Pseudomonas aeruginosa chronically infects the lungs of more than 80% of adult patients with cystic fibrosis (CF) and is a major contributor to the progression of disease pathology. P. aeruginosa requires iron for growth and has multiple iron uptake systems that have been studied in bacteria grown in laboratory culture. The purpose of this research was to determine which of these are active during infection in CF. RNA was extracted from 149 sputum samples obtained from 23 CF patients. Reverse transcription–quantitative real-time PCR (RT-qPCR) was used to measure the expression of P. aeruginosa genes encoding transport systems for the siderophores pyoverdine and pyochelin, for heme, and for ferrous ions. Expression of P. aeruginosa genes could be quantified in 89% of the sputum samples. Expression of genes associated with siderophore-mediated iron uptake was detected in most samples but was at low levels in some samples, indicating that other iron uptake mechanisms are active. Expression of genes encoding heme transport systems was also detected in most samples, indicating that heme uptake occurs during infection in CF. feoB expression was detected in all sputum samples, implying an important role for ferrous ion uptake by P. aeruginosa in CF. Our data show that multiple P. aeruginosa iron uptake mechanisms are active in chronic CF infection and that RT-qPCR of RNA extracted from sputum provides a powerful tool for investigating bacterial physiology during infection in CF.

ICAM-1 is a major receptor for ~60% of human rhinoviruses, and non-typeable Haemophilus influenzae, two major pathogens in COPD. Increased cell-surface expression of ICAM-1 in response to tobacco smoke exposure has been suggested. We have investigated epithelial ICAM-1 expression in both the large and small airways, and lung parenchyma in smoking-related chronic airflow limitation (CAL) patients. We evaluated epithelial ICAM-1 expression in resected lung tissue: 8 smokers with normal spirometry (NLFS); 29 CAL patients (10 small-airway disease; 9 COPD-smokers; 10 COPD ex-smokers); Controls (NC): 15 normal airway/lung tissues. Immunostaining with anti-ICAM-1 monoclonal antibody was quantified with computerized image analysis. The percent and type of cells expressing ICAM-1 in large and small airway epithelium and parenchyma were enumerated, plus percentage of epithelial goblet and submucosal glands positive for ICAM- 1. A major increase in ICAM-1 expression in epithelial cells was found in both large (p < 0.006) and small airways (p < 0.004) of CAL subjects compared to NC, with NLFS being intermediate. In the CAL group, both basal and luminal areas stained heavily for ICAM-1, so did goblet cells and sub-mucosal glands, however in either NC or NLFS subjects, only epithelial cell luminal surfaces stained. ICAM-1 expression on alveolar pneumocytes (mainly type II) was slightly increased in CAL and NLFS (p < 0.01). Pack-years of smoking correlated with ICAM-1 expression (r = 0.49; p < 0.03). Airway ICAM-1 expression is markedly upregulated in CAL group, which could be crucial in rhinoviral and NTHi infections. The parenchymal ICAM-1 is affected by smoking, with no further enhancement in CAL subjects.

Background: COPD is emerging as the third largest cause of human mortality worldwide after heart disease and stroke. Tobacco smoking, the primary risk factor for the development of COPD, induces increased expression of platelet-activating factor receptor (PAFr) in the lung epithelium. Nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae adhere to PAFr on the luminal surface of human respiratory tract epithelial cells. Objective: To investigate PAFr as a potential drug target for the prevention of infections caused by the main bacterial drivers of acute exacerbations in COPD patients, NTHi and S. pneumoniae. Methods: Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE). PAFr expression levels were determined using immunocytochemistry and quantitative polymerase chain reaction. The epithelial cells were challenged with either NTHi or S. pneumoniae labeled with fluorescein isothiocyanate, and bacterial adhesion was measured using immunofluorescence. The effect of a well-evaluated antagonist of PAFr, WEB-2086, on binding of the bacterial pathogens to BEAS-2B cells was then assessed. In silico studies of the tertiary structure of PAFr and the binding pocket for PAF and its antagonist WEB-2086 were undertaken. Results: PAFr expression by bronchial epithelial cells was upregulated by CSE, and significantly associated with increased bacterial adhesion. WEB-2086 reduced the epithelial adhesion by both NTHi and S. pneumoniae to levels observed for non-CSE-exposed cells. Furthermore, it was nontoxic toward the bronchial epithelial cells. In silico analyses identified a binding pocket for PAF/WEB-2086 in the predicted PAFr structure. Conclusion: WEB-2086 represents an innovative class of candidate drugs for inhibiting PAFr-dependent lung infections caused by the main bacterial drivers of smoking-related COPD.

Background Intravenous antibiotics for pulmonary exacerbations (PEs) of cystic fibrosis (CF) usually target Pseudomonas aeruginosa. Insights into the CF lung microbiome have questioned this approach. We used RT-qPCR to determine whether intravenous antibiotics reduced P. aeruginosa numbers and whether this correlated with improved lung function. We also investigated antibiotic effects on other common respiratory pathogens in CF. Methods Sputa were collected from patients when stable and again during a PE. Sputa were expectorated into a RNA preservation buffer for RNA extraction and preparation of cDNA. qPCR was used to enumerate viable P. aeruginosa as well as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Burkholderia cepacia complex and Aspergillus fumigatus. Results Fifteen CF patients were followed through 21 PEs. A complete set of serial sputum samples was unavailable for two patients (three separate PEs). P. aeruginosa numbers did not increase immediately prior to a PE, but numbers during intravenous antibiotic treatment were reduced ≥4-log in 6/18 and ≥1-log in 4/18 PEs. In 7/18 PEs, P. aeruginosa numbers changed very little with intravenous antibiotics and one patient demonstrated a ≥2-log increase in P. aeruginosa load. H. influenzae and S. pneumoniae were detected in ten and five PEs respectively, but with antibiotic treatment these bacteria rapidly became undetectable in 6/10 and 4/5 PEs, respectively. There was a negative correlation between P. aeruginosa numbers and FEV₁ during stable phase (r_s=0.75, p<0.05), and reductions in P. aeruginosa load with intravenous antibiotic treatment correlated with improved FEV₁ (r_s=0.52, p<0.05). Conclusions Exacerbations are not due to increased P. aeruginosa numbers in CF adults. However, lung function improvements correlate with reduced P. aeruginosa burden suggesting that current antibiotic treatment strategies remain appropriate in most patients. Improved understanding of PE characterised by unchanged P. aeruginosa numbers and minimal lung function improvement following treatment may allow better targeted therapies.