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Infection and Immunity, November 2008, p. 4959-4967, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00664-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Diminished ICAM-1 Expression and Impaired Pulmonary Clearance of Nontypeable Haemophilus influenzae in a Mouse Model of Chronic Obstructive Pulmonary Disease/Emphysema

Bing Pang,¹ Wenzhou Hong,^1, Shayla L. West-Barnette,^1, Nancy D. Kock,² and W. Edward Swords^1*

Departments of Microbiology and Immunology,¹ Pathology and Comparative Medicine, Wake Forest University Health Sciences, Winston-Salem, North Carolina²

Received 28 May 2008/ Returned for modification 2 July 2008/ Accepted 3 September 2008

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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.

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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 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 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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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.

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TABLE 1. NTHi strains used in this study

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-µl 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.

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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, x4 for left panels and x40 for right panels.

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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.

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 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 µm) 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 others (6). For visualization of NTHi bacteria, portions of fixed lung tissue samples were rinsed with 1x 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-µm 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.

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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 elastase-treated 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 postinfection. 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 time-point (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.

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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).

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 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 higher levels later after infection for the elastase treatment group.

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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).

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.

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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, x20 for left panels and x40 for right panels.

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-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 µm 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).

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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 elastase-treated 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, x20 for left set of panels in C and x100 for right set of panels.

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 phosphorylcholine-positive lipooligosaccharides (licD and lic^ON 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 elastase-treated 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.

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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 elastase-treated (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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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 infection, 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 lic^ON 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.

 
We acknowledge excellent technical assistance by Gayle Foster and helpful conversations with colleagues in the WFUHS Department of Microbiology and Immunology.

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).

 
* Corresponding author. Mailing address: 5101A Gray Building, Medical Center Boulevard, Winston-Salem, NC 27157. Phone: (336) 713-5049. Fax: (336) 716-9928. E-mail: wswords{at}wfubmc.edu

Published ahead of print on 15 September 2008.

Editor: J. N. Weiser

Present address: Department of Otolaryngology, Medical College of Wisconsin, Milwaukee, WI.

Present address: NIDCD/NIH, Bethesda, MD.

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Infection and Immunity, November 2008, p. 4959-4967, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00664-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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