绿脓菌素激活MAPKs、NF-KB信号通路诱导呼吸道上皮细胞IL-8表达

采用绿脓杆菌培养上清及绿脓菌素刺激人呼吸道上皮细胞株A549和SPC-A-1,用ELISA方法检测细胞IL-8分泌水平,并使用免疫印迹(Western blot)方法观察绿脓菌素对细胞内重要的炎症信号传导途径NF—κB及丝裂原激活蛋白激酶(MAPKs)的激活作用。实验发现,绿脓杆菌培养上清及绿脓菌素可诱导呼吸道上皮细胞株IL-8分泌增加,且具有剂量依赖效应。绿脓菌素刺激细胞可使细胞内IκB—α发生降解,同时使MAPK家族蛋白分子(ERK1／2、p38、JNK)发生磷酸化。MEK1／2(ERK1／2激酶)抑制剂U0126(10μmol／L)和p38MAPK抑制剂SB203580(10μmol／L)可降低绿脓菌素诱导A549细胞IL-8的合成。以上结果显示绿脓菌素通过MAPK信号传导通路增强呼吸道上皮细胞IL-8的表达;NF-κB通路也参与了绿脓菌素调控细胞IL-8表达的过程。     

Oxidative epithelial host defense is regulated by infectious and inflammatory stimuli

Epithelia express oxidative antimicrobial protection that uses lactoperoxidase (LPO), hydrogen peroxide (H₂O₂), and thiocyanate to generate the reactive hypothiocyanite. Duox1 and Duox2, found in epithelia, are hypothesized to provide H₂O₂ for use by LPO. To investigate the regulation of oxidative LPO-mediated host defense by bacterial and inflammatory stimuli, LPO and Duox mRNA were followed in differentiated primary human airway epithelial cells challenged with Pseudomonas aeruginosa flagellin or IFN-γ. Flagellin upregulated Duox2 mRNA 20-fold, but upregulated LPO mRNA only 2.5-fold. IFN-γ increased Duox2 mRNA 127-fold and upregulated LPO mRNA 10-fold. DuoxA2, needed for Duox2 activity, was also upregulated by flagellin and IFN-γ. Both stimuli increased H₂O₂ synthesis and LPO-dependent killing of P. aeruginosa. Reduction of Duox1 by siRNA showed little effect on basal H₂O₂ production, whereas Duox2 siRNA markedly reduced basal H₂O₂ production and resulted in an 8-fold increase in Nox4 mRNA. In conclusion, large increases in Duox2-mediated H₂O₂ production seem to be coordinated with increases in LPO mRNA and, without increased LPO, H₂O₂ levels in airway secretion are expected to increase substantially. The data suggest that Duox2 is the major contributor to basal H₂O₂ synthesis despite the presence of greater amounts of Duox1.     

Characterization of hydrogen peroxide production by Duox in bronchial epithelial cells exposed to Pseudomonas aeruginosa

Hydrogen peroxide production by the NADPH oxidase Duox1 occurs during activation of respiratory epithelial cells stimulated by purified bacterial ligands, such as lipopolysaccharide. Here, we characterize Duox activation using intact bacterial cells of several airway pathogens. We found that only Pseudomonas aeruginosa, not Burkholderia cepacia or Staphylococcus aureus, triggers H₂O₂ production in bronchial epithelial cells in a calcium-dependent but predominantly ATP-independent manner. Moreover, by comparing mutant Pseudomonas strains, we identify several virulence factors that participate in Duox activation, including the type-three secretion system. These data provide insight on Duox activation by mechanisms unique to P. aeruginosa.     

Regulation of interleukin-8 via an airway epithelial signaling cascade

Airways function as an innate immune organ against airborne bacteria that are inhaled and deposited in airways. One of the mechanisms of host defense is to recruit neutrophils into airways to clear the invaders. Airway epithelial cells produce neutrophil chemoattractant interleukin (IL)-8 in response to invading bacteria. In this study we show a signaling pathway on the plasma surface of human airway epithelial NCI-H292 cells that regulate IL-8 production in response to a model inflammatory stimulus, phorbol 12-myristate 13-acetate, and a pathophysiological stimulus, gram-negative bacterial lipopolysaccharide. First, we show that EGF receptor (EGFR) and MAP kinase ERK1/2 are involved in IL-8 expression by these stimuli. Second, we show that EGFR ligand transforming growth factor (TGF)-alpha mediates IL-8 production. Third, we show that tumor necrosis factor-alpha-converting enzyme (TACE) is required for IL-8 production by cleaving EGFR proligand proTGF-alpha into soluble TGF-alpha, activating EGFR. Last, we show that dual oxidase 1 (Duox1), a homolog of NADPH oxidase in airways, mediates TACE activation and IL-8 expression via generation of reactive oxygen species. In summary, we describe a signaling pathway, Duox1-TACE-TGF-alpha-EGFR, on the surface of airway epithelial (NCI-H292) cells that mediates airway epithelial defense against bacterial infection by producing IL-8. This pathway, which also regulates mucin production in human airways, provides mechanisms for killing foreign organisms and for their clearance.     

Induction of neutrophil apoptosis by the Pseudomonas aeruginosa exotoxin pyocyanin: a potential mechanism of persistent infection

Pseudomonas aeruginosa colonizes and infects human tissues, although the mechanisms by which the organism evades the normal, predominantly neutrophilic, host defenses are unclear. Phenazine products of P. aeruginosa can induce death in Caenorhabditis elegans. We hypothesized that phenazines induce death of human neutrophils, and thus impair neutrophil-mediated bacterial killing. We investigated the effects of two phenazines, pyocyanin and 1-hydroxyphenazine, upon apoptosis of neutrophils in vitro. Pyocyanin induced a concentration- and time-dependent acceleration of neutrophil apoptosis, with 50 microM pyocyanin causing a 10-fold induction of apoptosis at 5 h (p < 0.001), a concentration that has been documented in sputum from patients colonized with P. aeruginosa. 1-hydroxyphenazine was without effect. In contrast to its rapid induction of neutrophil apoptosis, pyocyanin did not induce significant apoptosis of monocyte-derived macrophages or airway epithelial cells at time points up to 24 h. Comparison of wild-type and phenazine-deleted strains of P. aeruginosa showed a highly significant reduction in neutrophil killing by the phenazine-deleted strain. In clinical isolates of P. aeruginosa pyocyanin production was associated with a proapoptotic effect upon neutrophils in culture. Pyocyanin-induced neutrophil apoptosis was not delayed either by treatment with LPS, a powerfully antiapoptotic bacterial product, or in neutrophils from cystic fibrosis patients. Pyocyanin-induced apoptosis was associated with rapid and sustained generation of reactive oxygen intermediates and subsequent reduction of intracellular cAMP. Treatment of neutrophils with either antioxidants or synthetic cAMP analogues significantly abrogated pyocyanin-induced apoptosis. We conclude that pyocyanin-induced neutrophil apoptosis may be a clinically important mechanism of persistence of P. aeruginosa in human tissue.     

Pyocyanin and its precursor phenazine-1-carboxylic acid increase IL-8 and intercellular adhesion molecule-1 expression in human airway epithelial cells by oxidant-dependent mechanisms

Pseudomonas aeruginosa secretes numerous factors that alter host cell function and may contribute to disease pathogenesis. Among recognized virulence factors is the redox-active phenazine pyocyanin. We have recently demonstrated that the precursor for pyocyanin, phenazine-1-carboxylic acid (PCA), increases oxidant formation and alters gene expression in human airway epithelial cells. We report in this work that PCA and pyocyanin increase expression of ICAM-1 both in vivo and in vitro. Moreover, phenazines enhanced cytokine-dependent increases in IL-8 and ICAM-1. Antioxidant intervention studies indicated both similarities and differences between PCA and pyocyanin. The thiol antioxidant N-acetyl cysteine, extracellular catalase, and inducible NO synthase inhibitors inhibited ICAM-1 and IL-8 increases in response to both phenazines. However, pyocyanin was significantly more sensitive to N-acetylcysteine inhibition. Interestingly, hydroxyl radical scavengers inhibited the response to pyocyanin, but not to PCA. These studies suggest that P. aeruginosa phenazines coordinately up-regulate chemokines (IL-8) and adhesion molecules (ICAM-1) by mechanisms that are, at least in part, oxidant dependent. However, results indicate that the mechanisms by which PCA and pyocyanin exert their effects are not identical, and not all antioxidant interventions are equally effective in inhibiting phenazine-mediated proinflammatory effects.     

Inactivation of the potent Pseudomonas aeruginosa cytotoxin pyocyanin by airway peroxidases and nitrite

Pyocyanin (1-hydroxy-N-methylphenazine, PCN) is a cytotoxic pigment and virulence factor secreted by the human bacterial pathogen, Pseudomonas aeruginosa. Here, we report that exposure of PCN to airway peroxidases, hydrogen peroxide (H(2)O(2)), and NaNO(2) generates unique mononitrated PCN metabolites (N-PCN) as revealed by HPLC/mass spectrometry analyses. N-PCN, in contrast to PCN, was devoid of antibiotic activity and failed to kill Escherichia coli and Staphylococcus aureus. Furthermore, in contrast to PCN, intratracheal instillation of N-PCN into murine lungs failed to induce a significant inflammatory response. Surprisingly, at a pH of ～7, N-PCN was more reactive than PCN with respect to NADH oxidation but resulted in a similar magnitude of superoxide production as detected by electron paramagnetic resonance and spin trapping experiments. When incubated with Escherichia coli or lung A549 cells, PCN and N-PCN both led to superoxide formation, but lesser amounts were detected with N-PCN. Our results demonstrate that PCN that has been nitrated by peroxidase/H(2)O(2)/NO(2)(-) systems possesses less cytotoxic/proinflammatory activity than native PCN. Yield of N-PCN was decreased by the presence of the competing physiological peroxidase substrates (thiocyonate) SCN(-) (myeloperoxidase, MPO, and lactoperoxidase, LPO) and Cl(-) (MPO), which with Cl(-) yielded chlorinated PCNs. These reaction products also showed decreased proinflammatory ability when instilled into the lungs of mice. These observations add important insights into the complexity of the pathogenesis of lung injury associated with Pseudomonas aeruginosa infections and provide additional rationale for exploring the efficacy of NO(2)(-) in the therapy of chronic Pseudomonas aeruginosa airway infection in cystic fibrosis.     

Positive signature-tagged mutagenesis in Pseudomonas aeruginosa: tracking patho-adaptive mutations promoting airways chronic infection

The opportunistic pathogen Pseudomonas aeruginosa can establish life-long chronic infections in the airways of cystic fibrosis (CF) patients. Persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. Several reports indicated that P. aeruginosa adapts by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. To validate this model of P. aeruginosa adaptation to CF airways and to identify novel genes involved in this microevolution, we designed a novel approach of positive-selection screening by PCR-based signature-tagged mutagenesis (Pos-STM) in a murine model of chronic airways infection. A systematic positive-selection scheme using sequential rounds of in vivo screenings for bacterial maintenance, as opposed to elimination, generated a list of genes whose inactivation increased the colonization and persistence in chronic airways infection. The phenotypes associated to these Pos-STM mutations reflect alterations in diverse aspects of P. aeruginosa biology which include lack of swimming and twitching motility, lack of production of the virulence factors such as pyocyanin, biofilm formation, and metabolic functions. In addition, Pos-STM mutants showed altered invasion and stimulation of immune response when tested in human respiratory epithelial cells, indicating that P. aeruginosa is prone to revise the interaction with its host during persistent lifestyle. Finally, sequence analysis of Pos-STM genes in longitudinally P. aeruginosa isolates from CF patients identified signs of patho-adaptive mutations within the genome. This novel Pos-STM approach identified bacterial functions that can have important clinical implications for the persistent lifestyle and disease progression of the airway chronic infection.     

Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.     

Phenazine-1-carboxylic acid,a secondary metabolite of Pseudomonas aeruginosa,alters expression of immunomodulatory proteins by human airway epithelial cells

Pseudomonas aeruginosa is a gram-negative bacterium that causes both acute and chronic lung disease in susceptible patient populations. P. aeruginosa secretes numerous proteins and secondary metabolites, many of which have biological effects that likely contribute to disease pathogenesis. An unidentified small-molecular-weight factor was previously reported to increase IL-8 release both in vitro and in vivo. To identify this factor, we subjected the <3-kDa fraction from P. aeruginosa-conditioned medium to HPLC analysis. A peak fraction that stimulated IL-8 release was found by mass spectrometry to have a molecular mass (MM) of 224 Da. On the basis of this MM and other biochemical properties, we hypothesized that the factor was phenazine-1-carboxylic acid (PCA). Subsequent studies and comparison with purified PCA confirmed this hypothesis. Purified PCA exhibited a number of biological effects in human airway epithelial cells, including increasing IL-8 release and ICAM-1 expression, as well as decreasing RANTES and monocyte chemoattractant protein-1 (MCP-1) release. PCA also increased intracellular oxidant formation as measured by electron paramagnetic resonance and by an intracellular oxidant-sensitive probe. Antioxidants inhibited PCA-dependent increases in IL-8 and ICAM-1, suggesting that oxidants contributed to these effects. However, in contrast to the related phenazine compound pyocyanin, PCA did not oxidize NAD(P)H at physiologically relevant pH, providing preliminary evidence that PCA and pyocyanin may have distinct redox chemistries within the cell. Thus PCA is a biologically active factor secreted by P. aeruginosa that has several activities that could alter the host immune and inflammatory response and thereby contribute to bacterial disease pathogenesis.     

Flagellin/TLR5 responses induce mucus hypersecretion by activating EGFR via an epithelial cell signaling cascades

Mucus hypersecretion is an important manifestation in patients with chronic inflammatory airway diseases. Excessive production of mucin leads to airway mucus obstruction and contributes to morbidity and mortality in these diseases. The molecular mechanisms underlying mucin overproduction, however, still remain largely unknown. Here, we report that the bacterium Pseudomonas aeruginosa (P. aeruginosa), an important human respiratory pathogen, induced MUC5AC mucin expression via an epithelial cell signaling cascade in human airway epithelial cells. The flagellin purified from P. aeruginosa up-regulated MUC5AC expression by activating its receptor Toll-like receptor 5 (TLR5) in 16HBE cells. This effect was inhibited by NADPH oxidase inhibitor (DPI), small interfering RNA of dual oxidase 2 (Duox2) and reactive oxygen species (ROS) scavengers (nPG and DMSO). Flagellin induced TGF-α release, and stimulated phosphorylated epidermal growth factor receptor (EGFR) and MUC5AC overproduction. These effects were prevented by EGFR and TGF-α neutralizing antibodies, metalloprotease inhibitors (GM6001 and TNF-α protease inhibitor-1) and specific knockdown of TNF-α-converting enzyme (TACE) with TACE siRNA. These findings may bring new insights into the molecular pathogenesis of P. aeruginosa infections and lead to novel therapeutic intervention for mucin overproduction in patients with P. aeruginosa infections.     

Multiple TLRs activate EGFR via a signaling cascade to produce innate immune responses in airway epithelium

Toll-like receptors (TLRs) are critical for the recognition of inhaled pathogens that deposit on the airway epithelial surface. The epithelial response to pathogens includes signaling cascades that activate the EGF receptor (EGFR). We hypothesized that TLRs communicate with EGFR via epithelial signaling to produce certain innate immune responses. Airway epithelium expresses the highest levels of TLR2, TLR3, TLR5, and TLR6, and here we found that ligands for these TLRs increased IL-8 and VEGF production in normal human bronchial epithelial cells. These effects were prevented by treatment with a selective inhibitor of EGFR phosphorylation (AG-1478), a metalloprotease (MP) inhibitor, a reactive oxygen species (ROS) scavenger, and an NADPH oxidase inhibitor. In an airway epithelial cell line (NCI-H292), TNF-alpha-converting enzyme (TACE) small interfering RNA (siRNA) was used to confirm that TACE is the MP involved in TLR ligand-induced IL-8 and VEGF production. We show that transforming growth factor (TGF)-alpha is the EGFR ligand in this signaling cascade by using TGF-alpha neutralizing antibody and by showing that epithelial production of TGF-alpha occurs in response to TLR ligands. Dual oxidase 1 (Duox1) siRNA was used to confirm that Duox1 is the NADPH oxidase involved in TLR ligand-induced IL-8 and VEGF production. We conclude that multiple TLR ligands induce airway epithelial cell production of IL-8 and VEGF via a Duox1--> ROS--> TACE--> TGF-alpha--> EGFR phosphorylation pathway. These results show for the first time that multiple TLRs in airway epithelial cells produce innate immune responses by activating EGFR via an epithelial cell signaling cascade.     

Premature cellular senescence induced by pyocyanin, a redox-active Pseudomonas aeruginosa toxin

Pseudomonas aeruginosa is an important nosocomial pathogen that can cause acute and chronic infection, particularly of the respiratory system. Pyocyanin is a major P. aeruginosa virulence factor that displays redox activity and induces oxidative stress in cellular systems. The effect of pyocyanin on replicating human pulmonary epithelial (A549) cells was investigated. Cells were exposed to pyocyanin for 24 h and their subsequent growth and development were followed for 7 days. Pyocyanin (5-10 microM) arrested cell growth and resulted in the development of a morphological phenotype consistent with cellular senescence, that is, an enlarged and flattened appearance. The senescent nature of these cells was supported by positive staining for increased lysosomal content and senescence-associated beta-galactosidase activity. All cells treated with pyocyanin (10 microM) converted to the senescent phenotype, which remained stable for up to 7 days. Exposure to pyocyanin at 25 microM or greater resulted in cell death due to apoptosis. A549 cells exposed to pyocyanin generated hydrogen peroxide in a dose-dependent manner and the senescence-inducing effect of pyocyanin was inhibited by the antioxidant, glutathione, suggesting the involvement of reactive oxygen species. The induction of premature cellular senescence by redox-active bacterial toxins may be a hitherto unrecognized aspect of infection pathology and a limiting factor in the tissue repair response to infection.     

Thiocyanate transport in resting and IL-4-stimulated human bronchial epithelial cells: role of pendrin and anion channels

SCN(-) (thiocyanate) is an important physiological anion involved in innate defense of mucosal surfaces. SCN(-) is oxidized by H(2)O(2), a reaction catalyzed by lactoperoxidase, to produce OSCN(-) (hypothiocyanite), a molecule with antimicrobial activity. Given the importance of the availability of SCN(-) in the airway surface fluid, we studied transepithelial SCN(-) transport in the human bronchial epithelium. We found evidence for at least three mechanisms for basolateral to apical SCN(-) flux. cAMP and Ca(2+) regulatory pathways controlled SCN(-) transport through cystic fibrosis transmembrane conductance regulator and Ca(2+)-activated Cl(-) channels, respectively, the latter mechanism being significantly increased by treatment with IL-4. Stimulation with IL-4 also induced the strong up-regulation of an electroneutral SCN(-)/Cl(-) exchange. Global gene expression analysis with microarrays and functional studies indicated pendrin (SLC26A4) as the protein responsible for this SCN(-) transport. Measurements of H(2)O(2) production at the apical surface of bronchial cells indicated that the extent of SCN(-) transport is important to modulate the conversion of this oxidant molecule by the lactoperoxidase system. Our studies indicate that the human bronchial epithelium expresses various SCN(-) transport mechanisms under resting and stimulated conditions. Defects in SCN(-) transport in the airways may be responsible for susceptibility to infections and/or decreased ability to scavenge oxidants.     

Pseudomonas aeruginosa pyocyanin directly oxidizes glutathione and decreases its levels in airway epithelial cells

Production of pyocyanin enhances Pseudomonas aeruginosa virulence. Many of pyocyanin's in vitro and in vivo cytotoxic effects on human cells appear to result from its ability to redox cycle. Pyocyanin directly accepts electrons from NADH or NADPH with subsequent electron transfer to oxygen, generating reactive oxygen species. Reduced glutathione (GSH) is an important cellular antioxidant, and it contributes to the regulation of redox-sensitive signaling systems. Using the human bronchial epithelial (HBE) and the A549 human type II alveolar epithelial cell lines, we tested the hypothesis that pyocyanin can deplete airway epithelial cells of GSH. Incubation of both cell types with pyocyanin led to a concentration-dependent loss of cellular GSH (up to 50%) and an increase in oxidized GSH (GSSG) in the HBE, but not A549 cells, at 24 h. An increase in total GSH, mostly as GSSG, was detected in the culture media, suggesting export of GSH or GSSG from the pyocyanin-exposed cells. Loss of GSH could be due to pyocyanin-induced H(2)O(2) formation. However, overexpression of catalase only partially prevented the pyocyanin-mediated decline in cellular GSH. Cell-free electron paramagnetic resonance studies revealed that pyocyanin directly oxidizes GSH, forming pyocyanin free radical and O(2)(-). Pyocyanin oxidized other thiol-containing compounds, cysteine and N-acetyl-cysteine, but not methionine. Thus GSH may enhance pyocyanin-induced cytotoxicity by functioning as an alternative source of reducing equivalents for pyocyanin redox cycling. Pyocyanin-mediated alterations in cellular GSH may alter epithelial cell functions by modulating redox sensitive signaling events.     

Role of the cystic fibrosis transmembrane conductance regulator in internalization of Pseudomonas aeruginosa by polarized respiratory epithelial cells

Pseudomonas aeruginosa is an important human pathogen, producing lung infection in individuals with cystic fibrosis (CF), patients who are ventilated and those who are neutropenic. The respiratory epithelium provides the initial barrier to infection. Pseudomonas aeruginosa can enter epithelial cells, although the mechanism of entry and the role of intracellular organisms in its life cycle are unclear. We devised a model of infection of polarized human respiratory epithelial cells with P. aeruginosa and investigated the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in adherence, uptake and IL-8 production by human respiratory epithelial cells. We found that a number of P. aeruginosa strains could invade and replicate within cells derived from a patient with CF. Intracellular bacteria did not produce host cell cytotoxicity over a period of 24 h. When these cells were transfected with wild-type CFTR, uptake of bacteria was significantly reduced and release of IL-8 following infection enhanced. We propose that internalized P. aeruginosa may play an important role in the pathogenesis of infection and that, by allowing greater internalization into epithelial cells, mutant CFTR results in an increased susceptibility of bronchial infection with this microbe.     

Epistatic roles for Pseudomonas aeruginosa MutS and DinB (DNA Pol IV) in coping with reactive oxygen species-induced DNA damage

Pseudomonas aeruginosa is especially adept at colonizing the airways of individuals afflicted with the autosomal recessive disease cystic fibrosis (CF). CF patients suffer from chronic airway inflammation, which contributes to lung deterioration. Once established in the airways, P. aeruginosa continuously adapts to the changing environment, in part through acquisition of beneficial mutations via a process termed pathoadaptation. MutS and DinB are proposed to play opposing roles in P. aeruginosa pathoadaptation: MutS acts in replication-coupled mismatch repair, which acts to limit spontaneous mutations; in contrast, DinB (DNA polymerase IV) catalyzes error-prone bypass of DNA lesions, contributing to mutations. As part of an ongoing effort to understand mechanisms underlying P. aeruginosa pathoadaptation, we characterized hydrogen peroxide (H(2)O(2))-induced phenotypes of isogenic P. aeruginosa strains bearing different combinations of mutS and dinB alleles. Our results demonstrate an unexpected epistatic relationship between mutS and dinB with respect to H(2)O(2)-induced cell killing involving error-prone repair and/or tolerance of oxidized DNA lesions. In striking contrast to these error-prone roles, both MutS and DinB played largely accurate roles in coping with DNA lesions induced by ultraviolet light, mitomycin C, or 4-nitroquinilone 1-oxide. Models discussing roles for MutS and DinB functionality in DNA damage-induced mutagenesis, particularly during CF airway colonization and subsequent P. aeruginosa pathoadaptation are discussed.     

Comparison of nasal and bronchial epithelial cells obtained from patients with COPD

For in vitro studies of airway pathophysiology, primary epithelial cells have many advantages over immortalised cell lines. Nasal epithelial cells are easier to obtain than bronchial epithelial cells and can be used as an alternative for in vitro studies. Our objective was to compare nasal and bronchial epithelial cells from subjects with COPD to establish if these cells respond similarly to pro-inflammatory stimuli. Cell cultures from paired nasal and bronchial brushings (21 subjects) were incubated with cigarette smoke extract (CSE) prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide. IL-6 and IL-8 were measured by ELISA and Toll-like receptor 4 (TLR-4) message and expression by RT-PCR and FACS respectively. IL-8 release correlated significantly between the two cell types. IL-6 secretion was significantly less from bronchial compared to nasal epithelial cells and secreted concentrations did not correlate. A 4 h CSE incubation was immunosuppressive for both nasal and bronchial cells, however prolonged incubation for 24 h was pro-inflammatory solely for the nasal cells. CSE reduced TLR-4 expression in bronchial cells only after 24 h, and was without effect on mRNA expression. In subjects with COPD, nasal epithelial cells cannot substitute for in vitro bronchial epithelial cells in airway inflammation studies.     

Flagellin-stimulated Cl- secretion and innate immune responses in airway epithelia: role for p38

Activation of an innate immune response in airway epithelia by the human pathogen Pseudomonas aeruginosa requires bacterial expression of flagellin. Addition of flagellin (10(-7) M) to airway epithelial cell monolayers (Calu-3, airway serous cell-like) increased Cl(-) secretion (I(Cl)) beginning after 3-10 min, reaching a plateau after 20-45 min at DeltaI(Cl) = 15-50 microA/cm(2). Similar, although 10-fold smaller, responses were observed in well-differentiated bronchial epithelial cultures. Flagellin stimulated I(Cl) in the presence of maximally stimulating doses of the purinergic agonist ATP, but had no effects following forskolin. IL-1beta (produced by both epithelia and neutrophils during infections) stimulated I(Cl) similar to flagellin. Flagellin-, IL-1beta-, ATP-, and forskolin-stimulated I(Cl) were inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) blockers GlyH101, CFTRinh172, and glibenclamide. Neither flagellin nor IL-1beta altered transepithelial fluxes of membrane-impermeant dextran (10 kDa) or lucifer yellow (mol wt = 457), but both activated p38, NF-kappaB, and IL-8 secretion. Blockers of p38 (SB-202190 and SB-203580) reduced flagellin- and IL-1beta-stimulated I(Cl) by 33-50% but had smaller effects on IL-8 and NF-kappaB. It is concluded that: 1) flagellin and IL-1beta activated p38, NF-kappaB, IL-8, and CFTR-dependent anion secretion without altering tight junction permeability; 2) p38 played a role in regulating I(Cl) and IL-8 but not NF-kappaB; and 3) p38 was more important in flagellin- than IL-1beta-stimulated responses. During P. aeruginosa infections, flagellin and IL-1beta are expected to increase CFTR-dependent ion and fluid flow into and bacterial clearance from the airways. In cystic fibrosis, the secretory response would be absent, but activation of p38, NF-kappaB, and IL-8 would persist.