Nonclassical Pathway ofPseudomonas aeruginosaDNA-Induced Interleukin-8 Secretion in Cystic Fibrosis Airway Epithelial Cells

Abstract

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.

Pseudomonal airway colonisation: risk factor for bronchiolitis obliterans syndrome after lung transplantation?

“…QSM have an intrinsic immunomodulatory capacity resulting in suppressed T-cell proliferation, neutrophil chemotaxis and chemokine (especially IL-8) or cytokine release from bronchial epithelial cells [41][42][43][44]. As the present author's have previously shown that transient airway colonisation after LTx is associated with airway inflammation [17], it is conceivable that chronic airway colonisation with P. aeruginosa causes IL-8-dependent neutrophilic airway inflammation (as confirmed by the present authors' current data; see supplementary material), eventually leading to a self-perpetuating cycle of airway damage and, subsequently, airway remodelling, as seen in chronic obstructive pulmonary disease [2], CF [13][14][15] and bronchiectasis [45]. Since neutrophilic airway inflammation is accepted as the main characteristic of chronic allograft rejection in LTx [46][47][48], it can be presumed that persistent airway colonisation with Pseudomonads facilitates the development of BOS after LTx.…”

“…Colonisation with mucoid or multiple-antibiotic resistant P. aeruginosa or B. cepacia is associated with a worse prognosis, particularly in cystic fibrosis (CF) patients [10][11][12]. It has been extensively shown that colonisation triggers expression of diverse cytokines by structural airway cells, inducing neutrophil recruitment and thereby perpetuating a cycle of airway inflammation and destruction [13][14][15][16][17][18]. However, whether pseudomonal airway colonisation after LTx occurs secondary to the airway remodelling in bronchiolitis obliterans syndrome (BOS), the major cause of late graft failure and death in long-term survivors after LTx [19], or similarly plays a primordial role in its aetiology and progression remains elusive [7,[20][21][22].…”

Elevated furin levels in human cystic fibrosis cells result in hypersusceptibility to exotoxin A–induced cytotoxicity

“…Mutations in CFTR have pleiotropic effects on the function of other ion transporters (2,3). Of particular importance is the CFTR-dependent regulation of the amiloride-sensitive epithelial sodium channel in respiratory epithelial cells (2,3), causing electrolyte and fluid hyperabsorption in the CF lung (2,4) and hyperacidification of intracellular organelles of CF lung epithelial cells including trans-Golgi network (TGN) and endosomal compartments (5)(6)(7)(8)(9)(10). In addition, there is an abundance of proposed defects related to pathology in CF, including defective phagosomal acidification in macrophages (11), altered P. aeruginosa uptake by respiratory epithelial cells (12), impaired glycosylation (13), enhanced Toll-like receptor activation (14), reduced levels of iNOS (15), and disrupted cholesterol transport (16).…”

Inhibition of Adhesion and Invasion of Pseudomonas aeruginosa to Lung Epithelial Cells: A Model of Cystic Fibrosis Infection

“…Dextran was applied at a concentration level of 50.0 mg/ml. MIC of ciprofloxacin and natural extracts against P. aeruginosa PAO1 and MCC of the same agents on A549 lung epithelial cells were determined according to CLSI criteria and the method described by Gillies et al [10], respectively. The results showed that all the tested natural extracts had relatively high concentrations of MICs against P. aeruginosa PAO1 and of MCC on A549 lung epithelial cells (Table 1).…”

“…MICs were determined according to the Clinical and Laboratory Standards Institute (CLSI, M7-A7 and M100-S16, Criteria for Pseudomonas aeruginosa). Cytotoxicity of the tested agents against the A549 lung epithelial cell line was determined using crystal violet nuclei staining method as described by Gillies, et al (1986) [10] with minor modifications. Briefly, confluent monolayer of A549 lung epithelial cell was incubated for 2 hrs with 200 µl of two-fold serial dilutions of different tested agents prepared in F12-K Medium, and then the cells were washed twice with phosphate-buffered saline (PBS).…”