scholarly journals Pseudomonas aeruginosa LasB protease impairs innate immunity in mice and humans by targeting a lung epithelial cystic fibrosis transmembrane regulator–IL-6–antimicrobial–repair pathway

Thorax ◽  
2017 ◽  
Vol 73 (1) ◽  
pp. 49-61 ◽  
Author(s):  
Vinciane Saint-Criq ◽  
Bérengère Villeret ◽  
Fabien Bastaert ◽  
Saadé Kheir ◽  
Aurélie Hatton ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Innate Immunity ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Ion Transport ◽  
Cell Lines ◽  
Virulence Factor ◽  
Cystic Fibrosis Transmembrane

BackgroundPseudomonas aeruginosa lung infections are a huge problem in ventilator-associated pneumonia, cystic fibrosis (CF) and in chronic obstructive pulmonary disease (COPD) exacerbations. This bacterium secretes virulence factors that may subvert host innate immunity.ObjectiveWe evaluated the effect of P. aeruginosa elastase LasB, an important virulence factor secreted by the type II secretion system, on ion transport, innate immune responses and epithelial repair, both in vitro and in vivo.MethodsWild-type (WT) or cystic fibrosis transmembrane conductance regulator (CFTR)-mutated epithelial cells (cell lines and primary cells from patients) were treated with WT or ΔLasB pseudomonas aeruginosa O1 (PAO1) secretomes. The effect of LasB and PAO1 infection was also assessed in vivo in murine models.ResultsWe showed that LasB was the most abundant protein in WT PAO1 secretomes and that it decreased epithelial CFTR expression and activity. In airway epithelial cell lines and primary bronchial epithelial cells, LasB degraded the immune mediators interleukin (IL)-6 and trappin-2, an important epithelial-derived antimicrobial molecule. We further showed that an IL-6/STAT3 signalling pathway was downregulated by LasB, resulting in inhibition of epithelial cell repair. In mice, intranasally instillated LasB induced significant weight loss, inflammation, injury and death. By contrast, we showed that overexpression of IL-6 and trappin-2 protected mice against WT-PAO1-induced death, by upregulating IL-17/IL-22 antimicrobial and repair pathways.ConclusionsOur data demonstrate that PAO1 LasB is a major P. aeruginosa secreted factor that modulates ion transport, immune response and tissue repair. Targeting this virulence factor or upregulating protective factors such as IL-6 or antimicrobial molecules such as trappin-2 could be beneficial in P. aeruginosa-infected individuals.

scholarly journals Factors Influencing Adeno-Associated Virus-Mediated Gene Transfer to Human Cystic Fibrosis Airway Epithelial Cells: Comparison with Adenovirus Vectors

1998 ◽  
Vol 72 (11) ◽  
pp. 8904-8912 ◽  
Author(s):  
S. Teramoto ◽  
J. S. Bartlett ◽  
D. McCarty ◽  
X. Xiao ◽  
R. J. Samulski ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Transfer ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Adeno Associated Virus ◽  
Differentiated Cells ◽  
Factors Influencing

ABSTRACT Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZgene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should impact successful gene delivery in CF patients.

scholarly journals Differential Sensitivity of Human Epithelial Cells to Pseudomonas aeruginosa Exoenzyme S

1999 ◽  
Vol 67 (7) ◽  
pp. 3494-3503 ◽  
Author(s):  
Eileen M. McGuffie ◽  
Jennifer E. Fraylick ◽  
Debra J. Hazen-Martin ◽  
Timothy S. Vincent ◽  
Joan C. Olson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Cell Function ◽  
Model Systems ◽  
Differential Sensitivity ◽  
Intrinsic Resistance ◽  
Exoenzyme S ◽  
Morphological Alterations

ABSTRACT Exoenzyme S (ExoS) is an ADP-ribosyltransferase produced and directly translocated into eukaryotic cells by the opportunistic pathogen Pseudomonas aeruginosa. Model systems that allow bacterial translocation of ExoS have found ExoS to have multiple effects on eukaryotic cell function, affecting DNA synthesis, actin cytoskeletal structure, and cell matrix adherence. To understand mechanisms underlying differences observed in cell sensitivities to ExoS, we examined the effects of bacterially translocated ExoS on multiple human epithelial cell lines. Of the cell lines examined, confluent normal kidney (NK) epithelial cells were most resistant to ExoS, while tumor-derived cell lines were highly sensitive to ExoS. Analysis of the mechanisms of resistance indicated that cell association as well as an intrinsic resistance to morphological alterations were associated with increased resistance to ExoS. Conversely, increased sensitivity to ExoS appeared to be linked to epithelial cell growth, with tumor cells capable of undergoing non-contact-inhibited, anchorage-independent growth all being sensitive to ExoS, and NK cells becoming sensitive to ExoS when subconfluent and growing. Consistent with the possibility that growth-related, actin-based structures are involved in sensitivity to ExoS, scanning electron microscopy revealed cellular extensions from sensitive, growing cells to bacteria, which were not readily evident in resistant cells. In all studies, the severity of effects of ExoS on cell function directly correlated with the degree of Ras modification, indicating that sensitivity to ExoS in some manner related to the efficiency of ExoS translocation and its ADP-ribosylation of Ras. Our results suggest that factors expressed by growing epithelial cells are required for the bacterial contact-dependent translocation of ExoS; as normal epithelial cells differentiate into polarized confluent monolayers, expression of these factors is altered, and cells in turn become more resistant to the effects of ExoS.

scholarly journals microRNA regulation of expression of the cystic fibrosis transmembrane conductance regulator gene

2011 ◽  
Vol 438 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Austin E. Gillen ◽  
Nehal Gosalia ◽  
Shih-Hsing Leir ◽  
Ann Harris
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Chloride Transport ◽  
Intestinal Cell ◽  
Transmembrane Conductance Regulator ◽  
Regulator Gene ◽  
Transmembrane Conductance ◽  
General Role ◽  
Cystic Fibrosis Transmembrane

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex temporal and spatial pattern of expression that is controlled by multiple cis-acting elements interacting with the basal promoter. Although significant progress has been made towards understanding these genomic elements, there have been no reports of post-transcriptional regulation of CFTR by miRNAs (microRNAs). In the present study, we identify two miRNAs, hsa-miR-145 and hsa-miR-494, which regulate CFTR expression by directly targeting discrete sites in the CFTR 3′ UTR (untranslated region). We show that at least 12 miRNAs are capable of repressing endogenous CFTR mRNA expression in the Caco-2 cell line. Ten of these also inhibit expression of a reporter construct containing the CFTR 3′ UTR in one or more cell lines, and five repress endogenous CFTR protein expression in Caco-2 cells. Moreover, at least three are expressed in primary human airway epithelial cells, where CFTR expression is maintained at low levels in comparison with intestinal cell lines. Three of the miRNAs that target CFTR, hsa-miR-384, hsa-miR-494 and hsa-miR-1246, also inhibit expression of a reporter carrying the Na+–K+–Cl− co-transporter SLC12A2 [solute carrier family 12 (Na+–K+–Cl− transporters), member 2] 3′ UTR, suggesting that these miRNAs may play a more general role in regulating chloride transport in epithelial cells.

Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype

1993 ◽  
Vol 264 (5) ◽  
pp. C1219-C1230 ◽  
Author(s):  
J. R. Yankaskas ◽  
J. E. Haizlip ◽  
M. Conrad ◽  
D. Koval ◽  
E. Lazarowski ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Ion Transport ◽  
Cell Lines ◽  
Cell Cultures ◽  
Airway Epithelial Cell ◽  
Airway Epithelial ◽  
Phenotypic Properties ◽  
E6 And E7 ◽  
Hpv 18

Human airway epithelial cell lines that retain phenotypic properties representative of the native tissue will be useful physiological models. Human papilloma viral (HPV) genes can immortalize human genital keratinocytes and breast and bronchial epithelia. We transfected cystic fibrosis (CF) and normal tracheobronchial epithelial cell cultures with DNA encoding the HPV-18 E6 and E7 genes and characterized phenotypic properties of resultant cell lines. Of the 11 CF clones isolated, 6 developed a polarized phenotype with vectorial ion transport and membrane-specific expression of histamine and purinergic receptors. The ion transport properties of these lines differed from the normal lines and approximated those of primary CF airway epithelial cell cultures more closely than do those of cell lines transformed with the simian virus 40 large T gene. When transplanted into denuded tracheal grafts, these cells can differentiate into ciliated and secretory phenotypes. We conclude that HPV-18 E6 and E7 genes are sufficient to transform human airway epithelial cells and that the resultant cell lines express differentiated phenotypic properties that approximate those of the native epithelium.

scholarly journals Lipoxin A4prevents tight junction disruption and delays the colonization of cystic fibrosis bronchial epithelial cells byPseudomonas aeruginosa

2016 ◽  
Vol 310 (11) ◽  
pp. L1053-L1061 ◽  
Author(s):  
Gerard Higgins ◽  
Coral Fustero Torre ◽  
Jean Tyrrell ◽  
Paul McNally ◽  
Brian J. Harvey ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Tight Junction ◽  
Cell Lines ◽  
Bronchial Epithelial Cell ◽  
Bacterial Invasion ◽  
Airway Epithelial ◽  
Bronchial Epithelial ◽  
Tight Junction Disruption

The specialized proresolution lipid mediator lipoxin A4(LXA4) is abnormally produced in cystic fibrosis (CF) airways. LXA4increases the CF airway surface liquid height and stimulates airway epithelial repair and tight junction formation. We report here a protective effect of LXA4(1 nM) against tight junction disruption caused by Pseudomonas aeruginosa bacterial challenge together with a delaying action against bacterial invasion in CF airway epithelial cells from patients with CF and immortalized cell lines. Bacterial invasion and tight junction integrity were measured by gentamicin exclusion assays and confocal fluorescence microscopy in non-CF (NuLi-1) and CF (CuFi-1) bronchial epithelial cell lines and in primary CF cultures, grown under an air/liquid interface, exposed to either a clinical or laboratory strains of P. aeruginosa. LXA4delayed P. aeruginosa invasion and transepithelial migration in CF and normal bronchial epithelial cell cultures. These protective effects of LXA4were inhibited by the ALX/FPR2 lipoxin receptor antagonist BOC-2. LXA4prevented the reduction in mRNA biosynthesis and protein abundance of the tight junction protein ZO-1 and reduced tight junction disruption induced by P. aeruginsosa inoculation. In conclusion, LXA4plays a protective role in bronchial epithelium by stimulating tight junction repair and by delaying and reducing the invasion of CF bronchial epithelial cells by P. aeruginsosa.

scholarly journals Pseudomonas aeruginosa-Induced Bleb-Niche Formation in Epithelial Cells Is Independent of Actinomyosin Contraction and Enhanced by Loss of Cystic Fibrosis Transmembrane-Conductance Regulator Osmoregulatory Function

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Amber L. Jolly ◽  
Desire Takawira ◽  
Olufolarin O. Oke ◽  
Sarah A. Whiteside ◽  
Stephanie W. Chang ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Opportunistic Pathogen ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Content Type ◽  
Bleb Formation ◽  
Niche Formation ◽  
Cystic Fibrosis Transmembrane

ABSTRACTThe opportunistic pathogenPseudomonas aeruginosacan infect almost any site in the body but most often targets epithelial cell-lined tissues such as the airways, skin, and the cornea of the eye. A common predisposing factor is cystic fibrosis (CF), caused by defects in the cystic fibrosis transmembrane-conductance regulator (CFTR). Previously, we showed that when P. aeruginosa enters epithelial cells it replicates intracellularly and occupies plasma membrane blebs. This phenotype is dependent on the type 3 secretion system (T3SS) effector ExoS, shown by others to induce host cell apoptosis. Here, we examined mechanisms for P. aeruginosa-induced bleb formation, focusing on its relationship to apoptosis and the CFTR. The data showed that P. aeruginosa-induced blebbing in epithelial cells is independent of actin contraction and is inhibited by hyperosmotic media (400 to 600 mOsM), distinguishing bacterially induced blebs from apoptotic blebs. Cells with defective CFTR displayed enhanced bleb formation upon infection, as demonstrated using bronchial epithelial cells from a patient with cystic fibrosis and a CFTR inhibitor, CFTR(Inh)-172. The defect was found to be correctable either by incubation in hyperosmotic media or by complementation with CFTR (pGFP-CFTR), suggesting that the osmoregulatory function of CFTR counters P. aeruginosa-induced bleb-niche formation. Accordingly, and despite their reduced capacity for bacterial internalization, CFTR-deficient cells showed greater bacterial occupation of blebs and enhanced intracellular replication. Together, these data suggest that P. aeruginosa bleb niches are distinct from apoptotic blebs, are driven by osmotic forces countered by CFTR, and could provide a novel mechanism for bacterial persistence in the host.IMPORTANCEPseudomonas aeruginosais an opportunistic pathogen problematic in hospitalized patients and those with cystic fibrosis (CF). Previously, we showed that P. aeruginosa can enter epithelial cells and replicate within them and traffics to the membrane blebs that it induces. This “bleb-niche” formation requires ExoS, previously shown to cause apoptosis. Here, we show that the driving force for bleb-niche formation is osmotic pressure, differentiating P. aeruginosa-induced blebs from apoptotic blebs. Either CFTR inhibition or CFTR mutation (as seen in people with CF) causes P. aeruginosa to make more bleb niches and provides an osmotic driving force for blebbing. CFTR inhibition also enhances bacterial occupation of blebs and intracellular replication. Since CFTR is targeted for removal from the plasma membrane when P. aeruginosa invades a healthy cell, these findings could relate to pathogenesis in both CF and healthy patient populations.

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