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*
Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and
Department of Anesthesiology and Critical Care Medicine, University of Tuebingen, Tuebingen, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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F508
Cftr allele or an allele with a Cftr stop
codon (S489X). Intranasal application achieved P.
aeruginosa lung infection in inbred C57BL/6 F508
Cftr mice, whereas F508 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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous attempts to demonstrate a defect in P. aeruginosa clearance using intranasal infection of neonatal transgenic CF mice were not successful (1), a failure we attributed to the noninbred nature of the mice we were using. Recent work (6) has shown that translocation of an intranasal inoculum of P. aeruginosa to the lungs immediately following infection was very low in neonatal mice compared with anesthetized adult mice, likely underlying our previous inability to use neonatal mice to compare P. aeruginosa clearance in wild-type and CF mice. Anesthetized adult mice were superior to unanesthetized neonatal mice in terms of translocation of the majority of an intranasal inoculum of P. aeruginosa to the lungs immediately after infection (6). Of importance was the finding that a proportion of the inoculum was found intracellularly within minutes of infection, providing evidence that uptake of P. aeruginosa by respiratory cells occurs in a situation of in vivo infection. Although Allewelt et al. (6) conducted these studies principally in inbred BALB/c mice, they mentioned that other mouse lines behaved comparably.
Recently, Chroneos et al. (7) reported no difference in the clearance of P. aeruginosa from the lungs of transgenic CF mice compared with mice with wild-type CFTR. However, they used a clinical mucoid isolate from a CF patient, strain FRD1, that does not express the complete LPS core, which is the bacterial ligand previously identified for P. aeruginosa internalization via CFTR (3, 8). In the present study, we used transgenic and wild-type CF mice in both an inbred (C57BL/6) and noninbred background to demonstrate that CFTR plays a critical role in clearing LPS-smooth nonmucoid P. aeruginosa from the lungs. We also associated clearance with cellular uptake of P. aeruginosa and further demonstrate that bacterial entry into an intact respiratory tract tissue, the trachea, can be readily observed in both mouse and monkey tissues, documenting that respiratory epithelial cell uptake of P. aeruginosa is part of the early interaction between host tissues and this organism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P. aeruginosa strain PAO1, a serogroup
O2/O5 strain sensitive to
chloramphenicol (grows only at 
3 µg chloramphenicol/ml) and
originally obtained from M. Vasil (University of Colorado,
Denver, CO) was used in these studies. Strains 149 and 324 are
LPS-smooth nonmucoid clinical isolates from CF patients early in the
course of infection. Strain 6294 is a clinical isolate from a patient
with ulcerative keratitis, and we have also demonstrated a role for
CFTR-mediated ingestion by corneal epithelial cells as part of the
pathogenic process in keratitis (9). Strain FRD1 is a
LPS-rough mucoid clinical isolate from a CF patient provided by D.
Ohman (Richmond, VA). Bacteria were seeded onto tryptic soy agar
and grown overnight at 37°C, then subcultured into tryptic soy broth,
and grown for 3 h at 37°C or overnight at 77°C with mild
rotation (200 rpm/min). Cells were pelleted, suspended, and diluted in
1% proteose peptone to an OD650 of 0.4,
equivalent to 
2 x 109 CFU/ml. Dilutions
were made in 1% proteose peptone, and the infection dose was
determined by plating serial dilutions on MacConkey plates BD
Biosciences (Cockeysville, MD).
Mouse strains and husbandry
Wild-type BALB/c and C57BL/6 mice were obtained commercially or
bred in our facility; transgenic CF mice were all bred in our facility
and all mice were housed in microisolater cages. Transgenic CF mice
included the following strains: C57BL/6 mice homozygous for the F508
allele of mouse Cftr
(B6.129S6-Cftrttm1Kth) (10),
noninbred homozygous F508 Cftr mice (11),
and doubly transgenic S489X Cftr-fatty acid binding protein
(FABP)huCftr mice (no murine Cftr protein
but expressing wild-type human (hu) CFTR protein in the
gastrointestinal tract from the huCFTR gene under the
control of the mouse FABP promoter) (12). The doubly
transgenic S489X Cftr-FABPhuCftr
mice were bred as homozygotes. The two F508
Cftr-transgenic CF mouse strains were maintained using
breeding pairs of heterozygous Cftr female mice and F508
Cftr homozygous male mice. Mouse genotype for the F508
Cftr mice was checked by PCR of DNA isolated from tails
using published protocols for each strain (10, 11) and by
phenotypic examination of tooth color, as described previously
(13). Drinking water was treated with 200 µg
gentamicin/ml to prevent growth of P. aeruginosa. Some of
the mice, both wild-type and transgenic CF, carried a mucoid
Enterobacter aerogenes in their upper respiratory tracts
that, when present, interfered with the detection of P.
aeruginosa in the lungs. The E. aerogenes could be
eliminated from the respiratory tract by treating the mice for 5 days
with 250 µg levofloxacin/ml drinking water. The levofloxacin was
removed at least 48 h before studies of P. aeruginosa
infection. We documented that there was insufficient residual
levofloxacin in the lung tissues to inhibit P. aeruginosa
growth by determining there was no difference in bacterial survival in
homogenates of lung tissues from levofloxacin-treated and
untreated mice.
Mouse infection models
Mice were anesthetized by i.p. injection of 0.2 ml of a mixture containing 65 mg/ml ketamine and 13 mg/ml xylazine in saline. For nasal infections, 10 µl of a bacterial suspension in 1% proteose peptone was applied onto each nostril giving a total dose of 1–2 x 107 CFU. The proteose peptone was used to avoid bacterial clumping and inaccurate delivery of an inoculum to an individual animal. For tracheal infection, anesthetized mice were placed on an apparatus that immobilized the canine teeth, positioning the mouse at a 60° angle. The animal’s tongue was pulled to a lateral position with tweezers to prevent the animal from swallowing the bacterial solution. A curved plastic microtube adapted to a microsyringe was used to administer 1–2 x 107 CFU in 20–50 µl at the opening of the trachea, being careful to avoid tracheal damage that could occur due to insertion of the plastic tube into the trachea.
Control trials were conducted to assess the success of infection by these two application routes. The anesthetized mice were sacrificed by cervical dislocation directly after the bacteria were applied. The trachea, lung, esophagus, and stomach were removed and homogenized in tryptic soy broth containing 0.5% Triton X-100. Serial dilutions were plated on MacConkey plates then incubated at 37°C overnight to determine the levels of bacteria in each tissue.
For the experimental evaluations, the animals were sacrificed by cervical dislocation at various time intervals after infection, with the majority of the studies being conducted 4.5 h after infection. The lungs were removed, weighed, and passed through a wire-mesh screen to obtain a single-cell suspension in tissue culture F12 medium (6). For determination of the total amount of bacteria in a tissue, including both extracellular and intracellular organisms, 0.5% Triton X-100 was added to a measured portion of the single-cell suspension. The amount of intracellular bacteria was determined by centrifuging another measured portion, resuspending the cells in F12 medium with 10% FCS containing 400 µg gentamicin/ml, and incubating the suspension for 1 h at 37°C. The cells were then washed three times in F12 medium to remove the antibiotic and then suspended in F12 medium with 0.5% Triton X-100 to release intracellular bacteria. Serial dilutions were plated on MacConkey plates and incubated at 37°C overnight.
Primate trachea infection
Tracheas from healthy rhesus monkeys (Macaca mulatta)
housed at the New England Regional Primate Research Center
(Southboro, MA) were obtained by surgical removal immediately after
animals were euthanized. The tracheas were placed in Ringer’s lactate
solution. Ex vivo infection was performed within 3–4 h after removal
of the tracheas. The tracheas were ligated surgically proximal of the
carina, then 5 x 107 CFU PAO1/ml in F12
medium was applied to the tracheal cavity, which was then ligated
distally of the larynx with silk 2.0 surgical sutures. The tracheas
were incubated from 3 to 18 h at 37°C in 95%
O2/5% CO2 in F12 medium.
At the end of the incubation period, the tracheas were cut into pieces
of 150–250 mg, weighed, sectioned longitudinally, and washed over a
40-min period by changing the medium at 5-min intervals. For
determination of the total number of bacteria, the samples were
incubated in F12 medium with 0.5% Triton X-100 for 30 min on ice and
then homogenized. For the assessment of intracellular bacteria, the
samples were placed in F12 medium containing 400 µg gentamicin/ml for
1 h. After the gentamicin was washed off, the pieces were
homogenized in F12 with 0.5% Triton X-100 and serial dilutions were
plated on MacConkey plates. The number of CFU was determined after
overnight incubation at 37°C.
Samples of uninfected tracheas were used for calibrating the weight (mg) of a portion of trachea to its surface area (mm2). The surface area of the samples weighing between 150 and 240 mg was estimated by measuring the sides of the sample and then calculating the area of a trapezoid. Plotting the surface area and the weight revealed a linear relationship (r2 = 0.873) in the range of the sample sizes used. The surface area of each sample after ex vivo infection with P. aeruginosa PAO1 was determined by interpolation of the linear regression fit for data containing surface area vs weight. The inoculum of a whole trachea at time 0 (N1 = CFU/mm2) and the total number of bacteria recovered from samples after various times of infection (N2 = CFU/mm2) were determined by normalizing the total CFU using the formula for the calculated surface area of the whole trachea or the sample, respectively. The ratio of N2:N1 was defined as the multiple of infection.
Confocal microscopy
Approximately 1 x 109 CFU of strain PAO1 were suspended in 10 ml PBS (0.1 M phosphate buffer, 0.15 M sodium chloride, pH 7.2), and 5 µl 10 mM stock solution of syto 17 (Molecular Probes, Eugene, OR) in water was added to stain the bacteria. These suspensions were incubated for 20 min in the dark at room temperature. The bacteria were then pelleted and washed eight times in PBS to remove unbound dye. The viability of the bacteria was unaffected by this labeling. BALB/c mice were infected intranasally with 108 CFU labeled bacteria. After 4.5 h the tracheas were removed, sectioned, and washed in F12 medium as described above. A dye that becomes fluorescent only when taken up by live mammalian cells, 5-(6)-carboxyfluorescein diacetate (5/6 CFDA; Molecular Probes), was added to the medium containing the tracheas to a final concentration of 1 µM and incubated for 30 min in the dark at room temperature. The acetate groups of the nonfluorescent 5/6 CFDA are cleaved by intracellular esterases after passive diffusion into the cells, resulting in a fluorescent signal emanating from within live eukaryotic tissues. The tracheas were washed three times to remove unbound dye. The tracheas were then placed in F12 medium and transferred into a 35-mm experimental chamber with a glass bottom (Plastek Cultureware, Ashland, MA). Confocal images were obtained with a Bio-Rad (Hercules, CA) MRC-1024/2P multiphoton instrument interfaced with a Zeiss (Oberkochen, Germany) Axiovert microscope using a 100 C-Apochromat/1.2 NA water immersion objective (Bio-Rad). The 5/6 CFDA was excited with the 488-nm line of a krypton-argon laser, and the emission was collected with a 522/DF 35-nm bandpass filter. Syto 17 was excited with a krypton-argon laser at 647 nm or in the multiphoton mode with a femptosecond-pulsed Ti-sapphire laser tuned to 680–825 nm, and the emissions were captured at 598 ± 40 nm (red color) or 680 ± 32 nm (blue color).
Scanning electron microscopy
BALB/c mice were infected intranasally as described above
(5 x 108 CFU). After 4.5 h of
incubation at 37°C, the tracheas were removed and washed as described
for the mouse infection model. Tracheas from rhesus monkeys were used
after 6 h of infection; noninfected samples were used as controls.
All samples were washed in F12 medium as described above then fixed in
2.5% glutaraldehyde in 100 mM cacodylate buffer, pH 7.2 at 4°C for
24 h. Then the tissue was washed for 1 h in this buffer with
three changes, postfixed in 1% osmium tetroxide in 100 mM cacodylate,
pH 7.2 for 2 h, and washed for 1 h in this buffer with three
changes of buffer. The sample was then dehydrated using a graded series
of ethanol concentrations. Next, the samples were critically point
dried from CO2 using a Tousimis (Rockville, MD)
Samdri PVT-3 critical-point drier. The dried samples were then
attached to a specimen mount with colloidal graphite, sputter coated
with 15 nm gold/palladium using a Tousimis Samsputter-2a coater, and
viewed on an Amray (Bedford, MA) AMR-1000 scanning electron
microscope.
Statistical analysis
ANOVA was used for multisample comparisons on parametric data, and Fisher probable least square differences (PLSD) was used for pairwise comparisons. Unpaired t tests or Mann-Whitney U tests were used for comparing two samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nasal application of P. aeruginosa to anesthetized adult BALB/c mice has recently been shown to be an effective means of studying acute P. aeruginosa pulmonary infection, because the bacteria are rapidly translocated to the lungs (6). Thus, it is a useful model for evaluating bacterial factors involved in the early establishment of infection (6) and host factors such as CFTR that contribute to innate immunity. However, when we evaluated lung infection levels following nasal application of P. aeruginosa to anesthetized transgenic CF mouse strains and compared the efficiency of translocation with that in wild-type mice, we found that CF mouse strains not in an inbred background variably translocated the inoculum to the lungs. Over half of these mice aspirated <1% of the applied inoculum, whereas others aspirated about as much of the inoculum as the wild-type mice. Plating serial dilutions of the gastrointestinal organs and the nasopharyngeal anatomic area of intranasally infected S489X-FABPhuCftr mice revealed that the applied bacteria were not swallowed but remained in the nasopharynx of these transgenic CF mice (data not shown). To be able to quantify bacterial concentrations in the respiratory tract from noninbred CF mice more accurately, and to compare actual lung infection levels with those in the wild-type strains, we established an alternative route of application by delivering the infectious inoculum to the tracheal opening in anesthetized mice. With this route of infection, the amounts of bacteria recovered from the lungs of the noninbred CF mouse strains immediately after infection (>60% of the inoculum) were similar to those found in the wild-type mouse strains (6).
Under these conditions, there were no differences in total CFU per gram lung tissue immediately after infection, with lung cell internalization of P. aeruginosa in wild-type mice comparable to that previously reported (6). In transgenic CF mice there was virtually no intracellular P. aeruginosa found immediately following infection (data not shown). Previous results (6) also documented that levels of P. aeruginosa in the lungs at time intervals ranging from 10 min to 6 h after infection showed increasing amounts of bacteria in wild-type mice; a similar result was found for transgenic CF mice (data not shown). By 6 h postinoculation, bloodstream infection can be detected in some mice (6), potentially confounding results due to the presence of bacteria in the lung vasculature. Although CF patients do not get bacteremic when chronically infected with serum-sensitive LPS-rough mucoid strains to which they also have high titers of Abs, the murine studies in this report were focused on early events in bacterial lung clearance using LPS-smooth nonmucoid strains representative of those that initially colonize CF patients. Therefore, a time interval of 4.5 h was chosen for further study because it appeared to be long enough after bacterial inoculations for differences in early aspects of clearance to be maximally manifest, allowing us to compare clearance mechanisms in the lungs of wild-type and transgenic CF mice.
We first compared the total and internalized amount of bacteria in the
lungs 4.5 h after tracheal infection of anesthetized wild-type
C57BL/6 mice with that in two strains of transgenic CF mice: noninbred
S489X FABPhuCftr mice and inbred
B6.129S6-Cftrttm1Kth. We found that lung cells of
wild-type C57BL/6 mice internalized 7.6 times more P.
aeruginosa PAO1 bacteria than did the C57BL/6 mice homozygous for
the F508 Cftr allele
(B6.129S6-Cftrttm1Kth mice) and 3.9 times more
bacteria than did the S489X-FABPhuCftr mice
(p < 0.001, ANOVA and Fisher PLSD; Fig. 1
A). The values in Fig. 1 are
expressed as the percentage of bacteria being internalized compared
with the total amount of bacteria found in the organs to account for
variation in aspiration of the inoculum among individual mice.
Coincident with the impaired internalization of P.
aeruginosa in the respiratory tract of the two transgenic CF mouse
strains, the total level of the infecting bacteria measured in the
lungs was increased 22-fold in homozygous F508 Cftr
C57BL/6 mice and 30-fold in
S489X-FABPhuCftr mice, whereas in wild-type
mice the increase in the bacterial burden over the inoculating dose was
only 10-fold (Fig. 1B; both CF strains p <
0.01, ANOVA and Fisher PLSD compared with wild-type mice).
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F508 Cftr allele using an intranasal
infection route. Inbred transgenic F508 Cftr mice
(B6.129S6-Cftrttm1Kth mice) were found to
translocate the majority of the P. aeruginosa intranasal
inoculum to the lungs (not shown). After 4.5 h of infection,
homozygous F508 Cftr mice had a 7.5-fold increase in the
CFU of P. aeruginosa in the lungs over the infecting
inoculum, whereas mice with at least one wild-type Cftr
allele had a 1.1- to 3.4-fold increase in CFU in the lung
(p < 0.01, ANOVA and Fisher PLSD, Fig. 2). In comparative terms, the P.
aeruginosa levels in the lungs of homozygous F508
Cftr C57BL/6 mice were increased by a factor of 6.8 compared
with that in heterozygous F508 Cftr C57BL/6 mice and by a
factor of 3.3 compared with that in wild-type C57BL/6 mice.
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F508 Cftr mouse.
Although these P. aeruginosa strains showed variability in
the overall level of internalization, the results were consistent with
the findings using strain PAO1. In all cases the intracellular
proportion of the organisms was significantly lower in CF mice and the
total bacterial burden significantly higher in the CF mice (Fig. 3). Thus, deficient clearance of P.
aeruginosa from the lungs and increased bacterial loads was
substantiated in three strains of transgenic CF mice with either a
F508 Cftr allele or null allele using four different
P. aeruginosa strains.
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F508 CFTR allele and the same
cell line transfected with a wild-type copy of CFTR. In two
different experiments there was little to no uptake of P.
aeruginosa strain FRD1 by either cell line when an inoculum of
2 x 106 CFU was applied to
105 cells (mean of <50 CFU of strain FRD1 per
105 cells with wild-type CFTR were recovered),
whereas 4 x 104 CFU of a comparable
inoculum of P. aeruginosa PAO1 was internalized. When
3.8 x 107 CFU of strain FRD1 was applied to
the tracheal opening of noninbred F508 Cftr mice and
wild-type controls there was no difference in internalized bacteria
4.5 h after infection, and no-significant difference in total CFU
in the lungs of the CF and wild-type mice (Fig. 4). Thus, we confirmed the finding of
Chroneos et al. (7) that there is no difference between CF
and wild-type mice in regard to clearance of strain FRD1, but also
showed this strain does not use CFTR to enter epithelial cells.
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We used confocal and scanning electron microscopy for qualitative
experiments visualizing attachment and internalization of
P. aeruginosa into cells of mice with wild-type CFTR.
The attachment and internalization of P. aeruginosa in vivo
in the course of a live infection was readily discerned under the
confocal microscope (Fig. 5). Bacteria
labeled with syto 17 could be visualized with the use of filters for
either blue or red light (Fig. 5A). Murine tissues labeled
with 5/6 CFDA gives off a green fluorophore when the 5/6 CFDA has been
cleaved inside of live cells to produce the active fluorophore (Fig. 5B). The composite photograph (Fig. 5C) clearly
showed the bacterial cells bound to the live tracheal tissue.
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D) and green-fluorescent tracheal cells (Fig. 5E) could be separately imaged, although the syto 17 dye
used to stain the P. aeruginosa also fluoresced in the green
channel under the conditions used. Confocal images were obtained in
0.5-µm steps starting from the apical surface of the trachea where
the attached bacteria were observed. In the results shown in Fig. 5, internalized bacteria were detected in scans 15 (7.5 µm from the
apical surface) to 19 (9.5 µm from the apical surface) from a total
of 23 scans; scans 14, 17, 19, and 21 are shown. The main location for
uptake of bacteria into epithelial cells appeared to be on the edge of
the transition of ciliated to nonciliated, possibly denuded, areas. Visualization of P. aeruginosa attachment and internalization in vivo by scanning electron microscopy
In vivo internalization of P. aeruginosa by
tracheal epithelial cells could also be shown by scanning electron
microscopy (Fig. 6). In addition, we
obtained images of epithelial cells loaded with bacteria and being shed
from the epithelial cell surface, findings supportive of the hypothesis
that this might be an important pathway for the clearance of P.
aeruginosa from the respiratory tract. The tracheal surface of
uninfected BALB/c mice showed a typical pattern of ciliated and
nonciliated cells (Fig. 6A). However, up to 5% of the
tracheal cell surface of infected mice was denuded (defined as loss of
ciliation along with the luminal epithelial cell layer). Artifacts
initiated by the fixation procedure that took place after removal of
the tissue from the mice were excluded by fixing the tracheas of some
infected and uninfected mice in situ, without removing the tissue from
the animal, after the mice were killed by cervical dislocation. No
difference was seen in these preparations compared with the
preparations of tissue that was fixed after removal from the animal.
Using scanning electron microscopy, we found bacteria were being
internalized mainly at the edge of the ciliated/nonciliated, denuded
areas (upper right portion of Fig. 6B). A typical
area is shown in Fig. 6, C and D, at two
different magnifications that focus in on an area of heavy adherence
and bacterial uptake by epithelial cells. Additional examples of
internalization of P. aeruginosa by tracheal epithelial
cells are shown in Fig. 6, E and F. Most of the
mammalian cells internalizing bacteria had lost parts or all of the
entire cilia. Cells heavily loaded with bacteria appeared to be
detached and lifting from the luminal cell layer (Fig. 6, D
and E). Clear examples of cells heavily laden with
surface-bound and internalized P. aeruginosa being moved by
cilia in the mucus layer were seen (Fig. 6G), as were
bacteria bound to smaller, unidentified particles (Fig. 6H).
Except for the rare appearance of single erythrocytes, no
morphologically distinct cell types, especially leukocytes, were
observed on the respiratory cell layer, although these other cell types
may have been present as unattached cells that were washed off during
tissue processing. Nonetheless, from these observations, we can
conclude that there is a significant interaction between P.
aeruginosa and respiratory epithelial cells during the course of
infection initiated by respiratory inhalation of bacteria. We were
unable to visualize this interaction in tissues taken from CF mice
because of the low levels of internalized P. aeruginosa
present in the tracheas.
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The epithelial cell-P. aeruginosa interaction was shown
not to be limited to murine tissues by the finding that P.
aeruginosa was internalized by rhesus monkey tracheal epithelial
cells 6 h after incubation with bacteria (Fig. 7). The uninfected tracheal epithelium
was composed principally of ciliated cells (Fig. 7A), and as
in the mice, the main spots of bacteria-cell interactions were at the
border of denuded and intact areas (Fig. 7B). We quantified
the number of attached cells and the percentage of internalized
bacteria per square millimeter after 3, 6, 12, and 18 h of
infection. Attachment of P. aeruginosa PAO1 was the
highest after 3 h of infection (30% ± 10% of the
inoculum), but few bacteria were found to be internalized after 3
h of infection. The maximum percentage of internalized bacteria was
found after 6 h of incubation (0.06% ± 0.01%); 10-fold fewer
bacteria were found internalized at 12 and 18 h postinfection. Use
of two other P. aeruginosa clinical isolates, 6294 and 149,
showed essentially identical kinetics for bacterial binding and
internalization by the monkey tracheas (0.06% and 0.04% internalized,
respectively, 6 h postinfection). Examples of bacterial binding
and internalization by the monkey tracheal epithelial cells are shown
in Fig. 7, C–F. It is likely that at the later
time intervals there were fewer attached and internalized bacteria
because the infected epithelial cells were lifted from the cell layer
and lost during tissue processing. Fig. 7G shows a
higher-power magnification of a cell in Fig. 7F that has
apparently curled up in the process of desquamating from the tracheal
surface. Fig. 7H shows monkey tracheal epithelial
cells with bound and internalized P. aeruginosa above
intact ciliated cells, possibly in the process of being removed by
mucociliary flow. This finding is consistent with the hypothesis that
uptake of P. aeruginosa by epithelial cells followed by
cellular desquamation is part of the mechanism for clearing these
organisms from the respiratory tract.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation and
production of cytokines and antimicrobial peptides thought to be
important in innate immunity to infection (18, 19, 20, 21). The
data presented in this study provide evidence in support of the
critical role in innate immunity of CFTR-mediated ingestion of P.
aeruginosa by showing that transgenic Cftr mice, either
lacking CFTR in the lung or homozygous for the F508 Cftr
allele have decreased internalization of P. aeruginosa in
the lung and increased levels of P. aeruginosa in the lung
tissue 4.5 h after infection. The use of a simple intranasal or
tracheal application of P. aeruginosa also provides clear
evidence that shortly after infection, P. aeruginosa gains
access to the respiratory epithelium to interact with cells and this
interaction is not dependent on previous injury to the tissue. Chroneos et al. (7) recently reported no difference in clearance of P. aeruginosa strain FRD-1 from the lungs of S489X-FABPhuCftr mice in comparison to wild-type mice, 24 h after a dose of 1.5 x 107 CFU delivered intratracheally. However, the strain they chose to use, FRD-1, is a mucoid LPS-rough clinical isolate typical of strains previously identified (3) as lacking the bacterial ligand for CFTR-mediated epithelial cell ingestion. In addition, others have reported that mucoid isolates of P. aeruginosa, which overexpress mucoid exopolysaccharide on their surface, enter epithelial cells significantly less well when compared with nonmucoid isogenic strains (22). Chroneos et al. (7) did not show that strain FRD-1 can enter epithelial cells via CFTR, and in this study we showed that strain FRD-1 did not enter a cell line with wild-type CFTR at a level detectable in this assay (<50 CFU internalized of 2 x 106 CFU added). We found, as did Chroneos et al. (7), no difference in clearance of strain FRD-1 from the lungs of wild-type or transgenic CF mice. Because P. aeruginosa strain FRD1 lacks the bacterial ligand for CFTR, we conclude that the lack of a difference in clearance of a P. aeruginosa strain FRD-1 between wild-type and Cftr mutant mice is actually highly supportive of our hypothesis.
Chroneos et al. (7) also studied strain PAO1, as we did, but they only compared clearance between wild-type mice and double-transgenic S489X-FABPhuCftr mice that had been further engineered to overexpress huCFTR in the lungs under the control of the surfactant protein C (SP-C) promoter (SP-ChuCFTR mice). They reported no difference in clearance of P. aeruginosa strain PAO1 when comparing these triple-transgenic mice and wild-type mice, and they also reported about a 7.7-fold increase in mRNA for CFTR in the Sp-ChuCFTR mice. However, mRNA levels are not informative about protein levels and overexpression of CFTR protein from levels achieved in wild-type mice may provide no additional advantage in bacterial clearance. In addition, the comparisons they made using strain PAO1 were conducted after 24 h of infection, a time interval that would not be representative of early lung clearance and wherein any data obtained would be confounded by the presence of P. aeruginosa in the lung vasculature following systemic spread (6). For strain PAO1, Chroneos et al. (7) did not compare clearance in the transgenic S489X-FABPhuCFTR mice lacking CFTR in the lung with wild-type mice.
Several hypotheses have been proposed to link the defects that give rise to mutated CFTR and hypersusceptibility to P. aeruginosa infection. These include decreased bacterial killing due to ineffective antimicrobial peptides (21, 23, 24), increased adherence of P. aeruginosa to respiratory epithelial cells of CF patients (25, 26), and decreased bacterial clearance from the periciliary layer above the epithelial cells due to dehydration (27, 28). Although all of these hypotheses include factors that could contribute to decreased removal of P. aeruginosa from the lungs of CF patients, none adequately explain the high-level association of P. aeruginosa infection with CF. Because we have shown previously that the CFTR-mediated internalization of P. aeruginosa by bronchial epithelial cells is specific to this respiratory pathogen (3), our hypothesis provides a molecular basis for the high-level association of this pathogenic organism with CF. We have proposed that epithelial cell desquamation of internalized P. aeruginosa is one of the mechanisms by which CFTR-mediated epithelial cell uptake promotes clearance of P. aeruginosa from the respiratory tract (1, 3). A similar mechanism has been proposed for elimination of Escherichia coli from the bladder (29). Although when viewed at a single time interval the overall level of lung cell internalization of P. aeruginosa is low, it is likely that over time a significant portion of the infectious inoculum becomes internalized. More relevant to human immunity to P. aeruginosa is the likelihood that natural exposure to P. aeruginosa is usually at levels much lower than that used to infect the mice. This would represent a situation where the absolute, not relative, level of internalization might be the key factor in high-level resistance to P. aeruginosa infection. The scanning electron micrographs obtained in this study provide evidence in an in vivo setting that epithelial cell ingestion of P. aeruginosa takes place in animals with wild-type CFTR alleles.
To ensure that P. aeruginosa interactions with the respiratory epithelium, including attachment and internalization, were comparable in primate and human tissues, we also used scanning electron microscopy to study the interaction of this microorganism with the tracheas of monkeys and compared the results with those from mice. The uninfected tracheal epithelial surface from both animals contained typical areas of mostly ciliated epithelial cells, although somewhat more ciliated cells were visible in the mouse tracheas. We were careful to document that these surfaces were undamaged by the fixation procedure. After infection with P. aeruginosa, up to 5% of the epithelial cell layer was disrupted, as characterized by the loss of cilia and denudation. P. aeruginosa appeared to interact with the tracheal cells in these areas, and bacterial attachment and internalization occurred primarily on the edge of the denuded areas. We also observed shedding of epithelial cells loaded with bacteria from the tracheal epithelial layer, apparently in the process of being lifted and cleared from the respiratory tract. The binding of P. aeruginosa to injured or regenerating areas of the epithelial surface has been observed in previous studies (30, 31, 32, 33). Our results are consistent with these observations but extend them by showing that P. aeruginosa itself, delivered either to an intact murine respiratory tract via nasal application or to an explanted monkey trachea, can cause sufficient damage to the epithelial surface to promote binding and epithelial cell uptake.
Overall, we present data that P. aeruginosa is internalized
by murine and primate epithelial cells with wild-type CFTR following in
vivo and in situ infection, respectively. We observed cells loaded with
P. aeruginosa being shed from the epithelial surface in
vivo, which provided a cellular explanation for one mechanism whereby
P. aeruginosa may be cleared following uptake. Transgenic CF
mice had impaired internalization of P. aeruginosa into
respiratory tissues following nasal or tracheal application of
bacteria, resulting in a significantly higher number of bacteria in the
lungs of the transgenic CF mouse strains. This occurred in both inbred
and outbred transgenic mouse lines homozygous for the F508 allele of
Cftr and in S489X
Cftr-FABPhuCftr mice, which have
no intact CFTR protein. Thus, we substantiated in an in vivo transgenic
mouse system that the lack of CFTR-mediated ingestion of P. aeruginosa
by respiratory epithelial cells leads to increased bacterial burdens in
the lungs. We conclude that effective clearance of P.
aeruginosa by CFTR-mediated internalization is crucial for
resistance to infection and that the lack of this host factor in
transgenic CF mice leads to increased bacterial levels in the lung, an
indicator of the basis for the hypersusceptibility of CF patients to
P. aeruginosa infection.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gerald B. Pier, Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115. E-mail address: gpier{at}channing.harvard.edu
3 Abbreviations used in this paper: CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; hu, human; 5/6 CFDA, 5-(6)-carboxyfluorescein diacetate; PLSD, probable least square differences; SP-C, surfactant protein C; FABP, fatty acid binding protein.
Received for publication January 21, 2001. Accepted for publication April 13, 2001.
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