The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing DeltaF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and DeltaF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on DeltaF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing DeltaF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The DeltaF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.     

Pseudomonas aeruginosa homoserine lactone triggers apoptosis and Bak/Bax‐independent release of mitochondrial cytochrome C in fibroblasts

Pseudomonas aeruginosa use N‐(3‐oxododecanoyl)‐homoserine lactone (C12) as a quorum‐sensing molecule to regulate gene expression in the bacteria. It is expected that in patients with chronic infections with P. aeruginosa, especially as biofilms, local [C12] will be high and, since C12 is lipid soluble, diffuse from the airways into the epithelium and underlying fibroblasts, capillary endothelia and white blood cells. Previous work showed that C12 has multiple effects in human host cells, including activation of apoptosis. The present work tested the involvement of Bak and Bax in C12‐triggered apoptosis in mouse embryo fibroblasts (MEF) by comparing MEF isolated from embryos of wild‐type (WT) and Bax^?/?/Bak^?/? (DKO) mice. In WT MEF C12 rapidly triggered (minutes to 2 h): activation of caspases 3/7 and 8, depolarization of mitochondrial membrane potential (Δψ_mito), release of cytochrome C from mitochondria into the cytosol, blebbing of plasma membranes, shrinkage/condensation of cells and nuclei and, subsequently, cell killing. A DKO MEF line that was relatively unaffected by the Bak/Bax‐dependent proapoptotic stimulants staurosporine and etoposide responded to C12 similarly to WT MEF: activation of caspase 3/7, depolarization of Δψ_mito and release of cytochrome C and cell death. Re‐expression of Bax or Bak in DKO MEF did not alter the WT‐like responses to C12 in DKO MEF. These data showed that C12 triggers novel, rapid proapoptotic Bak/Bax‐independent responses that include events commonly associated with activation of both the intrinsic pathway (depolarization of Δψ_mito and release of cytochrome C from mitochondria into the cytosol) and the extrinsic pathway (activation of caspase 8). Unlike the proapoptotic agonists staurosporine and etoposide that release cytochrome C from mitochondria, C12's effects do not require participation of either Bak or Bax.     

Does the {Delta}F508-CFTR mutation induce a proinflammatory response in human airway epithelial cells?

In the clinical setting, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene enhance the inflammatory response in the lung to Pseudomonas aeruginosa (P. aeruginosa) infection. However, studies on human airway epithelial cells in vitro have produced conflicting results regarding the effect of mutations in CFTR on the inflammatory response to P. aeruginosa, and there are no comprehensive studies evaluating the effect of P. aeruginosa on the inflammatory response in airway epithelial cells with the ΔF508/ΔF508 genotype and their matched CF cell line rescued with wild-type (wt)-CFTR. CFBE41o- cells (ΔF508/ΔF508) and CFBE41o- cells complemented with wt-CFTR (CFBE-wt-CFTR) have been used extensively as an experimental model to study CF. Thus the goal of this study was to examine the effect of P. aeruginosa on gene expression and cytokine/chemokine production in this pair of cells. P. aeruginosa elicited a more robust increase in cytokine and chemokine expression (e.g., IL-8, CXCL1, CXCL2 and TNF-α) in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells. These results demonstrate that CFBE41o- cells complemented with wt-CFTR mount a more robust inflammatory response to P. aeruginosa than CFBE41o-ΔF508/ΔF508-CFTR cells. Taken together with other published studies, our data demonstrate that there is no compelling evidence to support the view that mutations in CFTR induce a hyperinflammatory response in human airway epithelial cells in vivo. Although the lungs of patients with CF have abundant levels of proinflammatory cytokines and chemokines, because the lung is populated by immune cells and epithelial cells there is no way to know, a priori, whether airway epithelial cells in the CF lung in vivo are hyperinflammatory in response to P. aeruginosa compared with non-CF lung epithelial cells. Thus studies on human airway epithelial cell lines and primary cells in vitro that propose to examine the effect of mutations in CFTR on the inflammatory response to P. aeruginosa have uncertain clinical significance with regard to CF.     

Relation of exaggerated cytokine responses of CF airway epithelial cells to PAO1 adherence

In many model systems, cystic fibrosis (CF) phenotype airway epithelial cells in culture respond to P. aeruginosa with greater interleukin (IL)-8 and IL-6 secretion than matched controls. In order to test whether this excess inflammatory response results from the reported increased adherence of P. aeruginosa to the CF cells, we compared the inflammatory response of matched pairs of CF and non CF airway epithelial cell lines to the binding of GFP-PAO1, a strain of pseudomonas labeled with green fluorescent protein. There was no clear relation between GFP-PAO1 binding and cytokine production in response to PAO1. Treatment with exogenous aGM1 resulted in greater GFP-PAO1 binding to the normal phenotype compared to CF phenotype cells, but cytokine production remained greater from the CF cell lines. When cells were treated with neuraminidase, PAO1 adherence was equalized between CF and nonCF phenotype cell lines, but IL-8 production in response to inflammatory stimuli was still greater in CF phenotype cells. The polarized cell lines 16HBEo-Sense (normal phenotype) and Antisense (CF phenotype) cells were used to test the effect of disrupting tight junctions, which allows access of PAO1 to basolateral binding sites in both cell lines. IL-8 production increased from CF, but not normal, cells. These data indicate that increased bacterial binding to CF phenotype cells cannot by itself account for excess cytokine production in CF airway epithelial cells, encourage investigation of alternative hypotheses, and signal caution for therapeutic strategies proposed for CF that include disruption of tight junctions in the face of pseudomonas infection.     

Effects of cystic fibrosis transmembrane conductance regulator and DeltaF508CFTR on inflammatory response, ER stress, and Ca2+ of airway epithelia

We tested whether cystic fibrosis (CF) airway epithelia have larger innate immune responses than non-CF or cystic fibrosis transmembrane conductance regulator (CFTR)-corrected cells, perhaps resulting from ER stress due to retention of DeltaF508CFTR in the endoplasmic reticulum (ER) and activation of cytosolic Ca(2+) (Ca(i)) and nuclear factor (NF)-kappaB signaling. Adenovirus infections of a human CF (DeltaF508/DeltaF508) nasal cell line (CF15) provided isogenic comparisons of wild-type (wt) CFTR and DeltaF508CFTR. In the absence of bacteria, there were no or only small differences among CF15, CF15-lacZ (beta-galactosidase-expressing), CF15-wtCFTR (wtCFTR-corrected), and CF15-DeltaF508CFTR (to test ER retention of DeltaF508CFTR) cells in NF-kappaB activity, interleukin (IL)-8 secretion, Ca(i) responses, and ER stress. Non-CF and CF primary cultures of human bronchial epithelial cells (HBE) secreted IL-8 equivalently. Upon infection with Pseudomonas aeruginosa (PA) or flagellin (key activator for airway epithelia), CF15, CF15-lacZ, CF15-wtCFTR, and CF15DeltaF508CFTR cells exhibited equal PA binding, NF-kappaB activity, and IL-8 secretion; cells also responded similarly to flagellin when both CFTR (forskolin) and Ca(i) signaling (ATP) were activated. CF and non-CF HBE responded similarly to flagellin + ATP. Thapsigargin (Tg, releases ER Ca(2+)) increased flagellin-stimulated NF-kappaB and ER stress similarly in all cells. We conclude that ER stress, Ca(i), and NF-kappaB signaling and IL-8 secretion were unaffected by wt- or DeltaF508CFTR in control and during exposure to PA, flagellin, flagellin + ATP, or flagellin + ATP + forskolin. Tg, but not wt- or DeltaF508CFTR, triggered ER stress. Previous measurements showing hyperinflammatory responses in CF airway epithelia may have resulted from cell-specific, rather than CFTR- or DeltaF508CFTR-specific effects.     

Peroxisome proliferator-activated receptor-gamma in cystic fibrosis lung epithelium

The pathophysiology of cystic fibrosis (CF) inflammatory lung disease is not well understood. CF airway epithelial cells respond to inflammatory stimuli with increased production of proinflammatory cytokines as a result of increased NF-kappaB activation. Peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibits NF-kappaB activity and is reported to be reduced in CF. If PPARgamma participates in regulatory dysfunction in the CF lung, perhaps PPARgamma ligands might be useful therapeutically. Cell models of CF airway epithelium were used to evaluate PPARgamma expression and binding to NF-kappaB at basal and under conditions of inflammatory stimulation by Pseudomonas aeruginosa or TNFalpha/IL-1beta. An animal model of CF was used to evaluate the potential of PPARgamma agonists as therapeutic agents in vivo. In vitro, PPARgamma agonists reduced IL-8 and MMP-9 release from airway epithelial cells in response to PAO1 or TNFalpha/IL-1beta stimulation. Less NF-kappaB bound to PPARgamma in CF than normal cells, in two different assays; PPARgamma agonists abrogated this reduction. PPARgamma bound less to its target DNA sequence in CF cells. To test the importance of the reported PPARgamma inactivation by phosphorylation, we observed that inhibitors of ERK, but not JNK, were synergistic with PPARgamma agonists in reducing IL-8 secretion. In vivo, administration of PPARgamma agonists reduced airway inflammation in response to acute infection with P. aeruginosa in CF, but not wild-type, mice. In summary, PPARgamma inhibits the inflammatory response in CF, at least in part by interaction with NF-kappaB in airway epithelial cells. PPARgamma agonists may be therapeutic in CF.     

Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia.

The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms. Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P. aeruginosa alone. The interactions between P. aeruginosa biofilms and B. cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship. Using the Calgary Biofilm Device (CBD), members of all genomovars of the B. cepacia complex were shown to form biofilms, including those isolated from CF lungs. Mixed species biofilm formation between CF isolates of P. aeruginosa and B. cepacia was readily achieved using the CBD. Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition. Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure. Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B. cepacia complex. Characterization of the soluble inhibitor suggested pyocyanin as the active compound.     

Basal nucleotide levels, release, and metabolism in normal and cystic fibrosis airways

BACKGROUND: Cystic fibrosis (CF) is a syndrome caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. Despite advances in our understanding of the molecular pathogenesis of CF, the link between CFTR gene mutations and the pathogenesis of CF lung disease remains poorly defined. CFTR has been assigned a number of putative functions that may contribute to innate airway defense, including the regulation of adenosine 5'-triphosphate (ATP) release into the extracellular environment. Because extracellular ATP and uridine 5'-triphosphate (UTP) may regulate airway mucociliary clearance via interaction with luminal P2Y2 receptors, the loss of CFTR-mediated nucleotide release could explain the defect in CF airway defense. MATERIALS AND METHODS: We tested the physiologic importance of CFTR-mediated nucleotide release in vivo by directly measuring levels of ATP and UTP in nasal airway surface liquid from normal and CF subjects. Because these basal nucleotide levels reflect the net activities of nucleotide release and metabolic pathways, we also measured constitutive rates of nucleotide release and metabolism on well-differentiated normal and CF airway cultures in vitro. The measurement of ATP release rates were paralleled by in vivo studies employing continuous nasal perfusion in normal and CF subjects. Finally, the regulation of ATP release by isoproterenol and methacholine-stimulated submucosal gland secretion was tested. RESULTS: These studies revealed that steady-state ATP and UTP levels were similar in normal (470 +/- 131 nM and 37 +/- 7 nM, respectively) and CF (911 +/- 199 nM and 33 +/- 12 nM, respectively) subjects. The rates of both ATP release and metabolism were also similar in normal and CF airway epithelia both in vitro and in vivo. Airway submucosal glands did not secrete nucleotides, but rather, secreted a soluble nucleotidase in response to cholinergic stimuli. CONCLUSION: The concentration of ATP in airway surface liquid is in a range that is relevant for the activation of airway nucleotide receptors. However, despite this finding that suggests endogenous nucleotides may be important for the regulation of mucociliary clearance, our data do not support a role for CFTR in regulating extracellular nucleotide concentrations on airway surfaces.     

Transgenic cystic fibrosis mice exhibit reduced early clearance of Pseudomonas aeruginosa from the respiratory tract

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been proposed to be an epithelial cell receptor for Pseudomonas aeruginosa involved in bacterial internalization and clearance from the lung. We evaluated the role of CFTR in clearing P. aeruginosa from the respiratory tract using transgenic CF mice that carried either the DeltaF508 Cftr allele or an allele with a Cftr stop codon (S489X). Intranasal application achieved P. aeruginosa lung infection in inbred C57BL/6 DeltaF508 Cftr mice, whereas DeltaF508 Cftr and S489X Cftr outbred mice required tracheal application of the inoculum to establish lung infection. CF mice showed significantly less ingestion of LPS-smooth P. aeruginosa by lung cells and significantly greater bacterial lung burdens 4.5 h postinfection than C57BL/6 wild-type mice. Microscopy of infected mouse and rhesus monkey tracheas clearly demonstrated ingestion of P. aeruginosa by epithelial cells in wild-type animals, mostly around injured areas of the epithelium. Desquamating cells loaded with P. aeruginosa could also be seen in these tissues. No difference was found between CF and wild-type mice challenged with an LPS-rough mucoid isolate of P. aeruginosa lacking the CFTR ligand. Thus, transgenic CF mice exhibit decreased clearance of P. aeruginosa and increased bacterial burdens in the lung, substantiating a key role for CFTR-mediated bacterial ingestion in lung clearance of P. aeruginosa.     

Control of the proinflammatory state in cystic fibrosis lung epithelial cells by genes from the TNF-alphaR/NFkappaB pathway

BACKGROUND: Cystic fibrosis (CF) is the most common, lethal autosomal recessive disease affecting children in the United States and Europe. Extensive work is being performed to develop both gene and drug therapies. The principal mutation causing CF is in the CFTR gene ([Delta F508]CFTR). This mutation causes the mutant protein to traffic poorly to the plasma membrane, and degrades CFTR chloride channel activity. CPX, a candidate drug for CF, binds to mutant CFTR and corrects the trafficking deficit. CPX also activates mutant CFTR chloride channel activity. CF airways are phenotypically inundated by inflammatory signals, primarily contributed by sustained secretion of the proinflammatory cytokine interleukin 8 (IL-8) from mutant CFTR airway epithelial cells. IL-8 production is controlled by genes from the TNF-alphaR/NFkappaB pathway, and it is possible that the CF phenotype is due to dysfunction of genes from this pathway. In addition, because drug therapy with CPX and gene therapy with CFTR have the same common endpoint of raising the levels of CFTR, we have hypothesized that either approach should have a common genomic endpoint. MATERIALS AND METHODS: To test this hypothesis, we studied IL-8 secretion and global gene expression in IB-3 CF lung epithelial cells. The cells were treated by either gene therapy with wild-type CFTR, or by pharmacotherapy with the CFTR-surrogate drug CPX. CF cells, treated with either CFTR or CPX, were also exposed to Pseudomonas aeruginosa, a common chronic pathogen in CF patients. cDNA microarrays were used to assess global gene expression under the different conditions. A novel bioinformatic algorithm (GENESAVER) was developed to identify genes whose expression paralleled secretion of IL-8. RESULTS: We report here that IB3 CF cells secrete massive levels of IL-8. However, both gene therapy with CFTR and drug therapy with CPX substantially suppress IL-8 secretion. Nonetheless, both gene and drug therapy allow the CF cells to respond with physiologic secretion of IL-8 when the cells are exposed to P. aeruginosa. Thus, neither CFTR nor CPX acts as a nonspecific suppressor of IL-8 secretion from CF cells. Consistently, pharmacogenomic analysis indicates that CF cells treated with CPX greatly resemble CF cells treated with CFTR by gene therapy. Additionally, the same result obtains in the presence of P. aeruginosa. Classical hierarchical cluster analysis, based on similarity of global gene expression, also supports this conclusion. The GENESAVER algorithm, using the IL-8 secretion level as a physiologic variable, identifies a subset of genes from the TNF-alphaR/NFkappaB pathway that is expressed in phase with IL-8 secretion from CF epithelial cells. Certain other genes, previously known to be positively associated with CF, also fall into this category. Identified genes known to code for known inhibitors are expressed inversely, out of phase with IL-8 secretion. CONCLUSIONS: Wild-type CFTR and CPX both suppress proinflammatory IL-8 secretion from CF epithelial cells. The mechanism, as defined by pharmacogenomic analysis, involves identified genes from the TNF-alphaR/NFkappaB pathway. The close relationship between IL-8 secretion and genes from the TNF-alphaR/NFkappaB pathway suggests that molecular or pharmaceutical targeting of these novel genes may have strategic use in the development of new therapies for CF. From the perspective of global gene expression, both gene and drug therapy have similar genomic consequences. This is the first example showing equivalence of gene and drug therapy in CF, and suggests that a gene therapy-defined endpoint may prove to be a powerful paradigm for CF drug discovery. Finally, because the GENESAVER algorithm is capable of isolating disease-relevant genes in a hypothesis-driven manner without recourse to any a priori knowledge about the system, this new algorithm may also prove useful in applications to other genetic diseases.     

Pseudomonas aeruginosa and their small diffusible extracellular molecules inhibit Aspergillus fumigatus biofilm formation

Aspergillus fumigatus is often isolated from the lungs of cystic fibrosis (CF) patients, but unlike in severely immunocompromised individuals, the mortality rates are low. This suggests that competition from bacteria within the CF lung may be inhibitory. The purpose of this study was to investigate how Pseudomonas aeruginosa influences A. fumigatus conidial germination and biofilm formation. Aspergillus fumigatus biofilm formation was inhibited by direct contact with P. aeruginosa, but had no effect on preformed biofilm. A secreted heat-stable soluble factor was also shown to exhibit biofilm inhibition. Coculture of P. aeruginosa quorum-sensing mutants (PAO1:ΔLasI, PAO1:ΔLasR) did not significantly inhibit A. fumigatus biofilms (52.6-58.8%) to the same extent as that of the PA01 wild type (22.9-30.1%), both by direct and by indirect interaction (P<0.001). Planktonic and sessile inhibition assays with a series of short carbon chain molecules (decanol, decanoic acid and dodecanol) demonstrated that these molecules could both inhibit and disrupt biofilms in a concentration-dependent manner. Overall, this suggests that small diffusible and heat-stable molecules may be responsible for the competitive inhibition of filamentous fungal growth in polymicrobial environments such as the CF lung.     

Airway epithelial control of Pseudomonas aeruginosa infection in cystic fibrosis

Defective expression or function of the cystic fibrosis transmembrane conductance regulator (CFTR) underlies the hypersusceptibility of cystic fibrosis (CF) patients to chronic airway infections, particularly with Pseudomonas aeruginosa. CFTR is involved in the specific recognition of P. aeruginosa, thereby contributing to effective innate immunity and proper hydration of the airway surface layer (ASL). In CF, the airway epithelium fails to initiate an appropriate innate immune response, allowing the microbe to bind to mucus plugs that are then not properly cleared because of the dehydrated ASL. Recent studies have identified numerous CFTR-dependent factors that are recruited to the epithelial plasma membrane in response to infection and that are needed for bacterial clearance, a process that is defective in CF patients hypersusceptible to infection with this organism.     

Anaerobic culture conditions favor biofilm-like phenotypes in Pseudomonas aeruginosa isolates from patients with cystic fibrosis

Pseudomonas aeruginosa causes chronic infections in the lungs of cystic fibrosis (CF) individuals and remains the leading cause of morbidity and mortality associated with the disease. Biofilm growth and phenotypic diversification are factors thought to contribute to this organism's persistence. Most studies have focused on laboratory isolates such as strain PAO1, and there are relatively few reports characterizing the properties of CF strains, especially under decreased oxygen conditions such as occur in the CF lung. This study compared the phenotypic and functional properties of P. aeruginosa from chronically infected CF adults with those of strain PAO1 and other clinical non-CF isolates under aerobic and anaerobic culture conditions. The CF isolates overall displayed a reduced ability to form biofilms in standard in vitro short-term models. They also grew more slowly in culture, and exhibited decreased adherence to glass and decreased motilities (swimming, swarming and twitching). All of these characteristics were markedly accentuated by anaerobic growth conditions. Moreover, the CF strain phenotypes were not readily reversed by culture manipulations designed to encourage planktonic growth. The CF strains were thus inherently different from strain PAO1 and most of the other non-CF clinical P. aeruginosa isolates tested. In vitro models used to research CF isolate biofilm growth need to take the above properties of these strains into account.     

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.     

Neutrophil extracellular trap (NET)-mediated killing of Pseudomonas aeruginosa: evidence of acquired resistance within the CF airway, independent of CFTR

The inability of neutrophils to eradicate Pseudomonas aeruginosa within the cystic fibrosis (CF) airway eventually results in chronic infection by the bacteria in nearly 80 percent of patients. Phagocytic killing of P. aeruginosa by CF neutrophils is impaired due to decreased cystic fibrosis transmembrane conductance regulator (CFTR) function and virulence factors acquired by the bacteria. Recently, neutrophil extracellular traps (NETs), extracellular structures composed of neutrophil chromatin complexed with granule contents, were identified as an alternative mechanism of pathogen killing. The hypothesis that NET-mediated killing of P. aeruginosa is impaired in the context of the CF airway was tested. P. aeruginosa induced NET formation by neutrophils from healthy donors in a bacterial density dependent fashion. When maintained in suspension through continuous rotation, P. aeruginosa became physically associated with NETs. Under these conditions, NETs were the predominant mechanism of killing, across a wide range of bacterial densities. Peripheral blood neutrophils isolated from CF patients demonstrated no impairment in NET formation or function against P. aeruginosa. However, isogenic clinical isolates of P. aeruginosa obtained from CF patients early and later in the course of infection demonstrated an acquired capacity to withstand NET-mediated killing in 8 of 9 isolates tested. This resistance correlated with development of the mucoid phenotype, but was not a direct result of the excess alginate production that is characteristic of mucoidy. Together, these results demonstrate that neutrophils can kill P. aeruginosa via NETs, and in vitro this response is most effective under non-stationary conditions with a low ratio of bacteria to neutrophils. NET-mediated killing is independent of CFTR function or bacterial opsonization. Failure of this response in the context of the CF airway may occur, in part, due to an acquired resistance against NET-mediated killing by CF strains of P. aeruginosa.     

The role of the CFTR in susceptibility to Pseudomonas aeruginosa infections in cystic fibrosis

Recent molecular and cellular studies have shed new light on the basis for the susceptibility of cystic fibrosis (CF) patients to Pseudomonas aeruginosa infection. Changes in airway liquid composition and/or viscosity, enhanced bacterial binding to mucin and epithelial cell receptors, increased innate inflammation owing to disruptions in lipid metabolism and a role for the CFTR protein in bacterial ingestion and clearance have all been postulated. The high P. aeruginosa infection rate in CF patients can potentially be explained by the specificity of the interaction between the CFTR and P. aeruginosa.     

A Pseudomonas aeruginosa toxin that hijacks the host ubiquitin proteolytic system

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens.     

Cell culture models demonstrate that CFTR dysfunction leads to defective fatty acid composition and metabolism

Cystic fibrosis (CF) is associated with fatty acid alterations characterized by low linoleic and docosahexaenoic acid. It is not clear whether these fatty acid alterations are directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction or result from nutrient malabsorption. We hypothesized that if fatty acid alterations are a result of CFTR dysfunction, those alterations should be demonstrable in CF cell culture models. Two CF airway epithelial cell lines were used: 16HBE, sense and antisense CFTR cells, and C38/IB3-1 cells. Wild-type (WT) and CF cells were cultured in 10% fetal bovine serum (FBS) or 10% horse serum. Fatty acid levels were analyzed by GC-MS. Culture of both WT and CF cells in FBS resulted in very low linoleic acid levels. When cells were cultured in horse serum containing concentrations of linoleic acid matching those found in human plasma, physiological levels of linoleic acid were obtained and fatty acid alterations characteristic of CF tissues were then evident in CF compared with WT cells. Kinetic studies with radiolabeled linoleic acid demonstrated in CF cells increased conversion to longer and more-desaturated fatty acids such as arachidonic acid. In conclusion, these data demonstrate that CFTR dysfunction is associated with altered fatty acid metabolism in cultured airway epithelial cells.     

Up-regulation of AMP-activated kinase by dysfunctional cystic fibrosis transmembrane conductance regulator in cystic fibrosis airway epithelial cells mitigates excessive inflammation

AMP-activated kinase (AMPK) is a ubiquitous metabolic sensor that inhibits the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). To determine whether CFTR reciprocally regulates AMPK function in airway epithelia and whether such regulation is involved in lung inflammation, AMPK localization, expression, and activity and cellular metabolic profiles were compared as a function of CFTR status in CF and non-CF primary human bronchial epithelial (HBE) cells. As compared with non-CF HBE cells, CF cells had greater and more diffuse AMPK staining and had greater AMPK activity than their morphologically matched non-CF counterparts. The cellular [AMP]/[ATP] ratio was higher in undifferentiated than in differentiated non-CF cells, which correlated with AMPK activity under these conditions. However, this nucleotide ratio did not predict AMPK activity in differentiating CF cells. Inhibiting channel activity in non-CF cells did not affect AMPK activity or metabolic status, but expressing functional CFTR in CF cells reduced AMPK activity without affecting cellular [AMP]/[ATP]. Therefore, lack of functional CFTR expression and not loss of channel activity in CF cells appears to up-regulate AMPK activity in CF HBE cells, presumably through non-metabolic effects on upstream regulatory pathways. Compared with wild-type CFTR-expressing immortalized CF bronchial epithelial (CFBE) cells, DeltaF508-CFTR-expressing CFBE cells had greater AMPK activity and greater secretion of tumor necrosis factor-alpha and the interleukins IL-6 and IL-8. Further pharmacologic AMPK activation inhibited inflammatory mediator secretion in both wild type- and DeltaF508-expressing cells, suggesting that AMPK activation in CF airway cells is an adaptive response that reduces inflammation. We propose that therapies to activate AMPK in the CF airway may be beneficial in reducing excessive airway inflammation, a major cause of CF morbidity.