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Diminished ICAM-1 Expression and
Impaired Pulmonary Clearance of
Nontypeable Haemophilus influenzae in a
Mouse Model of Chronic Obstructive
Pulmonary Disease/Emphysema
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Bing Pang, Wenzhou Hong, Shayla L. West-Barnette, Nancy
D. Kock and W. Edward Swords
Infect. Immun. 2008, 76(11):4959. DOI: 10.1128/IAI.00664-08.
Published Ahead of Print 15 September 2008.
INFECTION AND IMMUNITY, Nov. 2008, p. 4959–4967
0019-9567/08/$08.00⫹0 doi:10.1128/IAI.00664-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 11
Diminished ICAM-1 Expression and Impaired Pulmonary Clearance of
Nontypeable Haemophilus influenzae in a Mouse Model of Chronic
Obstructive Pulmonary Disease/Emphysema䌤
Bing Pang,1 Wenzhou Hong,1† Shayla L. West-Barnette,1‡ Nancy D. Kock,2 and W. Edward Swords1*
Departments of Microbiology and Immunology,1 and Pathology and Comparative Medicine,2
Wake Forest University Health Sciences, Winston-Salem, North Carolina
The airways of patients with chronic obstructive pulmonary disease (COPD) are continually colonized with
bacterial opportunists like nontypeable Haemophilus influenzae (NTHi), and a wealth of evidence indicates that
changes in bacterial populations within the lung can influence the severity of COPD. In this study, we used a
murine model for COPD/emphysema to test the hypothesis that COPD affects pulmonary clearance. Mice were
treated with a pulmonary bolus of elastase, and as reported previously, the lungs of these mice were pathologically similar to those with COPD/emphysema at ⬃1 month posttreatment. Pulmonary clearance of NTHi
was significantly impaired in elastase-treated versus mock-treated mice. While histopathologic analysis revealed minimal differences in localized lung inflammation between the two groups, lower levels of intercellular
adhesion molecule 1 (ICAM-1) were observed for the airway epithelial surface of elastase-treated mice than for
those of control mice. Following infection, elastase-treated mice had lung pathology consistent with pneumonia
for as long as 72 h postinfection, whereas at the same time point, mock-treated mice had cleared NTHi and
showed little apparent pathology. Large aggregates of bacteria were observed within damaged lung tissue of the
elastase-treated mice, whereas sparse individual bacteria were observed in lungs of mock-treated mice at the
same time point postinfection. Additional infection studies showed that NTHi mutants with biofilm defects
were less persistent in the elastase-treated mice than the parent strain. These findings establish a model for
COPD-related infections and support the hypotheses that ICAM-1 promotes clearance of NTHi. Furthermore,
the data indicate that NTHi may form biofilms within the context of COPD-related infections.
healthy patients, but extends into the upper and lower airways
(23, 24, 29).
The composition of the bacterial population within the
COPD lung is extremely dynamic, with individual strains/clones
exhibiting variable persistence and with incoming strains supplanting other strains (35–39). Patients with COPD can be colonized by several different strains simultaneously, and the
length of persistence varies dramatically between the different
strains (30). Patient studies have demonstrated that some
strains of NTHi can persist within individual patients for
months or even years and that exacerbations of COPD are
significantly correlated with the acquisition of a new bacterial
strain (35, 36, 39), apparently independently of the bacterial
load (39) but in accordance with host-pathogen interactions
that may be specific to the individual bacterial strain (3). Notably, most animal models for pulmonary infection fail to
mimic the degree of bacterial persistence observed for human
patients. For example, it is well established that mice that
receive a pulmonary infection of NTHi reproducibly clear the
infection within 4 days postinoculation (33, 49, 50). There is a
need for an animal model that better reflects the persistent
infections that occur in the context of COPD.
One of the hallmarks of COPD/emphysema is tissue destruction by elastase released by neutrophils within the lung, resulting in pulmonary fibrin deposition and decreased lung volume
(41, 42). In prior studies, COPD-like conditions have been
established in mice by chronic exposure to cigarette smoke
(40–42) or by the introduction of a bolus of elastase into the
lung (15, 20, 32). In the latter model, mice treated with elastase
Chronic obstructive pulmonary disease (COPD) is a progressive lung disease that includes emphysema, chronic bronchitis, and bronchiectasis and is among the leading public
health problems worldwide (8, 34). COPD affects over 10 million adults in the United States alone (22) and is the fourth
leading cause of death in the United States (13). The estimated
total economic impact of COPD in the United States is over
$20 billion/year (44). While the primary cause of COPD is
smoking or exposure to other inhaled pollutants, the progression and severity of COPD may be promoted by opportunistic
airway infections (37). While this has been an area of some
controversy (9), it is undeniable that the management of
COPD is, at best, seriously complicated by bacterial and viral
infections (31). The agents that cause COPD-related airway
infections are found predominantly within the normal flora of
the nasopharynx and include nontypeable Haemophilus influenzae (NTHi), Streptococcus pneumoniae, Moraxella catarrhalis,
and Pseudomonas aeruginosa (25, 26, 37, 38). Patients may also
be chronically infected with H. haemolyticus, which does not
seem to be a significant pathogen but is associated with asymptomatic carriage (27). In patients with COPD, carriage of these
organisms is not limited to the nasopharynx, as is the case in
* Corresponding author. Mailing address: 5101A Gray Building,
Medical Center Boulevard, Winston-Salem, NC 27157. Phone: (336)
713-5049. Fax: (336) 716-9928. E-mail: [email protected].
† Present address: Department of Otolaryngology, Medical College
of Wisconsin, Milwaukee, WI.
‡ Present address: NIDCD/NIH, Bethesda, MD.
䌤
Published ahead of print on 15 September 2008.
4959
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Received 28 May 2008/Returned for modification 2 July 2008/Accepted 3 September 2008
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TABLE 1. NTHi strains used in this study
NTHi Strain
2019
2019
2019
2019
2019
2019
siaB strain
htrB strain
pgmB strain
licD strain
licON strain
Description
Bronchial isolate
Sialylated mutant
Lipid A acylation defect
Phosphoglucomutase mutant
Phosphorylcholine-negative mutant
Constitutive phosphorylcholinepositive mutant
Reference
or source
2
16
21
45
45
11
MATERIALS AND METHODS
Bacterial strains and growth conditions. All NTHi strains were cultivated on
supplemented brain-heart infusion (Difco) medium supplemented with NAD
(Sigma) and hemin (ICN), as described previously (45–48). NTHi 2019 is a
well-characterized strain that was originally isolated from the sputum of a patient
with chronic bronchitis (2), and all of the mutant strains were derived from this
strain background. A list of bacterial strains, along with primary references and
phenotypes, is provided in Table 1.
Elastase treatment. Healthy C57BL/6 mice were purchased from Charles
River Laboratories (Wilmington, MA). Mice were anesthetized with Avertin
(2,2,2-tribromoethanol), and a 50-␮l bolus of elastase (Sigma) suspended in
sterile PBS was instilled intratracheally into the lung. The amount of elastase
used was determined by dose-response experiments as the minimal amount of
enzyme necessary to generate lung damage consistent with COPD. Various doses
of elastase (1 to 9 units) were intratracheally instilled into mice (five/group), and
the mice were then euthanized 21 days posttreatment. Histopathologic analysis
revealed tissue fibrosis and reduction in airway space, consistent with COPD, at
3, 6, and 9 U of elastase (data not shown), with no apparent pathology in mice
that received vehicle (PBS) alone (Fig. 1 and 2). As 3 U of elastase was the
minimal dose needed to elicit COPD-like pathology, this was the amount chosen
for infection studies. Animals were allowed to recover for 21 days after elastase
treatment, before histopathology and/or infection studies were performed.
Infections. NTHi bacteria were harvested from overnight plate cultures and
suspended in PBS. Bacterial counts were estimated by optical density and suspended in PBS solution as described previously (45). The estimated bacterial
density was confirmed by plate count. Approximately 107 CFU was used to infect
mice (five animals/group). The mice were anesthetized as described before and
infected intratracheally, and the bacterial load in the inocula was confirmed by
plate count. At the times indicated, mice were euthanized, and their lungs were
FIG. 1. Elastase-treated mice have fibrotic lung damage consistent
with COPD. Mice were treated by nonsurgical intratracheal instillation
of elastase (see Materials and Methods) and allowed to recover for 21
days posttreatment. Lung tissue samples from euthanized mice receiving vehicle (PBS) (A), elastase (B), or heat-inactivated (HI) elastase
(C) were compared by hematoxylin-eosin staining of paraffin sections.
COPD-like pulmonary damage was observed for the elastase-treated
group that was not observed for either of the control groups. Magnification, ⫻4 for left panels and ⫻40 for right panels.
excised. For each animal, the left lung was homogenized and used for plate
count. Plate count data were analyzed by unpaired t test analysis with Welch’s
correction for unequal variance; groups with P values of less than 0.05 were
deemed significantly different from the control. The right lungs were fixed in 4%
paraformaldehyde-PBS for histopathology and cryosection. The elastase treatment and infection protocols were approved by the Wake Forest University
Health Sciences animal care and use committee.
Histopathology. Portions of fixed lung tissue were dehydrated and embedded
in paraffin according to standard methods. Sections (5 ␮m) were cut from
paraffin-embedded blocks with a microtome and mounted from warm water
(40°C) onto adhesive microscope slides. After serial deparaffinization and rehydration, tissue sections were stained with hematoxylin and eosin for histopathologic assessment. Stained slides were provided as a blinded set to a veterinary
pathologist (N.K.) and were scored for markers of inflammation (neutrophilic
influx, edema, etc.).
Immunostaining. To determine ICAM-1 expression, paraffin sections were
stained using monoclonal antibody 3E2, recognizing mouse ICAM-1 (BD Pharmingen) according to the manufacturer’s instructions, essentially as reported by
FIG. 2. Impaired pulmonary clearance of NTHi from elastase-treated
mice. Mice (five/group) were infected via the intratracheal route with
⬃107 CFU of NTHi 2019 and euthanized at the indicated times postinfection (see Materials and Methods). Symbols are CFU counts obtained
from homogenized lung tissue from mock-treated (filled circles) or elastase-treated mice (open circles). The dotted line indicates the lower limit
of detection, and symbols below that line indicate a mouse from which no
bacteria were recovered. Asterisks indicate groups in which bacterial
numbers were significantly higher than those in controls, as assessed by
nonparametric statistical analysis.
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exhibited lung damage consistent with COPD, including tissue
destruction within the lung, enlargement of airspaces, and fibrotic deposits within the lung alveolar spaces. We reasoned
that this model system could provide a better way to examine
bacterial clearance from the COPD lung. Therefore, we compared bacterial clearance following pulmonary infection with
NTHi in mice treated with elastase with that of controls
treated with vehicle (phosphate-buffered saline [PBS]) alone.
The results show that clearance of NTHi from the lung was
significantly impaired following elastase treatment, in accordance with the formation of large bacterial communities that
were not observed for the control mice. Furthermore, immunohistochemical analysis revealed diminished expression of intercellular adhesion molecule 1 (ICAM-1) on the airway epithelial surfaces of the elastase-treated mice following infection
compared to that of the control groups. We thus conclude that
pulmonary infections in mice with normal clearance may not
fully represent the host-pathogen interactions that determine
the outcome of infections in COPD and that the outcome of a
pulmonary bacterial infection in this setting may be determined by the interplay of host clearance events initiated by
ICAM-1 expression on the airway epithelium and by bacterial
persistence mechanisms that may include biofilm formation.
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others (6). For visualization of NTHi bacteria, portions of fixed lung tissue
samples were rinsed with 1⫻ PBS at room temperature and placed into a
Cryomold (Sakura Finetek USA, Torrance, CA). Tissue-Tek OCT compound
(Sakura Finetek USA, Torrance, CA) was added, and the blocks were frozen at
⫺70°C for 1 h. Serial 5-␮m sections were cut using an Accu-Edge low-profile
blade (Feather Safety Razor Co., Japan) at ⫺20°C and stored at ⫺70°C. Immunofluorescence staining was performed using rabbit antisera recognizing NTHi,
essentially as described previously (33). For both sets of sections, pixel quantization was performed for all tissue sections and is presented as the mean numbers of pixels for ICAM-1 or bacterial staining.
RESULTS
Pulmonary elastase treatment results in damage consistent
with COPD/emphysema and impaired bacterial clearance. To
elicit pulmonary fibrotic damage consistent with COPD, mice
were treated with a pulmonary bolus of elastase, vehicle (PBS),
or heat-inactivated elastase delivered via nonsurgical intratracheal instillation. After 21 days, mice in each group were euthanized, and their lungs were sectioned and stained with hematoxylin and eosin for histopathologic analysis. The results
clearly showed significant pulmonary damage in the elastasetreated mice, whereas there was minimal damage observed for
the control groups (Fig. 1).
The effect of this treatment on the clearance of NTHi strain
2019 from the lung was determined with pulmonary infection
studies using elastase-treated mice and mock-treated mice.
While bacterial counts obtained from lung homogenates were
comparable at an early time point (6 h postinfection), significantly higher numbers of bacteria were recovered from the
elastase-treated mice at 24 h, 48 h, and 72 h postinfection (Fig.
2). In the control group, the numbers of bacteria declined
significantly over time, and the majority of mice had pulmonary
bacterial loads below the threshold of detection by 72 h postin-
fection. Thus, we concluded that mice treated with elastase had
impaired pulmonary clearance of NTHi.
Histopathologic analysis of lung tissue from infected mice.
Lung tissue from each group of mice was embedded in paraffin,
sectioned, and stained for histopathologic assessment (as described in Materials and Methods). Stained slides were examined as a blinded set by a veterinary pathologist (N.K.) and
scored for parameters of airway inflammation, which were
compiled into a total score for inflammation. Figure 3 shows
representative images from each group at the different time
points postinfection. Notably, there was a dramatic loss of
alveolar lung tissue, along with fibrosis, observed for the elastase-treated mice even prior to infection (Fig. 3C to E). Localized tissue responses, including edema and cellular infiltrate
that included neutrophils, were observed for both groups of
animals. Total histopathology scores were compiled for each
group of sections as blinded sets. The only differences in scoring occurred with samples from the latest time point (72 h
postinfection), where inflammation remained notable in the
elastase-treated group. In contrast, all indicators of inflammation were decreased in the mock-treated group at this timepoint (Fig. 3F to G). As shown in Fig. 2, elastase-treated mice
failed to clear NTHi bacteria from the lung within 72 h postinfection. For the mock-treated mice, epithelial cells were observed sloughed into a bronchiolar lumen, and the interstitium
was mildly infiltrated by neutrophils. In contrast, the sections
from the elastase-treated mice showed severe pneumonia with
marked infiltration of neutrophils, causing consolidation of
the lung.
Histopathology scores. The stained sections were examined
as a blinded set and assigned scores (1–4) for markers of
inflammation. A composite score (1–10) was compiled based
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FIG. 3. Histopathologic assessment of lung tissue from infected animals. Lung tissue was sectioned and stained for histopathology analysis as
described in Materials and Methods. Each panel is a portion of a representative hematoxylin-eosin-stained section from the indicated group
(Control or Elastase group, left margin) at a given time point (Mock, 6 h, 24 h, 48 h, or 72 h).
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FIG. 4. Histopathology scores. Hematoxylin-eosin-stained sections from mock-treated (open bars) and elastase-treated (filled bars) mice were
examined as a blinded set and scored for markers of pulmonary inflammation (see Materials and Methods).
on the total scores. The results are depicted in Fig. 4. A significant increase in vascular degeneration was observed in the
elastase treatment group early after infection (Fig. 4A). Bronchial epithelial responses peaked later and to a greater degree
in the elastase treatment group (Fig. 4B), as was observed for
pneumonia (Fig. 4C), alveolar macrophages (Fig. 4D), and
airway inflammation (Fig. 4E). Similarly, the composite inflammation scores were higher for the elastase treatment
group at the later time points postinfection (Fig. 4F). No significant differences were noted in the number of total lymphocytes (data not shown). Taken together, these results indicate
a slower pulmonary inflammatory response that reached
VOL. 76, 2008
IMPAIRED BACTERIAL CLEARANCE IN MOUSE MODEL FOR COPD
4963
higher levels later after infection for the elastase treatment
group.
Diminished surface expression of ICAM-1 in elastase-treated
mice. ICAM-1 is expressed on the airway epithelial surface
under many different pulmonary inflammatory conditions and
serves to facilitate neutrophil recruitment into the lung (4, 7,
51, 52). Surface expression of ICAM-1 has been demonstrated to promote NTHi clearance from mouse lung (6),
although there are also indications that NTHi may utilize
ICAM-1 as a receptor for attachment to epithelial surfaces
(1). Therefore, we determined whether ICAM-1 expression
was altered in the elastase-treated mice. Paraffin sections of
lung tissue from infected mice were examined by immunostaining for ICAM-1. Figure 5 shows representative images
from light microscopic examination of tissue from each
group of animals, along with quantization of ICAM-1 staining as a percentage of total pixels in all sections. In the
control group, ICAM-1 expression increased significantly as
early as 6 h postinfection and remained elevated above the
baseline throughout the infection study. However, the level
of expression in the elastase-treated animals was markedly
lower at all time points and did not show an increase until 24
to 48 h postinfection. Therefore, based on these data, we
conclude that ICAM-1 expression is diminished and temporally changed in the elastase-treated mice.
Presence of multicellular NTHi bacterial communities
within the lungs of elastase-treated mice. To visualize NTHi
bacteria within the lungs of infected mice, cryosections were
prepared from mock-treated and elastase-treated mice at the
various time points after infection and stained with polyclonal
rabbit antisera directed against NTHi. For mock-treated animals, limited reactivity was observed that correlated in size
with individual bacteria dispersed throughout the lung tissue
taken 48 h postinfection (Fig. 6A). However, in the elastase-
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FIG. 5. Experimental COPD reduces the level and kinetics of immunohistochemical staining for ICAM-1 expression within lung tissue during
infection. ICAM-1 levels in paraffin sections of lung tissue were assessed by immunohistochemical staining with monoclonal antibody 3E2
(BD/Pharmingen) as described in Materials and Methods. Sections were counterstained with hematoxylin. Panels show representative sections
from the groups indicated at left. Graphs depict total ICAM-1 staining in all sections examined in the group as a percentage of total pixels.
Magnification, ⫻20 for left panels and ⫻40 for right panels.
4964
PANG ET AL.
INFECT. IMMUN.
treated animals, larger regions of reactivity were visible in
discrete locations within the lung tissue at this time point (Fig.
6B). Quantization of the fluorescence from microscopy images
from all infection groups showed an ⬃40-fold increase in bacterial density within the lung tissue of elastase-treated mice at
this time point (Fig. 6). Examination of sections at a higher
magnification revealed that these regions were more than 10
␮m in diameter, which correlates in size with a multicellular
community of NTHi bacteria (Fig. 6C). Moreover, as clearly
visible by differential interference contrast/Nomarski imaging,
the NTHi communities were present in damaged regions of the
lung with fibrotic deposits. Paired histopathologic staining of
serial sections immediately adjacent to those stained for immunofluorescence revealed the presence of neutrophils surrounding many of these communities (Fig. 6C).
Infection studies using mutant NTHi strains. To further
clarify the role(s) of specific persistence-related surface moieties in NTHi persistence within the elastase-treated mice, we
performed infection studies using a panel of mutant strains
(Fig. 7). For the purpose of comparison, we chose the 48-h
time point postinfection, as bacteria were consistently recovered from both the control and elastase-treated mice with
maximal differences at this time point. As in the initial studies
shown in Fig. 1, significantly higher numbers of NTHi 2019
bacteria were recovered from elastase-treated mice. However,
the counts from control and elastase-treated mice infected with
a sialylated (siaB) mutant strain were indistinguishable. Similarly, counts from mice infected with a “rough” mutant lacking
the oligosaccharide portion of the LOS moiety (pgmB mutant)
or with mutants with altered expression of phosphorylcholinepositive lipooligosaccharides (licD and licON mutants, the latter harboring an in-frame deletion of the CAAT repeat region
in licA), were indistinguishable between the control and elastase-treatment groups. However, mice infected with the NTHi
2019 htrB strain, which has an underacylated lipid A, had
significantly higher resistance to clearance in the elastasetreated group than in the control group, similar to the parental
strain. It is notable that all of the mutations that affected the
persistence of the elastase treatment group affected the oligosaccharide portion of the lipooligosaccharides on the NTHi
surface. Therefore, based on these data, we conclude that
moieties contained within the carbohydrate portion of the endotoxins on the NTHi surface promote persistence within elastase-treated mice. The implications of these data for the
role(s) of biofilms in the increased persistence phenotype will
be further outlined in the discussion.
DISCUSSION
While it is clear that patients with COPD/emphysema have
increased susceptibility to many respiratory pathogens, there
remains a need for a better understanding of the mechanisms
for this susceptibility. In this study, we adapted an existing
model of COPD for infection studies. The results clearly show
that following pulmonary treatment with elastase to elicit a
COPD-like condition, mice had a significant clearance defect
for NTHi bacteria compared to mock-treated mice. This clearance defect was correlated with a delayed expression of
ICAM-1 on the airway epithelial surfaces, a host response that
promotes the influx of neutrophils and the resolution of NTHi
infection (6). It is notable that the basal level of ICAM-1
expression observed for the elastase-treated mice was significantly higher than that observed for mock-treated mice.
ICAM-1 appears to play several roles in NTHi and viral infec-
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FIG. 6. Immunofluorescent staining reveals multicellular bacterial communities within lung tissue of elastase-treated animals. Lung tissue was
cryosectioned (see Materials and Methods) and stained with rabbit anti-H. influenzae sera (53) and fluorescent antibody conjugate (Jackson
Laboratories). Panels A and B show merged differential interference-contrast/fluorescent images from a mock-treated animal (A) and elastasetreated animal (B) 48 h postinfection. The graph depicts quantified bacterial staining as the percentage of total pixels and was obtained from
sections of tissue from all animals. Panels in C show sequential sections from the same tissue block stained to show the distribution of bacteria
(fluorescent image marked “NTHi”) and cellular infiltrate within the lung (light microscopy image marked “H & E”). Magnification, ⫻20 for left
set of panels in C and ⫻100 for right set of panels.
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4965
tion, including not only an essential role in the clearance of
pathogens but also as a receptor for bacterial and viral adherence (1). Thus, it is possible that the low-level, diffuse expression of ICAM-1 in the elastase-treated mice served to facilitate
bacterial adherence to the damaged epithelial surfaces within
the damaged regions of the lung, in addition to the observed
delay in pulmonary inflammation and neutrophilic influx
Therefore, our results may be consistent with multiple roles for
ICAM-1 expression in pulmonary infection with NTHi.
The presence of multicellular NTHi communities observed
within the lung tissue of the elastase-treated mice also merits
comment. Like many mucosal pathogens, NTHi bacteria form
biofilms during chronic infections, and we and other groups
have demonstrated that these biofilms are correlated with bacterial resistance to clearance in vivo (10, 11, 17–19, 48). Murphy and colleagues have demonstrated that NTHi peroxiredoxin is found within sputa and other samples from patients
with COPD (28). Since peroxiredoxin has increased expression
in NTHi biofilms, these results were suggestive of a biofilm
mode of growth for NTHi within the COPD lung. Our work
has demonstrated that the presence of specific lipooligosaccharide glycoforms containing sialic acid and phosphorylcholine
promotes biofilm formation in laboratory models, as well as in
animal models (10, 11, 48). Thus, the finding that the parental
strain has enhanced persistence within the elastase-treated
mouse lung, whereas sialylated and phosphorylcholine-deficient bacteria do not, is consistent with a key role for biofilm
formation in this phenotype. Likewise, the pgmB mutant, lacking all oligosaccharide structures, is sialylated and has a biofilm
defect in vivo (19). Alternative explanations for the persistence
defects observed for these strains include more efficient killing
by complement or other bactericidal host factors, which has
been reported for the siaB mutants (5, 12, 19). If this were the
case, one would expect a more severe defect in the control
group than was observed. On that note, the results obtained
with the htrB mutant are somewhat surprising given this
strain’s susceptibility to defensins in the lung (43, 46). Regardless, these data clearly point toward the oligosaccharide portion of the lipooligosaccharide as a determinant of resistance
to clearance in the mouse COPD model system. It is also
noteworthy that no enhancement was seen in the infection
studies with the licON mutant strain (Fig. 7). Whereas our prior
work has clearly demonstrated that this strain has increased
biofilm density (11), it is also clear that in some disease settings, not only the presence but the phase variation of the lic1
system is required (14). This may indicate that phosphorylcholine is advantageous only within certain windows of the disease
process. Furthermore, it should be noted that the requirements
for persistence and/or virulence within the lung and middle ear
may be subtly or even dramatically different.
In summary, we have used a mouse model for COPD to
demonstrate that ICAM-1 expression by the host and biofilm
formation by the pathogen are important in determining the
outcome of pulmonary infections with NTHi in the context of
experimental COPD in a murine model. As the availability of
a relevant animal model has been lacking for COPD-related
infections, the results of this study provide a means to test the
biofilm hypothesis as well as other fundamental hypotheses
regarding the role of opportunistic pathogens in the exacerbation of COPD.
ACKNOWLEDGMENTS
We acknowledge excellent technical assistance by Gayle Foster and
helpful conversations with colleagues in the WFUHS Department of
Microbiology and Immunology.
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FIG. 7. Clearance of NTHi mutants from mock-treated and elastase-treated mice. Mice were treated with elastase and infected as described
in the legend to Fig. 1. Symbols represent CFU counts recovered from homogenized lung tissue from mock-treated (filled circles) and elastasetreated (open circles) animals 48 h postinfection. The dotted line indicates the lower limit of detection, and symbols below that line indicate a
mouse from which no bacteria were recovered. Horizontal bars represent median values, and error bars represent standard errors of the mean for
each group. Asterisks indicate groups in which bacterial numbers were significantly higher than in controls, as assessed by nonparametric statistical
analysis.
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INFECT. IMMUN.
This work was supported by a grant from NIH/NIAID (AI054425; to
W.E.S.).
Shayla West-Barnette was supported by an individual NIH fellowship (AI061830).
23.
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Editor: J. N. Weiser
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