Pseudomonas infections Part 1

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Clin Invest, February 2002, Volume 109, Number 3, 317-325

Copyright ©2002 by the American Society for Clinical Investigation

Effects of reduced mucus oxygen concentration in airway Pseudomonas

infections of cystic fibrosis patients

The

University of North Carolina, Chapel Hill, Chapel Hill, North Carolina

27599, USA. Phone: ; Fax: ; E-mail:

rboucher@... for publication July 31, 2001, and accepted

in revised form December 19, 2001.

Current theories of CF pathogenesis predict different predisposing " local

environmental " conditions and sites of bacterial infection within CF

airways. Here we show that, in CF patients with established lung disease,

Psuedomonas aeruginosa was located within hypoxic mucopurulent masses in

airway lumens. In vitro studies revealed that CF-specific increases in

epithelial O2 consumption, linked to increased airway surface liquid

(ASL) volume absorption and mucus stasis, generated steep hypoxic

gradients within thickened mucus on CF epithelial surfaces prior to

infection. Motile P. aeruginosa deposited on CF airway surfaces

penetrated into hypoxic mucus zones and responded to this environment

with increased alginate production. With P. aeruginosa growth in oxygen

restricted environments, local hypoxia was exacerbated and frank

anaerobiosis, as detected in vivo, resulted. These studies indicate that

novel therapies for CF include removal of hypoxic mucus plaques and

antibiotics effective against P. aeruginosa adapted to anaerobic

environments.

    Introduction

Lung infections with Pseudomonas aeruginosa constitute the predominant

disease phenotype in cystic fibrosis (CF) patients (1). Despite a

vigorous and rapid influx of functional peripheral blood neutrophils into

infected CF airways (2) accompanied by the production of high titers of

specific Ab’s, P. aeruginosa infections become chronic (3), airways are

destroyed, and lung function declines. Several hypotheses have been

offered to explain the failure of mucosal defense and the high prevalence

of P. aeruginosa in the CF lung. Most hypotheses have focused on

bacterial infection of CF airway epithelia, mediated by an increased

binding of P. aeruginosa to the surfaces of CF airway epithelial cells

(4, 5) impaired internalization and killing of P. aeruginosa by CF airway

epithelia due to the absence of cystic fibrosis transmembrane conductance

regulator (CFTR) at the apical surface (6), or " high-salt " –mediated

defensin inactivation (7). In contrast, the reduced airway surface liquid

(ASL) volume (impaired mucus clearance) hypothesis predicts infection by

P. aeruginosa and other pathogens of stationary mucus adherent to airway

surfaces. As an approach to distinguish among these hypotheses, we sought

to identify the site of P. aeruginosa infection in freshly excised CF

airways, differentiating between the intraluminal (mucus) and epithelial

surface compartments. Based on our observations that P. aeruginosa

resided in the intraluminal contents, we asked what conditions confronted

P. aeruginosa in this microenvironment in vivo in CF patients, focusing

on O2 availability. We extended these studies to investigate in vitro

whether airway mucus hypoxia (O2 gradients) was present in thickened CF

mucus before infection and whether steep O2 gradients within mucus were

unique to the CF genotype. Finally, we explored the hypotheses that (a)

bacteria deposited on airway surfaces penetrate into hypoxic mucus, and

( P. aeruginosa responses to hypoxia, e.g., increased alginate

production, may favor its persistence in the CF lung.

    Methods

Study subjects: normal, CF, and disease controls. Lungs from seven CF

patients chronically infected with P. aeruginosa were obtained for

morphometric analyses after lung transplantation (five males and two

females, mean age 29.5 years; Cystic Fibrosis/Pulmonary Research and

Treatment Center, University of North Carolina, Chapel Hill, Chapel Hill,

North Carolina, USA) or after lobectomy (two females, mean age 8 years;

Service de Pédiatrie, Centre Hospitalier Lyon-Sud, Pierre-Bénite,

France). Nasal polyps from four CF patients (mean age 14.3 years; Ear,

Nose and Throat Clinic, Klinikum Ludwigshafen, Germany), and from four

non-CF individuals (mean age 46.5 years; Ear, Nose and Throat Clinic,

University of Tübingen, Germany) were used for spheroid cell cultures.

Cells for planar cell cultures were obtained from seven normal lung

transplant donors (four males, three females, mean age 42 ± 6 years),

eight CF lung transplant recipients (four males, three females, mean age

34 ± 3 years), and two primary ciliary dyskinesia (PCD) lung transplant

recipients (one male, one female, ages 15 and 50 years, respectively).

For in vivo oxygen partial pressure (pO2) measurements, six CF patients

(two males, four females, mean age 23.8 years; mean forced expiratory

volume in one second, 55.7% predicted) were studied. Data were

successfully obtained from three patients. Informed consent was obtained

from all patients and/or parents, and all parts of the study were

approved by the local ethical committees. Bacterial strains. PAO1 (8)

bacteria were grown for adhesion experiments in vitro overnight at 37°C

in 5 ml Trypticase soy broth (TSB; Oxoid Ltd., Basingstoke, United

Kingdom). A bacterial suspension (10–50 µl) of the overnight culture

(OD600nm 0.05) was inoculated into 5 ml fresh TSB medium and the bacteria

cultured until the OD600nm of 1.5 was reached. For confocal microscopy

studies, the P. aeruginosa strain ATCC 27853 was used. For alginate

measurements, 15 genetically different non-CF (environmental) strains of

P. aeruginosa as well as PAO1 were analyzed, and for growth in ASL, PAO1

and P. aeruginosa ATCC 700829 were tested. PAO1 grown in TSB was used for

O2 measurements. Microscopy of lung sections. Immediately after

resection, lung tissues were cut into 0.5-cm3 cubes and fixed in 2.5%

glutaraldehyde, 10% formaldehyde, or shock-frozen in liquid nitrogen. For

immunofluorescence, thin sections (5–10 µm) were prepared from

shock-frozen lung tissues and P. aeruginosa identified in

bronchi/bronchioli with polyclonal rabbit IgG specific for whole P.

aeruginosa cells; then incubation occurred with

indocarbocyanin-conjugated (Cy3-conjugated) goat anti-rabbit IgG

(Dianova, Hamburg, Germany), diluted 1:500. Eight sections from each of

nine separate CF lung samples (72 sections total) were analyzed for P.

aeruginosa location, using the KS300 Imaging System (Kontron Electronic

GmbH, Eching, Germany). For transmission electron microscopy (TEM),

segmental bronchi (6.5-mm sections) from nine CF lungs were postfixed in

OSO4, thin sections cut, and uracyl acetate/lead citrate stained. For

scanning electron micrographs, specimens were processed as described

previously (9). Squares (406) from 14 bronchi from two CF lungs were

analyzed for binding of P. aeruginosa to the epithelium. Adhesion of P.

aeruginosa to mucus adherent to primary nasal epithelial spheroids. The

spheroid cell culture system was used as described previously (9). To

collect secreted mucus, spheroids were incubated in DMEM/Ham’s F12 medium

(Life Technologies Inc., Heidelberg, Germany) depleted of antibiotics and

antimycotics for 5 days, supernatant collected by centrifugation (225 g),

and stored at –20°C until use. Spheroids (8 weeks; four CF and four

normal [NL]), suspended in DMEM/Ham’s F12 medium, were incubated with P.

aeruginosa at a cell/bacteria ratio of 1:100 for 2 hours/37°C/5% CO2. In

some experiments, mucus was removed from spheroids by prewashing with

PBS. After incubation with P. aeruginosa, spheroids were washed using a

cell strainer (Becton Dickinson, Heidelberg, Germany). Twenty to 30

spheroids per individual were analyzed for adherence of P. aeruginosa by

scanning electron microscopy (9). P. aeruginosa was incubated with

spheroid supernatants containing mucus for 2 hours at 37°C/5% CO2. After

washing, bacteria were fixed on coverslips, incubated with a mAb to human

mucins, washed, incubated with a Cy3-conjugated goat anti-mouse IgG (DAKO

Corp., Hamburg, Germany) for 40 minutes at 23°C, washed with water, and

embedded in Permafluor (Sigma Chemical Co., St. Louis, Missouri, USA).

Fiberoptic bronchoscopy. Fiberoptic bronchoscopy was performed as

described previously (2) with minor modifications. For in vivo pO2

measurements, a computerized type oxygen probe (length: 65 cm;

outer diameter: 2 mm; inner diameter: 0.4 mm; Licox pO2; GMS, Kiel,

Germany) was fixed to the tip of the bronchoscope and guided under video

control into right upper lobes obstructed with mucopurulent material.

Planar, primary bronchial culture system. Human airway epithelial cells

were obtained from freshly excised bronchi by protease digestion (10),

seeded directly on 12-mm Transwell Col membranes (Corning-Costar Corp.,

Cambridge, Massachusetts, USA) in modified bronchial epithelial growth

medium under air-liquid interface conditions and studied when fully

differentiated (2–5 weeks; transepithelial resistance of  350  cm2).

Measurement of ASL pO2–planar bronchial cultures. O2 microelectrodes were

purchased from Diamond General Development Corp. (Ann Arbor, Michigan,

USA). The O2 microelectrode and 3 M KCl reference electrode were advanced

into ASL with micromanipulators as described previously (11). Confocal

microscopy measurements of ASL/P. aeruginosa. PBS (30 µl) containing 2

mg/ml Texas Red-dextran (10 kDa; Molecular Probes Inc., Eugene, Oregon,

USA) was added to CF cultures 2–48 hours before the addition of bacteria

or fluorescent beads (1 µm; Molecular Probes Inc.) as described

previously (12). For all studies, perfluorocarbon (FC-77, 3M Co., St.

, Minnesota, USA) was added to the mucus surface to prevent ASL

evaporation. P. aeruginosa bacteria were suspended in 3 ml PBS (OD470 of

0.15 [107 CFU/ml]) and incubated with 5 µM SYTO 13 (Molecular Probes

Inc.) for 1 hour at 37°C. The bacterial suspension was washed once in

PBS, centrifuged, and the pellet resuspended in PBS (100 µl). Bacterial

growth and production of alginate by P. aeruginosa in aerobic and

anaerobic culture conditions. To determine whether P. aeruginosa is able

to grow in freshly harvested ASL from CF and NL well-differentiated

cultures (11) under aerobic and anaerobic (anaerobic chamber from Coy

Laboratory Products, Gross Lake, Michigan, USA) conditions, a small

number of bacteria (100–200 CFU/0.5 µl) of PAO1 or the environmental P.

aeruginosa strain (ATCC 700829) was added to 30 µl of ASL in parallel in

two titer plates. For these experiments, the bacteria were grown on sheep

blood agar overnight, suspended in PBS, with this suspension adjusted to

an OD470nm of 0.15, diluted 1:100 in PBS, and starved for 2 hours before

addition to ASL. Titer plates were incubated aerobically and

anaerobically for 72 hours at 37°C. To determine the number of bacteria

in ASL, samples were serially diluted and plated onto agar. We next

measured the alginate mass/bacterial protein mass under aerobic and

anaerobic conditions, using anaerobic jars and Anaerocult A (Merck KGaA,

Darmstadt, Germany), with strain PAO1 and 15 environmental P. aeruginosa

strains grown on Pseudomonas isolation agar (PIA). After 4 days of

growth, the bacteria were rinsed with water from the plates and the

alginate was measured by the carbazole assay (13). Uronic acids were

quantified using a standard curve of alginate purified from Macrocystis

pyrifera (Sigma Chemical Co.), followed by the BCA protein assay to

estimate bacterial protein mass (Pierce Chemical Co., Rockford, Illinois,

USA). In parallel, we visualized P. aeruginosa alginate by immunostaining

PAO1, grown on Columbia sheep blood agar and fixed on slides, with rabbit

antiserum specific for P. aeruginosa alginate followed by Cy3-labeled

goat anti-rabbit IgG (Dianova). Finally, we examined the role of nitrate

as a terminal electron acceptor in alginate production under aerobic

versus anaerobic conditions. PAO1 was grown on PIA agar plates (63 µM

nitrate without added nitrate) to which a range of KNO3 was added (10

µM–100 mM), and after 4 days of growth, alginate and bacterial protein

was quantitated as above. Measurements of ASL total nitrate

concentration. Total nitrate (nitrite/nitrate) concentrations in CF and

NL ASL (each CF/NL sample was obtained from pooled ASL collections from

cultures derived from ten or more different subjects) were measured in 10

µl aliquots using a Nitric Oxide Analyzer (Sievers Model 280b, Ionics

Instrument Business Group, Boulder, Colorado, USA). Bacteria and in vitro

measurements of pO2. Using the Licox oxygen probe, pO2 was measured at

37°C in suspensions of PAO1 grown aerobically in TSB (Oxoid Ltd.). The

pO2 was also measured in heat-inactivated washed bacterial cultures at

108 CFU/ml. Statistics. Unless otherwise stated, all data are presented

as mean ± SEM. ANOVA (followed by the Tukey test) was used as

appropriate. In the case of nonhomogeneity of variance, ANOVA followed by

either Dunn’s multiple comparison test, the Mann-Whitney U test, or the

Wilcoxon signed rank test were used.

Becki

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