependent activation of exotoxin A caused increased cell death in CF respiratory epithelial cells compared with genetically matched CF transmembrane conductance regulator-corrected cells. Thus elevated furin levels in CF respiratory epithelial cells contributes to bacterial toxin-induced cell death, fibrosis, and local immunosuppression. These data suggest that the use of furin inhibitors may represent a strategy for pharmacotherapy in CF.

Endosomal hyperacidification in cystic fibrosis (CF) respiratory epithelial cells is secondary to a loss of sodium transport control owing to a defective form of the CF transmembrane conductance regulator CFTR. Here, we show that endosomal hyperacidification can be corrected by activating the signalling cascade controlling sodium channels through cyclic GMP. Nitric oxide (NO) donors corrected the endosomal hyperacidification in CF cells. Stimulation of CF cells with guanylate cyclase agonists corrected the pH in endosomes. Exposure of CF cells to an inhibitor of cGMP‐specific phosphodiesterase PDE5, Sildenafil, normalized the endosomal pH. Treatment with Sildenafil reduced secretion by CF cells of the proinflammatory chemokine interleukin 8 following stimulation with Pseudomonas aeruginosa products. Thus, the endosomal hyperacidification and excessive proinflammatory response in CF are in part due to deficiencies in NO‐ and cGMP‐regulated processes and can be pharmacologically reversed using PDE5 inhibitors.

Pseudomonas aeruginosa is a critical colonizer of the respiratory tract in cystic fibrosis. The chronic infections with this microorganism contribute to excessive inflammation and progressive lung damage in cystic fibrosis patients. The full repertoire of Pseudomonas products that promote inflammation in the cystic fibrosis lung is not known. Here we show that P. aeruginosa DNA released from the bacterium, but not human DNA from epithelial cells or Escherichia coli DNA, displays proinflammatory properties and induces human respiratory epithelial cells to secrete interleukin-8 (IL-8), a key chemokine causing excessive neutrophil infiltration in the cystic fibrosis lung. IL-8 secretion was not due to an increase in NF-B-or activator protein-1-dependent IL-8 promoter transcription, but instead depended on p38 and Erk mitogen-activated protein kinases. No secretion of IL-8 was observed using conventional Toll-like receptor 9 ligands (CpG oligonucleotides), although it could be demonstrated that parts of the Toll-like receptor 9-signaling pathway were functional, since class B and C CpG oligonucleotide ligands stimulated production of RANTES chemokine. The IL-8 secretion in response to P. aeruginosa DNA was decreased by treatments that inhibit acidification of intracellular organelles, using chloroquine, a pH-neutralizing compound, or bafilomycin A1, an inhibitor of vacuolar H ؉ -ATPase. These data indicate that DNA released from P. aeruginosa during chronic infections may significantly contribute to the proinflammatory processes in cystic fibrosis. Our findings also show that treatments with drugs diminishing organellar acidification may reduce the inflammatory response in cystic fibrosis.